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Interaction between the 67 kilodalton metastasis-associated laminin receptor and laminin
Vittoria Cioce; Inger M K Margulies; Mark E Sobel; Vincent Castronovo
Interaction between the 67 kilodalton metastasis-associated laminin receptor and laminin. Normal and neoplastic cells interact with laminin via a variety of cell surface proteins. The specific binding sites on laminin for each particular cell surface laminin-binding protein have not yet been identified. In this study, the interaction between laminin and the high affinity metastasis-associated 67 kD laminin receptor (67 LR)
Laminin receptor on human breast carcinoma cells.
Terranova, V P; Rao, C N; Kalebic, T; Margulies, I M; Liotta, L A
Human MCF-7 breast carcinoma cells possess a receptor-like moiety on their surface that has a high binding affinity (Kd = 2 nM) for laminin, a glycoprotein localized in basement membranes. Laminin preferentially stimulates (8-fold) MCF-7 cells to attach to type IV (basement membrane) collagen, whereas fibronectin stimulates attachment only 2-fold for these cells on type I collagen. The attachment properties of two other human breast carcinoma cell lines to type IV collagen were also studied. The attachment of ZR-75-1 cells was stimulated 4-fold by laminin and 5-fold by fibronectin, whereas T47-D cell attachment was stimulated 2-fold by laminin and 7-fold by fibronectin. By employing protease-derived fragments of laminin, the major domains of the laminin molecule that participate in MCF-7 cell attachment to type IV collagen were identified. The whole laminin molecule has the configuration of a four-armed cross with three short arms and one long arm. A major cell-binding domain was found to reside near the intersection point of the short arms, and the type IV collagen-binding domain was associated with the globular end regions of the short arms. The receptor for laminin on the surface of these tumor cells may be involved in the initial interaction of tumor cells via laminin with the vascular basement membrane to facilitate invasion and subsequent promotion of metastasis. Images PMID:6300843
Presence of Laminin Receptors in Staphylococcus aureus
NASA Astrophysics Data System (ADS)
Lopes, J. D.; Dos Reis, M.; Brentani, R. R.
A characteristic feature of infection by Staphylococcus aureus is bloodstream invasion and widespread metastatic abscess formation. The ability to extravasate, which entails crossing the vascular basement membrane, appears to be critical for the organism's pathogenicity. Extravasation by normal and neoplastic mammalian cells has been correlated with the presence of specific cell surface receptors for the basement membrane glycoprotein laminin. Similar laminin receptors were found in Staphylococcus aureus but not in Staphylococcus epidermidis, a noninvasive pathogen. There were about 100 binding sites per cell, with an apparent binding affinity of 2.9 nanomolar. The molecular weight of the receptor was 50,000 and pI was 4.2. Eukaryotic laminin receptors were visualized by means of the binding of S. aureus in the presence of laminin. Prokaryotic and eukaryotic invasive cells might utilize similar, if not identical, mechanisms for invasion.
Laminin Polymerization Induces a Receptor-Cytoskeleton Network
Holly Colognato; Donald A. Winkelmann; Peter D. Yurchenco
The transition of laminin from a monomeric to a polymerized state is thought to be a crucial step in the development of basement membranes and in the case of skeletal muscle, mutations in laminin can result in severe muscular dystrophies with basement mem- brane defects. We have evaluated laminin polymer and receptor interactions to determine the requirements for laminin assembly
Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin.
Wewer, U M; Liotta, L A; Jaye, M; Ricca, G A; Drohan, W N; Claysmith, A P; Rao, C N; Wirth, P; Coligan, J E; Albrechtsen, R
The human laminin receptor was purified and molecularly cloned to investigate its biosynthetic regulation. Laminin receptor from normal and neoplastic tissue was preparatively affinity purified to homogeneity based on the high affinity of the receptor for laminin. The apparent molecular weight of the receptor from different carcinoma sources and from normal placental tissue is in the range of 68-72 kDa. Isoelectric focusing and two-dimensional gel electrophoresis indicated that the receptor protein consists of one major polypeptide chain with a pI value of 6.4 +/- 0.2. Laminin receptor cDNA clones were isolated after screening a human endothelial lambda gt11 cDNA library with a monoclonal antibody directed against a domain of the laminin receptor involved in ligand binding. Definitive identification of the cDNA clones was based on comparison of cDNA sequence with the amino acid sequence of a cyanogen bromide-generated octapeptide of purified placental laminin receptor. The laminin receptor mRNA is approximately 1700 bases long. The level of laminin receptor mRNA in a variety of human carcinoma-derived cell lines correlated with the number of laminin receptors on the cell surfaces of those cells. This suggests that the amount of laminin receptor mRNA may be a rate-limiting control step in the biosynthesis of the laminin receptor, and hence in the regulation of cellular attachment to basement membranes via laminin. Images PMID:2429301
67-kDa laminin receptor-dependent protein phosphatase 2A (PP2A) activation elicits melanoma-specific antitumor activity overcoming drug resistance.
Tsukamoto, Shuntaro; Huang, Yuhui; Umeda, Daisuke; Yamada, Shuhei; Yamashita, Shuya; Kumazoe, Motofumi; Kim, Yoonhee; Murata, Motoki; Yamada, Koji; Tachibana, Hirofumi
The Ras/Raf/MEK/ERK pathway has been identified as a major, druggable regulator of melanoma. Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, resulting in constitutive melanoma hyperproliferation. A selective BRAF inhibitor showed remarkable clinical activity in patients with mutated BRAF. Unfortunately, most patients acquire resistance to the BRAF inhibitor, highlighting the urgent need for new melanoma treatment strategies. Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) inhibits cell proliferation independently of BRAF inhibitor sensitivity, suggesting that increased understanding of the anti-melanoma activity of EGCG may provide a novel therapeutic target. Here, by performing functional genetic screening, we identified protein phosphatase 2A (PP2A) as a critical factor in the suppression of melanoma cell proliferation. We demonstrated that tumor-overexpressed 67-kDa laminin receptor (67LR) activates PP2A through adenylate cyclase/cAMP pathway eliciting inhibitions of oncoproteins and activation of tumor suppressor Merlin. Activating 67LR/PP2A pathway leading to melanoma-specific mTOR inhibition shows strong synergy with the BRAF inhibitor PLX4720 in the drug-resistant melanoma. Moreover, SET, a potent inhibitor of PP2A, is overexpressed on malignant melanoma. Silencing of SET enhances 67LR/PP2A signaling. Collectively, activation of 67LR/PP2A signaling may thus be a novel rational strategy for melanoma-specific treatment. PMID:25294877
Sasaki, Takako; F�ssler, Reinhard; Hohenester, Erhard
Laminin-1 is emerging as the key molecule in early embryonic basement membrane assembly. Here we review recent insights into its functions gained from the synergistic application of genetic and structural methods. PMID:15037599
Human neutrophil adherence to laminin in vitro. Evidence for a distinct neutrophil integrin receptor for laminin
We used mAbs against polymorphonuclear leukocyte (PMN) surface proteins to investigate the mechanisms by which stimulated human neutrophils (PMNs) adhere in vitro to laminin, the major glycoprotein of mammalian basement membrane. mAb IB4, which is directed against the common beta 2 chain of the CD11/CD18, only partially inhibited the adherence of PMA- stimulated PMNs to both laminin and to subendothelial matrices. In contrast, IB4 completely inhibited PMA-stimulated PMN adherence to gelatin, fibronectin, collagen IV, and endothelial cell monolayers. PMA- stimulated PMNs from a patient with severe congenital CD11/CD18 deficiency also adhered to laminin, but not to gelatin or endothelial cell monolayers. Therefore, PMA-stimulated PMNs adhere to laminin by both CD11/CD18-dependent and CD11/CD18-independent mechanisms. Expression of CD11/CD18-independent adherence to laminin was agonist dependent, occurring after stimulation with the calcium ionophore A23187 and recombinant TNF-alpha, but not with the chemotactic factors FMLP, platelet activating factor, or recombinant human C5a. Expression of CD11/CD18-independent adherence was also divalent cation dependent, occurring in the presence of Mg2+ but not Ca2+ as the sole added divalent cation. The mAbs AIIB2 and 13, which are directed against the beta 1 subunit of the VLA integrins, significantly inhibited the CD11/CD18-independent adherence of normal PMNs to laminin, and completely abolished the adherence of CD11/CD18-deficient PMNs to laminin. Both anti-beta 1 mAbs bound to PMNs, as demonstrated by flow cytometry, and immunoprecipitated a membrane molecule of Mr 130,000 daltons from 125I-labeled, detergent-solubilized PMNs. These data suggest that human PMNs possess beta 1 and beta 2 classes of integrins, and that both mediate PMN adherence. PMID:1969920
Laminin receptor on platelets is the integrin VLA6
Arnoud Sonnenberg; Piet W. Modderman; Frans Hogervorst
Adhesion of platelets to the subendothelial matrix of an injured vessel wall is an essential step in triggering the formation of a haemostatic plug. Fibronectin, collagen and laminin are three major components of the subendothelial matrix which support platelet adhesion1. Receptors for fibronectin and collagen have been identified on platelets and are included in the integrin family2-11. Here we report
Epigallocatechin Gallate (EGCG) Suppresses Lipopolysaccharide-Induced Toll-like Receptor 4 (TLR4) Activity via 67 kDa Laminin Receptor (67LR) in 3T3-L1 Adipocytes.
Bao, Suqing; Cao, Yanli; Zhou, Haicheng; Sun, Xin; Shan, Zhongyan; Teng, Weiping
Obesity-related insulin resistance is associated with chronic systemic low-grade inflammation, and toll-like receptor 4 (TLR4) regulates inflammation. We investigated the pathways involved in epigallocatechin gallate (EGCG) modulation of insulin and TLR4 signaling in adipocytes. Inflammation was induced in adipocytes by lipopolysaccharide (LPS). An antibody against the 67 kDa laminin receptor (67LR, to which EGCG exclusively binds) was used to examine the effect of EGCG on TLR4 signaling, and a TLR4/MD-2 antibody was used to inhibit TLR4 activity and to determine the insulin sensitivity of differentiated 3T3-L1 adipocytes. We found that EGCG dose-dependently inhibited LPS stimulation of adipocyte inflammation by reducing inflammatory mediator and cytokine levels (IKK?, p-NF-?B, TNF-?, and IL-6). Pretreatment with the 67LR antibody prevented EGCG inhibition of inflammatory cytokines, decreased glucose transporter isoform 4 (GLUT4) expression, and inhibited insulin-stimulated glucose uptake. TLR4 inhibition attenuated inflammatory cytokine levels and increased glucose uptake by reversing GLUT4 levels. These data suggest that EGCG suppresses TLR4 signaling in LPS-stimulated adipocytes via 67LR and attenuates insulin-stimulated glucose uptake associated with decreased GLUT4 expression. PMID:25732404
Laminin E8 alveolarization site: heparin sensitivity, cell surface receptors, and role in cell spreading
Laminin E8 alveolarization site: heparin sensitivity, cell surface receptors, and role in cell. Laurie, Roy C. Ogle, and Gordon W. Laurie. Laminin E8 alveolarization site: heparin sensitivity, cell for lung alveolar formation in vitro (M. L. Matter and G. W. Laurie. J. Cell Biol. 124: 1083-1090, 1994
Examination of the Role of the Urokinase Receptor in Human Colon Cancer Mediated Laminin Degradation1
Warren Schlechte; Genesio Murano; Douglas Boyd
The relevance of urokinase receptors to urokinase-mediated laminin degradation was investigated in cultured colon cancer. Six colon cancer cell lines degraded laminin in a plasminogen-dependent manner. The ability of the individual cell lines to cleave the glycoprotein correlated well (\\/• = 0.9242) with the amount of urokinase recovered from the cell surface by a mild acid treatment. A radioreceptor assay
Chemical inhibition of prometastatic lysyl-tRNA synthetase-laminin receptor interaction.
Kim, Dae Gyu; Lee, Jin Young; Kwon, Nam Hoon; Fang, Pengfei; Zhang, Qian; Wang, Jing; Young, Nicolas L; Guo, Min; Cho, Hye Young; Mushtaq, Ameeq Ul; Jeon, Young Ho; Choi, Jin Woo; Han, Jung Min; Kang, Ho Woong; Joo, Jae Eun; Hur, Youn; Kang, Wonyoung; Yang, Heekyoung; Nam, Do-Hyun; Lee, Mi-Sook; Lee, Jung Weon; Kim, Eun-Sook; Moon, Aree; Kim, Kibom; Kim, Doyeun; Kang, Eun Joo; Moon, Youngji; Rhee, Kyung Hee; Han, Byung Woo; Yang, Jee Sun; Han, Gyoonhee; Yang, Won Suk; Lee, Cheolju; Wang, Ming-Wei; Kim, Sunghoon
Lysyl-tRNA synthetase (KRS), a protein synthesis enzyme in the cytosol, relocates to the plasma membrane after a laminin signal and stabilizes a 67-kDa laminin receptor (67LR) that is implicated in cancer metastasis; however, its potential as an antimetastatic therapeutic target has not been explored. We found that the small compound BC-K-YH16899, which binds KRS, impinged on the interaction of KRS with 67LR and suppressed metastasis in three different mouse models. The compound inhibited the KRS-67LR interaction in two ways. First, it directly blocked the association between KRS and 67LR. Second, it suppressed the dynamic movement of the N-terminal extension of KRS and reduced membrane localization of KRS. However, it did not affect the catalytic activity of KRS. Our results suggest that specific modulation of a cancer-related KRS-67LR interaction may offer a way to control metastasis while avoiding the toxicities associated with inhibition of the normal functions of KRS. PMID:24212136
Chemical inhibition of prometastatic lysyl-tRNA synthetase�laminin receptor interaction
Kim, Dae Gyu; Lee, Jin Young; Kwon, Nam Hoon; Fang, Pengfei; Zhang, Qian; Wang, Jing; Young, Nicolas L.; Guo, Min; Cho, Hye Young; Mushtaq, AmeeqUl; Jeon, Young Ho; Choi, Jin Woo; Han, Jung Min; Kang, Ho Woong; Joo, Jae Eun; Hur, Youn; Kang, Wonyoung; Yang, Heekyoung; Nam, Do-Hyun; Lee, Mi-Sook; Lee, Jung Weon; Kim, Eun-Sook; Moon, Aree; Kim, Kibom; Kim, Doyeun; Kang, Eun Joo; Moon, Youngji; Rhee, Kyung Hee; Han, Byung Woo; Yang, Jee Sun; Han, Gyoonhee; Yang, Won Suk; Lee, Cheolju; Wang, Ming-Wei; Kim, Sunghoon
Lysyl-tRNA synthetase (KRS), a protein synthesis enzyme in the cytosol, relocates to the plasma membrane after a laminin signal and stabilizes a 67-kDa laminin receptor (67LR) that is implicated in cancer metastasis; however, its potential as an antimetastatic therapeutic target has not been explored. We found that the small compound BC-K-YH16899, which binds to KRS, impinged on interaction of KRS with 67LR and suppressed metastasis in 3 different mouse models. The compound inhibited KRS�67LR interaction in two ways. First, it directly blocked the association between KRS and 67LR. Second, it suppressed the dynamic movement of the N-terminal extension of KRS and reduced membrane localization of KRS. However, it did not affect the catalytic activity of KRS. Our results suggest that specific modulation of a cancer-related KRS�67LR interaction may offer a way to control metastasis while avoiding the toxicities associated with inhibition of the normal functions of KRS. PMID:24212136
Green tea polyphenols precondition against cell death induced by oxygen-glucose deprivation via stimulation of laminin receptor, generation of reactive oxygen species, and activation of protein kinase C?.
Gundimeda, Usha; McNeill, Thomas H; Elhiani, Albert A; Schiffman, Jason E; Hinton, David R; Gopalakrishna, Rayudu
As the development of synthetic drugs for the prevention of stroke has proven challenging, utilization of natural products capable of preconditioning neuronal cells against ischemia-induced cell death would be a highly useful complementary approach. In this study using an oxygen-glucose deprivation and reoxygenation (OGD/R) model in PC12 cells, we show that 2-day pretreatment with green tea polyphenols (GTPP) and their active ingredient, epigallocatechin-3-gallate (EGCG), protects cells from subsequent OGD/R-induced cell death. A synergistic interaction was observed between GTPP constituents, with unfractionated GTPP more potently preconditioning cells than EGCG. GTPP-induced preconditioning required the 67-kDa laminin receptor (67LR), to which EGCG binds with high affinity. 67LR also mediated the generation of reactive oxygen species (ROS) via activation of NADPH oxidase. An exogenous ROS-generating system bypassed 67LR to induce preconditioning, suggesting that sublethal levels of ROS are indeed an important mediator in GTPP-induced preconditioning. This role for ROS was further supported by the fact that antioxidants blocked GTPP-induced preconditioning. Additionally, ROS induced an activation and translocation of protein kinase C (PKC), particularly PKC? from the cytosol to the membrane/mitochondria, which was also blocked by antioxidants. The crucial role of PKC in GTPP-induced preconditioning was supported by use of its specific inhibitors. Preconditioning was increased by conditional overexpression of PKC? and decreased by its knock-out with siRNA. Collectively, these results suggest that GTPP stimulates 67LR and thereby induces NADPH oxidase-dependent generation of ROS, which in turn induces activation of PKC, particularly prosurvival isoenzyme PKC?, resulting in preconditioning against cell death induced by OGD/R. PMID:22879598
Follicular dendritic cells adhere to fibronectin and laminin fibers via their respective receptors.
Ogata, T; Yamakawa, M; Imai, Y; Takahashi, T
The expression of adhesion molecules on human tonsillar follicular dendritic cells (FDCs) in the secondary lymphoid follicle (LF) in vivo and in vitro was investigated using cryostat sections and cytospin preparations of FDCs isolated with a magnetic cell sorter, respectively. FDCs were immunochemically positive for Mac-1 (CD11b), sialyl-Lex (CD15s), CD22, integrin beta 1 (CD29), CD40, very late activation antigen (VLA)-alpha 3 (CD49c), VLA-alpha 5 (CD49e), VLA-alpha 6 (CD49f), intercellular adhesion molecule (ICAM)-3 (CD50), ICAM-1 (CD54), B7 (CD80), and vascular cell adhesion molecule (VCAM)-1 (CD106). With respect to ligands on B cells for these adhesion molecules, the CD11b-CD54, CD50-leukocyte function-associated molecule (LFA)-1 (CD11a/18), and CD106-VLA-4 (CD49d/29) interactions in the apical light (ALZ) and basal light (BLZ) zones; the CD15s-L-selectin (CD62L) and CD106-CD49d/29 interactions in the mantle zone; and the CD54-CD11a/18 interaction in the entire LF may participate in FDC-B cell adhesion. Namely, the adhesion molecules participating in FDC-B cell interactions may differ in each of the five zones. Furthermore, the immunochemical evidence that FDCs were fibronectin (VLA-5, CD49e/29) and laminin (VLA-6, CD49l/29) receptor-positive discussed above was confirmed by immunoelectron microscopy and binding assays. Immunoelectron microscopy revealed fibers surrounded by cytoplasmic FDC extensions that were CD29-, CD49e-, and CD49f-positive. In the binding assays, the numbers of FDCs bound to fibronectin- and laminin-coated dishes and LFs of cryostat sections of human tonsils were reduced markedly by pretreatment with monoclonal antibodies against CD29, CD49e, and CD49f. These data indicate clearly that FDCs bind to reticulin and laminin fibers in LFs via their respective receptors. PMID:8874197
Regulation of the laminin beta 1 (LAMB1), retinoic acid receptor beta, and bone morphogenetic protein 2 genes in mutant F9 teratocarcinoma cell lines partially deficient in cyclic AMP-dependent protein kinase activity.
Shen, J; Li, C; Gudas, L J
We stably transfected a gene encoding a dominant negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) into F9 cells and generated cell lines partially deficient in PKA activity (DN16 and DN19). In these cell lines, the retinoic acid (RA) receptor beta and laminin beta(1) chain (LAMB1) genes were regulated normally by RA alone, indicating that in the absence of exogenous modulation of cAMP levels, the PKA signaling pathway does not seem to play a major role in the RA-associated regulation of these genes. However, alterations in gene regulation were observed when the mutant cell lines were treated with a combination of RA and cAMP analogues. Moreover, in the DN16 cell line, which exhibits the lowest PKA activity among the mutant cell lines [22% of wild type (WT) at 1 microM cAMP], there was a significant decrease in the cAMP-associated activation of the LAMB1 gene DNase I hypersensitivity site 2 enhancer, as measured by chloramphenicol acetyl transferase assays. Using electrophoretic mobility shift assays, less protein binding was observed at one of the motifs (C2) within this enhancer region in the DN16 cells as compared to the F9 WT cells after treatment of the cells with RA and cAMP analogues for 24 h. Furthermore, no increase in C2 binding was observed when extracts from RA-treated F9 ST or DN16 cells were subjected to in vitro phosphorylation, suggesting that PKA is involved in the induction of the C2-binding protein in RA-treated cells. In contrast to the results with RA receptor beta and LAMB1, the effects of cAMP analogues on the RA-associated regulation of the bone morphogenetic protein 2 gene were not altered in the cell lines that exhibited reduced PKA activity. These results suggest that a partial reduction in PKA activity is not sufficient to abrogate the effects of cAMP analogues on all of the genes regulated by RA. PMID:9419418
Expression of Laminin Isoforms, Receptors and Binding Proteins Unique to Nucleus Pulposus Cells of Immature Intervertebral Disc
Chen, Jun; Jing, Liufang; Gilchrist, Christopher L; Richardson, William J; Fitch, Robert D; Setton, Lori A
Intervertebral disc (IVD) disorders are believed to be related to aging-related cell loss and phenotypic changes, as well as biochemical and structural changes in the extracellular matrix of the nucleus pulposus (NP) region. Previously, we found that the laminin ?1 chain was more highly expressed in immature NP porcine tissues, in parallel with the expression pattern for a laminin receptor, integrin ?6 subunit, as compared to adjacent anulus fibrosus region; suggesting that cell-matrix interactions may be unique to the immature NP. However, the identity of laminin isoforms specific to immature or mature NP tissues, their associated receptors and functional significance are still poorly understood. In this study, we evaluated the zonal-specific expression of the laminin chains, receptors (i.e. integrins) and other binding proteins in immature tissue and isolated cells of rat, porcine and human intervertebral disc, towards the goal of revealing features of cellular environment and cell-matrix interactions in the immature NP. Results from both immuno-histochemical staining and flow cytometry analysis found that NP cells expressed higher levels of the laminin ?5 chain, laminin receptors (integrin ?3, ?6, ?4 subunit and CD239) and related binding proteins (CD151), as compared to cells from adjacent anulus fibrosus. These differences suggest that laminin interactions with NP cells are distinct from that of the anulus fibrosus, and that laminins may be important contributors to region-specific IVD biology. The revealed laminin isoforms, their receptors and related binding proteins may be used as distinguishing features of these immature NP cells in the intervertebral disc. PMID:19863388
Characterization of a mouse laminin receptor gene homologous to the human blood group Lutheran gene
C�cile Rahuel; Yves Colin; Dominique Goossens; P. Gane; W. El Nemer; J. P. Cartron; C. Le Van Kim
The human Lutheran (Lu) blood group antigens are carried by two glycoproteins (gps) that belong to the immunoglobulin (Ig)\\u000a superfamily. These gps represent adhesion molecules that function as the unique erythroid receptors for laminin. We report\\u000a here the cloning and functional expression of the orthologous mouse Lu mRNA as well as the genomic organization of the mouse Lu gene. The
Laminin isoforms differentially regulate adhesion, spreading, proliferation, and ERK activation of h1 integrin-null cells
Campbell, Kevin P.
of h1 integrin-null cells Yamato Kikkawaa , Hao Yua , Elke Generscha , Noriko Sanzenb , Kiyotoshi The presence of many laminin receptors of the h1 integrin family on most cells makes it difficult to define the biological functions of other major laminin receptors such as integrin a6h4 and dystroglycan. We therefore
Downregulation of the non-integrin laminin receptor reduces cellular viability by inducing apoptosis in lung and cervical cancer cells.
Moodley, Kiashanee; Weiss, Stefan F T
The non-integrin laminin receptor, here designated the 37-kDa/67-kDa laminin receptor (LRP/LR), is involved in many physiologically relevant processes, as well as numerous pathological conditions. The overexpression of LRP/LR on various cancerous cell lines plays critical roles in tumour metastasis and angiogenesis. This study investigated whether LRP/LR is implicated in the maintenance of cellular viability in lung and cervical cancer cell lines. Here we show a significant reduction in cellular viability in the aforementioned cell lines as a result of the siRNA-mediated downregulation of LRP. This reduction in cellular viability is due to increased apoptotic processes, reflected by the loss of nuclear integrity and the significant increase in the activity of caspase-3. These results indicate that LRP/LR is involved in the maintenance of cellular viability in tumorigenic lung and cervix uteri cells through the blockage of apoptosis. Knockdown of LRP/LR by siRNA might represent an alternative therapeutic strategy for the treatment of lung and cervical cancer. PMID:23472084
P-cadherin signals through the laminin receptor ?6?4 integrin to induce stem cell and invasive properties in basal-like breast cancer cells
Vieira, Andr� Filipe; Ribeiro, Ana Sofia; Dion�sio, Maria Rita; Sousa, B�rbara; Nobre, Ana Rita; Albergaria, Andr�; Santiago-G�mez, Ang�lica; Mendes, Nuno; Gerhard, Ren�; Schmitt, Fernando; Clarke, Robert B.; Paredes, Joana
P-cadherin is a classical cell-cell adhesion molecule that, in contrast to E-cadherin, has a positive role in breast cancer progression, being considered a poor prognostic factor in this disease. In previous reports, we have shown that this protein induces cancer stem cell and invasive properties to basal-like breast cancer cells. Here, we clarify the downstream signaling pathways that are triggered by P-cadherin to mediate these effects. We demonstrated that P-cadherin inhibition led to a significant decreased adhesion of cancer cells to the basement membrane substrate laminin, as well as to a major reduction in the expression of the laminin receptor ?6?4 integrin. Remarkably, the expression of this heterodimer was required for the invasive capacity and increased mammosphere forming efficiency induced by P-cadherin expression. Moreover, we showed that P-cadherin transcriptionally up-regulates the ?6 integrin subunit expression and directly interacts with the ?4 integrin subunit. We still showed that P-cadherin downstream signaling, in response to laminin, involves the activation of focal adhesion (FAK), Src and AKT kinases. The association between the expression of P-cadherin, ?6?4 heterodimer and the active FAK and Src phosphorylated forms was validated in vivo. Our data establish that there is a crosstalk between P-cadherin and the laminin receptor ?6?4 integrin signaling pathway, which link has never been previously described. The activation of this heterodimer explains the stem cell and invasive properties induced by P-cadherin to breast cancer cells, pointing to a new molecular mechanism that may be targeted to counteract the effects induced by this adhesion molecule. PMID:24553076
P-cadherin signals through the laminin receptor ?6?4 integrin to induce stem cell and invasive properties in basal-like breast cancer cells.
Vieira, Andr� Filipe; Ribeiro, Ana Sofia; Dion�sio, Maria Rita; Sousa, B�rbara; Nobre, Ana Rita; Albergaria, Andr�; Santiago-G�mez, Ang�lica; Mendes, Nuno; Gerhard, Ren�; Schmitt, Fernando; Clarke, Robert B; Paredes, Joana
P-cadherin is a classical cell-cell adhesion molecule that, in contrast to E-cadherin, has a positive role in breast cancer progression, being considered a poor prognostic factor in this disease. In previous reports, we have shown that this protein induces cancer stem cell and invasive properties to basal-like breast cancer cells. Here, we clarify the downstream signaling pathways that are triggered by P-cadherin to mediate these effects. We demonstrated that P-cadherin inhibition led to a significant decreased adhesion of cancer cells to the basement membrane substrate laminin, as well as to a major reduction in the expression of the laminin receptor ?6?4 integrin. Remarkably, the expression of this heterodimer was required for the invasive capacity and increased mammosphere forming efficiency induced by P-cadherin expression. Moreover, we showed that P-cadherin transcriptionally up-regulates the ?6 integrin subunit expression and directly interacts with the ?4 integrin subunit. We still showed that P-cadherin downstream signaling, in response to laminin, involves the activation of focal adhesion (FAK), Src and AKT kinases. The association between the expression of P-cadherin, ?6?4 heterodimer and the active FAK and Src phosphorylated forms was validated in vivo. Our data establish that there is a crosstalk between P-cadherin and the laminin receptor ?6?4 integrin signaling pathway, which link has never been previously described. The activation of this heterodimer explains the stem cell and invasive properties induced by P-cadherin to breast cancer cells, pointing to a new molecular mechanism that may be targeted to counteract the effects induced by this adhesion molecule. PMID:24553076
Structural and functional analysis of the ovine laminin receptor gene ( RPSA ): Possible involvement of the LRP\\/LR protein in scrapie response
Ane Marcos-Carcavilla; Jorge H. Calvo; Carmen Gonz�lez; Carmen Serrano; Katayoun Moazami-Goudarzi; Pascal Laurent; Maud Bertaud; H�l�ne Hayes; Anne E. Beattie; Jaber Lyahyai; Inmaculada Mart�n-Burriel; Juan Mar�a Torres; Magdalena Serrano
Scrapie is a prion disease affecting sheep and goats. Susceptibility to this neurodegenerative disease shows polygenic variance.\\u000a The involvement of the laminin receptor (LRP\\/LR) in the metabolism and propagation of prions has previously been demonstrated.\\u000a In the present work, the ovine laminin receptor gene (RPSA) was isolated, characterized, and mapped to ovine chromosome OAR19q13. Real-time RT-PCR revealed a significant decrease
Laminin regulates PI3K basal localization and activation to sustain STAT5 activation
Spencer, Virginia A; Groesser, Dinah Levy
Extracellular matrix (ECM) is a key regulator of tissue morphogenesis and functional differentiation in the mammary gland. We showed recently that laminin-111 (LN1) together with prolactin induces ?-casein expression in mammary epithelial cells (MECs) by sustaining STAT5 activation. Others have shown that Rac1 is required for integrin-mediated STAT5 activation, but molecules upstream of Rac1 remain to be elucidated. Here, we show that exposure to three-dimensional (3D), laminin-rich ECM (LrECM) gels changes the localization of phosphoinositide 3-kinase (PI3K) in MECs from diffuse to basal, accompanied by the activation of the PI3K-Rac1 signaling pathway. We show by co-immunoprecipitation that Rac1 associates with STAT5 and that LrECM treatment enhances this interaction. Blocking PI3K with LY294002 inhibits LrECM-dependent Rac1 activation, attenuates sustained STAT5 phosphorylation and blocks ?-casein gene transcription. These results indicate that PI3K is a key mediator of the LN1-induced signaling cascade, which controls the activity of transcription factors essential for tissue-specific gene expression. PMID:20980837
Laminin and alpha-dystroglycan mediate acetylcholine receptor aggregation via a MuSK-independent pathway.
Montanaro, F; Gee, S H; Jacobson, C; Lindenbaum, M H; Froehner, S C; Carbonetto, S
Specific isoforms of laminin (LN) are concentrated at neuromuscular junctions (NMJs) where they may participate in synaptic organization or function. In myotubes from C2 cells, LN is concentrated within the majority of spontaneous acetylcholine receptor (AChR) aggregates. Neural agrin substantially increases this colocalization, suggesting that agrin can recruit LN into AChR aggregates. Addition of LN to C2 myotubes induces a more than twofold increase in the number of AChR aggregates. These aggregates have a larger size and are more dense than are those induced by agrin, suggesting that LN is involved in the growth and/or stabilization of AChR aggregates. Consistent with this hypothesis, an antiserum to LN reduces the size of individual AChR aggregates but increases their number. In C2 myotubes, extracellular matrix receptors containing the integrin beta1 subunit are poorly colocalized with AChR aggregates, suggesting that integrins may not be involved in LN-induced aggregation. In contrast, almost all AChR aggregates are associated with dystroglycan immunoreactivity, and monoclonal antibody (mAb) IIH6 against alpha-dystroglycan (alpha-DG), a LN and agrin receptor, causes a concentration-dependent inhibition of LN-induced aggregation. Moreover, S27 cells, which lack a functional alpha-DG, and two C2-derived cell lines expressing antisense DG mRNA fail to aggregate AChRs in response to LN. Finally, LN-induced AChR aggregation does not involve the phosphorylation of the muscle-specific tyrosine kinase receptor (MuSK) or the AChR beta subunit. We hypothesize that the interaction of LN with alpha-DG contributes to the growth and/or stabilization of AChR microaggregates into macroaggregates at the developing NMJ via a MuSK-independent mechanism. PMID:9454835
Laminin ?2-Mediated Focal Adhesion Kinase Activation Triggers Alport Glomerular Pathogenesis
Delimont, Duane; Dufek, Brianna M.; Meehan, Daniel T.; Zallocchi, Marisa; Gratton, Michael Anne; Phillips, Grady; Cosgrove, Dominic
It has been known for some time that laminins containing ?1 and ?2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the ?2 chain, but not those containing the ?1 chain activates focal adhesion kinase (FAK) on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of ?2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin ?2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages. PMID:24915008
Calcium channels link the muscle-derived synapse organizer laminin ?2 to Bassoon and CAST/Erc2 to organize presynaptic active zones
Chen, Jie; Billings, Sara E.; Nishimune, Hiroshi
Synapse formation requires the organization of presynaptic active zones, the synaptic vesicle release sites, in precise apposition to postsynaptic neurotransmitter receptor clusters; however, the molecular mechanisms responsible for these processes remain unclear. Here, we show that P/Q-type and N-type voltage-dependent calcium channels (VDCCs) play essential roles as scaffolding proteins in the organization of presynaptic active zones. The neuromuscular junction of double knockout mice for P/Q- and N-type VDCCs displayed a normal size, but had significantly reduced numbers of active zones and docked vesicles and featured an attenuation of the active zone proteins Bassoon, Piccolo, and CAST/Erc2. Consistent with this phenotype, direct interactions of the VDCC ?1b or ?4 subunits and the active zone-specific proteins Bassoon or CAST/Erc2 were confirmed by immunoprecipitation. A decrease in the number of active zones caused by a loss of presynaptic VDCCs resembled the pathological conditions observed in the autoimmune neuromuscular disorder Lambert�Eaton myasthenic syndrome (LEMS). At the synaptic cleft of double knockout mice, we also observed a decrease of the synaptic organizer laminin ?2 protein, an extracellular ligand of P/Q- and N-type VDCCs. However, the transcription level of laminin ?2 did not decrease in double knockout mice, suggesting that the synaptic accumulation of laminin ?2 protein required its interaction with presynaptic VDCCs. These results suggest that presynaptic VDCCs link the target-derived synapse organizer laminin ?2 to active zone proteins and function as scaffolding proteins to anchor active zone proteins to the presynaptic membrane. PMID:21228161
Evidence for the Participation of the Neuron-Specific CDK5 Activator P35 during Laminin-Enhanced Axonal Growth
Gabriela Paglini; Gustavo Pigino; Patricia Kunda; Gerardo Morfini; Ricardo Maccioni; Santiago Quiroga; Adriana Ferreira; Alfredo Caceres
Cultures of cerebellar macroneurons were used to study the pattern of expression, subcellular localization, and function of the neuronal cdk5 activator p35 during laminin-enhanced ax- onal growth. The results obtained indicate that laminin, an extracellular matrix molecule capable of selectively stimulating axonal extension and promoting MAP1B phosphorylation at a proline-directed protein kinase epitope, selectively stimulates p35 expression, increases its association
Construction and activity of a synthetic basement membrane with active laminin peptides and polysaccharides.
Yamada, Yuji; Hozumi, Kentaro; Nomizu, Motoyoshi
Cell-adhesive peptides derived from extracellular matrix (ECM) proteins are potential candidates for incorporating cell-binding activities into materials for tissue engineering. We have identified a number of cell adhesive peptides from laminins, which are major components of basement membrane ECM. Our goal is the development of synthetic basement membranes using the peptides on scaffolds. We review peptide�polysaccharide complexes, which were prepared by conjugation of the peptides to chitosan and alginate, and the biological activities of the resulting matrices. The peptide�polysaccharide matrices can also be used as a biomaterial for cell transplantation. These studies suggest that the peptide�polysaccharide complexes have the potential to mimic the multifunctional basement membrane and may be useful for tissue engineering. PMID:22003504
The 37kDa/67kDa Laminin Receptor acts as a receptor for A?42 internalization
Da Costa Dias, Bianca; Jovanovic, Katarina; Gonsalves, Danielle; Moodley, Kiashanee; Reusch, Uwe; Knackmuss, Stefan; Weinberg, Marc S.; Little, Melvyn; Weiss, Stefan F. T.
Neuronal loss is a major neuropathological hallmark of Alzheimer's disease (AD). The associations between soluble A? oligomers and cellular components cause this neurotoxicity. The 37?kDa/67?kDa laminin receptor (LRP/LR) has recently been implicated in A? pathogenesis. In this study the mechanism underlying the pathological role of LRP/LR was elucidated. F�rsters Resonance Energy Transfer (FRET) revealed that LRP/LR and A? form a biologically relevant interaction. The ability of LRP/LR to form stable associations with endogenously shed A? was confirmed by pull down assays and A?-ELISAs. Antibody blockade of this association significantly lowered A?42 induced apoptosis. Furthermore, antibody blockade and shRNA mediated downregulation of LRP/LR significantly hampered A?42 internalization. These results suggest that LRP/LR is a receptor for A?42 internalization, mediating its endocytosis and contributing to the cytotoxicity of the neuropeptide by facilitating intra-cellular A?42 accumulation. These findings recommend anti-LRP/LR specific antibodies and shRNAs as potential therapeutic tools for AD treatment. PMID:24990253
Division of labor among the alpha6beta4 integrin, beta1 integrins, and an E3 laminin receptor to signal morphogenesis and beta-casein expression in mammary epithelial cells.
Muschler, J; Lochter, A; Roskelley, C D; Yurchenco, P; Bissell, M J
Contact of cultured mammary epithelial cells with the basement membrane protein laminin induces multiple responses, including cell shape changes, growth arrest, and, in the presence of prolactin, transcription of the milk protein beta-casein. We sought to identify the specific laminin receptor(s) mediating the multiple cell responses to laminin. Using assays with clonal mammary epithelial cells, we reveal distinct functions for the alpha6beta4 integrin, beta1 integrins, and an E3 laminin receptor. Signals from laminin for beta-casein expression were inhibited in the presence of function-blocking antibodies against both the alpha6 and beta1 integrin subunits and by the laminin E3 fragment. The alpha6-blocking antibody perturbed signals mediated by the alpha6beta4 integrin, and the beta1-blocking antibody perturbed signals mediated by another integrin, the alpha subunit(s) of which remains to be determined. Neither alpha6- nor beta1-blocking antibodies perturbed the cell shape changes resulting from cell exposure to laminin. However, the E3 laminin fragment and heparin both inhibited cell shape changes induced by laminin, thereby implicating an E3 laminin receptor in this function. These results elucidate the multiplicity of cell-extracellular matrix interactions required to integrate cell structure and signaling and ultimately permit normal cell function. PMID:10473629
Phosphodiesterase 5 inhibitor acts as a potent agent sensitizing acute myeloid leukemia cells to 67-kDa laminin receptor-dependent apoptosis.
Kumazoe, Motofumi; Kim, Yoonhee; Bae, Jaehoon; Takai, Mika; Murata, Motoki; Suemasu, Yumi; Sugihara, Kaori; Yamashita, Shuya; Tsukamoto, Shuntaro; Huang, Yuhui; Nakahara, Kanami; Yamada, Koji; Tachibana, Hirofumi
(-)-Epigallocatechin-3-O-gallate (EGCG), a polyphenol in green tea, induces apoptosis in acute myeloid leukemia (AML) cells without affecting normal cells. In this study, we observed that cGMP acts as a cell death mediator of the EGCG-induced anti-AML effect through acid sphingomyelinase activation. EGCG activated the Akt/eNOS axis, a well-known mechanism in vascular cGMP upregulation. We also observed that a major cGMP negative regulator, phosphodiesterase 5, was overexpressed in AML cells, and PDE5 inhibitor, an anti-erectile dysfunction drug, synergistically enhanced the anti-AML effect of EGCG. This combination regimen killed AML cells via overexpressed 67-kDa laminin receptors. PMID:23916810
Polyphenols from green tea prevent antineuritogenic action of Nogo-A via 67-kDa laminin receptor and hydrogen peroxide.
Gundimeda, Usha; McNeill, Thomas H; Barseghian, Barsegh A; Tzeng, William S; Rayudu, David V; Cadenas, Enrique; Gopalakrishna, Rayudu
Axonal regeneration after injury to the CNS is hampered by myelin-derived inhibitors, such as Nogo-A. Natural products, such as green tea, which are neuroprotective and safe for long-term therapy, would complement ongoing various pharmacological approaches. In this study, using nerve growth factor-differentiated neuronal-like Neuroscreen-1 cells, we show that extremely low concentrations of unfractionated green tea polyphenol mixture (GTPP) and its active ingredient, epigallocatechin-3-gallate (EGCG), prevent both the neurite outgrowth-inhibiting activity and growth cone-collapsing activity of Nogo-66 (C-terminal domain of Nogo-A). Furthermore, a synergistic interaction was observed among GTPP constituents. This preventive effect was dependent on 67-kDa laminin receptor (67LR) to which EGCG binds with high affinity. The antioxidants N-acetylcysteine and cell-permeable catalase abolished this preventive effect of GTPP and EGCG, suggesting the involvement of sublethal levels of H2 O2 in this process. Accordingly, exogenous sublethal concentrations of H2 O2 , added as a bolus dose (5�?M) or more effectively through a steady-state generation (1-2�?M), mimicked GTPP in counteracting the action of Nogo-66. Exogenous H2 O2 mediated this action by bypassing the requirement of 67LR. Taken together, these results show for the first time that GTPP and EGCG, acting through 67LR and elevating intracellular sublethal levels of H2 O2 , inhibit the antineuritogenic action of Nogo-A. Currently, several agents are being evaluated for overcoming axonal growth inhibitors to promote functional recovery after stroke and spinal cord injury. Epigallocatechin-3-gallate (EGCG), present in green tea polyphenol mixture (GTPP), prevents antineuritogenic activity of Nogo-A, a myelin-derived axonal growth inhibitor. The preventive action of EGCG involves the cell-surface-associated 67-kDa laminin receptor and H2 O2 . GTPP may complement ongoing efforts to treat neuronal injuries.> PMID:25314656
Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis
Alqahtani, Fulwah; Mahdavi, Jafar; Wheldon, Lee M.; Vassey, Matthew; Pirinccioglu, Necmettin; Royer, Pierre-Joseph; Qarani, Suzan M.; Morroll, Shaun; Stoof, Jeroen; Holliday, Nicholas D.; Teo, Siew Y.; Oldfield, Neil J.; Wooldridge, Karl G.; Ala'Aldeen, Dlawer A. A.
The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C173) of Gal-3 or lysine (K166) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial�host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization. PMID:25274119
Activity-dependent retrograde laminin A signaling regulates synapse growth at Drosophila neuromuscular junctions
Tsai, Pei-I; Wang, Manyu; Kao, Hsiu-Hua; Cheng, Ying-Ju; Lin, Yu-Jing; Chen, Ruey-Hwa; Chien, Cheng-Ting
Retrograde signals induced by synaptic activities are derived from postsynaptic cells to potentiate presynaptic properties, such as cytoskeletal dynamics, gene expression, and synaptic growth. However, it is not known whether activity-dependent retrograde signals can also depotentiate synaptic properties. Here we report that laminin A (LanA) functions as a retrograde signal to suppress synapse growth at Drosophila neuromuscular junctions (NMJs). The presynaptic integrin pathway consists of the integrin subunit ?? and focal adhesion kinase 56 (Fak56), both of which are required to suppress crawling activity-dependent NMJ growth. LanA protein is localized in the synaptic cleft and only muscle-derived LanA is functional in modulating NMJ growth. The LanA level at NMJs is inversely correlated with NMJ size and regulated by larval crawling activity, synapse excitability, postsynaptic response, and anterograde Wnt/Wingless signaling, all of which modulate NMJ growth through LanA and ??. Our data indicate that synaptic activities down-regulate levels of the retrograde signal LanA to promote NMJ growth. PMID:23054837
Bovine Prion Is Endocytosed by Human Enterocytes via the 37 kDa/67 kDa Laminin Receptor
Morel, Etienne; Andrieu, Thibault; Casagrande, Fabrice; Gauczynski, Sabine; Weiss, Stefan; Grassi, Jacques; Rousset, Monique; Dormont, Dominique; Chambaz, Jean
Some forms of transmissible spongiform encephalopathies result from oral infection. We have thus analyzed the early mechanisms that could account for an uptake of infectious prion particles by enterocytes, the major cell population of the intestinal epithelium. Human Caco-2/TC7 enterocytes cultured on microporous filters were incubated with different prion strains and contaminated brain homogenates in the apical compartment. Internalization of infectious particles was analyzed by Western blotting and immunofluorescence. We observed internalization by enterocytes of prion particles from bovine spongiform encephalopathy brain homogenates but not from mouse-adapted scrapie-strain brain homogenates or purified bovine spongiform encephalopathy scrapie-associated fibrils. Bovine prion particles were internalized via endocytosis within minutes of infection and were associated with subapical vesicular structures related to early endosomes. The endocytosis of the infectious bovine PrPSc was reduced by preincubating the cells with an anti-LRP/LR blocking antibody, identifying the 37 kDa/67 kDa laminin receptor (LRP/LR), which is apically expressed in Caco-2/TC7 cells, as the receptor for the infectious prion protein. Altogether, our results underscore a potential role of enterocytes in the absorption of bovine prions during oral infection through specific LRP/LR-dependent endocytosis. PMID:16192638
Expression of the 67-kD laminin receptor, galectin-1, and galectin-3 in advanced human uterine adenocarcinoma
Fr�d�ric A Van Den Br�le; Crina Buicu; Andy Berchuck; Robert C Bast; Manuel Deprez; Fu-tong Liu; Douglas N. W Cooper; Claudette Pieters; Mark E Sobel; Vincent Castronovo
Alterations of tumor cell interactions with laminin, a basement membrane glycoprotein, are consistent features of the invasive and metastatic phenotype. Qualitative and quantitative changes in the expression of cell surface laminin-binding proteins have been correlated with the ability of cancer cells to cross basement membranes during the metastatic cascade. Such phenotypic modifications are usually associated with poor prognosis. In this
Receptor functions for the integrin VLA3: fibronectin, collagen, and laminin binding are differentially influenced by Arg-Gly-Asp peptide and by divalent cations
M. J. Elices; Lisa A. Urry; Martin E. Hemler
The capability of the integrin VLA-3 to function as a receptor for collagen (Coil), laminin (Lm), and fibronectin (Fn) was addressed using both whole cell adhesion assays and Iigand affinity columns. Analysis of VLA-3-mediated cell adhesion was facilitated by the use of a small cell lung carci- noma line (NCI-H69), which expresses VLA-3 but few other integrins. While VLA-3 interaction
Laminin receptor specific therapeutic gold nanoparticles ((AuNP)-Au-198-EGCg) show efficacy in treating prostate cancer
Shukla, Ravi; Chanda, Nripen; Zambre, Ajit; Katti, Kavita; Upendran, Anandhi; Kulkarni, Rajesh R.; Nune, Satish K.; Casteel, Stan W.; Smith, C. J.; Boote, Evan; Robertson, J. D.; Kan, Para; Engelbrecht, Hendrik; Watkinson, Lisa D.; Carmack, Terry L.; Lever, John R.; Cutler, Cathy; Caldwell, Charles; Kannan, Raghuraman; Katti, Kattesh V.
Systemic delivery of therapeutic agents to solid tumors is hindered by vascular and interstitial barriers. We hypothesized that prostate tumor specific epigallocatechingallate( EGCg) functionalized radioactive gold nanoparticles, when delivered intratumorally (IT), will circumvent transport barriers, resulting in targeted delivery of therapeutic payloads. The results described herein provide unequivocal validation of our hypothesis. We report the development of inherently therapeutic gold nanoparticles derived from Au-198 isotope; the range of 198Au ?-particle ( ~ 11 mm in tissue or ~1100 cell diameters) is sufficiently long to provide cross-fire effects of radiation dose delivered to cells within the prostate gland and short enough to minimize radiation dose to critical tissues near the periphery of the capsule. The formulation of biocompatible 198AuNPs utilizes the redox chemistry of prostate tumor specific phytochemical EGCg as it converts gold salt into gold nanoparticles and also selectively binds with excellent affinity to Laminin67R receptors which are over expressed in prostate tumor cells. Pharmacokinetic studies in PC-3 xenograft SCID mice showed ~72% retention of 198AuNP-EGCg in tumors 24 h after intratumoral administration. Therapeutic studies showed 80% reduction of tumor volumes after 28 days demonstrating significant inhibition of tumor growth compared to controls. This innovative �green nanotechnological�approach serves as a basis for designing target specific antineoplastic agents. This novel intratumorally injectable 198AuNP-EGCg nanotherapeutic agent may provide significant advances in oncology for use as an effective treatment for prostate and other solid tumors.
Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells
Horejs, Christine-Maria; Serio, Andrea; Purvis, Alan; Gormley, Adam J.; Bertazzo, Sergio; Poliniewicz, Anna; Wang, Alex J.; DiMaggio, Peter; Hohenester, Erhard; Stevens, Molly M.
The dynamic interplay between the extracellular matrix and embryonic stem cells (ESCs) constitutes one of the key steps in understanding stem cell differentiation in vitro. Here we report a biologically-active laminin-111 fragment generated by matrix metalloproteinase 2 (MMP2) processing, which is highly up-regulated during differentiation. We show that the ?1-chain�derived fragment interacts via ?3?1-integrins, thereby triggering the down-regulation of MMP2 in mouse and human ESCs. Additionally, the expression of MMP9 and E-cadherin is up-regulated in mouse ESCs�key players in the epithelial-to-mesenchymal transition. We also demonstrate that the fragment acts through the ?3?1-integrin/extracellular matrix metalloproteinase inducer complex. This study reveals a previously unidentified role of laminin-111 in early stem cell differentiation that goes far beyond basement membrane assembly and a mechanism by which an MMP2-cleaved laminin fragment regulates the expression of E-cadherin, MMP2, and MMP9. PMID:24706882
Aberrant Development of Thymocytes in Mice Lacking Laminin-2
Magner, William J.; Chang, Andrew C.; Owens, Jennie; Hong, M-J. P.; Brooks, Andrew
In previous in vitro studies, we proposed a role for the extracellular matrix component, laminin- 2, and its integrin receptor, VLA-6, in thymocyte development. The characterization of two dystrophic mouse strains with different defects in laminin-2 allowed us to examine this proposal in vivo. Mice deficient in laminin-2, dy/dy, show a significant reduction in thymus size and number of thymocytes compared to normal littermates. These mice also exhibited apparent alterations of thymic architecture. Examination of the CD4/CD8 populations in dy/dy thymi showed large relative increases in the DN (CD4-CD8-) and SP (CD4+CD8-, CD4-CD8+) populations and a significant decrease in the DP (CD4+CD8+) population. Further examination of the DN population for CD44 and CD25 expression showed a remarkable decrease in the more mature pre-T cell populations. Analysis of apoptosis in situ, and by flow cytometry, in dy/dy thymi revealed a significant increase in apoptotic DN thymocytes in the capsule and subcapsular regions. Interestingly, thymocyte development appeared to proceed normally in dystrophic mice expressing a mutant form of laminin-2, dy2J, as well as, in fetal and neonatal dy/dy mice. We propose that laminin-2 plays an active role in thymocyte development by delivering cell survival and differentiation signals at specific stages of development in young adult mice. PMID:11097211
Laminin receptor specific therapeutic gold nanoparticles (198AuNP-EGCg) show efficacy in treating prostate cancer.
Shukla, Ravi; Chanda, Nripen; Zambre, Ajit; Upendran, Anandhi; Katti, Kavita; Kulkarni, Rajesh R; Nune, Satish Kumar; Casteel, Stan W; Smith, Charles Jeffrey; Vimal, Jatin; Boote, Evan; Robertson, J David; Kan, Para; Engelbrecht, Hendrik; Watkinson, Lisa D; Carmack, Terry L; Lever, John R; Cutler, Cathy S; Caldwell, Charles; Kannan, Raghuraman; Katti, Kattesh V
Systemic delivery of therapeutic agents to solid tumors is hindered by vascular and interstitial barriers. We hypothesized that prostate tumor specific epigallocatechin-gallate (EGCg) functionalized radioactive gold nanoparticles, when delivered intratumorally (IT), would circumvent transport barriers, resulting in targeted delivery of therapeutic payloads. The results described herein support our hypothesis. We report the development of inherently therapeutic gold nanoparticles derived from the Au-198 isotope; the range of the (198)Au ?-particle (approximately 11 mm in tissue or approximately 1100 cell diameters) is sufficiently long to provide cross-fire effects of a radiation dose delivered to cells within the prostate gland and short enough to minimize the radiation dose to critical tissues near the periphery of the capsule. The formulation of biocompatible (198)AuNPs utilizes the redox chemistry of prostate tumor specific phytochemical EGCg as it converts gold salt into gold nanoparticles and also selectively binds with excellent affinity to Laminin67R receptors, which are over expressed in prostate tumor cells. Pharmacokinetic studies in PC-3 xenograft SCID mice showed approximately 72% retention of (198)AuNP-EGCg in tumors 24 h after intratumoral administration. Therapeutic studies showed 80% reduction of tumor volumes after 28 d demonstrating significant inhibition of tumor growth compared to controls. This innovative nanotechnological approach serves as a basis for designing biocompatible target specific antineoplastic agents. This novel intratumorally injectable (198)AuNP-EGCg nanotherapeutic agent may provide significant advances in oncology for use as an effective treatment for prostate and other solid tumors. PMID:22802668
Laminin receptor specific therapeutic gold nanoparticles (198AuNP-EGCg) show efficacy in treating prostate cancer
Shukla, Ravi; Chanda, Nripen; Zambre, Ajit; Upendran, Anandhi; Katti, Kavita; Kulkarni, Rajesh R.; Nune, Satish Kumar; Casteel, Stan W.; Smith, Charles Jeffrey; Vimal, Jatin; Boote, Evan; Robertson, J. David; Kan, Para; Engelbrecht, Hendrik; Watkinson, Lisa D.; Carmack, Terry L.; Lever, John R.; Cutler, Cathy S.; Caldwell, Charles; Kannan, Raghuraman; Katti, Kattesh V.
Systemic delivery of therapeutic agents to solid tumors is hindered by vascular and interstitial barriers. We hypothesized that prostate tumor specific epigallocatechin-gallate (EGCg) functionalized radioactive gold nanoparticles, when delivered intratumorally (IT), would circumvent transport barriers, resulting in targeted delivery of therapeutic payloads. The results described herein support our hypothesis. We report the development of inherently therapeutic gold nanoparticles derived from the Au-198 isotope; the range of the 198Au ?-particle (approximately 11�mm in tissue or approximately 1100�cell diameters) is sufficiently long to provide cross-fire effects of a radiation dose delivered to cells within the prostate gland and short enough to minimize the radiation dose to critical tissues near the periphery of the capsule. The formulation of biocompatible 198AuNPs utilizes the redox chemistry of prostate tumor specific phytochemical EGCg as it converts gold salt into gold nanoparticles and also selectively binds with excellent affinity to Laminin67R receptors, which are over expressed in prostate tumor cells. Pharmacokinetic studies in PC-3 xenograft SCID mice showed approximately 72% retention of 198AuNP-EGCg in tumors 24�h after intratumoral administration. Therapeutic studies showed 80% reduction of tumor volumes after 28�d demonstrating significant inhibition of tumor growth compared to controls. This innovative nanotechnological approach serves as a basis for designing biocompatible target specific antineoplastic agents. This novel intratumorally injectable 198AuNP-EGCg nanotherapeutic agent may provide significant advances in oncology for use as an effective treatment for prostate and other solid tumors. PMID:22802668
A simplified laminin nomenclature
Monique Aumailley; Leena Bruckner-Tuderman; William G. Carter; Rainer Deutzmann; David Edgar; Peter Ekblom; J�rgen Engel; Eva Engvall; Erhard Hohenester; Jonathan C. R. Jones; Hynda K. Kleinman; M. Peter Marinkovich; George R. Martin; Ulrike Mayer; Guerrino Meneguzzi; Jeffrey H. Miner; Kaoru Miyazaki; Manuel Patarroyo; Mats Paulsson; Vito Quaranta; Joshua R. Sanes; Takako Sasaki; Kiyotoshi Sekiguchi; Lydia M. Sorokin; Jan F. Talts; Karl Tryggvason; Jouni Uitto; Ismo Virtanen; Klaus von der Mark; Ulla M. Wewer; Yoshihiko Yamada; Peter D. Yurchenco
A simplification of the laminin nomenclature is presented. Laminins are multidomain heterotrimers composed of ?, ? and ? chains. Previously, laminin trimers were numbered with Arabic numerals in the order discovered, that is laminins-1 to -5. We introduce a new identification system for a trimer using three Arabic numerals, based on the ?, ? and ? chain numbers. For example,
PED/PEA-15 interacts with the 67 kD laminin receptor and regulates cell adhesion, migration, proliferation and apoptosis
Formisano, Pietro; Ragno, Pia; Pesapane, Ada; Alfano, Daniela; Alberobello, Anna Teresa; Rea, Vincenza Elena Anna; Giusto, Raffaella; Rossi, Francesca W; Beguinot, Francesco; Rossi, Guido; Montuori, Nunzia
Abstract Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kD (PED/PEA-15) is an anti-apoptotic protein whose expression is increased in several human cancers. In addition to apoptosis, PED/PEA-15 is involved in the regulation of other major cellular functions, including cell adhesion, migration, proliferation and glucose metabolism. To further understand the functions of this protein, we performed a yeast two-hybrid screening using PED/PEA-15 as a bait and identified the 67 kD high-affinity laminin receptor (67LR) as an interacting partner. 67 kD laminin receptor is a non-integrin cell-surface receptor for the extracellular matrix (ECM), derived from the dimerization of a 37 kD cytosolic precursor (37LRP). The 67LR is highly expressed in human cancers and widely recognized as a molecular marker of metastatic aggressiveness. The molecular interaction of PED/PEA-15 with 67LR was confirmed by pull-down experiments with recombinant His-tagged 37LRP on lysates of PED/PEA-15 transfected HEK-293 cells. Further, overexpressed or endogenous PED/PEA-15 was co-immunoprecipitated with 67LR in PED/PEA-15-transfected HEK-293 cells and in U-373 glioblastoma cells, respectively. PED/PEA-15 overexpression significantly increased 67LR-mediated HEK-293 cell adhesion and migration to laminin that, in turn, determined PED/PEA-15 phosphorylation both in Ser-104 and Ser-116, thus enabling cell proliferation and resistance to apoptosis. PED/PEA-15 ability to induce cell responses to ECM-derived signals through interaction with 67LR may be of crucial importance for tumour cell survival in a poor microenvironment, thus favouring the metastatic spread and colonization. PMID:21895963
Screening of integrin-binding peptides in a laminin peptide library derived from the mouse laminin ? chain short arm regions.
Katagiri, Fumihiko; Takagi, Masaharu; Nakamura, Minako; Tanaka, Yoichiro; Hozumi, Kentaro; Kikkawa, Yamato; Nomizu, Motoyoshi
Laminins, major components of basement membrane, consist of three different subunits, ?, ?, and ? chains, and so far, five ?, three ?, and three ? chains have been identified. We have constructed synthetic peptide libraries derived from the laminin sequences and identified various cell-adhesive peptides. Ten active peptides from the laminin ? chain sequences (?1-?5) were found to promote integrin-mediated cell adhesion. Previously, we found fourteen cell-adhesive peptides from the ?1 chain sequence but their receptors have not been analyzed. Here, we expanded the synthetic peptide library to add peptides from the short arm regions of the laminin ?2 and ?3 chains and screened for integrin-binding peptides. Twenty-seven peptides promoted human dermal fibroblast (HDF) attachment in a peptide-coated plate assay. The morphological appearance of HDFs on the peptide-coated plates differed depending on the peptides. B34 (REKYYYAVYDMV, mouse laminin ?1 chain, 255-266), B67 (IPYSMEYEILIRY, mouse laminin ?1 chain, 604-616), B2-105 (APNFWNFTSGRG, mouse laminin ?2 chain, 1081-1092), and B3-19 (GHLTGGKVQLNL, mouse laminin ?3 chain, 182-193) promoted HDF spreading and HDF attachment was inhibited by EDTA, suggesting that the peptides interact with integrins. Immunostaining analyses revealed that B67 induced well-organized actin stress fibers and focal contacts containing vinculin, however, B34, B2-105, and B3-19 did not exhibit stress fiber formation or focal contacts. The inhibition assay using anti-integrin antibodies indicated that B67 interacts with ?3, ?6, and ?1 integrins, and B34 and B3-19 interact with ?1 integrin. Based on adhesion analysis of peptides modified with an alanine scan and on switching analysis with the homologous inactive sequence B2-64 (LPRAMDYDLLLRW, mouse laminin ?2 chain, 618-630), the Glu(8) residue in the B67 peptide was critical for HDF adhesion. These findings are useful for identifying an integrin binding motif. The B67 peptide has potential for use as a molecular probe for integrins. PMID:24785228
Structure and function of an early divergent form of laminin in hydra: a structurally conserved ECM component that is essential for epithelial morphogenesis.
Zhang, Xiaoming; Fei, Kaiyin; Agbas, Abdulbaki; Yan, Li; Zhang, Jinsong; O'Reilly, Brooke; Deutzmann, Rainer; Sarras, Michael P
As a major component of the extracellular matrix (ECM), laminin has been found in many vertebrate and invertebrate organisms. Its molecular structure is very similar across species lines and its biological function in the ECM has been extensively studied. In an effort to study ECM structure and function in hydra, we have cloned a partial hydra laminin alpha chain and the full-length hydra laminin beta chain using ECM-enriched cDNA libraries. Analysis of deduced amino acid sequences indicated that both polypeptides have high sequence similarity to a number of invertebrate and vertebrate laminin alpha and beta subunits. Rotary shadow analysis of isolated hydra laminin indicates it has a heterotrimeric organization that is characteristic of vertebrate laminins. A putative integrin-class protein was also identified using a cell-binding peptide sequence from the laminin beta chain as an affinity probe, indicating that integrins are possible cell surface receptors in hydra. In agreement with previous results for the hydra laminin beta chain, in situ hybridization experiments revealed that hydra laminin alpha chain mRNA is restricted to endodermal cells. As with a number of other hydra ECM components, higher levels of laminin alpha chain mRNA are localized to regions where cell migration and differentiation are actively undertaken such as the base of tentacles, the peduncle region, buds, regenerating tentacles, and at the head end during regeneration. The role of laminin in morphogenesis was studied using an antisense approach and the results indicated that translation of the laminin alpha chain is required for head regeneration. PMID:12012231
A simplified laminin nomenclature.
Aumailley, Monique; Bruckner-Tuderman, Leena; Carter, William G; Deutzmann, Rainer; Edgar, David; Ekblom, Peter; Engel, J�rgen; Engvall, Eva; Hohenester, Erhard; Jones, Jonathan C R; Kleinman, Hynda K; Marinkovich, M Peter; Martin, George R; Mayer, Ulrike; Meneguzzi, Guerrino; Miner, Jeffrey H; Miyazaki, Kaoru; Patarroyo, Manuel; Paulsson, Mats; Quaranta, Vito; Sanes, Joshua R; Sasaki, Takako; Sekiguchi, Kiyotoshi; Sorokin, Lydia M; Talts, Jan F; Tryggvason, Karl; Uitto, Jouni; Virtanen, Ismo; von der Mark, Klaus; Wewer, Ulla M; Yamada, Yoshihiko; Yurchenco, Peter D
A simplification of the laminin nomenclature is presented. Laminins are multidomain heterotrimers composed of alpha, beta and gamma chains. Previously, laminin trimers were numbered with Arabic numerals in the order discovered, that is laminins-1 to -5. We introduce a new identification system for a trimer using three Arabic numerals, based on the alpha, beta and gamma chain numbers. For example, the laminin with the chain composition alpha5beta1gamma1 is termed laminin-511, and not laminin-10. The current practice is also to mix two overlapping domain and module nomenclatures. Instead of the older Roman numeral nomenclature and mixed nomenclature, all modules are now called domains. Some domains are renamed or renumbered. Laminin epidermal growth factor-like (LE) domains are renumbered starting at the N-termini, to be consistent with general protein nomenclature. Domain IVb of alpha chains is named laminin 4a (L4a), domain IVa of alpha chains is named L4b, domain IV of gamma chains is named L4, and domain IV of beta chains is named laminin four (LF). The two coiled-coil domains I and II are now considered one laminin coiled-coil domain (LCC). The interruption in the coiled-coil of beta chains is named laminin beta-knob (Lbeta) domain. The chain origin of a domain is specified by the chain nomenclature, such as alpha1L4a. The abbreviation LM is suggested for laminin. Otherwise, the nomenclature remains unaltered. PMID:15979864
Autocrine Transforming Growth Factor-?1 Activation Mediated by Integrin ?V?3 Regulates Transcriptional Expression of Laminin-332 in Madin-Darby Canine Kidney Epithelial Cells
Greciano, Patricia G.; Buschmann, Mary M.; Koch, Manuel; Matlin, Karl S.
Laminin (LM)-332 is an extracellular matrix protein that plays a structural role in normal tissues and is also important in facilitating recovery of epithelia from injury. We have shown that expression of LM-332 is up-regulated during renal epithelial regeneration after ischemic injury, but the molecular signals that control expression are unknown. Here, we demonstrate that in Madin-Darby canine kidney (MDCK) epithelial cells LM-332 expression occurs only in subconfluent cultures and is turned-off after a polarized epithelium has formed. Addition of active transforming growth factor (TGF)-?1 to confluent MDCK monolayers is sufficient to induce transcription of the LM ?3 gene and LM-332 protein expression via the TGF-? type I receptor (T?R-I) and the Smad2�Smad4 complex. Significantly, we show that expression of LM-332 in MDCK cells is an autocrine response to endogenous TGF-?1 secretion and activation mediated by integrin ?V?3 because neutralizing antibodies block LM-332 production in subconfluent cells. In confluent cells, latent TGF-?1 is secreted apically, whereas T?R-I and integrin ?V?3 are localized basolaterally. Disruption of the epithelial barrier by mechanical injury activates TGF-?1, leading to LM-332 expression. Together, our data suggest a novel mechanism for triggering the production of LM-332 after epithelial injury. PMID:20844080
Aumailley, Monique
Laminins are large molecular weight glycoproteins constituted by the assembly of three disulfide-linked polypeptides, the ?, ? and ? chains. The human genome encodes 11 genetically distinct laminin chains. Structurally, laminin chains differ by the number, size and organization of a few constitutive domains, endowing the various members of the laminin family with common and unique important functions. In particular, laminins are indispensable building blocks for cellular networks physically bridging the intracellular and extracellular compartments and relaying signals critical for cellular behavior, and for extracellular polymers determining the architecture and the physiology of basement membranes. PMID:23263632
Laminin deposition in the extracellular matrix: a complex picture emerges
Hamill, Kevin J.; Kligys, Kristina; Hopkinson, Susan B.; Jones, Jonathan C. R.
Summary Laminins are structural components of basement membranes. In addition, they are key extracellular-matrix regulators of cell adhesion, migration, differentiation and proliferation. This Commentary focuses on a relatively understudied aspect of laminin biology: how is laminin deposited into the extracellular matrix? This topic has fascinated researchers for some time, particularly considering the diversity of patterns of laminin that can be visualized in the matrix of cultured cells. We discuss current ideas of how laminin matrices are assembled, the role of matrix receptors in this process and how laminin-associated proteins modulate matrix deposition. We speculate on the role of signaling pathways that are involved in laminin-matrix deposition and on how laminin patterns might play an important role in specifying cell behaviors, especially directed migration. We conclude with a description of new developments in the way that laminin deposition is being studied, including the use of tagged laminin subunits that should allow the visualization of laminin-matrix deposition and assembly by living cells. PMID:19955338
Motility induction in breast carcinoma by mammary epithelial laminin 332 (laminin 5).
