text:
Keratinocyte growth factor-2 polynucleotides
US Patent & Trademark Office Database
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a Keratinocyte Growth Factor, sometimes hereinafter referred to as "KGF-2" also formerly known as Fibroblast Growth Factor 12 (FGF-12). This invention further relates to the therapeutic use of KGF-2 to promote or accelerate wound healing. This invention also relates to novel mutant forms of KGF-2 that show enhanced activity, increased stability, higher yield or better solubility.
Keratinocyte growth factor promotes melanosome transfer to keratinocytes.
Melanogenesis and melanosome transfer from the melanocytes to the neighboring keratinocytes are induced by ultraviolet radiation and modulated by autocrine and paracrine factors. Keratinocyte growth factor (KGF/fibroblast growth factor (FGF)7) is a paracrine mediator of human keratinocyte growth and differentiation. We evaluated the influence of KGF on melanosome transfer in co-cultures of keratinocytes and melanocytes. Immunofluorescence analysis using anti-tyrosinase and anti-human cytokeratin antibodies, phagocytic assays using fluorescent latex beads, and ultrastructural analysis indicated that KGF is able to induce melanosome transfer acting only on the recipient keratinocytes and as a consequence of a general role of KGF in the promotion of the phagocytic process. Inhibition of proteinase-activated receptor-2, to block the Rho-dependent phagocytic pathway, or of the Src family tyrosine kinases, to inhibit the Rac-dependent pathway, showed that KGF promotes phagocytosis through both mechanisms. Increased expression of the KGF receptor (KGFR) on the keratinocytes by transfection led to increased phagocytosis of latex beads following KGF treatment, suggesting that the KGF effect is directly mediated by KGFR expression and activation. Moreover, confocal microscopic analysis revealed that KGFR localize in phagosomes during KGF-induced phagocytosis, suggesting a direct role of the receptor in regulating both the early steps of uptake and the intracellular traffic of the phagosomes. PMID:16354189
Cardinali, Giorgia; Ceccarelli, Simona; Kovacs, Daniela; Aspite, Nicaela; Lotti, Lavinia Vittoria; Torrisi, Maria Rosaria; Picardo, Mauro
Issues within Keratinocyte Growth Factor (KGF) research
As the seventh member of Fibroblast Growth Factor (FGF) family, Keratinocyte Growth Factor (KGF or FGF-7) is observed to mediate\\u000a epithelial cell proliferation and differentiation in a variety of tissues. In this article, such following issues within KGF\\u000a research were reviewed, as (1) KGF functioning pathways: experimental results demonstrated the paracrine pathway of KGF played\\u000a main role in mesenchymal-epithelial interactions
Zheng Wen-jing; Yuan Li; Liu Xue-dong; Zheng Dong
Transforming Growth Factor Antagonizes Alveolar Type II Cell Proliferation Induced by Keratinocyte Growth Factor
Keratinocyte growth factor (KGF) is a mitogen for rat type II cells and also stimulates differentiation in vitro. Administration of KGF also protects the lung from a variety of injuries and subsequent development of fibrosis. Because transforming growth factor (TGF)- has been shown to inhibit epithelial cell proliferation and surfactant protein gene expression in other systems and is thought to
Feijie Zhang; Larry D. Nielsen; Joseph J. Lucas; Robert J. Mason
Trans Retinoic Acid Enhances the Growth Response of Epidermal Keratinocytes to Epidermal Growth Factor and Transforming Growth Factor Beta
Retinoids have been shown to either stimulate or inhibit epidermal keratinocyte proliferation. We have observed that in serum and growth factor free medium (basal medium), epidermal growth factor (EGF) and transforming growth factor alpha (TGF?) stimulated DNA synthesis in mouse epidermal keratinocyte cultures (mKC) in a time- and dose-dependent manner. Incubation with all-trans retinoic acid (RA) greatly enhanced the stimulatory
Philip S. Tong; Nancy N. Horowitz; Larry A. Wheeler
Stimulation of human keratinocyte growth by alginate oligosaccharides, a possible co-factor for epidermal growth factor in cell culture
Oligosaccharides, involved in regulation of plant developmental and defensive processes, were tested to determine their ability to enhance proliferation of human keratinocytes. A mixture of alginate oligosaccharides remarkably stimulated keratinocyte growth and [3H]thymidine uptake in the presence of epidermal growth factor (EGF). The activity was comparable to bovine pituitary extract, a common complement in keratinocyte culture, and additive on BPE-induced
Akira Kawada; Nozomi Hiura; Masakazu Shiraiwa; Shingo Tajima; Masataro Hiruma; Kenji Hara; Akira Ishibashi; Hidenari Takahara
Cortactin involvement in the keratinocyte growth factor and fibroblast growth factor 10 promotion of migration and cortical actin assembly in human keratinocytes
Keratinocyte growth factor (KGF/FGF7) and fibroblast growth factor 10 (FGF10/KGF2) regulate keratinocyte proliferation and differentiation by binding to the tyrosine kinase KGF receptor (KGFR). KGF induces keratinocyte motility and cytoskeletal rearrangement, whereas a direct role of FGF10 on keratinocyte migration is not clearly established. Here we analyzed the motogenic activity of FGF10 and KGF on human keratinocytes. Migration assays and immunofluorescence of actin cytoskeleton revealed that FGF10 is less efficient than KGF in promoting migration and exerts a delayed effect in inducing lamellipodia and ruffles formation. Both growth factors promoted phosphorylation and subsequent membrane translocation of cortactin, an F-actin binding protein involved in cell migration; however, FGF10-induced cortactin phosphorylation was reduced, more transient and delayed with respect to that promoted by KGF. Cortactin phosphorylation induced by both growth factors was Src-dependent, while its membrane translocation and cell migration were blocked by either Src and PI3K inhibitors, suggesting that both pathways are involved in KGF- and FGF10-dependent motility. Furthermore, siRNA-mediated downregulation of cortactin inhibited KGF- and FGF10-induced migration. These results indicate that cortactin is involved in keratinocyte migration promoted by both KGF and FGF10.
Ceccarelli, Simona [Dipartimento di Medicina Sperimentale, Universita di Roma 'La Sapienza', Viale Regina Elena 324, 00161, Rome (Italy); Cardinali, Giorgia [Istituto Dermatologico San Gallicano, IRCCS, Rome (Italy); Aspite, Nicaela [Istituto Dermatologico San Gallicano, IRCCS, Rome (Italy); Picardo, Mauro [Istituto Dermatologico San Gallicano, IRCCS, Rome (Italy); Marchese, Cinzia [Dipartimento di Medicina Sperimentale, Universita di Roma 'La Sapienza', Viale Regina Elena 324, 00161, Rome (Italy); Torrisi, Maria Rosaria [Dipartimento di Medicina Sperimentale, Universita di Roma 'La Sapienza', Viale Regina Elena 324, 00161, Rome (Italy); Azienda Ospedaliera Sant'Andrea, Rome (Italy); Mancini, Patrizia [Dipartimento di Medicina Sperimentale, Universita di Roma 'La Sapienza', Viale Regina Elena 324, 00161, Rome (Italy)]. E-mail: firstname.lastname@example.com
Keratinocyte growth factor is highly overexpressed in inflammatory bowel disease.
Recently we demonstrated an important function of keratinocyte growth factor (KGF) in wound re-epithelialization. As KGF is mitogenic for various epithelial cells, we speculated about a role of KGF in epithelial repair processes of other organs as seen in a variety of inflammatory diseases. Here we demonstrate a strikingly increased expression of KGF in surgical specimens from patients suffering from Crohn's disease and ulcerative colitis. The levels of KGF expression strongly correlated with the degree of inflammation as assessed by histological analysis of adjacent tissue and expression analysis of the pro-inflammatory cytokine interleukin-1 beta. The highest levels of KGF mRNA and protein were found in mesenchymal cells of the lamina propria, particularly in highly inflamed areas. As the KGF receptor is expressed in intestinal epithelial cells, KGF seems to act in a paracrine manner to stimulate proliferation of these cells. These data suggest a crucial role of KGF in epithelial repair after injury caused by inflammatory processes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6
Brauchle, M.; Madlener, M.; Wagner, A. D.; Angermeyer, K.; Lauer, U.; Hofschneider, P. H.; Gregor, M.; Werner, S.
Keratinocyte Growth Factor Enhances Maturation of Fetal Rat Lung Type II Cells
Keratinocyte growth factor (KGF) or fibroblast growth factor (FGF)-7, a peptide produced by stromal cells and in particular by lung mesenchyme, has recently been shown to influence early lung morphogenesis and to be a mitogen for fetal and adult alveolar type II cells. Although contradictory findings have been re- ported regarding its effects on surfactant protein expression, its effects on
Nadia Chelly; Oumel-Banine Mouhieddine-Gueddiche; Anne-Marie Barlier-Mur; Bernadette Chailley-Heu; Jacques R. Bourbon
Salivary leptin induces increased expression of growth factors in oral keratinocytes.
We investigated the influence of salivary leptin, purified by affinity chromatography, on the proliferation of human oral keratinocytes. Furthermore we determined the time- and dose-dependency of the incubation with salivary leptin on the production of epidermal growth factor (EGF) and keratinocyte growth factor (KGF), which are growth factors relevant to keratinocyte proliferation. The analysis was performed both intra- and extracellularly. The relationship between the three cytokines in cell proliferation was studied by successive blocking with specific antibodies. The incubation of oral keratinocytes with recombinant and native leptin led to a significantly increased, dose-dependent cell proliferation (P<0.001). A further significant increase in proliferation was observed after incubating the cells with sterile filtered saliva (P<0.001). The increase in proliferation could not be observed by simultaneous incubation with salivary leptin and specific antibodies against either leptin or KGF (P<0.001). We found a significant dose-dependency between leptin incubation and production of KGF and EGF at the RNA and protein level. Both cytokines were expressed intracellularly and released into the culture medium, where they could be quantified by ELISA. Furthermore, there was a dose- and time-dependent increase in the phosphorylation of STAT-1 and STAT-3, indicating that Ob-R(b) (the long form of the leptin receptor) expressed by the keratinocytes is functional. It is conceivable that the leptin-induced proliferation in keratinocytes is mediated by this signalling pathway. This is the first study to show a physiological role of salivary leptin as a growth factor for keratinocyte proliferation in the oral cavity. We could demonstrate its influence on the production of other growth factors essential for this proliferative effect. Based on the findings of our study we assume an important role for salivary leptin in wound healing within the vulnerable oral cavity. PMID:15821102
Gr�schl, M; Topf, H-G; Kratzsch, J; D�tsch, J; Rascher, W; Rauh, M
[Role of polypeptide growth factors in the regulation of epidermal keratinocyte proliferation].
The growth factors, cytokines, adhesive molecules and extracellular matrix components play the leading role in the processes of intercellular interactions. Literary data on structure and mechanisms of functioning of the growth factors and their receptors are summarised in the present review. Some aspects of regulatory functions of such growth factors as EGF, TGFalpha, TGFbeta, FGF, KGF, AR, and HGF in these processes in epidermis keratinocytes both in vivo and in vitro as example were also considered. PMID:11944330
Gusak, V K; Vasil'ev, R G; Zubov, D A; Slipchenko, I O; Korchak, O M; Popandopulo, A G
Silencing of Keratinocyte Growth Factor Receptor Restores 5Fluorouracil and Tamoxifen Efficacy on Responsive Cancer Cells
BackgroundKeratinocyte growth factor receptor (KGFR) is a splice variant of the FGFR2 gene expressed in epithelial cells. Activation of KGFR is a key factor in the regulation of physiological processes in epithelial cells such as proliferation, differentiation and wound healing. Alterations of KGFR signaling have been linked to the pathogenesis of different epithelial tumors. It has been also hypothesized that
Sabrina Rotolo; Simona Ceccarelli; Ferdinando Romano; Luigi Frati; Cinzia Marchese; Antonio Angeloni; Stefan Maas
Keratinocyte Growth Factor Modulates Alveolar Epithelial Cell Phenotype In Vitro : Expression of Aquaporin 5
We investigated the role of keratinocyte growth factor (KGF) in regulation of alveolar epithelial cell (AEC) phenotype in vitro. Effects of KGF on cell morphology, expression of surfactant apoproteins A, B, and C (SP-A, -B, and -C), and expression of aquaporin 5 (AQP5), a water channel present in situ on the apical surface of alveolar type I (AT1) cells but
Zea Borok; Richard L. Lubman; Spencer I. Danto; Xiao-Ling Zhang; Stephanie M. Zabski; Landon S. King; Douglas M. Lee; Peter Agre; Edward D. Crandall
PPARdelta enhances keratinocyte proliferation in psoriasis and induces heparin-binding EGF-like growth factor.
Psoriasis is a common skin disease involving keratinocyte proliferation and altered differentiation, as well as T-cell activation. Here, we show that altered gene transcription in psoriatic skin lesions is highly reproducible between independent data sets. Analysis of gene expression confirmed dysregulation in all expected functional categories, such as IFN signaling and keratinocyte differentiation, and allowed molecular fingerprinting of a previously characterized dendritic cell subset associated with psoriasis tumor necrosis factor alpha (TNF-alpha)- and inducible nitric oxide synthase (iNOS)-producing CD11b(INT) DC (Tip-DC). Unexpectedly, a large group of dysregulated transcripts was related to fatty acid signaling and adipocyte differentiation, exhibiting a pattern consistent with the activation of peroxisome proliferator-activated receptor delta (PPARdelta). PPARdelta itself was strongly induced in psoriasis in vivo. In primary keratinocytes, PPARdelta was induced by the transcription factor activator protein 1, in particular by junB, but not by canonical WNT signaling, in contrast to its regulation in colon carcinoma cells. Activation of PPARdelta enhanced proliferation of keratinocytes, while this was inhibited by knockdown of PPARdelta. Finally, heparin-binding EGF-like growth factor (HB-EGF), known to induce epidermal hyperplasia and itself overexpressed in psoriasis, was identified as a direct target gene of PPARdelta. The present data suggest that activation of PPARdelta is a major event in psoriasis, contributing to the hyperproliferative phenotype by induction of HB-EGF. PMID:17637826
Romanowska, Malgorzata; al Yacoub, Nadya; Seidel, Henrik; Donandt, Susanne; Gerken, Hannah; Phillip, Sandra; Haritonova, Nathalie; Artuc, Metin; Schweiger, Susann; Sterry, Wolfram; Foerster, John
Fibroblast growth factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous homeostasis
Fibroblast growth factors (FGFs) are master regulators of organogenesis and tissue homeostasis. In this study, we used different combinations of FGF receptor (FGFR)-deficient mice to unravel their functions in the skin. Loss of the IIIb splice variants of FGFR1 and FGFR2 in keratinocytes caused progressive loss of skin appendages, cutaneous inflammation, keratinocyte hyperproliferation, and acanthosis. We identified loss of FGF-induced expression of tight junction components with subsequent deficits in epidermal barrier function as the mechanism underlying the progressive inflammatory skin disease. The defective barrier causes activation of keratinocytes and epidermal ?? T cells, which produce interleukin-1 family member 8 and S100A8/A9 proteins. These cytokines initiate an inflammatory response and induce a double paracrine loop through production of keratinocyte mitogens by dermal cells. Our results identify essential roles for FGFs in the regulation of the epidermal barrier and in the prevention of cutaneous inflammation, and highlight the importance of stromal�epithelial interactions in skin homeostasis and disease.