Carpenter, Philip M; Dao, Anh V; Arain, Zahida S; Chang, Michelle K; Nguyen, Hoa P; Arain, Shehla; Wang-Rodriguez, Jessica; Kwon, Soon-Young; Wilczynski, Sharon P
Host interactions with tumor cells contribute to tumor progression by several means. This study was done to determine whether mammary epithelium could interact with breast carcinoma by producing substances capable of inducing motility in the cancer cells. Conditioned medium of immortalized 184A1 mammary epithelium collected in serum-free conditions induced dose-dependent motility in the MCF-7 breast carcinoma cell line by both a semiquantitative scattering assay and a Boyden chamber assay. Purification of the motility factor revealed that it was laminin 332 (formerly laminin 5) by mass spectroscopy. A Western blot of the 184A1 conditioned medium using a polyclonal antibody confirmed the presence of laminin 332 in the conditioned medium. Blockage of the motility with antibodies to the laminin 332 and its receptor components, alpha(3) and beta(1) integrins, provided further evidence that tumor cell motility was caused by the laminin 332 in the conditioned medium. Invasion of MCF-7, BT-20, and MDA-MB-435 S was induced by purified laminin 332 and 184A1 conditioned medium and blocked by an anti-alpha(3) integrin antibody. Staining of carcinoma in situ from breast cancer specimens revealed that laminin 332 in the myoepithelium adjacent to the preinvasive cells provided a source of laminin 332 that could potentially encourage the earliest steps of stromal invasion. In metaplastic breast carcinomas, the presence of laminin 332-producing cells coexpressing alpha(3) integrin and the greater metastatic potential of tumors with higher laminin 332 levels suggest that laminin 332 expression is associated with aggressive features in these human breast cancers. PMID:19351903
Impeded interaction between Schwann cells and axons in the absence of laminin alpha4.
Wallquist, Wilhelm; Plantman, Stefan; Thams, Sebastian; Thyboll, Jill; Kortesmaa, Jarkko; L�nnergren, Jan; Domogatskaya, Anna; Ogren, Sven Ove; Risling, M�rten; Hammarberg, Henrik; Tryggvason, Karl; Cullheim, Staffan
The Schwann cell basal lamina (BL) is required for normal myelination. Loss or mutations of BL constituents, such as laminin-2 (alpha2beta1gamma1), lead to severe neuropathic diseases affecting peripheral nerves. The function of the second known laminin present in Schwann cell BL, laminin-8 (alpha4beta1gamma1), is so far unknown. Here we show that absence of the laminin alpha4 chain, which distinguishes laminin-8 from laminin-2, leads to a disturbance in radial sorting, impaired myelination, and signs of ataxia and proprioceptive disturbances, whereas the axonal regenerative capacity is not influenced. In vitro studies show poor axon growth of spinal motoneurons on laminin-8, whereas it is extensive on laminin-2. Schwann cells, however, extend longer processes on laminin-8 than on laminin-2, and, in contrast to the interaction with laminin-2, solely use the integrin receptor alpha6beta1 in their interaction with laminin-8. Thus, laminin-2 and laminin-8 have different critical functions in peripheral nerves, mediated by different integrin receptors. PMID:15814800
Processing of Laminin5 and Its Functional Consequences: Role of Plasmin and Tissue-type Plasminogen Activator
Lawrence E. Goldfinger; M. Sharon Stack; Jonathan C. R. Jones
The laminin-5 component of the extracellu- lar matrices of certain cultured cells such as the rat epi- thelial cell line 804G and the human breast epithelial cell MCF-10A is capable of nucleating assembly of cell- matrix adhesive devices called hemidesmosomes when other cells are plated upon them. These matrices also impede cell motility. In contrast, cells plated onto the laminin-5-rich
Epigenetic activation of human kallikrein 13 enhances malignancy of lung adenocarcinoma by promoting N-cadherin expression and laminin degradation.
Chou, Ruey-Hwang; Lin, Sheng-Chieh; Wen, Hui-Chin; Wu, Cheng-Wen; Chang, Wun-Shaing Wayne
The tissue kallikrein (KLK) family contains 15 genes (KLK1-KLK15) tandemly arranged on chromosome 19q13.4 that forms the largest cluster of contiguous protease genes in the human genome. Here, we provide mechanistic evidence showing that the expression of KLK13, one of the most recently identified family members, is significantly up-regulated in metastatic lung adenocarcinoma. Whilst overexpression of KLK13 resulted in an increase in malignant cell behavior, knockdown of its endogenous gene expression caused a significant decrease in cell migratory and invasive properties. Functional studies further demonstrated that KLK13 is activated via demethylation of its upstream region. The elevated KLK13 protein then enhances the ability of tumor cells to degrade extracellular laminin that, subsequently, facilitates cell metastatic potential in the in vivo SCID mouse xenograft model. KLK13 was also found to induce the expression of N-cadherin to help promote tumor cell motility. Together, these results reveal the enhancing effects of KLK13 on tumor cell invasion and migration, and that it may serve as a diagnostic/prognostic marker and a potential therapeutic target for lung cancer. PMID:21596022
Crystal structure and cell surface anchorage sites of laminin ?1LG4-5
Harrison, David; Hussain, Sadaf-Ahmahni; Combs, Ariana C.; Ervasti, James M.; Yurchenco, Peter D.; Hohenester, Erhard
The laminin G-like (LG) domains of laminin-111, a glycoprotein widely expressed during embryogenesis, provide cell anchoring and receptor binding sites that are involved in basement membrane assembly and cell signaling. We now report the crystal structure of the laminin ?1 LG4-5 domains and provide a mutational analysis of heparin, ?-dystroglycan and galactosyl-sulfatide binding. The two domains of ?1LG4-5 are arranged in a V-shaped fashion similar to that observed with laminin ?2 LG4-5, but with a substantially different interdomain angle. Recombinant ?1LG4-5 binding to heparin, ?-dystroglycan and sulfatides was dependent upon both shared and unique contributions from basic residues distributed in several clusters on the surface of LG4. For heparin, the greatest contribution was detected from two clusters, 2719RKR and 2791KRK. Binding to ?-dystroglycan was particularly dependent on basic residues within 2719RKR, 2831RAR, and 2858KDR. Binding to galactosyl-sulfatide was most affected by mutations in 2831RAR and 2766KGRTK, but not in 2719RKR. The combined analysis of structure and activities reveal differences in LG domain interactions that should enable dissection of biological roles of different laminin ligands. PMID:17307732
Lutheran blood group glycoprotein and its newly characterized mouse homologue specifically binda5 chain-containing human laminin with high affinity
Stephen F. Parsons; Gloria Lee; Frances A. Spring; Thiebaut-Noel Willig; Luanne L. Peters; J. Aura Gimm; Michael J. A. Tanner; Narla Mohandas; David J. Anstee; Joel Anne Chasis
Lutheran blood group glycoproteins (Lu gps) are receptors for the extracellular matrix protein, laminin. Studies suggest that Lu gps may contribute to vaso- occlusion in sickle cell disease and it has recently been shown that sickle cells ad- here to laminin isoforms containing the a5 chain (laminin 10\\/11). Laminin a 5i s present in the subendothelium and is also a
The helper-component protease transmission factor of tobacco etch potyvirus binds specifically to an aphid ribosomal protein homologous to the laminin receptor precursor.
Fern�ndez-Calvino, Lourdes; Goytia, Elisa; L�pez-Abella, Dionisio; Giner, Ana; Urizarna, Mar�a; Vilaplana, Lluisa; L�pez-Moya, Juan Jos�
Potyviruses are plant pathogens transmitted by aphids in a non-persistent manner. During transmission, the virus-encoded factor helper-component protease (HCPro) is presumed to act as a molecular bridge, mediating the reversible retention of virions to uncharacterized binding sites in the vector mouthparts. Whilst the predicted interaction between HCPro and the coat protein (CP) of virions has been confirmed experimentally, the characterization of putative HCPro-specific receptors in aphids has remained elusive, with the exception of a report that described binding of HCPro of zucchini yellow mosaic virus to several cuticle proteins. To identify other aphid components that could play a role during transmission, this study used purified HCPro of tobacco etch virus (TEV) in far-Western blotting assays as bait to select interactors among proteins extracted from aphid heads. With this approach, new HCPro-interacting proteins were found, and several were identified after mass spectrometry analysis and searches in databases dedicated to aphid sequences. Among these interactors, a ribosomal protein S2 (RPS2) was chosen for further investigation due to its homology with the laminin receptor precursor, known to act as the receptor of several viruses. The specific interaction between RPS2 and TEV HCPro was confirmed after cloning and heterologous expression of the corresponding Myzus persicae gene. The possible involvement of RPS2 in the transmission process was further suggested by testing a variant of HCPro that was non-functional for transmission due to a mutation in the conserved KITC motif (EITC variant). This variant retained its ability to bind CP but failed to interact with RPS2. PMID:20631085
Linker molecules between laminins and dystroglycan ameliorate laminin-?2�deficient muscular dystrophy at all disease stages
Meinen, Sarina; Barzaghi, Patrizia; Lin, Shuo; Lochm�ller, Hanns; Ruegg, Markus A.
Mutations in laminin-?2 cause a severe congenital muscular dystrophy, called MDC1A. The two main receptors that interact with laminin-?2 are dystroglycan and ?7?1 integrin. We have previously shown in mouse models for MDC1A that muscle-specific overexpression of a miniaturized form of agrin (mini-agrin), which binds to dystroglycan but not to ?7?1 integrin, substantially ameliorates the disease (Moll, J., P. Barzaghi, S. Lin, G. Bezakova, H. Lochmuller, E. Engvall, U. Muller, and M.A. Ruegg. 2001. Nature. 413:302�307; Bentzinger, C.F., P. Barzaghi, S. Lin, and M.A. Ruegg. 2005. Matrix Biol. 24:326�332.). Now we show that late-onset expression of mini-agrin still prolongs life span and improves overall health, although not to the same extent as early expression. Furthermore, a chimeric protein containing the dystroglycan-binding domain of perlecan has the same activities as mini-agrin in ameliorating the disease. Finally, expression of full-length agrin also slows down the disease. These experiments are conceptual proof that linking the basement membrane to dystroglycan by specifically designed molecules or by endogenous ligands, could be a means to counteract MDC1A at a progressed stage of the disease, and thus opens new possibilities for the development of treatment options for this muscular dystrophy. PMID:17389231
A New Role for Laminins as Modulators of Protein Toxicity in Caenorhabditis elegans
Jensen, Louise Toft; M�ller, Tine H�rning; Larsen, Simon Asbj�rn; Jakobsen, Helle; Olsen, Anders
Summary Protein misfolding is a common theme in aging and several age-related diseases such as Alzheimer's and Parkinson's disease. The processes involved in the development of these diseases are many and complex. Here, we show that components of the basement membrane, particularly laminin, affect protein integrity of the muscle cells they support. We knocked down gene expression of epi-1, a laminin ?-chain, and found that this resulted in increased proteotoxicity in different Caenorhabditis elegans transgenic models expressing aggregating proteins in the body wall muscle. The effect could partially be rescued by decreased insulin-like signaling, known to slow the aging process and the onset of various age-related diseases. Our data points to an underlying molecular mechanism involving proteasomal degradation and HSP-16 chaperone activity. Furthermore, epi-1 depleted animals had altered synaptic function and displayed hypersensitivity to both levamisole and aldicarb, an acetylcholine receptor agonist and an acetylcholinesterase inhibitor, respectively. Our results implicate the basement membrane as an extracellular modulator of protein homeostasis in the adjacent muscle cells. This is in agreement with previous research showing that imbalance in neuromuscular signaling disturbs protein homeostasis in the postsynaptic cell. In our study, proteotoxicity may indeed be mediated by the neuromuscular junction which is part of the basement membrane, where laminins are present in high concentration, ensuring the proper microenvironment for neuromuscular signaling. Laminins are evolutionarily conserved and thus the basement membrane may play a much more causal role in protein misfolding diseases than currently recognized. PMID:22051349
The B16F10 Cell Receptor for a Metastasis-promoting Site on Laminin1 Is a Heparan Sulfate\\/Chondroitin Sulfate-containing Proteoglycan
Jean A. Engbring; Matthew P. Hoffman; Arezo J. Karmand; Hynda K. Kleinman
Exposure to AG73, a synthetic peptide (LQVQLSIR) from the COOH- terminal region of the laminin 1 chain, induces a malignant phenotype in B16F10 melanoma cells. Coinjection of this peptide with the cells results in an increase of lung tumors and also the formation of liver tumors in 50% of the mice (W. H. Kim et al., Int. J. Cancer, 77:
Laminin Mediates Tissue-specific Gene Expression in Mammary Epithelia
Streuli, Charles H; Schmidhauser, Christian; Bailey, Nina; Yurchenco, Peter; Skubitz, Amy P. N.; Roskelley, Calvin; Bissell, Mina J
Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.
Interaction of human laminin receptor with Sup35, the [PSI?] prion-forming protein from S. cerevisiae: a yeast model for studies of LamR interactions with amyloidogenic proteins.
Pampeno, Christine; Derkatch, Irina L; Meruelo, Daniel
The laminin receptor (LamR) is a cell surface receptor for extracellular matrix laminin, whereas the same protein within the cell interacts with ribosomes, nuclear proteins and cytoskeletal fibers. LamR has been shown to be a receptor for several bacteria and viruses. Furthermore, LamR interacts with both cellular and infectious forms of the prion protein, PrP(C) and PrP(Sc). Indeed, LamR is a receptor for PrP(C). Whether LamR interacts with PrP(Sc) exclusively in a capacity of the PrP receptor, or LamR specifically recognizes prion determinants of PrP(Sc), is unclear. In order to explore whether LamR has a propensity to interact with prions and amyloids, we examined LamR interaction with the yeast prion-forming protein, Sup35. Sup35 is a translation termination factor with no homology or functional relationship to PrP. Plasmids expressing LamR or LamR fused with the green fluorescent protein (GFP) were transformed into yeast strain variants differing by the presence or absence of the prion conformation of Sup35, respectively [PSI?] and [psi?]. Analyses by immunoprecipitation, centrifugal fractionation and fluorescent microscopy reveal interaction between LamR and Sup35 in [PSI?] strains. The presence of [PSI?] promotes LamR co-precipitation with Sup35 as well as LamR aggregation. In [PSI?] cells, LamR tagged with GFP or mCherry forms bright fluorescent aggregates that co-localize with visible [PSI?] foci. The yeast prion model will facilitate studying the interaction of LamR with amyloidogenic prions in a safe and easily manipulated system that may lead to a better understanding and treatment of amyloid diseases. PMID:24416454
Lutheran blood group glycoprotein and its newly characterized mouse homologue specifically bind alpha5 chain-containing human laminin with high affinity
S. F. Parsons; G Lee; F A Spring; T N Willig; L L Peters; J A Gimm; M J Tanner; N Mohandas; D J Anstee; J A Chasis
Lutheran blood group glycoproteins (Lu gps) are receptors for the extracellular matrix protein, laminin. Studies suggest that Lu gps may contribute to vaso-occlusion in sickle cell disease and it has recently been shown that sickle cells adhere to laminin isoforms containing the alpha5 chain (laminin 10\\/11). Laminin alpha5 is present in the subendothelium and is also a constituent of bone
Spatial control of neuronal cell attachment and differentiation on covalently patterned laminin oligopeptide substrates.
Ranieri, J P; Bellamkonda, R; Bekos, E J; Gardella, J A; Mathieu, H J; Ruiz, L; Aebischer, P
The spatial control of neuronal cell attachment and differentiation via specific receptor mediated interactions, may provide an effective means for the in vitro reconstruction of neuronal cell architecture. In this study, receptor-specific oligopeptide sequences derived from the extracellular matrix (ECM) molecule laminin, a potent neural cell attachment and differentiation promoter were covalently bound on fluorinated ethylene propylene (FEP) films. The degree of receptor-specific cell attachment and the ability to spatially control neurite outgrowth by covalently patterning the oligopeptide sequences on the FEP film surface were assessed. FEP films were first chemically activated with a Radio Frequency Glow Discharge (RFGD) process that covalently replaces the surface fluorine atoms with reactive hydroxyl groups. Oligopeptides containing the YIGSR sequence from the B1 chain of laminin and the water soluble oligopeptide containing the IKVAV sequence (CSRARKQAASIKVAVSADR) from the A chain were covalently bound to the hydroxylated FEP films. Electron Spectroscopy for Chemical Analysis (ESCA) verified the covalent attachment of the oligopeptides to the material surface. The degree of receptor mediated NG108-15 cell attachment on immobilized CDPGYIGSR films was determined using competitive binding media. A 78% reduction in cell attachment was observed on films containing CDPGYIGSR in the cell plating medium. Only a 23% reduction in cell attachment was noted on films plated in medium containing a mock CDPGYIGSK sequence. FEP films immobilized with the IKVAV oligopeptide sequence were shown to mediate PC12 cell attachment and a competitive binding medium also significantly attenuated cell attachment on the immobilized films. The spatial patterning of these oligopeptide sequences to the FEP surface was shown to localize cell attachment and neurite extension on the patterned pathways. The surrounding unmodified FEP surface was inhibitory in serum containing medium and prevented cellular interactions outside the oligopeptide modifications. The spatial immobilization of laminin oligopeptides on FEP films provides a means to organize the attachment and differentiation of neuronal cells in a receptor-specific manner. PMID:7747599
Laminin-6 assembles into multimolecular fibrillar complexes with perlecan and participates in mechanical-signal transduction via a dystroglycan-dependent, integrin-independent mechanism.
Jones, Jonathan C R; Lane, Kimberly; Hopkinson, Susan B; Lecuona, Emilia; Geiger, Robert C; Dean, David A; Correa-Meyer, Eduardo; Gonzales, Meredith; Campbell, Kevin; Sznajder, Jacob I; Budinger, Scott
Mechanical ventilation is a valuable treatment regimen for respiratory failure. However, mechanical ventilation (especially with high tidal volumes) is implicated in the initiation and/or exacerbation of lung injury. Hence, it is important to understand how the cells that line the inner surface of the lung [alveolar epithelial cells (AECs)] sense cyclic stretching. Here, we tested the hypothesis that matrix molecules, via their interaction with surface receptors, transduce mechanical signals in AECs. We first determined that rat AECs secrete an extracellular matrix (ECM) rich in anastamosing fibers composed of the alpha3 laminin subunit, complexed with beta1 and gamma1 laminin subunits (i.e. laminin-6), and perlecan by a combination of immunofluorescence microscopy and immunoblotting analyses. The fibrous network exhibits isotropic expansion when exposed to cyclic stretching (30 cycles per minute, 10% strain). Moreover, this same stretching regimen activates mitogen-activated-protein kinase (MAPK) in AECs. Stretch-induced MAPK activation is not inhibited in AECs treated with antagonists to alpha3 or beta1 integrin. However, MAPK activation is significantly reduced in cells treated with function-inhibiting antibodies against the alpha3 laminin subunit and dystroglycan, and when dystroglycan is knocked down in AECs using short hairpin RNA. In summary, our results support a novel mechanism by which laminin-6, via interaction with dystroglycan, transduces a mechanical signal initiated by stretching that subsequently activates the MAPK pathway in rat AECs. These results are the first to indicate a function for laminin-6. They also provide novel insight into the role of the pericellular environment in dictating the response of epithelial cells to mechanical stimulation and have broad implications for the pathophysiology of lung injury. PMID:15928048
Laminin-6 assembles into multimolecular fibrillar complexes with perlecan and participates in mechanical-signal transduction via a dystroglycan-dependent, integrin-independent mechanism
Jones, Jonathan C. R.; Lane, Kimberly; Hopkinson, Susan B.; Lecuona, Emilia; Geiger, Robert C.; Dean, David A.; Correa-Meyer, Eduardo; Gonzales, Meredith; Campbell, Kevin; Sznajder, Jacob I.; Budinger, Scott
Summary Mechanical ventilation is a valuable treatment regimen for respiratory failure. However, mechanical ventilation (especially with high tidal volumes) is implicated in the initiation and/or exacerbation of lung injury. Hence, it is important to understand how the cells that line the inner surface of the lung [alveolar epithelial cells (AECs)] sense cyclic stretching. Here, we tested the hypothesis that matrix molecules, via their interaction with surface receptors, transduce mechanical signals in AECs. We first determined that rat AECs secrete an extracellular matrix (ECM) rich in anastamosing fibers composed of the ?3 laminin subunit, complexed with ?1 and ?1 laminin subunits (i.e. laminin-6), and perlecan by a combination of immunofluorescence microscopy and immunoblotting analyses. The fibrous network exhibits isotropic expansion when exposed to cyclic stretching (30 cycles per minute, 10% strain). Moreover, this same stretching regimen activates mitogen-activated-protein kinase (MAPK) in AECs. Stretch-induced MAPK activation is not inhibited in AECs treated with antagonists to ?3 or ?1 integrin. However, MAPK activation is significantly reduced in cells treated with function-inhibiting antibodies against the ?3 laminin subunit and dystroglycan, and when dystroglycan is knocked down in AECs using short hairpin RNA. In summary, our results support a novel mechanism by which laminin-6, via interaction with dystroglycan, transduces a mechanical signal initiated by stretching that subsequently activates the MAPK pathway in rat AECs. These results are the first to indicate a function for laminin-6. They also provide novel insight into the role of the pericellular environment in dictating the response of epithelial cells to mechanical stimulation and have broad implications for the pathophysiology of lung injury. PMID:15928048
Syndrome Enhance Receptor Activation
Paraneoplastic Neurodegenerative; Scott W. Rogers
Background: Paraneoplastic syndromes are "remote" complications of cancer characterized clinically by neurological disease. The sera and cerebrospinal fluid (CSF) from patients with paraneoplastic neurological syndromes (PNS) frequently contain autoantibodies to illdefined neuronal antigens. We report here that neuronal glutamate receptors are targets for autoantibodies found in the serum from some patients with well-characterized PNS. Materials and Methods: We have analyzed the serum from seven patients with well-characterized PNS for the presence of autoreactive antibodies to non-NMDA glutamate receptor subunits. Autoantibodies were assessed using Western blot, immunohistochemistry, and immunocytochemistry. Whole-cell electrophysiological recordings were used to examine the effect of antibodies on glutamate receptors expressed by cortical neurons in culture. Results: Six of seven patients ' serum contained autoantibodies to the non-NMDA glutamate receptor (GluR) subunits GluRl, GluR4, and/or GluR5/6. No patient had autoantibodies to GluR2, and only one patient exhibited weak immunoreactivity to GluR3. Electrophysiological analysis demonstrated that the serum from four of the six GluR-antibody-positive patients enhanced glutamate-elicited currents on cultured cortical neurons but had no effect on receptor function alone. Enhancement of glutamate-elicited currents was also produced by affinity-purified antibody to GluR5. Conclusions: The occurrence of autoantibodies to specific neuronal neurotransmitter subunits in the sera of patients with PNS and the ability of these autoantibodies to modulate glutaminergic receptor function suggest that some paraneoplastic neurological injury could result from glutamate-mediated excitotoxicity.
Endothelial Cell Laminin Isoforms, Laminins 8 and 10, Play Decisive Roles in T Cell Recruitment across the Blood�Brain Barrier in Experimental Autoimmune Encephalomyelitis
Sixt, Michael; Engelhardt, Britta; Pausch, Friederike; Hallmann, Rupert; Wendler, Olaf; Sorokin, Lydia M.
An active involvement of blood�brain barrier endothelial cell basement membranes in development of inflammatory lesions in the central nervous system (CNS) has not been considered to date. Here we investigated the molecular composition and possible function of the extracellular matrix encountered by extravasating T lymphocytes during experimental autoimmune encephalomyelitis (EAE). Endothelial basement membranes contained laminin 8 (?4?1?1) and/or 10 (?5?1?1) and their expression was influenced by proinflammatory cytokines or angiostatic agents. T cells emigrating into the CNS during EAE encountered two biochemically distinct basement membranes, the endothelial (containing laminins 8 and 10) and the parenchymal (containing laminins 1 and 2) basement membranes. However, inflammatory cuffs occurred exclusively around endothelial basement membranes containing laminin 8, whereas in the presence of laminin 10 no infiltration was detectable. In vitro assays using encephalitogenic T cell lines revealed adhesion to laminins 8 and 10, whereas binding to laminins 1 and 2 could not be induced. Downregulation of integrin ?6 on cerebral endothelium at sites of T cell infiltration, plus a high turnover of laminin 8 at these sites, suggested two possible roles for laminin 8 in the endothelial basement membrane: one at the level of the endothelial cells resulting in reduced adhesion and, thereby, increased penetrability of the monolayer; and secondly at the level of the T cells providing direct signals to the transmigrating cells. PMID:11381080
Alumina-zirconia composites functionalized with laminin-1 and laminin-5 for dentistry: effect of protein adsorption on cellular response.
Vall�e, A; Faga, M G; Mussano, F; Catalano, F; Tolosano, E; Carossa, S; Altruda, F; Martra, G
The present paper describes a study on laminin interaction with the surface of two alumina-zirconia composites with different percentages of ZrO2, both with submicrometric grain size. As major molecules within the basement membrane (BM), laminins are important protein fragments for epithelial cell adhesion and migration. On the other hand, alumina-zirconia composites are very attractive materials for dental applications due to their esthetic and mechanical properties. X-Ray photoelectron spectroscopy and atomic force microscopy were used to study the adsorption of two types of laminin, laminin-1 (Ln-1) and laminin-5 (Ln-5), onto the ceramics surfaces. The in vitro cell response was determined by intracellular phosphorylation of major kinases. Ceramics samples functionalized with laminins showed better cellular activation than untreated specimens; furthermore, cellular activation was found to be greater for the composite with higher percentage in zirconia when functionalized with Ln-5, whereas the adsorption of Ln-1 resulted in a greater activation for the alumina-rich oxide. PMID:24216619
Laminin-8 (alpha4beta1gamma1) is synthesized by lymphoid cells, promotes lymphocyte migration and costimulates T cell proliferation.
Geberhiwot, T; Assefa, D; Kortesmaa, J; Ingerpuu, S; Pedraza, C; Wondimu, Z; Charo, J; Kiessling, R; Virtanen, I; Tryggvason, K; Patarroyo, M
Laminins are a growing family of large heterotrimeric proteins with cell adhesive and signalling functions. They are major components of basement membranes and are found in many organs, including the vasculature and other compartments of bone marrow, thymus, lymph nodes and spleen. However, expression, recognition and use of laminin isoforms by lymphoid cells are poorly understood. In the present study, lymphoid T cells (Jurkat) were found to synthesize laminin alpha4, beta1 and gamma1 mRNAs and polypeptides and to assemble the chains into laminin-8. Lymphoblastoid B (NAD-20) cells, lymphoid NK (NKL) cells and blood lymphocytes also contained laminin-8 and, after cell permeabilization, practically all blood lymphocytes reacted with mAbs to laminin beta1 and gamma1 chains. Following stimulation, blood lymphocytes secreted laminin-8, and this laminin isoform, but not laminin-10/11(alpha5beta1gamma1/alpha5beta2gamma1), promoted chemokine-induced migration of the cells. In an activation-dependent manner, purified blood CD4 T cells adhered to immobilized laminin-8 and laminin-10/11 by using alpha6beta1 integrin, but minimally to laminin-1 (alpha1beta1gamma1). Accordingly, laminin-8 and laminin-10/11, but not laminin-1, strongly costimulated proliferation of the T cells via the same integrin. Thus, lymphoid cells are able to synthesize and secrete complete laminin molecules. In addition, synthesis of laminin-8 and recognition of laminin-8 and -10/11 by lymphocytes indicate relevance of these laminin isoforms in lymphocyte physiology. PMID:11148143
Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.
Maciaszek, Jamie L; Partola, Kostyantyn; Zhang, Jing; Andemariam, Biree; Lykotrafitis, George
Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 �m(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding. PMID:25458578
Characterization of the binding of serum amyloid P to laminin.
Zahedi, K
Serum amyloid P (SAP) is a member of the pentraxin family. These are evolutionarily conserved proteins made up of five noncovalently bound identical subunits that are arranged in a flat pentameric disc. Although a variety of activities have been attributed to SAP and other pentraxins, their biological functions remain unclear. In humans SAP is a constitutive serum protein that is synthesized by hepatocytes. It is encoded by a single copy gene on chromosome 1. SAP is a component of all amyloid plaques and is also a normal component of a number of basement membranes including the glomerular basement membrane. The association and distribution of SAP within the glomerular basement membrane are altered or completely disrupted in a number of nephritides (e.g. Alport's Syndrome, type II membranoproliferative glomerulonephritis, and membranous glomerulonephritis). In the present study the binding of SAP to laminin was characterized. SAP binds to human laminin and merosin as well as mouse and rat laminins. The binding of SAP to mouse laminin is saturable and calcium-dependent. The Kd of this interaction is 2. 74 x 10(-7) M, with a SAP/laminin molar ratio of 1:7.1. Competition binding assays indicate that the binding of SAP to laminin is inhibited by both SAP and its analog, C-reactive protein, as well as phosphatidylethanolamine. In turbidity assays SAP enhanced the polymerization of laminin in a concentration-dependent manner. However, SAP did not alter the ability of laminin to serve as a cell adhesion substrate. Previous observations indicating that SAP binds to extracellular matrix components such as type IV collagen, proteoglycans, and fibronectin in concert with the data presented here suggest that SAP may play an important role in determining the structure of those basement membranes with which it is associated. PMID:8999915
Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity
NASA Astrophysics Data System (ADS)
Yao, Yao; Chen, Zu-Lin; Norris, Erin H.; Strickland, Sidney
Blood brain barrier (BBB) breakdown is not only a consequence of but also contributes to many neurological disorders, including stroke and Alzheimer�s disease. How the basement membrane (BM) contributes to the normal functioning of the BBB remains elusive. Here we use conditional knockout mice and an acute adenovirus-mediated knockdown model to show that lack of astrocytic laminin, a brain-specific BM component, induces BBB breakdown. Using functional blocking antibody and RNAi, we further demonstrate that astrocytic laminin, by binding to integrin ?2 receptor, prevents pericyte differentiation from the BBB-stabilizing resting stage to the BBB-disrupting contractile stage, and thus maintains the integrity of BBB. Additionally, loss of astrocytic laminin decreases aquaporin-4 (AQP4) and tight junction protein expression. Altogether, we report a critical role for astrocytic laminin in BBB regulation and pericyte differentiation. These results indicate that astrocytic laminin maintains the integrity of BBB through, at least in part, regulation of pericyte differentiation.
Laminin 5 expression protects against anoikis at aerogenous spread and lepidic growth of human lung adenocarcinoma.
Kodama, Keiji; Ishii, Gen'ichiro; Miyamoto, Shin'ichi; Goya, Masato; Zhang, Shi-Chuan; Sangai, Takafumi; Yoshikawa, Takeshi; Hasebe, Takahiro; Hitomi, Yoshiaki; Izumi, Keisuke; Ochiai, Atsushi
Adenocarcinoma of the lung is characterized by frequent aerogenous spread (AE) and advancement along the alveolar wall (BAC growth). To elucidate the mechanism of AE metastasis and BAC growth in human lung adenocarcinoma, we established an in vivo orthotopic animal model and an in vitro culture. Investigation of expression levels of integrins, laminins and Type IV collagens, which are the major regulating molecules for cell attachment and anoikis was carried out and a clear correlation between the expression level of laminin 5 (LN5) and the BAC growth was observed using an orthotopic animal model. Introduction of LN5 cDNA to A549 cells increased anoikis resistance in an expression dependent manner. Cells with LN5 overexpression resisted with anoikis after treatment with PI3K-Akt and ERK inhibitors. The amount of phosphorylated focal adhesion kinase (FAK) was also higher in LN5 overexpressing cells. Major tyrosine residues of the EGF receptor at 1068, 1086 and 1173, except at 1148, remained phosphorylated only in the LN5 overexpressing cells even without EGF stimulation, that indicates the ligand independent activation of EGF receptor. BAC growth ratio and AE was confirmed to be significantly correlated with LN5 expression in surgically resected human lung adenocarcinomas by immunohistochemistry. Our results indicate that the activation of the EGF receptor by overexpressing LN5-integrin-FAK signaling pathway may play a crucial role in BAC growth and AE metastasis in human lung adenocarcinoma. PMID:15856467
RSK Activation of Translation Factor eIF4B Drives Abnormal Increases of Laminin ?2 and MYC Protein during Neoplastic Progression to Squamous Cell Carcinoma
Degen, Martin; Barron, Patricia; Natarajan, Easwar
Overexpression of the basement membrane protein Laminin ?2 (Lam?2) is a feature of many epidermal and oral dysplasias and all invasive squamous cell carcinomas (SCCs). This abnormality has potential value as an immunohistochemical biomarker of premalignancy but its mechanism has remained unknown. We recently reported that Lam?2 overexpression in culture is the result of deregulated translation controls and depends on the MAPK-RSK signaling cascade. Here we identify eIF4B as the RSK downstream effector responsible for elevated Lam?2 as well as MYC protein in neoplastic epithelial cells. Premalignant dysplastic keratinocytes, SCC cells, and keratinocytes expressing the E6 oncoprotein of human papillomavirus (HPV) type 16 displayed MAPK-RSK and mTOR-S6K1 activation and overexpressed Lam?2 and MYC in culture. Immunohistochemical staining of oral dysplasias and SCCs for distinct, RSK- and S6K1-specific S6 phosphorylation events revealed that their respective upstream pathways become hyperactive at the same time during neoplastic progression. However, pharmacologic kinase inhibitor studies in culture revealed that Lam?2 and MYC overexpression depends on MAPK-RSK activity, independent of PI3K-mTOR-S6K1. eIF4B knockdown reduced Lam?2 and MYC protein expression, consistent with the known requirement for eIF4B to translate mRNAs with long, complex 5? untranslated regions (5?-UTRs). Accordingly, expression of a luciferase reporter construct preceded by the Lam?2 5?-UTR proved to be RSK-dependent and mTOR-independent. These results demonstrate that RSK activation of eIF4B is causally linked to elevated Lam?2 and MYC protein levels during neoplastic progression to invasive SCC. These findings have potential clinical significance for identifying premalignant lesions and for developing targeted drugs to treat SCC. PMID:24205356
Sialic acid-dependent recognition of laminin and fibrinogen by Aspergillus fumigatus conidia.
Bouchara, J P; Sanchez, M; Chevailler, A; Marot-Leblond, A; Lissitzky, J C; Tronchin, G; Chabasse, D
In an attempt to define the molecular basis of the adherence of Aspergillus fumigatus conidia to the host tissues, a step which might be mediated by the recognition of basement membrane laminin or fibrinogen, we analyzed the binding of these glycoproteins by flow cytometry and a microtiter plate adherence assay. Flow cytometry revealed that the binding of fluorescein isothiocyanate-labeled laminin to conidia was saturable and specific. Moreover, the ability of conidia to bind laminin increased with their maturation. Competition experiments showed a cross-reactivity between laminin and fibrinogen binding and a lack of interactions with glycosaminoglycans. In addition, the binding of laminin was not inhibited by the different adhesive synthetic peptides tested. Furthermore, the microtiter plate assay of adherence to chymotrypsin degradation products of laminin or fibrinogen purified by gel filtration suggested a unique binding site common to sequential degradation fragments or the presence of multiple binding sites on the two ligands. Therefore, the role of carbohydrates in the recognition process was investigated. Among the carbohydrates tested, constitutive of the conidial wall or of the oligosaccharide side chains of laminin and fibrinogen, only N-acetylneuraminic acid and sialyllactose inhibited the binding of these glycoproteins to conidia. In conclusion, these results strengthen the idea that the laminin and fibrinogen receptors in A. fumigatus are identical and suggest an interaction mediated by a sialic acid-specific lectin of the conidial wall. PMID:9199441
Laminin-based cell adhesion anchors microtubule plus ends to the epithelial cell basal cortex through LL5?/?
Hotta, Azusa; Kawakatsu, Tomomi; Nakatani, Tomoya; Sato, Toshitaka; Matsui, Chiyuki; Sukezane, Taiko; Akagi, Tsuyoshi; Hamaji, Tomoko; Grigoriev, Ilya; Akhmanova, Anna; Takai, Yoshimi
LL5? has been identified as a microtubule-anchoring factor that attaches EB1/CLIP-associating protein (CLASP)�bound microtubule plus ends to the cell cortex. In this study, we show that LL5? and its homologue LL5? (LL5s) colocalize with autocrine laminin-5 and its receptors, integrins ?3?1 and ?6?4, at the basal side of fully polarized epithelial sheets. Depletion of both laminin receptor integrins abolishes the cortical localization of LL5s, whereas LL5 depletion reduces the amount of integrin ?3 at the basal cell cortex. Activation of integrin ?3 is sufficient to initiate LL5 accumulation at the cell cortex. LL5s form a complex with the cytoplasmic tails of these integrins, but their interaction might be indirect. Analysis of the three-dimensional distribution of microtubule growth by visualizing EB1-GFP in epithelial sheets in combination with RNA interference reveals that LL5s are required to maintain the density of growing microtubules selectively at the basal cortex. These findings reveal that signaling from laminin�integrin associations attaches microtubule plus ends to the epithelial basal cell cortex. PMID:20513769
Laminin ?2 Chain-Positive Vessels and Epidermal Growth Factor in Lung Neuroendocrine Carcinoma
Vitolo, Domenico; Ciocci, Luciano; Deriu, Gloria; Spinelli, Silvia; Cortese, Stefania; Masuelli, Laura; Morrone, Stefania; Filice, Mary Jo; Coloni, Giorgio Furio; Natali, Pier Giorgio; Baroni, Carlo Davide
Capillaries expressing the laminin ?2 chain in basement membranes may be considered early developing vessels in normal and neoplastic human tissues. Therefore, we investigated whether up-regulation of this extracellular matrix protein favors transendothelial migration of neoplastic cells and then metastasis. In lung small and large cell neuroendocrine carcinomas, which exhibit a stronger metastatic tendency among carcinomas, laminin ?2 chain-positive vessels were more numerous than in carcinoid tumors and supraglottis, breast, and lung non-small cell carcinomas, suggesting a direct relationship between these vessels and metastasis. In vitro studies showed that epidermal growth factor (EGF) induced a more efficient migration of the AE-2 lung neuroendocrine carcinoma cell line through the purified laminin ?2 chain rather than through the laminin ?1 chain and fibronectin. AE-2 cells constitutively expressed all EGF receptors and the ?6?1 integrin, which is one of the laminin ?2 chain receptors. EGF up-regulated ?6?1 expression in several tumors. In this regard, we show that EGF increased the chemo-kinetic migration of AE-2 cells through EAHY endothelial monolayers, which was inhibited by the anti-?6 integrin chain monoclonal antibody. These data indicate that laminin ?2 chain and ?6?1 may be mutually involved in EGF-dependent migration of AE-2 cells and that laminin ?2 chain-positive vessels may favor metastasis of EGF-dependent tumors. PMID:16507913
Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment
Stable attachment of external epithelia to the basement membrane and underlying stroma is mediated by transmembrane proteins such as the integrin alpha6beta4 and bullous pemphigoid antigen 2 within the hemidesmosomes along the basolateral surface of the epithelial cell and their ligands that include a specialized subfamily of laminins. The laminin 5 molecule (previously termed kalinin/nicein/epiligrin) is a member of this epithelial-specific subfamily. Laminin 5 chains are not only considerably truncated within domains III-VI, but are also extensively proteolytically processed in vitro and in vivo. As a result, the domains expected to be required for the association of laminins with other basement membrane components are lacking in the mature laminin 5 molecule. Therefore, the tight binding of laminin 5 to the basement membrane may occur by a unique mechanism. To examine laminin 5 in tissue, we chose human amnion as the source, because of its availability and the similarity of the amniotic epithelial basement membrane with that of skin. We isolated the laminin 5 contained within the basement membrane of human amnion. In addition to monomeric laminin 5, we find that much of the laminin 5 isolated is covalently adducted with laminin 6 (alpha3beta1gamma1) and a novel laminin isotype we have termed laminin 7 (alpha3beta2gamma1). We propose that the association between laminin 5 and laminins 6 and 7 is a mechanism used in amnion to allow stable association of laminin 5 with the basement membrane. The beta2 chain is seen at the human amniotic epithelial-stromal interface and at the dermal-epidermal junction of fetal and adult bovine skin by immunofluorescence, but is not present, or only weakly present, in neonatal human skin. PMID:8601594
Activation of Metabotropic Glutamate Receptor 1 Accelerates NMDA Receptor Trafficking
Jian-yu Lan; Vytenis A. Skeberdis; Teresa Jover; Xin Zheng; Michael V. L. Bennett; R. Suzanne
Regulation of neuronal NMDA receptors (NMDARs) by group I metabotropic glutamate receptors (mGluRs) is known to play a critical role in synaptic transmission. The molecular mecha- nisms underlying mGluR1-mediated potentiation of NMDARs are as yet unclear. The present study shows that in Xenopus oocytes expressing recombinant receptors, activation of mGluR1 potentiates NMDA channel activity by recruitment of new channels to
The role of receptor structure in determining adenosine receptor activity
Mark E Olah; Gary L Stiles
Adenosine produces a wide variety of physiological effects through the activation of cell surface adenosine receptors (ARs). ARs are members of the G-protein-coupled receptor family, and currently, four subtypes, the A1AR, A2AAR, A2BAR, and A3AR, are recognized. This review focuses on the role of receptor structure in governing various facets of AR activity. Ligand-binding properties of ARs are primarily dictated
Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen
alpha 3 beta 1 (VLA-3), a member of the integrin family of cell adhesion receptors, may function as a receptor for fibronectin, laminin, and collagen. A partial cDNA clone (2.4 kb) for the human alpha 3 subunit was selected from an endothelial cell lambda gt11 cDNA library by specific antibody screening. Several overlapping cDNA clones were subsequently obtained, of a total length of 4.6 kb from various cDNA libraries. The reconstructed alpha 3 cDNA was expressed on the surface of chinese hamster ovary cells as detected by an alpha 3- specific mAb after transfection, suggesting that the cDNA is authentic. Within this sequence was an open reading frame, encoding for 1,051 amino acids, including a signal peptide of 32 residues, a long extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic segment (32 residues). Overall, the alpha 3 amino acid sequence was 25-37% similar to the other integrin alpha subunits that are cleaved, with most similarity to the alpha 6 sequence (37%), and less similarity to those alpha subunits that have I domains (15-20%, excluding the I domain sequence itself). Features most like those in other alpha subunits are (a) the positions of 18/19 cysteine residues, (b) three potential metal binding domains of the general structure DX(D/N)X(D/N)GXXD, and (c) the predicted transmembrane domain. The mass of alpha 3 calculated from its amino acid sequence is 113,505. The human alpha 3 sequence was 89% identical to hamster galactoprotein b3, and 70% similar to the chicken CSAT antigen band 2 protein partial sequence, suggesting that these two polypeptides are homologues of human alpha 3. PMID:1655803
Expression and localization of laminin 5, laminin 10, type IV collagen, and amelotin in adult murine gingiva.
Sawada, Takashi; Yamazaki, Takaki; Shibayama, Kazuko; Kumazawa, Kaido; Yamaguchi, Yoko; Ohshima, Mitsuhiro
The biochemical composition of the internal and external basal laminae in the junctional epithelium differs significantly, and the precise cellular origin of their respective molecules remains to be determined. In the present study, the expression and localization of three basement membrane-specific molecules-laminin 5 (?2 chain), type IV collagen (?1 chain), and laminin 10 (?5 chain)-and one tooth-specific molecule, amelotin, was analyzed in adult murine gingiva by using in situ hybridization and immunohistochemistry. The results showed that the outermost cells in junctional epithelium facing the tooth enamel strongly expressed laminin 5 mRNA, supporting the immunohistochemical staining data. This suggests that laminin 5 is actively synthesized in junctional epithelial cells and that the products are incorporated into the internal basal lamina to maintain firm epithelial adhesion to the tooth enamel throughout life. Conversely, no amelotin mRNA signals were detected in the junctional epithelial cells, suggesting that the molecules localized on the internal basal lamina are mainly derived from maturation-stage ameloblasts. Weak and sporadic expression of type IV collagen in addition to laminin 10 in the gingiva indicates that these molecules undergo turnover less frequently in adult animals. PMID:24338356
Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.
Chung, Heesung; Jung, Hyejung; Lee, Jung-Hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo
Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and ?-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. PMID:24951591
Anchorage mediated by integrin alpha6beta4 to laminin 5 (epiligrin) regulates tyrosine phosphorylation of a membrane-associated 80-kD protein
Detachment of basal keratinocytes from basement membrane signals a differentiation cascade. Two integrin receptors alpha6beta4 and alpha3beta1 mediate adhesion to laminin 5 (epiligrin), a major extracellular matrix protein in the basement membrane of epidermis. By establishing a low temperature adhesion system at 4 degrees C, we were able to examine the exclusive role of alpha6beta4 in adhesion of human foreskin keratinocyte (HFK) and the colon carcinoma cell LS123. We identified a novel 80-kD membrane-associated protein (p80) that is tyrosine phosphorylated in response to dissociation of alpha6beta4 from laminin 5. The specificity of p80 phosphorylation for laminin 5 and alpha6beta4 was illustrated by the lack of regulation of p80 phosphorylation on collagen, fibronectin, or poly-L-lysine surfaces. We showed that blocking of alpha3beta1 function using inhibitory mAbs, low temperature, or cytochalasin D diminished tyrosine phosphorylation of focal adhesion kinase but not p80 phosphorylation. Therefore, under our assay conditions, p80 phosphorylation is regulated by alpha6beta4, while motility via alpha3beta1 causes phosphorylation of focal adhesion kinase. Consistent with a linkage between p80 dephosphorylation and alpha6beta4 anchorage to laminin 5, we found that phosphatase inhibitor sodium vanadate, which blocked the p80 dephosphorylation, prevented the alpha6beta4-dependent cell anchorage to laminin 5 at 4degreesC. In contrast, adhesion at 37 degrees C via alpha3beta1 was unaffected. Furthermore, by in vitro kinase assay, we identified a kinase activity for p80 phosphorylation in suspended HFKs but not in attached cells. The kinase activity, alpha6beta4, and its associated adhesion structure stable anchoring contacts were all cofractionated in the Triton- insoluble cell fraction that lacks alpha3beta1. Thus, regulation of p80 phosphorylation, through the activities of p80 kinase and phosphatase, correlates with alpha6beta4-SAC anchorage to laminin 5 at 4 degrees C in epithelial cells of the skin and intestine. Transmembrane signaling through p80 is an early tyrosine phosphorylation event responsive to and possibly required for anchorage to laminin 5 by HFK and LS123 epithelial cells. PMID:8647901
Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts
Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of)] [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, So Young [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of) [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Jang, Hwan-Hee [Functional Food and Nutrition Division, Department of Agrofood Resources, Rural Development Administration, Suwon 441-853 (Korea, Republic of)] [Functional Food and Nutrition Division, Department of Agrofood Resources, Rural Development Administration, Suwon 441-853 (Korea, Republic of); Ryu, Sung Ho [Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk 790-784 (Korea, Republic of)] [Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, Beom Joon [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of) [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Lee, Taehoon G., E-mail: firstname.lastname@example.com [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of)
Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the {beta}1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.
The C-terminal region of laminin beta chains modulates the integrin binding affinities of laminins.
Taniguchi, Yukimasa; Ido, Hiroyuki; Sanzen, Noriko; Hayashi, Maria; Sato-Nishiuchi, Ryoko; Futaki, Sugiko; Sekiguchi, Kiyotoshi
Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (alpha, beta, and gamma), in which three laminin globular modules in the alpha chain and the Glu residue in the C-terminal tail of the gamma chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the beta chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the beta1 or beta2 chain toward a panel of laminin-binding integrins, and we found that beta2 chain-containing laminins (beta2-laminins) bound more avidly to alpha3beta1 and alpha7X2beta1 integrins than beta1 chain-containing laminins (beta1-laminins), whereas alpha6beta1, alpha6beta4, and alpha7X1beta1 integrins did not show any preference toward beta2-laminins. Because alpha3beta1 contains the "X2-type" variable region in the alpha3 subunit and alpha6beta1 and alpha6beta4 contain the "X1-type" region in the alpha6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between beta1-laminins and beta2-laminins. In support of this possibility, a putative X2-type variant of alpha6beta1 was produced and found to bind preferentially to beta2-laminins. Production of a series of swap mutants between the beta1 and beta2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by beta2-laminins. Taken together, the results provide evidence that the C-terminal region of beta chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins. PMID:19147489
Modulation of matrix metalloproteinase-9 secretion from tumor-associated macrophage-like cells by proteolytically processed laminin-332 (laminin-5).
Kamoshida, Go; Ogawa, Takashi; Oyanagi, Jun; Sato, Hiroki; Komiya, Eriko; Higashi, Shouichi; Miyazaki, Kaoru; Tsuji, Tsutomu
Macrophages infiltrating tumor tissues (tumor-associated macrophages, TAM) affect the malignant behaviors of tumor cells. We previously reported that monocytes were differentiated into TAM-like cells secreting matrix metalloproteinase (MMP)-9 by co-culture with tumor cells, and that cell adhesion to extracellular matrix (ECM) proteins played a critical role in the differentiation. In this study, we found that the monocyte differentiation was promoted by laminin-332 (laminin-5), a major epithelial ECM component. We also demonstrated that the proteolytic processing of the ?2 chain of laminin-332 was essential for its activity but that the N-terminal short arm of the ?2 chain inhibited MMP-9 secretion. These results indicate that the activity of laminin-332 for monocyte differentiation is dynamically regulated by the proteolytic processing of the ?2 chain. PMID:24292405
Laminin-based Nanomaterials for Peripheral Nerve Tissue Engineering
NASA Astrophysics Data System (ADS)
Neal, Rebekah Anne
Peripheral nerve transection occurs commonly in traumatic injury, causing motor and sensory deficits distal to the site of injury. One option for surgical repair is the nerve conduit. Conduits currently on the market are hollow tubes into which the nerve ends are sutured. Although these conduits fill the gap, they often fail due to the slow rate of regeneration over long gaps. To facilitate increased speed of regeneration and greater potential for functional recovery, the ideal conduit should provide biochemically relevant signals and physical guidance cues, thus playing an active role in peripheral nerve regeneration. In this dissertation, I fabricated laminin-1 and laminin-polycaprolactone (PCL) blend nanofibers that mimic the geometry and functionality of the peripheral nerve basement membrane. These fibers resist hydration in aqueous media and require no harsh chemical crosslinkers. Adhesion and differentiation of both neuron-like and neuroprogenitor cells is improved on laminin nanofibrous meshes over two-dimensional laminin substrates. Blend meshes with varying laminin content were characterized for composition, tensile properties, degradation rates, and bioactivity in terms of cell attachment and axonal elongation. I have established that 10% (wt) laminin content is sufficient to retain the significant neurite-promoting effects of laminin critical in peripheral nerve repair. In addition, I utilized modified collector plate design to manipulate electric field gradients during electrospinning for the fabrication of aligned nanofibers. These aligned substrates provide enhanced directional guidance cues to the regenerating axons. Finally, I replicated the clinical problem of peripheral nerve transection using a rat tibial nerve defect model for conduit implantation. When the lumens of conduits were filled with nanofiber meshes of varying laminin content and alignment, I observed significant recovery of sensory and motor function over six weeks. This recovery was supported by nerve conduction studies and electromyography which described impulse transmission, muscle stimulation, and foot twitch through the region of regeneration. These studies provide a firm foundation for the use of natural-synthetic blend electrospun nanofibers to enhance existing hollow nerve guidance conduits. The similarity in surgical technique and obvious benefit to the patient should lead to rapid translation into clinical application.
Laminin 332 Expression in Breast Carcinoma
Kwon, Soon-Young; Chae, Seoung W.; Wilczynski, Sharon P.; Arain, Ahmad; Carpenter, Philip M.
Laminin 332 (LN332) is a basally expressed extracellular matrix protein that enhances the migration and invasion of breast carcinoma cells. The goal of this study was to examine LN332 expression breast carcinoma. Triple negative breast carcinomas lack estrogen receptor (ER), progesterone receptor (PR) expression and HER2 positivity. Immunohistochemistry for ER, PR, HER2, and dual silver in situ hybridization for the HER2 gene were used to define the phenotype of 243 breast cancers in biopsies or arrays. Immunohistochemistry for LN332 revealed that 70 % of triple negative carcinomas stained for LN332. Cytokeratin 5/6 (CK5/6), epidermal growth factor receptor (EGFR) and p63 alone stained fewer triple negative breast carcinomas each, but the combination of LN332 and CK 5/6 or EGFR identified 92% of triple negative breast carcinoma.. Of the 163 non- triple negative cases, LN332 was expressed in only 15%. The identification of LN332 in triple negative breast carcinomas is consistent with gene profiling studies showing its expression among breast carcinomas with a basal phenotype. The observation that a pro-invasive protein such as LN332 is expressed in breast cancer suggests another mechanism by which the triple negative phenotype could be aggressive. PMID:22427740
Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly*S?
McKee, Karen K.; Capizzi, Stephanie; Yurchenco, Peter D.
Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the ?4 and ?5 subunits are expressed in ?2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (?LNNd) that adds a short arm terminus to laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the laminin coiled-coil dystroglycan and sulfatides. ?LNNd was found to mediate laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert ?-short arm deletion-mutant laminins Lm??LN and Lm??LN-L4b into polymerizing laminins. This protein enabled polymerization-deficient laminin but not an adhesion-deficient laminin lacking LG domains (Lm?LG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled Lm?LG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the ?LN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of laminin deficiency states. PMID:19189961
NMR constrained solution structures for laminin peptide 11. Analogs define structural requirements for inhibition of tumor cell invasion of basement membrane matrix.
Ostheimer, G J; Starkey, J R; Lambert, C G; Helgerson, S L; Dratz, E A
Peptide 11, CDPGYIGSR-NH2, is a segment of laminin which blocks tumor cell invasion. A high affinity laminin receptor in tumor cells is thought to be blocked by the carboxyl-terminal YIGSR, and conformational energy calculations suggest that the glycine in YIGSR allows an important conformational bend. We replaced the YIGSR glycine residue in peptide 11 with either D-alanine or L-alanine to allow or disfavor the proposed glycine bend. We found the Gly7-->D-Ala7 analog to be equal to peptide 11 in inhibiting tumor cell invasion of basement membrane matrix. The Gly7-->L-Ala7 analog was much less capable of invasion inhibition. Two-dimensional 1H-1H NMR was used to study the solution conformations of the peptide 11 analogs. NOESY experiments revealed close NH-NH contacts in peptide 11 and the D-Ala7 analog, but not in the L-Ala7 analog. Molecular dynamics generated low energy structures with excellent NOE agreement for peptide 11 and its analogs. Both peptide 11 and the D-Ala7 analog, but not the less active L-Ala7 analog, were predicted to have similar bends around Gly7 or D-Ala7. These results suggest that a bend in the YIGSR region of peptide 11 may be important for the binding of laminin to its metastasis-associated receptor. PMID:1460013
Lung-Specific Loss of ?3 Laminin Worsens Bleomycin-Induced Pulmonary Fibrosis.
Morales-Nebreda, Luisa I; Rogel, Micah R; Eisenberg, Jessica L; Hamill, Kevin J; Soberanes, Saul; Nigdelioglu, Recep; Chi, Monica; Cho, Takugo; Radigan, Kathryn A; Ridge, Karen M; Misharin, Alexander V; Woychek, Alex; Hopkinson, Susan; Perlman, Harris; Mutlu, Gokhan M; Pardo, Annie; Selman, Moises; Jones, Jonathan C R; Budinger, G R Scott
Laminins are heterotrimeric proteins that are secreted by the alveolar epithelium into the basement membrane, and their expression is altered in extracellular matrices from patients with pulmonary fibrosis. In a small number of patients with pulmonary fibrosis, we found that the normal basement membrane distribution of the ?3 laminin subunit was lost in fibrotic regions of the lung. To determine if these changes play a causal role in the development of fibrosis, we generated mice lacking the ?3 laminin subunit specifically in the lung epithelium by crossing mice expressing Cre recombinase driven by the surfactant protein C promoter (SPC-Cre) with mice expressing floxed alleles encoding the ?3 laminin gene (Lama3(fl/fl)). These mice exhibited no developmental abnormalities in the lungs up to 6 months of age, but, compared with control mice, had worsened mortality, increased inflammation, and increased fibrosis after the intratracheal administration of bleomycin. Similarly, the severity of fibrosis induced by an adenovirus encoding an active form of transforming growth factor-? was worse in mice deficient in ?3 laminin in the lung. Taken together, our results suggest that the loss of ?3 laminin in the lung epithelium does not affect lung development, but plays a causal role in the development of fibrosis in response to bleomycin or adenovirally delivered transforming growth factor-?. Thus, we speculate that the loss of the normal basement membrane organization of ?3 laminin that we observe in fibrotic regions from the lungs of patients with pulmonary fibrosis contributes to their disease progression. PMID:25188360
O-Mannosyl Phosphorylation of Alpha-Dystroglycan is Required for Laminin Binding
Yoshida-Moriguchi, Takako; Yu, Liping; Stalnaker, Stephanie H.; Davis, Sarah; Kunz, Stefan; Oldstone, Michael B.A.; Schachter, Harry; Wells, Lance; Campbell, Kevin P.
Alpha-dystroglycan is a cell-surface glycoprotein that acts as a receptor for both extracellular matrix proteins containing laminin-G domains and certain arenaviruses. Receptor binding is thought to be mediated by a post-translational modification, and defective binding with laminin underlies a subclass of congenital muscular dystrophy. Here, using mass spectrometry- and NMR-based structural analyses, we identified a phosphorylated O-mannosyl glycan on the mucin-like domain of recombinant alpha-dystroglycan, which was required for laminin binding. We demonstrated that patients with muscle-eye-brain disease and Fukuyama congenital muscular dystrophy, as well as mice with myodystrophy, commonly have defects in a post-phosphoryl modification of this phosphorylated O-linked mannose, and that this modification is mediated by the like-acetylglucosaminyltransferase (LARGE) protein. Our findings expand our understanding of the mechanisms that underlie congenital muscular dystrophy. PMID:20044576
Rethinking Molecular Mimicry in Rheumatic Heart Disease and Autoimmune Myocarditis: Laminin, Collagen IV, CAR, and B1AR as Initial Targets of Disease
Root-Bernstein, Robert
Rationale: Molecular mimicry theory (MMT) suggests that epitope mimicry between pathogens and human proteins can activate autoimmune disease. Group A streptococci (GAS) mimics human cardiac myosin in rheumatic heart disease (RHD) and coxsackie viruses (CX) mimic actin in autoimmune myocarditis (AM). But myosin and actin are immunologically inaccessible and unlikely initial targets. Extracellular cardiac proteins that mimic GAS and CX would be more likely. Objectives: To determine whether extracellular cardiac proteins such as coxsackie and adenovirus receptor (CAR), beta 1 adrenergic receptor (B1AR), CD55/DAF, laminin, and collagen IV mimic GAS, CX, and/or cardiac myosin or actin. Methods: BLAST 2.0 and LALIGN searches of the UniProt protein database were employed to identify potential molecular mimics. Quantitative enzyme-linked immunosorbent assay was used to measure antibody cross-reactivity. Measurements: Similarities were considered to be significant if a sequence contained at least 5 identical amino acids in 10. Antibodies were considered to be cross-reactive if the binding constant had a Kd less than 10-9 M. Main results: Group A streptococci mimics laminin, CAR, and myosin. CX mimics actin and collagen IV and B1AR. The similarity search results are mirrored by antibody cross-reactivities. Additionally, antibodies against laminin recognize antibodies against collagen IV; antibodies against actin recognize antibodies against myosin, and antibodies against GAS recognize antibodies against CX. Thus, there is both mimicry of extracellular proteins and antigenic complementarity between GAS-CX in RHD/AM. Conclusion: Rheumatic heart disease/AM may be due to combined infections of GAS with CX localized at cardiomyocytes that may produce a synergistic, hyperinflammatory response that cross-reacts with laminin, collagen IV, CAR, and/or B1AR. Epitope drift shifts the immune response to myosin and actin after cardiomyocytes become damaged. PMID:25191648
Notch Receptor Activation Inhibits Oligodendrocyte Differentiation
Songli Wang; Andrei D Sdrulla; Guy diSibio; Gay Bush; Donna Nofziger; Carol Hicks; Gerry Weinmaster; Ben A Barres
In this study, we show that oligodendrocyte differentiation is powerfully inhibited by activation of the Notch pathway. Oligodendrocytes and their precursors in the developing rat optic nerve express Notch1 receptors and, at the same time, retinal ganglion cells express Jagged1, a ligand of the Notch1 receptor, along their axons. Jagged1 expression is developmentally regulated, decreasing with a time course that
Differential Activation of Peroxisome Proliferator-activated Receptors by Eicosanoids
W. Bayona; Caleb B. Kallen; Heather P. Harding; Christina P. Ravera; Gerald McMahon; Myles Browni; Mitchell A. Lazar
Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate gene tran- scription in response to peroxisome proliferators and fatty acids. PPARs also play an important role in the regulation of adipocyte differentiation. It is unclear, however, what naturally occurring compounds activate each of the PPAR subtypes. To address this issue, a screening assay was established using heterologous fu- sions
A new nomenclature for the laminins.
Burgeson, R E; Chiquet, M; Deutzmann, R; Ekblom, P; Engel, J; Kleinman, H; Martin, G R; Meneguzzi, G; Paulsson, M; Sanes, J
The authors have adopted a new nomenclature for the laminins. They are numbered with arabic numerals in the order discovered. The previous A, B1 and B2 chains, and their isoforms, are alpha, beta and gamma, respectively, followed by an arabic numeral to identify the isoform. For example, the first laminin identified from the Engelbreth-Holm-Swarm tumor is laminin-1 with the chain composition alpha 1 beta 1 gamma 1. The genes for these chains are LAMA1, LAMB1 and LAMC1, respectively. PMID:7921537
Coupled expression and colocalization of 140K cell adhesion molecules, fibronectin, and laminin during morphogenesis and cytodifferentiation of chick lung cells
We have analyzed the expression and distribution of fibronectin, laminin, and the 140K cell adhesion molecules (140K complex) in embryonic chick lung cells by a combination of biochemical and immunofluorescent approaches. The 140K complex was identified by monoclonal antibody JG22E as a complex of glycoproteins averaging 140,000 Mr and has been implicated in vitro as a receptor for fibronectin and laminin. Our studies provide the first description that the 140K complex is developmentally regulated, and that the 140K complex appears to be involved in adhesion of epithelial and endothelial cells during morphogenesis. We have shown that the 140K complex is expressed in high quantity in embryonic lung cell types, but is markedly reduced in all of the differentiated cell types except smooth muscle. Embryonic lung cells are enriched in 140K complex on portions of cells in close proximity to areas rich in fibronectin. For example, during the formation of airways and alveolar tissues, 140K complex is concentrated at the basal surfaces of epithelial cells adjacent to fibronectin. Likewise, during the angiogenic invasion of capillaries into lung mesenchyme, the 140K complex becomes localized at sites on the basal surfaces of endothelial cells in close contact with fibronectin. Finally, cytodifferentiating lung smooth muscle cells show unusually high levels of 140K complex, fibronectin, and laminin that persist into the adult. In contrast to fibronectin, laminin is found to be uniformly distributed in the basement membranes of differentiating epithelial cells. It becomes prominent in adult alveolar epithelium and airway epithelium concomitant with a reduction or loss of 140K complex and fibronectin at cell-basement membrane attachment sites. Surprisingly, laminin is also present in a punctate pattern in the mesenchyme of early lung buds, however, laminin, fibronectin, and 140K complex are greatly reduced or lost during mesenchymal maturation. Our results are consistent with the active participation of the 140K complex in cell-to-matrix adhesion during morphogenesis of alveolar walls and cytodifferentiation of mesenchymal and smooth muscle cells. PMID:3528168
Androgen insensitivity syndrome: gonadal androgen receptor activity
Coulam, C.B.; Graham, M.L.; Spelsberg, T.C.
To determine whether abnormalities of the androgen receptor previously observed in skin fibroblasts from patients with androgen insensitivity syndrome also occur in the gonads of affected individuals, androgen receptor activity in the gonads of a patient with testicular feminization syndrome was investigated. Using conditions for optimal recovery of androgen receptor from human testes established by previous studies, we detected the presence of a high-affinity (dissociation constant . 3.2 X 10(-10) mol/L), low-capacity (4.2 X 10(-12) mol/mg DNA), androgen-binding protein when tritium-labeled R1881 was incubated at 4 degrees C with nuclear extracts from the gonads of control patients or from a patient with testicular feminization syndrome but not when incubated at 37 degrees C. Thus this patient has an androgen receptor with a temperature lability similar to that of receptors from normal persons.
Transglutaminase-mediated oligomerization of galectin-3 modulates human melanoma cell interactions with laminin.
van den Br�le, F A; Liu, F T; Castronovo, V
Tumor cell adhesion and migration to laminin are important events during invasion and metastatic spread. Galectin-3, a multifunctional member of the galectin family, binds specifically the poly-N-acetyllactosamine residues of laminin and has been implicated in tumor invasion and metastasis. Galectin-3 is multimerized by transglutaminase, an enzyme that catalyzes cross-linking between glutamine and other aminoacid residues. In this study, we examined the consequences of transglutaminase-mediated galectin-3 oligomerization on the interactions between cancer cells and laminin. We first demonstrated that human galectin-3 is cross-linked by guinea pig liver transglutaminase, forms oligomers, and incorporates the marker 5-(biotinamido) pentylamine. Expression of transglutaminase activity in the A375 and A2058 human melanoma cell extracts was revealed by its ability to induce galectin-3 oligomerization and 5-(biotinamido) pentylamine incorporation. Transglutaminase-treated galectin-3 did not affect adhesion or migration of the melanoma cells to laminin but consistently induced a significant increase of the percentage of cell spreading compared to the control (23.5 +/- 2.3%, vs. 10.6 +/- 1.9% at 180 min, p < 0.05), or to untreated galectin-3 or transglutaminase alone. Our study is the first demonstration that human galectin-3 is oligomerized by transglutaminase with, as a consequence, a specific effect of melanoma cell spreading on laminin. This phenomenon could be of significance in the modulation of cancer cell interactions with laminin during tumor invasion and metastasis. PMID:9791724
Using Nuclear Receptor Activity to Stratify Hepatocarcinogens
Shah, Imran; Houck, Keith; Judson, Richard S.; Kavlock, Robert J.; Martin, Matthew T.; Reif, David M.; Wambaugh, John; Dix, David J.