Yang, Jingxuan; Meyer, Michael; Muller, Anna-Katharina; Bohm, Friederike; Grose, Richard; Dauwalder, Tina; Verrey, Francois; Kopf, Manfred; Partanen, Juha; Bloch, Wilhelm; Ornitz, David M.
Keratinocyte growth factor receptor ligands target the receptor to different intracellular pathways.
The keratinocyte growth factor receptor (KGFR)/fibroblast growth factor receptor 2b is activated by high-affinity-specific interaction with two different ligands, keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)7 and FGF10/KGF2, which are characterized by an opposite requirement of heparan sulfate proteoglycans and heparin for binding to the receptor. We investigated here the possible different endocytic trafficking of KGFR, induced by the two ligands. Immunofluorescence and immunoelectron microscopy analysis showed that KGFR internalization triggered by either KGF or FGF10 occurs through clathrin-coated pits. Immunofluorescence confocal microscopy using endocytic markers as well as tumor susceptibility gene 101 (TSG101) silencing demonstrated that KGF drives KGFR to the degradative pathway, while FGF10 targets the receptor to the recycling endosomes. Biochemical analysis showed that KGFR is ubiquitinated and degraded after KGF treatment but not after FGF10 treatment, and that the alternative fate of KGFR might depend on the different ability of the receptor to phosphorylate the fibroblast growth factor receptor substrate 2 (FRS2) substrate and to recruit the ubiquitin ligase c-Cbl. The recycling endocytic pathway followed by KGFR upon FGF10 stimulation correlates with the higher mitogenic activity exerted by this ligand on epithelial cells compared with KGF, suggesting that the two ligands may play different functional roles through the regulation of the receptor endocytic transport. PMID:17944804
Belleudi, Francesca; Leone, Laura; Nobili, Valerio; Raffa, Salvatore; Francescangeli, Federica; Maggio, Maddalena; Morrone, Stefania; Marchese, Cinzia; Torrisi, Maria Rosaria
Ultraviolet Irradiation-Induces Epidermal Growth Factor Receptor (EGFR) Nuclear Translocation in Human Keratinocytes
Epidermal growth factor receptor (EGFR) plays a critical role in mediating ultraviolet (UV) irradiation-induced signal transduction and gene expression in human keratinocytes. EGFR activation results from increased phosphorylation on specific tyrosine residues in the C-terminal intracellular domain. It has recently been reported that following growth factor stimulation EGFR translocates from the surface membrane to the nucleus, where it may directly regulate gene transcription. We have investigated the ability of UV irradiation to induce EGFR nuclear translocation in human primary and HaCaT keratinocytes. UV irradiation caused rapid nuclear translocation of EGFR. Significant accumulation of EGFR in the nucleus was observed within fifteen minutes after UV irradiation exposure. Maximal translocation occurred at 30 minutes post UV irradiation, and resulted in a 10-fold increase in EGFR in the nucleus, as determined by Western blot analysis of nuclear extracts and confirmed by immunofluorescence. Inhibition of nuclear export by Leptomycin B did not alter UV irradiation-induced nuclear accumulation. EGFR tyrosine kinase inhibitor (PD169540) reduced UV irradiation-induced EGFR nuclear translocation 50%. Mutation of either tyrosine 1148 or tyrosine 1173 reduced nuclear translocation 70%, while mutation of tyrosine 1068 was without effect. In addition, over-expression of receptor type protein tyrosine phosphatase-kappa (RPTP-?), which specifically dephosphorylates EGFR tyrosines, decreased UV irradiation-induced EGFR nuclear translocation in human keratinocytes. These data demonstrate that UV irradiation stimulates rapid EGFR nuclear translocation, which is dependent on phosphorylation of specific EGFR tyrosine residues. EGFR nuclear translocation may act in concert with conventional signaling pathways to mediate UV irradiation-induced responses in human keratinocytes.
Xu, Yiru; Shao, Yuan; Zhou, Jin; Voorhees, John J.; Fisher, Gary J.
Arsenite and insulin exhibit opposing effects on epidermal growth factor receptor and keratinocyte proliferative potential
Previous work has suggested that arsenic exposure contributes to skin carcinogenesis by preserving the proliferative potential of human epidermal keratinocytes, thereby slowing the exit of putative target stem cells into the differentiation pathway. To find a molecular basis for this action, present work has explored the influence of arsenite on keratinocyte responses to epidermal growth factor (EGF). The ability of cultured keratinocytes to found colonies upon passaging several days after confluence was preserved by arsenite and EGF in an additive fashion, but neither was effective when the receptor tyrosine kinase activity was inhibited. Arsenite prevented the loss of EGF receptor protein and phosphorylation of tyrosine 1173, preserving its capability to signal. The level of nuclear {beta}-catenin was higher in cells treated with arsenite and EGF in parallel to elevated colony forming ability, and expression of a dominant negative {beta}-catenin suppressed the increase in both colony forming ability and yield of putative stem cells induced by arsenite and EGF. As judged by expression of three genes regulated by {beta}-catenin, this transcription factor had substantially higher activity in the arsenite/EGF-treated cells. Trivalent antimony exhibited the same effects as arsenite. A novel finding is that insulin in the medium induced the loss of EGF receptor protein, which was largely prevented by arsenite exposure.
Patterson, Timothy J. [Department of Environmental Toxicology, University of California, One Shields Avenue, Davis, CA 95616-8588 (United States); Rice, Robert H. [Department of Environmental Toxicology, University of California, One Shields Avenue, Davis, CA 95616-8588 (United States)]. E-mail: firstname.lastname@example.com
Stimulation of all epithelial elements during skin regeneration by keratinocyte growth factor
Keratinocyte growth factor (KGF), a recently discovered 18.9 kD member of the fibroblast growth factor family has been shown to selectively induce keratinocyte proliferation and differentiation in tissue culture. To explore its potential stimulating keratinocyte growth and differentiation in vivo, we analyzed for the influence of KGF on epithelial derived elements within a wound created through the cartilage on the rabbit ear. KGF accelerated reepithelialization (p = 0.004) and increased the thickness of the epithelium (p = 0.0005) when 4-40 micrograms/cm2 recombinant KGF was added at the time of wounding. The regenerating epidermis showed normal differentiation as detected by cytokeratin immunostaining. Remarkably, however, KGF stimulated proliferation and differentiation of early progenitor cells within hair follicles and sebaceous glands in the wound bed and adjacent dermis. There was a transient but highly significant increase in specific labeling of cycling cells in both basal and suprabasal layers that extended into the spinous layer of the regenerating epidermis. As an indication of specificity, the inflammatory cells and fibroblasts within the wound were not influenced by KGF. The results indicate that KGF is unique in its ability to accelerate reepithelialization and dermal regeneration by targeting multiple epithelial elements within the skin. These results suggest that KGF may induce specific epithelial progenitor cell lineages within the skin to proliferate and differentiate, and thus may be a critical determinant of regeneration of skin. Furthermore, these findings illustrate the potential capacity of this system to analyze epithelial differentiation programs and disorders of epidermis, dermal glandular elements, and hair follicles.
Progesterone-dependent expression of keratinocyte growth factor mRNA in stromal cells of the primate endometrium: keratinocyte growth factor as a progestomedin
In vitro studies have shown that keratinocyte growth factor (KGF, also known as FGF-7) is secreted by fibroblasts and is mitogenic specifically for epithelial cells. Therefore, KGF may be an important paracrine mediator of epithelial cell proliferation in vivo. Because stromal cells are thought to influence glandular proliferation in the primate endometrium, we investigated the hormonal regulation and cellular localization of KGF mRNA expression in the rhesus monkey uterus. Tissues were obtained both from naturally cycling monkeys in the follicular and luteal phases of the cycle, and from spayed monkeys that were either untreated or treated with estradiol (E2) alone, E2 followed by progesterone (P), E2 plus P, or E2 plus P plus an antiprogestin (RU 486). Northern blot analysis of total RNA with 32P- labeled probes revealed that the level of KGF mRNA in the endometrium was 70-100-fold greater in the luteal phase or after P treatment than in untreated, E2-treated, or follicular phase animals. Northern analysis also showed that KGF mRNA was present in the myometrium but was unaffected by hormonal state. RU 486 treatment prevented the P- induced elevation of endometrial KGF mRNA. P-dependent elevation of endometrial KGF expression was confirmed by measurement of KGF protein in tissue extracts using a two-site enzyme-linked immunosorbent assay. In situ hybridization with nonradioactive digoxigenin-labeled cDNA probes revealed that the KGF mRNA signal, which was present only in stromal and smooth muscle cells, was substantially increased by P primarily in the stromal cells located in the basalis region. Smooth muscle cells in the myometrium and the walls of the spiral arteries also expressed KGF mRNA, but the degree of this expression did not differ with hormonal state. P treatment led to increased proliferation in the glandular epithelium of the basalis region and to extensive growth of the spiral arteries. We conclude that the P-dependent increase in endometrial KGF resulted from a dual action of P: (a) a P- dependent induction of KGF expression in stromal cells, especially those in the basalis (zones III and IV), and (b) a P-dependent increase in the number of KGF-positive vascular smooth muscle cells caused by the proliferation of the spiral arteries. KGF is one of the first examples in primates of a P-induced, stromally derived growth factor that might function as a progestomedin.
Epidermal fatty acid-binding protein is increased in rat lungs following in vivo treatment with keratinocyte growth factor
Exogenous application of keratinocyte growth factor protects the lung against a variety of injurious stimuli. KGF-treatment leads to pronounced hyperplasia of alveolar epithelial type II cells and to stabilization of surfactant homeostasis after lung injury. Epidermal fatty acid-binding protein is involved in the synthesis of surfactant phospholipids and acts as an antioxidant scavenging reactive lipids. We treated adult rats with
Veronika Grau; Holger Garn; Julia Holler; Frank Rose; Sonja Bl�cher; Markus Hirschburger; Heinz Fehrenbach; Winfried Padberg
Alveolar epithelial type II cell apoptosis in vivo during resolution of keratinocyte growth factor-induced hyperplasia in the rat
Keratinocyte growth factor (KGF) induces rapid and transient hyperplasia of alveolar epithelial type II cells. We sought to determine components of the apoptotic process involved in the resolution of this hyperplasia and the fate of the apoptotic cells. Rats received intrabronchial instillation of 5 mg KGF\\/kg body weight or diluent. Lungs were fixed 1, 2, 3, 5, and 7 days
Heinz Fehrenbach; Michael Kasper; Roland Koslowski; Tianli Pan; Dieter Schuh; Martin M�ller; Robert J. Mason
Recombinant keratinocyte growth factor 1 in tobacco potentially promotes wound healing in diabetic rats.
Keratinocyte growth factor 1 (KGF1) is a growth factor that promotes epidermal cell proliferation, migration, differentiation, and wound repair. It is expressed at low levels in a form of inclusion body in E. coli. In order to increase its expression and activity, we produced tobacco plants expressing KGF1 via Agrobacterium-mediated transformation using a potato virus X (PVX)-based vector (pgR107). The vector contained the sequence encoding the KGF1 gene fused with a green florescence protein. The recombinant plasmid was introduced into leaf cells of Nicotiana benthamiana (a wild Australian tobacco) via Agrobacterium-mediated agroinfiltration. As determined by fluorescence and Western blot of leaf extracts, the KGF1 gene was correctly translated into the tobacco plants. The recombinant KGF1 was purified from plant tissues by heparin affinity chromatography, and cell proliferation in NIH/3T3 cells was stimulated by the purified KGF1. The purified KGF1 was also applied to the wounds of type-II diabetic rats. KGF1 had accumulated to levels as high as 530? ? g/g fresh weight in the leaves of agroinfected plants. We show that plant-derived KGF1 can promote the proliferation of NIH/3T3 cells and have significant effects on the type-II diabetic rat. The present findings indicated that KGF1 from tobacco maintains its biological activity, implying prospective industrial production in a plant bioreactor. PMID:24783215
Feng, Zhi-Guo; Pang, Shi-Feng; Guo, Ding-Jiong; Yang, Yue-Tao; Liu, Bin; Wang, Ji-Wei; Zheng, Ke-Qin; Lin, Yi
We have shown that autocrine proliferation of human keratinocytes (KC) is strongly dependent upon amphiregulin (AREG), whereas blockade of heparin-binding EGF-like growth factor (HB-EGF) inhibits KC migration in scratch wound assays. Here we demonstrate that expression of soluble HB-EGF (sHB-EGF) or full-length transmembrane HB-EGF (proHB-EGF), but not proAREG, results in profound increases in KC migration and invasiveness in monolayer culture. Coincident with these changes, HB-EGF significantly decreases mRNA expression of several epithelial markers including keratins 1, 5, 10, and 14, while increasing expression of markers of cellular motility including SNAI1, ZEB1, COX-2 and MMP1. Immunostaining revealed HB-EGF-induced expression of the mesenchymal protein vimentin and decreased expression of E-cadherin as well as nuclear translocation of ?-catenin. Suggestive of a trade-off between KC motility and proliferation, overexpression of HB-EGF also reduced KC growth by more than 90%. We also show that HB-EGF is strongly induced in regenerating epidermis after partial thickness wounding of human skin. Taken together, our data suggest that expression of HB-EGF in human KC triggers a migratory and invasive phenotype with many features of epithelial-mesenchymal transition (EMT), which may be beneficial in the context of cutaneous wound healing.
Stoll, Stefan W.; Rittie, Laure; Johnson, Jessica L.; Elder, James T.
Strengthening the Skin with Topical Delivery of Keratinocyte Growth Factor-1 Using a Novel DNA Plasmid
Fragile skin, susceptible to decubitus ulcers and incidental trauma, is a problem particularly for the elderly and for those with spinal cord injury. Here, we present a simple approach to strengthen the skin by the topical delivery of keratinocyte growth factor-1 (KGF-1) DNA. In initial feasibility studies with the novel minimalized, antibiotic-free DNA expression vector, NTC8385-VA1, the reporter genes luciferase and enhanced green fluorescent protein were delivered. Transfection was documented when luciferase expression significantly increased after transfection. Microscopic imaging of enhanced green fluorescent protein�transfected skin showed green fluorescence in hair follicles, hair shafts, and dermal and superficial epithelial cells. With KGF-1 transfection, KGF-1 mRNA level and protein production were documented with quantitative reverse transcriptase�polymerase chain reaction and immunohistochemistry, respectively. Epithelial thickness of the transfected skin in the KGF group was significantly increased compared with the control vector group (26?�?2 versus 16?�?4 �m) at 48 hours (P = 0.045). Dermal thickness tended to be increased in the KGF group (255?�?36 versus 162?�?16 �m) at 120 hours (P = 0.057). Biomechanical assessment showed that the KGF-1�treated skin was significantly stronger than control vector�transfected skin. These findings indicate that topically delivered KGF-1 DNA plasmid can increase epithelial thickness and strength, demonstrating the potential of this approach to restore compromised skin.
Dou, Chunqing; Lay, Frank; Ansari, Amir Mehdi; Rees, Donald J; Ahmed, Ali Karim; Kovbasnjuk, Olga; Matsangos, Aerielle E.; Du, Junkai; Hosseini, Sayed Mohammad; Steenbergen, Charles; Fox-Talbot, Karen; Tabor, Aaron T.; Williams, James A; Liu, Lixin; Marti, Guy P; Harmon, John W
Strengthening the skin with topical delivery of keratinocyte growth factor-1 using a novel DNA plasmid.
Fragile skin, susceptible to decubitus ulcers and incidental trauma, is a problem particularly for the elderly and for those with spinal cord injury. Here, we present a simple approach to strengthen the skin by the topical delivery of keratinocyte growth factor-1 (KGF-1) DNA. In initial feasibility studies with the novel minimalized, antibiotic-free DNA expression vector, NTC8385-VA1, the reporter genes luciferase and enhanced green fluorescent protein were delivered. Transfection was documented when luciferase expression significantly increased after transfection. Microscopic imaging of enhanced green fluorescent protein-transfected skin showed green fluorescence in hair follicles, hair shafts, and dermal and superficial epithelial cells. With KGF-1 transfection, KGF-1 mRNA level and protein production were documented with quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. Epithelial thickness of the transfected skin in the KGF group was significantly increased compared with the control vector group (26?�?2 versus 16?�?4 �m) at 48 hours (P = 0.045). Dermal thickness tended to be increased in the KGF group (255?�?36 versus 162?�?16 �m) at 120 hours (P = 0.057). Biomechanical assessment showed that the KGF-1-treated skin was significantly stronger than control vector-transfected skin. These findings indicate that topically delivered KGF-1 DNA plasmid can increase epithelial thickness and strength, demonstrating the potential of this approach to restore compromised skin. PMID:24434934
Dou, Chunqing; Lay, Frank; Ansari, Amir Mehdi; Rees, Donald J; Ahmed, Ali Karim; Kovbasnjuk, Olga; Matsangos, Aerielle E; Du, Junkai; Hosseini, Sayed Mohammad; Steenbergen, Charles; Fox-Talbot, Karen; Tabor, Aaron T; Williams, James A; Liu, Lixin; Marti, Guy P; Harmon, John W
Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes.
Flow cytometric analysis of primary ex vivo keratinocyte cultures demonstrated that stem cells, (beta 1 integrin+, keratin 1/keratin 10 [K1/K10-], proliferating cell nuclear antigen [PCNA-] [Bata-Csorgo, Zs., C. Hammerberg, J. J. Voorhees, and K. D. Cooper. 1993. J. Exp. Med. 178:1271-1281]) establish such cultures. This methodology also enabled the quantitation of synchronized recruitment of these cells from G0 into G1 of the cell cycle (PCNA expression), which preceded bright beta 1 integrin expression. (beta 1 integrinbright expression has been shown to be a characteristic feature of keratinocyte stem cells in culture (Jones, P. H., and F. M. Watt. 1993. Cell. 73:713-724). Using the above assay, we determined whether lesional T lymphocytes in psoriasis could be directly responsible for the induction of the stem cell hyperproliferation that is characteristic of this disease. Indeed, CD4+ T lymphocytes, cloned from lesional psoriatic skin and stimulated by immobilized anti-CD3 plus fibronectin, promoted psoriatic uninvolved keratinocyte stem cell proliferation via soluble factors. This induction appeared to be through accelerated recruitment of stem cells from their quiescent state (G0) into cell cycle. By contrast, normal keratinocyte stem cells exhibited no such growth stimulation. Supernatants exhibiting growth induction all contained high levels of GM-CSF and gamma-IFN, low IL-3 and TNF-alpha, and variable IL-4. Only anti-gamma-IFN antibody was able to neutralize growth stimulatory activity of the supernatants on psoriatic uninvolved keratinocyte stem cells. However, because recombinant gamma-IFN alone inhibited growth in this assay, these data suggest that, in psoriasis, gamma-IFN acts cooperatively with other growth factors in the immune induction of cell cycle progression by the normally quiescent stem cell keratinocytes. Images
Bata-Csorgo, Z; Hammerberg, C; Voorhees, J J; Cooper, K D
Keratinocyte growth factor-2 stimulates P-glycoprotein expression and function in intestinal epithelial cells
Intestinal P-glycoprotein (Pgp/multidrug resistance 1), encoded by the ATP-binding cassette B1 gene, is primarily involved in the transepithelial efflux of toxic metabolites and xenobiotics from the mucosa into the gut lumen. Reduced Pgp function and expression has been shown to be associated with intestinal inflammatory disorders. Keratinocyte growth factor-2 (KGF2) has emerged as a potential target for modulation of intestinal inflammation and maintenance of gut mucosal integrity. Whether KGF2 directly regulates Pgp in the human intestine is not known. Therefore, the present studies were undertaken to determine the modulation of Pgp by KGF2 using Caco-2 cells. Short-term treatment of Caco-2 cells with KGF2 (10 ng/ml, 1 h) increased Pgp activity (?2-fold, P < 0.05) as measured by verapamil-sensitive [3H]digoxin flux. This increase in Pgp function was associated with an increase in surface Pgp levels. The specific fibroblast growth factor receptor (FGFR) antagonist PD-161570 blocked the KGF2-mediated increase in Pgp activity. Inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD-98059 attenuated the stimulatory effects of KGF2 on Pgp activity. Small-interfering RNA knockdown of Erk1/2 MAPK blocked the increase in surface Pgp levels by KGF2. Long-term treatment with KGF2 (10 ng/ml, 24 h) also significantly increased PgP activity, mRNA, protein expression, and promoter activity. The long-term effects of KGF2 on Pgp promoter activity were also blocked by the FGFR antagonist and mediated by the Erk1/2 MAPK pathway. In conclusion, our findings define the posttranslational and transcriptional mechanisms underlying stimulation of Pgp function and expression by KGF2 that may contribute to the beneficial effects of KGF2 in intestinal inflammatory disorders.
Priyamvada, Shubha; Kumar, Anoop; Akhtar, Maria; Soni, Vikas; Anbazhagan, Arivarasu Natarajan; Alakkam, Anas; Alrefai, Waddah A.; Dudeja, Pradeep K.; Gill, Ravinder K.
Keratinocyte growth factor-induced hyperplasia of rat alveolar type II cells in vivo is resolved by differentiation into type I cells and by apoptosis
Keratinocyte growth factor-induced hyperplasia of rat alveolar type II cells in vivo is re- solved by differentiation into type I cells and by apoptosis. H. Fehrenbach, M. Kasper, T. Tschernig, T. Pan, D. Schuh, J.M. Shannon, M. Muller, R.J. Mason.#ERS Journals Ltd 1999. ABSTRACT: Keratinocyte growth factor (KGF) is a potent mitogen of alveolar epi- thelial type II cells (AEII).
H. Fehrenbach; M. Kasper; T. Tschernig; T. Pan; D. Schuh; J. M. Shannon; M. M�ller; R. J. Mason
Inhibition of sup 125 I-epidermal growth factor binding to cultured keratinocytes by antiproliferative molecules gamma interferon, cyclosporin A, and transforming growth factor-beta
The growth of cultured human keratinocytes (KC) is inhibited by gamma interferon (IFN-gamma), cyclosporin A and transforming growth factor-beta, but not by tumor necrosis factor. When these antiproliferative molecules were added to KC they induced a concentration and time-dependent inhibition of {sup 125}I-epidermal growth factor (I-EGF) binding. These anti-proliferative molecules primarily reduced the number of binding sites by approximately 25%-50% without affecting the binding affinity. Tumor necrosis factor did not influence the ligand binding by I-EGF. In parallel with the ability of the antiproliferative molecules to inhibit I-EGF binding, there was an increase in transforming growth factor-alpha production. These results suggest that several different antiproliferative molecules may share a common mechanism to inhibit cell growth by reducing I-EGF binding to KC.
Nickoloff, B.J.; Mitra, R.S. (Univ. of Michigan Medical Center, Ann Arbor (USA))
Recent advances in the epidermal growth factor receptor/ligand system biology on skin homeostasis and keratinocyte stem cell regulation.
The epidermal growth factor (EGF) receptor/ligand system stimulates multiple pathways of signal transduction, and is activated by various extracellular stimuli and inter-receptor crosstalk signaling. Aberrant activation of EGF receptor (EGFR) signaling is found in many tumor cells, and humanized neutralizing antibodies and synthetic small compounds against EGFR are in clinical use today. However, these drugs are known to cause a variety of skin toxicities such as inflammatory rash, skin dryness, and hair abnormalities. These side effects demonstrate the multiple EGFR-dependent homeostatic functions in human skin. The epidermis and hair follicles are self-renewing tissues, and keratinocyte stem cells are crucial for maintaining these homeostasis. A variety of molecules associated with the EGF receptor/ligand system are involved in epidermal homeostasis and hair follicle development, and the modulation of EGFR signaling impacts the behavior of keratinocyte stem cells. Understanding the roles of the EGF receptor/ligand system in skin homeostasis is an emerging issue in dermatology to improve the current therapy for skin disorders, and the EGFR inhibitor-associated skin toxicities. Besides, controlling of keratinocyte stem cells by modulating the EGF receptor/ligand system assures advances in regenerative medicine of the skin. We present an overview of the recent progress in the field of the EGF receptor/ligand system on skin homeostasis and regulation of keratinocyte stem cells. PMID:23819985
Nanba, Daisuke; Toki, Fujio; Barrandon, Yann; Higashiyama, Shigeki
Keratinocyte growth factor promotes epithelial survival and resolution in a human model of lung injury.
Rationale: Increasing epithelial repair and regeneration may hasten resolution of lung injury in patients with the acute respiratory distress syndrome (ARDS). In animal models of ARDS, keratinocyte growth factor (KGF) reduces injury and increases epithelial proliferation and repair. The effect of KGF in the human alveolus is unknown. Objectives: To test whether KGF can attenuate alveolar injury in a human model of ARDS. Methods: Volunteers were randomized to intravenous KGF (60 ?g/kg) or placebo for 3 days, before inhaling 50 ?g LPS. Six hours later, subjects underwent bronchoalveolar lavage (BAL) to quantify markers of alveolar inflammation and cell-specific injury. Measurements and Main Results: KGF did not alter leukocyte infiltration or markers of permeability in response to LPS. KGF increased BAL concentrations of surfactant protein D, matrix metalloproteinase (MMP)-9, IL-1Ra, granulocyte-macrophage colony-stimulating factor (GM-CSF), and C-reactive protein. In vitro, BAL fluid from KGF-treated subjects inhibited pulmonary fibroblast proliferation, but increased alveolar epithelial proliferation. Active MMP-9 increased alveolar epithelial wound repair. Finally, BAL from the KGF-pretreated group enhanced macrophage phagocytic uptake of apoptotic epithelial cells and bacteria compared with BAL from the placebo-treated group. This effect was blocked by inhibiting activation of the GM-CSF receptor. Conclusions: KGF treatment increases BAL surfactant protein D, a marker of type II alveolar epithelial cell proliferation in a human model of acute lung injury. Additionally, KGF increases alveolar concentrations of the antiinflammatory cytokine IL-1Ra, and mediators that drive epithelial repair (MMP-9) and enhance macrophage clearance of dead cells and bacteria (GM-CSF). Clinical trial registered with ISRCTN 98813895. PMID:24716610
Shyamsundar, Murali; McAuley, Daniel F; Ingram, Rebecca J; Gibson, David S; O'Kane, Donal; McKeown, Scott T; Edwards, Alex; Taggart, Cliff; Elborn, Joseph S; Calfee, Carolyn S; Matthay, Michael A; O'Kane, Cecilia M
The human homologue of the yeast CHL1 gene is a novel keratinocyte growth factor-regulated gene.
Keratinocyte growth factor (KGF) is a potent and specific mitogen for different types of epithelial cells, including keratinocytes of the skin. To gain insight into the mechanisms of KGF action in this tissue, we attempted to identify genes that are regulated by KGF in keratinocytes. Using the differential display reverse transcription polymerase chain reaction technology, a gene was identified which was strongly induced in these cells by treatment with KGF but not with serum growth factors or pro-inflammatory cytokines. This gene seems to be part of a multigene family as assessed by Southern blot analysis. Molecular cloning and sequencing of the full-length cDNA revealed a strong homology with the yeast CHL1 gene. The latter encodes a putative helicase, which is involved in correct chromosome transmission and cell cycle progression. Furthermore, the CHL1 gene product and the protein encoded by the novel KGF-regulated gene were identical in size, indicating that we had cloned the human CHL1 homologue. This finding suggests a novel and specific role of KGF in correct chromosome segregation and/or cell cycle progression. PMID:8798685
Frank, S; Werner, S
Keratinocyte growth factor modulates alveolar epithelial cell phenotype in vitro: expression of aquaporin 5.