Background Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic analysis of new in vitro human NR activity data on 309 environmental chemicals in relationship to their liver cancer-related chronic outcomes in rodents. Results The effects of 309 environmental chemicals on human constitutive androstane receptors (CAR/NR1I3), pregnane X receptor (PXR/NR1I2), aryl hydrocarbon receptor (AhR), peroxisome proliferator-activated receptors (PPAR/NR1C), liver X receptors (LXR/NR1H), retinoic X receptors (RXR/NR2B) and steroid receptors (SR/NR3) were determined using in vitro data. Hepatic histopathology, observed in rodents after two years of chronic treatment for 171 of the 309 chemicals, was summarized by a cancer lesion progression grade. Chemicals that caused proliferative liver lesions in both rat and mouse were generally more active for the human receptors, relative to the compounds that only affected one rodent species, and these changes were significant for PPAR (p0.001), PXR (p0.01) and CAR (p0.05). Though most chemicals exhibited receptor promiscuity, multivariate analysis clustered them into relatively few NR activity combinations. The human NR activity pattern of chemicals weakly associated with the severity of rodent liver cancer lesion progression (p0.05). Conclusions The rodent carcinogens had higher in vitro potency for human NR relative to non-carcinogens. Structurally diverse chemicals with similar NR promiscuity patterns weakly associated with the severity of rodent liver cancer progression. While these results do not prove the role of NR activation in human liver cancer, they do have implications for nuclear receptor chemical biology and provide insights into putative toxicity pathways. More importantly, these findings suggest the utility of in vitro assays for stratifying environmental contaminants based on a combination of human bioactivity and rodent toxicity. PMID:21339822
New GABA amides activating GABAA-receptors
Raster, Peter; Sp�th, Andreas; Bultakova, Svetlana; Gorostiza, Pau
Summary We have prepared a series of new and some literature-reported GABA-amides and determined their effect on the activation of GABAA-receptors expressed in CHO cells. Special attention was paid to the purification of the target compounds to remove even traces of GABA contaminations, which may arise from deprotection steps in the synthesis. GABA-amides were previously reported to be partial, full or superagonists. In our hands these compounds were not able to activate GABAA-receptor channels in whole-cell patch-clamp recordings. New GABA-amides, however, gave moderate activation responses with a clear structure�activity relationship suggesting some of these compounds as promising molecular tools for the functional analysis of GABAA-receptors. PMID:23503884
NMDA Receptor Activity in Neuropsychiatric Disorders
Lakhan, Shaheen E.; Caro, Mario; Hadzimichalis, Norell
N-Methyl-d-aspartate (NMDA) receptors play a variety of physiologic roles and their proper signaling is essential for cellular homeostasis. Any disruption in this pathway, leading to either enhanced or decreased activity, may result in the manifestation of neuropsychiatric pathologies such as schizophrenia, mood disorders, substance induced psychosis, Huntington�s disease, Alzheimer�s disease, and neuropsychiatric systemic lupus erythematosus. Here, we explore the notion that the overlap in activity of at least one biochemical pathway, the NMDA receptor pathway, may be the link to understanding the overlap in psychotic symptoms between diseases. This review intends to present a broad overview of those neuropsychiatric disorders for which alternations in NMDA receptor activity is prominent thus suggesting that continued direction of pharmaceutical intervention to this pathway may present a viable option for managing symptoms. PMID:23772215
Laminin Expression in Adult and Developing Retinae: Evidence of Two Novel CNS Laminins
Richard T. Libby; Marie-France Champliaud; Thomas Claudepierre; Yin Xu; Erin P. Gibbons; Manuel Koch; Robert E. Burgeson; Dale D. Hunter; William J. Brunken
Components of the extracellular matrix exert myriad effects on tissues throughout the body. In particular, the laminins, a family of heterotrimeric extracellular glycoproteins, have been shown to affect tissue development and integrity in such diverse organs as the kidney, lung, skin, and nervous system. Of these, we have focused on the roles that laminins play in the differentiation and maintenance
Signal transduction by protease-activated receptors
Soh, Unice JK; Dores, Michael R; Chen, Buxin; Trejo, JoAnn
The family of G protein-coupled receptors (GPCRs) constitutes the largest class of signalling receptors in the human genome, controlling vast physiological responses and are the target of many drugs. After activation, GPCRs are rapidly desensitized by phosphorylation and ?-arrestin binding. Most classic GPCRs are internalized through a clathrin, dynamin and ?-arrestin-dependent pathway and then recycled back to the cell surface or sorted to lysosomes for degradation. Given the vast number and diversity of GPCRs, different mechanisms are likely to exist to precisely regulate the magnitude, duration and spatial aspects of receptor signalling. The G protein-coupled protease-activated receptors (PARs) provide elegant examples of GPCRs that are regulated by distinct desensitization and endocytic sorting mechanisms, processes that are critically important for the spatial and temporal fidelity of PAR signalling. PARs are irreversibly activated through proteolytic cleavage and transmit cellular responses to extracellular proteases. Activated PAR1 internalizes through a clathrin- and dynamin-dependent pathway independent of ?-arrestins. Interestingly, PAR1 is basally ubiquitinated and deubiquitinated after activation and traffics from endosomes to lysosomes independent of ubiquitination. In contrast, ?-arrestins mediate activated PAR2 internalization and function as scaffolds that promote signalling from endocytic vesicles. Moreover, activated PAR2 is modified with ubiquitin, which facilitates lysosomal degradation. Activated PARs also adopt distinct active conformations that signal to diverse effectors and are likely regulated by different mechanisms. Thus, the identification of the molecular machinery important for PAR signal regulation will enable the development of new strategies to manipulate receptor signalling and will provide novel targets for the development of drugs. PMID:20423334
Alignment and composition of laminin�polycaprolactone nanofiber blends enhance peripheral nerve regeneration
Neal, Rebekah A.; Tholpady, Sunil S.; Foley, Patricia L.; Swami, Nathan; Ogle, Roy C.; Botchwey, Edward A.
Peripheral nerve transection occurs commonly in traumatic injury, causing deficits distal to the injury site. Conduits for repair currently on the market are hollow tubes; however, they often fail due to slow regeneration over long gaps. To facilitate increased regeneration speed and functional recovery, the ideal conduit should provide biochemically relevant signals and physical guidance cues, thus playing an active role in regeneration. To that end, laminin and laminin�polycaprolactone (PCL) blend nanofibers were fabricated to mimic peripheral nerve basement membrane. In vitro assays established 10% (wt) laminin content is sufficient to retain neurite-promoting effects of laminin. In addition, modified collector plate design to introduce an insulating gap enabled the fabrication of aligned nanofibers. The effects of laminin content and fiber orientation were evaluated in rat tibial nerve defect model. The lumens of conduits were filled with nanofiber meshes of varying laminin content and alignment to assess changes in motor and sensory recovery. Retrograde nerve conduction speed at 6 weeks was significantly faster in animals receiving aligned nanofiber conduits than in those receiving random nanofiber conduits. Animals receiving nanofiber-filled conduits showed some conduction in both anterograde and retrograde directions, whereas in animals receiving hollow conduits, no impulse conduction was detected. Aligned PCL nanofibers significantly improved motor function; aligned laminin blend nanofibers yielded the best sensory function recovery. In both cases, nanofiber-filled conduits resulted in better functional recovery than hollow conduits. These studies provide a firm foundation for the use of natural�synthetic blend electrospun nanofibers to enhance existing hollow nerve guidance conduits. PMID:22106069
Signaling pathways engaged by NK cell receptors: double concerto for activating receptors, inhibitory receptors and NK cells
Mathieu Blery; Eric Vely; Eric Vivier
Despite the absence of antigen-specific receptors at their surface, NK cells can selectively eliminate virus-infected cells, tumor cells and allogenic cells. A dynamic and precisely coordinated balance between activating and inhibitory receptors governs NK cell activation programs. Multiple activating and inhibitory NK cell surface molecules have been described, a group of them acting as receptors for MHC class I molecules.
Regeneration of Aplysia Bag Cell Neurons is Synergistically Enhanced by Substrate-Bound Hemolymph Proteins and Laminin
Hyland, Callen; Dufrense, Eric R.; Forscher, Paul
We have investigated Aplysia hemolymph as a source of endogenous factors to promote regeneration of bag cell neurons. We describe a novel synergistic effect between substrate-bound hemolymph proteins and laminin. This combination increased outgrowth and branching relative to either laminin or hemolymph alone. Notably, the addition of hemolymph to laminin substrates accelerated growth cone migration rate over ten-fold. Our results indicate that the active factor is either a high molecular weight protein or protein complex and is not the respiratory protein hemocyanin. Substrate-bound factor(s) from central nervous system-conditioned media also had a synergistic effect with laminin, suggesting a possible cooperation between humoral proteins and nervous system extracellular matrix. Further molecular characterization of active factors and their cellular targets is warranted on account of the magnitude of the effects reported here and their potential relevance for nervous system repair. PMID:24722588
Regeneration of Aplysia Bag Cell Neurons is Synergistically Enhanced by Substrate-Bound Hemolymph Proteins and Laminin
NASA Astrophysics Data System (ADS)
Hyland, Callen; Dufrense, Eric R.; Forscher, Paul
We have investigated Aplysia hemolymph as a source of endogenous factors to promote regeneration of bag cell neurons. We describe a novel synergistic effect between substrate-bound hemolymph proteins and laminin. This combination increased outgrowth and branching relative to either laminin or hemolymph alone. Notably, the addition of hemolymph to laminin substrates accelerated growth cone migration rate over ten-fold. Our results indicate that the active factor is either a high molecular weight protein or protein complex and is not the respiratory protein hemocyanin. Substrate-bound factor(s) from central nervous system-conditioned media also had a synergistic effect with laminin, suggesting a possible cooperation between humoral proteins and nervous system extracellular matrix. Further molecular characterization of active factors and their cellular targets is warranted on account of the magnitude of the effects reported here and their potential relevance for nervous system repair.
Fatty Acids Activate a Chimera of the Clofibric Acid-Activated Receptor and the Glucocorticoid Receptor
Martin Gottlicher; Eva Widmark; Qiao Li; Jan-Ake Gustafsson
Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a
Laminin isoforms in fetal and adult human adrenal cortex.
Virtanen, Ismo; Korhonen, Matti; Pet�j�niemi, Noora; Karhunen, Tuula; Thornell, Lars-Eric; Sorokin, Lydia M; Konttinen, Yrj� T
Laminin has been proposed to influence the function of human adrenal cortex. We have studied the distribution of laminin (Ln) chains using immunofluorescence in human fetal and adult adrenal cortex. In the fetal gland Ln alpha2- and alpha5-chains were weakly expressed in the definitive zone, whereas Ln alpha4-, beta1-, and gamma1-chains occurred around vessels. In the adult gland, Ln alpha2-, alpha5-, and gamma1-chains were found in epithelial basement membranes (BM) in all cortical zones, Ln alpha4-chain in vessels, Ln beta1-chain in outer zone, and Ln beta2-chain in the two inner zones of the cortex, respectively. Among the integrins in adult gland, integrin alpha(3)-subunit was confined to basal surfaces of cortical cells, alpha(6) to vessels, alpha(1) to the stroma, and alpha(2) diffusely to epithelial cells. Lutheran glycoprotein and dystroglycan occurred in the fetal gland diffusely in the definitive zone and throughout the epithelium in the adult. The isoform composition of BM of the adult adrenal gland is distinct, with Ln-2 and -10 in BM of the outer zone and Ln-4 and -11 in BM of the two inner zones. The results suggest that integrin alpha(3)beta(1) and Lutheran are candidate receptors for Ln-10 and -11, whereas dystroglycan probably binds Ln-2 and -4. PMID:14557481
Laminin/?1 integrin signal triggers axon formation by promoting microtubule assembly and stabilization
Lei, Wen-Liang; Xing, Shi-Ge; Deng, Cai-Yun; Ju, Xiang-Chun; Jiang, Xing-Yu; Luo, Zhen-Ge
Axon specification during neuronal polarization is closely associated with increased microtubule stabilization in one of the neurites of unpolarized neuron, but how this increased microtubule stability is achieved is unclear. Here, we show that extracellular matrix (ECM) component laminin promotes neuronal polarization via regulating directional microtubule assembly through ?1 integrin (Itgb1). Contact with laminin coated on culture substrate or polystyrene beads was sufficient for axon specification of undifferentiated neurites in cultured hippocampal neurons and cortical slices. Active Itgb1 was found to be concentrated in laminin-contacting neurites. Axon formation was promoted and abolished by enhancing and attenuating Itgb1 signaling, respectively. Interestingly, laminin contact promoted plus-end microtubule assembly in a manner that required Itgb1. Moreover, stabilizing microtubules partially prevented polarization defects caused by Itgb1 downregulation. Finally, genetic ablation of Itgb1 in dorsal telencephalic progenitors caused deficits in axon development of cortical pyramidal neurons. Thus, laminin/Itgb1 signaling plays an instructive role in axon initiation and growth, both in vitro and in vivo, through the regulation of microtubule assembly. This study has established a linkage between an extrinsic factor and intrinsic cytoskeletal dynamics during neuronal polarization. PMID:22430151
Activating Fc ? receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies
Bulliard, Yannick; Jolicoeur, Rose; Windman, Maurice; Rue, Sarah M.; Ettenberg, Seth; Knee, Deborah A.; Wilson, Nicholas S.; Dranoff, Glenn
Fc ? receptor (Fc?R) coengagement can facilitate antibody-mediated receptor activation in target cells. In particular, agonistic antibodies that target tumor necrosis factor receptor (TNFR) family members have shown dependence on expression of the inhibitory Fc?R, Fc?RIIB. It remains unclear if engagement of Fc?RIIB also extends to the activities of antibodies targeting immunoregulatory TNFRs expressed by T cells. We have explored the requirement for activating and inhibitory Fc?Rs for the antitumor effects of antibodies targeting the TNFR glucocorticoid-induced TNFR-related protein (GITR; TNFRSF18; CD357) expressed on activated and regulatory T cells (T reg cells). We found that although Fc?RIIB was dispensable for the in vivo efficacy of anti-GITR antibodies, in contrast, activating Fc?Rs were essential. Surprisingly, the dependence on activating Fc?Rs extended to an antibody targeting the non-TNFR receptor CTLA-4 (CD152) that acts as a negative regulator of T cell immunity. We define a common mechanism that correlated with tumor efficacy, whereby antibodies that coengaged activating Fc?Rs expressed by tumor-associated leukocytes facilitated the selective elimination of intratumoral T cell populations, particularly T reg cells. These findings may have broad implications for antibody engineering efforts aimed at enhancing the therapeutic activity of immunomodulatory antibodies. PMID:23897982
A Fractal Nature for Polymerized Laminin
Hochman-Mendez, Camila; Cantini, Marco; Moratal, David; Salmeron-Sanchez, Manuel; Coelho-Sampaio, Tatiana
Polylaminin (polyLM) is a non-covalent acid-induced nano- and micro-structured polymer of the protein laminin displaying distinguished biological properties. Polylaminin stimulates neuritogenesis beyond the levels achieved by ordinary laminin and has been shown to promote axonal regeneration in animal models of spinal cord injury. Here we used confocal fluorescence microscopy (CFM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize its three-dimensional structure. Renderization of confocal optical slices of immunostained polyLM revealed the aspect of a loose flocculated meshwork, which was homogeneously stained by the antibody. On the other hand, an ordinary matrix obtained upon adsorption of laminin in neutral pH (LM) was constituted of bulky protein aggregates whose interior was not accessible to the same anti-laminin antibody. SEM and AFM analyses revealed that the seed unit of polyLM was a flat polygon formed in solution whereas the seed structure of LM was highly heterogeneous, intercalating rod-like, spherical and thin spread lamellar deposits. As polyLM was visualized at progressively increasing magnifications, we observed that the morphology of the polymer was alike independently of the magnification used for the observation. A search for the Hausdorff dimension in images of the two matrices showed that polyLM, but not LM, presented fractal dimensions of 1.55, 1.62 and 1.70 after 1, 8 and 12 hours of adsorption, respectively. Data in the present work suggest that the intrinsic fractal nature of polymerized laminin can be the structural basis for the fractal-like organization of basement membranes in the neurogenic niches of the central nervous system. PMID:25296244
The heterotrimeric laminin coiled-coil domain exerts anti-adhesive effects and induces a pro-invasive phenotype.
Santos-Valle, Patricia; Guijarro-Mu�oz, Irene; Cuesta, Angel M; Alonso-Camino, Vanesa; Villate, Maider; Alvarez-Cienfuegos, Ana; Blanco, Francisco J; Sanz, Laura; Alvarez-Vallina, Luis
Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the ?1-, ?1-, and ?1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule. This domain was biologically active and not only failed to serve as a substrate for cell attachment, spreading and focal adhesion formation but also inhibited cell adhesion to laminin when added to cells in a soluble form at the time of seeding. Furthermore, gene array expression profiling in cells cultured in the presence of the laminin coiled-coil domain revealed up-regulation of genes involved in cell motility and invasion. These findings were confirmed by real-time quantitative PCR and zymography assays. In conclusion, this study shows for the first time that the laminin coiled-coil domain displays anti-adhesive functions and has potential implications for cell migration during matrix remodeling. PMID:22723936
Spongian diterpenoids inhibit androgen receptor activity
Yang, Yu Chi; Meimetis, Labros G; Tien, Amy H; Mawji, Nasrin R; Carr, Gavin; Wang, Jun; Andersen, Raymond J; Sadar, Marianne D
Androgen receptor (AR) is a ligand-activated transcription factor and a validated drug target for all stages of prostate cancer. Antiandrogens compete with physiological ligands for AR ligand-binding domain (LBD). High-throughput screening of a marine natural product library for small molecules that inhibit AR transcriptional activity yielded the furanoditerpenoid spongia-13(16),-14-dien-19-oic acid, designated terpene 1 (T1). Characterization of T1 and the structurally related semi-synthetic analogues (T2 and T3) revealed that these diterpenoids have antiandrogen properties that include inhibition of both androgen-dependent proliferation and AR transcriptional activity by a mechanism that involved competing with androgen for AR LBD and blocking essential N/C interactions required for androgen-induced AR transcriptional activity. Structure activity relationship analyses revealed some chemical features of T1 that are associated with activity and yielded T3 as the most potent analogue. In vivo, T3 significantly reduced the weight of seminal vesicles, which are an androgen-dependent tissue, thereby confirming T3�s on-target activity. The ability to create analogues of diterpenoids that have varying antiandrogen activity represents a novel class of chemical compounds for the analysis of AR ligand-binding properties and therapeutic development. PMID:23443807
How IGF-1 activates its receptor
Kavran, Jennifer M; McCabe, Jacqueline M; Byrne, Patrick O; Connacher, Mary Katherine; Wang, Zhihong; Ramek, Alexander; Sarabipour, Sarvenaz; Shan, Yibing; Shaw, David E; Hristova, Kalina; Cole, Philip A; Leahy, Daniel J
The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation. DOI: http://dx.doi.org/10.7554/eLife.03772.001 PMID:25255214
Sarcospan integration into laminin-binding adhesion complexes that ameliorate muscular dystrophy requires utrophin and ?7 integrin.
Marshall, Jamie L; Oh, Jennifer; Chou, Eric; Lee, Joy A; Holmberg, Johan; Burkin, Dean J; Crosbie-Watson, Rachelle H
Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in loss of the dystrophin-glycoprotein complex, a laminin receptor that connects the myofiber to its surrounding extracellular matrix. Utrophin, a dystrophin ortholog that is normally localized to the neuromuscular junction, is naturally upregulated in DMD muscle, which partially compensates for the loss of dystrophin. Transgenic overexpression of utrophin causes broad sarcolemma localization of utrophin, restoration of laminin binding and amelioration of disease in the mdx mouse model of DMD. We previously demonstrated that overexpression of sarcospan, a dystrophin- and utrophin-binding protein, ameliorates mdx muscular dystrophy. Sarcospan boosts levels of utrophin to therapeutic levels at the sarcolemma, where attachment to laminin is restored. However, understanding the compensatory mechanism is complicated by concomitant upregulation of ?7?1 integrin, which also binds laminin. Similar to the effects of utrophin, transgenic overexpression of ?7 integrin prevents DMD disease in mice and is accompanied by increased abundance of utrophin around the extra-synaptic sarcolemma. In order to investigate the mechanisms underlying sarcospan 'rescue' of muscular dystrophy, we created double-knockout mice to test the contributions of utrophin or ?7 integrin. We show that sarcospan-mediated amelioration of muscular dystrophy in DMD mice is dependent on the presence of both utrophin and ?7?1 integrin, even when they are individually expressed at therapeutic levels. Furthermore, we found that association of sarcospan into laminin-binding complexes is dependent on utrophin and ?7?1 integrin. PMID:25504048
Adhesion of membranes via actively switched receptors
Bartosz Rozycki; Reinhard Lipowsky; Thomas R. Weikl
We consider a theoretical model for membranes with adhesive receptors, or stickers, that are actively switched between two conformational states. In their 'on'-state, the stickers bind to ligands in an apposing membrane, whereas they do not interact with the ligands in their 'off'-state. We show that the adhesiveness of the membranes depends sensitively on the rates of the conformational switching process. This dependence is reflected in a resonance at intermediate switching rates, which can lead to large membrane separations and unbinding. Our results may provide insights into novel mechanisms for the controlled adhesion of biological or biomimetic membranes.
Monoclonal anti-mouse laminin antibodies: AL-1 reacts with laminin alpha1 chain, AL-2 with laminin beta1 chain, and AL-4 with the coiled-coil domain of laminin beta1 chain.
Sch�ele, Susanne; Sasaki, Takako; Arnal-Estap�, Anna; Durbeej, Madeleine; Ekblom, Peter
We analyzed the reactivity of three different commercially available rat monoclonal antibodies raised against mouse laminin-alpha1beta1gamma1 (laminin-111), AL-1, AL-2, and AL-4. Using ELISA assays, Western blot analysis and immunostainings we present refined epitope maps for these three laminin monoclonals. AL-1 reacted, as predicted with laminin alpha1 chain. AL-4 has also been marketed as an alpha1 chain specific probe, but we show here that AL-4 detects mouse laminin beta1 chain, in the distal part of the coiled-coil region. AL-2 was predicted to react with all three chains near the cross-region, but seems to primarily react with laminin beta1 chain. PMID:16631359
Laminin stimulates protein tyrosine dephosphorylation in PC12 cells.
Weeks, B S; Wilson, P J; Heffernan, C C; Gorra, V A; White, L E; Ahmad, A
Laminin stimulates neurite outgrowth in rat pheochromocytoma cells (PC12 cells). Here, we investigated laminin signal transduction mechanisms by adding the tyrosine kinase/phosphatase modulators, genistein, quercetin, aurin tricarboxylic acid (ATA), and vanadate to PC12 cells. At 10 microM both genistein and quercetin enhanced laminin-mediated neurite outgrowth by 1.7- and 2.3-fold, respectively, while at 10 microM, ATA inhibited laminin-mediated neurite outgrowth by 92%. Vanadate inhibited neurite outgrowth by 63% at 10 microM. Immunoblot analysis revealed four proteins of approximately 240, 22, 110, and 35 kDa, which were dephosphorylated on tyrosine residues in laminin-treated PC12 cells, but not in NIH 3T3 cells. These results demonstrate that laminin-mediated neurite outgrowth involves protein tyrosine dephosphorylation and suggests that this mechanism may have specificity to neuronal cells. PMID:10471391
Model for growth hormone receptor activation based on subunit rotation within a receptor dimer
Richard J Brown; Julian J Adams; Rebecca A Pelekanos; Yu Wan; William J McKinstry; Kathryn Palethorpe; Ruth M Seeber; Thea A Monks; Karin A Eidne; Michael W Parker; Michael J Waters
Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that
Identification of Gene Markers for Activation of the Nuclear Receptor Pregnane X Receptor
Many environmentally-relevant chemicals and drugs activate the nuclear receptor pregnane X receptor (PXR). Activation of PXR in the mouse liver can lead to increases in liver weight in part through increased hepatocyte replication similar to chemicals that activate other nuclear ...
Model for growth hormone receptor activation based on subunit rotation within a receptor dimer
Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J. (UWA); (St. Vincent); (Queensland)
Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.
Differential regulation of laminin b1 transgene expression in the neonatal and adult mouse brain.
Sharif, K A; Baker, H; Gudas, L J
Laminins are the major glycoproteins present in basement membrane, a type of extracellular matrix. We showed that the LAMB1 gene, which encodes the laminin beta1 subunit, is transcriptionally activated by retinoic acid in embryonic stem cells. However, little information is available concerning LAMB1 developmental regulation and spatial expression in the adult mouse brain. In this study we used transgenic mice expressing different lengths of LAMB1 promoter driving beta-galactosidase to investigate developmental and adult transcriptional regulation in the regions of the brain in which the laminin beta1 protein is expressed. CNS expression was not observed in transgenic mice carrying a 1.4LAMB1betagal construct. Mice carrying a 2.5LAMB1betagal construct expressed the LAMB1 transgene, as assayed by X-gal staining, only in the molecular layer of the neonatal cerebellum. In contrast, a 3.9LAMB1betagal transgene showed broad regional expression in the adult mouse brain, including the hippocampus, entorhinal cortex, colliculi, striatum, and substantia nigra. Similar expression patterns were observed for the endogenous laminin beta1 protein and for the 3.9LAMB1betagal transgene, analyzed with an antibody against the beta-galactosidase protein. The 3.9LAMB1betagal transgene expression in the hippocampal tri-synaptic circuit suggests a role for the LAMB1 gene in learning and memory. PMID:15207330
Transcriptional regulation of peroxisome proliferator-activated receptors and liver X receptors
Luis Villacorta; Minerva T. Garcia-Barrio; Yuqing E. Chen
Peroxisome proliferator-activated receptors (PPAR) and liver X receptors (LXR) regulate a plethora of biologic processes and\\u000a key metabolic and physiologic events. Deregulation of their transcription and activity is commonly associated with dyslipidemic\\u000a disorders, diabetes, cancer, and cardiovascular disease. This review addresses recent advances in our understanding of the\\u000a molecular mechanisms regulating transcription of these nuclear receptors. The heterogeneity of factors
Effects of ligand activation of peroxisome proliferator-activated receptor in human prostate cancer
Elisabetta Mueller; Matthew Smith; Pasha Sarraf; Todd Kroll; Anita Aiyer; Donald S. Kaufman; William Oh; George Demetri; William D. Figg; Xiao-Ping Zhou; Charis Eng; Bruce M. Spiegelman; Philip W. Kantoff
Peroxisome proliferator-activated receptor (PPAR) is a nuclear hormone receptor that plays a key role in the differentiation of adipocytes. Activation of this receptor in liposarcomas and breast and colon cancer cells also induces cell growth inhibition and differentiation. In the present study, we show that PPAR is expressed in human prostate adenocarcinomas and cell lines derived from these tumors. Activation
LG4-5 domains of laminin-2 binds ?-dystroglycan to allow myotube attachment and prevent anoikis
Munoz, Jesus; Zhou, YanWen; Jarrett, Harry W.
Poly(2-hydroxyethyl methacrylate) (PolyHEMA) prevents cell attachment was used here to study anoikis, the process where cells die when unattached or attached to an inappropriate matrix, in mouse C2C12 myotubes. A method was developed to efficiently embed proteins into PolyHEMA and the effect on cultured myotubes was determined. Myotubes grown on PolyHEMA-coated plates fail to attach to the surface and remain as rounded, suspended cells, undergo dramatic increases in apoptosis and necrosis, and the number of viable cells decreases,. Incorporation of merosin (laminin-211) or the short laminin globular (LG4-5) modules of the laminin ?2 chain C-terminus (called 2E3) that binds ?-dystroglycan diminishes both apoptosis and necrosis and increases viability while bovine serum albumin had a much lesser effect, showing the specificity of this effect for these matrix proteins. One sarcolemma receptor for laminin-binding is ?-dystroglycan. An antibody which binds ?-dystroglycan but which does not block laminin-binding (VIA4) had little effect on apoptosis or viability on merosin or 2E3 embedded plates while another antibody (IIH6) which specifically blocks binding dramatically decreased viability and increased apoptosis. When merosin or 2E3 are added to culture media rather than embedded on plates these can also increase viability and decrease apoptosis even though the cells remain in suspension, though the effect is not as great as found for the embedded proteins where the cells attach. Thus, we conclude that the binding of a small LG4-5 modules of laminin-211 to ?-dystroglycan is important in preventing anoikis and that attachment plus binding is necessary for maximal cell survival. PMID:19739104
Nociceptin activation of the human ORL 1 receptor expressed in Chinese hamster ovary cells: Functional homology with opioid receptors
Ahmad B Fawzi; Hongtao Zhang; Blair Weig; Brian Hawes; Michael P Graziano
Opioid receptor-like 1 (ORL1) receptor, a member of the superfamily of G-protein-coupled receptors has significant primary sequence homology to the ?-, ?-, and ?-opioid receptors. The ORL1 receptor is selectively activated by the recently discovered peptide nociceptin. To probe the functional homology amongst these receptors, a Chinese Hamster Ovary (CHO) cell line expressing the human ORL1 receptor has been characterized.
Peroxisome proliferator-activated receptors-alpha modulate dopamine cell activity through nicotinic receptors
Melis, Miriam; Carta, Stefano; Fattore, Liana; Tolu, Stefania; Yasar, Sevil; Goldberg, Steven R.; Fratta, Walter; Maskos, Uwe; Pistis, Marco
Background Modulation of midbrain dopamine neurons by nicotinic acetylcholine receptors (nAChRs) plays an important role in behavior, cognition, motivation and reward. Specifically, nAChRs containing ?2 subunits (?2-nAChRs) switch dopamine cells from a resting to an excited state. However, how ?2-nAChRs can be modulated and thereby dopamine firing activity be affected is still elusive. Because changes in dopamine cell activity are reflected in the dynamics of micro-circuits generating altered responses to stimuli/inputs, factors regulating their state are fundamental. Among these, endogenous ligands to the nuclear receptor-transcription factor peroxisome proliferator-activated receptors type-alpha (PPAR?) have been recently found to suppress nicotine-induced responses of dopamine neurons. Methods We used both in vitro and in vivo electrophysiological techniques together with behavioral analysis to investigate on the effects of modulation of PPAR? in Sprague Dawley rat and C57BLJ/6 mouse dopamine neurons, and their interactions with ?2-nAChRs. To this aim, we took advantage of a selective re-expression of ?2-nAChR exclusively in dopamine cells by stereotaxically injecting a lentiviral vector in the mouse ventral tegmental area. Results We found that activation of PPAR? decreases in vitro both dopamine cell activity and ventral tegmental area net output through negative modulation of ?2-nAChRs. Additionally, PPAR? activation in vivo reduces both the number of spontaneously active dopamine neurons and nicotine-induced increased locomotion. Conclusions Our combined findings suggest PPAR? ligands as important negative modulators of ?2-nAChRs on dopamine neurons. Thus, PPAR? ligands might prove beneficial in treating those disorders where dopamine dysfunction plays a prominent role, such as schizophrenia and nicotine addiction. PMID:20570248
Cell death sensitization of leukemia cells by opioid receptor activation
Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich
Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472
Constitutive Activity of the Androgen Receptor
Chan, Siu Chiu; Dehm, Scott M.
Prostate cancer (PCa) is the most frequently diagnosed cancer in the United States. The androgen receptor (AR) signaling axis is central to all stages of PCa pathophysiology and serves as the main target for endocrine-based therapy. The most advanced stage of the disease, castration resistant prostate cancer (CRPC), is presently incurable and accounts for most PCa mortality. In this review, we highlight the mechanisms by which the AR signaling axis can bypass endocrine-targeted therapies and drive progression of CRPC. These mechanisms include alterations in growth factor, cytokine, and inflammatory signaling pathways, altered expression or activity of transcriptional co-regulators, AR point mutations, and AR gene amplification leading to AR protein overexpression. Additionally, we will discuss the mechanisms underlying the synthesis of constitutively active AR splice variants (AR-Vs) lacking the COOH-terminal ligand binding domain, as well as the role and regulation of AR-Vs in supporting therapeutic resistance in CRPC. Finally, we summarize the ongoing development of inhibitors targeting discrete AR functional domains as well as the status of new biomarkers for monitoring the AR signaling axis in patients. PMID:24931201
Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function
Xu, Ren; Nelson, Celeste M.; Muschler, John L.; Veiseh, Mandana; Vonderhaar, Barbara K.; Bissell, Mina J.
Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We show that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and {beta}-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression.
Active nuclear receptors exhibit highly correlated AF-2 domain motions.
Teotico, Denise G; Frazier, Monica L; Ding, Feng; Dokholyan, Nikolay V; Temple, Brenda R S; Redinbo, Matthew R
Nuclear receptor ligand binding domains (LBDs) convert ligand binding events into changes in gene expression by recruiting transcriptional coregulators to a conserved activation function-2 (AF-2) surface. While most nuclear receptor LBDs form homo- or heterodimers, the human nuclear receptor pregnane X receptor (PXR) forms a unique and essential homodimer and is proposed to assemble into a functional heterotetramer with the retinoid X receptor (RXR). How the homodimer interface, which is located 30 A from the AF-2, would affect function at this critical surface has remained unclear. By using 20- to 30-ns molecular dynamics simulations on PXR in various oligomerization states, we observed a remarkably high degree of correlated motion in the PXR-RXR heterotetramer, most notably in the four helices that create the AF-2 domain. The function of such correlation may be to create "active-capable" receptor complexes that are ready to bind to transcriptional coactivators. Indeed, we found in additional simulations that active-capable receptor complexes involving other orphan or steroid nuclear receptors also exhibit highly correlated AF-2 domain motions. We further propose a mechanism for the transmission of long-range motions through the nuclear receptor LBD to the AF-2 surface. Taken together, our findings indicate that long-range motions within the LBD scaffold are critical to nuclear receptor function by promoting a mobile AF-2 state ready to bind coactivators. PMID:18617990
Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts.
Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G
The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the ?1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles. PMID:23111328
The Effect of Laminin-1-Doped Nanoroughened Implant Surfaces: Gene Expression and Morphological Evaluation
Schwartz-Filho, Humberto Osvaldo; Bougas, Kostas; Coelho, Paulo G.; Xue, Ying; Hayashi, Mariko; Faeda, Rafael Silveira; Marcantonio, Rosemary Adriana Chi�rici; Ono, Daisuke; Kobayashi, Fumio; Mustafa, Kamal; Wennerberg, Ann; Jimbo, Ryo
Aim. This study aimed to observe the morphological and molecular effect of laminin-1 doping to nanostructured implant surfaces in a rabbit model. Materials and Methods. Nanostructured implants were coated with laminin-1 (test; dilution, 100??g/mL) and inserted into the rabbit tibiae. Noncoated implants were used as controls. After 2 weeks of healing, the implants were removed and subjected to morphological analysis using scanning electron microscopy (SEM) and gene expression analysis using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Results. SEM revealed bony tissue attachment for both control and test implants. Real-time RT-PCR analysis showed that the expression of osteoblast markers RUNX-2, osteocalcin, alkaline phosphatase, and collagen I was higher (1.62-fold, 1.53-fold, 1.97-fold, and 1.04-fold, resp.) for the implants modified by laminin-1 relative to the control. All osteoclast markers investigated in the study presented higher expression on the test implants than controls as follows: tartrate-resistant acid phosphatase (1.67-fold), calcitonin receptor (1.35-fold), and ATPase (1.25-fold). The test implants demonstrated higher expression of inflammatory markers interleukin-10 (1.53-fold) and tumour necrosis factor-? (1.61-fold) relative to controls. Conclusion. The protein-doped surface showed higher gene expression of typical genes involved in the osseointegration cascade than the control surface. PMID:23304151
Combinatorial Fibronectin and Laminin Signaling Promote Highly Efficient Cardiac Differentiation of Human Embryonic Stem Cells
Sa, Silin; Wong, Lian
Abstract Cardiomyocytes (CMs) differentiated from human embryonic stem cells (hESCs) are a promising and potentially unlimited cell source for myocardial repair and regeneration. Recently, multiple methodologies�primarily based on the optimization of growth factors�have been described for efficient cardiac differentiation of hESCs. However, the role of extracellular matrix (ECM) signaling in CM differentiation has not yet been explored fully. This study examined the role of ECM signaling in the efficient generation of CMs from both H7 and H9 ESCs. The hESCs were differentiated on ECM substrates composed of a range of fibronectin (FN) and laminin (LN) ratios and gelatin and evaluated by the fluorescence activated cell scanning (FACS) analysis on day 14. Of the ECM substrates examined, the 70:30 FN:LN reproducibly generated the greatest numbers of CMs from both hESC lines. Moreover, the LN receptor integrin ?4 (ITGB4) and FN receptor integrin ?5 (ITGB5) genes, jointly with increased phosphorylated focal adhension kinase and phosphorylated extracellular signal-regulated kinases (p-ERKs), were up-regulated over 13-fold in H7 and H9 cultured on 70:30 FN:LN compared with gelatin. Blocking studies confirmed the role of all these molecules in CM specification, suggesting that the 70:30 FN:LN ECM promotes highly efficient differentiation of CMs through the integrin-mediated MEK/ERK signaling pathway. Lastly, the data suggest that FN:LN-induced signaling utilizes direct cell-to-cell signaling from distinct ITGB4+ and ITGB5+ cells. PMID:25126479
The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor
Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W.; Reynolds, Ross; Engel, James Douglas; Xu, H. Eric (Van Andel); (Michigan-Med)
Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.
Activation of purinergic receptor subtypes modulates odor sensitivity.
Hegg, Colleen C; Greenwood, Denise; Huang, Wei; Han, Pengcheng; Lucero, Mary T
Purinergic nucleotides, including ATP and adenosine, are important neuromodulators of peripheral auditory and visual sensory systems (Thorne and Housley, 1996). ATP released by the olfactory epithelium (OE) after noxious stimuli provides a physiological source for a neuromodulatory substance independent of efferent innervation. Here we show that multiple subtypes of purinergic receptors are differentially expressed in olfactory receptor neurons and sustentacular support cells. Activation of purinergic receptors evoked inward currents and increases in intracellular calcium in cultured mouse olfactory receptor neurons. A mouse olfactory epithelial slice preparation and confocal imaging were used to measure changes in intracellular calcium in response to odors, purinergic receptor (P2R) agonists, or combined odor + P2R agonists. Pharmacological studies show that both P2Y and P2X receptor activation by exogenous and endogenous ATP significantly reduces odor responsiveness. Moreover, purinergic receptor antagonists increase the odor-evoked calcium transient, providing direct evidence that endogenous ATP modulates odor sensitivity via activation of multiple purinergic receptor subtypes in olfactory receptor neurons. Odor activation of G-protein-coupled receptors results in increased cAMP production, opening of cyclic nucleotide-gated channels, influx of Ca2+ and Na+, depolarization of the membrane, and activation of voltage- and Ca2+-gated ion channels. On-cell current-clamp recordings of olfactory receptor neurons from neonatal mouse slices revealed that ATP reduced cyclic nucleotide-induced electrical responses. These data also support the idea that ATP modulates odor sensitivity in mammalian olfactory neurons. Peripheral ATP-mediated odor suppression is a novel mechanism for reduced olfactory sensitivity during exposure to olfactotoxins and may be a novel neuroprotective mechanism. PMID:12967991
Activation of Purinergic Receptor Subtypes Modulates Odor Sensitivity
Hegg, Colleen C.; Greenwood, Denise; Huang, Wei; Han, Pengcheng; Lucero, Mary T.
Purinergic nucleotides, including ATP and adenosine, are important neuromodulators of peripheral auditory and visual sensory systems (Thorne and Housley, 1996). ATP released by the olfactory epithelium (OE) after noxious stimuli provides a physiological source for a neuromodulatory substance independent of efferent innervation. Here we show that multiple subtypes of purinergic receptors are differentially expressed in olfactory receptor neurons and sustentacular support cells. Activation of purinergic receptors evoked inward currents and increases in intracellular calcium in cultured mouse olfactory receptor neurons. A mouse olfactory epithelial slice preparation and confocal imaging were used to measure changes in intracellular calcium in response to odors, purinergic receptor (P2R) agonists, or combined odor + P2R agonists. Pharmacological studies show that both P2Y and P2X receptor activation by exogenous and endogenous ATP significantly reduces odor responsiveness. Moreover, purinergic receptor antagonists increase the odor-evoked calcium transient, providing direct evidence that endogenous ATP modulates odor sensitivity via activation of multiple purinergic receptor subtypes in olfactory receptor neurons. Odor activation of G-protein-coupled receptors results in increased cAMP production, opening of cyclic nucleotide-gated channels, influx of Ca2+and Na+, depolarization of the membrane, and activation of voltage- and Ca2+-gated ion channels. On-cell current-clamp recordings of olfactory receptor neurons from neonatal mouse slices revealed that ATP reduced cyclic nucleotide-induced electrical responses. These data also support the idea that ATP modulates odor sensitivity in mammalian olfactory neurons. Peripheral ATP-mediated odor suppression is a novel mechanism for reduced olfactory sensitivity during exposure to olfactotoxins and may be a novel neuroprotective mechanism. PMID:12967991
PDGF enhances IRES-mediated translation of Laminin B1 by cytoplasmic accumulation of La during epithelial to mesenchymal transition.
Petz, Michaela; Them, Nicole C C; Huber, Heidemarie; Mikulits, Wolfgang
The extracellular matrix protein Laminin B1 (LamB1) regulates tumor cell migration and invasion. Carcinoma cells acquire invasive properties by epithelial to mesenchymal transition (EMT), which is a fundamental step in dissemination of metastatic cells from the primary tumor. Recently, we showed that enhanced translation of LamB1 upon EMT of malignant hepatocytes is mediated by an internal ribosome entry site (IRES). We demonstrated that the IRES transacting factor La binds the minimal IRES motif and positively modulates IRES activity of LamB1. Here, we show that platelet-derived growth factor (PDGF) enhances IRES activity of LamB1 by the increasing cytoplasmic localization of La during EMT. Accordingly, cells expressing dominant negative PDGF receptor display reduced cytoplasmic accumulation of La and show no elevation of IRES activity or endogenous LamB1 levels after stimulation with PDGF. Furthermore, La-mediated regulation of LamB1 IRES activity predominantly depends on MAPK/ERK signaling downstream of PDGF. Notably, LamB1 expression is not significantly downregulated by the impairment of the translation initiation factor eIF4E. In vivo, knockdown of La associated with decreased LamB1 expression and reduced tumor growth. Together, these data suggest that PDGF is required for the cytoplasmic accumulation of La that triggers IRES-dependent translation of LamB1 during EMT. PMID:22904067
Characterization of peroxisome proliferator-activiated receptor alpha (PPARalpha)-independent effects of PPARalpha activators in the rodent liver: Di(2-ethylehexyl) phthalate activates the constitutive activated receptor
Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). Recent studies indicate that the plasticizer di-2-ethylhexyl ph...
Study on expression of laminin in patients with intractable epilepsy.
Wu, Yuan; Feng, Yun; Pang, Jia-Rong; Tang, Mei; Liu, Xiu-Ying; Li, Jia-Quan; Wang, Xue-Feng
In this study, we examined differences in serum laminin expression in patients with intractable epilepsy. Our results suggest that elevated laminin may contribute to the pathogenesis of intractable epilepsy. ELISA and western blots were used to measure laminin in the serum of 30 intractable epilepsy patients, 46 nonintractable epilepsy patients, and 20 normal subjects. By ELISA, serum laminin levels were greater in intractable epilepsy patients (177.396 +/- 30.602) and nonintractable epilepsy patients (121.915 +/- 35.215) than in normal control subjects (67.474 +/- 7.197); laminin was significantly greater in the intractable epilepsy group than in the nonintractable epilepsy group. In western blots, the optical density ratio of laminin to ss-actin was 0.871 +/- 0.032 for the intractable epilepsy group, 0.686 +/- 0.017 for the nonintractable epilepsy group, and 0.385 +/- 0.024 for the normal control group. The optical density ratios of the intractable and nonintractable epilepsy groups were higher than those for the normal control group, and the intractable epilepsy group was even greater than the nonintractable epilepsy group. Thus, laminin is significantly increased in epilepsy patients, and this increase is more profound in intractable epilepsy patients. PMID:19916850
The ? opioid receptor agonist SNC80 selectively activates heteromeric ?-? opioid receptors.
Metcalf, Matthew D; Yekkirala, Ajay S; Powers, Michael D; Kitto, Kelley F; Fairbanks, Carolyn A; Wilcox, George L; Portoghese, Philip S
Coexpressed and colocalized ?- and ?-opioid receptors have been established to exist as heteromers in cultured cells and in vivo. However the biological significance of opioid receptor heteromer activation is less clear. To explore this significance, the efficacy of selective activation of opioid receptors by SNC80 was assessed in vitro in cells singly and coexpressing opioid receptors using a chimeric G-protein-mediated calcium fluorescence assay, SNC80 produced a substantially more robust response in cells expressing ?-? heteromers than in all other cell lines. Intrathecal SNC80 administration in ?- and ?-opioid receptor knockout mice produced diminished antinociceptive activity compared with wild type. The combined in vivo and in vitro results suggest that SNC80 selectively activates ?-? heteromers to produce maximal antinociception. These data contrast with the current view that SNC80 selectively activates ?-opioid receptor homomers to produce antinociception. Thus, the data suggest that heteromeric ?-? receptors should be considered as a target when SNC80 is employed as a pharmacological tool in vivo. PMID:22860219
Laminin-5 and type I collagen promote adhesion and osteogenic differentiation of animal serum-free expanded human mesenchymal stromal cells.
Mittag, Falk; Falkenberg, Eva-Maria; Janczyk, Alexandra; G�tze, Marco; Felka, Tino; Aicher, Wilhelm K; Kluba, Torsten
Mesenchymal stromal cells (MSC) are differentiation competent cells and may generate, among others, mature osteoblasts or chondrocytes in vitro and in vivo. Laminin-5 and type I collagen are important components of the extracellular matrix. They are involved in a variety of cellular and extracellular activities including cell attachment and osteogenic differentiation of MSC. MSC were isolated and expanded using media conforming good medical practice (GMP)-regulations for medical products. Cells were characterized according to the defined minimal criteria for multipotent MSC. MTT- and BrdU-assays were performed to evaluate protein-dependent (laminin-5, laminin-1, type I collagen) metabolic activity and proliferation of MSC. MSC-attachment assays were performed using protein-coated culture plates. Osteogenic differentiation of MSC was measured by protein-dependant mineralization and expression of osteogenic marker genes (osteopontin, alkaline phophatase, Runx2) after three, seven and 28 days of differentiation. Marker genes were identified using quantitative reverse-transcription polymerase chain reaction. Expansion of MSC in GMP-conforming media yielded vital cells meeting all minimal criteria for MSC. Attachment assay revealed a favorable binding of MSC to laminin-5 and type I collagen at a protein concentration of 1-5 fmol/�L. Compared to plastic, osteogenic differentiation was significantly increased by laminin-5 after 28 days of culture (P<0.04). No significant differences in gene expression patterns were observed. We conclude that laminin-5 and type I collagen promote attachment, but laminin-1 and laminin-5 promote osteogenic differentiation of MSC. This may influence future clinical applications. PMID:23589764
Activities of nicotinic acetylcholine receptors modulate neurotransmission and synaptic architecture
Oda, Akira; Tanaka, Hidekazu
The cholinergic system is involved in a broad spectrum of brain function, and its failure has been implicated in Alzheimer's disease. Acetylcholine transduces signals through muscarinic and nicotinic acetylcholine receptors, both of which influence synaptic plasticity and cognition. However, the mechanisms that relate the rapid gating of nicotinic acetylcholine receptors to persistent changes in brain function have remained elusive. Recent evidence indicates that nicotinic acetylcholine receptors activities affect synaptic morphology and density, which result in persistent rearrangements of neural connectivity. Further investigations of the relationships between nicotinic acetylcholine receptors and rearrangements of neural circuitry in the central nervous system may help understand the pathogenesis of Alzheimer's disease. PMID:25657733
Visualization of Chemokine Receptor Activation in Transgenic Mice Reveals Peripheral Activation of CCR2 Receptors in States of Neuropathic Pain
Jung, Hosung; Bhangoo, Sonia; Banisadr, Ghazal; Freitag, Caroline; Ren, Dongjun; White, Fletcher A.; Miller, Richard J.
CCR2 chemokine receptor signaling has been implicated in the generation of diverse types of neuropathology, including neuropathic pain. For example, ccr2 knock-out mice are resistant to the establishment of neuropathic pain, and mice overexpressing its ligand, monocyte chemoattractant protein-1 (MCP1; also known as CCL2), show enhanced pain sensitivity. However, whether CCR2 receptor activation occurs in the central or peripheral nervous system in states of neuropathic pain has not been clear. We developed a novel method for visualizing CCR2 receptor activation in vivo by generating bitransgenic reporter mice in which the chemokine receptor CCR2 and its ligand MCP1 were labeled by the fluorescent proteins enhanced green fluorescent protein and monomeric red fluorescent protein-1, respectively. CCR2 receptor activation under conditions such as acute inflammation and experimental autoimmune encephalomyelitis could be faithfully visualized by using these mice. We examined the status of CCR2 receptor activation in a demyelination injury model of neuropathic pain and found that MCP1-induced CCR2 receptor activation mainly occurred in the peripheral nervous system, including the injured peripheral nerve and dorsal root ganglia. These data explain the rapid antinociceptive effects of peripherally administered CCR2 antagonists under these circumstances, suggesting that CCR2 antagonists may ameliorate pain by inhibiting CCR2 receptor activation in the periphery. The method developed here for visualizing CCR2 receptor activation in vivo may be extended to G-protein-coupled receptors (GPCRs) in general and will be valuable for studying intercellular GPCR-mediated communication in vivo. PMID:19553445
Isolation and Characterization of Rat Schwannoma Neurite-promoting Factor: Evidence that the Factor Contains Laminin
Rat RN22 schwannoma cells in vitro release into their growth medium a macromolecular factor that, when bound to polyornithine-coated culture substrata, will stimulate neuritic regeneration from axotomized peripheral and central neu- rons. During the purification of this factor, the neurite-pro- moting activity co-purifies with laminin immunoreactivity as measured by an enzyme-linked immunoadsorbant assay. The purified factor has an immunoreactivity per
Migration of breast epithelial cells on Laminin5: differential role of integrins in normal and transformed cell types
George E. Plopper; Susan Z. Domanico; Vincenzo Cirulli; William B. Kiosses; Vito Quaranta
We examined the role of Laminin-5 (Ln-5) an extracellular matrix component of breast gland basement membrane, in supporting migration of normal (HUMEC), immortalized (MCF-10A), and malignant breast epithelial cells that exhibit different degrees of metastatic potential (MDA-MB-435>MDA-MB-231>MCF-7). HUMEC, MCF-10A, and MCF-7 cells all adhered to purified Ln-5 through the a3�1 integrin receptor in adhesion assays. However, HUMEC and MCF-10A cells
Inhibiting Proteasomal Proteolysis Sustains Estrogen Receptor� Activation
Estrogen receptor- (ER) is a ligand-dependent transcription factor that mediates physiological re- sponses to 17-estradiol (E2). Ligand binding rap- idly down-regulates ER levels through proteaso- mal proteolysis, but the functional impact of receptor degradation on cellular responses to E2 has not been fully established. In this study, we investigated the effect of blocking the ubiquitin- proteasome pathway on ER-mediated transcrip-
Human mast cell activation through Fc receptors and Toll-like receptors
Yoshimichi Okayama; Shigeru Okumura; Hisashi Tomita; Hiroko Katayama; Keisuke Yuki; Shinji Kagaya; Jun-ichi Kashiwakura; Hirohisa Saito
Mast cells express high-affinity IgE receptors (Fc ? RI) on their surface and can be activated to secrete a variety of biologically active mediators by cross-linking of receptor-bound IgE. Recent studies in animal models indicate that mouse mast cells may play a protective role in host defense against bacteria through the pro- duction of tumor necrosis factor- ? , mainly
Mode of peroxisome proliferator-activated receptor ? activation by luteolin.
Puhl, Ana C; Bernardes, Amanda; Silveira, Rodrigo L; Yuan, Jing; Campos, J�ssica L O; Saidemberg, Daniel M; Palma, Mario S; Cvoro, Aleksandra; Ayers, Stephen D; Webb, Paul; Reinach, Peter S; Skaf, Munir S; Polikarpov, Igor
The peroxisome proliferator-activated receptor ? (PPAR?) is a target for treatment of type II diabetes and other conditions. PPAR? full agonists, such as thiazolidinediones (TZDs), are effective insulin sensitizers and anti-inflammatory agents, but their use is limited by adverse side effects. Luteolin is a flavonoid with anti-inflammatory actions that binds PPAR? but, unlike TZDs, does not promote adipocyte differentiation. However, previous reports suggested variously that luteolin is a PPAR? agonist or an antagonist. We show that luteolin exhibits weak partial agonist/antagonist activity in transfections, inhibits several PPAR? target genes in 3T3-L1 cells (LPL, ORL1, and CEBP?) and PPAR?-dependent adipogenesis, but activates GLUT4 to a similar degree as rosiglitazone, implying gene-specific partial agonism. The crystal structure of the PPAR? ligand-binding domain (LBD) reveals that luteolin occupies a buried ligand-binding pocket (LBP) but binds an inactive PPAR? LBD conformer and occupies a space near the ?-sheet region far from the activation helix (H12), consistent with partial agonist/antagonist actions. A single myristic acid molecule simultaneously binds the LBP, suggesting that luteolin may cooperate with other ligands to bind PPAR?, and molecular dynamics simulations show that luteolin and myristic acid cooperate to stabilize the ?-loop among H2', H3, and the ?-sheet region. It is noteworthy that luteolin strongly suppresses hypertonicity-induced release of the pro-inflammatory interleukin-8 from human corneal epithelial cells and reverses reductions in transepithelial electrical resistance. This effect is PPAR?-dependent. We propose that activities of luteolin are related to its singular binding mode, that anti-inflammatory activity does not require H12 stabilization, and that our structure can be useful in developing safe selective PPAR? modulators. PMID:22391103
Laminin-421 produced by lymphatic endothelial cells induces chemotaxis for human melanoma cells.
Saito, Noriko; Hamada, Jun-ichi; Furukawa, Hiroshi; Tsutsumida, Arata; Oyama, Akihiko; Funayama, Emi; Saito, Akira; Tsuji, Tsutomu; Tada, Mitsuhiro; Moriuchi, Tetsuya; Yamamoto, Yuhei
Melanoma has a high tendency to metastasize to lymph nodes, which is one of the clinicopathological factors to indicate poor prognosis. Recent investigations have shown the importance of lymphangiogenesis in lymph node metastasis in a variety of human tumors including melanoma. However, molecular mechanism of lymphatic metastasis is still poorly defined. We examined influence of interactions between normal lymphatic endothelial cells (LECs) and melanoma cells on cell migration. Medium conditioned with LEC (LEC-CM) contained chemotactic and chemokinetic activities for human melanoma cell lines. The chemotactic activity was fractionated in more than 100 kDa, and inactivated by heat-treatment. The chemotactic activity of LEC-CM was abolished by immunodepletion with anti-laminin-1 antibody. And immunoprecipitation and Western blot analyses revealed that LEC-CM contained laminin-421. When melanoma C8161 cells were treated with function-blocking antibodies to integrin alpha3 or alpha6, their chemotactic responses to LEC-CM were markedly reduced. Furthermore, the knock-down of tetraspanin CD151 weakened the chemotactic responses of C8161 and MeWo cells to LEC-CM. These data suggest that laminin-421 secreted by LEC possibly facilitates lymphatic metastasis through the induction of chemotaxis of melanoma cells. PMID:19508413
Neuronal cell attachment to fluorinated ethylene propylene films with covalently immobilized laminin oligopeptides YIGSR and IKVAV. II.
Ranieri, J P; Bellamkonda, R; Bekos, E J; Vargo, T G; Gardella, J A; Aebischer, P
Material surfaces that can mediate cellular interactions by the coupling of specific cell membrane receptors may allow for the design of a biomaterial that can control cell attachment, differentiation, and tissue organization. Cell adhesion proteins have been shown to contain minimum oligopeptide sequences that are recognized by cell surface receptors and can be covalently immobilized on material surfaces. In this study, cell attachment to fluorinated ethylene propylene (FEP) films functionalized with the laminin-derived oligopeptides, YIGSR and a 19-mer IKVAV-containing sequence, was assessed using NG108-15 neuroblastoma and PC12 cells. A radiofrequency glow discharge (RFGD) process that replaces the FEP surface fluorine atoms with reactive hydroxyl functionalities was used to activate the film surfaces. The oligopeptides were then covalently coupled to the surface by their C-terminus using a standard nucleophilic substitution reaction. The covalent attachment of the oligopeptides to the FEP surface was verified using electron spectroscopy for chemical analysis (ESCA). Receptor-mediated NG108-15 cell attachment on the YIGSR-modified films was determined using competitive binding assays. Average cell attachment on the oligopeptide immobilized films in medium containing soluble CDPGYIGSR was reduced by approximately a factor of 2, compared to cell attachment in serum-free medium alone. No significant decrease in cell attachment was noted in medium containing the mock oligopeptide sequence CDPGYIGSK. FEP films immobilized with the 19-mer IKVAV sequence demonstrated a higher percentage of receptor mediated cell attachment on the film surfaces.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7593015
Tie2 and Eph Receptor Tyrosine Kinase Activation and Signaling
Barton, William A.; Dalton, Annamarie C.; Seegar, Tom C.M.; Himanen, Juha P.; Nikolov, Dimitar B.
The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition. PMID:24478383
Enhanced Acetaminophen Toxicity by Activation of the Pregnane X Receptor
Grace L. Guo; Jeff S. Moffit; Christopher J. Nicol; Jerrold M. Ward; Lauren A. Aleksunes; Angela L. Slitt; Steven A. Kliewer; Jose E. Manautou; Frank J. Gonzalez
The pregnane X receptor (PXR) is a ligand-activated transcrip- tion factor and member of the nuclear receptor superfamily. Acti- vation of PXR represents an important mechanism for the induction of cytochrome P450 3A (CYP3A) enzymes that can convert acetaminophen (APAP) to its toxic intermediate metabo- lite, N-acetyl-p-benzoquinone imine (NAPQI). Therefore, it was hypothesized that activation of PXR plays a major
Activation of the Mineralocorticoid Receptor Increases Striatin Levels
Luminita H. Pojoga; Patricia Coutinho; Alicia Rivera; Tham M. Yao; Enrique R. Maldonado; Rodeler Youte; Gail K. Adler; Jonathan Williams; Alexander Turchin; Gordon H. Williams; Jose R. Romero
BackgroundAldosterone (ALDO), a critical regulator of sodium homeostasis, mediates its effects via activation of the mineralocorticoid receptor (MR) through mechanisms that are not entirely clear. Striatin, a membrane associated protein, interacts with estrogen receptors in endothelial cells.MethodsWe studied the effects of MR activation in vitro and in vivo on striatin levels in vascular tissue.ResultsWe observed that dietary sodium restriction was
5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation
Samarajeewa, Anshula; Goldemann, Lolita; Vasefi, Maryam S.; Ahmed, Nawaz; Gondora, Nyasha; Khanderia, Chandni; Mielke, John G.; Beazely, Michael A.
The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and G?s-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both G?s and G?12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands. PMID:25426041
Activation of the p75 neurotrophin receptor through conformational rearrangement of disulphide-linked receptor dimers
Vilar, Mar�al; Charalampopoulos, Ioannis; Kenchappa, Rajappa S.; Simi, Anastasia; Karaca, Esra; Reversi, Alessandra; Choi, Soyoung; Bothwell, Mark; Mingarro, Ismael; Friedman, Wilma J.; Schiavo, Giampietro; Bastiaens, Philippe I. H.; Verveer, Peter J.; Carter, Bruce D.; Ib��ez, Carlos F.
SUMMARY Ligand-mediated dimerization has emerged as a universal mechanism of growth factor receptor activation. Recent structural studies have shown that neurotrophins interact with dimers of the p75 neurotrophin receptor (p75NTR), but the actual mechanism of receptor activation has remained elusive. Here we show that p75NTR forms disulphide-linked dimers independently of neurotrophin binding through the highly conserved Cys257 in its transmembrane domain. Mutation of Cys257 abolished neurotrophin-dependent receptor activity but did not affect downstream signaling by the p75NTR/NgR/Lingo-1 complex in response to MAG, indicating the existence of distinct, ligand-specific activation mechanisms for p75NTR. FRET experiments revealed a close association of p75NTR intracellular domains that was transiently disrupted by conformational changes induced upon NGF binding. Although mutation of Cys257 did not alter the oligomeric state of p75NTR, the mutant receptor was no longer able to propagate conformational changes to the cytoplasmic domain upon ligand binding. We propose that neurotrophins activate p75NTR by a novel mechanism involving rearrangement of disulphide-linked receptor subunits. PMID:19376068
Activation of the p75 neurotrophin receptor through conformational rearrangement of disulphide-linked receptor dimers.
Vilar, Mar�al; Charalampopoulos, Ioannis; Kenchappa, Rajappa S; Simi, Anastasia; Karaca, Esra; Reversi, Alessandra; Choi, Soyoung; Bothwell, Mark; Mingarro, Ismael; Friedman, Wilma J; Schiavo, Giampietro; Bastiaens, Philippe I H; Verveer, Peter J; Carter, Bruce D; Ib��ez, Carlos F
Ligand-mediated dimerization has emerged as a universal mechanism of growth factor receptor activation. Neurotrophins interact with dimers of the p75 neurotrophin receptor (p75(NTR)), but the mechanism of receptor activation has remained elusive. Here, we show that p75(NTR) forms disulphide-linked dimers independently of neurotrophin binding through the highly conserved Cys(257) in its transmembrane domain. Mutation of Cys(257) abolished neurotrophin-dependent receptor activity but did not affect downstream signaling by the p75(NTR)/NgR/Lingo-1 complex in response to MAG, indicating the existence of distinct, ligand-specific activation mechanisms for p75(NTR). FRET experiments revealed a close association of p75(NTR) intracellular domains that was transiently disrupted by conformational changes induced upon NGF binding. Although mutation of Cys(257) did not alter the oligomeric state of p75(NTR), the mutant receptor was no longer able to propagate conformational changes to the cytoplasmic domain upon ligand binding. We propose that neurotrophins activate p75(NTR) by a mechanism involving rearrangement of disulphide-linked receptor subunits. PMID:19376068
M5 Receptor Activation Produces Opposing Physiological Outcomes in Dopamine Neurons Depending on the Receptor's Location
Foster, Daniel J.; Gentry, Patrick R.; Lizardi-Ortiz, Jose E.; Bridges, Thomas M.; Wood, Michael R.; Niswender, Colleen M.; Sulzer, David; Lindsley, Craig W.; Xiang, Zixiu
Of the five muscarinic receptor subtypes, the M5 receptor is the only one detectable in midbrain dopaminergic neurons, making it an attractive potential therapeutic target for treating disorders in which dopaminergic signaling is disrupted. However, developing an understanding of the role of M5 in regulating midbrain dopamine neuron function has been hampered by a lack of subtype-selective compounds. Here, we extensively characterize the novel compound VU0238429 and demonstrate that it acts as a positive allosteric modulator with unprecedented selectivity for the M5 receptor. We then used VU0238429, along with M5 knock-out mice, to elucidate the role of this receptor in regulating substantia nigra pars compacta (SNc) neuron physiology in both mice and rats. In sagittal brain slices that isolate the SNc soma from their striatal terminals, activation of muscarinic receptors induced Ca2+ mobilization and inward currents in SNc dopamine neurons, both of which were potentiated by VU0238429 and absent in M5 knock-out mice. Activation of M5 also increased the spontaneous firing rate of SNc neurons, suggesting that activation of somatodendritic M5 increases the intrinsic excitability of SNc neurons. However, in coronal slices of the striatum, potentiation of M5 with VU0238429 resulted in an inhibition in dopamine release as monitored with fast scan cyclic voltammetry. Accordingly, activation of M5 can lead to opposing physiological outcomes depending on the location of the receptor. Although activation of somatodendritic M5 receptors on SNc neurons leads to increased neuronal firing, activation of M5 receptors in the striatum induces an inhibition in dopamine release. PMID:24573284
The structural biology of growth factor receptor activation.
Harmer, Nicholas J; Chirgadze, Dima; Hyun Kim, Kyung; Pellegrini, Luca; Blundell, Tom L
Stimulation of cells by growth factors triggers cascades of signalling that result in cellular responses such as growth, differentiation, migration and survival. Many growth factors signal through receptor tyrosine kinases, leading to dimerization, trans-phosphorylation and activation of tyrosine kinases that phosphorylate components further downstream of the signal transduction cascade. Using insulin-like growth factor, nerve growth factor, hepatocyte growth factor and fibroblast growth factor as examples, we show that the globular architecture of the growth factors is essential for receptor binding. We describe how nerve growth factor (NGF) is a symmetrical dimer that binds four storage proteins (two alpha-NGF and two gamma-NGF) to give a symmetrical hetero-hexameric 7SNGF organised around the beta-NGF dimer. It binds the extracellular domains of two receptor molecules in a similar way, so dimerising the receptor. Hepatocyte growth factor/scatter factor (HGF/SF) probably binds its receptor as a dimer stabilised by interactions with heparan sulfate, and fibroblast growth factor (FGF) binds its receptor as a dimer cross-linked by heparan sulfate. Surprisingly, insulin and insulin-like growth factor (IGF) bind in the monomeric form to receptors that are already covalent dimers. We propose that, in general, weak binary interactions between growth factor and individual domains of receptors are enhanced by cooperative interactions with further receptor domains, and sometimes other components like heparan, to give rise to specific multi-protein/domain complexes. PMID:12646390
Keratinocyte Differentiation is Stimulated by Activators of the Nuclear Hormone Receptor PPAR?
Karen Hanley; Yan Jiang; Shan Shan He; Mark Friedman; Peter M. Elias; Daniel D. Bikle; Mary L. Williams; Kenneth R. Feingold
Peroxisome proliferator activated receptors (PPAR) belong to the superfamily of nuclear hormone receptors that heterodimerize with the retinoid X receptor and regulate transcription of several genes involved in lipid metabolism and adipocyte differentiation. Because of the role of 1,25-dihydroxyvitamin D3 and retinoic acid working through similar receptors (the vitamin D receptor and retinoic acid receptor, respectively) on keratinocyte differentiation, we
Vitamin D receptor activation and survival in chronic kidney disease
C P Kovesdy; K Kalantar-Zadeh
Replacement of activated vitamin D has been the cornerstone of therapy for secondary hyperparathyroidism (SHPT). Recent findings from several large observational studies have suggested that the benefits of vitamin D receptor activators (VDRA) may extend beyond the traditional parathyroid hormone (PTH)-lowering effect, and could result in direct cardiovascular and metabolic benefits. The advent of several new analogs of the activated
Modulation of ?-Catenin Signaling by Glucagon Receptor Activation
Harikumar, Kaleeckal G.; Zylstra-Diegel, Cassandra R.; Wang, Liren; Mowry, Laura E.; Miller, Laurence J.; Williams, Bart O.; Xu, H. Eric
The glucagon receptor (GCGR) is a member of the class B G protein�coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA) pathway, activation of GCGR also induced ?-catenin stabilization and activated ?-catenin�mediated transcription. Activation of ?-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R) and glucagon-like peptide 1 (GLP-1R) receptors. Since low-density-lipoprotein receptor�related protein 5 (Lrp5) is an essential co-receptor required for Wnt protein mediated ?-catenin signaling, we examined the role of Lrp5 in glucagon-induced ?-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced ?-catenin stabilization and TCF promoter�mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1) or by expression of the Lrp5 extracellular domain blocked glucagon-induced ?-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and ?-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations. PMID:22438981
Activation and allosteric modulation of a muscarinic acetylcholine receptor
Kruse, Andrew C.; Ring, Aaron M.; Manglik, Aashish; Hu, Jianxin; Hu, Kelly; Eitel, Katrin; H�bner, Harald; Pardon, Els; Valant, Celine; Sexton, Patrick M.; Christopoulos, Arthur; Felder, Christian C.; Gmeiner, Peter; Steyaert, Jan; Weis, William I.; Garcia, K. Christopher; Wess, J�rgen; Kobilka, Brian K.
Despite recent advances in crystallography of G protein-coupled receptors (GPCRs), little is known about the mechanism of their activation process, as only the ?2 adrenergic receptor (?2AR) and rhodopsin have been crystallized in fully active conformations. Here, we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the ?2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously binding the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into activation mechanism and allosteric modulation of muscarinic receptors. PMID:24256733
ACE activity is modulated by kinin B2 receptor.
Sabatini, Regiane A; Guimar�es, Paola B; Fernandes, Liliam; Reis, Felipe C G; Bersanetti, Patricia A; Mori, Marcelo A; Navarro, Alberto; Hilzendeger, Aline M; Santos, Edson L; Andrade, Maria C C; Chagas, Jair R; Pesquero, Jorge L; Casarini, Dulce E; Bader, Michael; Carmona, Adriana K; Pesquero, Jo�o B
Angiotensin-converting enzyme (ACE) is an ectoprotein able to modulate the activity of a plethora of compounds, among them angiotensin I and bradykinin. Despite several decades of research, new aspects of the mechanism of action of ACE have been elucidated, expanding our understanding of its role not only in cardiovascular regulation but also in different areas. Recent findings have ascribed an important role for ACE/kinin B(2) receptor heterodimerization in the pharmacological properties of the receptor. In this work, we tested the hypothesis that this interaction also affects ACE enzymatic activity. ACE catalytic activity was analyzed in Chinese hamster ovary cell monolayers coexpressing the somatic form of the enzyme and the receptor coding region using as substrate the fluorescence resonance energy transfer peptide Abz-FRK(Dnp)P-OH. Results show that the coexpression of the kinin B(2) receptor leads to an augmentation in ACE activity. In addition, this effect could be blocked by the B(2) receptor antagonist icatibant. The hypothesis was also tested in endothelial cells, a more physiological system, where both proteins are naturally expressed. Endothelial cells from genetically ablated kinin B(2) receptor mice showed a decreased ACE activity when compared with wild-type mice cells. In summary, this is the first report showing that the ACE/kinin B(2) receptor interaction modulates ACE activity. Taking into account the interplay among ACE, ACE inhibitors, and kinin receptors, we believe that these results will shed new light into the arena of the controversial search for the mechanism controlling these interactions. PMID:18212275
Assessment of seminal plasma laminin in fertile and infertile men
Mohamed R. El-Dakhly; Gamil A. Tawadrous; Taymour Mostafa; Mohamed M. F. Roaia; Abdel R. M. El-Nashar; Shedeed A. Shedeed; Ihab I. Kamel; Amal A. Aziz; Yasser El-Mohtaseb
Aim:To assess laminin levels in the seminal plasma of infertile and fertile men, and to analyze the correlation of laminin levels with sperm count, age, sperm motility and semen volume.Methods:One hundred and twenty-five recruited men were equally divided into five groups according to their sperm concentration and clinical examination: fertile normozoospermia, oligoasthenozoospermia, non-obstructive azoospermia (NOA), obstructive azoospermia (OA) and congenital
Histidine(7.36(305)) in the conserved peptide receptor activation domain of the gonadotropin releasing hormone receptor couples peptide binding and receptor activation.
Mayevu, Nkateko M I; Choe, Han; Abagyan, Ruben; Seong, Jae Young; Millar, Robert P; Katz, Arieh A; Flanagan, Colleen A
Transmembrane helix seven residues of G protein-coupled receptors (GPCRs) couple agonist binding to a conserved receptor activation mechanism. Amino-terminal residues of the GnRH peptide determine agonist activity. We investigated GnRH interactions with the His(7.36(305)) residue of the GnRH receptor, using functional and computational analysis of modified GnRH receptors and peptides. Non-polar His(7.36(305)) substitutions decreased receptor affinity for GnRH four- to forty-fold, whereas GnRH signaling potency was more decreased (~150-fold). Uncharged polar His(7.36(305)) substitutions decreased GnRH potency, but not affinity. [2-Nal(3)]-GnRH retained high affinity at receptors with non-polar His(7.36(305)) substitutions, supporting a role for His(7.36(305)) in recognizing Trp(3) of GnRH. Compared with GnRH, [2-Nal(3)]-GnRH potency was lower at the wild type GnRH receptor, but unchanged or higher at mutant receptors. Results suggest that His(7.36(305)) of the GnRH receptor forms two distinct interactions that determine binding to Trp(3) and couple agonist binding to the conserved transmembrane domain network that activates GPCRs. PMID:25583361
Functions of the extracellular histidine residues of receptor activity-modifying proteins vary within adrenomedullin receptors
Kuwasako, Kenji [Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan)], E-mail: email@example.com; Kitamura, Kazuo; Nagata, Sayaka [Circulation and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan); Kato, Johji [Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan)
Receptor activity-modifying protein (RAMP)-2 and -3 chaperone calcitonin receptor-like receptor (CRLR) to the plasma membrane, where together they form heterodimeric adrenomedullin (AM) receptors. We investigated the contributions made by His residues situated in the RAMP extracellular domain to AM receptor trafficking and receptor signaling by co-expressing hCRLR and V5-tagged-hRAMP2 or -3 mutants in which a His residue was substituted with Ala in HEK-293 cells. Flow cytometric analysis revealed that hRAMP2-H71A mediated normal hCRLR surface delivery, but the resultant heterodimers showed significantly diminished [{sup 125}I]AM binding and AM-evoked cAMP production. Expression of hRAMP2-H124A and -H127A impaired surface delivery of hCRLR, which impaired or abolishing AM binding and receptor signaling. Although hRAMP3-H97A mediated full surface delivery of hCRLR, the resultant heterodimers showed impaired AM binding and signaling. Other His residues appeared uninvolved in hCRLR-related functions. Thus, the His residues of hRAMP2 and -3 differentially govern AM receptor function.
Peroxisome Proliferator-Activated Receptors and Progression of Colorectal Cancer
Wang, Dingzhi; DuBois, Raymond N.
The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. These receptors are also ligand-dependent transcription factors responsible for the regulation of cellular events that range from glucose and lipid homeostases to cell differentiation and apoptosis. The importance of these receptors in lipid homeostasis and energy balance is well established. In addition to these metabolic and anti-inflammatory properties, emerging evidence indicates that PPARs can function as either tumor suppressors or accelerators, suggesting that these receptors are potential candidates as drug targets for cancer prevention and treatment. However, conflicting results have emerged regarding the role of PPARs on colon carcinogenesis. Therefore, further investigation is warranted prior to considering modulation of PPARs as an efficacious therapy for colorectal cancer chemoprevention and treatment. PMID:18551185
Enhanced laminin adsorption on nanowires compared to flat surfaces.
Hammarin, Greger; Persson, Henrik; Dabkowska, Aleksandra P; Prinz, Christelle N
Semiconductor nanowires are widely used to interface living cells, and numerous nanowire-based devices have been developed to manipulate or sense cell behavior. We have, however, little knowledge on the nature of the cell-nanowire interface. Laminin is an extracellular matrix protein promoting cell attachment and growth. Here, we used a method based on fluorescence microscopy and measured the relative amount of laminin adsorbed on nanowires compared to flat surfaces. The amount of adsorbed laminin per surface area is up to 4 times higher on 55nm diameter gallium phosphide nanowires compared to the flat gallium phosphide surface between the nanowires. We show that this enhanced adsorption on nanowires cannot be attributed to electrostatic effects, nor to differences in surface chemistry, but possibly to pure geometrical effects, as increasing the nanowire diameter results in a decreased amount of adsorbed protein. The increased adsorption of laminin on nanowires may explain the exceptionally beneficial properties of nanowire substrates for cellular growth reported in the literature since laminin is often used as surface coating prior to cell cultures in order to promote cell growth, and also because primary cell suspensions contain endogenous laminin. PMID:25024109
Hedgehog Signaling and Laminin Play Unique and Synergistic Roles in Muscle Development
Peterson, Matthew T.; Henry, Clarissa A.
Hedgehog (Hh) signaling and laminin-111, a basement membrane protein, are required for early muscle development. Hh signaling specifies different populations of muscle fibers and laminin-111 is critical for early muscle morphogenesis. However, additional requirements for Hh signaling and laminin during later phases of muscle development are not known. Furthermore, interactions between Hh signaling and laminin in this context are unknown. We used laminin gamma1 mutant zebrafish and cyclopamine to block Hh signal transduction separately and in combination to investigate their functions and interactions. We found that both Hh signaling and laminin are required for normal myosin chain expression. In addition, Hh signaling and laminin act synergistically during fast-twitch fiber elongation: fast muscle cells do not elongate in embryos deficient for both Hh signaling and laminin. Finally, we present evidence which suggests that Hh signaling is indirectly required via slow fiber specification for recovery of fast fiber elongation in laminin gamma1 mutant embryos. PMID:20063418
Cloning, constitutive activity and expression profiling of two receptors related to relaxin receptors in Drosophila melanogaster.
Van Hiel, Matthias B; Vandersmissen, Hans Peter; Proost, Paul; Vanden Broeck, Jozef
Leucine-rich repeat containing G protein-coupled receptors (LGRs) comprise a cluster of transmembrane proteins, characterized by the presence of a large N-terminal extracellular domain. This receptor group can be classified into three subtypes. Belonging to the subtype C LGRs are the mammalian relaxin receptors LGR7 (RXFP1) and LGR8 (RXFP2), which mediate important reproductive and other processes. We identified two related receptors in the genome of the fruit fly and cloned their open reading frames into an expression vector. Interestingly, dLGR3 demonstrated constitutive activity at very low doses of transfected plasmid, whereas dLGR4 did not show any basal activity. Both receptors exhibited a similar expression pattern during development, with relatively high transcript levels during the first larval stage. In addition, both receptors displayed higher expression in male adult flies as compared to female flies. Analysis of the tissue distribution of both receptor transcripts revealed a high expression of dLGR3 in the female fat body, while the expression of dLGR4 peaked in the midgut of both the wandering and adult stage. PMID:25064813
Coupling of Receptor Conformation and Ligand Orientation Determine Graded Activity
Bruning, John B.; Parent, Alex A.; Gil, German; Zhao, Min; Nowak, Jason; Pace, Margaret C.; Smith, Carolyn L.; Afonine, Pavel V.; Adams, Paul D.; Katzenellenbogen, John A.; Nettles, Kendall W.
SUMMARY Small molecules stabilize specific protein conformations from a larger ensemble, enabling molecular switches that control diverse cellular functions. We show here that the converse also holds true, where the conformational state of the estrogen receptor can direct distinct orientations of the bound ligand. �Gain of allostery� mutations that mimic the effects of ligand in driving protein conformation allowed crystallization of the partial agonist ligand WAY-169916 with both the canonical active and inactive conformations of the estrogen receptor. The intermediate transcriptional activity induced by WAY169916 is associated with the ligand binding differently to the active and inactive conformations of the receptor. Analyses of a series of chemical derivatives demonstrated that altering the ensemble of ligand binding orientations changes signaling output. The coupling of different ligand binding orientations to distinct active and inactive protein conformations defines a novel mechanism for titrating allosteric signaling activity. PMID:20924370
Dynamic regulation of Drosophila nuclear receptor activity in vivo
Palanker, Laura; Necakov, Aleksandar S.; Sampson, Heidi M.; Ni, Ruoyu; Hu, Chun; Thummel, Carl S.; Krause, Henry M.
Nuclear receptors are a large family of transcription factors that play major roles in development, metamorphosis, metabolism and disease. To determine how, where and when nuclear receptors are regulated by small chemical ligands and/or protein partners, we have used a �ligand sensor� system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live developing animals. Transgenic lines were established that express the ligand binding domain of each nuclear receptor fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, the fusion proteins show tissue- and stage-specific patterns of activation. We show that these responses accurately reflect the presence of endogenous and exogenously added hormone, and that they can be modulated by nuclear receptor partner proteins. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. We also see dynamic changes in activation that are indicative of sweeping changes in ligand and/or co-factor production. The screening of a small compound library using this system identified the angular psoralen angelicin and the insect growth regulator fenoxycarb as activators of the Ultraspiracle (USP) ligand-binding domain. These results demonstrate the utility of this system for the functional dissection of nuclear receptor pathways and for the development of new receptor agonists and antagonists that can be used to modulate metabolism and disease and to develop more effective means of insect control. PMID:16914501
A Potential Role of Progestin-Induced Laminin-5/?6-Integrin Signaling in the Formation of Side Branches in the Mammary Gland
Meyer, Gabriele; Leipprandt, Jeffrey; Xie, Jianwei; Aupperlee, Mark D.
Mammary organoids from adult mice produce tubules, analogous to mammary ducts in vivo, in response to hepatocyte growth factor (HGF) when cultured in collagen gels. The combination of HGF plus progestin (R5020) causes reduced tubule number and length. We hypothesized that the inhibitory effect on tubulogenesis was due to progestin-mediated alteration of HGF/c-Met signaling. Using molecular inhibitors and short hairpin RNA, it was determined that HGF activation of Ras-related C3 botulinum toxin substrate (Rac1) was required for the formation of cytoplasmic extensions, the first step of tubulogenesis, and that Rac1 activity was Src kinase (Src) and focal adhesion kinase (FAK) dependent. The highly novel finding was that R5020 reduced tubulogenesis by up-regulating and increasing extracellular laminin and ?6-integrin ligation to reduce activation of the Src, focal adhesion kinase, and Rac1 pathway. Receptor activator of nuclear factor-?B ligand, another progesterone-induced paracrine factor, did not replicate this effect of R5020. The inhibitory effect of R5020 on tubulogenesis was likely mediated through progesterone receptor (PR) isoform A (PRA), because PRA is the predominant PR isoform expressed in the organoids, and the progestin-induced effect was prevented by the PR antagonist RU486. These results provide a plausible mechanism that explains progestin/PRA-mediated blunting of HGF-induced tubulogenesis in vitro and is proposed to be relevant to progesterone/PRA-induced side-branching in vivo during pregnancy. PMID:22910029
Flavonoids as dietary regulators of nuclear receptor activity
Avior, Yishai; Bomze, David; Ramon, Ory
Metabolic diseases such as obesity, type II diabetes, and dyslipidemia are a rising cause of mortality worldwide. The progression of many metabolic diseases is fundamentally regulated on the transcriptional level by a family of ligand-activated transcription factors, called nuclear receptors, which detect and respond to metabolic changes. Their role in maintaining metabolic homeostasis makes nuclear receptors an important pharmaceutical and dietary target. This review will present the growing evidence that flavonoids, natural secondary plant metabolites, are important regulators of nuclear receptor activity. Structural similarities between flavonoids and cholesterol derivatives combined with the promiscuous nature of most nuclear receptors provide a wealth of possibilities for pharmaceutical and dietary modulation of metabolism. While the challenges of bringing flavonoid-derived therapeutics to the market are significant, we consider this rapidly growing field to be an essential aspect of the functional food initiative and an important mine for pharmaceutical compounds. PMID:23598551
Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid�Activated Receptor
Kruse, Schoen W; Suino-Powell, Kelly; Zhou, X. Edward; Kretschman, Jennifer E; Reynolds, Ross; Vonrhein, Clemens; Xu, Yong; Wang, Liliang; Tsai, Sophia Y; Tsai, Ming-Jer; Xu, H. Eric
The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 � crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix ?10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation. PMID:18798693
Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid?Activated Receptor
Kruse, Schoen W.; Suino-Powell, Kelly; Zhou, X. Edward; Kretschman, Jennifer E.; Reynolds, Ross; Vonrhein, Clemens; Xu, Yong; Wang, Liliang; Tsai, Sophia Y.; Tsai, Ming-Jer; Xu, H. Eric (Baylor); (Van Andel); (Globel Phasing); (Grand Valley)
The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 {angstrom} crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix {alpha}10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.
Glycine Receptor ?2 Subunit Activation Promotes Cortical Interneuron Migration
Avila, Ariel; Vidal, P�a�M.; Dear, T.�Neil; Harvey, Robert�J.; Rigo, Jean-Michel; Nguyen, Laurent
Summary Glycine receptors (GlyRs) are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in�vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the ?2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR ?2 activation that control cortical tangential migration during embryogenesis. PMID:23954789
Activation of Proteinase-Activated Receptor2 by Human Kallikrein-Related Peptidases
Kristina Stefansson; Maria Brattsand; Dirk Roosterman; Cordula Kempkes; Georgeta Bocheva; Martin Steinhoff; Torbj�rn Egelrud
Proteinase-activated receptor-2 (PAR2) is a seven transmembrane spanning, G-protein-coupled receptor, present on the membrane of many cell types including keratinocytes. In skin, PAR2 is suggested to play a regulatory role during inflammation, epidermal barrier function, and pruritus. PAR2 is activated by trypsin-like proteases by a unique mechanism where cleavage of the receptor leads to the release of a small peptide,
Direct detection of guidance receptor activity during border cell migration.
Janssens, Katrien; Sung, Hsin-Ho; R�rth, Pernille
Guidance receptor signaling is crucial for steering migrating cells. Despite this, we generally lack direct measurements of such signaling. Border cells in Drosophila migrate as a tightly associated group, but dynamically, with front and rear cells exchanging places. They use the receptor tyrosine kinase (RTK) PDGF/VEGF receptor (PVR) as a guidance receptor, perceiving the attractant Pvf1. Here we determine the spatial distribution of PVR signaling by generating an antibody that specifically detects activated PVR in situ. PVR activity is very low in migrating border cells, due to strong activity of cellular phosphatases. Measurements of signal at the cell cortex show variability but a strong bias for both total active PVR and specific activity of PVR to be elevated at the front versus side of the leading cell, often with several-fold difference in signal levels. This polarized active PVR signal requires the E3 ubiquitin ligase Cbl and the recycling regulator Rab11, indicating a dependency on receptor trafficking. The endogenous ligand gradient contributes to shaping of signaling by increasing the specific activity of PVR toward the source in front cells. Surprisingly, signaling is also elevated at the back versus the side of rear cells. This distally polarized distribution of active PVR is ligand independent. Thus the actual guidance signal transmitted in border cells appears to integrate perceived ligand distribution with cell polarity or cell orientation with respect to the cluster. A general implication is that both group configuration and extrinsic cues can directly modulate guidance receptor signaling during collective cell migration. PMID:20368415
Interactions of drugs active at opiate receptors and drugs active at alpha 2-receptors on various test systems.
Browning, S; Lawrence, D; Livingston, A; Morris, B
1 The actions of the opiate receptor drugs, morphine, methionine-enkephalin (Met-enkephalin) and naloxone were compared with the actions of the alpha 2-receptor drugs, clonidine, xylazine and yohimbine on analgesic tests, in vitro bioassay (guinea-pig ileum and mouse vas deferens), and radioligand displacement studies on rat brain membrane preparations. 2. Both opiate and alpha 2-agonist drugs showed analgesic activity but whilst the alpha 2-agonist analgesic activity was antagonized by only alpha 2-antagonists, the analgesic activity of morphine was antagonized by both naloxone and yohimbine. In the in vitro tests, both groups of agonists inhibited electrically evoked activity; however in these experiments only antagonism of opiates by naloxone and alpha 2-agonists by yohimbine could be shown and it is concluded that in these systems the activity of the alpha 2-agonists is mediated via the presynaptic alpha 2-receptors only. 3 In the radioligand studies the drugs acting at alpha 2-receptors were active in the micromolar range at displacing labelled opioid ligands but opiates did not displace labelled alpha 2-ligands. 4 It is concluded that drugs which act on alpha 2-receptors interfere with the in vivo analgesic effects of opiates and weakly displace opioid radioligand binding, but opioids do not affect alpha 2 agonist analgesia and do not appear to displace alpha 2-agonist radioligand binding. PMID:6128044
Regulation of Proteome Maintenance Gene Expression by Activators of Peroxisome Proliferator-Activated Receptor a (PPARa)
The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARa) is activated by a large number of xenobiotic and hypolipidemic compounds called peroxisome proliferator chemicals (PPC). One agonist of PPARa (WY-14,643) regulates responses in the mouse liver to chemic...
Steroid signaling activation and intracellular localization of sex steroid receptors
Giraldi, Tiziana; Giovannelli, Pia; Di Donato, Marzia; Migliaccio, Antimo; Auricchio, Ferdinando
In addition to stimulating gene transcription, sex steroids trigger rapid, non-genomic responses in the extra-nuclear compartment of target cells. These events take place within seconds or minutes after hormone administration and do not require transcriptional activity of sex steroid receptors. Depending on cell systems, activation of extra-nuclear signaling pathways by sex steroids fosters cell cycle progression, prevents apoptosis, leads to epigenetic modifications and increases cell migration through cytoskeleton changes. These findings have raised the question of intracellular localization of sex steroid receptors mediating these responses. During the past years, increasing evidence has shown that classical sex steroid receptors localized in the extra-nuclear compartment or close to membranes of target cells induce these events. The emerging picture is that a process of bidirectional control between signaling activation and sex steroid receptor localization regulates the outcome of hormonal responses in target cells. This mechanism ensures cell cycle progression in estradiol-treated breast cancer cells, and its derangement might occur in progression of human proliferative diseases. These findings will be reviewed here together with unexpected examples of the relationship between sex steroid receptor localization, signaling activation and biological responses in target cells. We apologize to scientists whose reports are not mentioned or extensively discussed owing to space limitations. PMID:21234121
Influence of laminin substratum on cell proliferation and CALC I gene expression in medullary thyroid carcinoma C cell lines.
Lekmine, F; Lausson, S; Pidoux, E; Segond, N; Roos, B; Treilhou-Lahille, F; Jeanne, A
Medullary thyroid carcinoma (MTC) originates from C cells, which secrete calcitonin (CT) and CT gene-related peptide (CGRP), the two splice peptide products of the CALC I gene. Normal and hyperplastic C cells are intrafollicular, in contact with the basement membrane (BM) that is maintained around the differentiated tumors. To investigate the relationships between MTC evolution and BM constituents, we examined the modifications induced by laminin-1 and -2 (merosin), two isoforms colocalized in the follicular BM, on three MTC cell lines: murine rMTC 6-23 and CA-77 cells, and human TT cells. Laminin exerted a mitogenic activity on rMTC 6-23 and on TT cells, causing a concurrent decrease in both CT and CGRP mRNA levels and production of the peptides. Conversely, laminin reduced the proliferation rate and enhanced CGRP synthesis and secretion in CA-77 cells. This antiproliferative response, which coincides with an increase in differentiation markers, is comparable to that reported in normal cells and also in the neoplastic Caco-2 cell line. This suggests that laminin could exert opposite effects depending on the stage of tumor evolution. PMID:10619409
Platelet protease-activated receptor antagonism in cardiovascular medicine.
Wiisanen, Matthew E; Moliterno, David J
Ischemic heart disease remains the number one cause of death in the world despite advances in invasive and pharmacologic therapies. An ongoing area of research is the central role of platelets in atherothrombosis. Many therapeutic strategies have been developed over the last few decades affecting different platelet receptors to alter platelet-mediated thrombosis including targeting the receptors for thromboxane A(2), adenosine diphosphate, and fibrinogen. However, despite the use of pharmacologic agents directed at these pathways, residual morbidity and mortality still exist. Therefore, identifying agents that more favorably balance a reduction in ischemic events while minimizing bleeding events is an ongoing mission. Thrombin is known to be the most potent stimulant of platelet-mediated thrombosis whose action on the platelet is through a family of receptors known as the protease-activated receptors (PARs). Activation through the PAR-1 receptor, in particular, results in an early and intense response by the platelet to thrombin, and it is the primary thrombin receptor on platelets, thus making it a potentially desirable target for therapy. Most recently, two PAR-1 antagonists, atopaxar and vorapaxar, have been tested in clinical trials. Generally, the results show a reduction in ischemic event rates, but an increase in bleeding event rates. This article will summarize the current state of the literature and consider the role these drugs might play in the future for the prevention of ischemic heart disease events. PMID:22781741
Neurotransmitter GABA activates muscle but not ?7 nicotinic receptors.
Dionisio, Leonardo; Berg�, Ignacio; Bravo, Mat�as; Esandi, Mar�a Del Carmen; Bouzat, Cecilia
Cys-loop receptors are neurotransmitter-activated ion channels involved in synaptic and extrasynaptic transmission in the brain and are also present in non-neuronal cells. As GABAA and nicotinic receptors (nAChR) belong to this family, we explored by macroscopic and single-channel recordings whether the inhibitory neurotransmitter GABA has the ability to activate excitatory nAChRs. GABA differentially activates nAChR subtypes. It activates muscle nAChRs, with maximal peak currents of about 10% of those elicited by acetylcholine (ACh) and 15-fold higher EC50 with respect to ACh. At the single-channel level, the weak agonism is revealed by the requirement of 20-fold higher concentration of GABA for detectable channel openings, a major population of brief openings, and absence of clusters of openings when compared with ACh. Mutations at key residues of the principal binding-site face of muscle nAChRs (?Y190 and ?G153) affect GABA activation similarly as ACh activation, whereas a mutation at the complementary face (?G57) shows a selective effect for GABA. Studies with subunit-lacking receptors show that GABA can activate muscle nAChRs through the ?/? interface. Interestingly, single-channel activity elicited by GABA is similar to that elicited by ACh in gain-of-function nAChR mutants associated to congenital myasthenic syndromes, which could be important in the progression of the disorders due to steady exposure to serum GABA. In contrast, GABA cannot elicit single-channel or macroscopic currents of ?7 or the chimeric ?7-serotonin-type 3 receptor, a feature important for preserving an adequate excitatory/inhibitory balance in the brain as well as for avoiding activation of non-neuronal receptors by serum GABA. PMID:25492812
Activation mechanism of solubilized epidermal growth factor receptor tyrosine kinase.
Ge, Gaoxiang; Wu, Jing; Wang, Yan; Lin, Qishui
Dimerization of epidermal growth factor receptor (EGFR) leads to the activation of its tyrosine kinase. To elucidate whether dimerization is responsible for activation of the intracellular tyrosine kinase domain or just plays a role in the stabilization of the active form, the activated status of wild-type EGFR moiety in the heterodimer with kinase activity-deficient mutant receptors was investigated. The kinase activity of the wild-type EGFR was partially activated by EGF in the heterodimer with intracellular domain deletion (sEGFR) or ATP binding-deficient mutant (K721A) EGFRs, while the wild-type EGFR in the heterodimer of wild-type and phosphate transfer activity-deficient mutant receptor D813N could be fully activated. After treatment with EGF, the ATP binding affinity and the V(max) of the wild-type EGFR increased. In the presence of sEGFR, a similar increase in the affinity for ATP was observed, but V(max) did not change. A two-step activation mechanism for EGFR was proposed: upon binding of EGF, the affinity for ATP increased and then, as a result of interaction between the neighboring tyrosine kinase domain, V(max) increased. PMID:11798160
TGF-?1 activates two distinct type I receptors in neurons
K�nig, Hans-Georg; K�gel, Donat; Rami, Abdelhaq; Prehn, Jochen H.M.
Transforming growth factor-?s (TGF-?s) are pleiotropic cytokines involved in development and maintenance of the nervous system. In several neural lesion paradigms, TGF-?1 exerts potent neuroprotective effects. Neurons treated with TGF-?1 activated the canonical TGF-? receptor I/activin-like kinase receptor 5 (ALK5) pathway. The transcription factor nuclear factor-?B (NF-?B) plays a fundamental role in neuroprotection. Treatment with TGF-?1 enhanced NF-?B activity in gelshift and reporter gene analyses. However, ectopic expression of a constitutively active ALK5 failed to mimic these effects. ALK1 has been described as an alternative TGF-? receptor in endothelial cells. Interestingly, we detected significant basal expression of ALK1 and its injury-induced up-regulation in neurons. Treatment with TGF-?1 also induced a pronounced increase in downstream Smad1 phosphorylation. Overexpression of a constitutively active ALK1 mimicked the effect of TGF-?1 on NF-?B activation and neuroprotection. Our data suggest that TGF-?1 simultaneously activates two distinct receptor pathways in neurons and that the ALK1 pathway mediates TGF-?1�induced NF-?B survival signaling. PMID:15781474
Molecular Kinetics of Nerve Growth Factor Receptor Trafficking and Activation*
Jullien, J�r�me; Guili, Vincent; Reichardt, Louis F.; Rudkin, Brian B.
A growing body of evidence indicates a close relationship between tyrosine kinase receptor trafficking and signaling. Biochemical and molecular analyses of the expression, fate, and kinetics of membrane trafficking of the nerve growth factor (NGF) receptor TrkA were performed in PC12 cells. Pulse-chase experiments indicate that TrkA is synthesized as a 110-kDa N-glycosylated precursor that leads to the mature 140-kDa form of the receptor with a half-life of conversion of ?24 � 0.5 min. Neuraminidase digestion shows that modification of the carbohydrate moiety of the receptor by sialylation occurs during maturation. The 140-kDa form is rapidly translocated to the cell surface as assessed by cell surface biotinylation performed on intact PC12 cells. Mature receptor half-life is ?138 � 4 min and is shortened to 86 � 8 min by NGF treatment. Flow cytometric analysis indicates that NGF induces clearing of this receptor from the cell surface within minutes of treatment. The addition of NGF decreases the half-life of cell surface gp140TrkA from 100 to 35 min and leads to enhanced lysosomal degradation of the receptor. The process of NGF-induced TrkA internalization is clearly affected by interfering with ligand binding to p75NTR. An analysis of receptor activation kinetics also shows that receptor signaling primarily takes place from an intracellular location. Together, these data show that the primary effect of NGF treatment is a p75NTR-modulated decrease in TrkA transit time at the cell surface. PMID:12055187
Influence of Peroxisome Proliferator-activated Receptor aaa on Ubiquinone Biosynthesis
Omiecinski, Curtis
of catalase activity with aminotriazole, the amount of ubiquinone was not increased, suggestingInfluence of Peroxisome Proliferator-activated Receptor aaa on Ubiquinone Biosynthesis Mikael proliferators was investigated using peroxisome proliferator activated receptor a (PPARa)- null mice
Qualitative alterations in laminin expression in experimental lupus nephritis.
Kootstra, C. J.; Bergijk, E. C.; Veninga, A.; Prins, F. A.; de Heer, E.; Abrahamson, D. R.; Bruijn, J. A.
Previous studies have revealed quantitative alterations in laminin-1 expression at the mRNA and protein levels during the development of glomerulonephritis and glomerulosclerosis in chronic graft-versus-host disease in mice, a model for lupus nephritis. We have now studied the qualitative alterations in laminin expression with two monoclonal antibodies that recognize epitopes on either the E8 or the P1 fragment of laminin-1. Both of these fragments are involved in cell-matrix and matrix-matrix interactions. In normal glomeruli these laminin epitopes are present only in the mesangial matrix; during embryogenesis, however, they are also present in the glomerular basement membrane. The distribution of laminin epitopes was first studied by using immunofluorescence in kidneys of mice with graft-versus-host disease at different points in time after disease induction. Reflection contrast and immunoelectron microscopy were performed after in vivo injection of the horseradish peroxidase-coupled monoclonal antibodies. In glomeruli of mice 8 weeks after disease induction, both injected antibodies bound specifically in electron-dense immune deposits in the mesangium and subepithelially along the glomerular basement membrane as well as in the expanded mesangial matrix. At 11 and 12 weeks after disease induction, when focal and segmental glomerulosclerosis had developed, the antibodies additionally bound in the matrix subendothelially along the glomerular basement membrane and at the periphery of end-stage sclerotic lesions. To study changes in the distribution of laminin epitopes over time, mice were injected with either monoclonal antibody before induction of graft-versus-host disease. The antibodies were detected 8 and 12 weeks later in the mesangial matrix of mice with lupus nephritis. Once segmental glomerulosclerosis had developed, the antibodies were additionally detected within the thickened glomerular capillary wall. The specific binding of anti-laminin monoclonal antibodies in electron-dense immune deposits further substantiates the hypothesis that anti-laminin autoantibodies participate in glomerular immune complex formation in this model, as suggested by earlier studies. Furthermore, our results show that the distribution of glomerular laminin epitopes in the matrix is altered during the development of glomerular disease. These changes in the structure of the glomerular basement membrane may contribute to the abnormal cell-matrix and matrix-matrix interactions during the development of glomerular disease in this model for lupus nephritis. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 Figure 8 PMID:7543736
Laminin Functionalized Biomimetic Nanofibers For Nerve Tissue Engineering
Junka, Radoslaw; Valmikinathan, Chandra M; Kalyon, Dilhan M; Yu, Xiaojun
Large-gap peripheral nerve injuries present a significant challenge for nerve regeneration due to lack of suitable grafts, insufficient cell penetration, and repair. Biomimetic nanofibrous scaffolds, functionalized on the surface with extracellular matrix proteins, can lead to novel therapies for repair and regeneration of damaged peripheral nerves. Here, nanofibrous scaffolds electrospun from blends of poly(caprolactone) (PCL) and chitosan were fabricated. Taking advantage of the amine groups on the chitosan, the surface of the scaffolds were functionalized with laminin by carbodiimide based crosslinking. Crosslinking allowed laminin to be attached to the surfaces of the PCL-chitosan nanofibers at relatively high concentrations that were not possible using conventional adsorption methods. The nanofibrous meshes were tested for wettability, mechanical properties and cell attachment and proliferation. Blending of chitosan with PCL provided more favorable surfaces for attachment of Schwann cells due to the reduction of the contact angle in comparison to neat PCL. Proliferation rates of Schwann cells grown on PCL-chitosan scaffolds with crosslinked laminin were significantly higher than the rates for PCL-chitosan nanofibrous matrices with adsorbed laminin. PCL-chitosan scaffolds with modified surfaces via crosslinking of laminin could potentially serves as versatile substrates with excellent mechanical and surface properties for in vivo cell delivery for nerve tissue engineering applications. PMID:24083073
Expression of laminin beta1 in hippocampi of patients with intractable epilepsy.
Wu, Yuan; Wang, Xue-feng; Mo, Xue-an; Sun, Hong-bin; Li, Jin-mei; Zeng, Yan; Lin, Tao; Yuan, Jie; Xi, Zhi-qin; Zhu, Xi; Zheng, Jin-ou
We investigated laminin beta1 expression in the hippocampi of patients with intractable epilepsy and explored the role of laminin beta1 in the pathogenesis of this condition. Fluorescence quantitative PCR, immunofluorescence, immunohistochemistry and Western blotting were used to measure laminin beta1 expression in surgically removed hippocampi of patients with intractable epilepsy, and the results were compared with control hippocampi. Fluorescence quantitative PCR showed increased expression of laminin beta1 mRNA in patient hippocampi compared with control tissues. Immunohistochemical staining demonstrated that laminin beta1 protein expression was significantly increased in patient hippocampi, and immunofluorescence microscopy showed accumulation of laminin beta1 in the cell membrane and cytoplasm of patient hippocampi. These findings were confirmed by Western blotting of protein preparations from patient hippocampi. Elevated expression of laminin beta1 mRNA and protein in the hippocampus suggests that laminin beta1 may play a role in the development of epileptic seizures in patients with intractable epilepsy. PMID:18691630
Mechanisms of Activation of Receptor Tyrosine Kinases: Monomers or Dimers
Maruyama, Ichiro N.
Receptor tyrosine kinases (RTKs) play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR) family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights. PMID:24758840
Studies on an insulin-stimulated insulin receptor serine kinase activity: separation of the kinase activity from the insulin receptor and its reconstitution back to the insulin receptor.
Asamoah, K A; Atkinson, P G; Carter, W G; Sale, G J
In cells insulin stimulates autophosphorylation of the insulin receptor on tyrosine and its phosphorylation on serine and threonine by poorly characterized kinases. Recently we have achieved co-purification of the insulin receptor with insulin-stimulated insulin receptor serine kinase activity. We now show that the co-purified serine kinase activity can be removed by NaCl washing and reconstituted by adding back the NaCl eluate. Reconstitution enabled higher serine phosphorylation than achieved with the co-purified preparation. Myelin basic protein was discovered to be a potent substrate for insulin-stimulated serine phosphorylation by the co-purified preparation, with the activity responsible having similar properties to the serine kinase activity towards the receptor. Myelin basic protein was also phosphorylated on serine by the NaCl eluate. Myelin basic protein phosphorylated by the co-purified preparation or the NaCl eluate gave the same set of phosphoserine peptides. The major myelin basic protein serine kinase activity in the NaCl eluate co-purified exactly on Mono Q with the activity that restored insulin-stimulated insulin receptor serine phosphorylation. These results provide strong evidence for the true separation of the serine kinase from the insulin receptor and the distinctiveness of the serine kinase activity from the insulin receptor tyrosine kinase and mitogen-activated protein kinases. The procedures developed for the isolation of the serine kinase and the establishment of an effective in vitro substrate should allow purification of the kinase. The protocols also provide flexible systems for identifying the functions of the insulin-stimulated serine phosphorylations and the respective kinase(s). PMID:8948451
Differential effects of quercetin glycosides on GABAC receptor channel activity.
Kim, Hyeon-Joong; Lee, Byung-Hwan; Choi, Sun-Hye; Jung, Seok-Won; Kim, Hyun-Sook; Lee, Joon-Hee; Hwang, Sung-Hee; Pyo, Mi-Kyung; Kim, Hyoung-Chun; Nah, Seung-Yeol
Quercetin, a representative flavonoid, is a compound of low molecular weight found in various colored plants and vegetables. Quercetin shows a wide range of neuropharmacological activities. In fact, quercetin naturally exists as monomer-(quercetin-3-O-rhamnoside) (Rham1), dimer-(Rutin), or trimer-glycosides [quercetin-3-(2(G)-rhamnosylrutinoside)] (Rham2) at carbon-3 in fruits and vegetables. The carbohydrate components are removed after ingestion into gastrointestinal systems. The role of the glycosides attached to quercetin in the regulation of ?-aminobutyric acid class C (GABAC) receptor channel activity has not been determined. In the present study, we examined the effects of quercetin glycosides on GABAC receptor channel activity by expressing human GABAC alone in Xenopus oocytes using a two-electrode voltage clamp technique and also compared the effects of quercetin glycosides with quercetin. We found that GABA-induced inward current (I GABA ) was inhibited by quercetin or quercetin glycosides. The inhibitory effects of quercetin and its glycosides on I GABA were concentration-dependent and reversible in the order of Rutin�?�quercetin�?�Rham 1�>�Rham 2. The inhibitory effects of quercetin and its glycosides on I GABA were noncompetitive and membrane voltage-insensitive. These results indicate that quercetin and its glycosides regulate GABAC receptor channel activity through interaction with a different site from that of GABA, and that the number of carbohydrate attached to quercetin might play an important role in the regulation of GABAC receptor channel activity. PMID:24895146
Androgen Deprivation by Activating the Liver X Receptor
Hoon Lee, Jung; Gong, Haibiao; Khadem, Shaheen; Lu, Yi; Gao, Xiang; Li, Song; Zhang, Jian; Xie, Wen
Prostate cancer is the most commonly diagnosed and the second leading cause of cancer death in men. The androgens-androgen receptor signaling plays an important role in normal prostate development, as well as in prostatic diseases, such as benign hyperplasia and prostate cancer. Accordingly, androgen ablation has been the most effective endocrine therapy for hormone-dependent prostate cancer. Here, we report a novel nuclear receptor-mediated mechanism of androgen deprivation. Genetic or pharmacological activation of the liver X receptor (LXR) in vivo lowered androgenic activity by inducing the hydroxysteroid sulfotransferase 2A1, an enzyme essential for the metabolic deactivation of androgens. Activation of LXR also inhibited the expression of steroid sulfatase in the prostate, which may have helped to prevent the local conversion of sulfonated androgens back to active metabolites. Interestingly, LXR also induced the expression of selected testicular androgen synthesizing enzymes. At the physiological level, activation of LXR in mice inhibited androgen-dependent prostate regeneration in castrated mice. Treatment with LXR agonists inhibited androgen-dependent proliferation of prostate cancer cells in a LXR- and sulfotransferase 2A1-dependent manner. In summary, we have revealed a novel function of LXR in androgen homeostasis, an endocrine role distinct to the previously known sterol sensor function of this receptor. LXR may represent a novel therapeutic target for androgen deprivation, and may aid in the treatment and prevention of hormone-dependent prostate cancer. PMID:18450964
Glucocorticoid receptor inactivation and activation by phosphorylation mechanisms.
Pratt, W B; Sando, J J; Nielsen, C J
The specific glucocorticoid binding capacity of cytosol preparations is rapidly lost on incubation at 25 degrees in the absence of ligand. We have examined this process in cell-free preparations from rat thymus, rat liver and mouse fibroblasts (L 929 cells), and we have found that the unoccupied receptor is inactivated by endogenous enzymes to a form that does not bind steroids. The inactivation can be prevented by inhibitors of phosphatase action such as molybdate, fluoride and glucose-1-phosphate. On the basis of this type of evidence we propose that the receptor activity of cytosol can be rendered inactive by a dephosphorylation process. We have now been able to partially reactivate the receptor in both L cell and rat thymus cytosols in an ATP dependent manner. If fibroblast (L cell) cytosol is preincubated to permit receptor inactivation by endogenous enzyme, further inactivation can be prevented by the addition of 10 mM molybdate and reactivation of the binding capacity can be obtained by adding 5 to 10 mM ATP in addition to molybdate. ATP dependent activation is prevented with EDTA and this block is overcome by added magnesium. ADP, CTP, GTP, and UTP are inactive. After inactivating the glucocorticoid binding capacity of rat thymocyte cytosol by incubation for 45 minutes at 25 degrees, considerable reactivation is obtained by addition of dithiothreitol and ATP. This system does not absolutely require the presence of a phosphatase inhibitor in order to show activation. Thymocyte cytosol can also be activated to a steroid binding state by addition of DTT and heat-treated (90 degrees for 15 min.) cytosol from a variety of cell types. The heat-treated cytosol contains ATP, reducing equivalents, and a relatively small molecular weight heat-stable activator(s) that potentiates the reactivation process. Maximum receptor activation is obtained by adding dithiothreitol, heat-stable factor, ATP, and molybdate to the inactivated thymocyte cytosol. PMID:224677
Memory retrieval requires ongoing protein synthesis and NMDA receptor activity-mediated AMPA receptor trafficking.
Lopez, Jo�lle; Gamache, Karine; Schneider, Rilla; Nader, Karim
Whereas consolidation and reconsolidation are considered dynamic processes requiring protein synthesis, memory retrieval has long been considered a passive readout of previously established plasticity. However, previous findings suggest that memory retrieval may be more dynamic than previously thought. This study therefore aimed at investigating the molecular mechanisms underlying memory retrieval in the rat. Infusion of protein synthesis inhibitors (rapamycin or anisomycin) in the amygdala 10 min before memory retrieval transiently impaired auditory fear memory expression, suggesting ongoing protein synthesis is required to enable memory retrieval. We then investigated the role of protein synthesis in NMDA receptor activity-mediated AMPA receptor trafficking. Coinfusion of an NMDA receptor antagonist (ifenprodil) or infusion of an AMPA receptor endocytosis inhibitor (GluA23Y) before rapamycin prevented this memory impairment. Furthermore, rapamycin transiently decreased GluA1 levels at the postsynaptic density (PSD), but did not affect extrasynaptic sites. This effect at the PSD was prevented by an infusion of GluA23Y before rapamycin. Together, these data show that ongoing protein synthesis is required before memory retrieval is engaged, and suggest that this protein synthesis may be involved in the NMDAR activity-mediated trafficking of AMPA receptors that takes place during memory retrieval. PMID:25673841
The role of LamininB2 ( LanB2 ) during mesoderm differentiation in Drosophila
Georg Wolfstetter; Anne Holz
In Drosophila, four genes encode for laminin subunits and the formation of two laminin heterotrimers has been postulated. We report the\\u000a identification of mutations in the Drosophila LamininB2 (LanB2) gene that encodes for the only laminin ? subunit and is found in both heterotrimers. We describe their effects on embryogenesis,\\u000a in particular the differentiation of visceral tissues with respect to
Cinnamaldehyde suppresses Toll-like receptor 4 activation mediated through the inhibition of receptor oligomerization
Technology Transfer Automated Retrieval System (TEKTRAN)
Toll-like receptors (TLRs) play a critical role in induction of innate immune and inflammatory responses by recognizing invading pathogens. TLRs have two major downstream signaling pathways, MyD88- and TRIF-dependent pathways leading to the activation of NF'B and IRF3 and the expression of inflammat...
Liver X Receptor and Peroxisome Proliferator-Activated Receptor Agonist from Cornus alternifolia
He, Yang-Qing; Ma, Guo-Yi; Peng, Jiang-nan; Ma, Zhan-Ying; Hamann, Mark T.
Background Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptors superfamily and are transcription factors activated by specific ligands. Liver X receptors (LXR) belong to the nuclear hormone receptors and have been shown to play an important role in cholesterol homeostasis. From the previous screening of several medicinal plants for potential partial PPAR? agonists, the extracts of Cornus alternifolia were found to exhibit promising bioactivity. In this paper, we report the isolation and structural elucidation of four new compounds and their potential as ligands for PPAR. Methods The new compounds were extracted from the leaves of Cornus alternifolia and fractionated by high-performance liquid chromatography. Their structures were elucidated on the basis of spectroscopic evidence and analysis of their hydrolysis products. Results Three new iridoid glycosides including an iridolactone, alternosides A-C (1�3), a new megastigmane glycoside, cornalternoside (4) and 10 known compounds, were obtained from the leaves of Cornus alternifolia. Kaempferol-3-O-?-glucopyranoside (5) exhibited potent agonistic activities for PPAR?, PPAR? and LXR with EC50 values of 0.62, 3.0 and 1.8 ? M, respectively. Conclusions We isolated four new and ten known compounds from Cornus alternifolia, and one known compound showed agonistic activities for PPAR?, PPAR? and LXR. General significance Compound 1 is the first example of a naturally occurring iridoid glycoside containing a ?-glucopyranoside moiety at C-6. PMID:22353334
Protease-activated receptor-1 antagonists: focus on SCH 530348.
Sugunaraj, Jaya Prakash; Mehta, Vimal; Kalra, Ankur; Sukhija, Rishi; Palaniswamy, Chandrasekar
Currently available antiplatelet agents have shown improved short- and long-term clinical outcomes but are associated with increased bleeding risk, and the rates of recurrent ischemic events still remain high. Selective inhibition of protease-activated receptor-1 for thrombin represents a potential novel strategy to reduce ischemic events without increasing the risk of bleeding. Two protease-activated receptor-1 antagonists are currently being evaluated in clinical trials: SCH 530348 and E5555. Results of phase II trials have shown that SCH 530348, when added to standard antiplatelet therapy, was well tolerated and not associated with increased bleeding risk. Two large-scale phase III trials assessing the efficacy of SCH 530348 in addition to the standard of care are currently ongoing. This review provides an outline of the current status of understanding on platelet thrombin-receptor antagonist SCH 530348, focusing on its pharmacologic properties and clinical development. PMID:21248619
Structural basis for selective activation of ABA receptors
Peterson, Francis C.; Burgie, E. Sethe; Park, Sang-Youl; Jensen, Davin R.; Weiner, Joshua J.; Bingman, Craig A.; Chang, Chia-En A.; Cutler, Sean R.; Phillips, Jr., George N.; Volkman, Brian F. (MCW); (UW); (UCR)
Changing environmental conditions and lessening fresh water supplies have sparked intense interest in understanding and manipulating abscisic acid (ABA) signaling, which controls adaptive responses to drought and other abiotic stressors. We recently discovered a selective ABA agonist, pyrabactin, and used it to discover its primary target PYR1, the founding member of the PYR/PYL family of soluble ABA receptors. To understand pyrabactin's selectivity, we have taken a combined structural, chemical and genetic approach. We show that subtle differences between receptor binding pockets control ligand orientation between productive and nonproductive modes. Nonproductive binding occurs without gate closure and prevents receptor activation. Observations in solution show that these orientations are in rapid equilibrium that can be shifted by mutations to control maximal agonist activity. Our results provide a robust framework for the design of new agonists and reveal a new mechanism for agonist selectivity.
Cofactoring and Dimerization of Proteinase-Activated Receptors
Lin, Huilan; Liu, Allen P.; Smith, Thomas H.
Proteinase-activated receptors (PARs) are G protein�coupled receptors that transmit cellular responses to extracellular proteases and have important functions in vascular physiology, development, inflammation, and cancer progression. The established paradigm for PAR activation involves proteolytic cleavage of the extracellular N terminus, which reveals a new N terminus that functions as a tethered ligand by binding intramolecularly to the receptor to trigger transmembrane signaling. Most cells express more than one PAR, which can influence the mode of PAR activation and signaling. Clear examples include murine PAR3 cofactoring of PAR4 and transactivation of PAR2 by PAR1. Thrombin binds to and cleaves murine PAR3, which facilitates PAR4 cleavage and activation. This process is essential for thrombin signaling and platelet activation, since murine PAR3 cannot signal alone. Although PAR1 and PAR4 are both competent to signal, PAR1 is able to act as a cofactor for PAR4, facilitating more rapid cleavage and activation by thrombin. PAR1 can also facilitate PAR2 activation through a different mechanism. Cleavage of the PAR1 N terminus by thrombin generates a tethered ligand domain that can bind intermolecularly to PAR2 to activate signaling. Thus, PARs can regulate each other�s activity by localizing thrombin when in complex with PAR3 and PAR4 or by cleaved PAR1, providing its tethered ligand domain for PAR2 activation. The ability of PARs to cofactor or transactivate other PARs would necessitate that the two receptors be in close proximity, likely in the form of a heterodimer. Here, we discuss the cofactoring and dimerization of PARs and the functional consequences on signaling. PMID:24064459
Endocannabinoid tone versus constitutive activity of cannabinoid receptors
Howlett, Allyn C; Reggio, Patricia H; Childers, Steven R; Hampson, Robert E; Ulloa, Nadine M; Deutsch, Dale G
This review evaluates the cellular mechanisms of constitutive activity of the cannabinoid (CB) receptors, its reversal by inverse agonists, and discusses the pitfalls and problems in the interpretation of the research data. The notion is presented that endogenously produced anandamide (AEA) and 2-arachidonoylglycerol (2-AG) serve as autocrine or paracrine stimulators of the CB receptors, giving the appearance of constitutive activity. It is proposed that one cannot interpret inverse agonist studies without inference to the receptors' environment vis-�-vis the endocannabinoid agonists which themselves are highly lipophilic compounds with a preference for membranes. The endocannabinoid tone is governed by a combination of synthetic pathways and inactivation involving transport and degradation. The synthesis and degradation of 2-AG is well characterized, and 2-AG has been strongly implicated in retrograde signalling in neurons. Data implicating endocannabinoids in paracrine regulation have been described. Endocannabinoid ligands can traverse the cell's interior and potentially be stored on fatty acid-binding proteins (FABPs). Molecular modelling predicts that the endocannabinoids derived from membrane phospholipids can laterally diffuse to enter the CB receptor from the lipid bilayer. Considering that endocannabinoid signalling to CB receptors is a much more likely scenario than is receptor activation in the absence of agonist ligands, researchers are advised to refrain from assuming constitutive activity except for experimental models known to be devoid of endocannabinoid ligands. LINKED ARTICLES This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7 PMID:21545414
A homogenous assay to monitor the activity of the insulin receptor using Bioluminescence Resonance Energy Transfer
Tarik Issad; Nicolas Boute; Karine Pernet
Insulin exerts its biological effects through a plasma membrane receptor that possesses a tyrosine kinase activity. Binding of insulin to its receptor induces a conformational change that stimulates the autophosphorylation of the receptor on tyrosine residues. This autophosphorylation stimulates the tyrosine kinase activity of the receptor toward intracellular substrates involved in the transmission of the signal. The discovery of pharmacological
Paris-Sud XI, Université de
1 LIVER X RECEPTOR (LXR) ACTIVATION NEGATIVELY REGULATES VISFATIN EXPRESSION IN MACROPHAGES Thérèse Communications 2011;404(1):458-62" DOI : 10.1016/j.bbrc.2010.12.002 #12;2 ABSTRACT Adipose tissue macrophages. Liver X Receptor (LXR) and Peroxisome Proliferator-Activated Receptor (PPAR) are nuclear receptors
Rat Receptor-Activity-Modifying Proteins (RAMPs) for Adrenomedullin\\/CGRP Receptor: Cloning and Upregulation in Obstructive Nephropathy
Tetsuya Nagae; Masashi Mukoyama; Akira Sugawara; Kiyoshi Mori; Kensei Yahata; Masato Kasahara; Takayoshi Suganami; Hisashi Makino; Yuriko Fujinaga; Tetsuro Yoshioka; Issei Tanaka; Kazuwa Nakao
Adrenomedullin (AM) is a potent vasorelaxing peptide originally isolated pheochromocytoma. Recently, a family of receptor-activity-modifying proteins (RAMPs 1�3) were identified in humans. Associated with the calcitonin receptor-like receptor (CRLR), RAMP2 or RAMP3 may function as the AM receptor. Here we cloned rat RAMP family, analyzed their distribution in rat tissues, and examined regulation of their expression in the kidney using
Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-?
Robert T. Nolte; G. Bruce Wisely; Stefan Westin; Jeffery E. Cobb; Millard H. Lambert; Riki Kurokawa; Michael G. Rosenfeldk; Timothy M. Willson; Christopher K. Glass; Michael V. Milburn
The peroxisome proliferator-activated receptor-? (PPAR-?) is a ligand-dependent transcription factor that is important in adipocyte differentiation and glucose homeostasis and which depends on interactions with co-activators, including steroid receptor co-activating factor-1 (SRC-1). Here we present the X-ray crystal structure of the human apo-PPAR-? ligand-binding domain (LBD), at 2.2 ? resolution; this structure reveals a large binding pocket, which may explain
Laminin-111 improves muscle repair in a mouse model of merosin-deficient congenital muscular dystrophy
Van Ry, Pam M.; Minogue, Priscilla; Hodges, Bradley L.; Burkin, Dean J.
Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a severe and fatal muscle-wasting disease with no cure. MDC1A patients and the dyW?/? mouse model exhibit severe muscle weakness, demyelinating neuropathy, failed muscle regeneration and premature death. We have recently shown that laminin-111, a form of laminin found in embryonic skeletal muscle, can substitute for the loss of laminin-211/221 and prevent muscle disease progression in the dyW?/? mouse model. What is unclear from these studies is whether laminin-111 can restore failed regeneration to laminin-?2-deficient muscle. To investigate the potential of laminin-111 protein therapy to improve muscle regeneration, laminin-111 or phosphate-buffered saline-treated laminin-?2-deficient muscle was damaged with cardiotoxin and muscle regeneration quantified. Our results show laminin-111 treatment promoted an increase in myofiber size and number, and an increased expression of ?7?1 integrin, Pax7, myogenin and embryonic myosin heavy chain, indicating a restoration of the muscle regenerative program. Together, our results show laminin-111 restores muscle regeneration to laminin-?2-deficient muscle and further supports laminin-111 protein as a therapy for the treatment of MDC1A. PMID:24009313
Induction of a Basement Membrane Glycoprotein in Embryonic Kidney: Possible Role of Laminin in Morphogenesis
Peter Ekblom; Kari Alitalo; Antti Vaheri; Rupert Timpl; Lauri Saxen
The glycoprotein laminin is found exclusively in the basement membranes of adult tissues, not in the mesenchymal stroma. We studied the appearance and distribution of laminin during the early formation of kidney tubules in mouse embryos and in an in vitro transfilter model system. In immunofluorescence using affinity-purified antibodies, the distribution of laminin showed a clear correlation, both spatially and
Platelet-activating factor (PAF) receptor and genetically engineered PAF receptor mutant mice
Satoshi Ishii; Takao Shimizu
Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid mediator. Although PAF was initially recognized for its potential to induce platelet aggregation and secretion, intense investigations have elucidated potent biological actions of PAF in a broad range of cell types and tissues, many of which also produce the molecule. PAF acts by binding to a unique G-protein-coupled seven transmembrane receptor.
Extrasynaptic targeting of NMDA receptors following D1 dopamine receptor activation and cocaine self-administration
Ortinski, Pavel I.; Turner, Jill R.; Pierce, R. Christopher
We previously showed that after repeated exposure to cocaine, D1-like dopamine receptor (D1DR) stimulation reverses plastic changes of AMPA receptor-mediated signaling in the nucleus accumbens shell. However, there is little information on the impact of cocaine self-administration on D1-NMDA receptor interactions in this brain region. Here, we assessed whether cocaine self-administration alters the effects of D1DR stimulation on synaptic and extrasynaptic NMDA receptors (NMDARs) using whole-cell patch-clamp recordings. In slices from cocaine-na�ve rats, pre-treatment with a D1DR agonist decreased synaptic NMDAR receptor-mediated currents and increased the contribution of extrasynaptic NMDARs. In contrast, neither cocaine self-administration alone nor cocaine experience followed by D1DR stimulation had an effect on synaptic or extrasynaptic NMDAR signaling. Activation of extrasynaptic NMDARs relies on the availability of extracellular glutamate, which is regulated primarily by glutamate transporters. In cocaine-experienced animals, administration of a glutamate re-uptake blocker, DL-threo-?-benzyloxyaspartic acid (TBOA), revealed increased extrasynaptic NMDAR activity and stronger baseline activity of glutamate uptake transporters relative to cocaine-na�ve rats. In cocaine-na�ve rats, the D1DR-mediated increase in extrasynaptic NMDAR signaling was independent of the activity of glutamate re-uptake transporters. Taken together, these results indicate that cocaine experience blunts the influence of D1DRs on synaptic and extrasynaptic NMDAR signaling. Additionally, prior cocaine self-administration limits activation of the extrasynaptic NMDAR pool by increasing glutamate re-uptake. These findings outline a pattern of adaptive interactions between D1DRs and NMDARs in the nucleus accumbens shell and demonstrate up-regulation of extrasynaptic NMDAR signaling as a novel consequence of cocaine self-administration. PMID:23719812
Activation mechanism of the heterodimeric GABA(B) receptor.
Pin, Jean-Philippe; Kniazeff, Julie; Binet, Virginie; Liu, Jianfeng; Maurel, Damien; Galvez, Thierry; Duthey, B�atrice; Havlickova, Michaela; Blahos, Jaroslav; Pr�zeau, Laurent; Rondard, Philippe
The GABA(B) receptor was the first heteromeric G-protein coupled receptor (GPCR) identified. Indeed, both GABA(B1) and GABA(B2) subunits appear necessary to get a functional GABA(B) receptor. Soon after the cloning of both subunits, it was demonstrated that GABA(B2) was required for GABA(B1) to reach the cell surface. However, even a mutated GABA(B1) able to reach the cell surface is not functional alone despite its ability to bind GABA(B) ligands. This clearly demonstrated that GABA(B2) is not only required for the correct trafficking of GABA(B1) but also for the correct functioning of the receptor. In the present review article, we will summarize our actual knowledge of the specific role of each subunit in ligand recognition, intramolecular transduction, G-protein activation and allosteric modulation. We will show that the GABA(B) receptor is an heterodimer (not an hetero-oligomer), that agonists bind in GABA(B1), whereas GABA(B2) controls agonist affinity and is responsible for G-protein coupling. Finally, we will show that the recently identified positive allosteric modulator CGP7930 acts as a direct activator of the heptahelical domain of GABA(B2), being therefore the first GABA(B2) ligand identified so far. PMID:15451400
Adenosine kinase inhibitors attenuate opiate withdrawal via adenosine receptor activation
Gary B. Kaplan; Tara Sharon Coyle
Previous studies have demonstrated a role for adenosine in mediating opiate effects. This study examines the effects of indirect activation of adenosine receptors, via treatment with adenosine kinase inhibitors, on the expression of opiate withdrawal in mice. Mice receive chronic morphine treatment via implantation of subcutaneous morphine pellets (75 mg) for 72 h. Mice then receive parenteral treatment with adenosine
Inhibition of the Androgen Receptor Activity by Coprinus comatus Substances
Nesly Dotan; Solomon P. Wasser; Jamal Mahajna
Prostatic adenocarcinoma is the second leading cause of death from cancer in Western men. The common prostate cancer treatments are effective in the early stages; however, advanced prostate cancer is resilient to most of these treatments. Altered androgen receptor (AR) activity caused by point mutations or signaling mechanisms that regulate AR function has been proposed as a key mechanism in
Activation of ?2-adrenergic receptor stimulates ?-secretase activity and accelerates amyloid plaque formation
Yanxiang Ni; Xiaohui Zhao; Guobin Bao; Lin Zou; Lin Teng; Zhu Wang; Min Song; Jiaxiang Xiong; Yun Bai; Gang Pei
Amyloid plaque is the hallmark and primary cause of Alzheimer disease. Mutations of presenilin-1, the ?-secretase catalytic subunit, can affect amyloid-? (A?) production and Alzheimer disease pathogenesis. However, it is largely unknown whether and how ?-secretase activity and amyloid plaque formation are regulated by environmental factors such as stress, which is mediated by receptors including ?2-adrenergic receptor (?2-AR). Here we
Lysophosphatidylserine analogues differentially activate three LysoPS receptors.
Uwamizu, Akiharu; Inoue, Asuka; Suzuki, Kensuke; Okudaira, Michiyo; Shuto, Akira; Shinjo, Yuji; Ishiguro, Jun; Makide, Kumiko; Ikubo, Masaya; Nakamura, Sho; Jung, Sejin; Sayama, Misa; Otani, Yuko; Ohwada, Tomohiko; Aoki, Junken
Lysophosphatidylserine (1-oleoyl-2 R-lysophosphatidylserine, LysoPS) has been shown to have lipid mediator-like actions such as stimulation of mast cell degranulation and suppression of T lymphocyte proliferation, although the mechanisms of LysoPS actions have been elusive. Recently, three G protein-coupled receptors (LPS1/GPR34, LPS2/P2Y10 and LPS3/GPR174) were found to react specifically with LysoPS, raising the possibility that LysoPS serves as a lipid mediator that exerts its role through these receptors. Previously, we chemically synthesized a number of LysoPS analogues and evaluated them as agonists for mast-cell degranulation. Here, we used a transforming growth factor-? (TGF?) shedding assay to see if these LysoPS analogues activated the three LysoPS receptors. Modification of the serine moiety significantly reduced the ability of the analogues to activate the three LysoPS receptors, whereas modification of other parts resulted in loss of activity in receptor-specific manner. We found that introduction of methyl group to serine moiety (1-oleoyl-lysophosphatidylallothreonine) and removal of sn-2 hydroxyl group (1-oleoyl-2-deoxy-LysoPS) resulted in reduction of reactivity with LPS1 and LPS3, respectively. Accordingly, we synthesized a LysoPS analogue with the two modifications (1-oleoyl-2-deoxy-lysophosphatidylallothreonine) and found it to be an LPS2-selective agonist. These pharmacological tools will definitely help to identify the biological roles of these LysoPS receptors. PMID:25320102
Delta-opioid receptors activate ERK/MAP kinase via integrin-stimulated receptor tyrosine kinases.
Eisinger, Daniela A; Ammer, Hermann
Integrin-mediated cell adherence to extracellular matrix proteins results in stimulation of ERK1/2 activity, a mechanism involving focal adhesion tyrosine kinases (pp125FAK, Pyk-2) and epidermal growth factor receptors (EGFRs). G protein-coupled receptors (GPCRs) may also mediate ERK1/2 activation in an integrin-dependent manner, the underlying signaling mechanism of which still remains unclear. Here we demonstrate that the delta-opioid receptor (DOR), a typical GPCR, stimulates ERK1/2 activity in HEK293 cells via integrin-mediated transactivation of EGFR function. Inhibition of integrin signaling by RGDT peptides, cytochalasin, and by keeping the cells in suspension culture both blocked [D-Ala(2), D-Leu(5)]enkephalin (DADLE)- and etorphine-stimulated ERK1/2 activity. Integrin-dependent ERK1/2 activation does not involve FAK/Pyk-2, because over-expression of the FAK/Pyk-2 inhibitor SOCS-3 failed to attenuate DOR signaling. Exposure of the cells to the EGFR inhibitors AG1478 and BPIQ-I blocked DOR-mediated ERK1/2 activation. Because RGDT peptides also prevented DOR-mediated EGFR activation, the present findings indicate that in HEK293 cells DOR-stimulated ERK1/2 activity is mediated by integrin-stimulated EGFRs. Further studies with the phospholipase C (PLC) inhibitors U73122 and ET-18-OCH(3) revealed that opioid-stimulated integrin activation is sensitive to PLC. In contrast, integrin-mediated transactivation of EGFR function appears to be dependent on PKC-delta, as indicated by studies with rottlerin and siRNA knock-down. A similar ERK1/2 signaling pathway was observed for NG108-15 cells, a neuronal cell line endogenously expressing the DOR. In these cells, the nerve growth factor TrkA receptor replaces the EGFR in connecting DOR-activated integrins to the Ras/Raf/ERK1/2 pathway. Together, these data describe an alternative ERK1/2 signaling pathway in which the DOR transactivates the growth factor receptor associated mitogen-activated protein kinase cascade in an integrin-dependent manner. PMID:18804531
Distinct changes in the laminin composition of basement membranes in human seminiferous tubules during development and degeneration.
Virtanen, I.; Lohi, J.; Tani, T.; Korhonen, M.; Burgeson, R. E.; Lehto, V. P.; Leivo, I.
We studied the distribution of laminin (Ln) chains and their integrin (Int) receptors in normal developing and adult and in atrophied human testes by using immunohistochemistry. Immunostaining for EHS Ln and type IV collagen was used to identify basement membranes (BMs). In the BM of seminiferous epithelium of fetal testis, a panel of monoclonal antibodies showed immunoreactivity for Ln alpha 1-, alpha 2-, beta 1-, beta 2- and gamma 1-chains, suggestive of the presence of Lns 1 to 3. In BM of adult seminiferous epithelium with active spermatogenesis, immunoreactivity for Ln beta 2- and gamma 1-chains was found but not for Ln alpha-chains, suggesting a complex of Ln chains not compatible with any known trimers. Instead, with polyclonal Ln antiserum and monoclonal antibody to type IV collagen, a distinct BM-like reactivity was seen. In atrophied testes, prominent immunoreactivities for Ln chains, compatible with Lns 1 to 3, were seen in the thickened BM of seminiferous tubules, hence suggestive of reappearance of fetal Lns. Among the subunits of Ln-binding Int receptors in fetal seminiferous tubules, a strong immunoreactivity for Int beta 1- and Int alpha 6-subunits was seen throughout the seminiferous epithelium, other Int subunits being found in interstitial cells. In the adult and atrophied testes, immunoreactivities for Int beta 1- and Int alpha 6-subunits were seen to be confined to the basal aspect of the seminiferous epithelium whereas immunoreactivities for Int alpha 1-, alpha 2-, alpha 3- and beta 4-subunits were seen in the myoid cells. The results show that both maturation and degenerative changes of human testes are accompanied by distinct changes in the Ln expression of BM of seminiferous epithelium, which appears to accompany epithelial differentiation of the Sertoli cells. Furthermore, they suggest the presence of a novel Ln trimer in BM of adult human seminiferous tubules. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:9094997
The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A
Sato, Shoko, E-mail: firstname.lastname@example.com [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Shirakawa, Hitoshi, E-mail: email@example.com [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Tomita, Shuhei, E-mail: firstname.lastname@example.com [Division of Molecular Pharmacology, Department of Pathophysiological and Therapeutic Science, Yonago 683-8503 (Japan); Tohkin, Masahiro, E-mail: firstname.lastname@example.com [Department of Medical Safety Science, Graduate School of Pharmaceutical Science, Nagoya City University, Nagoya 267-8603 (Japan); Gonzalez, Frank J., E-mail: email@example.com [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Komai, Michio, E-mail: email@example.com [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan)
Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein�protein interactions with GR. - Highlights: � Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. � Human metallothionein gene is regulated by the AHR and GR interaction. � AHR�GR complex binds to glucocorticoid response element in metallothionein gene. � We demonstrated a novel transcriptional mechanism via AHR and GR interaction.
Activation of axonal receptors by GABA spillover increases somatic firing.
Pugh, Jason R; Jahr, Craig E
Axons can be depolarized by ionotropic receptors and transmit subthreshold depolarizations to the soma by passive electrical spread. This raises the possibility that axons and axonal receptors can participate in integration and firing in neurons. Previously, we have shown that exogenous GABA depolarizes cerebellar granule cell axons through local activation of GABA(A) receptors (GABA(A)Rs) and the soma through electrotonic spread of the axonal potential resulting in increased firing. We show here that excitability of granule cells is also increased by release of endogenous GABA from molecular layer interneurons (MLIs) and spillover activation of parallel fiber GABA(A)Rs in mice and rats. Changes in granule cell excitability were assessed by excitability testing after activation of MLIs with channelrhodopsin or electrical stimulation in the molecular layer. In granule cells lacking an axon, excitability was not changed, suggesting that axonal receptors are required. To determine the distance over which subthreshold potentials may spread, we estimated the effective axonal electrical length constant (520 ?m) by excitability testing and focal uncaging of RuBi-GABA on the axon at varying distances from the soma. These data suggest that GABA(A)R-mediated axonal potentials can participate in integration and firing of cerebellar granule cells. PMID:24155298
Activation of Axonal Receptors by GABA Spillover Increases Somatic Firing
Pugh, Jason R.
Axons can be depolarized by ionotropic receptors and transmit subthreshold depolarizations to the soma by passive electrical spread. This raises the possibility that axons and axonal receptors can participate in integration and firing in neurons. Previously, we have shown that exogenous GABA depolarizes cerebellar granule cell axons through local activation of GABAA receptors (GABAARs) and the soma through electrotonic spread of the axonal potential resulting in increased firing. We show here that excitability of granule cells is also increased by release of endogenous GABA from molecular layer interneurons (MLIs) and spillover activation of parallel fiber GABAARs in mice and rats. Changes in granule cell excitability were assessed by excitability testing after activation of MLIs with channelrhodopsin or electrical stimulation in the molecular layer. In granule cells lacking an axon, excitability was not changed, suggesting that axonal receptors are required. To determine the distance over which subthreshold potentials may spread, we estimated the effective axonal electrical length constant (520 ?m) by excitability testing and focal uncaging of RuBi�GABA on the axon at varying distances from the soma. These data suggest that GABAAR-mediated axonal potentials can participate in integration and firing of cerebellar granule cells. PMID:24155298
Protease-activated-receptor-2 affects protease-activated-receptor-1-driven breast cancer.