We investigated the role of keratinocyte growth factor (KGF) in regulation of alveolar epithelial cell (AEC) phenotype in vitro. Effects of KGF on cell morphology, expression of surfactant apoproteins A, B, and C (SP-A, -B, and -C), and expression of aquaporin 5 (AQP5), a water channel present in situ on the apical surface of alveolar type I (AT1) cells but not expressed in alveolar type II (AT2) cells, were evaluated in AECs grown in primary culture. Observations were made on AEC monolayers grown in serum-free medium without KGF (control) or grown continuously in the presence of KGF (10 ng/ml) from either Day 0 (i.e., the time of plating) or Day 4 or 6 through Day 8 in culture. AECs monolayers express AQP5 only on their apical surfaces as determined by cell surface biotinylation studies. Control AECs grown in the absence of KGF through Day 8 express increasing levels of AQP5, consistent with transition toward the AT1 cell phenotype. Exposure of AECs to KGF from Day 0 results in decreased AQP5 expression, retention of a cuboidal morphology, and greater numbers of lamellar bodies relative to control on Day 8 in culture. AECs treated with KGF from Day 4 or 6 exhibit a decrease in AQP5 expression through subsequent days in culture, as well as an increase in expression of surfactant apoproteins. These data, showing that KGF both prevents and reverses the increase in AQP5 (and decrease in surfactant apoprotein) expression that accompanies progression of the AT2 toward the AT1 cell phenotype, support the concepts that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible and that KGF may play a major role in modulating AEC phenotype. PMID:9533944
Borok, Z; Lubman, R L; Danto, S I; Zhang, X L; Zabski, S M; King, L S; Lee, D M; Agre, P; Crandall, E D
Keratinocyte growth factor enhances barrier function without altering claudin expression in primary alveolar epithelial cells
Keratinocyte growth factor (KGF) has efficacy in several experimental models of lung injury; however, the mechanisms underlying KGF's protective effect remain incompletely understood. This study was undertaken to determine whether KGF augments barrier function in primary rat alveolar epithelial cells grown in culture, specifically whether KGF alters tight junction function via claudin expression. KGF significantly increased alveolar epithelial barrier function in culture as assessed by transepithelial electrical resistance (TER) and paracellular permeability. Fluorescence-activated cell sorting of freshly isolated type 1 (AT1) and type 2 (AT2) cells followed by quantitative real-time RT-PCR revealed that more than 97% of claudin mRNA transcripts in these cells were for claudins-3, -4, and -18. Using cultured AT2 cells, we then examined the effect of KGF on the protein levels of the claudins with the highest mRNA levels: -3, -4, -5, -7, -12, -15, and -18. KGF did not alter the levels of any of the claudins tested, nor of zona occludens-1 (ZO-1) or occludin. Moreover, localization of claudins-3, -4, -18, and ZO-1 was unchanged. KGF did induce a marked increase in the apical perijunctional F-actin ring. Actin depolymerization with cytochalasin D blocked the KGF-mediated increase in TER without significantly changing TER in control cells. Together, these data support a novel mechanism by which KGF enhances alveolar barrier function, modulation of the actin cytoskeleton. In addition, these data demonstrate the complete claudin expression profile for AT1 and AT2 cells and indicate that claudins-3, -4, and -18 are the primary claudins expressed in these cell types.
LaFemina, Michael J.; Rokkam, Deepti; Chandrasena, Anita; Pan, Jue; Bajaj, Anisha; Johnson, Meshell
Keratinocyte Growth Factor Induces Gene Expression Signature Associated with Suppression of Malignant Phenotype of Cutaneous Squamous Carcinoma Cells
Keratinocyte growth factor (KGF, fibroblast growth factor-7) is a fibroblast-derived mitogen, which stimulates proliferation of epithelial cells. The expression of KGF by dermal fibroblasts is induced following injury and it promotes wound repair. However, the role of KGF in cutaneous carcinogenesis and cancer progression is not known. We have examined the role of KGF in progression of squamous cell carcinoma (SCC) of the skin. The expression of KGF receptor (KGFR) mRNA was lower in cutaneous SCCs (n?=?6) than in normal skin samples (n?=?6). Expression of KGFR mRNA was detected in 6 out of 8 cutaneous SCC cell lines and the levels were downregulated by 24-h treatment with KGF. KGF did not stimulate SCC cell proliferation, but it reduced invasion of SCC cells through collagen. Gene expression profiling of three cutaneous SCC cell lines treated with KGF for 24 h revealed a specific gene expression signature characterized by upregulation of a set of genes specifically downregulated in SCC cells compared to normal epidermal keratinocytes, including genes with tumor suppressing properties (SPRY4, DUSP4, DUSP6, LRIG1, PHLDA1). KGF also induced downregulation of a set of genes specifically upregulated in SCC cells compared to normal keratinocytes, including genes associated with tumor progression (MMP13, MATN2, CXCL10, and IGFBP3). Downregulation of MMP-13 and KGFR expression in SCC cells and HaCaT cells was mediated via ERK1/2. Activation of ERK1/2 in HaCaT cells and tumorigenic Ha-ras-transformed HaCaT cells resulted in downregulation of MMP-13 and KGFR expression. These results provide evidence, that KGF does not promote progression of cutaneous SCC, but rather suppresses the malignant phenotype of cutaneous SCC cells by regulating the expression of several genes differentially expressed in SCC cells, as compared to normal keratinocytes.
Toriseva, Mervi; Ala-aho, Risto; Peltonen, Sirkku; Peltonen, Juha; Grenman, Reidar; Kahari, Veli-Matti
Keratinocyte Growth Factor Decreases Pulmonary Edema, Transforming Growth Factor-Beta and Platelet-Derived Growth Factor-BB Expression, and Alveolar Type II Cell Loss in Bleomycin-Induced Lung Injury
Keratinocyte growth factor (KGF), a potent growth factor for type II pneumocytes and Clara cells, has been shown to prevent the end-stage pulmonary fibrosis and mortality in a rat model of bleomycin-induced lung injury. In this study, protective effects of KGF were explored during the earlier course of bleomycin-induced lung injury by studying protein exudation in alveolar edema fluids, pulmonary
Eunhee S. Yi; Moses Salgado; Scott Williams; Seong-Jin Kim; Eliezer Masliah; Songmei Yin; Thomas R. Ulich
Effects of keratinocyte growth factor on skin epithelial differentiation of human amnion epithelial cells.
The aim of the present study was to determine the effects of KGF on the differentiation of cultured human amnion epithelial cells (HAECs) towards skin keratinocyte. HAECs at passage 1 were cultured in medium HAM's F12: Dulbecco's Modified Eagles Medium (1:1) supplemented with different concentrations of KGF (0, 5, 10, 20, 30 and 50ng/ml KGF). Dose-response of KGF on HAECs was determined by morphological assessment; growth kinetic evaluation; immunocytochemical analysis; stemness and epithelial gene expression quantification with two step real time RT-PCR. KGF promotes the proliferation of HAECs with maximal effect observed at 10ng/ml KGF. However, KGF decreased the stemness genes expression: Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4, FZD-9 and BST-1. KGF also down-regulates epithelial genes expression: CK3, CK18, CK19, Integrin-?1, p63 and involucrin in cultured HAECs. No significant difference on the gene expression was detected for each Nestin, ABCG-2, CK1 and CK14 in KGF-treated HAECs. Immunocytochemical analysis for both control and KGF-treated HAECs demonstrated positive staining against CK14 and CK18 but negative staining against involucrin. The results suggested that KGF stimulates an early differentiation of HAECs towards epidermal cells. Differentiation of KGF-treated HAECs to corneal lineage is unfavourable. Therefore, further studies are needed to elucidate the roles of KGF in the differentiation of HAECs towards skin keratinocytes. PMID:23273814
Fatimah, Simat Siti; Tan, Geok Chin; Chua, Kienhui; Tan, Ay Eeng; Nur Azurah, Abdul Ghani; Hayati, Abdul Rahman
Keratinocyte growth factor-induced proliferation of rat airway epithelium is restricted to Clara cells in vivo
ABSTRACT: Keratinocyte growth,factor (KGF) is a potent mitogen,of pulmonary bronchial and alveolar epithelial cells. However, it is unclear which type(s) of airway epithelial cells (AEC) proliferate(s) in response to KGF. AEC proliferation was,induced in rats by either endobronchial,instillation of 5 mg recombinant,human,(rHu) KGF per kg body,weight or by adenoviral transfer of the human,KGF gene (Ad5-HuKGF). Alterations in terminal airway AEC
H. Fehrenbach; A. Fehrenbach; T. Pan; M. Kasperz; R. J. Mason
PPAR? Enhances Keratinocyte Proliferation in Psoriasis and Induces Heparin-Binding EGF-Like Growth Factor
Psoriasis is a common skin disease involving keratinocyte proliferation and altered differentiation, as well as T-cell activation. Here, we show that altered gene transcription in psoriatic skin lesions is highly reproducible between independent data sets. Analysis of gene expression confirmed dysregulation in all expected functional categories, such as IFN signaling and keratinocyte differentiation, and allowed molecular fingerprinting of a previously
Malgorzata Romanowska; Nadya al Yacoub; Henrik Seidel; Susanne Donandt; Hannah Gerken; Sandra Phillip; Nathalie Haritonova; Metin Artuc; Susann Schweiger; Wolfram Sterry; John Foerster
Angiopoietin-related growth factor (AGF) supports adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells through interaction with RGD-binding integrins
Angiopoietin-related growth factor (AGF) is a newly identified member of angiopoietin-related proteins (ARPs)/angiopoietin-like proteins (Angptls). AGF has been considered as a novel growth factor in accelerating cutaneous wound healing, as it is capable of stimulating keratinocytes proliferation as well as angiogenesis. But in our paper, we demonstrate that AGF stimulates keratinocytes proliferation only at high protein concentration, however, it can potently promote adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells. Furthermore, we confirm that the adhesion and migration cellular events are mediated by RGD-binding integrins, most possibly the {alpha}{sub v}-containing integrins, by in vitro inhibition assays using synthetic competitive peptides. Our results strongly suggest that AGF is an integrin ligand as well as a mitogenic growth factor and theoretically participates in cutaneous wound healing in a more complex mechanism.
Zhang Yueqing [Shanghai Institute for Biological Sciences, Graduate school of Chinese Academy of Sciences, 500 Cao Bao Road, Shanghai 200233 (China); Hu Xiaobo [Shanghai Institute for Biological Sciences, Graduate school of Chinese Academy of Sciences, 500 Cao Bao Road, Shanghai 200233 (China); Tian Ruiyang [Shanghai Institute for Biological Sciences, Graduate school of Chinese Academy of Sciences, 500 Cao Bao Road, Shanghai 200233 (China); Wei Wangui [Shanghai Institute for Biological Sciences, Graduate school of Chinese Academy of Sciences, 500 Cao Bao Road, Shanghai 200233 (China); Hu Wei [Shanghai Institute for Biological Sciences, Graduate school of Chinese Academy of Sciences, 500 Cao Bao Road, Shanghai 200233 (China); Chen Xia [Shanghai Institute for Biological Sciences, Graduate school of Chinese Academy of Sciences, 500 Cao Bao Road, Shanghai 200233 (China); Han Wei [Shanghai Institute for Biological Sciences, Graduate school of Chinese Academy of Sciences, 500 Cao Bao Road, Shanghai 200233 (China); Chen Huayou [Shanghai Institute for Biological Sciences, Graduate school of Chinese Academy of Sciences, 500 Cao Bao Road, Shanghai 200233 (China); Gong Yi [Shanghai Institute for Biological Sciences, Graduate school of Chinese Academy of Sciences, 500 Cao Bao Road, Shanghai 200233 (China)]. E-mail: email@example.com
The Effects of Repeated Doses of Keratinocyte Growth Factor on Cell Proliferation in the Cellular Hierarchy of the Crypts of the Murine Small Intestine1
Keratinocyte growth factor (KGF) administered on a daily basis for 3 or more days can result in dramatic changes in tissue architecture, particularly the thickness in oral epithelia, and can afford protection against the cytotoxic effects of radiation on the clonogenic stem cells in the crypts. This protection of intestinal stem cells (increased numbers of surviving crypts) is reflected in
Christopher S. Potten; Julie A. O'Shea; Catherine L. Farrell; Karen Rex; Catherine Booth
Chemical modification of recombinant human keratinocyte growth factor 2 with polyethylene glycol improves biostability and reduces animal immunogenicity.
Recombinant human keratinocyte growth factor 2 (rhKGF-2) is a member of fibroblast growth factor protein family currently being investigated for its promising significant effects in treating epithelial damage. Molecular modification with polyethylene glycol (PEGylation) is an effective approach to improve protein biostability and decrease protein immunogenic activity. In this study, we modified rhKGF-2 through PEGylation at N-terminal residue using 20 kDa PEG-phenyl-isothiocyanate (PIT-PEG20K). PEGylated rhKGF-2 is then purified to near homogeneity by Sephadex G-25 gel filtration followed by a Heparin Sepharose TM CL-6B affinity chromatography. This PEGylated rhKGF-2 retained about 60% of mitogenic activity compared to the non-modified rhKGF-2. Its relative thermal stability at normal physiological temperature and structural stability were significantly enhanced. Moreover, the immunogenicity of PEGylated rhKGF-2 in mice is significant decreased compared to non-modified rhKGF-2. These results suggest that PEGylation of rhKGF-2 could be a more effective approach to the pharmacological and therapeutic application of rhKGF-2. PMID:19477206
Huang, Zhifeng; Ni, Chunyan; Chu, Yanhui; Wang, Shanshan; Yang, Shulin; Wu, Xiaoping; Wang, Xiaojie; Li, Xiaokun; Feng, Wenke; Lin, Shaoqiang
Keratinocyte growth factor (KGF) can replace testosterone in the ductal branching morphogenesis of the rat ventral prostate.
Prostatic growth occurs through ductal elongation and branching into the mesenchyme. Ductal branching morphogenesis in the prostate is elicited by androgens via mesenchymal-epithelial interactions mediated by paracrine influences from mesenchyme. The role of keratinocyte growth factor (KGF) was investigated in the developing prostate as KGF has been suggested to be a paracrine acting factor. KGF transcripts were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in neonatal rat ventral prostates (VPs) in vivo, in VPs cultured in vitro, and in isolated VP mesenchyme. KGF receptor was detected in VP's by RT-PCR and was localized specifically to the epithelium by in situ hybridization. KGF was investigated as a potential paracrine mediator during androgen-induced prostatic development by examining neonatal rat VPs cultured for 6 days under serum-free conditions using a basal medium supplemented only with insulin and transferrin. When testosterone (10(-9) to 10(-8) M) was added to the basal medium, VPs grew and underwent ductal branching morphogenesis similar to that in situ. Neutralization of endogenous KGF with a monoclonal antibody to KGF (anti-KGF) or a soluble KGF receptor peptide inhibited androgen-stimulated VP growth (DNA content) and reduced the number of ductal end buds after 6 days of culture. When KGF (50 or 100 ng/ml) was added to the basal medium in the absence of testosterone, VP growth and ductal branching morphogenesis were stimulated. The number of ductal end buds was about 70% of that obtained with an optimal dose of testosterone (10(-8)M), and DNA content of VP's cultured with 100 ng/ml KGF was equivalent to that of glands cultured with testosterone. The stimulatory effect of KGF was partially blocked by cyproterone acetate, a steroidal anti-androgen. These data imply that KGF plays an important role as a mesenchymal paracrine mediator of androgen-induced epithelial growth and ductal branching morphogenesis in the rat VP. PMID:8946242
Sugimura, Y; Foster, B A; Hom, Y K; Lipschutz, J H; Rubin, J S; Finch, P W; Aaronson, S A; Hayashi, N; Kawamura, J; Cunha, G R
Keratinocyte growth factor and thiazolidinediones and linolenic acid differentiate characterized mammary fat pad adipose stem cells isolated from prepubertal Korean black goat to epithelial and adipogenic lineage.