Jaber, Mohammad; Maoz, Miriam; Kancharla, Arun; Agranovich, Daniel; Peretz, Tamar; Grisaru-Granovsky, Sorina; Uziely, Beatrice; Bar-Shavit, Rachel
Mammalian protease-activated-receptor-1 and -2 (PAR1 and PAR2) are activated by proteases found in the flexible microenvironment of a tumor and play a central role in breast cancer. We propose in the present study that PAR1 and PAR2 act together as a functional unit during malignant and physiological invasion processes. This notion is supported by assessing pro-tumor functions in the presence of short hairpin; shRNA knocked-down hPar2 or by the use of a truncated PAR2 devoid of the entire cytoplasmic tail. Silencing of hPar2 by shRNA-attenuated thrombin induced PAR1 signaling as recapitulated by inhibiting the assembly of Etk/Bmx or Akt onto PAR1-C-tail, by thrombin-instigated colony formation and invasion. Strikingly, shRNA-hPar2 also inhibited the TFLLRN selective PAR1 pro-tumor functions. In addition, while evaluating the physiological invasion process of placenta extravillous trophoblast (EVT) organ culture, we observed inhibition of both thrombin or the selective PAR1 ligand; TFLLRNPNDK induced EVT invasion by shRNA-hPar2 but not by scrambled shRNA-hPar2. In parallel, when a truncated PAR2 was utilized in a xenograft mouse model, it inhibited PAR1-PAR2-driven tumor growth in vivo. Similarly, it also attenuated the interaction of Etk/Bmx with the PAR1-C-tail in vitro and decreased markedly selective PAR1-induced Matrigel invasion. Confocal images demonstrated co-localization of PAR1 and PAR2 in HEK293T cells over-expressing YFP-hPar2 and HA-hPar1. Co-immuno-precipitation analyses revealed PAR1-PAR2 complex formation but no PAR1-CXCR4 complex was formed. Taken together, our observations show that PAR1 and PAR2 act as a functional unit in tumor development and placenta-uterus interactions. This conclusion may have significant consequences on future breast cancer therapeutic modalities and improved late pregnancy outcome. PMID:24177339
?-Apo-13-carotenone regulates retinoid X receptor transcriptional activity through tetramerization of the receptor.
Sun, Jian; Narayanasamy, Sureshbabu; Curley, Robert W; Harrison, Earl H
Retinoid X receptor (RXR?) is activated by 9-cis-retinoic acid (9cRA) and regulates transcription as a homodimer or as a heterodimer with other nuclear receptors. We have previously demonstrated that ?-apo-13-carotenone, an eccentric cleavage product of ?-carotene, antagonizes the activation of RXR? by 9cRA in mammalian cells overexpressing this receptor. However, the molecular mechanism of ?-apo-13-carotenone's modulation on the transcriptional activity of RXR? is not understood and is the subject of this report. We performed transactivation assays using full-length RXR? and reporter gene constructs (RXRE-Luc) transfected into COS-7 cells, and luciferase activity was examined. ?-Apo-13-carotenone was compared with the RXR? antagonist UVI3003. The results showed that both ?-apo-13-carotenone and UVI3003 shifted the dose-dependent RXR? activation by 9cRA. In contrast, the results of assays using a hybrid Gal4-DBD:RXR?LBD receptor reporter cell assay that detects 9cRA-induced coactivator binding to the ligand binding domain demonstrated that UVI3003 significantly inhibited 9cRA-induced coactivator binding to RXR?LBD, but ?-apo-13-carotenone did not. However, both ?-apo-13-carotenone and UVI3003 inhibited 9-cRA induction of caspase 9 gene expression in the mammary carcinoma cell line MCF-7. To resolve this apparent contradiction, we investigated the effect of ?-apo-13-carotenone on the oligomeric state of purified recombinant RXR?LBD. ?-Apo-13-carotenone induces tetramerization of the RXR?LBD, although UVI3003 had no effect on the oligomeric state. These observations suggest that ?-apo-13-carotenone regulates RXR? transcriptional activity by inducing the formation of the "transcriptionally silent" RXR? tetramer. PMID:25324544
Androgen receptor activation by polychlorinated biphenyls
Casati, Lavinia; Sendra, Ramon; Poletti, Angelo; Negri-Cesi, Paola; Celotti, Fabio
The exposure to environmental endocrine disrupting compounds (EDC), as polychlorinated biphenyls (PCBs), widely diffused in the environment may produce epigenetic changes that affect the endocrine system. We found that PCBs activate AR transcriptional activity and that this effect is potentiated by the demethylase Jarid1b, a histone demethylase that catalyzes the removal of trimethylation of lysine 4 on histone H3 (H3K4me3), induced by PCB. The aim of the present study was to investigate the effect of the treatment of cultured cells (HEK293) with a mixture of the most diffused environmental PCBs and, also with dihydrotestosterone (DHT), on the functional interaction between AR and Jarid1b. Although the effect induced by DHT on the AR transactivation was considerably higher, the PCB mixture produced an AR-mediated transactivation in a dose-dependent manner. Cotransfection with plasmids expressing Jarid1b and various AR isoforms containing polyglutamine tracts (polyQ tracts) of different lengths showed that Jarid1b potentiates the AR transcriptional activity induced by PCBs but only with the shortest AR isoform. The potentiating effect of Jarid1b on the AR is mediated by a direct interaction of the enzyme with the AR promoter. In fact, utilizing constructs containing AR promoters with a different length and a luciferase reporter gene, we showed that the effect of PCBs, but not of DHT, needs the presence of Jarid1b and of at least two DNA binding sites for Jarid1b. PMID:23907094
Human peroxisome proliferator-activated receptor mRNA and protein expression during development
The peroxisome proliferator-activated receptors (PPAR) are nuclear hormone receptors that regulate lipid and glucose homeostasis and are important in reproduction and development. PPARs are targets ofpharmaceuticals and are also activated by environmental contaminants, including ...
Peroxisome proliferator-activated receptor ? and colorectal cancer
Dai, Yun; Wang, Wei-Hong
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily and ligand-activated transcription factors. PPAR? plays an important role in adipocyte differentiation, lipid storage and energy dissipation in adipose tissue, and is involved in the control of inflammatory reactions as well as in glucose metabolism through the improvement of insulin sensitivity. Growing evidence has demonstrated that activation of PPAR? has an antineoplastic effect in tumors, including colorectal cancer. High expression of PPAR? is detected in human colon cancer cell lines and adenocarcinoma. This review describes the molecular mechanisms by which PPAR? regulates tumorigenesis in colorectal cancer, and examines current clinical trials evaluating PPAR? agonists as therapeutic agents for colorectal cancer. PMID:21160824
Protease-activated receptors: novel PARtners in innate immunity.
Shpacovitch, V; Feld, M; Bunnett, N W; Steinhoff, M
Protease-activated receptors (PARs) belong to a family of G protein-coupled receptors activated by serine proteases via proteolytic cleavage. PARs are expressed on epithelial cells, endothelial cells, and leukocytes, indicating a role in controlling barrier function against external danger. During inflammation, microorganisms as well as host immune cells release various proteases activating PARs. Thus, PARs can be viewed as an integral component of the host antimicrobial alarm system. When stimulated, PARs regulate various functions of leukocytes in vivo and in vitro, revealing a novel pathway by which proteases affect innate immune responses. Understanding protease-immune interactions could lead to novel strategies for the treatment of infectious and immune-related diseases. PMID:17977790
Oxytocin induces social communication by activating arginine-vasopressin V1a receptors and not oxytocin receptors.
Song, Zhimin; McCann, Katharine E; McNeill, John K; Larkin, Tony E; Huhman, Kim L; Albers, H Elliott
Arginine-vasopressin (AVP) and oxytocin (OT) and their receptors are very similar in structure. As a result, at least some of the effects of these peptides may be the result of crosstalk between their canonical receptors. The present study investigated this hypothesis by determining whether the induction of flank marking, a form of social communication in Syrian hamsters, by OT is mediated by the OT receptor or the AVP V1a receptor. Intracerebroventricular (ICV) injections of OT or AVP induced flank marking in a dose-dependent manner although the effects of AVP were approximately 100 times greater than those of OT. Injections of highly selective V1a receptor agonists but not OT receptor agonists induced flank marking, and V1a receptor antagonists but not OT receptor antagonists significantly inhibited the ability of OT to induce flank marking. Lastly, injection of alpha-melanocyte-stimulating hormone (?-MSH), a peptide that stimulates OT but not AVP release, significantly increased odor-induced flank marking, and these effects were blocked by a V1a receptor antagonist. These data demonstrate that OT induces flank marking by activating AVP V1a and not OT receptors, suggesting that the V1a receptor should be considered to be an OT receptor as well as an AVP receptor. PMID:25173438
Acute 5-HT7 receptor activation increases NMDA-evoked currents and differentially alters NMDA receptor subunit phosphorylation and trafficking in hippocampal neurons
Background N-methyl-D-aspartate (NMDA) receptors are regulated by several G protein-coupled receptors (GPCRs) as well as receptor tyrosine kinases. Serotonin (5-HT) type 7 receptors are expressed throughout the brain including the thalamus and hippocampus. Long-term (2�24�h) activation of 5-HT7 receptors promotes the expression of neuroprotective growth factor receptors, including the platelet-derived growth factor (PDGF) ? receptors which can protect neurons against NMDA-induced neurotoxicity. Results In contrast to long-term activation of 5-HT7 receptors, acute (5�min) treatment of isolated hippocampal neurons with the 5-HT7 receptor agonist 5-carboxamidotryptamine (5-CT) enhances NMDA-evoked peak currents and this increase in peak currents is blocked by the 5-HT7 receptor antagonist, SB 269970. In hippocampal slices, acute 5-HT7 receptor activation increases NR1 NMDA receptor subunit phosphorylation and differentially alters the phosphorylation state of the NR2B and NR2A subunits. NMDA receptor subunit cell surface expression is also differentially altered by 5-HT7 receptor agonists: NR2B cell surface expression is decreased whereas NR1 and NR2A surface expression are not significantly altered. Conclusions In contrast to the negative regulatory effects of long-term activation of 5-HT7 receptors on NMDA receptor signaling, acute activation of 5-HT7 receptors promotes NMDA receptor activity. These findings highlight the potential for temporally differential regulation of NMDA receptors by the 5-HT7 receptor. PMID:23672716
Laminin isoforms: biological roles and effects on the intracellular distribution of nuclear proteins in intestinal epithelial cells
Natacha Turck; Isabelle Gross; Patrick Gendry; Jeanne Stutzmann; Jean-No�l Freund; Mich�le Kedinger; Patricia Simon-Assmann; Jean-Francois. Launay
Laminins are structurally and functionally major components of the extracellular matrix. Four isoforms of laminins (laminin-1, -2, -5 and -10) are expressed in a specific pattern along the crypt�villus axis of the intestine. Previous works indicated that expression of these isoforms is developmentally regulated and that laminins could modulate the behaviour of intestinal cells, but the exact role of each
CINPA1 Is an Inhibitor of Constitutive Androstane Receptor That Does Not Activate Pregnane X Receptor.
Cherian, Milu T; Lin, Wenwei; Wu, Jing; Chen, Taosheng
Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ?70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 �M; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. PMID:25762023
Mite and Cockroach Allergens Activate Protease-Activated Receptor 2 and Delay Epidermal Permeability Barrier Recovery
Se Kyoo Jeong; Hyun Jeong Kim; Jong-Kyung Youm; Sung Ku Ahn; Eung Ho Choi; Myung Hyun Sohn; Kyu-Earn Kim; Jeong Hee Hong; Dong Min Shin; Seung Hun Lee
Protease-activated receptor-2 (PAR-2) is known to be involved in epidermal permeability barrier function homeostasis. PAR-2 activation occurs after barrier disruption and further activation of PAR-2 by activating peptide significantly delays barrier recovery rate. Cockroach and house dust mite allergens, both known to be associated with the development of asthma, allergic rhinitis, and atopic dermatitis, have protease activity, which can activate
Laminin-111-derived peptide-hyaluronate hydrogels as a synthetic basement membrane.
Yamada, Yuji; Hozumi, Kentaro; Katagiri, Fumihiko; Kikkawa, Yamato; Nomizu, Motoyoshi
We have identified a number of cell-adhesive peptides from laminins, a major component of basement membranes. Cell-adhesive peptides derived from basement membrane proteins are potential candidates for incorporating cell-binding activities into scaffold materials for tissue engineering. Our goal is development of a chemically synthetic basement membrane using laminin-derived cell-adhesive peptides and polymeric materials. In this study, we used hyaluronic acid (HA) as a scaffold material and laminin-derived cell-adhesive peptides, A99 (AGTFALRGDNPQG, binds to integrin ?v?3), AG73 (RKRLQVQLSIRT, binds to syndecans), and an A99/AG73 mixture (molar ratio = 9:1) conjugated to two-dimensional (2D) HA matrices. As a result, it was found that the 2D A99/AG73-HA matrices have strong biological functions, such as cell attachment, cell spreading, and neurite outgrowth, similar to that of basement membrane extract (BME)-coated plates. Next, we developed three-dimensional (3D) peptide-HA matrices using the A99/AG73 mixture. The 3D A99/AG73-HA matrices promoted cell spreading and improved cell viability and collagen gene expression. Further, PC12 neurite extension was observed in the 3D A99/AG73-HA matrices. These biological activities of the 3D A99/AG73-HA matrices were similar to those of the 3D BME matrices. These results suggest that the peptide-HA matrices are useful as 2D and 3D matrices and can be applied for tissue engineering as a synthetic basement membrane. PMID:23764113
An integrative framework of the skin receptors activation: mechanoreceptors activity patterns versus "labeled lines".
Zeveke, Alexander V; Efes, Ekaterina D; Polevaya, Sofia A
The paper presents a review of electrophysiological data which indicate the integrative mechanisms of information coded in the human and animal peripheral skin receptors. The activity of the skin sensory receptors was examined by applying various natural stimuli. It was revealed that numerous identical receptors respond to various stimuli (mechanical, temperature, and pain ones), but the spike patterns of these receptors were found to be specific for each stimulus. The description of characteristic structures of spike patterns in the cutaneous nerve fibers in response to five major modalities, namely: "touch", "pain", "vibration/breath", "cold", and "heat", is being presented. The recordings of the cutaneous physical state revealed a correlation between the patterns of spatiotemporal skin deformation and the receptors activity. A rheological state of the skin can be changed either in response to external temperature variation or by the sympathetic pilomotor activation. These results indicate that the skin sensory receptors activity may be considered as an integrative process. It depends not only on the receptors themselves, but also on the changes in the surrounding tissue and on the adaptive influence of the central nervous system. A new framework for the sensory channel system related to the skin is proposed on the basis of experimental results. PMID:23621456
"Mirror image" antagonists of thrombin-induced platelet activation based on thrombin receptor structure.
Hung, D T; Vu, T K; Wheaton, V I; Charo, I F; Nelken, N A; Esmon, N; Esmon, C T; Coughlin, S R
Platelet activation by thrombin plays a critical role in hemostasis and thrombosis. Based on structure-activity studies of a cloned platelet thrombin receptor, we designed two "mirror image" antagonists of thrombin and thrombin receptor function. First, "uncleavable" peptides mimicking the receptor domain postulated to interact with thrombin were found to be potent thrombin inhibitors. Second, proteolytically inactive mutant thrombins designed to bind but not cleave the thrombin receptor were found to be specific antagonists of receptor activation by thrombin. The effectiveness of these designed antagonists in blocking thrombin-induced platelet activation suggests a model for thrombin-receptor interaction and possible strategies for the development of novel antithrombotic agents. PMID:1310695
A novel disulfide pattern in laminin-type epidermal growth factor-like (LE) modules of laminin ?1 and ?1 chains.
Kalkhof, Stefan; Witte, Konstanze; Ihling, Christian H; M�ller, Mathias Q; Keller, Manuel V; Haehn, Sebastian; Smyth, Neil; Paulsson, Mats; Sinz, Andrea
In-depth mass spectrometric analysis of disulfide bond patterns in recombinant mouse laminin ?1 and ?1 chain N-terminal fragments comprising the laminin N-terminal (LN) domain and the first four laminin epidermal growth factor-like (LE) domains revealed a novel disulfide pattern for LE domains. This showed a (2-3, 4-5, 6-7, 8-1) connectivity with the last cysteine of one LE domain being connected to the first cysteine of the following LE domain. The same pattern was also found in E4, the N-terminal ?1 chain fragment derived by elastase digestion of mouse EHS tumor laminin-111, showing that this pattern occurs in native laminin. The strictly linear pattern with an interdomain disulfide has not been described previously for EGF domains. The N-terminal portions of laminin short arms, consisting of the LN domain and LE domains 1-4, are essential for laminin-laminin self-interactions, whereas the internal LE domains 7-9 in the laminin ?1 chain harbor the nidogen binding site and have a conventional disulfide pattern. This suggests that LE domains differing in function also differ in their disulfide patterns. PMID:20731416
The Laminin 511/521 Binding Site on the Lutheran Blood Group Glycoprotein is Located at theFlexible Junction of Ig Domains 2 and 3
Mankelow, Tosti J.; Burton, Nicholas; Stedansdottir, Fanney O.; Spring, Frances A.; Parsons, Stephen F.; Pesersen, Jan S.; Oliveira, Cristiano L.P.; Lammie, Donna; Wess, Timothy; Mohandas, Narla; Chasis, Joel A.; Brady, R. Leo; Anstee, David J.
The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the {alpha}5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vasocclusive events that are an important cause of morbidity in sickle cell disease. Using X-ray crystallography, small angle X-ray scattering and site directed mutagenesis we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease.
Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators
Isabelle Issemann; Stephen Green
We have cloned a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. The receptor homologue is activated by a diverse class of rodent hepatocarcinogens that causes proliferation of peroxisomes. Identification of a peroxisome proliferator-activated receptor should help elucidate the mechanism of the hypolipidaemic effect of these hepatocarcinogens and aid evaluation of their potential carcinogenic risk to man.
Expression of laminin subunits in congenital muscular dystrophy
C. A. Sewry; J. Philpot; D. Mahony; L. A. Wilson; F. Muntoni; V. Dubowitz
The expression of laminin subunits M, A, B1 and B2 was studied immunocytochemically in 25 cases of classical congenital muscular dystrophy (CMD), 11 hypotonic infants, 20 cases of a variety of inherited and acquired neuromuscular disorders, and 11 controls. Merosin, as indicated by labelling for the M chain, was deficient in 12 (48%) of the cases of classical CMD. Seven
Dopamine-2 receptor activation suppresses PACAP expression in gonadotrophs.
Winters, Stephen J; Ghooray, Dushan T; Yang, Rong Q; Holmes, Joshua B; O'Brien, Andrew Rw; Morgan, Jay; Moore, Joseph P
Pituitary adenylate cyclase-activating polypeptide (PACAP) is expressed at a high level in the fetal pituitary and decreases profoundly between embryonic day 19 and postnatal day 1 (PN1), with a further decrease from PN1 to PN4. In this series of experiments, we investigated the hypothesis that dopamine 2 receptor (Drd2) activation interrupts a cAMP-dependent feed-forward loop that maintains PACAP expression at a high level in the fetal pituitary. Using single-cell RT-PCR of pituitary cell cultures from newborn rats, Drd2 mRNA was identified in gonadotrophs that were also positive for PACAP mRNA. PACAP expression in pituitary cultures from embryonic day 19 rats was suppressed by the PACAP6-38 antagonist and by the Drd2 agonist bromocriptine. Increasing concentrations of bromocriptine inhibited cAMP production as well as cAMP signaling based on cAMP response element-luciferase activity, decreased PACAP promoter activity, and decreased PACAP mRNA levels in ?T3-1 gonadotroph cells. Furthermore, blockade of dopamine receptors by injecting haloperidol into newborn rat pups partially reversed the developmental decline in pituitary PACAP mRNA that occurs between PN1 and PN4. These results provide evidence that dopamine receptor signaling regulates PACAP expression under physiological conditions and lend support to the hypothesis that a rise in hypothalamic dopamine at birth abrogates cAMP signaling in fetal gonadotrophs to interrupt a feed-forward mechanism that maintains PACAP expression at a high level in the fetal pituitary. We propose that this perinatal decline in pituitary PACAP reduces pituitary follistatin which permits GnRH receptors and FSH-? to increase to facilitate activation of the neonatal gonad. PMID:24823390
NMDA receptor activation strengthens weak electrical coupling in mammalian brain.
Turecek, Josef; Yuen, Genevieve S; Han, Victor Z; Zeng, Xiao-Hui; Bayer, K Ulrich; Welsh, John P
Electrical synapses are formed by gap junctions and permit electrical coupling, which shapes the synchrony of neuronal ensembles. Here, we provide a direct demonstration of receptor-mediated strengthening of electrical coupling in mammalian brain. Electrical coupling in the inferior olive of rats was strengthened by activation of NMDA-type glutamate receptors (NMDARs), which were found at synaptic loci and at extrasynaptic loci 20-100�nm proximal to gap junctions. Electrical coupling was strengthened by pharmacological and synaptic activation of NMDARs, whereas costimulation of ionotropic non-NMDAR glutamate receptors transiently antagonized the effect of NMDAR activation. NMDAR-dependent strengthening (1) occurred despite increased input conductance, (2) induced Ca(2+)-influx microdomains near dendritic spines, (3) required activation of the Ca(2+)/calmodulin-dependent protein-kinase II, (4) was restricted to neurons that were weakly coupled, and (5) thus strengthened coupling, mainly between nonadjacent neurons. This provided a mechanism to expand the synchronization of rhythmic membrane potential oscillations by chemical neurotransmitter input. PMID:24656255
Activation of Penile Proadipogenic Peroxisome Proliferator-Activated Receptor ? with an Estrogen: Interaction with Estrogen Receptor Alpha during Postnatal Development
Mansour, Mahmoud M.; Goyal, Hari O.; Braden, Tim D.; Dennis, John C.; Schwartz, Dean D.; Judd, Robert L.; Bartol, Frank F.; Coleman, Elaine S.; Morrison, Edward E.
Exposure to the estrogen receptor alpha (ER?) ligand diethylstilbesterol (DES) between neonatal days 2 to 12 induces penile adipogenesis and adult infertility in rats. The objective of this study was to investigate the in vivo interaction between DES-activated ER? and the proadipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPAR?). Transcripts for PPARs ?, ?, and ? and ?1a splice variant were detected in Sprague-Dawley normal rat penis with PPAR? predominating. In addition, PPAR?1b and PPAR?2 were newly induced by DES. The PPAR? transcripts were significantly upregulated with DES and reduced by antiestrogen ICI 182, 780. At the cellular level, PPAR? protein was detected in urethral transitional epithelium and stromal, endothelial, neuronal, and smooth muscular cells. Treatment with DES activated ER? and induced adipocyte differentiation in corpus cavernosum penis. Those adipocytes exhibited strong nuclear PPAR? expression. These results suggest a biological overlap between PPAR? and ER? and highlight a mechanism for endocrine disruption. PMID:18769493
Brown, Rachel C.; Wu, Ling; Hicks, Kali; O�Neil, Roger G.
Objective To identify plasma membrane ion channels mediating calcium influx at the blood-brain barrier in response to disrupting stimuli. Methods We examined the expression and function of candidate transient receptor potential channels using RT-PCR, Fura-2 calcium imaging and permeability assays. Results Immortalized mouse brain microvessel endothelial cells expressed multiple transient receptor potential isoforms: transient receptor potential C1, C2, C4, and C7, M2, M3, M4 and M7, and V2 and V4. Similar profiles were observed in freshly isolated cerebral microvessels and primary cultured rat brain endothelial cells. Thrombin-stimulated calcium influx in brain endothelial cells was blocked by transient receptor potential C inhibitors. Transient receptor potential V activating stimuli also increased intracellular calcium. This increase was inhibited by a transient receptor potential V blocker or by removal of extracellular calcium. Barrier integrity was compromised by thrombin, hypo-osmolar stress, and PMA treatment. The increase in barrier permeability induced by transient receptor potential V activators was blocked by transient receptor potential V inhibition, while thrombin effects were inhibited by transient receptor potential C inhibitors. Conclusions These results demonstrate that transient receptor potential C and transient receptor potential V channels mediate calcium influx at the blood-brain barrier, and as a consequence, may modulate barrier integrity. PMID:18464164
Associate editor: A.L. Morrow Regulation of GABAA receptor trafficking, channel activity,
Luscher, Bernhard
density; PP, protein phosphatase; RACK, receptor for activated C-kinase; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TGN, trans-Golgi network; TM, transmembrane; VGCC, voltage-gated Ca2, the function of receptor-associated proteins in trafficking of GABAA receptors to and from synapses
NMDA-type glutamate receptors participate in reduction of food intake following hindbrain melanocortin receptor activation.
Campos, Carlos A; Ritter, Robert C
Hindbrain injection of a melanocortin-3/4 receptor agonist, MTII, reduces food intake primarily by reducing meal size. Our previously reported results indicate that N-methyl-D-aspartate-type glutamate receptors (NMDAR) in the nucleus of the solitary tract (NTS) play an important role in the control of meal size and food intake. Therefore, we hypothesized that activation of NTS NMDARs contribute to reduction of food intake in response to fourth ventricle or NTS injection of MTII. We found that coinjection of a competitive NMDAR antagonist (d-CPP-ene) with MTII into the fourth ventricle or directly into the NTS of adult male rats attenuated MTII-induced reduction of food intake. Hindbrain NMDAR antagonism also attenuated MTII-induced ERK1/2 phosphorylation in NTS neurons and prevented synapsin I phosphorylation in central vagal afferent endings, both of which are cellular mechanisms previously shown to participate in hindbrain melanocortinergic reduction of food intake. Together, our results indicate that NMDAR activation significantly contributes to reduction of food intake following hindbrain melanocortin receptor activation, and it participates in melanocortinergic signaling in NTS neural circuits that mediate reduction of food intake. PMID:25394828
Peroxisome Proliferator-Activated Receptors in HBV-Related Infection
Dubuquoy, Laurent; Louvet, Alexandre; Hollebecque, Antoine; Mathurin, Philippe; Dharancy, S�bastien
Thirty years after its discovery, the hepatitis B virus (HBV) still remains a major global public health problem. Worldwide, two billion subjects have been infected, 350 million have a chronic infection and more than 600 000 die annually of HBV-related liver disease or hepatocellular carcinoma; new infections occur because of the presence of a large reservoir of chronic carriers of the virus. Since a decade several studies describe the interrelations between HBV and nuclear receptors and more particularly the peroxisome proliferator-activated receptors (PPARs). After a brief introduction, this review will make a rapid description of HBV incidence and biology. Then a report of the literature on the role of PPARs on viral transcription and replication will be developed. Finally, the role of HBV on PPAR? expression and activity will be discussed. Concluding remarks and perspectives will close this review. PMID:19365584
Peroxisome Proliferator-Activated Receptors and Acute Lung Injury
Paola, Rosanna Di; Cuzzocrea, Salvatore
Peroxisome proliferator-activated receptors are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily. PPARs regulate several metabolic pathways by binding to sequence-specific PPAR response elements in the promoter region of target genes, including lipid biosynthesis and glucose metabolism. Recently, PPARs and their respective ligands have been implicated as regulators of cellular inflammatory and immune responses. These molecules are thought to exert anti-inflammatory effects by negatively regulating the expression of proinflammatory genes. Several studies have demonstrated that PPAR ligands possess anti-inflammatory properties and that these properties may prove helpful in the treatment of inflammatory diseases of the lung. This review will outline the anti-inflammatory effects of PPARs and PPAR ligands and discuss their potential therapeutic effects in animal models of inflammatory lung disease. PMID:17710233
Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo
Kono, Mari; Tucker, Ana E.; Tran, Jennifer; Bergner, Jennifer B.; Turner, Ewa M.; Proia, Richard L.
Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a ?-arrestin/protease fusion protein. Activated S1P1 recruits the ?-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease. PMID:24667638
IP receptor-dependent activation of PPAR? by stable prostacyclin analogues
Falcetti, Emilia; Flavell, David M.; Staels, Bart; Tinker, Andrew; Haworth, Sheila. G.; Clapp, Lucie H.
Stable prostacyclin analogues can signal through cell surface IP receptors or by ligand binding to nuclear peroxisome proliferator-activated receptors (PPARs). So far these agents have been reported to activate PPAR? and PPAR? but not PPAR?. Given PPAR? agonists and prostacyclin analogues both inhibit cell proliferation, we postulated that the IP receptor might elicit PPAR? activation. Using a dual luciferase reporter gene assay in HEK-293 cells stably expressing the IP receptor or empty vector, we found that prostacyclin analogues only activated PPAR? in the presence of the IP receptor. Moreover, the novel IP receptor antagonist, RO1138452, but not inhibitors of the cyclic AMP pathway, prevented activation. Likewise, the anti-proliferative effects of treprostinil observed in IP receptor expressing cells, were partially inhibited by the PPAR? antagonist, GW9662. We conclude that PPAR? is activated through the IP receptor via a cyclic AMP-independent mechanism and contributes to the anti-growth effects of prostacyclin analogues. PMID:17624303
Dopamine Activates Noradrenergic Receptors in the Preoptic Area
Cornil, C. A.; Balthazart, J.; Motte, P.; Massotte, L.; Seutin, V.
Dopamine (DA) facilitates male sexual behavior and modulates aromatase activity in the quail preoptic area (POA). Aromatase neurons in the POA receive dopaminergic inputs, but the anatomical substrate that mediates the behavioral and endocrine effects of DA is poorly understood. Intracellular recordings showed that 100 ?M DA hyperpolarizes most neurons in the medial preoptic nucleus (80%) by a direct effect, but depolarizes a few others (10%). DA-induced hyperpolarizations were not blocked by D1 or D2 antagonists (SCH-23390 and sulpiride). Extracellular recordings confirmed that DA inhibits the firing of most cells (52%) but excites a few others (24%). These effects also were not affected by DA antagonists (SCH-23390 and sulpiride) but were blocked by ?2-(yohimbine) and ?1-(prazosin) noradrenergic receptor antagonists, respectively. Two dopamine-?-hydroxylase (DBH) inhibitors (cysteine and fusaric acid) did not block the DA-induced effects, indicating that DA is not converted into norepinephrine (NE) to produce its effects. The pKB of yohimbine for the receptor involved in the DA- and NE-induced inhibitions was similar, indicating that the two monoamines interact with the same receptor. Together, these results demonstrate that the effects of DA in the POA are mediated mostly by the activation of ?2 (inhibition) and ?1 (excitation) adrenoreceptors. This may explain why DA affects the expression of male sexual behavior through its action in the POA, which contains high densities of ?2-noradrenergic but limited amounts of DA receptors. This study thus clearly demonstrates the existence of a cross talk within CNS catecholaminergic systems between a neurotransmitter and heterologous receptors. PMID:12417657
Insect Repellents: Modulators of Mosquito Odorant Receptor Activity
Jonathan D. Bohbot; Joseph C. Dickens; Mark A. Frye
BackgroundDEET, 2-undecanone (2-U), IR3535 and Picaridin are widely used as insect repellents to prevent interactions between humans and many arthropods including mosquitoes. Their molecular action has only recently been studied, yielding seemingly contradictory theories including odorant-dependent inhibitory and odorant-independent excitatory activities on insect olfactory sensory neurons (OSNs) and odorant receptor proteins (ORs).Methodology\\/Principal FindingsHere we characterize the action of these repellents
Laminin-?1 impairs spatial learning through inhibition of ERK/MAPK and SGK1 signaling.
Yang, Ying C; Ma, Yun L; Liu, Wen T; Lee, Eminy H Y
Laminin is a major structural element of the basal lamina consisting of an ?-chain, a ?-chain, and a ?-chain arranged in a cross-like structure, with their C-terminal inter-coiled. Laminin is abundantly expressed in the hippocampus of mature brain and is implicated in several psychiatric disorders, but its possible role involved in learning and memory function is not known. This issue was examined here. Our results revealed that water maze training significantly decreased laminin-?1 (LB1) expression in the rat hippocampal CA1 area. Transfection of LB1 WT plasmid to hippocampal CA1 neurons impaired water maze performance in rats. Meanwhile, it decreased the phosphorylation level of ERK/MAPK and protein kinase serum- and glucocorticoid-inducible kinase-1 (SGK1). By contrast, knockdown of endogenous LB1 expression using RNA interference (LB1 siRNA) enhanced water maze performance. Meanwhile, it increased the phosphorylation level of ERK/MAPK and SGK1. The enhancing effect of LB1 siRNA on spatial learning and on the phosphorylation of ERK/MAPK and SGK1 was blocked by co-treatment with the MEK inhibitor U0126 at a concentration that did not apparently affect spatial learning and ERK/MAPK phosphorylation alone. Further, the enhancing effect of LB1 siRNA on spatial learning and SGK1 phosphorylation was similarly blocked by co-transfection with SGK1 siRNA at a concentration that did not markedly affect spatial learning and SGK1 expression alone. These results together indicate that LB1 negatively regulates spatial learning in rats. In addition, LB1 impairs spatial learning through decreased activation of the ERK/MAPK-SGK1 signaling pathway in the rat hippocampus. PMID:21849984
Laminin-?1 Impairs Spatial Learning through Inhibition of ERK/MAPK and SGK1 Signaling
Yang, Ying C; Ma, Yun L; Liu, Wen T; Lee, Eminy HY
Laminin is a major structural element of the basal lamina consisting of an ?-chain, a ?-chain, and a ?-chain arranged in a cross-like structure, with their C-terminal inter-coiled. Laminin is abundantly expressed in the hippocampus of mature brain and is implicated in several psychiatric disorders, but its possible role involved in learning and memory function is not known. This issue was examined here. Our results revealed that water maze training significantly decreased laminin-?1 (LB1) expression in the rat hippocampal CA1 area. Transfection of LB1 WT plasmid to hippocampal CA1 neurons impaired water maze performance in rats. Meanwhile, it decreased the phosphorylation level of ERK/MAPK and protein kinase serum- and glucocorticoid-inducible kinase-1 (SGK1). By contrast, knockdown of endogenous LB1 expression using RNA interference (LB1 siRNA) enhanced water maze performance. Meanwhile, it increased the phosphorylation level of ERK/MAPK and SGK1. The enhancing effect of LB1 siRNA on spatial learning and on the phosphorylation of ERK/MAPK and SGK1 was blocked by co-treatment with the MEK inhibitor U0126 at a concentration that did not apparently affect spatial learning and ERK/MAPK phosphorylation alone. Further, the enhancing effect of LB1 siRNA on spatial learning and SGK1 phosphorylation was similarly blocked by co-transfection with SGK1 siRNA at a concentration that did not markedly affect spatial learning and SGK1 expression alone. These results together indicate that LB1 negatively regulates spatial learning in rats. In addition, LB1 impairs spatial learning through decreased activation of the ERK/MAPK�SGK1 signaling pathway in the rat hippocampus. PMID:21849984
Characterization of Peroxisome Proliferator�Activated Receptor ?�Independent Effects of PPAR? Activators in the Rodent Liver: Di-(2-ethylhexyl) phthalate also Activates the Constitutive-Activated Receptor
Ren, Hongzu; Aleksunes, Lauren M.; Wood, Carmen; Vallanat, Beena; George, Michael H.; Klaassen, Curtis D.; Corton, J. Christopher
Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator�activated receptor (PPAR) ?. Recent studies indicate that the plasticizer di-(2-ethylhexyl) phthalate (DEHP) increased the incidence of liver tumors in PPAR?-null mice. We hypothesized that some PPC, including DEHP, induce transcriptional changes independent of PPAR? but dependent on other nuclear receptors, including the constitutive-activated receptor (CAR) that mediates phenobarbital (PB) effects on hepatocyte growth and liver tumor induction. To determine the potential role of CAR in mediating effects of PPC, a meta-analysis was performed on transcript profiles from published studies in which rats and mice were exposed to PPC and compared the profiles to those produced by exposure to PB. Valproic acid, clofibrate, and DEHP in rat liver and DEHP in mouse liver induced genes, including Cyp2b family members that are known to be regulated by CAR. Examination of transcript changes by Affymetrix ST 1.0 arrays and reverse transcription-PCR in the livers of DEHP-treated wild-type, PPAR?-null, and CAR-null mice demonstrated that (1) most (?94%) of the transcriptional changes induced by DEHP were PPAR?-dependent, (2) many PPAR?-independent genes overlapped with those regulated by PB, (3) induction of genes Cyp2b10, Cyp3a11, and metallothionine-1 by DEHP was CAR dependent but PPAR?-independent, and (4) induction of a number of genes (Cyp8b1, Gstm4, and Gstm7) was independent of both CAR and PPAR?. Our results indicate that exposure to PPAR? activators including DEHP leads to activation of multiple nuclear receptors in the rodent liver. PMID:19850644
Constitutive activities in the thyrotropin receptor: regulation and significance.
Kleinau, Gunnar; Biebermann, Heike
The thyroid-stimulating hormone receptor (TSHR, or thyrotropin receptor) is a family A G protein-coupled receptor. It not only binds thyroid-stimulating hormone (TSH, or thyrotropin) but also interacts with autoantibodies under pathological conditions. The TSHR and TSH are essential for thyroid growth and function and thus for all thyroid hormone-associated physiological superordinated processes, including metabolism and development of the central nervous system. In vitro studies have found that the TSHR permanently stimulates ligand-independent (constitutive) activation of Gs, which ultimately leads to intracellular cAMP accumulation. Furthermore, a vast variety of constitutively activating mutations of TSHR-at more than 50 different amino acid positions-have been reported to enhance basal signaling. These lead in vivo to a "gain-of-function" phenotype of nonautoimmune hyperthyroidism or toxic adenomas. Moreover, many naturally occurring inactivating mutations are known to cause a "loss-of-function" phenotype, resulting in resistance to thyroid hormone or hyperthyrotropinemia. Several of these mutations are also characterized by impaired basal signaling, and these are designated here as "constitutively inactivating mutations" (CIMs). More than 30 amino acid positions with CIMs have been identified so far. Moreover, the permanent TSHR signaling capacity can also be blocked by inverse agonistic antibodies or small drug-like molecules, which both have a potential for clinical usage. In this chapter, information on constitutive activity in the TSHR is described, including up- and downregulation, linked protein conformations, physiological and pathophysiological conditions, and related intracellular signaling. PMID:24931193
?-Aminobutyric acid type A (GABAA) receptor activation modulates tau phosphorylation.
Nyk�nen, Niko-Petteri; Kysenius, Kai; Sakha, Prasanna; Tammela, P�ivi; Huttunen, Henri J
Abnormal phosphorylation and aggregation of the microtubule-associated protein Tau are hallmarks of various neurodegenerative diseases, such as Alzheimer disease. Molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. We have developed a novel live cell reporter system based on protein-fragment complementation assay to study dynamic changes in Tau phosphorylation status. In this assay, fusion proteins of Tau and Pin1 (peptidyl-prolyl cis-trans-isomerase 1) carrying complementary fragments of a luciferase protein serve as a sensor of altered protein-protein interaction between Tau and Pin1, a critical regulator of Tau dephosphorylation at several disease-associated proline-directed phosphorylation sites. Using this system, we identified several structurally distinct GABA(A) receptor modulators as novel regulators of Tau phosphorylation in a chemical library screen. GABA(A) receptor activation promoted specific phosphorylation of Tau at the AT8 epitope (Ser-199/Ser-202/Thr-205) in cultures of mature cortical neurons. Increased Tau phosphorylation by GABA(A) receptor activity was associated with reduced Tau binding to protein phosphatase 2A and was dependent on Cdk5 but not GSK3? kinase activity. PMID:22235112
Estrogen Receptor ? (ER?) Activity Modulates Synaptic Signaling and Structure
Srivastava, Deepak P.; Woolfrey, Kevin M.; Liu, Feng; Brandon, Nicholas J.; Penzes, Peter
Brain-synthesized estrogen has been shown to influence synaptic structure, function, and cognitive processes. However, the molecular mechanisms underlying the rapid effects of estrogen on the dendritic spines of cortical neurons are not clear. Estrogen receptor ? (ER?) is expressed in cortical neurons, and ER? knockout mice display impaired performance in cortically mediated processes, suggesting that signaling via this receptor has profound effects on cortical neuron function. However, the effect of rapid signaling via ER? on dendritic spines, and the signaling pathways initiated by this receptor in cortical neurons, are unknown. Here we show that activation of ER? with the specific agonist WAY-200070 results in increased spine density, and PSD-95 accumulation in membrane regions. Activation of ER? by WAY-200070 also resulted in the phosphorylation of p21-activated kinase (PAK) and ERK1/2 in cultured cortical neurons, suggesting a mechanism for the regulation of the actin cytoskeleton. Moreover, we found that aromatase, an enzyme critical for estrogen production, is present at presynaptic termini, supporting a role for brain-synthesized estrogen as a neuromodulator in the cortex. These results implicate ER? signaling in controlling dendritic spine morphology, in part via a PAK/ERK1/2-dependent pathway, and provide mechanistic insight into the rapid cellular effects of estrogen on brain function. PMID:20926671
Retinoic acid induces neurogenesis by activating both retinoic acid receptors (RARs) and peroxisome proliferator-activated receptor ?/? (PPAR?/?).
Yu, Shuiliang; Levi, Liraz; Siegel, Ruth; Noy, Noa
Retinoic acid (RA) regulates gene transcription by activating the nuclear receptors retinoic acid receptor (RAR) and peroxisome proliferator-activated receptor (PPAR) ?/? and their respective cognate lipid-binding proteins CRABP-II and FABP5. RA induces neuronal differentiation, but the contributions of the two transcriptional pathways of the hormone to the process are unknown. Here, we show that the RA-induced commitment of P19 stem cells to neuronal progenitors is mediated by the CRABP-II/RAR path and that the FABP5/PPAR?/? path can inhibit the process through induction of the RAR repressors SIRT1 and Ajuba. In contrast with its inhibitory activity in the early steps of neurogenesis, the FABP5/PPAR?/? path promotes differentiation of neuronal progenitors to mature neurons, an activity mediated in part by the PPAR?/? target gene PDK1. Hence, RA-induced neuronal differentiation is mediated through RAR in the early stages and through PPAR?/? in the late stages of the process. The switch in RA signaling is accomplished by a transient up-regulation of RAR? concomitantly with a transient increase in the CRABP-II/FABP5 ratio at early stages of differentiation. In accordance with these conclusions, hippocampi of FABP5-null mice display excess accumulation of neuronal progenitor cells and a deficit in mature neurons versus wild-type animals. PMID:23105114
Single-molecule optical methods analyzing receptor tyrosine kinase activation in living cells.
Chung, Inhee; Mellman, Ira
Receptor tyrosine kinase activity is typically measured by diverse biochemical methods detecting the amount of phosphorylation of proteins within a cell lysate. In this chapter, we present biophysical methods that allow for studying the activation process of single receptors, in particular the human epidermal growth factor receptor (EGFR) family, in live cells. We describe optical tracking of quantum dot (QD)-labeled single receptors using the total internal reflection fluorescence microscopy (TIRFM), and initial steps of data analysis to identify the time-dependent variation of single-receptor diffusion, which can be widely applied to studying activation of various cell surface receptors. PMID:25319887
Methylthioadenosine Reprograms Macrophage Activation through Adenosine Receptor Stimulation
Keyel, Peter A.; Romero, Matthew; Wu, Wenbo; Kwak, Daniel H.; Zhu, Qin; Liu, Xinyu; Salter, Russell D.
Regulation of inflammation is necessary to balance sufficient pathogen clearance with excessive tissue damage. Central to regulating inflammation is the switch from a pro-inflammatory pathway to an anti-inflammatory pathway. Macrophages are well-positioned to initiate this switch, and as such are the target of multiple therapeutics. One such potential therapeutic is methylthioadenosine (MTA), which inhibits TNF? production following LPS stimulation. We found that MTA could block TNF? production by multiple TLR ligands. Further, it prevented surface expression of CD69 and CD86 and reduced NF-KB signaling. We then determined that the mechanism of this action by MTA is signaling through adenosine A2 receptors. A2 receptors and TLR receptors synergized to promote an anti-inflammatory phenotype, as MTA enhanced LPS tolerance. In contrast, IL-1? production and processing was not affected by MTA exposure. Taken together, these data demonstrate that MTA reprograms TLR activation pathways via adenosine receptors to promote resolution of inflammation. PMID:25117662
Methylthioadenosine reprograms macrophage activation through adenosine receptor stimulation.
Keyel, Peter A; Romero, Matthew; Wu, Wenbo; Kwak, Daniel H; Zhu, Qin; Liu, Xinyu; Salter, Russell D
Regulation of inflammation is necessary to balance sufficient pathogen clearance with excessive tissue damage. Central to regulating inflammation is the switch from a pro-inflammatory pathway to an anti-inflammatory pathway. Macrophages are well-positioned to initiate this switch, and as such are the target of multiple therapeutics. One such potential therapeutic is methylthioadenosine (MTA), which inhibits TNF? production following LPS stimulation. We found that MTA could block TNF? production by multiple TLR ligands. Further, it prevented surface expression of CD69 and CD86 and reduced NF-KB signaling. We then determined that the mechanism of this action by MTA is signaling through adenosine A2 receptors. A2 receptors and TLR receptors synergized to promote an anti-inflammatory phenotype, as MTA enhanced LPS tolerance. In contrast, IL-1? production and processing was not affected by MTA exposure. Taken together, these data demonstrate that MTA reprograms TLR activation pathways via adenosine receptors to promote resolution of inflammation. PMID:25117662
HPV binding assay to Laminin-332/integrin ?6?4 on human keratinocytes.
Brendle, Sarah A; Christensen, Neil D
Human papillomaviruses (HPVs) have been shown to bind to Laminin-332 (Ln-332) on the extracellular matrix (ECM) secreted by human keratinocytes. The assay described here is an important tool to study HPV receptor binding to the ECM. The assay can also be modified to study the receptors required for HPV infection and for binding to tissues. We previously showed that Ln-332 is essential for the binding of HPV11 to human keratinocytes and that infectious entry of HPV11 requires ?6?4 integrin for the transfer of HPV11 from ECM to host cells (Culp et al., J Virol 80:8940-8950, 2006). We also demonstrated that several of the high-risk HPV types (16, 18, 31 and 45) bind to Ln-332 and/or other components of the ECM in vitro (Broutian et al., J Gen Virol 91:531-540, 2010). The exact binding and internalization mechanism(s) for HPV are still under investigation. A better understanding of these mechanisms will aid in the design of therapeutics against HPVs and ultimately help prevent many cancers. In this chapter, we describe the HPV binding assay to Ln-332/integrin ?6?4 on human keratinocytes (ECM). We also present data and suggestions for modifying the assay for testing the specificity of HPV for receptors (by blocking receptors) and binding to human tissues (basement membrane, BM) in order to study binding mechanisms. PMID:25348297
Insect Repellents: Modulators of Mosquito Odorant Receptor Activity
Bohbot, Jonathan D.; Dickens, Joseph C.
Background DEET, 2-undecanone (2-U), IR3535 and Picaridin are widely used as insect repellents to prevent interactions between humans and many arthropods including mosquitoes. Their molecular action has only recently been studied, yielding seemingly contradictory theories including odorant-dependent inhibitory and odorant-independent excitatory activities on insect olfactory sensory neurons (OSNs) and odorant receptor proteins (ORs). Methodology/Principal Findings Here we characterize the action of these repellents on two Aedes aegypti ORs, AaOR2 and AaOR8, individually co-expressed with the common co-receptor AaOR7 in Xenopus oocytes; these ORs are respectively activated by the odors indole (AaOR2) and (R)-(?)-1-octen3-ol (AaOR8), odorants used to locate oviposition sites and host animals. In the absence of odorants, DEET activates AaOR2 but not AaOR8, while 2-U activates AaOR8 but not AaOR2; IR3535 and Picaridin do not activate these ORs. In the presence of odors, DEET strongly inhibits AaOR8 but not AaOR2, while 2-U strongly inhibits AaOR2 but not AaOR8; IR3535 and Picaridin strongly inhibit both ORs. Conclusions/Significance These data demonstrate that repellents can act as olfactory agonists or antagonists, thus modulating OR activity, bringing concordance to conflicting models. PMID:20725637
Endothelial mineralocorticoid receptor activation enhances endothelial protein C receptor and decreases vascular thrombosis in mice.
Lagrange, J�r�my; Li, Zhenlin; Fassot, C�line; Bourhim, Mustapha; Louis, Huguette; Nguyen Dinh Cat, Aur�lie; Parlakian, Ara; Wahl, Denis; Lacolley, Patrick; Jaisser, Fr�deric; Regnault, V�ronique
Previous studies have shown that aldosterone, which activates the mineralocorticoid receptor (MR), promotes thrombosis in animal models. Our objective was to determine whether MR activation/expression in the vascular endothelium could modify thrombotic risk in vivo and to examine thrombin generation at the surface of aortic endothelial cells (HAECs). MR was conditionally overexpressed in vivo in vascular endothelial cells in mice (MR-EC mice) or stimulated with aldosterone in HAECs. Thrombosis after ferric chloride injury was delayed in MR-EC mice compared with controls as well as in wild-type FVB/NRj mice treated with aldosterone (60 ?g/kg/d for 21 d). Thrombin generation in platelet-poor plasma did not differ between MR-EC mice and controls. In MR-EC mice, aortic endothelial cell protein C receptor (EPCR) expression was increased. Aldosterone (10(-8) M) attenuated thrombin generation at the surface of cultured HAECs, and this effect was associated with up-regulation of expression of EPCR, which promotes formation of activated protein C. Aldosterone increases EPCR expression via a transcriptional mechanism involving interaction of MR with the specificity protein 1 site. These findings demonstrate that MR activation acts on endothelial cells to protect against thrombosis in physiological conditions and that MR-mediated EPCR overexpression drives this antithrombotic property through enhancing protein C activation. PMID:24451386
Distinct receptor activity-modifying protein domains differentially modulate interaction with calcitonin receptors.
Udawela, Madhara; Christopoulos, George; Tilakaratne, Nanda; Christopoulos, Arthur; Albiston, Anthony; Sexton, Patrick M
Calcitonin receptors (CTRs) dimerize with receptor activity-modifying proteins (RAMPs) to generate high-affinity amylin (AMY) receptors; however, the relative contribution of individual RAMP domains to the formation of AMY receptors is poorly understood. We have used chimeras between RAMP1 and RAMP2 that specifically exchanged the N-terminal, transmembrane, or C-terminal domain and examined these in assays of [(125)I]amylin binding or peptide-induced cAMP signaling in COS-7 cells transiently transfected with wild-type or chimeric RAMPs and human CTRa. The specificity of peptides in competition for [(125)I]amylin binding was principally dictated by the N-terminal domain present in the chimeras; however, the maximal level of binding induced was dictated by the transmembrane domain present. This extended previous data (Zumpe et al., 2000) to provide a distinction between the transmembrane domain and the C terminus in this function. In contrast to the effects on binding, each of the RAMP domains played a role in the signaling phenotype of the receptors. In particular, the potency of calcitonin gene-related peptide (CGRP) was most influenced by the C-terminal domain present, in which the presence of the RAMP1 C-terminal domain led to increased potency over CTRa alone, whereas chimeras with the RAMP2 C-terminal domain did not induce increased CGRP potency. The data provide additional support for the importance of the N terminus in determining binding affinity but reveal a prominent role of the transmembrane domain in the strength of amylin binding and a unique role for the C terminus in signaling by peptides to stimulate cAMP production. PMID:16531504
Receptor-interacting protein 140 is a repressor of the androgen receptor activity
Carascossa, Sophie; Gobinet, J�r�me; Georget, Virginie; Lucas, Annick; Badia, Eric; Castet, Audrey; White, Roger; Nicolas, Jean-Claude; Cavaill�s, Vincent; Jalaguier, St�phan Sj
The androgen receptor (AR) is a ligand-activated transcription factor which controls growth and survival of prostate cancer cells. In the present study, we investigated the regulation of AR activity by the receptor interacting protein RIP140. We first showed that RIP140 could be co-immunoprecipitated with the receptor when co-expressed in 293T cells. This interaction appeared physiologically relevant since ChIP assays revealed that under R1881 treatment, RIP140 could be recruited to the PSA encoding gene in LNCaP cells. In vitro GST pull-down assays evidenced that the carboxy-terminal domain of AR could interact with different regions of RIP140. By means of fluorescent proteins we demonstrated that ligand-activated AR was not only able to translocate to the nucleus but also to relocate RIP140 from very structured nuclear foci to a diffuse pattern. Overexpression of RIP140 strongly repressed AR-dependent transactivation by preferentially targeting the ligand binding domain-dependent activity. Moreover, disruption of RIP140 expression induced AR overactivation thus revealing RIP140 as a strong AR repressor. We analysed its mechanism of transrepression and first demonstrated that different regions of RIP140 could mediate AR-dependent repression. We then showed that the carboxy-terminal end of RIP140 could reverse transcriptional intermediary factor TIF2-dependent overactivation of AR. The use of mutants of RIP140 allowed us to suggest that CtBP played no role in RIP140-dependent inhibition of AR activity whereas HDACs partly regulated that transrepression. Finally, we provided evidence for a stimulation of RIP140 mRNA expression in LNCaP cells under androgen treatment, further emphasizing the role of RIP140 in androgen signalling. PMID:16527872
Selenoprotein W controls epidermal growth factor receptor surface expression, activation and degradation via receptor ubiquitination.
Alkan, Zeynep; Duong, Frank L; Hawkes, Wayne C
Epidermal growth factor (EGF) receptor (EGFR) is the founding member of the ErbB family of growth factor receptors that modulate a complex network of intracellular signaling pathways controlling growth, proliferation, differentiation, and motility. Selenoprotein W (SEPW1) is a highly conserved, diet-regulated 9kDa thioredoxin-like protein required for normal cell cycle progression. We report here that SEPW1 is required for EGF-induced EGFR activation and that it functions by suppressing EGFR ubiquitination and receptor degradation. SEPW1 depletion inhibited EGF-dependent cell cycle entry in breast and prostate epithelial cells. In prostate cells, SEPW1 depletion decreased EGFR auto-phosphorylation, while SEPW1 overexpression increased EGFR auto-phosphorylation. SEPW1 depletion increased the rate of EGFR degradation, which decreased total and surface EGFR and suppressed EGF-dependent EGFR endocytosis, EGFR dimer formation, and activation of EGF-dependent pathways. EGFR ubiquitination was increased in SEPW1-depleted cells - in agreement with the increased rate of EGFR degradation, and suggests that SEPW1 suppresses EGFR ubiquitination. Ubiquitination-directed lysozomal degradation controls post-translational EGFR expression and is dysregulated in many cancers. Thus, suppression of EGFR ubiquitination by SEPW1 may be related to the putative increase in cancer risk associated with high selenium intakes. Knowledge of the mechanisms underlying SEPW1's regulation of EGFR ubiquitination may reveal new opportunities for nutritional cancer prevention or cancer drug development. PMID:25721765
Liver X Receptor ? and Peroxisome Proliferator-Activated Receptor ? regulate cholesterol transport in cholangiocytes
Xia, Xuefeng; Jung, Dongju; Webb, Paul; Zhang, Aijun; Zhang, Bin; Li, Lifei; Ayers, Stephen D.; Gabbi, Chiara; Ueno, Yoshiyuki; Gustafsson, Jan-�ke; Alpini, Gianfranco; Moore, David D.; LeSage, Gene D.
Nuclear receptors (NRs) play crucial roles in regulation of hepatic cholesterol synthesis, metabolism and conversion to bile acids, but their actions in cholangiocytes have not been examined. In this study, we investigated the roles of NRs in cholangiocyte physiology and cholesterol metabolism and flux. We examined the expression of NRs and other genes involved in cholesterol homeostasis in freshly isolated and cultured rodent cholangiocytes and found that these cells express a specific subset of NRs which includes Liver X Receptor ? (LXR?) and Peroxisome Proliferator-Activated Receptor ? (PPAR?). Activation of LXR? and/or PPAR? in cholangiocytes induces ATP-binding cassette cholesterol transporter A1 (ABCA1) and increases cholesterol export at the basolateral compartment in polarized cultured cholangiocytes. In addition, PPAR? induces Niemann Pick C1 Like L1 (NPC1L1), which imports cholesterol into cholangiocytes and is expressed on the apical cholangiocyte membrane, via specific interaction with a PPRE within the NPC1L1 promoter. Based on these studies, we propose that (i) LXR? and PPAR? coordinate NPC1L1/ABCA1 dependent vectorial cholesterol flux from bile through cholangiocytes and (ii) manipulation of these processes may influence bile composition with important applications in cholestatic liver disease and gallstone disease, serious health concerns for humans. PMID:22729460
Murine atopic dermatitis responds to peroxisome proliferator-activated receptor ?, ?/?(but not ?), and liver-X-receptor activators
Hatano, Yutaka; Man, Mao-Qiang; Uchida, Yoshikazu; Crumrine, Debra; Mauro, Theodora M.; Feingold, Kenneth R.; Elias, Peter M.; Holleran, Walter M.
Background Atopic dermatitis (AD) is a chronic inflammatory dermatosis now increasingly linked to mutations that alter the structure and function of the stratum corneum (SC). Activators of peroxisome proliferators-activated receptor (PPAR)?, ?/?, ? and liver-X-receptor (LXR) regulate epidermal protein and lipid production, leading to superior barrier function. Additionally, some of these activators exhibit potent anti-hyperplastic and anti-inflammatory activity in irritant contact dermatitis and acute allergic contact dermatitis mouse models. Objective We evaluated the efficacy of PPAR/LXR activation in a hapten (oxazolone [Ox])-induced atopic dermatitis-like model (Ox-AD) in hairless mice. Methods Ox-AD was established with ten Ox challenges (every-other day) on the flank. After the establishment of Ox-AD, twice daily topical application with individual PPAR/LXR activators was then performed for 4 days, with continued Ox challenges every other day. The efficacy of topical PPAR/LXR activators to reduce parameters of Ox-AD was assessed physiologically, morphologically and immunologically. Results Certain topical activators of PPAR?, PPAR?/?, and LXR, but not activators of PPAR?, reversed the clinical dermatosis, significantly improved barrier function, and increased SC hydration in Ox-AD mice. In addition, the same activators, but again not PPAR?, largely reversed the immunologic abnormalities in Ox-AD mice, including the elevated TH2 markers, such as tissue eosinophil/mast cell density, serum TARC levels, density of CRTH2-positive lymphocytes (but not serum IgE levels), and reduced IL-1? and TNF? activation, despite on-going hapten challenges. Conclusion These results suggest that topical applications of certain activators/ligands of PPAR?, ?/?, and LXR could be useful for the treatment of AD in humans. PMID:19818482
Laminins containing the ?2 and ?3 chains regulate astrocyte migration and angiogenesis in the retina
Gnanaguru, Gopalan; Bachay, Galina; Biswas, Saptarshi; Pinz�n-Duarte, Germ�n; Hunter, Dale D.; Brunken, William J.
Pathologies of retinal blood vessels are among the major causes of blindness worldwide. A key cell type that regulates retinal vascular development is the astrocyte. Generated extrinsically to the retina, astrocytes migrate into the retina through the optic nerve head. Even though there is a strong correlation between astrocyte distribution and retinal vascular development, the factors that guide astrocytes into the retina remain unclear. In this study, we show that astrocytes migrate within a laminin-containing basement membrane - the inner limiting membrane. Genetic deletion of the laminin ?2 and ?3 chains affects astrocyte migration and spatial distribution. We show that laminins act as haptotactic factors in vitro in an isoform-specific manner, inducing astrocyte migration and promoting astrocyte differentiation. The addition of exogenous laminins to laminin-null retinal explants rescues astrocyte migration and spatial patterning. Furthermore, we show that the loss of laminins reduces ?1 integrin expression in astrocytes. Culturing laminin-null retinal astrocytes on laminin substrates restores focal localization of ?1 integrin. Finally, we show that laminins containing ?2 and ?3 chains regulate subsequent retinal blood vessel growth and maintain vascular integrity. These in vivo and in vitro studies demonstrate clearly that laminins containing ?2 and ?3 chains are indispensable for migration and spatial organization of astrocytes and that they play a crucial role during retinal angiogenesis in vivo. PMID:23571221
Liver X Receptors Regulate the Transcriptional Activity of the Glucocorticoid Receptor: Implications for the Carbohydrate Metabolism
Nader, Nancy; Ng, Sinnie Sin Man; Wang, Yonghong; Abel, Brent S.; Chrousos, George P.; Kino, Tomoshige
GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase), a key enzyme of gluconeogenesis, and suppress the immune system through the glucocorticoid receptor (GR). The liver X receptors (LXRs), on the other hand, bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR), and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. Since the actions of these receptors overlap with each other, we evaluated the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXR?/RXR? binding to GREs. Endogenous LXR?/RXR? bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that administration of LXR agonists may be beneficial in glucocorticoid treatment- or stress-associated dysmetabolic states by directly and gene-specifically attenuating the transcriptional activity of the GR on glucose and/or lipid metabolism. PMID:22457708
Aggregin: a platelet ADP receptor that mediates activation.
Colman, R W
ADP is known to induce platelet shape change, aggregation, and exposure of fibrinogen binding sites as well as inhibit stimulated adenylate cyclase. The platelet is unique in that its purinergic receptor prefers ADP over ATP, which functions as a competitive antagonist. The affinity reagent, 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), has been used to covalently label a single membrane protein, aggregin, on the external platelet surface with mol wt of 100 kDa. Concomitant with incorporation of FSBA, ADP-induced shape change, aggregation, and fibrinogen binding is inhibited. FSBA is also a weak agonist at short times and high concentration, which suggests that prior noncovalent binding to aggregin takes place before covalent modification. Aggregin differs from platelet glycoprotein IIIa in its physical and immunochemical properties. Aggregin is distinct from the receptor coupled to adenylate cyclase. Using FSBA as a probe, platelet aggregation by thromboxane A2 analogs and collagen was shown to be dependent on ADP but not the shape change induced by these agonists. Binding to aggregin is required for epinephrine-induced aggregation. In turn, epinephrine increases the affinity of ADP for its receptor. Thrombin at concentrations greater than 2 nM (0.2 units/ml) stimulates platelet aggregation independent of ADP, but by raising cytoplasmic Ca2+ it activates platelet calpain, which in turn cleaves aggregin. Thus aggregin, in addition to serving as the ADP receptor linked to shape change and aggregation, plays a role in fibrinogen receptor latency that is relieved entirely by ADP binding to or proteolysis of aggregin. PMID:2407587
Trace Amine Associated Receptor 1 Signaling in Activated Lymphocytes
Panas, Michael W.; Xie, Zhihua; Panas, Helen N.; Hoener, Marius C.; Vallender, Eric J.; Miller, Gregory M.
Although most research to date on Trace Amine Associated Receptor 1 (TAAR1) has focused on its role in the brain, it has been recognized since its discovery in 2001 that TAAR1 mRNA is expressed in peripheral tissues as well, suggesting that this receptor may play a role in non-neurological pathways. This study reports TAAR1 expression, signaling and functionality in rhesus monkey lymphocytes. We detected a high level of TAAR1 protein in immortalized rhesus monkey B cell lines and a significant upregulation of TAAR1 protein expression in rhesus monkey lymphocytes following PHA treatment. Through screening a wide range of signaling pathways for their upregulation following TAAR1 activation by its potent agonist methamphetamine, we identified two transcription factors, CREB and NFAT, which are commonly associated with immune activation. Furthermore, we observed a TAAR1-dependent phosphorylation of PKA and PKC following treatment with methamphetamine in transfected HEK293 cells, immortalized rhesus monkey B cells and PHA-activated rhesus monkey lymphocytes. Accordingly, the high levels of TAAR1 that we observed on lymphocytes are inducible and fully functional, capable of transmitting a signal likely via PKA and PKC activation following ligand binding. More importantly, an increase in TAAR1 receptor expression is concomitant with lymphocyte immune activation, suggesting a possible role for TAAR1 in the generation or regulation of an immune response. TAAR1 is emerging as a potential therapeutic target, with regard to its ability to modulate brain monoamines. The current data raises the possibility that TAAR1-targeted drugs may also alter immune function. PMID:22038157
Aldosterone-specific activation of cardiomyocyte mineralocorticoid receptor in vivo.
Messaoudi, Smail; Gravez, Basile; Tarjus, Antoine; Pelloux, V�ronique; Ouvrard-Pascaud, Antoine; Delcayre, Claude; Samuel, Janelise; Launay, Jean-Marie; Sierra-Ramos, Catalina; Alvarez de la Rosa, Diego; Cl�ment, Karine; Farman, Nicolette; Jaisser, Fr�deric
Inappropriate mineralocorticoid receptor (MR) activation is involved in cardiac diseases. Whether and how aldosterone is involved in the deleterious effects of cardiac mineralocorticoid activation is still unclear. Mice overexpressing MR in cardiomyocytes and their controls were treated for 7 days with aldosterone, and cardiac transcriptome was analyzed. Aldosterone regulated 265 genes in cardiomyocyte-targeted MR overexpression mice. Forty three of these genes were also differentially expressed between untreated cardiomyocyte-targeted MR overexpression and controls mice, thus representing putative aldosterone-regulated genes in cardiomyocytes. Among these genes, we focused on connective tissue growth factor (CTGF). In vivo, in cardiomyocyte-targeted MR overexpression mice, aldosterone (but not corticosterone) induced CTGF expression (mRNA and protein) in cardiomyocytes. Ex vivo, aldosterone induced the binding of mineralocorticoid receptor to CTGF promoter and increased the expression of its transcript. Aldosterone induction of CTGF synthesis in cardiomyocytes seems pathologically relevant as the increase in CTGF observed in a model of heart failure (transverse aortic constriction) in rats was prevented by eplerenone, a mineralocorticoid receptor blocker. This study demonstrates that aldosterone specifically regulates gene expression in cardiomyocytes despite large prevalence of glucocorticoids in plasma. PMID:23297371
Activation of MT2 melatonin receptors in rat suprachiasmatic nucleus phase advances the circadian clock
Gillette, Martha U.
Activation of MT2 melatonin receptors in rat suprachiasmatic nucleus phase advances the circadian. Dubocovich. Activation of MT2 melatonin receptors in rat suprachiasmatic nucleus phase advances the circadian the melatonin receptor type(s) (MT1 or MT2) mediating circadian clock resetting by melatonin in the mammalian
Dopamine and noradrenaline receptor stimulation: Reversal of reserpine-induced suppression of motor activity
Nils-Erik And�n; Ulf Str�mbom; Torgny H. Svensson
The motor activity of reserpine treated mice was recorded after drug treatments causing stimulation of dopamine or noradrenaline receptors or both. The dopamine receptor stimulating agent apomorphine elicited an activation with stereotypies whereas the noradrenaline receptor stimulating agent clonidine was inefficient. Combined treatment with apomorphine and clonidine induced marked stimulation with jumping. Biochemically, clonidine did not significantly interfere with the
An Orphan Nuclear Receptor Activated by Pregnanes Defines a Novel Steroid Signaling Pathway
Steven A. Kliewer; John T. Moore; Laura Wade; Jeff L. Staudinger; Michael A. Watson; Stacey A. Jones; David D. McKee; Beverly B. Oliver; Timothy M. Willson; Rolf H. Zetterstrom; Thomas Perlmann; J�rgen M Lehmann
Steroid hormones exert profound effects on differentiation, development, and homeostasis in higher eukaryotes through interactions with nuclear receptors. We describe a novel orphan nuclear receptor, termed the pregnane X receptor (PXR), that is activated by naturally occurring steroids such as pregnenolone and progesterone, and synthetic glucocorticoids and antiglucocorticoids. PXR exists as two isoforms, PXR.1 and PXR.2, that are differentially activated
A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins
To examine the function of the alpha 6 beta 4 integrin we have determined its ligand-binding ability and overexpressed two potentially dominant negative mutant beta 4 subunits, lacking either the cytoplasmic or extracellular domain, in bladder epithelial 804G cells. The results of cell adhesion and radioligand-binding assays showed that alpha 6 beta 4 is a receptor for several laminin isoforms, including laminin 1, 2, 4, and 5. Overexpression of the tail-less or head-less mutant beta 4 subunit did not suppress alpha 6 beta 4-mediated adhesion to laminins, as both types of transfectants adhered to these ligands in the presence of blocking anti-beta 1 antibodies as well as the controls. However, immunofluorescence experiments indicated that the endogenous alpha 6 beta 4 integrin and other hemidesmosomal markers were not concentrated in hemidesmosomes in cells overexpressing tail- less beta 4, while the distribution of these molecules was not altered in cells overexpressing the head-less subunit. Electron microscopic studies confirmed that cells overexpressing tail-less beta 4 had a drastically reduced number of hemidesmosomes, while cells expressing the head-less subunit had a normal number of these structures. Thus, expression of a tail-less, but not a head-less mutant beta 4 subunit leads to a dominant negative effect on hemidesmosome assembly without suppressing initial adhesion to laminins. We conclude that the alpha 6 beta 4 integrin binds to several laminins and plays an essential role in the assembly and/or stability of hemidesmosomes, that alpha 6 beta 4- mediated adhesion and hemidesmosome assembly have distinct requirements, and that it is possible to use a dominant negative approach to selectively interfere with a specific function of an integrin. PMID:7721947
A highly efficient and sensitive screening method for trans-activation activity of estrogen receptors
Zhao, Huimin
describe a highly efficient and sensitive yeast-based screening method for isolating human estrogen of estrogen receptor to the growth rate of yeast cells. We used this method to screen a library of human ERaA highly efficient and sensitive screening method for trans-activation activity of estrogen
Feedback control of T-cell receptor activation.