The study was conducted to know and investigate the mechanism involved during mesenchymal to epithelial transition to unravel questions related to mammary gland development in prepubertal Korean black goat. We, therefore, biopsied mammary fat pad and isolated adipose cells and characterized with stemness factors (CD34, CD13, CD44, CD106, and vimentin) immunologically and through their genetic expression. Furthermore, characterized cells were differentiated to adipogenic (thiazolidinediones and ?-linolenic acid) and epithelial (keratinocyte growth factor) lineages. Thiazolidinediones/or in combination with ?-linolenic acid demonstrated significant upregulation of adipo-Q, PPAR-?, CEBP-?, LPL, and resistin. Adipose stem cells in induction mixture (5�?g/ml insulin, 1�?g/ml hydrocortisone, and 10�ng/ml epidermal growth factor) and subsequent treatment with 10�ng/ml keratinocyte growth factor revealed their trans-differentiating ability to epithelial lineage. From 2�d onwards, the cells under keratinocyte growth factor influenced cells to assume rectangular (2-4�d) to cuboidal (8-10�d) shapes. Ayoub-Shklar stain developed brownish-red pigment in the transformed cells. Though, expressions of K8 and K18 were noted to be highly significant (p?0.01) but expressions of epithelial membrane antigens and epithelial specific antigens were also significant (p?0.05) compared to 0�d. Conclusively, epithelial transformations of mammary adipose stem cells would add up knowledge to develop therapeutic regimen to deal with mammary tissue injury and diseases. PMID:24101555
Reza, A M M T; Shiwani, S; Singh, N K; Lohakare, J D; Lee, S J; Jeong, D K; Han, J Y; Rengaraj, D; Lee, B W
Keratinocyte growth factor down-regulates expression of the sucrase-isomaltase gene in Caco-2 intestinal epithelial cells.
The molecular mechanisms that regulate the proliferation and differentiation of intestinal mucosal epithelial cells are not well understood. Keratinocyte growth factor (KGF) is an epithelial cell-specific growth factor that may be involved in the maintenance of mucosal epithelial populations and in mediating epithelial repair after injury. The sucrase-isomaltase (SI) gene, which encodes an enterocyte brush border disaccharidase, has served as a model for study of intestinal-specific gene expression and differentiation. KGF down-regulated SI mRNA and protein expression in Caco-2 intestinal epithelial cells but not the expression of other brush border enzymes. The down-regulation was dose- and time-dependent and specifically blocked by anti-KGF antibodies. Transfection experiments using SI promoter constructs demonstrated that KGF decreased SI gene transcription. In contrast, the stability of SI mRNA was not affected by incubation of Caco-2 cells with KGF. Electrophoretic mobility shift analysis demonstrated that binding of nuclear proteins to the SI footprint (SIF) 3 and SIF4 regulatory elements within the SI promoter region was increased in Caco-2 cells that had been incubated with KGF. In transfection experiments using a construct in which tandem copies of the SIF4-binding site were inserted upstream of the SV40 promoter and luciferase gene, incubation with KGF resulted in a significant decrease in luciferase activity. However, transfection with a similar construct containing tandem copies of SIF3 had no significant effect on SV40 promoter activity following KGF treatment. SIF4 may bind E4BP4, a previously identified transcriptional repressor protein. This factor may in part mediate the decrease in SI transcription by KGF in Caco-2 cells. PMID:9837912
Zhou, J; Wu, K; Fernandes, C L; Cheng, A L; Finch, P W
Epidermal growth factor receptor signalling in keratinocyte biology: implications for skin toxicity of tyrosine kinase inhibitors.
The epidermal growth factor receptor (EGFR) and its ligands have been long recognized as centrally involved in the growth and repair process of epithelia, as well as in carcinogenesis. In addition, the EGFR has been demonstrated to be importantly involved in the control of inflammatory responses. During this last decade, a number of highly specific agents targeting this system have become an integral component of pharmacologic strategies against many solid malignancies. These drugs have led to increased patient survival and made therapy more tolerant when compared to conventional cytotoxic drugs. Nonetheless, their use is associated with a constellation of toxic effects on the skin, including follicular pustules, persistent inflammation, xerosis and pruritus, and enhanced susceptibility to infections. This dramatic impairment of skin homoeostasis underscores the centrality of the EGFR-ligand system in the whole skin immune system. So far, no mechanism-based approaches are available to specifically counteract the adverse effects of anti-EGFR drugs or any other class of tyrosine kinase inhibitors. Only the knowledge of the cellular and molecular events underlying these adverse effects in humans, combined with in vitro/in vivo models able to mimic these toxic responses, may guide the development of mechanism-based treatment or prevention strategies. PMID:24770552
Pastore, Saveria; Lulli, Daniela; Girolomoni, Giampiero
A protective role for keratinocyte growth factor in a murine model of chemotherapy and radiotherapy-induced mucositis
Purpose: To evaluate the activity of palifermin (rHuKGF) in a murine model of mucosal damage induced by a radiotherapy/chemotherapy (RT/CT) regimen mimicking treatment protocols used in head-and-neck cancer patients. Methods and Materials: A model of mucosal damage induced by RT/CT was established by injecting female BDF1 mice with cisplatin (10 mg/kg) on Day 1; 5-fluorouracil (40 mg/kg/day) on Days 1-4, and irradiation (5 Gy/day) to the head and neck on Days 1-5. Palifermin was administered subcutaneously on Days -2 to 0 (5 mg/kg/day) and on Day 5 (5 mg/kg). Evaluations included body weight, organ weight, keratinocyte growth factor receptor expression, epithelial thickness, and cellular proliferation. Results: Initiation of the radiochemotherapeutic regimen resulted in a reduction in body weight in control animals. Palifermin administration suppressed weight loss and resulted in increased organ weight (salivary glands and small intestine), epithelial thickness (esophagus and tongue), and cellular proliferation (tongue and salivary glands). Conclusions: Administration of palifermin before RT/CT promotes cell proliferation and increases in epithelial thickness in the oral mucosa, salivary glands, and digestive tract. Palifermin administration before and after RT/CT mitigates weight loss and a trophic effect on the intestinal mucosa and salivary glands, suggesting that palifermin use should be investigated further in the RT/CT settings, in which intestinal mucositis and salivary gland dysfunction are predominant side effects of cytotoxic therapy.
Borges, Luis [Departments of Hematology, Cancer Biology, and Pathology, Amgen Inc., Thousand Oaks, CA (United States)]. E-mail: firstname.lastname@example.com; Rex, Karen L. [Departments of Hematology, Cancer Biology, and Pathology, Amgen Inc., Thousand Oaks, CA (United States); Chen, Jennifer N. [Departments of Hematology, Cancer Biology, and Pathology, Amgen Inc., Thousand Oaks, CA (United States); Wei, Ping [Departments of Hematology, Cancer Biology, and Pathology, Amgen Inc., Thousand Oaks, CA (United States); Kaufman, Stephen [Departments of Hematology, Cancer Biology, and Pathology, Amgen Inc., Thousand Oaks, CA (United States); Scully, Sheila [Departments of Hematology, Cancer Biology, and Pathology, Amgen Inc., Thousand Oaks, CA (United States); Pretorius, James K. [Departments of Hematology, Cancer Biology, and Pathology, Amgen Inc., Thousand Oaks, CA (United States); Farrell, Catherine L. [Departments of Hematology, Cancer Biology, and Pathology, Amgen Inc., Thousand Oaks, CA (United States)
Differential induction of phosphatidylcholine hydrolysis, diacylglycerol formation and protein kinase C activation by epidermal growth factor and transforming growth factor-alpha in normal human skin fibroblasts and keratinocytes.
We have investigated coupling between the epidermal growth factor (EGF) receptor and the phospholipase C (PLC)/protein kinase C (PKC) signal-transduction system in normal skin fibroblasts and keratinocytes, for which EGF and transforming growth factor alpha (TGF-alpha) are mitogenic. EGF and TGF-alpha induced a rapid increase in tyrosine phosphorylation of the EGF receptor, in both fibroblasts and keratinocytes, but failed to induce tyrosine phosphorylation of PLC-gamma 1 or detectable phosphoinositide hydrolysis, as measured by two sensitive assays. In fibroblasts, EGF induced phosphatidylcholine (PC) hydrolysis, resulting in increased diacylglycerol (DAG). In contrast, in keratinocytes, there was no detectable PC hydrolysis or elevation of DAG in response to EGF or TGF-alpha. EGF and TGF-alpha activated PKC in fibroblasts, as evidenced by increased phosphorylation of a specific cellular PKC substrate (myristoylated alanine-rich C-kinase substrate, 'MARCKS'). In keratinocytes, TGF-alpha and EGF induced only a modest increase in MARCKS protein phosphorylation. This apparent modest activation of PKC, in the absence of detectable DAG formation, may have been mediated by arachidonic acid, which was released from keratinocytes in response to TGF-alpha, and has been shown to stimulate PKC activity in vitro. These data demonstrate that (1) in dermal fibroblasts and keratinocytes, which express normal levels of EGF receptors, EGF receptor activation is not coupled to tyrosine phosphorylation of PLC-gamma 1 or PtdIns hydrolysis, suggesting that these events are not required for the mitogenic activity of EGF or TGF-alpha in these cells, (2) coupling of EGF receptor to PC hydrolysis is cell-type specific, and (3) in skin fibroblasts, DAG, formed through EGF-induced PC hydrolysis, is capable of activating PKC. Images Figure 3 Figure 4 Figure 6
Reynolds, N J; Talwar, H S; Baldassare, J J; Henderson, P A; Elder, J T; Voorhees, J J; Fisher, G J
Infection of Keratinocytes with Trichophytum rubrum Induces Epidermal Growth Factor-Dependent RNase 7 and Human Beta-Defensin-3 Expression
Human keratinocytes are able to express various antimicrobial peptides (AMP) to protect the skin from exaggerated microbial colonization and infection. Recently, in vitro growth-inhibiting activity of the skin-derived AMP psoriasin, RNase 7 and human beta-defensin (hBD)-2 against dermatophytes such as Trichophyton (T.) rubrum have been reported. To evaluate whether keratinocytes are able to respond to T. rubrum infection by an induced expression of AMP we exposed primary keratinocytes to living conidia of T. rubrum. This led to conidia germination and mycelial growth which was paralleled by a strong gene induction of the skin-derived AMP RNase 7 and hBD-3. Gene expression of the AMP psoriasin (S100A7) and hBD-2 were only slightly induced. The T. rubrum-mediated RNase 7 gene induction was accompanied by increased secretion of RNase 7. Parallel treatment of the keratinocytes with T. rubrum and the cytokine combination IL-17A/IFN-? resulted in synergistic induction of RNase 7 and hBD-3 expression. Since patients receiving therapy by inhibition of the epidermal growth factor receptor (EGFR) more often suffer from dermatophytoses we investigated whether EGFR may be involved in the T. rubrum-mediated RNase 7 and hBD-3 induction. Primary keratinocytes incubated with an EGFR blocking antibody as well as with the EGFR antagonist AG1478 showed a significantly diminished RNase 7 and hBD-3 induction upon exposure of the keratinocytes to T. rubrum indicating that EGFR is involved in the T. rubrum-mediated induction of RNase 7 and hBD-3. The growth of T. rubrum in vitro was inhibited by hBD-3 in a dose-dependent manner suggesting that hBD-3 may contribute to cutaneous innate defense against T. rubrum. Taken together our data indicate that keratinocytes are able to initiate a fast defense response towards T. rubrum by the increased expression of AMP active against T. rubrum. A dysregulation of AMP may contribute to chronic and recurring dermatophytoses.
Rademacher, Franziska; Schroder, Lena; Brasch, Jochen; Harder, Jurgen
Infection of Keratinocytes with Trichophytum rubrum Induces Epidermal Growth Factor-Dependent RNase 7 and Human Beta-Defensin-3 Expression.
Human keratinocytes are able to express various antimicrobial peptides (AMP) to protect the skin from exaggerated microbial colonization and infection. Recently, in vitro growth-inhibiting activity of the skin-derived AMP psoriasin, RNase 7 and human beta-defensin (hBD)-2 against dermatophytes such as Trichophyton (T.) rubrum have been reported. To evaluate whether keratinocytes are able to respond to T. rubrum infection by an induced expression of AMP we exposed primary keratinocytes to living conidia of T. rubrum. This led to conidia germination and mycelial growth which was paralleled by a strong gene induction of the skin-derived AMP RNase 7 and hBD-3. Gene expression of the AMP psoriasin (S100A7) and hBD-2 were only slightly induced. The T. rubrum-mediated RNase 7 gene induction was accompanied by increased secretion of RNase 7. Parallel treatment of the keratinocytes with T. rubrum and the cytokine combination IL-17A/IFN-? resulted in synergistic induction of RNase 7 and hBD-3 expression. Since patients receiving therapy by inhibition of the epidermal growth factor receptor (EGFR) more often suffer from dermatophytoses we investigated whether EGFR may be involved in the T. rubrum-mediated RNase 7 and hBD-3 induction. Primary keratinocytes incubated with an EGFR blocking antibody as well as with the EGFR antagonist AG1478 showed a significantly diminished RNase 7 and hBD-3 induction upon exposure of the keratinocytes to T. rubrum indicating that EGFR is involved in the T. rubrum-mediated induction of RNase 7 and hBD-3. The growth of T. rubrum in vitro was inhibited by hBD-3 in a dose-dependent manner suggesting that hBD-3 may contribute to cutaneous innate defense against T. rubrum. Taken together our data indicate that keratinocytes are able to initiate a fast defense response towards T. rubrum by the increased expression of AMP active against T. rubrum. A dysregulation of AMP may contribute to chronic and recurring dermatophytoses. PMID:24747887
Firat, Yasemin Helene; Simanski, Maren; Rademacher, Franziska; Schr�der, Lena; Brasch, Jochen; Harder, J�rgen
Cooperative effects in vitro on fibroblast and keratinocyte functions related to wound healing by transforming growth factor-beta and a low molecular weight fraction from hemolyzed blood.