Chan, Cliburn; Stark, Jaroslav; George, Andrew J. T.
The specificity and sensitivity of T-cell recognition is vital to the immune response. Ligand engagement with the T-cell receptor (TCR) results in the activation of a complex sequence of signalling events, both on the cell membrane and intracellularly. Feedback is an integral part of these signalling pathways, yet is often ignored in standard accounts of T-cell signalling. Here we show, using a mathematical model, that these feedback loops can explain the ability of the TCR to discriminate between ligands with high specificity and sensitivity, as well as provide a mechanism for sustained signalling. The model also explains the recent counter-intuitive observation that endogenous 'null' ligands can significantly enhance T-cell signalling. Finally, the model may provide an archetype for receptor switching based on kinase-phosphatase switches, and thus be of interest to the wider signalling community. PMID:15255048
Trans-activation between 7TM domains: implication in heterodimeric GABAB receptor activation.
Monnier, Carine; Tu, Haijun; Bourrier, Emmanuel; Vol, Claire; Lamarque, Laurent; Trinquet, Eric; Pin, Jean-Philippe; Rondard, Philippe
Seven-transmembrane domain (7TM) receptors have important functions in cell-cell communication and can assemble into dimers or oligomers. Such complexes may allow specific functional cross-talk through trans-activation of interacting 7TMs, but this hypothesis requires further validation. Herein, we used the GABAB receptor, which is composed of two distinct subunits, GABAB1, which binds the agonist, and GABAB2, which activates G proteins, as a model system. By using a novel orthogonal-labelling approach compatible with time-resolved FRET and based on ACP- and SNAP-tag technologies to verify the heterodimerization of wild-type and mutated GABAB subunits, we demonstrate the existence of a direct allosteric coupling between the 7TMs of GABAB heterodimers. Indeed, a GABAB receptor, in which the GABAB2 extracellular domain was deleted, was still capable of activating G proteins. Furthermore, synthetic ligands for the GABAB2 7TM could increase agonist affinity at the GABAB1 subunit in this mutated receptor. In addition to bringing new information on GABAB receptor activation, these data clearly demonstrate the existence of direct trans-activation between the 7TM of two interacting proteins. PMID:21063387
Trans-activation between 7TM domains: implication in heterodimeric GABAB receptor activation
Monnier, Carine; Tu, Haijun; Bourrier, Emmanuel; Vol, Claire; Lamarque, Laurent; Trinquet, Eric; Pin, Jean-Philippe; Rondard, Philippe
Seven-transmembrane domain (7TM) receptors have important functions in cell�cell communication and can assemble into dimers or oligomers. Such complexes may allow specific functional cross-talk through trans-activation of interacting 7TMs, but this hypothesis requires further validation. Herein, we used the GABAB receptor, which is composed of two distinct subunits, GABAB1, which binds the agonist, and GABAB2, which activates G proteins, as a model system. By using a novel orthogonal-labelling approach compatible with time-resolved FRET and based on ACP- and SNAP-tag technologies to verify the heterodimerization of wild-type and mutated GABAB subunits, we demonstrate the existence of a direct allosteric coupling between the 7TMs of GABAB heterodimers. Indeed, a GABAB receptor, in which the GABAB2 extracellular domain was deleted, was still capable of activating G proteins. Furthermore, synthetic ligands for the GABAB2 7TM could increase agonist affinity at the GABAB1 subunit in this mutated receptor. In addition to bringing new information on GABAB receptor activation, these data clearly demonstrate the existence of direct trans-activation between the 7TM of two interacting proteins. PMID:21063387
An Improved Ivermectin-activated Chloride Channel Receptor for Inhibiting Electrical Activity in Defined Neuronal Populations*
Lynagh, Timothy; Lynch, Joseph W.
The ability to silence the electrical activity of defined neuronal populations in vivo is dramatically advancing our understanding of brain function. This technology may eventually be useful clinically for treating a variety of neuropathological disorders caused by excessive neuronal activity. Several neuronal silencing methods have been developed, with the bacterial light-activated halorhodopsin and the invertebrate allatostatin-activated G protein-coupled receptor proving the most successful to date. However, both techniques may be difficult to implement clinically due to their requirement for surgically implanted stimulus delivery methods and their use of nonhuman receptors. A third silencing method, an invertebrate glutamate-gated chloride channel receptor (GluClR) activated by ivermectin, solves the stimulus delivery problem as ivermectin is a safe, well tolerated drug that reaches the brain following systemic administration. However, the limitations of this method include poor functional expression, possibly due to the requirement to coexpress two different subunits in individual neurons, and the nonhuman origin of GluClR. Here, we describe the development of a modified human ?1 glycine receptor as an improved ivermectin-gated silencing receptor. The crucial development was the identification of a mutation, A288G, which increased ivermectin sensitivity almost 100-fold, rendering it similar to that of GluClR. Glycine sensitivity was eliminated via the F207A mutation. Its large unitary conductance, homomeric expression, and human origin may render the F207A/A288G ?1 glycine receptor an improved silencing receptor for neuroscientific and clinical purposes. As all known highly ivermectin-sensitive GluClRs contain an endogenous glycine residue at the corresponding location, this residue appears essential for exquisite ivermectin sensitivity. PMID:20308070
Peroxisome Proliferator-Activated Receptors (PPAR) and the Mitochondrial Aldehyde Dehydrogenase
Omiecinski, Curtis
Peroxisome Proliferator-Activated Receptors (PPAR) and the Mitochondrial Aldehyde Dehydrogenase. Peters, Robert A. Harris, and Mark Stewart Background: The aldehyde dehydrogenase 2 (ALDH2) promoter Receptor, Liver, Peroxisome Proliferator, Aldehyde Dehydrogenase. THE PEROXISOME proliferator
Role of peroxisome proliferator-activated receptors in mechanisms of rejection in heart transplantation
Binello, Emanuela
Peroxisome proliferator-activated receptors (PPARs) belong to a nuclear receptor superfamily; two major isoforms, PPAR? and PPAR[gamma], are primarily involved in lipid and glucose homeostasis. However, evidence also ...
Activation of family C G-protein-coupled receptors by the tripeptide glutathione.
Wang, Minghua; Yao, Yi; Kuang, Donghui; Hampson, David R
The Family C G-protein-coupled receptors include the metabotropic glutamate receptors, the gamma-aminobutyric acid, type B (GABAB) receptor, the calcium-sensing receptor (CaSR), which participates in the regulation of calcium homeostasis in the body, and a diverse group of sensory receptors that encompass the amino acid-activated fish 5.24 chemosensory receptor, the mammalian T1R taste receptors, and the V2R pheromone receptors. A common feature of Family C receptors is the presence of an amino acid binding site. In this study, a preliminary in silico analysis of the size and shape of the amino acid binding pocket in selected Family C receptors suggested that some members of this family could accommodate larger ligands such as peptides. Subsequent screening and docking experiments identified GSH as a potential ligand or co-ligand at the fish 5.24 receptor and the rat CaSR. These in silico predictions were confirmed using an [3H]GSH radioligand binding assay and a fluorescence-based functional assay performed on wild-type and chimeric receptors. Glutathione was shown to act as an orthosteric agonist at the 5.24 receptor and as a potent enhancer of calcium-induced activation of the CaSR. Within the mammalian receptors, this effect was specific to the CaSR because GSH neither directly activated nor potentiated other Family C receptors including GPRC6A (the putative mammalian homolog of the fish 5.24 receptor), the metabotropic glutamate receptors, or the GABAB receptor. Our findings reveal a potential new role for GSH and suggest that this peptide may act as an endogenous modulator of the CaSR in the parathyroid gland where this receptor is known to control the release of parathyroid hormone, and in other tissues such as the brain and gastrointestinal tract where the role of the calcium receptor appears to subserve other, as yet unknown, physiological functions. PMID:16455645
Activation of mineralocorticoid receptor in salt-sensitive hypertension.
Ayuzawa, Nobuhiro; Fujita, Toshiro
The impaired capacity of the kidney to excrete sodium plays an essential role in the development of hypertension. Adrenal corticosteroids control renal handling of sodium by regulating tubular sodium reabsorption in the distal nephron where both mineralocorticoid receptors (MR) and glucocorticoid receptors are expressed. In addition, cell type- and segment-specific expression of 11?-HSD2 and sodium transporters such as Na-Cl cotransporter (NCC), epithelial sodium channel (ENaC), and pendrin/Na(+)-driven Cl(-)/HCO3 (-) exchanger (NDCBE) builds a distinctive model of sodium transport in the aldosterone-sensitive distal nephron. Aberrant MR activation in the distal nephron triggers salt-sensitive hypertension and hypokalemia through inappropriate sodium reabsorption and potassium secretion. However, MR activity is not necessarily modulated by the ligand alone. Recently, several lines of evidence revealed alternative mechanisms that regulate the activity of MR in a ligand-independent manner or through ligand binding modulation. This review summarizes the disorders related to MR activation in individual tubular cells and highlights the renal mechanism of salt-sensitive hypertension and new approaches for the prevention and treatment of this disease. PMID:25903070
Turecek, Josef; Yuen, Genevieve S.; Han, Victor Z.; Zeng, Xiao-Hui; Bayer, K. Ulrich; Welsh, John P.
SUMMARY Electrical synapses are formed by gap junctions and permit electrical coupling that shapes the synchrony of neuronal ensembles. Here, we provide the first direct demonstration of receptormediated strengthening of electrical coupling in mammalian brain. Electrical coupling in the inferior olive of rats was strengthened by activation of NMDA-type glutamate-receptors (NMDARs), which were found at synaptic loci and at extrasynaptic loci 20�100 nm proximal to gap junctions. Electrical coupling was strengthened by pharmacological and synaptic activation of NMDARs, while co-stimulation of ionotropic non-NMDAR glutamate-receptors transiently antagonized the effect of NMDAR activation. NMDAR-dependent strengthening (i) occurred despite increased input conductance, (ii) induced Ca2+-influx microdomains near dendritic spines, (iii) required activation of the Ca2+/calmodulin-dependent protein-kinase II, (iv) was restricted to neurons that were weakly coupled, and thus, (v) strengthened coupling mainly between non-adjacent neurons. This provided a mechanism to expand the synchronization of rhythmic membrane potential oscillations by chemical neurotransmitter input. PMID:24656255
Thiophene-Core Estrogen Receptor Ligands Having Superagonist Activity
Min, Jian; Wang, Pengcheng; Srinivasan, Sathish; Nwachukwu, Jerome C.; Guo, Pu; Huang, Minjian; Carlson, Kathryn E.; Katzenellenbogen, John A.; Nettles, Kendall W.; Zhou, Hai-Bing
To probe the importance of the heterocyclic core of estrogen receptor (ER) ligands, we prepared a series of thiophene-core ligands by Suzuki cross-coupling of aryl boronic acids with bromo-thiophenes, and we assessed their receptor binding and cell biological activities. The disposition of the phenol substituents on the thiophene core, at alternate or adjacent sites, and the nature of substituents on these phenols all contribute to binding affinity and subtype selectivity. Most of the bis(hydroxyphenyl)-thiophenes were ER? selective, whereas the tris(hydroxyphenyl)-thiophenes were ER? selective; analogous furan-core compounds generally have lower affinity and less selectivity. Some diarylthiophenes show distinct superagonist activity in reporter gene assays, giving maximal activities 2�3 times that of estradiol, and modeling suggests that these ligands have a different interaction with a hydrogen-bonding residue in helix-11. Ligand-core modification may be a new strategy for developing ER ligands whose selectivity is based on having transcriptional activity greater than that of estradiol. PMID:23586645
High constitutive activity of native H3 receptors regulates histamine neurons in brain
S�verine Morisset; Agn�s Rouleau; Xavier Ligneau; Florence Gbahou; Jo�l Tardivel-Lacombe; Holger Stark; Walter Schunack; C. Robin Ganellin; Jean-Michel Arrang
Some G-protein-coupled receptors display `constitutive activity', that is, spontaneous activity in the absence of agonist. This means that a proportion of the receptor population spontaneously undergoes an allosteric transition, leading to a conformation that can bind G proteins. The process has been shown to occur with recombinant receptors expressed at high density, and\\/or mutated, but also non-mutated recombinant receptors expressed
Preferential Binding of an Odor Within Olfactory Receptors: A Precursor to Receptor Activation
Using computational methods, which allow mechanistic insights at a molecular level, we explored the olfactory receptor (OR)�odor interactions for 2 mouse ORs, S79 and S86. Both ORs have been previously experimentally, functionally characterized. The odors used were mostly carboxylic acids, which differed in chain length, substituents on the primary carbon atom-chain and degree of unsaturation. These odors elicited varied activation responses from both ORs. Our studies revealed that both receptors have 2 distinct binding sites. Preferential binding in 1 of the 2 sites is correlated with OR activation. The activating odorants: nonanedioic acid, heptanoic acid, and octanoic acid for OR S79 and nonanoic acid for OR S86 preferentially bind in the region bound by transmembranes (TMs [helical domains]) III, IV, V, and VI. The non excitatory odorants heptanol for S79 and heptanoic acid for S86 showed a greater likelihood of binding in the region bound by TMs I, II, III, and VII. Nanosecond-scale molecular dynamics simulations of the physiologically relevant conditions of docked OR�odorant complexes enabled us to quantitatively assess the roles of individual OR amino acids in odor binding. Amino acid�odorant contact maps and distance determinations over the course of the simulations lend support to our conclusions. PMID:24398973
Serotonin 5-HT2C receptor agonist promotes hypophagia via downstream activation of melanocortin 4 receptors.
Lam, Daniel D; Przydzial, Magdalena J; Ridley, Simon H; Yeo, Giles S H; Rochford, Justin J; O'Rahilly, Stephen; Heisler, Lora K
The neurotransmitter serotonin (5-hydroxytryptamine) is a well-established modulator of energy balance. Both pharmacological and genetic evidence implicate the serotonin 2C receptor (5-HT(2C)R) as a critical receptor mediator of serotonin's effects on ingestive behavior. Here we characterized the effect of the novel and selective 5-HT(2C)R agonist BVT.X on energy balance in obese and lean mice and report that BVT.X significantly reduces acute food intake without altering locomotor activity or oxygen consumption. In an effort to elucidate the mechanism of this effect, we examined the chemical phenotype of 5-HT(2C)R-expressing neurons in a critical brain region affecting feeding behavior, the arcuate nucleus of the hypothalamus. We show that 5-HT(2C)Rs are coexpressed with neurons containing proopiomelanocortin, known to potently affect appetite, in the arcuate nucleus of the hypothalamus of the mouse. We then demonstrate that prolonged infusion with BVT.X in obese mice significantly increases Pomc mRNA and reduces body weight, percent body fat, and initial food intake. To evaluate the functional importance of melanocortin circuitry in the effect of BVT.X on ingestive behavior, we assessed mice with disrupted melanocortin pathways. We report that mice lacking the melanocortin 4 receptor are not responsive to BVT.X-induced hypophagia, demonstrating that melanocortins acting on melanocortin 4 receptor are a requisite downstream pathway for 5-HT(2C)R agonists to exert effects on food intake. The data presented here not only indicate that the novel 5-HT(2C)R agonist BVT.X warrants further investigation as a treatment for obesity but also elucidate specific neuronal pathways potently affecting energy balance through which 5-HT(2C)R agonists regulate ingestive behavior. PMID:18039773
NMDA receptor subunit expression and PAR2 receptor activation in colospinal afferent neurons (CANs) during inflammation induced visceral hypersensitivity
Suckow, Shelby K; Caudle, Robert M
Background Visceral hypersensitivity is a clinical observation made when diagnosing patients with functional bowel disorders. The cause of visceral hypersensitivity is unknown but is thought to be attributed to inflammation. Previously we demonstrated that a unique set of enteric neurons, colospinal afferent neurons (CANs), co-localize with the NR1 and NR2D subunits of the NMDA receptor as well as with the PAR2 receptor. The aim of this study was to determine if NMDA and PAR2 receptors expressed on CANs contribute to visceral hypersensitivity following inflammation. Recently, work has suggested that dorsal root ganglion (DRG) neurons expressing the transient receptor potential vanilloid-1 (TRPV1) receptor mediate inflammation induced visceral hypersensitivity. Therefore, in order to study CAN involvement in visceral hypersensitivity, DRG neurons expressing the TRPV1 receptor were lesioned with resiniferatoxin (RTX) prior to inflammation and behavioural testing. Results CANs do not express the TRPV1 receptor; therefore, they survive following RTX injection. RTX treatment resulted in a significant decrease in TRPV1 expressing neurons in the colon and immunohistochemical analysis revealed no change in peptide or receptor expression in CANs following RTX lesioning as compared to control data. Behavioral studies determined that both inflamed non-RTX and RTX animals showed a decrease in balloon pressure threshold as compared to controls. Immunohistochemical analysis demonstrated that the NR1 cassettes, N1 and C1, of the NMDA receptor on CANs were up-regulated following inflammation. Furthermore, inflammation resulted in the activation of the PAR2 receptors expressed on CANs. Conclusion Our data show that inflammation causes an up-regulation of the NMDA receptor and the activation of the PAR2 receptor expressed on CANs. These changes are associated with a decrease in balloon pressure in response to colorectal distension in non-RTX and RTX lesioned animals. Therefore, these data suggest that CANs contribute to visceral hypersensitivity during inflammation. PMID:19772634
Expression of laminin gamma 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter.
Levavasseur, F; Li�tard, J; Ogawa, K; Th�ret, N; Burbelo, P D; Yamada, Y; Guillouzo, A; Cl�ment, B
Laminin gamma 1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin gamma 1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin gamma 1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5' flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin gamma 1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that A M(r) 60,000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures. PMID:8611150
Five Layers of Receptor Signaling in ?? T-Cell Differentiation and Activation
Ribeiro, S�rgio T.; Ribot, Julie C.; Silva-Santos, Bruno
The contributions of ?? T-cells to immunity to infection or tumors critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate ?? T-cell physiology. The understanding of how environmental signals are integrated by ?? T-cells is critical for their manipulation in clinical settings. Here, we discuss how different classes of surface receptors impact on human and murine ?? T-cell differentiation, activation, and expansion. In particular, we review the role of five receptor types: the T-cell receptor (TCR), costimulatory receptors, cytokine receptors, NK receptors, and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of ?? T-cells; the cytokine receptors IL-2R, IL-7R, and IL-15R, which drive functional differentiation and expansion of ?? T-cells; the NK receptor NKG2D and its contribution to ?? T-cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control ?? T-cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signaling in ?? T-cell differentiation and activation, and discuss its implications for the manipulation of ?? T-cells in immunotherapy. PMID:25674089
Five Layers of Receptor Signaling in ?? T-Cell Differentiation and Activation.
Ribeiro, S�rgio T; Ribot, Julie C; Silva-Santos, Bruno
The contributions of ?? T-cells to immunity to infection or tumors critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate ?? T-cell physiology. The understanding of how environmental signals are integrated by ?? T-cells is critical for their manipulation in clinical settings. Here, we discuss how different classes of surface receptors impact on human and murine ?? T-cell differentiation, activation, and expansion. In particular, we review the role of five receptor types: the T-cell receptor (TCR), costimulatory receptors, cytokine receptors, NK receptors, and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of ?? T-cells; the cytokine receptors IL-2R, IL-7R, and IL-15R, which drive functional differentiation and expansion of ?? T-cells; the NK receptor NKG2D and its contribution to ?? T-cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control ?? T-cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signaling in ?? T-cell differentiation and activation, and discuss its implications for the manipulation of ?? T-cells in immunotherapy. PMID:25674089
Dopamine receptor activation increases HIV entry into primary human macrophages.
Gaskill, Peter J; Yano, Hideaki H; Kalpana, Ganjam V; Javitch, Jonathan A; Berman, Joan W
Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. PMID:25268786
Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages
Gaskill, Peter J.; Yano, Hideaki H.; Kalpana, Ganjam V.; Javitch, Jonathan A.; Berman, Joan W.
Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. PMID:25268786
A new mechanism for growth hormone receptor activation of JAK2, and implications for related cytokine receptors
Waters, Michael J; Brooks, Andrew J; Chhabra, Yash
The growth hormone receptor was the first cytokine receptor to be cloned and crystallized, and provides a valuable exemplar for activation of its cognate kinase, JAK2. We review progress in understanding its activation mechanism, in particular the molecular movements made by this constitutively dimerized receptor in response to ligand binding, and how these lead to a separation of JAK-binding Box1 motifs. Such a separation leads to removal of the pseudokinase inhibitory domain from the kinase domain of a partner JAK2 bound to the receptor, and vice versa, leading to apposition of the kinase domains and transactivation. This may be a general mechanism for class I cytokine receptor action. PMID:25101218
Mode of action framework analysis for receptor-mediated toxicity: the Peroxisome Proliferator-Activated Receptor alpha (PPARa) as a case study
Therapeutic hypolipidemic agents and industrial chemicals that cause peroxisome proliferation and induce liver tumors in rodents activate the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARa). Research has elucidated the cellular and molecular events by w...
Peroxisome Proliferator-Activated Receptor (PPAR)-?\\/? Stimulates Differentiation and Lipid Accumulation in Keratinocytes
Matthias Schmuth; Christopher M. Haqq; William J. Cairns; Julie C. Holder; Sheri Dorsam; Sandra Chang; Peggy Lau; Ashley J. Fowler; Gary Chuang; Arthur H. Moser; Barbara E. Brown; Man Mao-Qiang; Yoshikazu Uchida; Kristina Schoonjans; Johan Auwerx; P. Chambon; Timothy M. Willson; Peter M. Elias; Kenneth R. Feingold
Peroxisome proliferator-activated receptor (PPAR) are nuclear hormone receptors that are activated by endogenous lipid metabolites. Previous studies have demonstrated that PPAR-? activation stimulates keratinocyte differentiation in vitro and in vivo, is anti-inflammatory, and improves barrier homeostasis. Recent studies have shown that PPAR-?\\/? activation induces keratinocyte differentiation in vitro. This study demonstrated that topical treatment of mice with a selective PPAR-?\\/?
Peroxisome Proliferator-Activated Receptors in Diabetic Nephropathy
Kume, Shinji; Uzu, Takashi; Isshiki, Keiji; Koya, Daisuke
Diabetic nephropathy is a leading cause of end-stage renal disease, which is increasing in incidence worldwide, despite intensive treatment approaches such as glycemic and blood pressure control in patients with diabetes mellitus. New therapeutic strategies are needed to prevent the onset of diabetic nephropathy. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear transcription factors that play important roles in lipid and glucose homeostases. These agents might prevent the progression of diabetic nephropathy, since PPAR agonists improve dyslipidemia and insulin resistance. Furthermore, data from murine models suggest that PPAR agonists also have independent renoprotective effects by suppressing inflammation, oxidative stress, lipotoxicity, and activation of the renin-angiotensin system. This review summarizes data from clinical and experimental studies regarding the relationship between PPARs and diabetic nephropathy. The therapeutic potential of PPAR agonists in the treatment of diabetic nephropathy is also discussed. PMID:19277201
D-Serine Regulation of NMDA Receptor Activity
NSDL National Science Digital Library
Herman Wolosker (Technion-Israel Institute of Technology; Department of Biochemistry REV)
The N-Methyl-D-aspartate�type glutamate receptor (NMDAR) plays a key role in several important processes involving the nervous system, including brain development, synaptic plasticity, and learning. Unlike other neurotransmitter receptors, which are activated by individual neurotransmitters, activation of NMDARs requires the binding of a coagonist (D-serine or glycine) in addition to glutamate. Although previously considered an "unnatural" amino acid, D-serine is a key regulator of NMDAR activity and may be the main physiological ligand at the coagonist site. D-Serine is synthesized in the mammalian brain and is enriched in astrocytes, a class of glial cells that ensheath synapses in the brain. Astrocytes physiologically affect NMDAR neurotransmission by releasing D-serine, suggesting that D-serine acts as a gliotransmitter. However, recent findings indicate that D-serine signaling does not depend solely on glia, because D-serine and its biosynthetic enzyme are also present in substantial amounts in neurons. Here, we discuss these new findings, which begin to shed light on the relative roles of glia and neurons in D-serine signaling.
T cell antigen receptor activation and actin cytoskeleton remodeling
Kumari, Sudha; Curado, Silvia; Mayya, Viveka
T cells constitute a crucial arm of the adaptive immune system and their optimal function is required for a healthy immune response. After the initial step of T cell-receptor (TCR) triggering by antigenic peptide complexes on antigen presenting cell (APC), the T cell exhibits extensive cytoskeletal remodeling. This cytoskeletal remodeling leads to formation of an �immunological synapse� [1] characterized by regulated clustering, segregation and movement of receptors at the interface. Synapse formation regulates T cell activation and response to antigenic peptides and proceeds via feedback between actin cytoskeleton and TCR signaling. Actin polymerization participates in various events during the synapse formation, maturation, and eventually its disassembly. There is increasing knowledge about the actin effectors that couple TCR activation to actin rearrangements [2, 3], and how defects in these effectors translate into impairment of T cell activation. In this review we aim to summarize and integrate parts of what is currently known about this feedback process. In addition, in light of recent advancements in our understanding of TCR triggering and translocation at the synapse, we speculate on the organizational and functional diversity of microfilament architecture in the T cell. PMID:23680625
Molecular Details of the Activation of the ? Opioid Receptor
Shim, Jihyun; Coop, Andrew; MacKerell, Alexander D.
Molecular details of ? opioid receptor activations were obtained using molecular dynamics simulations of the receptor in the presence of 3 agonists, 3 antagonists, a partial agonist and on the constitutively active T279K mutant. Agonists have a higher probability of direct interactions of their basic nitrogen (N) with Asp147 as compared to antagonists, indicating that direct ligand-Asp147 interactions modulate activation. Medium size substituents on the basic N of antagonists lead to steric interactions that perturb N-Asp147 interactions, while additional favorable interactions occur with larger basic N substituents, such as in N-phenethylnormorphine, restoring N-Asp147 interactions, leading to agonism. With the orvinols, the increased size of the C19 substituent in buprenorphine over diprenorphine leads increased interactions with residues adjacent to Asp147, partially overcoming the presence of the cyclopropyl N substituent, such that buprenorphine is a partial agonist. Results also indicate different conformational properties of the intracellular regions of the transmembrane helices in agonists versus antagonists. PMID:23758404
Activity of lingual, laryngeal and oesophageal receptors in conscious sheep.
Falempin, M; Rousseau, J P
Vagal afferent impulse traffic has been studied in conscious sheep by electromyographic recording from the motor units of the sterno-cleido-mastoid (s.c.m.) muscle reinnervated by sensory vagal axons. Units which responded during movements of the tongue and during the pharyngolaryngeal and oesophageal stages of swallowing were chosen for this study. Lingual units showed a phasic discharge bearing a temporal relation to movements of the tongue during licking of the lips or chewing of a bolus before swallowing. Laryngeal units had no spontaneous activity. A discharge occurred with the ascending movement of the larynx during swallowing. Oesophageal units did not exhibit any tonic activity. They fired only at the time of primary or secondary oesophageal peristalsis. The oesophageal units showed a bimodal distribution. The oesophageal receptors are more concentrated at the beginning and the end of the thoracic oesophagus. During primary peristalsis, the afferent discharge was reinforced in only 57% of the cases when sheep swallowed a bolus (pellets or inflated balloons). When the discharge was reinforced, its increase ceased as volumes of the bolus were increased from 20 to 40 ml. During local oesophageal contractions, the afferent discharge was only present when the inflated balloon was located at the site of the receptor. It was enhanced at the time the primary peristaltic wave passed over the balloon. Inflation of a second balloon cranially in the oesophagus led to abolition of the activity of the unit at the caudal site though the distension there was maintained. PMID:6707965
Methamphetamine Increases Locomotion and Dopamine Transporter Activity in Dopamine D5 Receptor-Deficient Mice
Hayashizaki, Seiji; Hirai, Shinobu; Ito, Yumi; Honda, Yoshiko; Arime, Yosefu; Sora, Ichiro; Okado, Haruo; Kodama, Tohru; Takada, Masahiko
Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine receptor subtypes in mammals. The functional relevance of D5 dopamine receptors in the central nervous system is not well understood. To determine the functional relevance of D5 dopamine receptors, we created D5 dopamine receptor-deficient mice and then used these mice to assess the roles of D5 dopamine receptors in the behavioral response to methamphetamine. Interestingly, D5 dopamine receptor-deficient mice displayed increased ambulation in response to methamphetamine. Furthermore, dopamine transporter threonine phosphorylation levels, which regulate amphetamine-induced dopamine release, were elevated in D5 dopamine receptor-deficient mice. The increase in methamphetamine-induced locomotor activity was eliminated by pretreatment with the dopamine transporter blocker GBR12909. Taken together, these results suggest that dopamine transporter activity and threonine phosphorylation levels are regulated by D5 dopamine receptors. PMID:24155877
A novel receptor involved in T-cell activation
Benjamin G. Cocks; Chia-Chun J. Chang; Hans Yssel; Jan E. de Vries; Gregorio Aversa
OPTIMAL T-cell activation and T-cell expansion require triggering by T-cell antigen receptors and co-stimulatory signals provided by accessory cells1�-3. A major co-stimulatory pathway involves crosslinking the CD28 molecule on T cells by its ligands CD80 or CD86 expressed on antigen-presenting cells4�-7. But recent studies8,9 on CD28-deficient mice have indicated that CD28 is not required for all T-cell responses and that
Artificial masculinization in tilapia involves androgen receptor activation.
Golan, Matan; Levavi-Sivan, Berta
Estrogens have a pivotal role in natural female sexual differentiation of tilapia while lack of steroids results in testicular development. Despite the fact that androgens do not participate in natural sex differentiation, synthetic androgens, mainly 17-?-methyltestosterone (MT) are effective in the production of all-male fish in aquaculture. The sex inversion potency of synthetic androgens may arise from their androgenic activity or else as inhibitors of aromatase activity. The current study is an attempt to differentiate between the two alleged activities in order to evaluate their contribution to the sex inversion process and aid the search for novel sex inversion agents. In the present study, MT inhibited aromatase activity, when applied in vitro as did the non-aromatizable androgen dihydrotestosterone (DHT). In comparison, exposure to fadrozole, a specific aromatase inhibitor, was considerably more effective. Androgenic activity of MT was evaluated by exposure of Sciaenochromis fryeri fry to the substance and testing for the appearance of blue color. Flutamide, an androgen antagonist, administered concomitantly with MT, reduced the appearance of the blue color and the sex inversion potency of MT in a dose-dependent manner. In tilapia, administration of MT, fadrozole or DHT resulted in efficient sex inversion while flutamide reduced the sex inversion potency of all three compounds. In the case of MT and DHT the decrease in sex inversion efficiency caused by flutamide is most likely due to the direct blocking of the androgen binding to its cognate receptor. The negative effect of flutamide on the efficiency of the fadrozole treatment may indicate that the masculinizing activity of fadrozole may be attributed to excess, un-aromatized, androgens accumulated in the differentiating gonad. The present study shows that when androgen receptors are blocked, there is a reduction in the efficiency of sex inversion treatments. Our results suggest that in contrast to natural sex differentiation, during sex inversion treatments, androgens, either endogenous or exogenous, participate in inducing testicular differentiation. PMID:24815887
The Environmental Estrogen, Nonylphenol, Activates the Constitutive Androstane Receptor
Hernandez, Juan P.; Huang, Wendong; Chapman, Laura M.; Chua, Steven; Moore, David D.; Baldwin, William S.
Nonylphenol (NP) and its parent compounds, the nonylphenol ethoxylates are some of the most prevalent chemicals found in U.S. waterways. NP is also resistant to biodegradation and is a known environmental estrogen, which makes NP a chemical of concern. Our data show that NP also activates the constitutive androstane receptor (CAR), an orphan nuclear receptor important in the induction of detoxification enzymes, including the P450s. Transactivation assays demonstrate that NP increases murine CAR (mCAR) transcriptional activity, and NP treatment can overcome the inhibitory effects of the inverse agonist, androstanol, on mCAR activation. Treatment of wild-type (CAR +/+) mice with NP at 50 or 75 mg/kg/day increases Cyp2b protein expression in a dose-dependent manner as demonstrated by Western blotting, and was confirmed by quantitative reverse transcription�PCR of Cyp2b10 transcript levels. CAR-null (CAR ?/?) mice show no increased expression of Cyp2b following NP treatment, indicating that CAR is required for NP-mediated Cyp2b induction. In addition, NP increases the translocation of CAR into the nucleus, which is the key step in the commencement of CAR's transcriptional activity. NP also induced CYP2B6 in primary human hepatocytes, and increased Cyp2b10 messenger RNA and protein expression in humanized CAR mice, indicating that NP is an activator of human CAR as well. In conclusion, NP is a CAR activator, and this was demonstrated in vitro with transactivation assays and in vivo with transgenic CAR mouse models. PMID:17483497
Structural determinants of the insulin receptor-related receptor activation by alkali.
Deyev, Igor E; Mitrofanova, Alla V; Zhevlenev, Egor S; Radionov, Nikita; Berchatova, Anastasiya A; Popova, Nadezhda V; Serova, Oxana V; Petrenko, Alexander G
IRR is a member of the insulin receptor (IR) family that does not have any known agonist of a peptide nature but can be activated by mildly alkaline medium and was thus proposed to function as an extracellular pH sensor. IRR activation by alkali is defined by its N-terminal extracellular region. To reveal key structural elements involved in alkali sensing, we developed an in vitro method to quantify activity of IRR and its mutants. Replacing the IRR L1C domains (residues 1-333) or L2 domain (residues 334-462) or both with the homologous fragments of IR reduced the receptor activity to 35, 64, and 7% percent, respectively. Within L1C domains, five amino acid residues (Leu-135, Gly-188, Arg-244, and vicinal His-318 and Lys-319) were identified as IRR-specific by species conservation analysis of the IR family. These residues are exposed and located in junctions between secondary structure folds. The quintuple mutation of these residues to alanine had the same negative effect as the entire L1C domain replacement, whereas none of the single mutations was as effective. Separate mutations of these five residues and of L2 produced partial negative effects that were additive. The pH dependence of cell-expressed mutants (L1C and L2 swap, L2 plus triple LGR mutation, and L2 plus quintuple LGRHK mutation) was shifted toward alkalinity and, in contrast with IRR, did not show significant positive cooperativity. Our data suggest that IRR activation is not based on a single residue deprotonation in the IRR ectodomain but rather involves synergistic conformational changes at multiple points. PMID:24121506
Structural Determinants of the Insulin Receptor-related Receptor Activation by Alkali*
Deyev, Igor E.; Mitrofanova, Alla V.; Zhevlenev, Egor S.; Radionov, Nikita; Berchatova, Anastasiya A.; Popova, Nadezhda V.; Serova, Oxana V.; Petrenko, Alexander G.
IRR is a member of the insulin receptor (IR) family that does not have any known agonist of a peptide nature but can be activated by mildly alkaline medium and was thus proposed to function as an extracellular pH sensor. IRR activation by alkali is defined by its N-terminal extracellular region. To reveal key structural elements involved in alkali sensing, we developed an in vitro method to quantify activity of IRR and its mutants. Replacing the IRR L1C domains (residues 1�333) or L2 domain (residues 334�462) or both with the homologous fragments of IR reduced the receptor activity to 35, 64, and 7% percent, respectively. Within L1C domains, five amino acid residues (Leu-135, Gly-188, Arg-244, and vicinal His-318 and Lys-319) were identified as IRR-specific by species conservation analysis of the IR family. These residues are exposed and located in junctions between secondary structure folds. The quintuple mutation of these residues to alanine had the same negative effect as the entire L1C domain replacement, whereas none of the single mutations was as effective. Separate mutations of these five residues and of L2 produced partial negative effects that were additive. The pH dependence of cell-expressed mutants (L1C and L2 swap, L2 plus triple LGR mutation, and L2 plus quintuple LGRHK mutation) was shifted toward alkalinity and, in contrast with IRR, did not show significant positive cooperativity. Our data suggest that IRR activation is not based on a single residue deprotonation in the IRR ectodomain but rather involves synergistic conformational changes at multiple points. PMID:24121506
B Cell Activation Triggered by the Formation of the Small Receptor Cluster: A Computational Study
Hat, Beata; Kazmierczak, Bogdan; Lipniacki, Tomasz
We proposed a spatially extended model of early events of B cell receptors (BCR) activation, which is based on mutual kinase-receptor interactions that are characteristic for the immune receptors and the Src family kinases. These interactions lead to the positive feedback which, together with two nonlinearities resulting from the double phosphorylation of receptors and Michaelis-Menten dephosphorylation kinetics, are responsible for the system bistability. We demonstrated that B cell can be activated by a formation of a tiny cluster of receptors or displacement of the nucleus. The receptors and Src kinases are activated, first locally, in the locus of the receptor cluster or the region where the cytoplasm is the thinnest. Then the traveling wave of activation propagates until activity spreads over the whole cell membrane. In the models in which we assume that the kinases are free to diffuse in the cytoplasm, we found that the fraction of aggregated receptors, capable to initiate B cell activation decreases with the decreasing thickness of cytoplasm and decreasing kinase diffusion. When kinases are restricted to the cell membrane - which is the case for most of the Src family kinases - even a cluster consisting of a tiny fraction of total receptors becomes activatory. Interestingly, the system remains insensitive to the modest changes of total receptor level. The model provides a plausible mechanism of B cells activation due to the formation of small receptors clusters collocalized by binding of polyvalent antigens or arising during the immune synapse formation. PMID:21998572
A restricted population of CB1 cannabinoid receptors with neuroprotective activity
Chiarlone, Anna; Bellocchio, Luigi; Bl�zquez, Cristina; Resel, Eva; Soria-G�mez, Edgar; Cannich, Astrid; Ferrero, Jos� J.; Sagredo, Onintza; Benito, Cristina; Romero, Juli�n; S�nchez-Prieto, Jos�; Lutz, Beat; Fern�ndez-Ruiz, Javier; Galve-Roperh, Ismael; Guzm�n, Manuel
The CB1 cannabinoid receptor, the main molecular target of endocannabinoids and cannabis active components, is the most abundant G protein-coupled receptor in the mammalian brain. Of note, CB1 receptors are expressed at the synapses of two opposing (i.e., GABAergic/inhibitory and glutamatergic/excitatory) neuronal populations, so the activation of one and/or another receptor population may conceivably evoke different effects. Despite the widely reported neuroprotective activity of the CB1 receptor in animal models, the precise pathophysiological relevance of those two CB1 receptor pools in neurodegenerative processes is unknown. Here, we first induced excitotoxic damage in the mouse brain by (i) administering quinolinic acid to conditional mutant animals lacking CB1 receptors selectively in GABAergic or glutamatergic neurons, and (ii) manipulating corticostriatal glutamatergic projections remotely with a designer receptor exclusively activated by designer drug pharmacogenetic approach. We next examined the alterations that occur in the R6/2 mouse, a well-established model of Huntington disease, upon (i) fully knocking out CB1 receptors, and (ii) deleting CB1 receptors selectively in corticostriatal glutamatergic or striatal GABAergic neurons. The data unequivocally identify the restricted population of CB1 receptors located on glutamatergic terminals as an indispensable player in the neuroprotective activity of (endo)cannabinoids, therefore suggesting that this precise receptor pool constitutes a promising target for neuroprotective therapeutic strategies. PMID:24843137
Zebrafish mutants identify an essential role for laminins in notochord formation.
Parsons, Michael J; Pollard, Steven M; Sa�de, Leonor; Feldman, Benjamin; Coutinho, Pedro; Hirst, Elizabeth M A; Stemple, Derek L
Basement membranes are thought to be essential for organ formation, providing the scaffold on which individual cells organize to form complex tissues. Laminins are integral components of basement membranes. To understand the development of a simple vertebrate organ, we have used positional cloning to characterize grumpy and sleepy, two zebrafish loci known to control notochord formation, and find that they encode laminin beta1 and laminin gamma1, respectively. Removal of either chain results in the dramatic loss of laminin 1 staining throughout the embryo and prevents formation of the basement membrane surrounding the notochord. Notochord cells fail to differentiate and many die by apoptosis. By transplantation, we demonstrate that, for both grumpy and sleepy, notochord differentiation can be rescued by exogenous sources of the missing laminin chain, although notochordal sources are also sufficient for rescue. These results demonstrate a clear in vivo requirement for laminin beta1 and laminin gamma1 in the formation of a specific vertebrate organ and show that laminin or the laminin-dependent basement membrane is essential for the differentiation of chordamesoderm to notochord. PMID:12070089
Spatial and temporal control of laminin-332 and -511 expressions during hair morphogenesis.
Imanishi, Hisayoshi; Tsuruta, Daisuke; Tateishi, Chiharu; Sugawara, Koji; Kobayashi, Hiromi; Ishii, Masamitsu; Kishi, Kazuo
Hair is one of the smallest organs, but has many important functions to mammals. Hair morphogenesis occurs through the reciprocal exchange of epithelial and mesenchymal signals. There are some reports about the expression of laminin-511 and -332 during hair morphogenesis, but are no reports of the chronological expression and function of laminin-511 and its counter regulator laminin-332 during hair morphogenesis. Our results of immunoblotting revealed that laminin-332 proteins were detected at stage 0 and downregulated during stage 1 to stage 2, and then recovered at stage 3. However, laminin ?5 expression was constant throughout stages 0-3. According to the results of semi-quantitative RT-PCR, the mRNA expression of all laminin-332 subunits increased gradually from stage 0 to stage 2, while the mRNA expression of all laminin-511 subunits remained constant from stage 0 to stage 3. Our results suggest that the proper expression of laminin-332 and laminin-511 may regulate appropriate hair morphogenesis. PMID:23529140
Muscarinic Receptor Activation Elicits Sustained, Recurring Depolarizations in Reticulospinal Neurons
Smetana, R. W.; Alford, S.; Dubuc, R.
In lampreys, brain stem reticulospinal (RS) neurons constitute the main descending input to the spinal cord and activate the spinal locomotor central pattern generators. Cholinergic nicotinic inputs activate RS neurons, and consequently, induce locomotion. Cholinergic muscarinic agonists also induce locomotion when applied to the brain stem of birds. This study examined whether bath applications of muscarinic agonists could activate RS neurons and initiate motor output in lampreys. Bath applications of 25 ?M muscarine elicited sustained, recurring depolarizations (mean duration of 5.0 � 0.5 s recurring with a mean period of 55.5 � 10.3 s) in intracellularly recorded rhombencephalic RS neurons. Calcium imaging experiments revealed that muscarine induced oscillations in calcium levels that occurred synchronously within the RS neuron population. Bath application of TTX abolished the muscarine effect, suggesting the sustained depolarizations in RS neurons are driven by other neurons. A series of lesion experiments suggested the caudal half of the rhombencephalon was necessary. Microinjections of muscarine (75 ?M) or the muscarinic receptor (mAchR) antagonist atropine (1 mM) lateral to the rostral pole of the posterior rhombencephalic reticular nucleus induced or prevented, respectively, the muscarinic RS neuron response. Cells immunoreactive for muscarinic receptors were found in this region and could mediate this response. Bath application of glutamatergic antagonists (6-cyano-7-nitroquinoxaline-2,3-dione/D-2-amino-5-phosphonovaleric acid) abolished the muscarine effect, suggesting that glutamatergic transmission is needed for the effect. Ventral root recordings showed spinal motor output coincides with RS neuron sustained depolarizations. We propose that unilateral mAchR activation on specific cells in the caudal rhombencephalon activates a circuit that generates synchronous sustained, recurring depolarizations in bilateral populations of RS neurons. PMID:17344371
The MT2 receptor stimulates axonogenesis and enhances synaptic transmission by activating Akt signaling.
Liu, D; Wei, N; Man, H-Y; Lu, Y; Zhu, L-Q; Wang, J-Z
The MT2 receptor is a principal type of G protein-coupled receptor that mainly mediates the effects of melatonin. Deficits of melatonin/MT2 signaling have been found in many neurological disorders, including Alzheimer's disease, the most common cause of dementia in the elderly, suggesting that preservation of the MT2 receptor may be beneficial to these neurological disorders. However, direct evidence linking the MT2 receptor to cognition-related synaptic plasticity remains to be established. Here, we report that the MT2 receptor, but not the MT1 receptor, is essential for axonogenesis both in vitro and in vivo. We find that axon formation is retarded in MT2 receptor knockout mice, MT2-shRNA electroporated brain slices or primary neurons treated with an MT2 receptor selective antagonist. Activation of the MT2 receptor promotes axonogenesis that is associated with an enhancement in excitatory synaptic transmission in central neurons. The signaling components downstream of the MT2 receptor consist of the Akt/GSK-3?/CRMP-2 cascade. The MT2 receptor C-terminal motif binds to Akt directly. Either inhibition of the MT2 receptor or disruption of MT2 receptor-Akt binding reduces axonogenesis and synaptic transmission. Our data suggest that the MT2 receptor activates Akt/GSK-3?/CRMP-2 signaling and is necessary and sufficient to mediate functional axonogenesis and synaptic formation in central neurons. PMID:25501601
Antitussive activity of Withania somnifera and opioid receptors.
Nos�lov�, Gabriela; Sivov�, Veronika; Ray, Bimalendu; Fra?ov�, So?a; Ondrejka, Igor; Fle�kov�, Dana
Arabinogalactan is a polysaccharide isolated from the roots of the medicinal plant Withania somnifera L. It contains 65% arabinose and 18% galactose. The aim of the present study was to evaluate the antitussive activity of arabinogalactan in conscious, healthy adult guinea pigs and the role of the opioid pathway in the antitussive action. A polysaccharide extract was given orally in a dose of 50 mg/kg. Cough was induced by an aerosol of citric acid in a concentration 0.3 mol/L, generated by a jet nebulizer into a plethysmographic chamber. The intensity of cough response was defined as the number of cough efforts counted during a 3-min exposure to the aerosol. The major finding was that arabinogalactan clearly suppressed the cough reflex; the suppression was comparable with that of codeine that was taken as a reference drug. The involvement of the opioid system was tested with the use of a blood-brain barrier penetrable, naloxone hydrochloride, and non-penetrable, naloxone methiodide, to distinguish between the central and peripheral mu-opioid receptor pathways. Both opioid antagonists acted to reverse the arabinogalactan-induced cough suppression; the reversion was total over time with the latter antagonist. We failed to confirm the presence of a bronchodilating effect of the polysaccharide, which could be involved in its antitussive action. We conclude that the polysaccharide arabinogalactan from Withania somnifera has a distinct antitussive activity consisting of cough suppression and that this action involves the mu-opioid receptor pathways. PMID:25252908
[Activators, receptors and signal transduction pathways of blood platelets].
Shaturny?, V I; Shakhidzhanov, S S; Sveshnikova, A N; Panteleev, M A
Platelet participation in hemostatic plug formation requires transition into an activated state (or, rather, variety of states) upon action of agonists like ADP, thromboxane A , collagen, thrombin, and others. The mechanisms of action for different agonists, their receptors and signaling pathways associated with them, as well as the mechanisms of platelet response inhibition are the subject of the present review. Collagen exposed upon vessel wall damage induced initial platelet attachment and start of thrombus formation, which involves numerous processes such as aggregation, activation of integrins, granule secretion and increase of intracellular Ca2+. Thrombin, ADP, thromboxane A , and ATP activated platelets that were not initially in contact with the wall and induce additional secretion of activating substances. Vascular endothelium and secretory organs also affect platelet activation, producing both positive (adrenaline) an d negative (prostacyclin, nitric oxide) regulators, thereby determining the relation of activation and inhibition signals, which plays a significant role in the formation of platelet aggregate under normal and pathological conditions. The pathways of platelet signaling are still incompletely understood, and their exploration presents an important objective both for basic cell biology and for the development of new drugs, the methods of diagnostics and of treatment of hemostasis disorders. PMID:24837309
Differential Superactivation of Adenylyl Cyclase Isozymes after Chronic Activation of the CB1 Cannabinoid Receptor1
Vogel, Zvi
Cannabinoid Receptor1 MAN-HEE RHEE, IGAL NEVO, TOMER AVIDOR-REISS, RIVKA LEVY, and ZVI VOGEL Department observed on chronic activation of the CB1 cannabinoid receptor. Moreover, using COS-7 cells cotrans- fected and especially G dimers in the cannabinoid-induced superactivation of AC. Two cannabinoid receptor subtypes, CB1
Tonic GABAA receptor conductance in medial subnucleus of the tractus solitarius neurons is inhibited by activation of ?-opioid receptors.
Herman, Melissa A; Gillis, Richard A; Vicini, Stefano; Dretchen, Kenneth L; Sahibzada, Niaz
Our laboratory previously reported that gastric activity is controlled by a robust GABA(A) receptor-mediated inhibition in the medial nucleus of the tractus solitarius (mNTS) (Herman et al. 2009), and that ?-opioid receptor activation inhibits gastric tone by suppression of this GABA signaling (Herman et al. 2010). These data raised two questions: 1) whether any of this inhibition was due to tonic GABA(A) receptor-mediated conductance in the mNTS; and 2) whether ?-opioid receptor activation suppressed both tonic and phasic GABA signaling. In whole cell recordings from rat mNTS neurons, application of three GABA(A) receptor antagonists (gabazine, bicuculline, and picrotoxin) produced a persistent reduction in holding current and decrease in population variance or root mean square (RMS) noise, suggesting a blockade of tonic GABA signaling. Application of gabazine at a lower concentration abolished phasic currents, but had no effect on tonic currents or RMS noise. Application of the ?-subunit preferring agonist gaboxadol (THIP) produced a dose-dependent persistent increase in holding current and RMS noise. Pretreatment with tetrodotoxin prevented the action of gabazine, but had no effect on the THIP-induced current. Membrane excitability was unaffected by the selective blockade of phasic inhibition, but was increased by blockade of both phasic and tonic currents. In contrast, activation of tonic currents decreased membrane excitability. Application of the ?-opioid receptor agonist DAMGO produced a persistent reduction in holding current that was not observed following pretreatment with a GABA(A) receptor antagonist and was not evident in mice lacking the ?-subunit. These data suggest that mNTS neurons possess a robust tonic inhibition that is mediated by GABA(A) receptors containing the ?-subunit, that determines membrane excitability, and that is partially regulated by ?-opioid receptors. PMID:22114164
Diabetes or peroxisome proliferator-activated receptor alpha agonist increases mitochondrial thioesterase I activity in heart
Technology Transfer Automated Retrieval System (TEKTRAN)
Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a transcriptional regulator of the expression of mitochondrial thioesterase I (MTE-I) and uncoupling protein 3 (UCP3), which are induced in the heart at the mRNA level in response to diabetes. Little is known about the regulation of pr...
Canonical Wnt signalling regulates epithelial patterning by modulating levels of laminins in zebrafish appendages
Nagendran, Monica; Arora, Prateek; Gori, Payal; Mulay, Aditya; Ray, Shinjini; Jacob, Tressa; Sonawane, Mahendra
The patterning and morphogenesis of body appendages � such as limbs and fins � is orchestrated by the activities of several developmental pathways. Wnt signalling is essential for the induction of limbs. However, it is unclear whether a canonical Wnt signalling gradient exists and regulates the patterning of epithelium in vertebrate appendages. Using an evolutionarily old appendage � the median fin in zebrafish � as a model, we show that the fin epithelium exhibits graded changes in cellular morphology along the proximo-distal axis. This epithelial pattern is strictly correlated with the gradient of canonical Wnt signalling activity. By combining genetic analyses with cellular imaging, we show that canonical Wnt signalling regulates epithelial cell morphology by modulating the levels of laminins, which are extracellular matrix components. We have unravelled a hitherto unknown mechanism involved in epithelial patterning, which is also conserved in the pectoral fins � evolutionarily recent appendages that are homologous to tetrapod limbs. PMID:25519245
Canonical Wnt signalling regulates epithelial patterning by modulating levels of laminins in zebrafish appendages.
Nagendran, Monica; Arora, Prateek; Gori, Payal; Mulay, Aditya; Ray, Shinjini; Jacob, Tressa; Sonawane, Mahendra
The patterning and morphogenesis of body appendages - such as limbs and fins - is orchestrated by the activities of several developmental pathways. Wnt signalling is essential for the induction of limbs. However, it is unclear whether a canonical Wnt signalling gradient exists and regulates the patterning of epithelium in vertebrate appendages. Using an evolutionarily old appendage - the median fin in zebrafish - as a model, we show that the fin epithelium exhibits graded changes in cellular morphology along the proximo-distal axis. This epithelial pattern is strictly correlated with the gradient of canonical Wnt signalling activity. By combining genetic analyses with cellular imaging, we show that canonical Wnt signalling regulates epithelial cell morphology by modulating the levels of laminins, which are extracellular matrix components. We have unravelled a hitherto unknown mechanism involved in epithelial patterning, which is also conserved in the pectoral fins - evolutionarily recent appendages that are homologous to tetrapod limbs. PMID:25519245
The activation of liver X receptors inhibits toll-like receptor-9-induced foam cell formation.
Sorrentino, Rosalinda; Morello, Silvana; Chen, Shuang; Bonavita, Eduardo; Pinto, Aldo
Toll-like receptors (TLRs) are related to foam cell formation (FCF), key event in the establishment/progression of atherosclerosis. The activation of TLR2 and TLR4 can increase FCF. The aim of this study was to evaluate the role of TLR9 in FCF. Murine macrophages were treated with CpG-ODN, TLR9 agonist, and oxidized particles of LDL (Paz-PC) and FCF was analyzed by means of Oil Red O staining. The administration of CpG-ODN plus Paz-PC onto macrophages increased the amount of lipid droplets, correlated to increased levels of tumor necrosis factor (TNF)-alpha, IFNbeta, and IP-10. The underlying mechanism by which TLR9 ligation influenced Paz-PC in the FCF was NF-kappaB- and IRF7-dependent, as observed by higher levels of phosphorylated IkappaBalpha, increased nuclear translocation of the p65 subunit, lower levels of the total IKKalpha protein and higher release of interferon-dependent cytokines, such as IP-10. Liver X receptors (LXRs) regulate lipid cellular transport and negatively modulate TLR-dependent signaling pathways. Indeed, the addition of GW3965, synthetic LXRs agonist, significantly reduced FCF after CpG-ODN plus Paz-PC stimulation. In this condition, we observed decreased levels of the nuclear translocation of the p65 subunit, related to the higher presence of LXRalpha into the nucleus. TNF-alpha, IP-10, and IFNbeta levels were reduced by the administration of GW3965 following CpG-ODN and Paz-PC treatment. In conclusion, the activation of TLR9 facilitates the formation of foam cells in an NF-kappaB- and IRF7-dependent manner, countered by the activation of LXRs. This study further support LXRs as potential anti-atherosclerotic target. PMID:20049870
Activation of EphA Receptors Mediates the Recruitment of the Adaptor Protein Slap, Contributing to the Downregulation of N-Methyl-d-Aspartate Receptors
Semerdjieva, Sophia; Abdul-Razak, Hayder H.; Salim, Sharifah S.; Y��ez-Mu�oz, Rafael J.; Chen, Philip E.; Tarabykin, Victor
Regulation of the activity of N-methyl-d-aspartate receptors (NMDARs) at glutamatergic synapses is essential for certain forms of synaptic plasticity underlying learning and memory and is also associated with neurotoxicity and neurodegenerative diseases. In this report, we investigate the role of Src-like adaptor protein (Slap) in NMDA receptor signaling. We present data showing that in dissociated neuronal cultures, activation of ephrin (Eph) receptors by chimeric preclustered eph-Fc ligands leads to recruitment of Slap and NMDA receptors at the sites of Eph receptor activation. Interestingly, our data suggest that prolonged activation of EphA receptors is as efficient in recruiting Slap and NMDA receptors as prolonged activation of EphB receptors. Using established heterologous systems, we examined whether Slap is an integral part of NMDA receptor signaling. Our results showed that Slap does not alter baseline activity of NMDA receptors and does not affect Src-dependent potentiation of NMDA receptor currents in Xenopus oocytes. We also demonstrate that Slap reduces excitotoxic cell death triggered by activation of NMDARs in HEK293 cells. Finally, we present evidence showing reduced levels of NMDA receptors in the presence of Slap occurring in an activity-dependent manner, suggesting that Slap is part of a mechanism that homeostatically modulates the levels of NMDA receptors. PMID:23382070
Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1) by Diacylglycerol (DAG)
Dong Ho Woo; Sung Jun Jung; Mei Hong Zhu; Chul-Kyu Park; Yong Ho Kim; Seog Bae Oh; C Justin Lee
The capsaicin receptor, known as transient receptor potential channel vanilloid subtype 1 (TRPV1), is activated by a wide range of noxious stimulants and putative ligands such as capsaicin, heat, pH, anandamide, and phosphorylation by protein kinase C (PKC). However, the identity of endogenous activators for TRPV1 under physiological condition is still debated. Here, we report that diacylglycerol (DAG) directly activates
Technology Transfer Automated Retrieval System (TEKTRAN)
Pterostilbene, a stilbenoid antioxidant found in blueberries, grapes, other small fruits, and in woody plants was shown to activate the peroxisome proliferator-activated receptor alpha (PPAR alpha) isoform. This nuclear receptor is proposed to mediate the activity of lipid-lowering drugs such as th...
Modulation of Toll-Like Receptor Activity by Leukocyte Ig-Like Receptors and Their Effects during Bacterial Infection
Pilsbury, Louise E.; Allen, Rachel L.; Vordermeier, Martin
Toll-like receptors (TLRs) are a potent trigger for inflammatory immune responses. Without tight regulation their activation could lead to pathology, so it is imperative to extend our understanding of the regulatory mechanisms that govern TLR expression and function. One family of immunoregulatory proteins which can provide a balancing effect on TLR activity are the Leukocyte Ig-like receptors (LILRs), which act as innate immune receptors for self-proteins. Here we describe the LILR family, their inhibitory effect on TLR activity in cells of the monocytic lineage, their signalling pathway, and their antimicrobial effects during bacterial infection. Agents have already been identified which enhances or inhibits LILR activity raising the future possibility that modulation of LILR function could be used as a means to modulate TLR activity. PMID:20634939
CB2 cannabinoid receptor activation produces antinociception by stimulating peripheral release of endogenous opioids
Mohab M. Ibrahim; Frank Porreca; Josephine Lai; Phillip J. Albrecht; Frank L. Rice; Alla Khodorova; Gudarz Davar; Alexandros Makriyannis; Todd W. Vanderah; Heriberto P. Mata; T. Philip Malan Jr.
CB2 cannabinoid receptor-selective agonists are promising candidates for the treatment of pain. CB2 receptor activation inhibits acute, inflammatory, and neuropathic pain responses but does not cause central nervous system (CNS) effects, consistent with the lack of CB2 receptors in the normal CNS. To date, there has been virtually no information regarding the mechanism of CB2 receptor-mediated inhibition of pain responses.
Structure-activity relationships of phenylalkylamines as agonist ligands for 5-HT(2A) receptors.
Blaazer, Antoni R; Smid, Pieter; Kruse, Chris G
Agonist activation of central 5-HT(2A) receptors results in diverse effects, such as hallucinations and changes of consciousness. Recent findings indicate that activation of the 5-HT(2A) receptor also leads to interesting physiological responses, possibly holding therapeutic value. Selective agonists are needed to study the full therapeutic potential of this receptor. 5-HT(2A) ligands with agonist profiles are primarily derived from phenylalkylamines, indolealkylamines, and certain piperazines. Of these, phenylalkylamines, most notably substituted phenylisopropylamines, are considered the most selective agonists for 5-HT(2) receptors. This review summarizes the structure-activity relationships (SAR) of phenylalkylamines as agonist ligands for 5-HT(2A) receptors. Selectivity is a central theme, as is selectivity for the 5-HT(2A) receptor and for its specific signaling pathways. SAR data from receptor affinity studies, functional assays, behavioral drug discrimination as well as human studies are discussed. PMID:18666267
Purinergic signaling negatively regulates activity of an olfactory receptor in an odorant-dependent manner.
Yu, Y; Zhang, C
Extracellular purines and pyrimidines are important signaling molecules that mediate diverse biological functions via cell surface purinergic receptors. Although purinergic modulation to olfactory activity has been reported, cell-specific expression and action of purinergic receptors deserve further exploration. We physiologically characterized expression of purinergic receptors in a set of olfactory sensory neurons that are responsive to both acetophenone and benzaldehyde (AB-OSNs). Sparsely distributed in the most ventral olfactory receptor zone, AB-OSNs were activated by P2 purinergic receptor agonists but not by P1 purinergic receptor agonist adenosine. Both P2X-selective agonist ?,?-methylene ATP and P2Y-selective agonist uridine 5'-triphosphate (UTP) were stimulatory to AB-OSNs, indicating expression of both P2X and P2Y purinergic receptors in AB-OSNs. Pharmacological characterization of receptor specificity using various P2X and P2Y agonists and antagonists illustrated that P2X1 and P2Y2 receptors played major roles in purinergic signaling in AB-OSNs. Interestingly, the results of purinergic modulation to acetophenone-evoked responses were different from those to benzaldehyde-evoked responses within the same neurons. Activation of P2X1 receptors had more profound inhibitory effects on benzaldehyde-evoked intracellular calcium elevation than on acetophenone-evoked responses within the same neurons, and the reverse was true when P2Y2 receptors were activated. Cross-adaptation data showed that acetophenone and benzaldehyde bound to the same olfactory receptor. Thus, our study has demonstrated that purinergic signaling of P2X and P2Y receptors has different effects on olfactory transduction mediated by a defined olfactory receptor and the consequences of purinergic modulation of olfactory activity might depend on stereotypic structures of the odorant-receptor complex. PMID:24928349
Modulation of transient receptor vanilloid 1 activity by transient receptor potential ankyrin 1.
Spahn, Viola; Stein, Christoph; Z�llner, Christian
Transient receptor potential vanilloid 1 (TRPV1) is a nonselective ligand-gated cation channel responding to noxious heat, protons, and chemicals such as capsaicin. TRPV1 is expressed in sensory neurons and plays a critical role in pain associated with tissue injury, inflammation, and nerve lesions. Transient receptor potential ankyrin 1 (TRPA1) is coexpressed with TRPV1. It is activated by compounds that cause a burning sensation (e.g., mustard oil) and, indirectly, by components of the inflammatory milieu eliciting nociceptor excitation and pain hypersensitivity. Previous studies indicate an interaction of TRPV1 and TRPA1 signaling pathways. Here we sought to examine the molecular mechanisms underlying such interactions in nociceptive neurons. We first excluded physical interactions of both channels using radioligand binding studies. By microfluorimetry, electrophysiological experiments, cAMP measurements, and site-directed mutagenesis we found a sensitization of TRPV1 after TRPA1 stimulation with mustard oil in a calcium and cAMP/protein kinase A (PKA)-dependent manner. TRPA1 stimulation enhanced TRPV1 phosphorylation via the putative PKA phosphorylation site serine 116. We also detected calcium-sensitive increased TRPV1 activity after TRPA1 activation in dorsal root ganglion neurons. The inhibition of TRPA1 by HC-030031 (1,2,3,6-tetrahydro-1,3-dimethyl-N-[4-(1-methylethyl)phenyl]-2,6-dioxo-7H-purine-7-acetamide, 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide) after its initial stimulation (and the calcium-insensitive TRPA1 mutant D477A) still showed increased capsaicin-induced TRPV1 activity. This excludes a calcium-induced additive TRPA1 current after TRPV1 stimulation. Our study shows sensitization of TRPV1 via activation of TRPA1, which involves adenylyl cyclase, increased cAMP, subsequent translocation and activation of PKA, and phosphorylation of TRPV1 at PKA phosphorylation residues. This suggests that cross-sensitization of TRP channels contributes to enhanced pain sensitivity in inflamed tissues. PMID:24275229
Activation and Regulation of Purinergic P2X Receptor Channels
Coddou, Claudio; Yan, Zonghe; Obsil, Tomas; Huidobro-Toro, J. Pablo
Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions. PMID:21737531
Enhancer transcripts mark active estrogen receptor binding sites
Hah, Nasun; Murakami, Shino; Nagari, Anusha; Danko, Charles G.; Kraus, W. Lee
We have integrated and analyzed a large number of data sets from a variety of genomic assays using a novel computational pipeline to provide a global view of estrogen receptor 1 (ESR1; a.k.a. ER?) enhancers in MCF-7 human breast cancer cells. Using this approach, we have defined a class of primary transcripts (eRNAs) that are transcribed uni- or bidirectionally from estrogen receptor binding sites (ERBSs) with an average transcription unit length of ?3�5 kb. The majority are up-regulated by short treatments with estradiol (i.e., 10, 25, or 40 min) with kinetics that precede or match the induction of the target genes. The production of eRNAs at ERBSs is strongly correlated with the enrichment of a number of genomic features that are associated with enhancers (e.g., H3K4me1, H3K27ac, EP300/CREBBP, RNA polymerase II, open chromatin architecture), as well as enhancer looping to target gene promoters. In the absence of eRNA production, strong enrichment of these features is not observed, even though ESR1 binding is evident. We find that flavopiridol, a CDK9 inhibitor that blocks transcription elongation, inhibits eRNA production but does not affect other molecular indicators of enhancer activity, suggesting that eRNA production occurs after the assembly of active enhancers. Finally, we show that an enhancer transcription �signature� based on GRO-seq data can be used for de novo enhancer prediction across cell types. Together, our studies shed new light on the activity of ESR1 at its enhancer sites and provide new insights about enhancer function. PMID:23636943
Domains for activation and inactivation in G protein-coupled receptors--a mutational analysis of constitutive activity of the adenosine A2B receptor.
Peeters, Miriam C; Li, Qilan; Elands, Rachel; van Westen, Gerard J P; Lenselink, Eelke B; M�ller, Christa E; IJzerman, Adriaan P
G protein-coupled receptors (GPCRs) are a major drug target and can be activated by a range of stimuli, from photons to proteins. Most, if not all, GPCRs also display a basal level of biological response in the absence of such a stimulus. This level of so-called constitutive activity results from a delicate energy equilibrium that exists between the active and the inactive state of the receptor and is the first determinant in the GPCR activation mechanism. Here we describe new insights in specific regions of the adenosine A2B receptor that are essential in activation and inactivation. We developed a new screening method using the MMY24 S. Cerevisiae strain by which we were able to screen for constitutively inactive mutants receptors (CIMs). We applied this screening method on a mutagenic library of the adenosine A2B receptor, where random mutations were introduced in transmembrane domains four and five (TM4 and TM5) linked by extracellular loop 2 (EL2). The screen resulted in the identification of 22 single and double mutant receptors, all showing a decrease in constitutive activity as well as in agonist potency. By comparing these results with a previous screen of the same mutagenic library for constitutively active mutant receptors (CAMs), we discovered specific regions in this G protein-coupled receptor involved in either inactivation or activation or both. The results suggest the activation mechanism of GPCRs to be much less restricted to sites of high conservation or direct interaction with the ligand or G protein and illustrate how dynamic the activation process of GPCRs is. PMID:25179146
The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation
Hansen, Guido; Hercus, Timothy R.; McClure, Barbara J.; Stomski, Frank C.; Dottore, Mara; Powell, Jason; Ramshaw, Hayley; Woodcock, Joanna M.; Xu, Yibin; Guthridge, Mark; McKinstry, William J.; Lopez, Angel F.; Parker, Michael W. (SVIMR-A); (Hanson)
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific {alpha} subunit and a {beta}c subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.
Peroxisome Proliferator-Activated Receptor-? in Thyroid Autoimmunity
Ferrari, Silvia Martina; Fallahi, Poupak; Vita, Roberto; Benvenga, Salvatore
Peroxisome proliferator-activated receptor- (PPAR-) ? expression has been shown in thyroid tissue from patients with thyroiditis or Graves' disease and furthermore in the orbital tissue of patients with Graves' ophthalmopathy (GO), such as in extraocular muscle cells. An increasing body of evidence shows the importance of the (C-X-C motif) receptor 3 (CXCR3) and cognate chemokines (C-X-C motif) ligand (CXCL)9, CXCL10, and CXCL11, in the T helper 1 immune response and in inflammatory diseases such as thyroid autoimmune disorders. PPAR-? agonists show a strong inhibitory effect on the expression and release of CXCR3 chemokines, in vitro, in various kinds of cells, such as thyrocytes, and in orbital fibroblasts, preadipocytes, and myoblasts from patients with GO. Recently, it has been demonstrated that rosiglitazone is involved in a higher risk of heart failure, stroke, and all-cause mortality in old patients. On the contrary, pioglitazone has not shown these effects until now; this favors pioglitazone for a possible use in patients with thyroid autoimmunity. However, further studies are ongoing to explore the use of new PPAR-? agonists in the treatment of thyroid autoimmune disorders. PMID:25722716
Retinoid X receptor and peroxisome proliferator-activated receptor-gamma agonists cooperate to inhibit matrix metalloproteinase gene expression
Peter S Burrage; Adam C Schmucker; Yanqing Ren; Michael B Sporn; Constance E Brinckerhoff
INTRODUCTION: We recently described the ability of retinoid X receptor (RXR) ligand LG100268 (LG268) to inhibit interleukin-1-beta (IL-1-?)-driven matrix metalloproteinase-1 (MMP-1) and MMP-13 gene expression in SW-1353 chondrosarcoma cells. Other investigators have demonstrated similar effects in chondrocytes treated with rosiglitazone, a ligand for peroxisome proliferator-activated receptor-gamma (PPAR?), for which RXR is an obligate dimerization partner. The goals of this study
RSUME Enhances Glucocorticoid Receptor SUMOylation and Transcriptional Activity
Druker, Jimena; Liberman, Ana C.; Antunica-Noguerol, Mar�a; Gerez, Juan; Paez-Pereda, Marcelo; Rein, Theo; I�iguez-Lluh�, Jorge A.; Holsboer, Florian
Glucocorticoid receptor (GR) activity is modulated by posttranslational modifications, including phosphorylation, ubiquitination, and SUMOylation. The GR has three SUMOylation sites: lysine 297 (K297) and K313 in the N-terminal domain (NTD) and K721 within the ligand-binding domain. SUMOylation of the NTD sites mediates the negative effect of the synergy control motifs of GR on promoters with closely spaced GR binding sites. There is scarce evidence on the role of SUMO conjugation to K721 and its impact on GR transcriptional activity. We have previously shown that RSUME (RWD-containing SUMOylation enhancer) increases protein SUMOylation. We now demonstrate that RSUME interacts with the GR and increases its SUMOylation. RSUME regulates GR transcriptional activity and the expression of its endogenous target genes, FKBP51 and S100P. RSUME uncovers a positive role for the third SUMOylation site, K721, on GR-mediated transcription, demonstrating that GR SUMOylation acts positively in the presence of a SUMOylation enhancer. Both mutation of K721 and small interfering RNA-mediated RSUME knockdown diminish GRIP1 coactivator activity. RSUME, whose expression is induced under stress conditions, is a key factor in heat shock-induced GR SUMOylation. These results show that inhibitory and stimulatory SUMO sites are present in the GR and at higher SUMOylation levels the stimulatory one becomes dominant. PMID:23508108
RSUME enhances glucocorticoid receptor SUMOylation and transcriptional activity.
Druker, Jimena; Liberman, Ana C; Antunica-Noguerol, Mar�a; Gerez, Juan; Paez-Pereda, Marcelo; Rein, Theo; I�iguez-Lluh�, Jorge A; Holsboer, Florian; Arzt, Eduardo
Glucocorticoid receptor (GR) activity is modulated by posttranslational modifications, including phosphorylation, ubiquitination, and SUMOylation. The GR has three SUMOylation sites: lysine 297 (K297) and K313 in the N-terminal domain (NTD) and K721 within the ligand-binding domain. SUMOylation of the NTD sites mediates the negative effect of the synergy control motifs of GR on promoters with closely spaced GR binding sites. There is scarce evidence on the role of SUMO conjugation to K721 and its impact on GR transcriptional activity. We have previously shown that RSUME (RWD-containing SUMOylation enhancer) increases protein SUMOylation. We now demonstrate that RSUME interacts with the GR and increases its SUMOylation. RSUME regulates GR transcriptional activity and the expression of its endogenous target genes, FKBP51 and S100P. RSUME uncovers a positive role for the third SUMOylation site, K721, on GR-mediated transcription, demonstrating that GR SUMOylation acts positively in the presence of a SUMOylation enhancer. Both mutation of K721 and small interfering RNA-mediated RSUME knockdown diminish GRIP1 coactivator activity. RSUME, whose expression is induced under stress conditions, is a key factor in heat shock-induced GR SUMOylation. These results show that inhibitory and stimulatory SUMO sites are present in the GR and at higher SUMOylation levels the stimulatory one becomes dominant. PMID:23508108
Defective Muscle Basement Membrane and Lack of M-Laminin in the Dystrophic dy\\/dy Mouse
Hong Xu; Peter Christmas; Xiao-Rong Wu; Ulla M. Wewer; Eva Engvall
M-laminin is a major member of the laminin family of basement membrane proteins. It is prominently expressed in striated muscle and peripheral nerve. M-laminin is deficient in patients with the autosomal recessive Fukuyama congenital muscular dystrophy but is normal in patients with the sex-linked Duchenne and Becker muscular dystrophies. We have examined M-laminin expression in mice with autosomal recessive muscular
Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X2-green fluorescent protein receptors
Khakh, Baljit S.; Smith, W. Bryan; Chiu, Chi-Sung; Ju, Donghong; Davidson, Norman; Lester, Henry A.
ATP-gated P2X2 receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X2 receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X2-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X2-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X2-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X2 receptor distribution on the seconds time scale. PMID:11296257
Receptor-Mediated Activation of Heterotrimeric G-Proteins in
Devreotes, Peter
-proteincoupled receptors (GPCRs). Excited receptors catalyze the ex- change of guanosine triphosphate (GTP) for guanosine and free complexes to signal to downstream effec- tors. The intrinsic guanosine triphosphatase (GTPase
Regulation of laminin and COUP-TF expression in extraembryonic endodermal cells.
Murray, P; Edgar, D
Laminin expression and the subsequent deposition of a basement membrane by primitive endoderm cells is necessary for early mammalian development. We demonstrate that the transcription factors COUP-TF I and II are up-regulated in primitive endoderm cells faster than LAMB1 and LAMC1, and that either COUP-TF is sufficient to induce expression of these laminin genes. PMID:11231078
Immunohistochemical localization of laminin and type IV collagen in human cutaneous sensory nerve formations
J. A. Vega; I. Esteban; F. J. Naves; M. E. Valle; L. Malinovsky
We used immunohistochemical techniques and monoclonal antibodies to localize two basement membrane components (laminin and type IV collagen) in the nerves and sensory nerve formations, or corpuscles, supplying human digital skin. Furthermore, neurofilament proteins, S-100 protein and epithelial membrane antigen were studied in parallel. In dermal nerve trunks, immunostaining for laminin and type IV collagen was found to be co-localized
"Mirror image" antagonists of thrombin-induced platelet activation based on thrombin receptor structure.
Hung, D T; Vu, T K; Wheaton, V I; Charo, I F; Nelken, N A; Esmon, N; Esmon, C T; Coughlin, S R
Platelet activation by thrombin plays a critical role in hemostasis and thrombosis. Based on structure-activity studies of a cloned platelet thrombin receptor, we designed two "mirror image" antagonists of thrombin and thrombin receptor function. First, "uncleavable" peptides mimicking the receptor domain postulated to interact with thrombin were found to be potent thrombin inhibitors. Second, proteolytically inactive mutant thrombins designed to bind but not cleave the thrombin receptor were found to be specific antagonists of receptor activation by thrombin. The effectiveness of these designed antagonists in blocking thrombin-induced platelet activation suggests a model for thrombin-receptor interaction and possible strategies for the development of novel antithrombotic agents. Images PMID:1310695
EGF activates its receptor by removing interactions that autoinhibit ectodomain dimerization.
Ferguson, Kathryn M; Berger, Mitchell B; Mendrola, Jeannine M; Cho, Hyun Soo; Leahy, Daniel J; Lemmon, Mark A
Epidermal growth factor (EGF) receptor is the prototype of the ErbB (HER) family receptor tyrosine kinases (RTKs), which regulate cell growth and differentiation and are implicated in many human cancers. EGF activates its receptor by inducing dimerization of the 621 amino acid EGF receptor extracellular region. We describe the 2.8 A resolution crystal structure of this entire extracellular region (sEGFR) in an unactivated state. The structure reveals an autoinhibited configuration, where the dimerization interface recently identified in activated sEGFR structures is completely occluded by intramolecular interactions. To activate the receptor, EGF binding must promote a large domain rearrangement that exposes this dimerization interface. This contrasts starkly with other RTK activation mechanisms and suggests new approaches for designing ErbB receptor antagonists. PMID:12620237
Beta-arrestin inhibits CAMKKbeta-dependent AMPK activation downstream of protease-activated-receptor-2
Background Proteinase-activated-receptor-2 (PAR2) is a seven transmembrane receptor that can activate two separate signaling arms: one through G?q and Ca2+ mobilization, and a second through recruitment of ?-arrestin scaffolds. In some cases downstream targets of the G?q/Ca2+ signaling arm are directly inhibited by ?-arrestins, while in other cases the two pathways are synergistic; thus ?-arrestins act as molecular switches capable of modifying the signal generated by the receptor. Results Here we demonstrate that PAR2 can activate adenosine monophosphate-activated protein kinase (AMPK), a key regulator of cellular energy balance, through Ca2+-dependent Kinase Kinase ? (CAMKK?), while inhibiting AMPK through interaction with ?-arrestins. The ultimate outcome of PAR2 activation depended on the cell type studied; in cultured fibroblasts with low endogenous ?-arrestins, PAR2 activated AMPK; however, in primary fat and liver, PAR2 only activated AMPK in ?-arrestin-2-/- mice. ?-arrestin-2 could be co-immunoprecipitated with AMPK and CAMKK? under baseline conditions from both cultured fibroblasts and primary fat, and its association with both proteins was increased by PAR2 activation. Addition of recombinant ?-arrestin-2 to in vitro kinase assays directly inhibited phosphorylation of AMPK by CAMKK? on Thr172. Conclusions Studies have shown that decreased AMPK activity is associated with obesity and Type II Diabetes, while AMPK activity is increased with metabolically favorable conditions and cholesterol lowering drugs. These results suggest a role for ?-arrestin in the inhibition of AMPK signaling, raising the possibility that ?-arrestin-dependent PAR2 signaling may act as a molecular switch turning a positive signal to AMPK into an inhibitory one. PMID:20858278
Trigeminal Medullary Dorsal Horn Neurons Activated by Nasal Stimulation Coexpress AMPA, NMDA, and NK1 Receptors
McCulloch, P. F.; DiNovo, K. M.; Westerhaus, D. J.; Vizinas, T. A.; Peevey, J. F.; Lach, M. A.; Czarnocki, P.
Afferent information initiating the cardiorespiratory responses during nasal stimulation projects from the nasal passages to neurons within the trigeminal medullary dorsal horn (MDH) via the anterior ethmoidal nerve (AEN). Central AEN terminals are thought to release glutamate to activate the MDH neurons. This study was designed to determine which neurotransmitter receptors (AMPA, kainate, or NMDA glutamate receptor subtypes or the Substance P receptor NK1) are expressed by these activated MDH neurons. Fos was used as a neuronal marker of activated neurons, and immunohistochemistry combined with epifluorescent microscopy was used to determine which neurotransmitter receptor subunits were coexpressed by activated MDH neurons. Results indicate that, during nasal stimulation with ammonia vapors in urethane-anesthetized Sprague-Dawley rats, activated neurons within the superficial MDH coexpress the AMPA glutamate receptor subunits GluA1 (95.8%) and GluA2/3 (88.2%), the NMDA glutamate receptor subunits GluN1 (89.1%) and GluN2A (41.4%), and NK1 receptors (64.0%). It is therefore likely that during nasal stimulation the central terminals of the AEN release glutamate and substance P that then produces activation of these MDH neurons. The involvement of AMPA and NMDA receptors may mediate fast and slow neurotransmission, respectively, while NK1 receptor involvement may indicate activation of a nociceptive pathway. PMID:24967301
Differential effects of exercise on brain opioid receptor binding and activation in rats.
Arida, Ricardo Mario; Gomes da Silva, S�rgio; de Almeida, Alexandre Aparecido; Cavalheiro, Esper Abr�o; Zavala-Tecuapetla, Cecilia; Brand, Serge; Rocha, Luisa
Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTP?S under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms. PMID:25330347
Novel protein interactors of urokinase-type plasminogen activator receptor.
Mekkawy, Ahmed H; De Bock, Charles E; Lin, Zhen; Morris, David L; Wang, Yao; Pourgholami, Mohammad H
The urokinase-type plasminogen activator receptor (uPAR) has been implicated in tumor growth and metastasis. The crystal structure of uPAR revealed that the external surface is largely free to interact with a number of proteins. Additionally, due to absence of an intracellular cytoplasmic protein domain, many of the biological functions of uPAR necessitate interactions with other proteins. Here, we used yeast two-hybrid screening of breast cancer cDNA library to identify hSpry1 and HAX1 proteins as putative candidate proteins that interact with uPAR bait constructs. Interaction between these two candidates and uPAR was confirmed by GST-pull down, co-immunoprecipitation assays and confocal microscopy. These novel interactions that have been identified may also provide further evidence that uPAR can interact with a number of other proteins which may influence a range of biological functions. PMID:20696135
Peroxisome Proliferator-Activated Receptors in HCV-Related Infection
Dharancy, S�bastien; Lemoine, Maud; Mathurin, Philippe; Serfaty, Lawrence; Dubuquoy, Laurent
The topic of peroxisome proliferator-activated receptors has been developed in the field of hepatology allowing envisaging therapeutic strategies for the most frequent chronic liver diseases such as chronic infection with hepatitis C virus (HCV). PPARs contribute to wide physiological processes within the liver such as lipid/glucid metabolisms, inflammatory response, cell differentiation, and cell cycle. In vitro experiments and animal studies showed that PPAR? discloses anti-inflammatory property, and PPAR? discloses anti-inflammatory, antifibrogenic, and antiproliferative properties in the liver. Experimental and human studies showed impaired PPARs expression and function during HCV infection. The available nonhepatotoxic agonists of PPARs may constitute a progress in the therapeutic management of patients chronically infected with HCV. PMID:19343188
Peroxisome proliferator-activated receptors and hepatitis C virus
Eslam, M.; Khattab, M. A.; Harrison, S. A.
The prevalence of type 2 diabetes mellitus and insulin resistance are higher among people chronically infected with hepatitis C (CHC) when compared with the general population and people with other causes of chronic liver disease. Both insulin resistance and diabetes are associated with adverse outcomes across all stages of CHC, including the liver transplant population. CHC is also associated with the development of hepatic steatosis, a common histological feature present in approximately 55% (32�81%) of cases. There is a complex interrelationship between insulin resistance and hepatic steatosis and both are postulated to aggravate each other. The peroxisome proliferator-activated receptors (PPARs) are nuclear factors involved in the regulation of glucose, lipid homeostasis, inflammatory response, cell differentiation, and cell cycle. The relationship between hepatitis C virus replication and PPARs has been the focus of recent study. Given the availability of potent agonists, PPARs may represent a novel pharmacological target in the treatment of CHC. PMID:22043232
Regulation of GABA Receptor Activity by Neurosteroids and Phosphorylation
NSDL National Science Digital Library
Jeffrey Tasker (Tulane University; Department of Cell and Molecular Biology REV)
These two animations show two models for how neurosteroids regulate the flow of chloride ions (Cl-) through ionotropic gamma-aminobutyric acid (GABA) receptors. In the first model, binding of the neurosteroid allows a protein kinase C (PKC) phosphorylation site to become accessible. Phosphorylation of the channel increases flux through the channel. In the second model, phosphorylation by PKC allows the neurosteroid to bind and increase flux through the channel. The animations have two parts: (i) a diagrammatic representation of the sequence of events at the channel in the membrane and (ii) a representative current trace of data obtained using electrophysiological techniques. These animations would be useful in teaching how allosteric modulators (neurosteroids) and covalent modulators (kinases) can work together as regulators of protein activity.
Neurokinin-1 receptor activation in Botzinger complex evokes bradypnoea.
Fong, Angelina Y; Potts, Jeffrey T
In the present study, we examined the role of the neurokinin-1 receptor (NK1R) in the modulation of respiratory rhythm in a functionally identified bradypnoeic region of the ventral respiratory group (VRG) in the in situ arterially perfused juvenile rat preparation. In electrophysiologically and functionally identified bradypnoeic sites corresponding to the B�tzinger complex (B�tC), microinjection of the selective NK1R agonist [Sar(9)-Met(O(2))(11)]-substance P (SSP) produced a significant reduction in phrenic frequency mediated exclusively by an increase in expiratory duration (T(E)). The reduction was characterized by a significant increase in postinspiratory (post-I) duration with no effect on either late-expiratory duration (E2) or inspiratory duration (T(I)). In contrast, in a functionally identified tachypnoeic region, corresponding to the preB�tzinger complex (Pre-B�tC), control microinjection of SSP elicited tachypnoea. Pretreatment with the NK1R antagonist CP99994 in the B�tC significantly attenuated the bradypnoeic response to SSP injection and blunted the increase in T(E) duration. This effect of SSP mimicked the extension of T(E) produced by activation of the Hering-Breuer reflex. Therefore, we hypothesized that activation of NK1Rs in the B�tC is requisite for the expiratory-lengthening effect of the Hering-Breuer reflex. Unilateral electrical stimulation of the cervical vagus nerve produced bradypnoea by exclusively extending T(E). Ipsilateral blockade of NK1Rs by CP99994 following blockade of the contralateral B�tC by the GABA(A) receptor agonist muscimol significantly reduced the extension of T(E) produced by vagal stimulation. Results from the present study demonstrate that selective activation of NK1Rs in a functionally identified bradypnoeic region of the VRG can depress respiratory frequency by selectively lengthening post-I duration and provide evidence that endogenous activation of NK1Rs in the B�tC appears to be involved in the expiratory-lengthening effect of the Hering-Breuer reflex. In conclusion, our findings demonstrate that selective activation of NK1Rs in discrete regions of the VRG can exert functionally diverse effects on breathing. PMID:16825299
Tonic Activation of NMDA Receptors by Ambient Glutamate Enhances Excitability of Neurons
P. Sah; S. Hestrin; R. A. Nicoll
Voltage clamp recordings and noise analysis from pyramidal cells in hippocampal slices indicate that N-methyl-D-aspartate (NMDA) receptors are tonically active. On the basis of the known concentration of glutamate in the extracellular fluid, this tonic action is likely caused by the ambient glutamate level. NMDA receptors are voltage-sensitive, thus background activation of these receptors imparts a regenerative electrical property to
Activation of GABAA receptors containing the �4 subunit by GABA and pentobarbital
Gustav Akk; John Bracamontes; Joe Henry Steinbach
The activation properties of GABAA receptors containing ?4?2?2 and ?4?2? subunits were examined in the presence of GABA or pentobarbital. The receptors were expressed trans- iently in HEK 293 cells, and the electrophysiological experiments were carried out using cell- attached single-channel patch clamp or whole-cell macroscopic recordings. The data show that GABA is a stronger activator of ?4?2?2 receptors than
Transcriptional activation of the Drosophila ecdysone receptor by insect and plant ecdysteroids
Keith D Baker; James T Warren; Carl S Thummel; Lawrence I Gilbert; David J Mangelsdorf
A number of insect ecdysteroids, plant ecdysteroids and juvenoids were assayed for their ability to activate Drosophila nuclear receptors in transfected tissue culture cells. Discrete modifications to 20-hydroxyecdysone, the apparent natural ligand for the ecdysone receptor (EcR), conferred dramatic changes on the transcriptional activity of this receptor, suggesting that other biologically relevant EcR ligands may exist. Conversely, none of the
Peroxisome proliferator-activated receptor-? agonist troglitazone protects against nondiabetic glomerulosclerosis in rats
Li-Jun Ma; Carmelita Marcantoni; Macrae F. Linton; Sergio Fazio; Agnes B. Fogo
Peroxisome proliferator-activated receptor-? agonist troglitazone protects against nondiabetic glomerulosclerosis in rats.BackgroundPeroxisome proliferator-activated receptor-? (PPAR?) is a member of the nuclear receptor superfamily of ligand-dependent transcriptional factors with beneficial effects in diabetes mediated by improved insulin sensitivity and lipid metabolism, but potential adverse effects in atherosclerosis by promoting in vitro foam cell formation. We explored whether a PPAR? agonist, troglitazone (TGL),
Analysis of the Heat Shock Response in Mouse Liver Reveals Transcriptional Dependence on the Nuclear Receptor Peroxisome Proliferator-Activated Receptor alpha �(PPARa)
BACKGROUND: The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. Many of these genes are also transcriptionally regulated by h...
Investigating real-time activation of adenosine receptors by bioluminescence resonance energy transfer technique
NASA Astrophysics Data System (ADS)
Huang, Yimei; Yang, Hongqin; Zheng, Liqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen
Adenosine receptors play important roles in many physiological and pathological processes, for example regulating myocardial oxygen consumption and the release of neurotransmitters. The activations of adenosine receptors have been studied by some kinds of techniques, such as western blot, immunohistochemistry, etc. However, these techniques cannot reveal the dynamical response of adenosine receptors under stimulation. In this paper, bioluminescence resonance energy transfer technique was introduced to study the real-time activation of adenosine receptors by monitoring the dynamics of cyclic adenosine monophosphate (cAMP) level. The results showed that there were significant differences between adenosine receptors on real-time responses under stimulation. Moreover, the dynamics of cAMP level demonstrated that competition between adenosine receptors existed. Taken together, our study indicates that monitoring the dynamics of cAMP level using bioluminescence resonance energy transfer technique could be one potential approach to investigate the mechanism of competitions between adenosine receptors.
Characterization and partial purification of solubilized active opiate receptors from toad brain.
Ruegg, U T; Cuenod, S; Hiller, J M; Gioannini, T; Howells, R D; Simon, E J
Opiate receptors have been solubilized from toad brain membranes in active form by using digitonin. Between 40% and 50% of the stereospecific binding activity present in toad brain membranes is recoverable in the ultracentrifugal supernatant of digitonin extracts. Binding of opiates to the solubilized receptor is enhanced 4- to 5-fold by decreasing digitonin concentration to 0.1% or less prior to binding. The solubilized receptor is similar to the membrane-bound receptor in its affinity for various ligands and its sensitivity to heat, trypsin, and N-ethylmaleimide. Moreover, the sodium effect seen in membrane-bound receptor is retained in the solubilized preparation. Both membrane-bound and soluble toad receptors show weak binding of enkephalins, suggesting that they are predominantly of the mu type. The solubilized opiate receptor has an approximate molecular weight of 350,000-400,000. Purification of up to 20-fold has been achieved by gel filtration on Sepharose CL-6B. PMID:6270689
Dysregulated Fc receptor function in active rheumatoid arthritis.
Magnusson, Sofia E; Wennerberg, Erik; Matt, Peter; Lindqvist, Ulla; Kleinau, Sandra
Given the critical role of Fc gamma receptors (Fc?R) as primary targets for autoantibody-mediated effects an important issue is how the Fc?R pathway is affected in autoimmune disorders. Here we investigated the Fc?R function in monocytes from rheumatoid arthritis (RA) patients in relation to immunoglobulin levels and disease activity. Peripheral blood was obtained from 30 RA patients with clinical acute joint synovitis (active RA), 28 RA patients with no clinical signs of acute joint synovitis (non-active RA) and 34 healthy controls. Prior the functional studies the monocytes were characterized of their Fc?RI (CD64), II (CD32), IIb (CD32b) and III (CD16) expression as well as their cell surface bound IgG. The monocytic Fc?R function was assessed by binding of human IgG1 and IgG3 immune complexes (IC) and TNF secretion in vitro. IgG anti-citrullinated peptide antibodies (ACPA) were analyzed in the plasma. We found that monocytes from active RA patients had increased levels of Fc?RI, II and cell surface IgG concurrently with impaired Fc?R function. This was evident by reduced IgG1-IC binding and decreased TNF secretion in response to IgG3-IC. In contrast, monocytes from non-active RA patients displayed a normal Fc?R function and had increased Fc?RIIb expression together with elevated Fc?RI, II and cell surface IgG. The ACPA levels did not differ in active and non-active RA patients but correlated with the monocytic Fc?RIII expression in the patients. In conclusion, active RA patients display a dysregulated Fc?R function that may represent a novel phenotypic and likely pathogenetic marker for active RA. A disease and Fc?R function controlling effect is suggested by the increased inhibitory Fc?RIIb in non-active RA. PMID:25194756
Activated Protein C Enhances Human Keratinocyte Barrier Integrity via Sequential Activation of Epidermal Growth Factor Receptor and Tie2*
Xue, Meilang; Chow, Shu-Oi; Dervish, Suat; Chan, Yee-Ka Agnes; Julovi, Sohel M.; Jackson, Christopher J.
Keratinocytes play a critical role in maintaining epidermal barrier function. Activated protein C (APC), a natural anticoagulant with anti-inflammatory and endothelial barrier protective properties, significantly increased the barrier impedance of keratinocyte monolayers, measured by electric cell substrate impedance sensing and FITC-dextran flux. In response to APC, Tie2, a tyrosine kinase receptor, was rapidly activated within 30 min, and relocated to cell-cell contacts. APC also increased junction proteins zona occludens, claudin-1 and VE-cadherin. Inhibition of Tie2 by its peptide inhibitor or small interfering RNA abolished the barrier protective effect of APC. Interestingly, APC did not activate Tie2 through its major ligand, angiopoietin-1, but instead acted by binding to endothelial protein C receptor, cleaving protease-activated receptor-1 and transactivating EGF receptor. Furthermore, when activation of Akt, but not ERK, was inhibited, the barrier protective effect of APC on keratinocytes was abolished. Thus, APC activates Tie2, via a mechanism requiring, in sequential order, the receptors, endothelial protein C receptor, protease-activated receptor-1, and EGF receptor, which selectively enhances the PI3K/Akt signaling to enhance junctional complexes and reduce keratinocyte permeability. PMID:21173154
Odorant receptor-mediated sperm activation in disease vector mosquitoes
Pitts, R. Jason; Liu, Chao; Zhou, Xiaofan; Malpartida, Juan C.; Zwiebel, Laurence J.
Insects, such as the malaria vector mosquito, Anopheles gambiae, depend upon chemoreceptors to respond to volatiles emitted from a range of environmental sources, most notably blood meal hosts and oviposition sites. A subset of peripheral signaling pathways involved in these insect chemosensory-dependent behaviors requires the activity of heteromeric odorant receptor (OR) ion channel complexes and ligands for numerous A. gambiae ORs (AgOrs) have been identified. Although AgOrs are expressed in nonhead appendages, studies characterizing potential AgOr function in nonolfactory tissues have not been conducted. In the present study, we explore the possibility that AgOrs mediate responses of spermatozoa to endogenous signaling molecules in A. gambiae. In addition to finding AgOr transcript expression in testes, we show that the OR coreceptor, AgOrco, is localized to the flagella of A. gambiae spermatozoa where Orco-specific agonists, antagonists, and other odorant ligands robustly activate flagella beating in an Orco-dependent process. We also demonstrate Orco expression and Orco-mediated activation of spermatozoa in the yellow fever mosquito, Aedes aegypti. Moreover, we find Orco localization in testes across distinct insect taxa and posit that OR-mediated responses in spermatozoa may represent a general characteristic of insect reproduction and an example of convergent evolution. PMID:24550284
A Mechanism of Intracellular P2X Receptor Activation*
Sivaramakrishnan, Venketesh; Fountain, Samuel J.
P2X receptors (P2XRs) are ATP-activated calcium-permeable ligand-gated ion channels traditionally viewed as sensors of extracellular ATP during diverse physiological processes including pain, inflammation, and taste. However, in addition to a cell surface residency P2XRs also populate the membranes of intracellular compartments, including mammalian lysosomes, phagosomes, and the contractile vacuole (CV) of the amoeba Dictyostelium. The function of intracellular P2XRs is unclear and represents a major gap in our understanding of ATP signaling. Here, we exploit the genetic versatility of Dictyostelium to investigate the effects of physiological concentrations of ATP on calcium signaling in isolated CVs. Within the CV, an acidic calcium store, P2XRs are orientated to sense luminal ATP. Application of ATP to isolated vacuoles leads to luminal translocation of ATP and release of calcium. Mechanisms of luminal ATP translocation and ATP-evoked calcium release share common pharmacology, suggesting that they are linked processes. The ability of ATP to mobilize stored calcium is reduced in vacuoles isolated from P2XAR knock-out amoeba and ablated in cells devoid of P2XRs. Pharmacological inhibition of luminal ATP translocation or depletion of CV calcium attenuates CV function in vivo, manifesting as a loss of regulatory cell volume decrease following osmotic swelling. We propose that intracellular P2XRs regulate vacuole activity by acting as calcium release channels, activated by translocation of ATP into the vacuole lumen. PMID:22736763
Botulinum Neurotoxin Devoid of Receptor Binding Domain Translocates Active Protease
Fischer, Audrey; Mushrush, Darren J.; Lacy, D. Borden; Montal, Mauricio
Clostridium botulinum neurotoxin (BoNT) causes flaccid paralysis by disabling synaptic exocytosis. Intoxication requires the tri-modular protein to undergo conformational changes in response to pH and redox gradients across endosomes, leading to the formation of a protein-conducting channel. The ?50 kDa light chain (LC) protease is translocated into the cytosol by the ?100 kDa heavy chain (HC), which consists of two modules: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). Here we exploited the BoNT modular design to identify the minimal requirements for channel activity and LC translocation in neurons. Using the combined detection of substrate proteolysis and single-channel currents, we showed that a di-modular protein consisting only of LC and TD was sufficient to translocate active protease into the cytosol of target cells. The RBD is dispensable for cell entry, channel activity, or LC translocation; however, it determined a pH threshold for channel formation. These findings indicate that, in addition to its individual functions, each module acts as a chaperone for the others, working in concert to achieve productive intoxication. PMID:19096517
Botulinum neurotoxin devoid of receptor binding domain translocates active protease.
Fischer, Audrey; Mushrush, Darren J; Lacy, D Borden; Montal, Mauricio
Clostridium botulinum neurotoxin (BoNT) causes flaccid paralysis by disabling synaptic exocytosis. Intoxication requires the tri-modular protein to undergo conformational changes in response to pH and redox gradients across endosomes, leading to the formation of a protein-conducting channel. The approximately 50 kDa light chain (LC) protease is translocated into the cytosol by the approximately 100 kDa heavy chain (HC), which consists of two modules: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). Here we exploited the BoNT modular design to identify the minimal requirements for channel activity and LC translocation in neurons. Using the combined detection of substrate proteolysis and single-channel currents, we showed that a di-modular protein consisting only of LC and TD was sufficient to translocate active protease into the cytosol of target cells. The RBD is dispensable for cell entry, channel activity, or LC translocation; however, it determined a pH threshold for channel formation. These findings indicate that, in addition to its individual functions, each module acts as a chaperone for the others, working in concert to achieve productive intoxication. PMID:19096517
Odorant receptor-mediated sperm activation in disease vector mosquitoes.
Pitts, R Jason; Liu, Chao; Zhou, Xiaofan; Malpartida, Juan C; Zwiebel, Laurence J
Insects, such as the malaria vector mosquito, Anopheles gambiae, depend upon chemoreceptors to respond to volatiles emitted from a range of environmental sources, most notably blood meal hosts and oviposition sites. A subset of peripheral signaling pathways involved in these insect chemosensory-dependent behaviors requires the activity of heteromeric odorant receptor (OR) ion channel complexes and ligands for numerous A. gambiae ORs (AgOrs) have been identified. Although AgOrs are expressed in nonhead appendages, studies characterizing potential AgOr function in nonolfactory tissues have not been conducted. In the present study, we explore the possibility that AgOrs mediate responses of spermatozoa to endogenous signaling molecules in A. gambiae. In addition to finding AgOr transcript expression in testes, we show that the OR coreceptor, AgOrco, is localized to the flagella of A. gambiae spermatozoa where Orco-specific agonists, antagonists, and other odorant ligands robustly activate flagella beating in an Orco-dependent process. We also demonstrate Orco expression and Orco-mediated activation of spermatozoa in the yellow fever mosquito, Aedes aegypti. Moreover, we find Orco localization in testes across distinct insect taxa and posit that OR-mediated responses in spermatozoa may represent a general characteristic of insect reproduction and an example of convergent evolution. PMID:24550284
Complement activation triggered by chondroitin sulfate released by thrombin receptor-activated platelets
Summary Background Chondroitin sulfate (CS) is a glycosaminoglycan released by activated platelets. Objective Herewe test the hypothesis that CS released by activated platelets can trigger complement activation in the fluid phase. Methods and results Thrombin receptor-activating peptide (TRAP)-6 was used to activate platelets in platelet-rich plasma and blood, anticoagulated with the thrombin inhibitor lepirudin. TRAP activation induced fluid-phase complement activation, as reflected by the generation of C3a and sC5b-9, which could be attenuated by the C3 inhibitor compstatin. Chondroitinase ABC treatment of supernatants from activated platelets totally inhibited the activation, indicating that platelet-derived CS had initiated the complement activation. Furthermore, addition of purified CS to plasma strongly triggered complement activation. C1q was identified as the recognition molecule, as it bound directly to CS, and CS-triggered complement activation could be restored in C1q-depleted serum by adding purified C1q. TRAP activation of whole blood increased the expression of CD11b on leukocytes and generation of leukocyte�platelet complexes. It was demonstrated that these leukocyte functions were dependent on C3 activation and signaling via C5a, as this expression could be inhibited by compstatin and by a C5aR antagonist. Conclusions We conclude that platelets trigger complement activation in the fluid phase by releasing CS, which leads to inflammatory signals mediated by C5a. PMID:18503629
Activation of Cannabinoid Receptor 2 Enhances Osteogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells
Sun, Yong-Xin; Xu, Ai-Hua; Yang, Yang; Zhang, Jia-Xing; Yu, Ai-Wen
Bone marrow derived mesenchymal stem cells (BM-MSCs) are considered as the most promising cells source for bone engineering. Cannabinoid (CB) receptors play important roles in bone mass turnover. The aim of this study is to test if activation of CB2 receptor by chemical agonist could enhance the osteogenic differentiation and mineralization in bone BM-MSCs. Alkaline phosphatase (ALP) activity staining and real time PCR were performed to test the osteogenic differentiation. Alizarin red staining was carried out to examine the mineralization. Small interference RNA (siRNA) was used to study the role of CB2 receptor in osteogenic differentiation. Results showed activation of CB2 receptor increased ALP activity, promoted expression of osteogenic genes, and enhanced deposition of calcium in extracellular matrix. Knockdown of CB2 receptor by siRNA inhibited ALP activity and mineralization. Results of immunofluorescent staining showed that phosphorylation of p38 MAP kinase is reduced by knocking down of CB2 receptor. Finally, bone marrow samples demonstrated that expression of CB2 receptor is much lower in osteoporotic patients than in healthy donors. Taken together, data from this study suggested that activation of CB2 receptor plays important role in osteogenic differentiation of BM-MSCs. Lack of CB2 receptor may be related to osteoporosis. PMID:25685815
Significance of AT1 Receptor Independent Activation of Mineralocorticoid Receptor in Murine Diabetic Cardiomyopathy
Nagatomo, Yuji; Meguro, Tomomi; Ito, Hiroyuki; Koide, Kimi; Anzai, Toshihisa; Fukuda, Keiichi; Ogawa, Satoshi; Yoshikawa, Tsutomu
Background Diabetes mellitus (DM) has deleterious influence on cardiac performance independent of coronary artery disease and hypertension. The objective of the present study was to investigate the role of the renin-angiotensin-aldosterone system, especially angiotensin II type 1a receptor (AT1aR) and mineralocorticoid receptor (MR) signaling, in left ventricular (LV) dysfunction induced by diabetes mellitus (DM). Methods and Results DM was induced by intraperitoneal injection of streptozotocin (200 mg/kg BW) in wild-type (WT) or AT1aR knockout (KO) male mice, and they were bred during 6 or 12 weeks. Some KO mice were administered the MR antagonist eplerenone (100 mg/kg body weight). At 6 weeks, LV diastolic function was impaired in WT-DM, but preserved in KO-DM. At that time point MR mRNA expression was upregulated, NADPH oxidase subunit (p47phox) and glutathione peroxidase (GPx1) mRNA expression were upregulated, the staining intensities of LV tissue for 4-hydroxy-2-nonenal was stronger in immunohistochemistry, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive cells was increased, Bcl-2 protein expression was significantly downregulated, and the expression of SERCA2a and phosphorylated phospholamban was depressed in WT-DM, while these changes were not seen in KO-DM. At 12 weeks, however, these changes were also noted in KO-DM. Eplerenone arrested those changes. The plasma aldosterone concentration was elevated in WT-DM but not in KO-DM at 6 weeks. It showed 3.7-fold elevation at 12 weeks even in KO-DM, which suggests �aldosterone breakthrough� phenomenon. However, the aldosterone content in LV tissue was unchanged in KO-DM. Conclusions DM induced diastolic dysfunction was observed even in KO at 12 weeks, which was ameliorated by minelarocorticoid receptor antagonist, eplerenone. AT1-independent MR activation in the LV might be responsible for the pathogenesis of diabetic cardiomyopathy. PMID:24664319
Synergistic activation of the human orphan nuclear receptor SHP gene promoter by basic helix-loop-helix protein E2A and orphan nuclear receptor SF1
Han-Jong Kim; Joon-Young Kim; Yun-Yong Park; Hueng-Sik Choi
The orphan nuclear receptor small heterodimer partner (SHP; NR0B2) is an unusual orphan nuclear receptor that lacks a conventional DNA-binding domain and acts as a modulator of transcriptional activities of a number of nuclear receptors. We have previously reported that the orphan nuclear receptor ERRg activates the SHP promoter. In this study, we have found that basic helix-loop-helix (bHLH) transcription
Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation.
Miner, Jeffrey H; Li, Cong; Mudd, Jacqueline L; Go, Gloriosa; Sutherland, Ann E
Laminins are components of all basement membranes and have well demonstrated roles in diverse developmental processes, from the peri-implantation period onwards. Laminin 1 (alpha1beta1gamma1) is a major laminin found at early stages of embryogenesis in both embryonic and extraembryonic basement membranes. The laminin gamma1 chain has been shown by targeted mutation to be required for endodermal differentiation and formation of basement membranes; Lamc1(-/-) embryos die within a day of implantation. We report the generation of mice lacking laminin alpha1 and laminin beta1, the remaining two laminin 1 chains. Mutagenic insertions in both Lama1 and Lamb1 were obtained in a secretory gene trap screen. Lamb1(-/-) embryos are similar to Lamc1(-/-) embryos in that they lack basement membranes and do not survive beyond embryonic day (E) 5.5. However, in Lama1(-/-) embryos, the embryonic basement membrane forms, the embryonic ectoderm cavitates and the parietal endoderm differentiates, apparently because laminin 10 (alpha5beta1gamma1) partially compensates for the absent laminin 1. However, such compensation did not occur for Reichert's membrane, which was absent, and the embryos died by E7. Overexpression of laminin alpha5 from a transgene improved the phenotype of Lama1(-/-) embryos to the point that they initiated gastrulation, but this overexpression did not rescue Reichert's membrane, and trophoblast cells did not form blood sinuses. These data suggest that both the molecular composition and the integrity of basement membranes are crucial for early developmental events. PMID:15102706
Regional differences in the expression of laminin isoforms during mouse neural tube development
Copp, Andrew J.; Carvalho, Rita; Wallace, Adam; Sorokin, Lydia; Sasaki, Takako; Greene, Nicholas D.E.; Ybot-Gonzalez, Patricia
Many significant human birth defects originate around the time of neural tube closure or early during post-closure nervous system development. For example, failure of the neural tube to close generates anencephaly and spina bifida, faulty cell cycle progression is implicated in primary microcephaly, while defective migration of neuroblasts can lead to neuronal migration disorders such as lissencephaly. At the stage of neural tube closure, basement membranes are becoming organised around the neuroepithelium, and beneath the adjacent non-neural surface ectoderm. While there is circumstantial evidence to implicate basement membrane dynamics in neural tube and surface ectodermal development, we have an incomplete understanding of the molecular composition of basement membranes at this stage. In the present study, we examined the developing basement membranes of the mouse embryo at mid-gestation (embryonic day 9.5), with particular reference to laminin composition. We performed in situ hybridization to detect the mRNAs of all eleven individual laminin chains, and immunohistochemistry to identify which laminin chains are present in the basement membranes. From this information, we inferred the likely laminin variants and their tissues of origin: that is, whether a given basement membrane laminin is contributed by epithelium, mesenchyme, or both. Our findings reveal major differences in basement composition along the body axis, with the rostral neural tube (at mandibular arch and heart levels) exhibiting many distinct laminin variants, while the lumbar level where the neural tube is just closing shows a much simpler laminin profile. Moreover, there appears to be a marked difference in the extent to which the mesenchyme contributes laminin variants to the basement membrane, with potential contribution of several laminins rostrally, but no contribution caudally. This information paves the way towards a mechanistic analysis of basement membrane laminin function during early neural tube development in mammals. PMID:21524702
5-Hydroxytryptamine receptor activity of the dopamine receptor agonist fenoldopam in canine tracheal smooth muscle.
Gretler, D D; Jones, K C; Murphy, M B
Fenoldopam is a new vasodilator undergoing clinical trials for the treatment of hypertensive emergencies. Its pharmacologic effects result from activation of vascular dopamine-1 receptors. In canine tracheal smooth muscle strips, fenoldopam caused a concentration- and calcium-dependent increase in tension, which was not antagonized by atropine, indomethacin or the dopamine-1 receptor antagonist, SCH 23390. The EC50 (1.89 x 10(-6) M) exceeded that of serotonin or acetylcholine (8.38 x 10(-8) and 8.25 x 10(-8) M, respectively). Maximum tension was similar for fenoldopam and serotonin (11.6 +/- 1.5 g, n = 7 and 13.8 +/- 0.8 g, n = 24; P greater than .2) and considerably greater for acetylcholine (20.5 +/- 1.3 g, n = 14; P less than .005). The serotonin antagonists ketanserin and methysergide reversed completely the effect of fenoldopam (5 x 10(-7) M) with IC50 values of 2.5 x 10(-9) and 2.7 x 10(-9) M, respectively. Phentolamine, rauwolscine and chlorpheniramine were also effective, but they were less potent (IC50 values 6.6 x 10(-8), 1.0 x 10(-7) and 1.7 x 10(-7) M, respectively). By contrast, only very high concentrations (IC50, 5.3 x 10(-5) M) of terazosin produced an inhibition of fenoldopam-induced tension increases. The effect of antagonists could be overcome by increasing the fenoldopam concentration. Experiments performed on strips precontracted with serotonin (5 x 10(-8) M) revealed a very similar order of potency for the five antagonists. The addition of serotonin did not increase the tension produced by supramaximal concentrations of fenoldopam (and vice-versa), whereas acetylcholine increased tension further.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1346635
Selective Activation of Liver X Receptors by Acanthoic Acid-Related Diterpenes
Theodorakis, Emmanuel
Selective Activation of Liver X Receptors by Acanthoic Acid-Related Diterpenes Paqui G. Traves structurally related to acanthoic acid in macrophage functions. We found that diterpenes with different substitutions at the C4 position in ring A are potent activators of liver X receptors (LXR and LXR ) in both
Circ Res . Author manuscript Liver X receptor activation stimulates iron export in human alternative
Paris-Sud XI, Université de
68 MR macrophages+ + accumulate oxidized lipids, which activate Liver X Receptors (LXR and LXRCirc Res . Author manuscript Page /1 12 Liver X receptor activation stimulates iron export in human alternative macrophages Gael Bories 1 2 , Sophie Colin 1 2 , Jonathan Vanhoutte 1 2 , Bruno Derudas 1 2
Heterologous Activation of Protein Kinase C Stimulates Phosphorylation of -Opioid Receptor at Serine 344, Resulting
Tian, Weidong
Heterologous Activation of Protein Kinase C Stimulates Phosphorylation of -Opioid Receptor of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our
Redistribution of the fibrinogen receptor of human platelets after surface activation
J. C. Loftus; R. M. ALBRECHT
We investigated the whole cell distribution of the platelet membrane receptor for fibrinogen in surface-activated human platelets. Fibrinogen-labeled colloidal gold was used in conjunction with platelet whole mount preparations to visualize directly the fibrinogen receptor. Unstimulated platelets fail to bind fibrinogen, and binding was minimal in the stages of activation immediately following adhesion. The amount of fibrinogen bound per platelet
?1-adrenergic receptors activate two distinct signaling pathways in striatal neurons
Meitzen, John; Luoma, Jessie I.; Stern, Christopher M.; Mermelstein, Paul G.
Monoamine action in the dorsal striatum and nucleus accumbens plays essential roles in striatal physiology. Although research often focuses on dopamine and its receptors, norepinephrine and adrenergic receptors are also crucial in regulating striatal function. While noradrenergic neurotransmission has been identified in the striatum, little is known regarding the signaling pathways activated by ?-adrenergic receptors in this brain region. Using cultured striatal neurons, we characterized a novel signaling pathway by which activation of ?1-adrenergic receptors leads to the rapid phosphorylation of cAMP Response Element Binding Protein (CREB), a transcription-factor implicated as a molecular switch underlying long-term changes in brain function. Norepinephrine-mediated CREB phosphorylation requires ?1-adrenergic receptor stimulation of a receptor tyrosine kinase, ultimately leading to the activation of a Ras/Raf/MEK/MAPK/MSK signaling pathway. Activation of ?1-adrenergic receptors also induces CRE-dependent transcription and increased c-fos expression. In addition, stimulation of ?1-adrenergic receptors produces cAMP production, but surprisingly, ?1-adrenergic receptor activation of adenylyl cyclase was not functionally linked to rapid CREB phosphorylation. These findings demonstrate that activation of ?1-adrenergic receptors on striatal neurons can stimulate two distinct signaling pathways. These adrenergic actions can produce long-term changes in gene expression, as well as rapidly modulate cellular physiology. By elucidating the mechanisms by which norepinephrine and ?1-adrenergic receptor activation affects striatal physiology, we provide the means to more fully understand the role of monoamines in modulating striatal function, specifically how norepinephrine and ?1-adrenergic receptors may affect striatal physiology. PMID:21143600
Functional selectivity induced by mGlu4 receptor positive allosteric modulation and concomitant activation of Gq coupled receptors
Yin, Shen; Zamorano, Rocio; Conn, P. Jeffrey; Niswender, Colleen M.
Metabotropic glutamate receptors (mGlus) are a group of Family C Seven Transmembrane Spanning Receptors (7TMRs) that play important roles in modulating signaling transduction, particularly within the central nervous system. mGlu4 belongs to a subfamily of mGlus that is predominantly coupled to Gi/o G proteins. We now report that the ubiquitous autacoid and neuromodulator, histamine, induces substantial glutamate-activated calcium mobilization in mGlu4-expressing cells, an effect which is observed in the absence of co-expressed chimeric G proteins. This strong induction of calcium signaling downstream of glutamate activation of mGlu4 depends upon the presence of H1 histamine receptors. Interestingly, the potentiating effect of histamine activation does not extend to other mGlu4-mediated signaling events downstream of Gi/o G proteins, such as cAMP inhibition, suggesting that the presence of Gq coupled receptors such as H1 may bias normal mGlu4-mediated Gi/o signaling events. When the activity induced by small molecule positive allosteric modulators of mGlu4 is assessed, the potentiated signaling of mGlu4 is further biased by histamine toward calcium-dependent pathways. These results suggest that Gi/o-coupled mGlus may induce substantial, and potentially unexpected, calcium-mediated signaling events if stimulation occurs concomitantly with activation of Gq receptors. Additionally, our results suggest that signaling induced by small molecule positive allosteric modulators may be substantially biased when Gq receptors are co-activated. This article is part of a Special Issue entitled �mGluR� PMID:22426233
The N Terminus of the Adhesion G Protein-coupled Receptor GPR56 Controls Receptor Signaling Activity*S
Hall, Randy A
The N Terminus of the Adhesion G Protein-coupled Receptor GPR56 Controls Receptor Signaling of Medicine, Atlanta, Georgia GPR56 is an adhesion G protein-coupled receptor that plays a key role in cortical development. Mutations to GPR56 in humans cause malformations of the cerebral cortex, but little
Treating the metabolic syndrome: telmisartan as a peroxisome proliferator-activated receptor-gamma activator.
Kurtz, T W
Hypertension commonly occurs as part of a genetically complex disorder of carbohydrate and lipid metabolism known as the metabolic syndrome. Most current antihypertensive drugs appear ineffective against the metabolic syndrome, which is a strong predictor of cardiovascular disease and death in affected patients. Angiotensin II can influence the activity of certain genes and cellular and biochemical pathways that may contribute to the pathogenesis of the metabolic syndrome. However, as a class, angiotensin II receptor blockers (ARBs) have proven only minimally to modestly effective in ameliorating the disturbances in carbohydrate and lipid metabolism that characterise the metabolic syndrome. Recent preclinical studies indicate that the ARB telmisartan acts as a selective peroxisome proliferators-activated receptor-gamma (PPARgamma) modulator when tested at concentrations that might be achievable with oral doses recommended for treatment of hypertension; this property does not appear to be shared by other ARBs. PPARgamma is a nuclear receptor that influences the expression of multiple genes involved in carbohydrate and lipid metabolism and is an attractive therapeutic target for the prevention and control of insulin resistance, type 2 diabetes and atherosclerosis. In cellular transactivation assays, telmisartan functioned as a partial agonist of PPARgamma and achieved 25-30% of maximal receptor activation attained with conventional PPARgamma ligands. Preclinical and clinical studies indicate that administration of telmisartan can improve carbohydrate and lipid metabolism without causing the side effects that accompany full PPARgamma activators. If the preliminary data are supported by the results of ongoing large-scale clinical studies, telmisartan could have a central role in the prevention and treatment of metabolic syndrome, diabetes and atherosclerosis. PMID:15868121
Activation of Peroxisome Proliferator-Activated Receptor Protects the Heart From Ischemia\\/Reperfusion Injury
Tian-li Yue; Weike Bao; Beat M. Jucker; Juan-li Gu; Anne M. Romanic; Peter J. Brown; Jianqi Cui; Douglas T. Thudium; Rogely Boyce; Cynthia L. Burns-Kurtis; Rosanna C. Mirabile; Karpagam Aravindhan; Eliot H. Ohlstein
Background�Peroxisome proliferator-activated receptor- (PPAR-) is expressed in the heart and regulates genes involved in myocardial fatty acid oxidation (FAO). The role of PPAR- in acute ischemia\\/reperfusion myocardial injury remains unclear. Methods and Results�The coronary arteries of male mice were ligated for 30 minutes. After reperfusion for 24 hours, ischemic and infarct sizes were determined. A highly selective and potent PPAR-
Activation of N-methyl-d-aspartate receptor downregulates inflammasome activity and liver inflammation via a ?-arrestin-2 pathway.
Farooq, Ahmad; Hoque, Rafaz; Ouyang, Xinshou; Farooq, Ahsan; Ghani, Ayaz; Ahsan, Kaimul; Guerra, Mateus; Mehal, Wajahat Zafar
Activation of the cytosolic inflammasome machinery is responsible for acute and chronic liver inflammation, but little is known about its regulation. The N-methyl-d-aspartate (NMDA) receptor families are heterotetrameric ligand-gated ion channels that are activated by a range of metabolites, including aspartate, glutamate, and polyunsaturated fatty acids. In the brain NMDA receptors are present on neuronal and nonneuronal cells and regulate a diverse range of functions. We tested the role of the NMDA receptor and aspartate in inflammasome regulation in vitro and in models of acute hepatitis and pancreatitis. We demonstrate that the NMDA receptor is present on Kupffer cells, and their activation on primary mouse and human cells limits inflammasome activation by downregulating NOD-like receptor family, pyrin domain containing 3 and procaspase-1. The NMDA receptor pathway is active in vivo, limits injury in acute hepatitis, and can be therapeutically further activated by aspartate providing protection in acute inflammatory liver injury. Downregulation of inflammasome activation by NMDA occurs via a ?-arrestin-2 NF-k? and JNK pathway and not via Ca(2+) mobilization. We have identified the NMDA receptor as a regulator of inflammasome activity in vitro and in vivo. This has identified a new area of immune regulation associated by metabolites that may be relevant in a diverse range of conditions, including nonalcoholic steatohepatitis and total parenteral nutrition-induced immune suppression. PMID:25104498
Prenylation inhibitors stimulate both estrogen receptor ? transcriptional activity through AF-1 and AF-2 and estrogen receptor ? transcriptional activity
Cestac, Philippe; Sarrabayrouse, Guillaume; M�dale-Giamarchi, Claire; Rochaix, Philippe; Balaguer, Patrick; Favre, Gilles; Faye, Jean-Charles; Doisneau-Sixou, Sophie
Introduction We showed in a previous study that prenylated proteins play a role in estradiol stimulation of proliferation. However, these proteins antagonize the ability of estrogen receptor (ER) ? to stimulate estrogen response element (ERE)-dependent transcriptional activity, potentially through the formation of a co-regulator complex. The present study investigates, in further detail, how prenylated proteins modulate the transcriptional activities mediated by ER? and by ER?. Methods The ERE-?-globin-Luc-SV-Neo plasmid was either stably transfected into MCF-7 cells or HeLa cells (MELN cells and HELN cells, respectively) or transiently transfected into MCF-7 cells using polyethylenimine. Cells deprived of estradiol were analyzed for ERE-dependent luciferase activity 16 hours after estradiol stimulation and treatment with FTI-277 (a farnesyltransferase inhibitor) or with GGTI-298 (a geranylgeranyltransferase I inhibitor). In HELN cells, the effect of prenyltransferase inhibitors on luciferase activity was compared after transient transfection of plasmids coding either the full-length ER?, the full-length ER?, the AF-1-deleted ER? or the AF-2-deleted ER?. The presence of ER? was then detected by immunocytochemistry in either the nuclei or the cytoplasms of MCF-7 cells. Finally, Clostridium botulinum C3 exoenzyme treatment was used to determine the involvement of Rho proteins in ERE-dependent luciferase activity. Results FTI-277 and GGTI-298 only stimulate ERE-dependent luciferase activity in stably transfected MCF-7 cells. They stimulate both ER?-mediated and ER?-mediated ERE-dependent luciferase activity in HELN cells, in the presence of and in the absence of estradiol. The roles of both AF-1 and AF-2 are significant in this effect. Nuclear ER? is decreased in the presence of prenyltransferase inhibitors in MCF-7 cells, again in the presence of and in the absence of estradiol. By contrast, cytoplasmic ER? is mainly decreased after treatment with FTI-277, in the presence of and in the absence of estradiol. The involvement of Rho proteins in ERE-dependent luciferase activity in MELN cells is clearly established. Conclusions Together, these results demonstrate that prenylated proteins (at least RhoA, RhoB and/or RhoC) antagonize the ability of ER? and ER? to stimulate ERE-dependent transcriptional activity, potentially acting through both AF-1 and AF-2 transcriptional activities. PMID:15642170
Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity
Pigott, Craig R.; Ellar, David J.
Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death. PMID:17554045
Impact of human autoantibodies on ?1-adrenergic receptor conformation, activity, and internalization
Bornholz, Beatrice; Weidtkamp-Peters, Stefanie; Schmitmeier, Stephanie; Seidel, Claus A. M.; Herda, Lars R.; Felix, Stephan B.; Lemoine, Horst; Hescheler, J�rgen; Nguemo, Filomain; Sch�fer, Christoph; Christensen, Morten O.; Mielke, Christian; Boege, Fritz
Aims Autoantibodies against second extracellular loops of ?1-adrenergic receptors frequent in dilated cardiomyopathy confer myocardial dysfunction presumably via cAMP stimulation. Here, we investigate the autoantibody impact on receptor conformation and function. Methods and results IgG was prepared from patients with dilated cardiomyopathy, matched healthy donors (10 each) or commercial IgG preparations (2). IgG binding to ?1-adrenergic receptor peptides was detected in 5 of 10 patients and 2 of 10 controls. IgG colocalization with the native receptor was detected in 8 of 10 patients and 1 of 10 controls (10 of 10 patients and 7 of 10 controls at >30 mg IgG/L). All IgGs exhibiting receptor colocalization triggered changes in receptor conformation (determined with fluorescent sensors) not stringently correlated to cAMP stimulation, suggesting the induction of more or less active receptor conformations. Receptor-activating IgG was detected in 8 of 10 patients but only 1 of 10 controls. In addition, IgG from 8 of 10 patients and 3 of 10 controls attenuated receptor internalization (measured by total internal reflection fluorescence microscopy). IgG-inducing inactive receptor conformations had no effect on subsequent cAMP stimulation by isoproterenol. IgG-inducing active receptor conformations dampened or augmented subsequent cAMP stimulation by isoproterenol, depending on whether receptor internalization was attenuated or not. Corresponding IgG effects on the basal beating rate and chronotropic isoproterenol response of embryonic human cardiomyocytes were observed. Conclusions (i) Autoantibodies trigger conformation changes in the ?1-adrenergic receptor molecule. (ii) Some also attenuate receptor internalization. (iii) Combinations thereof increase the basal beating rate of cardiomyocytes and optionally entail dampening of their chronotropic catecholamine responses. (iv) The latter effects seem specific for patient autoantibodies, which also have higher levels. PMID:23208588
Development of a bioluminescence resonance energy transfer (BRET) for monitoring estrogen receptor alpha activation
Elisa Michelini; Mara Mirasoli; Matti Karp; Marko Virta; Aldo Roda
Estrogen receptor (ER) is a ligand-activated transcriptional factor, able to dimerize after activation and to bind specific DNA sequences (estrogen response elements), thus activating gene target transcription. Since ER homo- and hetero-dimerization (giving a-a and a-b isoforms) is a fundamental step for receptor activation, we developed an assay for detecting compounds that induce human ERa homo-dimerization based on bioluminescence resonance
The Wnt receptor Frizzled-4 modulates ADAM13 metalloprotease activity.
Abbruzzese, Genevieve; Gorny, Anne-Kathrin; Kaufmann, Lilian T; Cousin, H�l�ne; Kleino, Iivari; Steinbeisser, Herbert; Alfandari, Dominique
Cranial neural crest (CNC) cells are a transient population of stem cells that originate at the border of the neural plate and the epidermis, and migrate ventrally to contribute to most of the facial structures including bones, cartilage, muscles and ganglia. ADAM13 is a cell surface metalloprotease that is essential for CNC cell migration. Here, we show in Xenopus laevis embryos that the Wnt receptor Fz4 binds to the cysteine-rich domain of ADAM13 and negatively regulates its proteolytic activity in vivo. Gain of Fz4 function inhibits CNC cell migration and can be rescued by gain of ADAM13 function. Loss of Fz4 function also inhibits CNC cell migration and induces a reduction of mature ADAM13, together with an increase in the ADAM13 cytoplasmic fragment that is known to translocate into the nucleus to regulate gene expression. We propose that Fz4 associates with ADAM13 during its transport to the plasma membrane to regulate its proteolytic activity. PMID:25616895
Characterization of Peroxisome Proliferator-Activated Receptor a (PPARa) -Independent Effects of PPARa Activators in the Rodent Liver: Di-(2-ethylhexyl) phthalate Also Activates the Constitutive Activated Receptor
Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferatoractivated receptor alpha (PPARa). Recent studies indicate that one such PPC, the plasticizer di2- et...
A selective metabotropic glutamate receptor 7 agonist: Activation of receptor signaling via an allosteric site modulates stress parameters in vivo
Mitsukawa, Kayo; Yamamoto, Rina; Ofner, Silvio; Nozulak, Joachim; Pescott, Oliver; Lukic, Snezana; Stoehr, Natacha; Mombereau, Cedric; Kuhn, Rainer; McAllister, Kevin H.; van der Putten, Herman; Cryan, John F.; Flor, Peter J.
Metabotropic glutamate receptor (mGluR) subtypes (mGluR1 to mGluR8) act as important pre- and postsynaptic regulators of neurotransmission in the CNS. These receptors consist of two domains, an extracellular region containing the orthosteric agonist site and a transmembrane heptahelical domain involved in G protein activation and recognition of several recently synthesized pharmacological modulators. The presynaptic receptor mGluR7 shows the highest evolutionary conservation within the family, but no selective pharmacological tool was known. Here we characterize an mGluR7-selective agonist, N,N?-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), which directly activates receptor signaling via an allosteric site in the transmembrane domain. At transfected mammalian cells expressing mGluR7, AMN082 potently inhibits cAMP accumulation and stimulates GTP?S binding (EC50-values, 64-290 nM) with agonist efficacies comparable with those of l-2-amino-4-phosphonobutyrate (L-AP4) and superior to those of l-glutamate. AMN082 (?10 ?M) failed to show appreciable activating or inhibitory effects at other mGluR subtypes and selected ionotropic GluRs. Chimeric receptor studies position the binding site of AMN082 in the transmembrane region of mGluR7, and we demonstrate that this allosteric agonist has little, if any, effect on the potency of orthosteric ligands. Here we provide evidence for full agonist activity mediated by the heptahelical domain of family 3 G protein-coupled receptors (which have mGluR-like structure) that may lead to drug development opportunities. Further, AMN082 is orally active, penetrates the blood-brain barrier, and elevates the plasma stress hormones corticosterone and corticotropin in an mGluR7-dependent fashion. Therefore, AMN082 is a valuable tool for unraveling the role of mGluR7 in stress-related CNS disorders. PMID:16339898
Nicotine Activation of ?4* Receptors: Sufficient for Reward, Tolerance, and Sensitization
NASA Astrophysics Data System (ADS)
Tapper, Andrew R.; McKinney, Sheri L.; Nashmi, Raad; Schwarz, Johannes; Deshpande, Purnima; Labarca, Cesar; Whiteaker, Paul; Marks, Michael J.; Collins, Allan C.; Lester, Henry A.
The identity of nicotinic receptor subtypes sufficient to elicit both the acute and chronic effects of nicotine dependence is unknown. We engineered mutant mice with ?4 nicotinic subunits containing a single point mutation, Leu9' --> Ala9' in the pore-forming M2 domain, rendering ?4* receptors hypersensitive to nicotine. Selective activation of ?4* nicotinic acetylcholine receptors with low doses of agonist recapitulates nicotine effects thought to be important in dependence, including reinforcement in response to acute nicotine administration, as well as tolerance and sensitization elicited by chronic nicotine administration. These data indicate that activation of ?4* receptors is sufficient for nicotine-induced reward, tolerance, and sensitization.
Adrenergic ?2 Receptor Activation Stimulates Anti-Inflammatory Properties of Dendritic Cells In Vitro
Nijhuis, Laurens E.; Olivier, Brenda J.; Dhawan, Shobit; Hilbers, Francisca W.; Boon, Louis; Wolkers, Monika C.; Samsom, Janneke N.; de Jonge, Wouter J.
Vagal nerve efferent activation has been shown to ameliorate the course of many inflammatory disease states. This neuro-modulatory effect has been suggested to rest on acetylcholine receptor (AChR) activation on tissue macrophages or dendritic cells (DCs). In more recent studies, vagal anti-inflammatory activity was shown involve adrenergic, splenic, pathways. Here we provide evidence that the adrenergic, rather than cholinergic, receptor activation on bone marrow derived DCs results in enhanced endocytosis uptake, enhanced IL-10 production but a decreased IL-6, IL-12p70 and IL-23 production. In antigen specific T cell stimulation assays, adrenergic ?2 receptor activation on bone marrow DCs led to an enhanced potential to induce Foxp3 positive suppressive Treg cells. These effects were independent of IL10-R activation, TGF? release, or retinoic acid (RA) secretion. Hence, adrenergic receptor ?2 activation modulates DC function resulting in skewing towards anti-inflammatory T cell phenotypes. PMID:24465481
MAPK/ERK-Dependent Translation Factor Hyperactivation and Dysregulated Laminin ?2 Expression in Oral Dysplasia and Squamous Cell Carcinoma
Degen, Martin; Natarajan, Easwar; Barron, Patricia; Widlund, Hans R.; Rheinwald, James G.
Lesions displaying a variety of dysplastic changes precede invasive oral and epidermal squamous cell carcinoma (SCC); however, there are no histopathological criteria for either confirming or staging premalignancy. SCCs and dysplasias frequently contain cells that abnormally express the ?2 subunit of laminin-332. We developed cell culture models to investigate ?2 dysregulation. Normal human keratinocytes displayed density-dependent repression of ?2, whereas premalignant keratinocytes and SCC cells overexpressed ?2 and secreted laminin assembly intermediates. Neoplastic cells had hyperactive EGFR/MAPK(ERK) signaling coordinate with overexpressed ?2, and EGFR and MEK inhibitors normalized ?2 expression. Keratinocytes engineered to express HPV16 E6 or activated mutant HRAS, cRAF1, or MEK1 lost density repression of ?2 and shared with neoplastic cells signaling abnormalities downstream of ERK, including increased phosphorylation of S6 and eIF4 translation factors. Notably, qPCR results revealed that ?2 overexpression was not accompanied by increased ?2 mRNA levels, consistent with ERK-dependent, eIF4B-mediated translation initiation of the stem-looped, 5?-untranslated region of ?2 mRNA in neoplastic cells. Inhibitors of MEK, but not of TORC1/2, blocked S6 and eIF4B phosphorylation and ?2 overexpression. Immunostaining of oral dysplasias identified ?2 overexpression occurring within fields of basal cells that had elevated p-S6 levels. These results reveal a causal relationship between ERK-dependent translation factor activation and laminin ?2 dysregulation and identify new markers of preinvasive neoplastic change during progression to SCC. PMID:22546478
Activation mechanism of the heterodimeric GABA B receptor
Jean-Philippe Pin; Julie Kniazeff; Virginie Binet; Jianfeng Liu; Damien Maurel; Thierry Galvez; B�atrice Duthey; Michaela Havlickova; Jaroslav Blahos; Laurent Pr�zeau; Philippe Rondard
The GABAB receptor was the first heteromeric G-protein coupled receptor (GPCR) identified. Indeed, both GABAB1 and GABAB2 subunits appear necessary to get a functional GABAB receptor. Soon after the cloning of both subunits, it was demonstrated that GABAB2 was required for GABAB1 to reach the cell surface. However, even a mutated GABAB1 able to reach the cell surface is not
Receptor-Drug Interaction: Europium Employment for Studying the Biochemical Pathway of G-Protein-Coupled Receptor Activation
Antonio, Colabufo Nicola; Grazia, Perrone Maria; Marialessandra, Contino; Francesco, Berardi; Roberto, Perrone
In medicinal chemistry field, the biochemical pathways, involved in 7-transmembrane domains G-protein coupled receptors (GPCRs) activation, are commonly studied to establish the activity of ligands towards GPCRs. The most studied steps are the measurement of activated GTP-? subunit and stimulated intracellular cAMP. At the present, many researchers defined agonist or antagonist activity of potential GPCRs drugs employing [35S]GTP?S or [3H]cAMP as probes. Recently, the corresponding lanthanide labels Eu-GTP and Eu-cAMP as alternative to radiochemicals have been developed because they are highly sensitive, easy to automate, easily synthesized, they display a much longer shelf-life and they can be used in multilabel experiments. In the present review, the receptor-drug interaction by europium employment for studying the biochemical pathway of GPCR activation has been focused. Moreover, comparative studies between lanthanide label probes and the corresponding radiolabeled compounds have been carried out. PMID:18350113
High-resolution crystal structure of human protease-activated receptor 1.