Blood or its constituents, respectively, contain a.o. substances such as TGF-beta (transforming growth factor-beta), PDGF (platelet-derived growth factor) and other factors, which beneficially influence wound healing. Patients with perivascular disturbances and consequently with inadequate supply of the affected tissue cells often suffer from poor healing of dermal wounds. Here, various cellular functions in situations of poor supply and related to wound healing, such as proliferation, colony formation, and migration of fibroblasts, and of monolayer formation by keratinocytes were emulated in vitro by supplementing cultures with reduced amounts of serum. Computer-aided image analysis allowed to quantify the cellular reactions under normal and serum-deprived medium conditions and under the influence of a low molecular weight fraction manufactured by dialysis of hemolyzed calf blood (HD, Solcoseryl) and of TGF-beta. Both preparations are in use for the treatment of poorly healing wounds. While HD preferentially normalized the reduced viability of fibroblasts, the keratinocyte activity was enhanced by TGF-beta. Restoration of fibroblast and keratinocyte functions proved most effective when combining HD with TGF-beta. PMID:7945526
Miltenburger, H G; Baschong, W; H�rner, V; Marx, G
Keratinocyte Growth Factor and Glucocorticoid Induction of Human Peroxiredoxin 6 Gene Expression Occur by Independent Mechanisms That Are Synergistic
Abstract Aims: Peroxiredoxin 6 (Prdx6), a 1-cys Prdx has both peroxidase and phospholipase A2 activities, protecting against oxidative stress and regulating pulmonary surfactant phospholipid metabolism. This study determined the mechanism by which keratinocyte growth factor (KGF) and the glucocorticoid analogue, dexamethasone (Dex), induce increased Prdx6 expression. Results: Transcriptional activation by KGF in both A549 lung adenocarcinoma cells and rat lung alveolar epithelial type II (ATII) cells utilizes an antioxidant response element (ARE), located between 357 and 349 nucleotides before the PRDX6 translational start, that is also necessary for upregulation of the human PRDX6 promoter in response to oxidative stress. Activation is mediated by binding of the transcription factor, Nrf2, to the ARE as shown by experiments using siRNA against Nrf2 and by transfecting ATII cells isolated from lungs of Nrf2 null mice. KGF triggers the migration of Nrf2 from cytoplasm to nucleus where it binds to the PRDX6 promoter as shown by chromatin immunoprecipitation assays. Activation of transcription by Dex occurs through a glucocorticoid response element located about 750 nucleotides upstream of the PRDX6 translational start. Innovation: This study demonstrates that KGF can activate an ARE in a promoter without reactive oxygen species involvement and that KGF and Dex can synergistically activate the PRDX6 promoter and protect cells from oxidative stress. Conclusion: These two different activators work through different DNA elements. Their combined effect on transcription of the reporter gene is synergistic; however, at the protein level, the combined effect is additive and protects cells from oxidative damage. Antioxid. Redox Signal. 20, 391�402.
Chowdhury, Ibrul; Fisher, Aron B.; Christofidou-Solomidou, Melpo; Gao, Ling; Tao, Jain-Qin; Sorokina, Elena M.; Lien, Yu-Chin; Bates, Sandra R.
Keratinocyte growth factor-2 and autologous serum potentiate the regenerative effect of mesenchymal stem cells in cornea damage in rats
AIM To investigate the healing process after severe corneal epithelial damage in rats treated with mesenchymal stem cells (MSCs) cultured with or without keratinocyte growth factor (KGF-2) and autologous serum (AS) on amniotic membrane (AM). Many patients are blind and devastated by severe ocular surface diseases due to limbal stem cell deficiency. Bone marrow-derived MSCs are potential sources for cell-based tissue engineering to repair or replace the corneal tissue, having the potential to differentiate to epithelial cells. METHODS The study included 5 groups each including 10 female �Sprague Dawley� rats in addition to 20 male rats used as bone marrow donors. Group I rats received AM+MSCs, Group II rats AM+MSCs cultured with KGF-2, Group III rats AM+MSCs cultured with KGF-2+AS, Group IV rats only AM and Group V rats, none. AS was derived from blood drawn from male rats and bone marrow was obtained from the femur and tibia bones of the same animals. Therapeutic effect was evaluated with clinical, histopathological and immunohistochemical assessment. MSC engraftment was demonstrated via detection of donor genotype (Y+) in the recipient tissue (X) with polymerase chain reaction. RESULTS Corneal healing was significantly better in Groups I-III rats treated with MSC transplantation compared to Group IV and Group V rats with supportive treatment only. The best results were obtained in Group III rats with 90% transparency, 70% lack of neovascularization, and 100% epithelium damage limited to less than 1/4 of cornea. CONCLUSION We suggest that culture of MSCs with KGF-2 and AS on AM is effective in corneal repair in case of irreversible damage to limbal stem cells.
P?narl?, Ferda Alpaslan; Okten, Gulsen; Beden, Umit; F?sg?n, Tunc; Kefeli, Mehmet; Kara, Nurten; Duru, Feride; Tomak, Leman
Keratinocyte growth factor prevents radiation damage to salivary glands by expansion of the stem/progenitor pool.
Irradiation of salivary glands during radiotherapy treatment of patients with head and neck cancer evokes persistent hyposalivation. This results from depletion of stem cells, which renders the gland incapable of replenishing saliva to produce acinar cells. The aim of this study was to investigate whether it is possible to expand the salivary gland stem/progenitor cell population, thereby preventing acinar cell depletion and subsequent gland dysfunction after irradiation. To induce cell proliferation, keratinocyte growth factor (DeltaN23-KGF, palifermin) was administered to C57BL/6 mice for 4 days before and/or after local irradiation of salivary glands. Salivary gland vitality was quantified by in vivo saliva flow rates, morphological measurements, and a newly developed in vitro salisphere progenitor/stem cell assay. Irradiation of salivary glands led to a pronounced reduction in the stem cells of the tissues, resulting in severe hyposalivation and a reduced number of acinar cells. DeltaN23-KGF treatment for 4 days before irradiation indeed induced salivary gland stem/progenitor cell proliferation, increasing the stem and progenitor cell pool. This did not change the relative radiation sensitivity of the stem/progenitor cells, but, as a consequence, an absolute higher number of stem/progenitor cells and acinar cells survived after radiation. Postirradiation treatment with DeltaN23-KGF also improved gland function, and this effect was much more pronounced in DeltaN23-KGF pretreated animals. Post-treatment with DeltaN23-KGF seemed to act through accelerated expansion of the pool of progenitor/stem cells that survived the irradiation treatment. Overall, our data indicate that DeltaN23-KGF is a promising drug to enhance the number of salivary gland progenitor/stem cells and consequently prevent radiation-induced hyposalivation. Disclosure of potential conflicts of interest is found at the end of this article. PMID:18669914
Lombaert, Isabelle M A; Brunsting, Jeanette F; Wierenga, Pieter K; Kampinga, Harm H; de Haan, Gerald; Coppes, Robert P
Reduction of radiochemotherapy-induced early oral mucositis by recombinant human keratinocyte growth factor (palifermin): Experimental studies in mice
Purpose: To study the effect of recombinant human keratinocyte growth factor (rHuKGF or palifermin) on oral mucositis induced by radiochemotherapy in a mouse model. Methods and Materials: Cis-diamminedichloroplatinum (cisplatin) and/or 5-fluorouracil were given before single dose irradiation, combined with palifermin before or after the treatment, or both. Daily fractionated irradiation for 2 weeks was followed by graded test doses. With additional chemotherapy in Week 1, palifermin was given before radiotherapy and at the end of the first week, or additionally at the end of Week 2. Radiochemotherapy in Week 2 was combined with palifermin at the end of Weeks 1 and 2, Weeks 1, 2, and 3, or additionally before radiotherapy. Ulceration of mouse tongue mucosa was analyzed as the endpoint. Results: The dose associated with ulcer induction in 50% of the mice (ED{sub 50}) for single-dose irradiation was 11.5 {+-} 0.7 Gy. Palifermin increased the ED{sub 50} to about 19 Gy in all protocols tested. Similar values were observed when chemotherapy was added before irradiation. With fractionated irradiation, palifermin increased the ED{sub 50} for test irradiation from 5.7 {+-} 1.5 Gy to 12-15 Gy, depending on the administration protocol. With chemotherapy in Week 1, two palifermin injections had no significant effect, but a third injection increased the ED{sub 50} to 13 Gy. With chemotherapy in Week 2, all palifermin protocols resulted in ED{sub 50} values of 13-14 Gy. Conclusion: A marked increase in oral mucosal radiation tolerance by palifermin was found, which was preserved in combinations with chemotherapy using cisplatin and/or 5-fluorouracil.
Doerr, Wolfgang [Klinik und Poliklinik fuer Strahlentherapie und Radioonkologie, Medical Faculty Carl Gustav Carus, University of Technology, Dresden (Germany) and Experimentelles Zentrum, Medical Faculty Carl Gustav Carus, University of Technology, Dresden (Germany)]. E-mail: email@example.com; Baessler, Stefan; Reichel, Sandra [Klinik und Poliklinik fuer Strahlentherapie und Radioonkologie, Medical Faculty Carl Gustav Carus, University of Technology, Dresden (Germany); Spekl, Kathrin [Klinik und Poliklinik fuer Strahlentherapie und Radioonkologie, Medical Faculty Carl Gustav Carus, University of Technology, Dresden (Germany); Experimentelles Zentrum, Medical Faculty Carl Gustav Carus, University of Technology, Dresden (Germany)
Effects of recombinant human keratinocyte growth factor on surfactant, plasma, and liver phospholipid homeostasis in hyperoxic neonatal rats.
Respiratory distress and bronchopulmonary dysplasia (BPD) are major problems in preterm infants that are often addressed by glucocorticoid treatment and increased oxygen supply, causing catabolic and injurious side effects. Recombinant human keratinocyte growth factor (rhKGF) is noncatabolic and antiapoptotic and increases surfactant pools in immature lungs. Despite its usefulness in injured neonatal lungs, the mechanisms of improved surfactant homeostasis in vivo and systemic effects on lipid homeostasis are unknown. We therefore exposed newborn rats to 85% vs. 21% oxygen and treated them systemically with rhKGF for 48 h before death at 7 days. We determined type II pneumocyte (PN-II) proliferation, surfactant protein (SP) mRNA expression, and the pulmonary metabolism of individual phosphatidylcholine (PC) species using [D(9)-methyl]choline and tandem mass spectrometry. In addition, we assessed liver and plasma lipid metabolism, addressing PC synthesis de novo, the liver-specific phosphatidylethanolamine methyl transferase (PEMT) pathway, and triglyceride concentrations. rhKGF was found to maintain PN-II proliferation and increased SP-B/C expression and surfactant PC in both normoxic and hyperoxic lungs. We found increased total PC together with decreased [D(9)-methyl]choline enrichment, suggesting decreased turnover rather than increased secretion and synthesis as the underlying mechanism. In the liver, rhKGF increased PC synthesis, both de novo and via PEMT, underlining the organotypic differences of rhKGF actions on lipid metabolism. rhKGF increased the hepatic secretion of newly synthesized polyunsaturated PC, indicating improved systemic supply with choline and essential fatty acids. We suggest that rhKGF has potential as a therapeutic agent in neonates by improving pulmonary and systemic PC homeostasis. PMID:22323656
Raith, Marco; Schaal, Katharina; Koslowski, Roland; Fehrenbach, Heinz; Poets, Christian F; Schleicher, Erwin; Bernhard, Wolfgang
Severe ulceration with impaired induction of growth factors and cytokines in keratinocytes after trichloroacetic acid application on TRPV1-deficient mice.
Transient receptor potential vanilloid 1 (TRPV1) is a highly polymodal TRP channel activated by various stimuli, including capsaicin, heat and acids. TRPV1 expression can be detected widely but is highest in sensory neurons and its activation alerts the body to noxious signals via neurogenic pain. Although TRPV1 is reportedly localized in the epidermis, it remains unclear how TRPV1 is involved in the chemical peeling processes with cytotoxic acids. Therefore, in this study, the role of TRPV1 on the effects of trichloroacetic acid (TCA) peeling was assessed using TRPV1-deficient mice. Following the confirmation of TRPV1 expression in murine keratinocytes with reverse transcription-polymerase chain reaction and immunohistochemistry, the effects of TCA on TRPV1-deficient mouse skin were compared with those on wild-type mouse skin. Our results indicated that TRPV1 expression was not required for TCA-induced DNA damage, as shown by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, but was indispensable for the TCA-induced production of distinct growth factors and cytokines by keratinocytes. Ulceration after TCA peeling was actually more severe in the absence of TRPV1, suggesting that the TRPV1-mediated epidermal production of growth factors and cytokines affected the damaging and healing processes of TCA-peeled skin to induce rejuvenation. PMID:22858856
Li, Hong-jin; Kanazawa, Nobuo; Kimura, Ayako; Kaminaka, Chikako; Yonei, Nozomi; Yamamoto, Yuki; Furukawa, Fukumi
Insulin-like growth factor-binding protein-7 (IGFBP7) transcript: A-to-I editing events in normal and cancerous human keratinocytes.
Non-melanoma skin cancers (NMSC) are the most common malignancies in caucasians worldwide. Insulin-like growth factor-binding protein-7 (IGFBP7) was suggested to function as a tumor suppressor gene in several cancers, and to play a role in the proliferation of keratinocytes. A-to-I RNA editing is a post-transcriptional mechanism frequently used to expand and diversify transcriptome and proteome repertoire in eukaryotic cells. A-to-I RNA editing can alter codons, substitute amino acids and affect protein sequence, structure, and function. Two editing sites were identified within the IGFBP7 transcript. To evaluate the expression and editing of IGFBP7 mRNA in NMSC compared to normal epidermis. We examined the expression and mRNA editing level of IGFBP7 in 22 basal cell carcinoma (BCC), 15 squamous cell carcinoma (SCC), and 18 normal epidermis samples that were surgically removed from patients by the Mohs Micrographic Surgery procedure. We studied the effect of IGFBP7 editing on an immortalized HaCaT keratinocyte cell model. IGFBP7 mRNA is over expressed in BCC and SCC compared to normal epidermis. Moreover, the IGFBP7 transcript is highly edited in normal epidermis, but its editing is significantly reduced in BCC and SCC. The edited form of IGFBP7 can inhibit proliferation and induce senescence in cultured keratinocytes. This study describes for the first time A-to-I editing in the coding sequence of a tumor suppressor gene in humans, and suggests that IGFBP7 editing serves as a fine-tuning mechanism to maintain the equilibrium between proliferation and senescence in normal skin. PMID:23543219
Hochberg, Malka; Gilead, Leon; Markel, Gal; Nemlich, Yael; Feiler, Yulia; Enk, Claes David; Denichenko, Polina; Karni, Rotem; Ingber, Arieh
Plant Polyphenols Regulate Chemokine Expression and Tissue Repair in Human Keratinocytes Through Interaction with Cytoplasmic and Nuclear Components of Epidermal Growth Factor Receptor System
Abstract Aims: To evaluate mechanisms underlying modulation of inflammatory chemokines in primary human keratinocytes (normal human epidermal keratinocytes) and repair-related processes in wound models by plant polyphenols (PPs) with antioxidant and superoxide scavenging properties (verbascoside [Vb], resveratrol [Rv], polydatin [Pd], quercetin [Qr], and rutin). Results: Epidermal growth factor receptor (EGFR)-controlled chemokines CXCL8/interleukin 8 (IL-8), CCL2/monocyte chemotactic protein-1 (MCP-1), and CXCL10/interferon gamma-produced protein of 10?kDa (IP-10) were modulated by transforming growth factor alpha (TGF-?) and by the tumor necrosis factor alpha/interferon gamma combination (T/I). EGFR phosphorylation, nuclear translocation, and downstream cytoplasmic signaling pathways (extracellular regulation kinase [ERK]1/2, p38, STAT3, and PI-3K) were studied. All PPs did not affect TGF-?-induced STAT3 phosphorylation, whereas they suppressed T/I-activated NFkappaB and constitutive and T/I-induced but not TGF-?-induced ERK1/2 phosphorylation. Vb and Qr suppressed total EGFR phosphorylation, but they synergized with TGF-? to enhance nuclear accumulation of phosphorylated EGFR. Vb strongly inhibited TGF-?-induced p38 phosphorylation and T/I-induced NFkappaB and activator protein-1 (AP-1) binding to DNA. Vb was an effective inhibitor of T/I-stimulated chemokine synthesis, and it accelerated scratch wound healing in vitro. Anti-inflammatory and wound healing activities of Vb were confirmed in vivo in the full-thickness excision wound. Although Pd and Rv did not affect EGFR activation/translocation, they and Qr synergized with TGF-? and T/I in the induction of IL-8 transcription/synthesis while opposing enhanced MCP-1 and IP-10 transcription/synthesis connected with pharmacologically impaired EGFR functioning. Innovation: PPs perturb the EGFR system in human keratinocytes, and this effect may be implicated in the regulation of inflammatory and repair-related processes in the skin. Conclusion: Anti-inflammatory and wound healing effects of PPs depend on their interaction with EGFR-controlled cytoplasmic and nuclear pathways rather than on their direct redox properties. Antioxid. Redox Signal. 16, 314�328.