Zhang, Cheng; Srinivasan, Yoga; Arlow, Daniel H; Fung, Juan Jose; Palmer, Daniel; Zheng, Yaowu; Green, Hillary F; Pandey, Anjali; Dror, Ron O; Shaw, David E; Weis, William I; Coughlin, Shaun R; Kobilka, Brian K
Protease-activated receptor 1 (PAR1) is the prototypical member of a family of G-protein-coupled receptors that mediate cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the amino-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the heptahelical bundle of the receptor to affect G-protein activation. Here we report the 2.2 � resolution crystal structure of human PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a small molecule binds virtually irreversibly to inhibit receptor activation by the tethered ligand of PAR1. In contrast to deep, solvent-exposed binding pockets observed in other peptide-activated G-protein-coupled receptors, the vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. Protease-activated receptors are important targets for drug development. The structure reported here will aid the development of improved PAR1 antagonists and the discovery of antagonists to other members of this receptor family. PMID:23222541
Acutely increasing ?GABAA receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus
Whissell, Paul D.; Eng, Dave; Lecker, Irene; Martin, Loren J.; Wang, Dian-Shi; Orser, Beverley A.
Extrasynaptic ?-aminobutyric acid type A (GABAA) receptors that contain the ? subunit (?GABAA receptors) are expressed in several brain regions including the dentate gyrus (DG) and CA1 subfields of the hippocampus. Drugs that increase ?GABAA receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the ?GABAA receptor�preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing ?GABAA receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p.) on memory performance in wild-type (WT) and ?GABAA receptor null mutant (Gabrd?/?) mice. Additionally, the effects of THIP on long-term potentiation (LTP), a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd?/? mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd?/? mice, an effect that was blocked by GABAA receptor antagonist bicuculline. Thus, acutely increasing ?GABAA receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing ?GABAA receptor activity. PMID:24062648
Laminin isoforms: biological roles and effects on the intracellular distribution of nuclear proteins in intestinal epithelial cells
Turck, Natacha [INSERM, Unit 381, 67200 Strasbourg (France); University Louis Pasteur, Strasbourg, F-67200 (France); Gross, Isabelle [INSERM, Unit 381, 67200 Strasbourg (France); University Louis Pasteur, Strasbourg, F-67200 (France); Gendry, Patrick [INSERM, Unit 381, 67200 Strasbourg (France); University Louis Pasteur, Strasbourg, F-67200 (France); Stutzmann, Jeanne [INSERM, Unit 381, 67200 Strasbourg (France); University Louis Pasteur, Strasbourg, F-67200 (France); Freund, Jean-Noel [INSERM, Unit 381, 67200 Strasbourg (France); University Louis Pasteur, Strasbourg, F-67200 (France); Kedinger, Michele [INSERM, Unit 381, 67200 Strasbourg (France); University Louis Pasteur, Strasbourg, F-67200 (France); Simon-Assmann, Patricia [INSERM, Unit 381, 67200 Strasbourg (France); University Louis Pasteur, Strasbourg, F-67200 (France); Launay, Jean-Francois [INSERM, Unit 381, 67200 Strasbourg (France) and University Louis Pasteur, Strasbourg, F-67200 (France)]. E-mail: email@example.com
Laminins are structurally and functionally major components of the extracellular matrix. Four isoforms of laminins (laminin-1, -2, -5 and -10) are expressed in a specific pattern along the crypt-villus axis of the intestine. Previous works indicated that expression of these isoforms is developmentally regulated and that laminins could modulate the behaviour of intestinal cells, but the exact role of each isoform remained unclear. Here, we report the first systematic analysis of the cellular functions of the four isoforms using the human colon adenocarcinoma Caco2/TC7 cell line as a model. We compared the respective abilities of each isoform to modulate adhesion, proliferation and differentiation of intestinal epithelial cells. We found that the isoforms were functionally distinct, with laminin-10 being the most adhesive substratum, laminin-2, laminin-5 and laminin-10 enhancing cellular proliferation and at the opposite, laminin-1 stimulating intestinal cell differentiation. To begin to characterise the molecular events induced by the different isoforms, we examined by immunofluorescence the intracellular distribution of several nuclear proteins, recently highlighted by a nuclear proteomic approach. We observed clear nucleocytoplasmic redistribution of these proteins, which depended on the laminin isoform. These results provide evidence for a distinct functional role of laminins in intestinal cell functions characterised by specific localisation of nuclear proteins.
cAMP-dependent protein kinase A activity modulates topiramate potentiation of GABA(A) receptors.
Simeone, Timothy A; Wilcox, Karen S; White, H Steve
Activation of cAMP-dependent protein kinase A (PKA) prevents inhibition of non-NMDA glutamate receptors by the anticonvulsant topiramate. Using two-electrode voltage-clamp techniques, we demonstrate that PKA activity also modulates topiramate potentiation of recombinant GABA(A) receptors expressed in Xenpus laevis oocytes. PKA activators, dibutyryl-cAMP and forskolin, attenuate topiramate potentiation, whereas the PKA inhibitor H-89 increases topiramate potentiation. Thus, endogenous PKA activity and receptor phosphorylation states may contribute to topiramate treatment efficacy. PMID:21665439
Activation of Peroxisome Proliferator-Activated Receptor Stimulates the Proliferation of Human Breast and Prostate Cancer Cell Lines
Ruth L. Stephen; Mattias C. U. Gustafsson; Morag Jarvis; Roger Tatoud; Barry R. Marshall; Deborah Knight; Ewa Ehrenborg; Adrian L. Harris; C. Roland Wolf; Colin N. A. Palmer
The nuclear receptor peroxisome proliferator-activated receptor (PPAR\\/ (NR1C2)) has been implicated in colorectal carcinogenesis by various molecular genetic observations. These observations have recently been supported by studies of activation of PPAR by pharmacological agents. Here we present the first report of the stimulation of breast and prostate cancer cell growth using PPAR selective agonists. Activation of PPAR with compound F
[Modulation of GABA- and kainate-activated currents by metabotropic receptors in isolated rat cortical neurons].
Amakhin, D V; Popov, V A; Veselkin, N P
Whole-cell patch-clamp recordings from isolated neurons from rat prefrontal cortex have been made to study GABAb and mGluR receptor modulation of currents induced by applications of GABA and kainate. The GABAb-receptor antagonist CGP-55845 (5 microM) enhanced the peak by 26 +/- 13% (n = 6) but had no effect on the steady-state of GABA-activated current. Bath application of GABAb-receptor agonist baclofen (50 microM) enhanced the GABAa currents by 9 +/- 2% (n = 8). Kainate-activated currents were not affected by baclofen. Both GABA-activated currents and kainate-activated currents were not affected by trans-ACPD (MGluR agonist). These results suggest that in cortex postsynaptic response of GABAa-receptors can be modulated by GABAb-receptors. PMID:25697024
Activation of NTS A2a adenosine receptors differentially resets baroreflex control of renal vs. adrenal sympathetic nerve activity.
Ichinose, Tomoko K; O'Leary, Donal S; Scislo, Tadeusz J
The role of nucleus of solitary tract (NTS) A(2a) adenosine receptors in baroreflex mechanisms is controversial. Stimulation of these receptors releases glutamate within the NTS and elicits baroreflex-like decreases in mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA), whereas inhibition of these receptors attenuates HR baroreflex responses. In contrast, stimulation of NTS A(2a) adenosine receptors increases preganglionic adrenal sympathetic nerve activity (pre-ASNA), and the depressor and sympathoinhibitory responses are not markedly affected by sinoaortic denervation and blockade of NTS glutamatergic transmission. To elucidate the role of NTS A(2a) adenosine receptors in baroreflex function, we compared full baroreflex stimulus-response curves for HR, RSNA, and pre-ASNA (intravenous nitroprusside/phenylephrine) before and after bilateral NTS microinjections of selective adenosine A(2a) receptor agonist (CGS-21680; 2.0, 20 pmol/50 nl), selective A(2a) receptor antagonist (ZM-241385; 40 pmol/100 nl), and nonselective A(1) + A(2a) receptor antagonist (8-SPT; 1 nmol/100 nl) in urethane/alpha-chloralose anesthetized rats. Activation of A(2a) receptors decreased the range, upper plateau, and gain of baroreflex-response curves for RSNA, whereas these parameters all increased for pre-ASNA, consistent with direct effects of the agonist on regional sympathetic activity. However, no resetting of baroreflex-response curves along the MAP axis occurred despite the marked decreases in baseline MAP. The antagonists had no marked effects on baseline variables or baroreflex-response functions. We conclude that the activation of NTS A(2a) adenosine receptors differentially alters baroreflex control of HR, RSNA, and pre-ASNA mostly via non-baroreflex mechanism(s), and these receptors have virtually no tonic action on baroreflex control of these sympathetic outputs. PMID:19202001
Modulatory effects of L-carnitine on glucocorticoid receptor activity.
Manoli, Irini; De Martino, Massimo U; Kino, Tomoshige; Alesci, Salvatore
L-carnitine (3-hydroxy-4-N,N,N-trimethylaminobutyrate) is a conditionally essential nutrient with a major role in cellular energy metabolism. It is available in the United States as both a prescription drug and an over-the-counter nutritional supplement. Accumulating evidence from both animal and human studies indicates that pharmacologic doses of L-carnitine (LCAR) have immunomodulatory effects resembling those of glucocorticoids (GC). On the other hand, in contrast to GC, which cause bone loss, LCAR seems to have positive effects on bone metabolism. To explore the molecular bases of this GC-like activity of LCAR, we investigated its effects on glucocorticoid receptor (GR)-modulated cytokine release ex vivo, and on the transcriptional activity, intracellular trafficking, and binding of GR in vitro. At high noncytotoxic doses, LCAR (a) suppressed the lipopolysaccharide-stimulated release of tumor necrosis factor alpha and interleukin-12 from primary human monocytes in a GC-like fashion, (b) stimulated the transcriptional activity of GR on the GC-responsive promoters, (c) triggered nuclear translocation of green fluorescent protein (GFP)-fused GR, and (d) reduced the whole cell binding of [(3)H]-dexamethasone to GR. These results suggest that LCAR is a "nutritional modulator" of the GR, by acting as an agonist-like compound. Since LCAR appears to have positive effects on bone metabolism, in contrast to GC, LCAR may share some of the therapeutic properties of GC, particularly on the immune system, but not their deleterious side effects on some of other organs/tissues. Thus, LCAR is potentially a useful alternative compound of GC in particular therapeutic situations. The clinical and therapeutic implications of these findings, as well as a better understanding of their mechanisms, warrant further research. PMID:15591012
Biological Effects of Thyrotropin Receptor Activation on Human Orbital Preadipocytes
Zhang, Lei; Baker, Glynn; Janus, D.; Paddon, C.; Fuhrer, D.; Ludgate, M.
Purpose Thyrotropin receptor (TSHR) expression is upregulated in the orbits of Graves? ophthalmopathy (GO) patients, the majority of whom have TSHR stimulating antibodies. We have investigated the biological effects of TSHR activation in vitro, in adipose tissue - the site of orbital TSHR expression. Methods Activating mutant TSHR (TSHR*) or wild type (WT), were introduced into human orbital preadipocytes using retroviral vectors. Their proliferation (Coulter counting), basal cAMP accumulation (radio-immunoassay), spontaneous and PPAR? induced adipogenesis (quantitative oil red O staining) were assessed and compared with non-modified cells. QRT-PCR was used to measure transcripts of C/EBP�, PPAR? and LPL (early, intermediate & late markers of adipogenesis) and for UCP-1 (brown adipose tissue, BAT). Results Expression of TSHR* significantly inhibited the proliferation of pre- adipocytes and produced an increase in unstimulated cAMP of 200 to 600%. Basal lipid levels were significantly increased in TSHR* (127 to 275%) compared with non-modified (100%) or WT (104 to 187%) expressing cells. This was accompanied by 2 to 10 fold increases in early-intermediate markers and UCP-1 transcripts (2 to 8 fold); LPL was at the limit of detection. In non-modified cells, adipogenesis produced significant increases in transcripts of all markers, including LPL (?30 fold). This was not the case in TSHR* expressing cells, which also displayed 67 to 84% reduction in lipid levels, Conclusions TSHR activation stimulates early differentiation (favouring BAT formation?) but renders preadipocytes refractory to PPAR? induced adipogenesis. In neither case do lipid-containing vacuoles accumulate suggesting that terminal stages of differentiation have been inhibited. PMID:17122103
Phospholipase C Activity Affinity Purifies with the Torpedo Nicotinic Acetylcholine Receptor*
Labriola, Jonathan M.; daCosta, Corrie J. B.; Wang, Shuzhi; Figeys, Daniel; Smith, Jeffrey C.; Sturgeon, R. Michel; Baenziger, John E.
Nicotinic acetylcholine receptors mediate fast synaptic transmission by fluxing ions across the membrane in response to neurotransmitter binding. We show here that during affinity purification of the nicotinic acetylcholine receptor from Torpedo, phosphatidic acid, but not other anionic or zwitterionic phospholipids, is hydrolyzed to diacylglycerol. The phospholipase C activity elutes with the acetylcholine receptor and is inhibited by a lipid phosphate phosphohydrolase inhibitor, sodium vanadate, but not a phosphatidate phosphohydrolase inhibitor, N-ethylmaleimide. Further, the hydrolysis product of phosphatidic acid, diacylglycerol, enhances the functional capabilities of the acetylcholine receptor in the presence of anionic lipids. We conclude that a phospholipase C activity, which appears to be specific for phosphatidic acid, is associated with the nicotinic acetylcholine receptor. The acetylcholine receptor may directly or indirectly influence lipid metabolism in a manner that enhances its own function. PMID:20133947
The impact of laminin on 3D neurite extension in collagen gels
NASA Astrophysics Data System (ADS)
Swindle-Reilly, Katelyn E.; Papke, Jason B.; Kutosky, Hannah P.; Throm, Allison; Hammer, Joshua A.; Harkins, Amy B.; Kuntz Willits, Rebecca
The primary goal of this research was to characterize the effect of laminin on three-dimensional (3D) neurite growth. Gels were formed using type I collagen at concentrations of 0.4-2.0 mg mL-1 supplemented with laminin at concentrations of 0, 1, 10, or 100 �g mL-1. When imaged with confocal microscopy, laminin was shown to follow the collagen fibers; however, the addition of laminin had minimal effect on the stiffness of the scaffolds at any concentration of collagen. Individual neurons dissociated from E9 chick dorsal root ganglia were cultured in the gels for 24 h, and neurite lengths were measured. For collagen gels without laminin, a typical bimodal response of neurite outgrowth was observed, with increased growth at lower concentrations of collagen gel. However, alteration of the chemical nature of the collagen gel by the laminin additive shifted, or completely mitigated, the bimodal neurite growth response seen in gels without laminin. Expression of integrin subunits, ?1, ?3, ?6 and ?1, were confirmed by PCR and immunolabeling in the 3D scaffolds. These results provide insight into the interplay between mechanical and chemical environment to support neurite outgrowth in 3D. Understanding the relative impact of environmental factors on 3D nerve growth may improve biomaterial design for nerve cell regeneration.
Folliculostellate Cells Are Required for Laminin Release from Gonadotrophs in Rat Anterior Pituitary
Tsukada, Takehiro; Fujiwara, Ken; Horiguchi, Kotaro; Azuma, Morio; Ramadhani, Dini; Tofrizal, Alimuddin; Batchuluun, Khongorzul; Maliza, Rita; Syaidah, Rahimi; Kikuchi, Motoshi; Yashiro, Takashi
The anterior pituitary gland is organized tissue comprising hormone-producing cells and folliculostellate (FS) cells. FS cells interconnect to form a meshwork, and their cytoplasmic processes are anchored by a basement membrane containing laminin. Recently, we developed a three-dimensional (3D) cell culture that reproduces this FS cell architecture. In this study of the novel function of FS cells, we used transgenic rats that express green fluorescent protein in FS cells for the 3D culture. Anterior pituitary cells were cultured with different proportions of FS cells (0%, 5%, 10%, and 20%). Anterior pituitary cells containing 5�20% FS cells formed round/oval cell aggregates, whereas amorphous cell aggregates were formed in the absence of FS cells. Interestingly, immunohistochemistry showed laminin-immunopositive cells instead of extracellular laminin deposition in FS cell-deficient cell aggregates. Double-immunostaining revealed that these laminin-immunopositive cells were gonadotrophs. Laminin mRNA expression did not differ in relation to the presence or absence of FS cells. When anterior pituitary cells with no FS cells were cultured with FS cell-conditioned medium, the proportion of laminin-immunopositive cells was lower than in control. These results suggest that a humoral factor from FS cells is required for laminin release from gonadotrophs.
Laminin modified infection-preventing collagen membrane containing silver sulfadiazine-hyaluronan microparticles.
Lee, Jong-Eun; Park, Jong-Chul; Lee, Kwang Hoon; Oh, Sang Ho; Suh, Hwal
The newly developed laminin modified infection-preventing collagen membrane consists of a 3 component laminate, comprising 2 outer collagen layers and a central laminin layer. The 2 outer collagen layers (dense and porous layers) were fabricated by air-drying and freeze drying, respectively, and the laminin layer was formed by a straightforward liquid coating method. In addition, hyaluronan based microparticles containing silver sulfadiazine (AgSD) were incorporated into the 2 collagen layers (AgSD content 50 microg/cm2). Laminin coated collagen surfaces did not promote fibroblast attachment but showed a retarded fibroblast proliferation rate and an increased rate of collagen synthesis versus pure collagen surfaces. In an animal study, a laminin coating on a nonmedicated collagen membrane significantly increased both wound size reduction and vessel proliferation 7 days after application versus polyurethane film. Interestingly, the laminin coated AgSD medicated collagen membrane demonstrated higher wound size reduction and vessel proliferation and lower inflammation than the polyurethane control, suggesting that the laminin AgSD medicated collagen membrane substantially improves dermal wound healing. PMID:12072108
Drosophila laminins act as key regulators of basement membrane assembly and morphogenesis
Urbano, Jose M.; Torgler, Catherine N.; Molnar, Cristina; Tepass, Ulrich; L�pez-Varea, Ana; Brown, Nicholas H.; de Celis, Jose F.; Mart�n-Bermudo, Maria D.
Laminins are heterotrimeric molecules found in all basement membranes. In mammals, they have been involved in diverse developmental processes, from gastrulation to tissue maintenance. The Drosophila genome encodes two laminin ? chains, one ? and one ?, which form two distinct laminin trimers. So far, only mutations affecting one or other trimer have been analysed. In order to study embryonic development in the complete absence of laminins, we mutated the gene encoding the sole laminin ? chain in Drosophila, LanB1, so that no trimers can be made. We show that LanB1 mutant embryos develop until the end of embryogenesis. Electron microscopy analysis of mutant embryos reveals that the basement membranes are absent and the remaining extracellular material appears disorganised and diffuse. Accordingly, abnormal accumulation of major basement membrane components, such as Collagen IV and Perlecan, is observed in mutant tissues. In addition, we show that elimination of LanB1 prevents the normal morphogenesis of most organs and tissues, including the gut, trachea, muscles and nervous system. In spite of the above structural roles for laminins, our results unravel novel functions in cell adhesion, migration and rearrangement. We propose that while an early function of laminins in gastrulation is not conserved in Drosophila and mammals, their function in basement membrane assembly and organogenesis seems to be maintained throughout evolution. PMID:19906841
Quantitative impedimetric NPY-receptor activation monitoring and signal pathway profiling in living cells.
te Kamp, Verena; Lindner, Ricco; Jahnke, Heinz-Georg; Krinke, Dana; Kostelnik, Katja B; Beck-Sickinger, Annette G; Robitzki, Andrea A
Label-free and non-invasive monitoring of receptor activation and identification of the involved signal pathways in living cells is an ongoing analytic challenge and a great opportunity for biosensoric systems. In this context, we developed an impedance spectroscopy-based system for the activation monitoring of NPY-receptors in living cells. Using an optimized interdigital electrode array for sensitive detection of cellular alterations, we were able for the first time to quantitatively detect the NPY-receptor activation directly without a secondary or enhancer reaction like cAMP-stimulation by forskolin. More strikingly, we could show that the impedimetric based NPY-receptor activation monitoring is not restricted to the Y1-receptor but also possible for the Y2- and Y5-receptor. Furthermore, we could monitor the NPY-receptor activation in different cell lines that natively express NPY-receptors and proof the specificity of the observed impedimetric effect by agonist/antagonist studies in recombinant NPY-receptor expressing cell lines. To clarify the nature of the observed impedimetric effect we performed an equivalent circuit analysis as well as analyzed the role of cell morphology and receptor internalization. Finally, an antagonist based extensive molecular signal pathway analysis revealed small alterations of the actin cytoskeleton as well as the inhibition of at least L-type calcium channels as major reasons for the observed NPY-induced impedance increase. Taken together, our novel impedance spectroscopy based NPY-receptor activation monitoring system offers the opportunity to identify signal pathways as well as for novel versatile agonist/antagonist screening systems for identification of novel therapeutics in the field of obesity and cancer. PMID:25239555
Peroxisomal bifunctional enzyme binds and activates the activation function-1 region of the peroxisome proliferator-activated receptor alpha.
Juge-Aubry, C E; Kuenzli, S; Sanchez, J C; Hochstrasser, D; Meier, C A
The transcriptional activity of peroxisome proliferator-activated receptors (PPARs), and of nuclear hormone receptors in general, is subject to modulation by cofactors. However, most currently known co-activating proteins interact in a ligand-dependent manner with the C-terminal ligand-regulated activation function (AF)-2 domain of nuclear receptors. Since PPARalpha exhibits a strong constitutive transactivating function contained within an N-terminal AF-1 region, it can be speculated that a different set of cofactors might interact with this region of PPARs. An affinity purification approach was used to identify the peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (bifunctional enzyme, BFE) as a protein which strongly and specifically interacted with the N-terminal 92 amino acids of PPARalpha. Protein-protein interaction assays with the cloned BFE confirmed this interaction, which could be mapped to amino acids 307-514 of the BFE and the N-terminal 70 amino acids of PPARalpha. Moreover, transient transfection experiments in hepatoma cells revealed a 2.2-fold increase in the basal and ligand-stimulated transcriptional activity of PPARalpha in the presence of BFE. This stimulatory effect is preferentially observed for the PPARalpha isoform and it is significantly stronger (4.8-fold) in non-hepatic cells, which presumably express lower levels of endogenous BFE. Hence, the BFE represents the first known cofactor capable of activating the AF-1 domain of PPAR without requiring additional regions of this receptor. These data are compatible with a model whereby the PPAR-regulated BFE is able to modulate its own expression through an enhancement of the activity of PPARalpha, representing a novel peroxisomal-nuclear feed-forward regulatory loop. PMID:11139388
Activation of cannabinoid receptor 1 inhibits increased bladder activity induced by nerve growth factor.
Wang, Zun-Yi; Wang, Peiqing; Bjorling, Dale E
Nerve growth factor (NGF) is an important mediator of inflammatory pain, in part by sensitizing afferent nerve fibers, and expression of NGF is increased during bladder inflammation. We investigated whether intravesical instillation of the selective cannabinoid receptor 1 (CB1) agonist arachidonyl-2'-chloroethylamide (ACEA) affects NGF-induced increased bladder activity in female C57BL/6J wild-type (WT) mice. We also examined the effects of intravesical NGF in female fatty acid amide hydrolase knock-out (FAAH KO) mice. We found that CB1 and tyrosine kinase A (trkA, the high-affinity NGF receptor) were present in L6 dorsal root ganglion (DRG) afferent neurons and in bladders of both genotypes. Intravesical NGF increased bladder activity that was inhibited by intravesical ACEA in WT mice. The inhibitory effects of ACEA were reversed by the selective CB1 antagonist AM 251. Intravesical NGF failed to affect bladder activity in FAAH KO mice, and treatment with AM251, restored the stimulatory effects of NGF on the bladder in FAAH KO mice. These results indicate that activation of CB1 inhibits increased bladder activity induced by NGF. PMID:25575795
The expression of glucocorticoid receptor is negatively regulated by active androgen receptor signaling in prostate tumors.
Xie, Ning; Cheng, Helen; Lin, Dong; Liu, Liangliang; Yang, Ou; Jia, Li; Fazli, Ladan; Gleave, Martin E; Wang, Yuzhuo; Rennie, Paul; Dong, Xuesen
The glucocorticoid and androgen receptors (GR and AR) can commonly regulate up to 50% of their target genes in prostate cancer (PCa) cells. GR expression is stimulated by castration therapy, which has been proposed to be one mechanism that compensates for AR signaling blockade and promotes castration-resistant PCa (CRPC) progression. However, whether GR functions as a driver for CRPC or a marker reflecting AR activity remains unclear. Here, we applied PCa tissue microarrays to show that GR protein levels were elevated by castration therapy, but reduced to pre-castration levels when tumors were at the CRPC stage. Using subrenal capsule xenograft models, we showed that GR expression was inversely correlated with AR and PSA expressions. GR expression levels are not associated with tumor invasion and metastasis phenotypes. In castration-resistant C4-2 xenografts expressing AR shRNA, regressing tumors induced by AR knockdown expressed higher levels of GR and lower levels of PSA than non-regressing tumors. Immunoblotting and real-time PCR assays further showed that AR knockdown or AR antagonists increased GR expression at both mRNA and protein levels. ChIP combined with DNA sequencing techniques identified a negative androgen responsive element (nARE) 160K base pairs upstream of the GR gene. Gel shift assays confirmed that AR directly interacted with the nARE and luciferase assays demonstrated that the nARE could mediate transcription repression by ligand-activated AR. In conclusion, GR expression is negatively regulated by AR signaling and may serve as a marker for AR signaling in prostate tumors. PMID:25138562
Potentiation of N-methyl- d-aspartate receptor-mediated neuronal injury during methamphetamine withdrawal in vitro requires co-activation of IP 3 receptors
Katherine J. Smith; Tracy R. Butler; Rachel L. Self; Brittany B. Braden; Mark A. Prendergast
Recent findings suggest that methamphetamine (METH) functions acutely to inhibit N-methyl-d-aspartate (NMDA) receptor function. Protracted withdrawal from METH exposure may increase the sensitivity of NMDA receptors to agonist exposure, promoting neuronal excitability. However, the relevance of METH effects on NMDA receptor activity with regard to neuronal viability has not been fully studied. The present studies examined the effects of protracted
Terminal Differentiation of Human Liposarcoma Cells Induced by Ligands for Peroxisome Proliferator-Activated Receptor gamma and the Retinoid X Receptor
Peter Tontonoz; Samuel Singer; Barry M. Forman; Pasha Sarraf; Jonathan A. Fletcher; Christopher D. M. Fletcher; Regina P. Brun; Elisabetta Mueller; Soner Altiok; Heather Oppenheim; Ronald M. Evans; Bruce M. Spiegelman
Induction of terminal differentiation represents a promising therapeutic approach to certain human malignancies. The peroxisome proliferator-activated receptor gamma (PPARgamma ) and the retinoid X receptor alpha (RXRalpha ) form a heterodimeric complex that functions as a central regulator of adipocyte differentiation. Natural and synthetic ligands for both receptors have been identified. We demonstrate here that PPARgamma is expressed at high
The oxidative stress mediator 4-hydroxynonenal is an intracellular agonist of the nuclear receptor peroxisome proliferator-activated receptor-?/? (PPAR?/?)
Coleman, Jeffrey D.; Prabhu, K. Sandeep; Thompson, Jerry T.; Reddy, P. Sreenivasula; Peters, Jeffrey M.; Peterson, Blake R.; Reddy, C. Channa; Vanden Heuvel, John P.
Liver insufficiency and damage is a major cause of death and disease worldwide and may result from exposure to environmental toxicants, specific combinations or dosages of pharmaceuticals and microbial metabolites. The generation of reactive intermediates, in particular 4-hydroxynonenal (4-HNE), is a common event in liver damage caused by a variety of hepatotoxic drugs and solvents. The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that are involved in the transcriptional regulation of lipid metabolism as well as other biological functions. Importantly, we have observed that the PPAR?/??/? mouse is more susceptible to chemically-induced hepatotoxicity than its wildtype counterpart, and our objective in this study was to elucidate the mechanism(s) by which PPAR?/? confers protection to hepatocytes. We hypothesized that PPAR?/? plays a protective role by responding to toxic lipids and altering gene expression accordingly. In support, oxidized-VLDL and constituents including 13-S-hydroxyoctadeca-dienoic acid (13(S)-HODE) and 4-HNE are PPAR?/? ligands. A structure-activity relationship was established where 4-HNE and 4-hydroperoxynonenal (4-HpNE) enhanced the activity of the PPAR?/? subtype while 4-hyroxy-hexenal (4-HHE), 4-oxo-2-Nonenal (4-ONE), and trans-4,5-epoxy-2(E)-decenal did not activate this receptor. Increasing PPAR?/? activity with a synthetic agonist decreased sensitivity of hepatocytes to 4-HNE and other toxic agents, whereas inhibition of this receptor had the opposite result. Gene expression microarray analysis identified several important PPAR?/?-regulated detoxification enzymes involved in 4-HNE metabolism that are regulated at the transcript level. This research established 4-HNE as an endogenous modulator of PPAR?/? activity and raises the possibility that agonists of this nuclear receptor may be utilized to prevent or treat liver disease associated with oxidative damage. PMID:17382197
Regulation of Proteome Maintenance Gene Expression by Activators of Peroxisome Proliferator-Activated Receptor ?
Ren, Hongzu; Vallanat, Beena; Brown-Borg, Holly M.; Currie, Richard; Corton, J. Christopher
The nuclear receptor peroxisome proliferator-activated receptor ? (PPAR?) is activated by a large number of xenobiotic and hypolipidemic compounds called peroxisome proliferator chemicals (PPCs). One agonist of PPAR? (WY-14,643) regulates responses in the mouse liver to chemical stress in part by altering expression of genes involved in proteome maintenance (PM) including protein chaperones in the heat shock protein (Hsp) family and proteasomal genes (Psm) involved in proteolysis. We hypothesized that other PPAR? activators including diverse hypolipidemic and xenobiotic compounds also regulate PM genes in the rat and mouse liver. We examined the expression of PM genes in rat and mouse liver after exposure to 7 different PPCs (WY-14,643, clofibrate, fenofibrate, valproic acid, di-(2-ethylhexyl) phthalate, perfluorooctanoic acid, and perfluorooctane sulfonate) using Affymetrix microarrays. In rats and mice, 174 or 380?PM genes, respectively, were regulated by at least one PPC. The transcriptional changes were, for the most part, dependent on PPAR?, as most changes were not observed in similarly treated PPAR?-null mice and the changes were not consistently observed in rats treated with activators of the nuclear receptors CAR or PXR. In rats and mice, PM gene expression exhibited differences compared to typical direct targets of PPAR? (e.g., Cyp4a family members). PM gene expression was usually delayed and in some cases, it was transient. Dose-response characterization of protein expression showed that Hsp86 and Hsp110 proteins were induced only at higher doses. These studies demonstrate that PPAR?, activated by diverse PPC, regulates the expression of a large number of genes involved in protein folding and degradation and support an expanded role for PPAR? in the regulation of genes that protect the proteome. PMID:21318169
Regulation of Proteome Maintenance Gene Expression by Activators of Peroxisome Proliferator-Activated Receptor ?.
Ren, Hongzu; Vallanat, Beena; Brown-Borg, Holly M; Currie, Richard; Corton, J Christopher
The nuclear receptor peroxisome proliferator-activated receptor ? (PPAR?) is activated by a large number of xenobiotic and hypolipidemic compounds called peroxisome proliferator chemicals (PPCs). One agonist of PPAR? (WY-14,643) regulates responses in the mouse liver to chemical stress in part by altering expression of genes involved in proteome maintenance (PM) including protein chaperones in the heat shock protein (Hsp) family and proteasomal genes (Psm) involved in proteolysis. We hypothesized that other PPAR? activators including diverse hypolipidemic and xenobiotic compounds also regulate PM genes in the rat and mouse liver. We examined the expression of PM genes in rat and mouse liver after exposure to 7 different PPCs (WY-14,643, clofibrate, fenofibrate, valproic acid, di-(2-ethylhexyl) phthalate, perfluorooctanoic acid, and perfluorooctane sulfonate) using Affymetrix microarrays. In rats and mice, 174 or 380?PM genes, respectively, were regulated by at least one PPC. The transcriptional changes were, for the most part, dependent on PPAR?, as most changes were not observed in similarly treated PPAR?-null mice and the changes were not consistently observed in rats treated with activators of the nuclear receptors CAR or PXR. In rats and mice, PM gene expression exhibited differences compared to typical direct targets of PPAR? (e.g., Cyp4a family members). PM gene expression was usually delayed and in some cases, it was transient. Dose-response characterization of protein expression showed that Hsp86 and Hsp110 proteins were induced only at higher doses. These studies demonstrate that PPAR?, activated by diverse PPC, regulates the expression of a large number of genes involved in protein folding and degradation and support an expanded role for PPAR? in the regulation of genes that protect the proteome. PMID:21318169
Chronic activation of sigma-1 receptor evokes nociceptive activation of trigeminal nucleus caudalis in rats.
Pyun, Kihyun; Son, Ji Seon; Kwon, Young Bae
Primary headache disorders, including migraine, are thought to be mediated by prolonged nociceptive activation of the trigeminal nucleus caudalis (TNC), but the precise mechanisms are poorly understood. Our past studies demonstrated that sigma-1 receptors (Sig-1R) facilitate spinal nociceptive transmission in several pain models. Based on these findings, this study asked if chronic activation of Sig-1R by intracisternal administration of the selective Sig-1R agonist, PRE084, produced TNC neuronal activation as a migraine trigger in rats. A single infusion of PRE084 (10, 50, 100, 500 nmol) significantly increased the number of Fos immunoreactive neurons (Fos-IR) in TNC, which BD1047 (a Sig-1R antagonist) reversed. Chronic infusion of PRE084 (100 nmol for 1, 3, 7 and 14 days) time-dependently elevated Fos-IR in TNC. The number of Fos-IR elevation from day 7 of infusion was comparable with a single capsaicin infusion as a headache model. Increase in face grooming/scratching behavior was evident from day 7, and peaked at day 14 of chronic PRE084 infusion, which was correlated with ?FosB elevation and phosphorylation of extracellular signal-regulated kinase, and the NMDA receptor NR1 subunit in TNC. Following 14 days of PRE084 infusion, the number of Fos-IR increased until day 7 after final infusion. Moreover, by day 14, Fos-IR associated with PRE084 infusion was significantly reversed by NMDA receptor antagonist MK801, rather than BD1047. These findings indicated that chronic activation of Sig-1R could evoke prolonged neuronal activation in the trigeminovascular system. PMID:24992726
Receptor activity modifying protein-3 mediates the protumorigenic activity of lysyl oxidase-like protein-2.
Brekhman, Vera; Lugassie, Jennie; Zaffryar-Eilot, Shelly; Sabo, Edmond; Kessler, Ofra; Smith, Victoria; Golding, Hana; Neufeld, Gera
Lysyl oxidase-like protein-2 (LOXL2) induces epithelial to mesenchymal transition and promotes invasiveness. To understand the mechanisms involved, we examined the effect of LOXL2 overexpression in MCF-7 cells on gene expression. We found that LOXL2 up-regulated the expression of receptor activity modifying protein-3 (RAMP3). Expression of RAMP3 in MDA-MB-231 cells in which LOXL2 expression was inhibited restored vimentin expression, invasiveness, and tumor development. Inhibition of RAMP3 expression in MDA-MB-231 cells mimicked the effects produced by inhibition of LOXL2 expression and was accompanied by inhibition of p38 phosphorylation. LOXL2 overexpression in these cells did not restore invasiveness, suggesting that RAMP3 functions downstream to LOXL2. LOXL2 and RAMP3 are strongly coexpressed in human colon, breast, and gastric carcinomas but not in normal colon or gastric epithelial cells. RAMP3 associates with several G-protein-coupled receptors forming receptors for peptides, such as adrenomedullin and amylin. We hypothesized that RAMP3 could function as a transducer of autocrine signals induced by such peptides. However, the proinvasive effects of RAMP3 could not be abrogated following inhibition of the expression or activity of these peptides. Our experiments suggest that the protumorigenic effects of LOXL2 are partially mediated by RAMP3 and that RAMP3 inhibitors may function as antitumorigenic agents. - PMID:20802105
NMDA receptor activation requires remodelling of intersubunit contacts within ligand-binding heterodimers
William F. Borschel; Swetha E. Murthy; Eileen M. Kasperek; Gabriela K. Popescu
Two classes of glutamate-activated channels mediate excitation at central synapses: N-methyl-D-aspartic acid (NMDA) receptors and non-NMDA receptors. Despite substantial structural homology, each class generates signals with characteristic kinetics and mediates distinct synaptic functions. In non-NMDA receptors, the strength of intersubunit contacts within ligand-binding domains is inversely correlated with functional desensitization. Here we test how the strength of these contacts affects
Genetic Program of Neuronal Differentiation and Growth Induced by Specific Activation of NMDA Receptors
Cristina A. Ghiani; Luis Beltran-Parrazal; Daniel M. Sforza; Jemily S. Malvar; Akop Seksenyan; Ruth Cole; Desmond J. Smith; Andrew Charles; Pedro A. Ferchmin; Jean de Vellis
Glutamate and its receptors are expressed very early during development and may play important roles in neurogenesis, synapse\\u000a formation and brain wiring. The levels of glutamate and activity of its receptors can be influenced by exogenous factors,\\u000a leading to neurodevelopmental disorders. To investigate the role of NMDA receptors on gene regulation in a neuronal model,\\u000a we used primary neuronal cultures
New insight into active muscarinic receptors with the novel radioagonist [�H]iperoxo.
Schrage, Ramona; Holze, Janine; Kl�ckner, Jessica; Balkow, Aileen; Klause, Anne S; Schmitz, Anna-Lena; De Amici, Marco; Kostenis, Evi; Tr�nkle, Christian; Holzgrabe, Ulrike; Mohr, Klaus
Activation of G protein-coupled receptors involves major conformational changes of the receptor protein ranging from the extracellular transmitter binding site to the intracellular G protein binding surface. GPCRs such as the muscarinic acetylcholine receptors are commonly probed with radioantagonists rather than radioagonists due to better physicochemical stability, higher affinity, and indifference towards receptor coupling states of the former. Here we introduce tritiated iperoxo, a superagonist at muscarinic M? receptors with very high affinity. In membrane suspensions of transfected CHO-cells, [�H]iperoxo - unlike the common radioagonists [�H]acetylcholine and [�H]oxotremorine M - allowed labelling of each of the five muscarinic receptor subtypes in radioagonist displacement and saturation binding studies. [�H]iperoxo revealed considerable differences in affinity between the even- and the odd-numbered muscarinic receptor subtypes with affinities for the M? and M? receptor in the picomolar range. Probing ternary complex formation on the M? receptor, [�H]iperoxo dissociation was not influenced by an archetypal allosteric inverse agonist, reflecting activation-related rearrangement of the extracellular loop region. At the inner side of M?, the preferred Gi protein acted as a positive allosteric modulator of [�H]iperoxo binding, whereas Gs and Gq were neutral in spite of their robust coupling to the activated receptor. In intact CHO-hM? cells, endogenous guanylnucleotides promoted receptor/G protein-dissociation resulting in low-affinity agonist binding which, nevertheless, was still reported by [�H]iperoxo. Taken together, the muscarinic superagonist [�H]iperoxo is the best tool currently available for direct probing activation-related conformational transitions of muscarinic receptors. PMID:24863257
Mechanisms involved in VPAC receptors activation and regulation: lessons from pharmacological and mutagenesis studies.
Langer, Ingrid
Vasoactive intestinal peptide (VIP) plays diverse and important role in human physiology and physiopathology and their receptors constitute potential targets for the treatment of several diseases such as neurodegenerative disorder, asthma, diabetes, and inflammatory diseases. This article reviews the current knowledge regarding the two VIP receptors, VPAC(1) and VPAC(2), with respect to mechanisms involved in receptor activation, G protein coupling, signaling, regulation, and oligomerization. PMID:23115557
Receptor Isoforms Mediate Opposing Proliferative Effects through Gbg-Activated p38 or Akt Pathways
The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst2 receptor isoforms, which couple to identical Ga subunit
Sigma-1 receptor activation prevents intracellular calcium dysregulation in cortical neurons during in vitro ischemia.
Katnik, Christopher; Guerrero, Waldo R; Pennypacker, Keith R; Herrera, Yelenis; Cuevas, Javier
Sigma receptors are putative targets for neuroprotection following ischemia; however, little is known on their mechanism of action. One of the key components in the demise of neurons following ischemic injury is the disruption of intracellular calcium homeostasis. Fluorometric calcium imaging was used to examine the effects of sigma receptor activation on changes in intracellular calcium concentrations ([Ca(2+)](i)) evoked by in vitro ischemia in cultured cortical neurons from embryonic rats. The sigma receptor agonist, 1,3-di-o-tolyl-guanidine (DTG), was shown to depress [Ca(2+)](i) elevations observed in response to ischemia induced by sodium azide and glucose deprivation. Two sigma receptor antagonists, metaphit [1-(1-(3-isothiocyanatophenyl)-cyclohexyl)-piperidine] and BD-1047 (N-[2-3,4-dichlorophenyl)-ethyl]-N-methyl-2-(dimethylamino)ethylamine), were shown to blunt the ability of DTG to inhibit ischemia-evoked increases in [Ca(2+)](i), revealing that the effects are mediated by activation of sigma receptors and not via the actions of DTG on nonspecific targets such as N-methyl-d-aspartate receptors. DTG inhibition of ischemia-induced increases in [Ca(2+)](i) was mimicked by the sigma-1 receptor-selective agonists, carbetapentane, (+)-pentazocine and PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], but not by the sigma-2-selective agonist, ibogaine, showing that activation of sigma-1 receptors is responsible for the effects. In contrast, DTG, carbetapentane, and ibogaine blocked spontaneous, synchronous calcium transients observed in our preparation at concentrations consistent with sigma receptor-mediated effects, indicating that both sigma-1 and sigma-2 receptors regulate events that affect [Ca(2+)](i) in cortical neurons. Our studies show that activation of sigma receptors can ameliorate [Ca(2+)](i) dysregulation associated with ischemia in cortical neurons and, thus, identify one of the mechanisms by which these receptors may exert their neuroprotective properties. PMID:16988055
Aberrant expression of laminin-332 promotes cell proliferation and cyst growth in ARPKD
Dang, Suparna; Marinkovich, M. Peter; Lazarova, Zelmira; Yoder, Bradley; Torres, Vicente E.; Wallace, Darren P.
Basement membrane abnormalities have often been observed in kidney cysts of polycystic kidney disease (PKD) patients and animal models. There is an abnormal deposition of extracellular matrix molecules, including laminin-?3,?3,?2 (laminin-332), in human autosomal dominant PKD (ADPKD). Knockdown of PKD1 paralogs in zebrafish leads to dysregulated synthesis of the extracellular matrix, suggesting that altered basement membrane assembly may be a primary defect in ADPKD. In this study, we demonstrate that laminin-332 is aberrantly expressed in cysts and precystic tubules of human autosomal recessive PKD (ARPKD) kidneys as well as in the kidneys of PCK rats, an orthologous ARPKD model. There was aberrant expression of laminin-?2 as early as postnatal day 2 and elevated laminin-332 protein in postnatal day 30, coinciding with the formation and early growth of renal cysts in PCK rat kidneys. We also show that a kidney cell line derived from Oak Ridge polycystic kidney mice, another model of ARPKD, exhibited abnormal lumen-deficient and multilumen structures in Matrigel culture. These cells had increased proliferation rates and altered expression levels of laminin-332 compared with their rescued counterparts. A function-blocking polyclonal antibody to laminin-332 significantly inhibited their abnormal proliferation rates and rescued their aberrant phenotype in Matrigel culture. Furthermore, abnormal laminin-332 expression in cysts originating from collecting ducts and proximal tubules as well as in precystic tubules was observed in a human end-stage ADPKD kidney. Our results suggest that abnormal expression of laminin-332 contributes to the aberrant proliferation of cyst epithelial cells and cyst growth in genetic forms of PKD. PMID:24370592
Aberrant expression of laminin-332 promotes cell proliferation and cyst growth in ARPKD.
Vijayakumar, Soundarapandian; Dang, Suparna; Marinkovich, M Peter; Lazarova, Zelmira; Yoder, Bradley; Torres, Vicente E; Wallace, Darren P
Basement membrane abnormalities have often been observed in kidney cysts of polycystic kidney disease (PKD) patients and animal models. There is an abnormal deposition of extracellular matrix molecules, including laminin-?3,?3,?2 (laminin-332), in human autosomal dominant PKD (ADPKD). Knockdown of PKD1 paralogs in zebrafish leads to dysregulated synthesis of the extracellular matrix, suggesting that altered basement membrane assembly may be a primary defect in ADPKD. In this study, we demonstrate that laminin-332 is aberrantly expressed in cysts and precystic tubules of human autosomal recessive PKD (ARPKD) kidneys as well as in the kidneys of PCK rats, an orthologous ARPKD model. There was aberrant expression of laminin-?2 as early as postnatal day 2 and elevated laminin-332 protein in postnatal day 30, coinciding with the formation and early growth of renal cysts in PCK rat kidneys. We also show that a kidney cell line derived from Oak Ridge polycystic kidney mice, another model of ARPKD, exhibited abnormal lumen-deficient and multilumen structures in Matrigel culture. These cells had increased proliferation rates and altered expression levels of laminin-332 compared with their rescued counterparts. A function-blocking polyclonal antibody to laminin-332 significantly inhibited their abnormal proliferation rates and rescued their aberrant phenotype in Matrigel culture. Furthermore, abnormal laminin-332 expression in cysts originating from collecting ducts and proximal tubules as well as in precystic tubules was observed in a human end-stage ADPKD kidney. Our results suggest that abnormal expression of laminin-332 contributes to the aberrant proliferation of cyst epithelial cells and cyst growth in genetic forms of PKD. PMID:24370592
Stable, covalent attachment of laminin to microposts improves the contractility of mouse neonatal cardiomyocytes.
Ribeiro, Alexandre J S; Zaleta-Rivera, Kathia; Ashley, Euan A; Pruitt, Beth L
The mechanical output of contracting cardiomyocytes, the muscle cells of the heart, relates to healthy and disease states of the heart. Culturing cardiomyocytes on arrays of elastomeric microposts can enable inexpensive and high-throughput studies of heart disease at the single-cell level. However, cardiomyocytes weakly adhere to these microposts, which limits the possibility of using biomechanical assays of single cardiomyocytes to study heart disease. We hypothesized that a stable covalent attachment of laminin to the surface of microposts improves cardiomyocyte contractility. We cultured cells on polydimethylsiloxane microposts with laminin covalently bonded with the organosilanes 3-glycidoxypropyltrimethoxysilane and 3-aminopropyltriethoxysilane with glutaraldehyde. We measured displacement of microposts induced by the contractility of mouse neonatal cardiomyocytes, which attach better than mature cardiomyocytes to substrates. We observed time-dependent changes in contractile parameters such as micropost deformation, contractility rates, contraction and relaxation speeds, and the times of contractions. These parameters were affected by the density of laminin on microposts and by the stability of laminin binding to micropost surfaces. Organosilane-mediated binding resulted in higher laminin surface density and laminin binding stability. 3-glycidoxypropyltrimethoxysilane provided the highest laminin density but did not provide stable protein binding with time. Higher surface protein binding stability and strength were observed with 3-aminopropyltriethoxysilane with glutaraldehyde. In cultured cardiomyocytes, contractility rate, contraction speeds, and contraction time increased with higher laminin stability. Given these variations in contractile function, we conclude that binding of laminin to microposts via 3-aminopropyltriethoxysilane with glutaraldehyde improves contractility observed by an increase in beating rate and contraction speed as it occurs during the postnatal maturation of cardiomyocytes. This approach is promising for future studies to mimic in vivo tissue environments. PMID:25133578
Palmitoylation of human proteinase-activated receptor-2 differentially regulates receptor-triggered ERK1/2 activation, calcium signalling and endocytosis.
Botham, Andrew; Guo, Xiaodan; Xiao, Yu Pei; Morice, Alyn H; Compton, Steven J; Sadofsky, Laura R
hPAR(2) (human proteinase-activated receptor-2) is a member of the novel family of proteolytically activated GPCRs (G-protein-coupled receptors) termed PARs (proteinase-activated receptors). Previous pharmacological studies have found that activation of hPAR(2) by mast cell tryptase can be regulated by receptor N-terminal glycosylation. In order to elucidate other post-translational modifications of hPAR(2) that can regulate function, we have explored the functional role of the intracellular cysteine residue Cys(361). We have demonstrated, using autoradiography, that Cys(361) is the primary palmitoylation site of hPAR(2). The hPAR(2)C361A mutant cell line displayed greater cell-surface expression compared with the wt (wild-type)-hPAR(2)-expressing cell line. hPAR(2)C361A also showed a decreased sensitivity and efficacy (intracellular calcium signalling) towards both trypsin and SLIGKV. In stark contrast, hPAR(2)C361A triggered greater and more prolonged ERK (extracellular-signal-regulated kinase) phosphorylation compared with that of wt-hPAR(2) possibly through Gi, since pertussis toxin inhibited the ability of this receptor to activate ERK. Finally, flow cytometry was utilized to assess the rate and extent of receptor internalization following agonist challenge. hPAR(2)C361A displayed faster internalization kinetics following trypsin activation compared with wt-hPAR(2), whereas SLIGKV had a negligible effect on internalization for either receptor. In conclusion, palmitoylation plays an important role in the regulation of PAR(2) expression, agonist sensitivity, desensitization and internalization. PMID:21627585
Inhibition of Proliferation and Estrogen Receptor Signaling by Peroxisome Proliferator-activated Receptor Ligands in Uterine Leiomyoma1
Kevin D. Houston; John A. Copland; Russell R. Broaddus; Marco M. Gottardis; Susan M. Fischer; Cheryl Lyn Walker
Peroxisome proliferator-activated receptor (PPAR) is an important signaling molecule in cells of mesenchymal origin, inducing differentiation and regulating cell proliferation in several cell types such as vascular smooth muscle cells. Leiomyomas arise from smooth muscle cells of the uterine myometrium with an incidence rate as high as 70% in women of reproductive age. PPAR signaling has not been characterized in
Dynamic conformational switching in the chemokine ligand is essential for G-protein-coupled receptor activation
Joseph, Prem Raj B.; Sawant, Kirti V.; Isley, Angela; Pedroza, Mesias; Garofalo, Roberto P.; Richardson, Ricardo M.; Rajarathnam, Krishna
Chemokines mediate diverse functions from organogenesis to mobilizing leucocytes, and are unusual agonists for class-A GPCRs (G-protein-coupled receptors) because of their large size and multi-domain structure. The current model for receptor activation, which involves interactions between chemokine N-loop and receptor N-terminal residues (Site-I) and between chemokine N-terminal and receptor extracellular loop/transmembrane residues (Site-II), fails to describe differences in ligand/receptor selectivity and the activation of multiple signalling pathways. In the present study, we show in neutrophil-activating chemokine CXCL8 that the highly conserved GP (glycine-proline) motif located distal to both N-terminal and N-loop residues couples Site-I and Site-II interactions. Mutations in the GP motif caused various differences from native-like function to complete loss of activity that could not be correlated with the specific mutation, receptor affinity or subtype, or a specific signalling pathway. NMR studies indicated that the GP motif does not influence Site-I interactions, but molecular dynamics simulations suggested that this motif dictates substates of the CXCL8 conformational ensemble. We conclude that the GP motif enables diverse receptor functions by controlling cross-talk between Site-I and Site-II, and further propose that the repertoire of chemokine functions is best described by a conformational ensemble model in which a network of long-range coupled indirect interactions mediate receptor activity. PMID:24032673
Ligands Raise the Constraint That Limits Constitutive Activation in G Protein-coupled Opioid Receptors*
Vezzi, Vanessa; Onaran, H. Ongun; Molinari, Paola; Guerrini, Remo; Balboni, Gianfranco; Cal�, Girolamo; Costa, Tommaso
Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in ? (DOP) and ? (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4�5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the �two state� extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form. PMID:23836900
Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells
Abeyweera, Thushara P.; Merino, Ernesto
Natural killer (NK) lymphocytes use a variety of activating receptors to recognize and kill infected or tumorigenic cells during an innate immune response. To prevent targeting healthy tissue, NK cells also express numerous inhibitory receptors that signal through immunotyrosine-based inhibitory motifs (ITIMs). Precisely how signals from competing activating and inhibitory receptors are integrated and resolved is not understood. To investigate how ITIM receptor signaling impinges on activating pathways, we developed a photochemical approach for stimulating the inhibitory receptor KIR2DL2 during ongoing NK cell�activating responses in high-resolution imaging experiments. Photostimulation of KIR2DL2 induces the rapid formation of inhibitory receptor microclusters in the plasma membrane and the simultaneous suppression of microclusters containing activating receptors. This is followed by the collapse of the peripheral actin cytoskeleton and retraction of the NK cell from the source of inhibitory stimulation. These results suggest a cell biological basis for ITIM receptor signaling and establish an experimental framework for analyzing it. PMID:21339333
A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation
Technology Transfer Automated Retrieval System (TEKTRAN)
Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...
Protease-activated receptor-2 inhibits BK channel activity in bronchopulmonary sensory neurons.
Moss, Charles R; Gilbert, Carolyn A; Gabriel, Sabry A; Gu, Qihai
Activation of protease-activated receptor-2 (PAR2) contributes to airway inflammation and airway hypersensitivity, the hallmark features of allergic asthma; and a neurogenic mechanism involving hypersensitivity of bronchopulmonary sensory nerves has been indicated. Large-conductance Ca(2+)-activated potassium (BK) channels are known to play an important role in shaping neuronal excitability. The aim of this study was to investigate the potential regulation of BK channel activities by PAR2 activation in vagal bronchopulmonary sensory neurons. Our results showed that pretreatment with PAR2-activating peptide (PAR2-AP; 100?M, 120s), but not its control peptide PAR2-RP, significantly reduced BK current density in these neurons. Inhibition of phospholipase C, PKC, PKA or MEK/ERK signaling pathway did not prevent the suppression of BK current by PAR2 activation; whereas intracellular application of Ca(2+) chelator BAPTA-AM completely abolished the PAR2 regulation of BK current. In addition, our results demonstrated that activation of PAR2 increased excitability of bronchopulmonary sensory neurons, in a similar manner as displayed by a direct BK channel blockade. In summary, our data suggest that suppression of BK channel activity contributes to PAR2 activation-induced hyperexcitability of vagal bronchopulmonary sensory neurons. PMID:25578948
Inhibition of androgen receptor and ?-catenin activity in prostate cancer.
Lee, Eugine; Madar, Aviv; David, Gregory; Garabedian, Michael J; Dasgupta, Ramanuj; Logan, Susan K
Androgen receptor (AR) is the major therapeutic target in aggressive prostate cancer. However, targeting AR alone can result in drug resistance and disease recurrence. Therefore, simultaneous targeting of multiple pathways could in principle be an effective approach to treating prostate cancer. Here we provide proof-of-concept that a small-molecule inhibitor of nuclear ?-catenin activity (called C3) can inhibit both the AR and ?-catenin-signaling pathways that are often misregulated in prostate cancer. Treatment with C3 ablated prostate cancer cell growth by disruption of both ?-catenin/T-cell factor and ?-catenin/AR protein interaction, reflecting the fact that T-cell factor and AR have overlapping binding sites on ?-catenin. Given that AR interacts with, and is transcriptionally regulated by ?-catenin, C3 treatment also resulted in decreased occupancy of ?-catenin on the AR promoter and diminished AR and AR/?-catenin target gene expression. Interestingly, C3 treatment resulted in decreased AR binding to target genes accompanied by decreased recruitment of an AR and ?-catenin cofactor, coactivator-associated arginine methyltransferase 1 (CARM1), providing insight into the unrecognized function of ?-catenin in prostate cancer. Importantly, C3 inhibited tumor growth in an in vivo xenograft model and blocked renewal of bicalutamide-resistant sphere-forming cells, indicating the therapeutic potential of this approach. PMID:24019458
Soluble urokinase plasminogen activator receptor during allogeneic stem cell transplantation.
Haastrup, E; Andersen, J; Ostrowski, S R; H�yer-Hansen, G; Jacobsen, N; Heilmann, C; Ullum, H; M�ller, K
Previous studies have found that soluble urokinase plasminogen activation receptor (suPAR) increases during inflammatory and malignant illness and elevated suPAR levels may be associated with poor clinical outcome. The purpose of this study was to investigate plasma levels of suPAR during the course of allogeneic stem cell transplantation (SCT). Twenty SCT patients were included in the study. suPAR was measured by ELISA in daily taken plasma samples during the pretransplant conditioning with chemotherapy and weekly for 1 month after infusion of the graft. suPAR levels before the start of the conditioning were significantly elevated when compared to those of healthy controls. During the conditioning in particular treatment with antithymocyte globulin was associated with significantly increased suPAR levels (P = 0.012). At day +7 after infusion of the graft, suPAR levels had decreased to pretreatment levels. High suPAR levels at day 0 were associated with increased mortality (P = 0.011). The present study found increased suPAR levels during the conditioning in SCT patients. Further, the data indicated that increased suPAR levels may be associated with increased mortality, suggesting suPAR as a candidate for further studies as an outcome predictor in SCT. PMID:21223347
CIRCRESAHA/2013/301656/R2 Liver X Receptor (LXR) activation stimulates iron export in human alternative macrophages
Boyer, Edmond
atherosclerotic plaques, CD68+MR+ macrophages accumulate oxidized lipids, which activate Liver X Receptors (LXR LXR liver X receptor LXRE LXR response element M1 classically activated macrophages M2 alternativelyCIRCRESAHA/2013/301656/R2 1 Liver X Receptor (LXR) activation stimulates iron export in human
Delta Opioid Receptors Regulate Temporoammonic-Activated Feedforward Inhibition to the Mouse CA1 Hippocampus
Rezai, Xavier; Kieffer, Brigitte L.
The opioid system influences learning and memory processes. However, neural mechanisms underlying the modulation of hippocampal activity by opioid receptors remain largely unknown. Here, we compared how mu and delta receptors operate within the mouse CA1 network, and used knock-in mice expressing functional delta opioid receptors fused to the green fluorescent protein (DOR-eGFP) to determine how delta opioid receptor-expressing interneurons integrate within the hippocampal circuitry. Through whole cell patch-clamp recording of CA1 pyramidal neurons from wild-type and DOR-eGFP mice, we found that mu and delta receptors both modulate spontaneous GABAergic inhibition received by these cells. Interestingly, mu but not delta receptor activation decreased the feed-forward inhibitory input evoked by Schaffer collateral stimulation. However, mu and delta agonists modulated GABAergic feed-forward inhibition when evoked upon stimulation of the temporoammonic pathway. In addition, anterograde tracing using biotinylated dextran amine injected into the entorhinal cortex of DOR-eGFP mice suggests the existence of synaptic contacts between temporoammonic afferents and delta receptor-expressing interneurons processes in CA1. Altogether, our data demonstrate a distinct modulatory role of the hippocampal network activity by mu and delta opioid receptors, and show for the first time that delta receptor-expressing interneurons in the CA1 are recruited by the temporoammonic pathway rather than the Schaffer collateral. PMID:24260157
Insulin receptor binding and protein kinase activity in muscles of trained rats
Dohm, G.L.; Sinha, M.K.; Caro, J.F.
Exercise has been shown to increase insulin sensitivity, and muscle is quantitatively the most important tissue of insulin action. Since the first step in insulin action is the binding to a membrane receptor, the authors postulated that exercise training would change insulin receptors in muscle and in this study they have investigated this hypothesis. Female rats initially weighing approx. 100 g were trained by treadmill running for 2 h/day, 6 days/wk for 4 wk at 25 m/min (0 grade). Insulin receptors from vastus intermedius muscles were solubilized by homogenizing in a buffer containing 1% Triton X-100 and then partially purified by passing the soluble extract over a wheat germ agglutinin column. The 4 wk training regimen resulted in a 65% increase in citrate synthase activity in red vastus lateralis muscle, indicating an adaptation to exercise ( SVI). Insulin binding by the partially purified receptor preparations was approximately doubled in muscle of trained rats at all insulin concentrations, suggesting an increase in the number of receptors. Training did not alter insulin receptor structure as evidenced by electrophoretic mobility under reducing and nonreducing conditions. Basal insulin receptor protein kinase activity was higher in trained than untrained animals and this was likely due to the greater number of receptors. However, insulin stimulation of the protein kinase activity was depressed by training. These results demonstrate that endurance training does alter receptor number and function in muscle and these changes may be important in increasing insulin sensitivity after exercise training.
Harikumar, Kaleeckal G.; Simms, John; Christopoulos, George; Sexton, Patrick M.; Miller, Laurence J.
The three receptor activity-modifying proteins (RAMPs) have been recognized as being important for the trafficking and function of a subset of family B G protein-coupled receptors, although the structural basis for this has not been well established. In the current work, we use morphological fluorescence techniques, bioluminescence resonance energy transfer, and bimolecular fluorescence complementation to demonstrate that the secretin receptor associates specifically with RAMP3, but not with RAMP1 or RAMP2. We use truncation constructs, peptide competition experiments, and chimeric secretin-GLP1 receptor constructs to establish that this association is structurally-specific, dependent on the intramembranous region of the RAMP and TM6 and TM7 of this receptor. There were no observed changes in secretin-stimulated cAMP, intracellular calcium, ERK1/2 phosphorylation, or receptor internalization in receptor-bearing COS or CHO-K1 cells in the presence or absence of exogenous RAMP transfection, although the secretin receptor trafficks normally to the cell surface in these cells in a RAMP-independent manner, resulting in both free and RAMP-associated receptor on the cell surface. RAMP3 association with this receptor was shown to be capable of rescuing a receptor mutant (G241C) that is normally trapped intracellularly in the biosynthetic machinery. Similarly, secretin receptor expression had functional effects on adrenomedullin activity, with increasing secretin receptor expression competing for RAMP3 association with the calcitonin receptor-like receptor to yield a functional adrenomedullin receptor. These data provide important new insights into the structural basis for RAMP3 interaction with a family B G protein-coupled receptor, potentially providing a highly selective target for drug action. This may be representative of similar interactions between other members of this receptor family and RAMP proteins. PMID:19886671
A CBP Integrator Complex Mediates Transcriptional Activation and AP1 Inhibition by Nuclear Receptors
Yasutomi Kamei; Lan Xu; Thorsten Heinzel; Joseph Torchia; Riki Kurokawa; Bernd Gloss; Sheng-Cai Lin; Richard A Heyman; David W Rose; Christopher K Glass; Michael G Rosenfeld
Nuclear receptors regulate gene expression by direct activation of target genes and inhibition of AP-1. Here we report that, unexpectedly, activation by nuclear receptors requires the actions of CREB-binding protein (CBP) and that inhibition of AP-1 activity is the apparent result of competition for limiting amounts of CBP\\/p300 in cells. Utilizing distinct domains, CBP directly interacts with the ligand-binding domain
The transcriptional co-activator p\\/CIP binds CBP and mediates nuclear-receptor function
Joseph Torchia; David W. Rose; Juan Inostroza; Yasutomi Kamei; Stefan Westin; Christopher K. Glass; Michael G. Rosenfeld
The functionally conserved proteins CBP and p300 act in conjunction with other factors to activate transcription of DNA. A new factor, p\\/CIP, has been discovered that is present in the cell as a complex with CBP and is required for transcriptional activity of nuclear receptors and other CBP\\/p300-dependent transcription factors. The highly related nuclear-receptor co-activator protein NCoA-1 is also specifically
Respiratory syncytial virus represses glucocorticoid receptor-mediated gene activation.
Hinzey, Adam; Alexander, Jacob; Corry, Jacqueline; Adams, Kathleen M; Claggett, Amanda M; Traylor, Zachary P; Davis, Ian C; Webster Marketon, Jeanette I
Respiratory syncytial virus (RSV) is a common cause of bronchiolitis in infants. Although antiinflammatory in nature, glucocorticoids have been shown to be ineffective in the treatment of RSV-induced bronchiolitis and wheezing. In addition, the effectiveness of glucocorticoids at inhibiting RSV-induced proinflammatory cytokine production in cell culture has been questioned. In this study, we have investigated the effect of RSV infection on glucocorticoid-induced gene activation in lung epithelium-derived cells. We show that RSV infection inhibits dexamethasone induction of three glucocorticoid receptor (GR)-regulated genes (glucocorticoid-inducible leucine zipper, FK506 binding protein, and MAPK phosphatase 1) in A549, BEAS-2B cells, and primary small airway epithelial cells. UV irradiation of the virus prevents this repression, suggesting that viral replication is required. RSV is known to activate the nuclear factor ?B (NF?B) pathway, which is mutually antagonistic towards the GR pathway. However, specific inhibition of NF?B had no effect on the repression of GR-induced genes by RSV infection, indicating that RSV repression of GR is independent of NF?B. RSV infection of A549 cells does not alter GR protein levels or GR nuclear translocation but does reduce GR binding to the promoters of the glucocorticoid responsive genes analyzed in this study. Repression of GR by RSV infection may account for the apparent clinical ineffectiveness of glucocorticoids in RSV bronchiolitis therapy. In addition, this data adds to our previously published data suggesting that GR may be a general target for infectious agents. Identifying the mechanisms through which this suppression occurs may lead to the development of novel therapeutics. PMID:21190962
Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2
Kuwasako, Kenji, E-mail: email@example.com [Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan)] [Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan); Kitamura, Kazuo; Nagata, Sayaka; Hikosaka, Tomomi [Division of Circulation and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan)] [Division of Circulation and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan); Kato, Johji [Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan)] [Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan)
Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [{sup 125}I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser{sup 449} to Ser{sup 467} were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.
Glycogen content regulates peroxisome proliferator activated receptor-? (PPAR-?) activity in rat skeletal muscle.
Philp, Andrew; MacKenzie, Matthew G; Belew, Micah Y; Towler, Mhairi C; Corstorphine, Alan; Papalamprou, Angela; Hardie, D Grahame; Baar, Keith
Performing exercise in a glycogen depleted state increases skeletal muscle lipid utilization and the transcription of genes regulating mitochondrial ?-oxidation. Potential candidates for glycogen-mediated metabolic adaptation are the peroxisome proliferator activated receptor (PPAR) coactivator-1? (PGC-1?) and the transcription factor/nuclear receptor PPAR-?. It was therefore the aim of the present study to examine whether acute exercise with or without glycogen manipulation affects PGC-1? and PPAR-? function in rodent skeletal muscle. Twenty female Wistar rats were randomly assigned to 5 experimental groups (n = 4): control [CON]; normal glycogen control [NG-C]; normal glycogen exercise [NG-E]; low glycogen control [LG-C]; and low glycogen exercise [LG-E]). Gastrocnemius (GTN) muscles were collected immediately following exercise and analyzed for glycogen content, PPAR-? activity via chromatin immunoprecipitation (ChIP) assays, AMPK ?1/?2 kinase activity, and the localization of AMPK and PGC-1?. Exercise reduced muscle glycogen by 47 and 75% relative to CON in the NG-E and LG-E groups, respectively. Exercise that started with low glycogen (LG-E) finished with higher AMPK-?2 activity (147%, p<0.05), nuclear AMPK-?2 and PGC-1?, but no difference in AMPK-?1 activity compared to CON. In addition, PPAR-? binding to the CPT1 promoter was significantly increased only in the LG-E group. Finally, cell reporter studies in contracting C2C12 myotubes indicated that PPAR-? activity following contraction is sensitive to glucose availability, providing mechanistic insight into the association between PPAR-? and glycogen content/substrate availability. The present study is the first to examine PPAR-? activity in skeletal muscle in response to an acute bout of endurance exercise. Our data would suggest that a factor associated with muscle contraction and/or glycogen depletion activates PPAR-? and initiates AMPK translocation in skeletal muscle in response to exercise. PMID:24146969
Phorbol ester stimulates secretory activity while inhibiting receptor-activated aminopyrine uptake by gastric glands
Brown, M.R.; Chew, C.S.
Both cyclic AMP-dependent and -independent secretagogues stimulate pepsinogen release, respiration and H/sup +/ secr