Pastore, Saveria; Lulli, Daniela; Fidanza, Paolo; Potapovich, Alla I.; Kostyuk, Vladimir A.; De Luca, Chiara; Mikhal'Chik, Elena
Platelet Derived Growth Factor (PDGF) Responsive Epidermis Formed from Human Keratinocytes Transduced with the PDGF? Receptor Gene
Platelet-derived growth factor is a major proliferative and migratory stimulus for connective tissue cells during the initiation of skin repair processes. In response to injury, locally produced platelet-derived growth factor is secreted by a diversity of cutaneous cell types whereas target activity is confined to cells of mesenchymal origin, e.g. dermal fibroblasts and smooth muscle cells. Although epidermal cells contribute
Ola Rollman; Uffe B Jensen; Arne �stman; Lars Bolund; Sigr�n M G�stafsd�ttir; Thomas G Jensen
A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: Activation of proteinase-activated receptor 1 and epidermal growth factor receptor
Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR{sub 1}), and by PAR{sub 1} inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR{sub 1}-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.
Gao, Lin; Chao, Lee [Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425-2211 (United States)] [Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425-2211 (United States); Chao, Julie, E-mail: email@example.com [Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425-2211 (United States)] [Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425-2211 (United States)
Telomerase Rescues the Expression Levels of Keratinocyte Growth Factor and Insulin-like Growth Factor-II in Senescent Human Fibroblasts
Changes in expression levels of various cytokines, growth factors, and related genes were examined by reverse transcriptase polymerase chain reaction in a normal human fibroblast cell strain, TIG-3, along with in vitro aging. The expression levels of KGF and IGF-II were decreased with proliferative aging but not by growth arrest of young cells. In telomere-elongated cells prepared by transfection with
Yukari Kanzaki; Fumikazu Onoue; Fuyuki Ishikawa; Toshinori Ide
Telomerase rescues the expression levels of keratinocyte growth factor and insulin-like growth factor-II in senescent human fibroblasts.
Changes in expression levels of various cytokines, growth factors, and related genes were examined by reverse transcriptase polymerase chain reaction in a normal human fibroblast cell strain, TIG-3, along with in vitro aging. The expression levels of KGF and IGF-II were decreased with proliferative aging but not by growth arrest of young cells. In telomere-elongated cells prepared by transfection with human telomerase reverse transcriptase cDNA, high expression levels of these two genes were maintained, suggesting a causal relation between telomere shortening and reduced expression of KGF and IGF-II. The expression level of HGF was high in both growing and growth-arrested young cells but low in both senescent and telomere-elongated cells. The expression levels of follistatin and HB-EGF were high in both young growing and telomere-elongated cells but low in both senescent and growth-arrested young cells, indicating a growth-dependent expression. Expression levels of FGF-1, FGF-2, VEGF, BMP-3, and amphiregulin did not change with proliferative aging, growth arrest of young cells, or telomere elongation and life-span extension. PMID:12243757
Kanzaki, Yukari; Onoue, Fumikazu; Ishikawa, Fuyuki; Ide, Toshinori
Keratinocyte growth factor in inflammatory bowel disease. Increased mRNA transcripts in ulcerative colitis compared with Crohn's disease in biopsies and isolated mucosal myofibroblasts.
Inflammation in the gastrointestinal tract is associated with increased epithelial cell proliferation. Keratinocyte growth factor (KGF) is an epithelial cell mitogen widely expressed by mesenchymal cells subjacent to the epithelial cells. In this study, we have investigated the expression and distribution of KGF in normal and diseased (Crohn's disease and ulcerative colitis(UC)) intestine by quantitative competitive reverse-transcriptase polymerase chain reaction in whole biopsies and purified lamina propria myofibroblasts and by in situ hybridization. Analysis of whole mucosal biopsies reveals significantly higher numbers of KGF mRNA transcripts in UC compared with Crohn's colitis and control colon (P < 0.001). KGF transcripts were also elevated in Crohn's ileitis compared with normal ileum. In situ hybridization showed a marked increase in cells expressing KGF mRNA throughout the lamina propria in both UC and Crohn's tissue. In Crohn's disease, positively hybridizing cells were only rarely seen in the submucosa but were abundant around the bases of the crypts and were not associated with lymphoid aggregates. In purified mucosal myofibroblasts, increased (15- to 20-fold) KGF mRNA expression was seen in UC compared with control and Crohn's tissue. These results confirm and extend earlier studies showing that KGF transcripts are elevated in inflammatory bowel disease, but they show for the first time that transcripts are higher in UC than Crohn's disease because of increased production by mucosal mesenchymal cells. Images Figure 2 Figure 3
Bajaj-Elliott, M.; Breese, E.; Poulsom, R.; Fairclough, P. D.; MacDonald, T. T.
Keratinocyte growth factor and dexamethasone plus elevated cAMP levels synergistically support pluripotent stem cell differentiation into alveolar epithelial type II cells.
Alveolar epithelial type II (ATII)-like cells can be generated from murine embryonic stem cells (ESCs), although to date, no robust protocols applying specific differentiation factors are established. We hypothesized that the keratinocyte growth factor (KGF), an important mediator of lung organogenesis and primary ATII cell maturation and proliferation, together with dexamethasone, 8-bromoadenosine-cAMP, and isobutylmethylxanthine (DCI), which induce maturation of primary fetal ATII cells, also support the alveolar differentiation of murine ESCs. Here we demonstrate that the above stimuli synergistically potentiate the alveolar differentiation of ESCs as indicated by increased expression of the surfactant proteins (SP-) C and SP-B. This effect is most profound if KGF is supplied not only in the late stage, but at least also during the intermediate stage of differentiation. Our results indicate that KGF most likely does not enhance the generation of (mes)endodermal or NK2 homeobox 1 (Nkx2.1) expressing progenitor cells but rather, supported by DCI, accelerates further differentiation/maturation of respiratory progeny in the intermediate phase and maturation/proliferation of emerging ATII cells in the late stage of differentiation. Ultrastructural analyses confirmed the presence of ATII-like cells with intracellular composite and lamellar bodies. Finally, induced pluripotent stem cells (iPSCs) were generated from transgenic mice with ATII cell-specific lacZ reporter expression. Again, KGF and DCI synergistically increased SP-C and SP-B expression in iPSC cultures, and lacZ expressing ATII-like cells developed. In conclusion, ATII cell-specific reporter expression enabled the first reliable proof for the generation of murine iPSC-derived ATII cells. In addition, we have shown KGF and DCI to synergistically support the generation of ATII-like cells from ESCs and iPSCs. Combined application of these factors will facilitate more efficient generation of stem cell-derived ATII cells for future basic research and potential therapeutic application. PMID:23176317
Schmeckebier, Sabrina; Mauritz, Christina; Katsirntaki, Katherina; Sgodda, Malte; Puppe, Verena; Duerr, Julia; Schubert, Susanne C; Schmiedl, Andreas; Lin, Qiong; Pale?ek, Jiri; Draeger, Gerald; Ochs, Matthias; Zenke, Martin; Cantz, Tobias; Mall, Marcus A; Martin, Ulrich
AMPK regulation of the growth of cultured human keratinocytes
AMP kinase (AMPK) is a fuel sensing enzyme that responds to cellular energy depletion by increasing processes that generate ATP and inhibiting others that require ATP but are not acutely necessary for survival. In the present study, we examined the relationship between AMPK activation and the growth (proliferation) of cultured human keratinocytes and assessed whether the inhibition of keratinocyte growth by vitamin D involves AMPK activation. In addition, we explored whether the inhibition of keratinocyte proliferation as they approach confluence could be AMPK-related. Keratinocytes were incubated for 12 h with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside (AICAR). At concentrations of 10{sup -4} and 10{sup -3} M, AICAR inhibited keratinocyte growth by 50% and 95%, respectively, based on measurements of thymidine incorporation into DNA. It also increased AMPK and acetyl CoA carboxylase phosphorylation (P-AMPK and P-ACC) and decreased the concentration of malonyl CoA confirming that AMPK activation had occurred. Incubation with the thiazolidinedione, troglitazone (10{sup -6} M) caused similar alterations in P-AMPK, P-ACC, and cell growth. In contrast, the well known inhibition of keratinocyte growth by 1,25-dihydroxyvitamin D{sub 3} (10{sup -7} and 10{sup -6} M) was not associated with changes in P-AMPK or P-ACC. Like most cells, the growth of keratinocytes diminished as they approached confluence. Thus, it was of note that we found a progressive increase in P-AMPK (1.5- to 2-fold, p < 0.05) as keratinocytes grown in control medium went from 25% to 100% confluence. In conclusion, the data are consistent with the hypothesis that activation of AMPK acts as a signal to diminish the proliferation of cultured keratinocytes as they approach confluence. They also suggest that AMPK activators, such as AICAR and troglitazone, inhibit keratinocyte growth and that the inhibition of cell growth by 1,25-dihydroxyvitamin D{sub 3} is AMPK-independent.
Saha, Asish K. [Endocrinology, Diabetes and Nutrition, Department of Medicine, Boston University School of Medicine, Boston, MA (United States)]. E-mail: firstname.lastname@example.com; Persons, Kelly [Endocrinology, Diabetes and Nutrition, Department of Medicine, Boston University School of Medicine, Boston, MA (United States); Safer, Joshua D. [Endocrinology, Diabetes and Nutrition, Department of Medicine, Boston University School of Medicine, Boston, MA (United States); Luo Zhijun [Endocrinology, Diabetes and Nutrition, Department of Medicine, Boston University School of Medicine, Boston, MA (United States); Holick, Michael F. [Endocrinology, Diabetes and Nutrition, Department of Medicine, Boston University School of Medicine, Boston, MA (United States); Ruderman, Neil B. [Endocrinology, Diabetes and Nutrition, Department of Medicine, Boston University School of Medicine, Boston, MA (United States)
Surfactant metabolism and anti-oxidative capacity in hyperoxic neonatal rat lungs: effects of keratinocyte growth factor on gene expression in vivo.
Development of preterm infant lungs is frequently impaired resulting in bronchopulmoary dysplasia (BPD). BPD results from interruption of physiologic anabolic intrauterine conditions, the inflammatory basis and therapeutic consequences of premature delivery, including increased oxygen supply for air breathing. The latter requires surfactant, produced by alveolar type II (AT II) cells to lower surface tension at the pulmonary air:liquid interface. Its main components are specific phosphatidylcholine (PC) species including dipalmitoyl-PC, anionic phospholipids and surfactant proteins. Local antioxidative enzymes are essential to cope with the pro-inflammatory side effects of normal alveolar oxygen pressures. However, respiratory insufficiency frequently requires increased oxygen supply. To cope with the injurious effects of hyperoxia to epithelia, recombinant human keratinocyte growth factor (rhKGF) was proposed as a surfactant stimulating, non-catabolic and epithelial-protective therapeutic. The aim of the present study was to examine the qualification of rhKGF to improve expression parameters of lung maturity in newborn rats under hyperoxic conditions (85% O(2) for 7 days). In response to rhKGF proliferating cell nuclear antigen mRNA, as a feature of stimulated proliferation, was elevated. Similarly, the expressions of ATP-binding cassette protein A3 gene, a differentiation marker of AT II cells and of peroxiredoxin 6, thioredoxin and thioredoxin reductase, three genes involved in oxygen radical protection were increased. Furthermore, mRNA levels of acyl-coA:lysophosphatidylcholine acyltransferase 1, catalyzing dipalmitoyl-PC synthesis by acyl remodeling, and adipose triglyceride lipase, considered as responsible for fatty acid supply for surfactant PC synthesis, were elevated. These results, together with a considerable body of other confirmative evidence, suggest that rhKGF should be developed into a therapeutic option to treat preterm infants at risk for impaired lung development. PMID:23100171
Koslowski, Roland; Kasper, Michael; Schaal, Katharina; Knels, Lilla; Lange, Marco; Bernhard, Wolfgang
A novel solid-phase site-specific PEGylation enhances the in vitro and in vivo biostabilty of recombinant human keratinocyte growth factor 1.
Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl(4)-induced injury in rats compared to rhKGF-1. PMID:22574160
Huang, Zhifeng; Zhu, Guanghui; Sun, Chuanchuan; Zhang, Jingui; Zhang, Yi; Zhang, Youting; Ye, Chaohui; Wang, Xiaojie; Ilghari, Dariush; Li, Xiaokun
Transforming growth factor-beta regulation of bone morphogenetic protein-1/procollagen C-proteinase and related proteins in fibrogenic cells and keratinocytes.
Transforming growth factor-beta1 (TGF-beta1) induces increased extracellular matrix deposition. Bone morphogenetic protein-1 (BMP-1) also plays key roles in regulating vertebrate matrix deposition; it is the procollagen C-proteinase (PCP) that processes procollagen types I-III, and it may also mediate biosynthetic processing of lysyl oxidase and laminin 5. Here we show that BMP-1 is itself up-regulated by TGF-beta1 and that secreted BMP-1, induced by TGF-beta1, is either processed to an active form or remains as unprocessed proenzyme, in a cell type-dependent manner. In MG-63 osteosacrcoma cells, TGF-beta1 elevated levels of BMP-1 mRNA approximately 7-fold and elevated levels of mRNA for mammalian tolloid (mTld), an alternatively spliced product of the BMP1 gene, to a lesser extent. Induction of RNA was dose- and time-dependent and cycloheximide-inhibitable. Secreted BMP-1 and mTld, induced by TGF-beta1 in MG-63 and other fibrogenic cell cultures, were predominantly in forms in which proregions had been removed to yield activated enzyme. TGF-beta1 treatment also induced procollagen N-proteinase activity in fibrogenic cultures, while expression of the procollagen C-proteinase enhancer (PCPE), a glycoprotein that stimulates PCP activity, was unaffected. In contrast to fibrogenic cells, keratinocytes lacked detectable PCPE under any culture conditions and were induced by TGF-beta1 to secrete BMP-1 and mTld predominantly as unprocessed proenzymes. PMID:9228090
Lee, S; Solow-Cordero, D E; Kessler, E; Takahara, K; Greenspan, D S
Growth of human keratinocytes and fibroblasts on bacterial cellulose film.
Thin films of bacterial cellulose (BC) from a nata de coco culture system were developed, characterized, and investigated for the growth of human keratinocytes and fibroblasts. The average pore diameter and total surface area of the dried BC films estimated by BET were 224 A and 12.62 m(2)/g, respectively. With an film thickness of 0.12 mm, the average tensile strength and break strain of the dried films were 5.21 MPa and 3.75%, whereas those of the wet films were 1.56 MPa and 8.00%, respectively. The water absorption capacity of air-dried film was 5.09 g water/g dried films. For uses in the therapy of skin wounds, the potential biological mechanism of action of BC film was evaluated by using human keratinocytes and fibroblasts. Our results were the first direct demonstration that BC film supported the growth, spreading, and migration of human keratinocytes but not those of human fibroblasts. Expressions of E-cadherin and the alpha-3 chain of laminin confirmed the phenotype of human keratinocytes on BC film. PMID:16889398
Sanchavanakit, Neeracha; Sangrungraungroj, Wunwisa; Kaomongkolgit, Ruchadaporn; Banaprasert, Tanom; Pavasant, Prasit; Phisalaphong, Muenduen
Effect of Adenovector-Mediated Gene Transfer of Keratinocyte Growth Factor on the Proliferation of Alveolar Type II Cells In Vitro and In Vivo
Alveolar type II cell proliferation occurs after lung injury and is thought to minimize the subsequent fibrotic response. Kerati- nocyte growth factor (KGF) has been shown to be a potent growth factor for rat alveolar type II cells. In this study, we created a replication-deficient, recombinant human type 5 ade- novirus vector expressing human KGF (Ad5-KGF) to produce alveolar type
Osamu Morikawa; Thomas A. Walker; Larry D. Nielsen; Tianli Pan; James L. Cook; Robert J. Mason
Human keratinocytes produce the complement inhibitor factor H: synthesis is regulated by interferon-gamma
Locally synthesized complement is believed to play an important role in host defense and inflammation at organ level. In the epidermis, keratinocytes have so far been shown to synthesize two complement components, C3 and factor B. Here, we studied the synthesis of factor H by human keratinocytes. We also studied the regulation of factor H synthesis in keratinocytes by several
K. K. Timar; M. C. Pasch; N. H. van den Bosch; H. Jarva; S. Junnikkala; S. Meri; J. D. Bos; S. S. Asghar
Human keratinocytes produce the complement inhibitor factor H: Synthesis is regulated by interferon-?
Locally synthesized complement is believed to play an important role in host defense and inflammation at organ level. In the epidermis, keratinocytes have so far been shown to synthesize two complement components, C3 and factor B. Here, we studied the synthesis of factor H by human keratinocytes. We also studied the regulation of factor H synthesis in keratinocytes by several
Krisztina K. Tim�r; Marcel C. Pasch; Norbert H. A. van den Bosch; Hanna Jarva; Sami Junnikkala; Seppo Meri; Jan D. Bos; Syed S. Asghar
Human keratinocytes produce the complement inhibitor factor H: Synthesis is regulated by interferon-gamma
Locally synthesized complement is believed to play an important role in host defense and inflammation at organ level. In the epidermis, keratinocytes have so far been shown to synthesize two complement components, 0 and factor B. Here, we studied the synthesis of factor H by human keratinocytes. We also studied the regulation of factor H synthesis in keratinocytes by several
K. K. Timar; M. C. Pasch; Bosch van den N. H. A; H. Jarva; S. Junnikkala; S. Meri; J. D. Bos; S. S. Asghar
Nerve growth factor levels in cultured human skin cells: effect of gestation and viral transformation
Extracts of cultured human keratinocytes and fibroblasts were assayed for nerve growth factor-like immunoreactivity (NGF) by a specific enzyme-linked immunoabsorbant assay. NGF levels were higher in primary cultured keratinocytes than in freshly isolated keratinocytes or culture through multiple passages. Viral transformation of keratinocytes with the human papilloma virus (HPV 16) significantly increased NGF levels, whilst transformation with the simian virus
P. Anand; P. Foley; H. A. Navsaria; D. Sinicropi; R. E. Williams-Chestnut; I. M. Leigh
Salivary trefoil factor 3 enhances migration of oral keratinocytes.
Trefoil factor 3 (TFF3) is a member of the mammalian TFF family. Trefoil factors are secreted onto mucosal surfaces of the entire body and exert different effects according to tissue location. Trefoil factors may enhance mucosal healing by modulating motogenic activity, inhibiting apoptosis, and promoting angiogenesis. Trefoil factor 3 is secreted from the submandibular gland and is present in whole saliva. The aim of this study was to assess the migratory and proliferative effects of TFF3 on primary oral human keratinocytes and oral cancer cell lines. The addition of TFF3 increased the migration of both normal oral keratinocytes and the cancer cell line D12, as evaluated by a two-dimensional scratch assay. By contrast, no increase in proliferation or energy metabolism was observed after stimulation with TFF3. Trefoil factor 3-enhanced migration was found to be driven partly by the extracellular signal-related kinase (Erk1/2) pathway, as shown by addition of the mitogen-activated protein kinase (MAPK) inhibitor PD 98059. Previous functional studies on trefoil peptides have all been based on cells from monolayered epithelium like the intestinal mucosa; this is the first report to show that normal and cancerous keratinocytes from stratified epithelium respond to TFF stimuli. Taken together, salivary TFF3 is likely to contribute to oral wound healing. PMID:18353006
Storesund, Trond; Hayashi, Katsuhiko; Kolltveit, Kristin M; Bryne, Magne; Schenck, Karl
[Keratinocyte-fibroblast interactions: I. Production by the keratinocytes of soluble factors stimulating the proliferation of normal human skin fibroblasts].
Epidermal cells produce IL-1 and contra IL-1 which respectively stimulate and decrease fibroblast proliferation. In order to get better insight into the nature of keratinocyte-fibroblast interactions, we have analyzed the effect of soluble factors produced by unstimulated normal human keratinocytes and keratinocyte cell lines on the proliferation of normal human dermal fibroblasts. The results were compared to these obtained with human recombinant IL-1 and IL-2. We observed that: 1) normal keratinocytes (monolayers or stratified) released in the culture medium a factor stimulating fibroblast proliferation by 45 to 160%; 2) supernatants of unstimulated PAM 212 keratinocyte cell line also contained the stimulatory activity; 3) addition of IL-1 beta to the culture medium at concentration ranging from 0.1 to 1.000 U/ml induced a dose-dependent increase in fibroblast proliferation, whereas IL-2 was ineffective; 4) gel filtration analysis (ACA 54) of serum-free supernatant showed that the activity could be eluted from 3 peaks (Mw ranging from 10 to 20 kD). The present data show that unstimulated normal human keratinocytes produce soluble factor(s) (maybe related to IL-1) which stimulate human dermal fibroblast proliferation and which could be of major importance in the modulation of fibroblast metabolism, in vivo. PMID:2616203
Delaporte, E; Croute, F; Vincent, C; Bonnefoy, J Y; Robert, J; Thivolet, J; Nicolas, J F
Cell type specific interleukin-6 induced responses in tumor keratinocytes and stromal fibroblasts are essential for invasive growth.
Interleukin-6 (IL-6) is one of the major inflammatory interleukins that has been linked to cancer progression. In our model for human skin squamous cell carcinoma (SCC), IL-6 expression is strongly upregulated upon progression from benign tumors to highly malignant, metastasizing SCCs. We now demonstrate that IL-6 promotes malignant and invasive tumor growth in human skin SCCs by inducing cell type specific cytokine profiles in tumor keratinocytes and stromal fibroblasts, activating the latter towards a tumor associated fibroblast (TAF) phenotype. In three-dimensional organotypic cocultures in vitro invasive growth of IL-6 overexpressing tumor keratinocytes, is associated with increased expression of matrix metalloproteinase-2 (MMP-2), MMP-14 and tissue inhibitor of metalloproteinases-2, and clearly depends on IL-6 activated fibroblasts. IL-6-induced secretion of monocyte chemotactic protein-1 (MCP-1) in tumor keratinocytes and of hepatocyte growth factor in fibroblasts is crucial for regulating expression and activation of MMP-2. This functional role of IL-6 is confirmed in vivo. Here MMP-14 and MMP-2 expression occur exclusively in surface transplants of IL-6 overexpressing keratinocytes and fibroblasts are identified as important source of MMP-2. Our data indicate that tumor keratinocytes derived IL-6 activates stromal fibroblasts towards a TAF phenotype, promoting tumor invasion via enhanced expression and activation of MMP-2. PMID:23165423
Depner, Sofia; Lederle, Wiltrud; Gutschalk, Claudia; Linde, Nina; Zajonz, Alexandra; Mueller, Margareta M
BAG-1 enhances cell-cell adhesion, reduces proliferation and induces chaperone-independent suppression of hepatocyte growth factor-induced epidermal keratinocyte migration
Cell motility is important in maintaining tissue homeostasis, facilitating epithelial wound repair and in tumour formation and progression. The aim of this study was to determine whether BAG-1 isoforms regulate epidermal cell migration in in vitro models of wound healing. In the human epidermal cell line HaCaT, endogenous BAG-1 is primarily nuclear and increases with confluence. Both transient and stable p36-Bag-1 overexpression resulted in increased cellular cohesion. Stable transfection of either of the three human BAG-1 isoforms p36-Bag-1 (BAG-1S), p46-Bag-1 (BAG-1M) and p50-Bag-1 (BAG-1L) inhibited growth and wound closure in serum-containing medium. However, in response to hepatocyte growth factor (HGF) in serum-free medium, BAG-1S/M reduced communal motility and colony scattering, but BAG-1L did not. In the presence of HGF, p36-Bag-1 transfectants retained proliferative response to HGF with no change in ERK1/2 activation. However, the cells retained E-cadherin localisation at cell-cell junctions and exhibited pronounced cortical actin. Point mutations in the BAG domain showed that BAG-1 inhibition of motility is independent of its function as a chaperone regulator. These findings are the first to suggest that BAG-1 plays a role in regulating cell-cell adhesion and suggest an important function in epidermal cohesion.
Hinitt, C.A.M.; Wood, J.; Lee, S.S. [Oral and Dental Science, University of Bristol, Lower Maudlin Street, Bristol, BS1 2LY (United Kingdom)] [Oral and Dental Science, University of Bristol, Lower Maudlin Street, Bristol, BS1 2LY (United Kingdom); Williams, A.C. [Cellular and Molecular Medicine, University of Bristol, School of Medical Sciences, University Walk, Bristol, BS8 1TD (United Kingdom)] [Cellular and Molecular Medicine, University of Bristol, School of Medical Sciences, University Walk, Bristol, BS8 1TD (United Kingdom); Howarth, J.L.; Glover, C.P.; Uney, J.B. [The Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol, BS1 3NY (United Kingdom)] [The Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol, BS1 3NY (United Kingdom); Hague, A., E-mail: firstname.lastname@example.com [Oral and Dental Science, University of Bristol, Lower Maudlin Street, Bristol, BS1 2LY (United Kingdom)
Recombinant adeno-associated virus type 2-mediated gene transfer into human keratinocytes is influenced by both the ubiquitin\\/proteasome pathway and epidermal growth factor receptor tyrosine kinase
Efficient gene delivery into keratinocytes is a prerequisite for successful skin gene therapy. Vectors based on recombinant adeno-associated virus type 2 (rAAV-2) offer several promising features that make them attractive for cutaneous applications. However, highly efficient gene delivery may be hampered by different cellular factors, including lack of viral receptors, impairment of cytoplasmic trafficking or limitations in viral second-strand synthesis.
Markus Braun-Falco; Angelika Eisenried; Hildegard B�ning; Johannes Ring
Expression of paired-like homeodomain transcription factor 2c (PITX2c) in epidermal keratinocytes
Paired-like homeodomain transcription factor 2 (PITX2) has been implicated as one of the genes responsible for Rieger syndrome. It has been also shown to play a central role during development. In this study, we investigated the functional role of PITX2 in keratinocyte differentiation. RT-PCR analysis showed that PITX2c isoform was predominantly expressed in a differentiation-dependent manner. Consistent with, immunohistochemical staining showed that PITX2 expression was increased in the upper layer of epidermis. When PITX2c was overexpressed in cultured keratinocytes by a recombinant adenovirus, the differentiation markers such as involucrin and loricrin were significantly increased at both mRNA and protein levels. In addition, PITX2c overexpression led to the decrease of cell growth, concomitantly with the upregulation of cell cycle-related genes p21. To investigate the effect of PITX2c in vivo, we microinjected PITX2c expression vector into zebrafish embryo. Interestingly, overexpression of PITX2c in zebrafish embryo led to the formation of horn-like structure and thickening of epidermis, together with the increase of keratin 8 (K8) expression. These results suggest that PITX2c has a role in proliferation and differentiation of epidermal keratinocytes.
Shi, Ge [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of) [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Department of Dermatology, The First Affiliated Hospital, Guangxi Traditional Chinese Medical University, Guangxi, Nanning, 530023 (China); Sohn, Kyung-Cheol; Choi, Tae-Young; Choi, Dae-Kyoung; Lee, Sang-Sin [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of) [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Ou, Bai-sheng [Department of Dermatology, The First Affiliated Hospital, Guangxi Traditional Chinese Medical University, Guangxi, Nanning, 530023 (China)] [Department of Dermatology, The First Affiliated Hospital, Guangxi Traditional Chinese Medical University, Guangxi, Nanning, 530023 (China); Kim, Sooil; Lee, Young Ho [Department of Anatomy, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of)] [Department of Anatomy, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Yoon, Tae-Jin [Department of Dermatology, School of Medicine, Gyeongsang National University, Jinju, 660-702 (Korea, Republic of) [Department of Dermatology, School of Medicine, Gyeongsang National University, Jinju, 660-702 (Korea, Republic of); Institute of Health Sciences, School of Medicine, Gyeongsang National University, Jinju, 660-702 (Korea, Republic of); Kim, Seong-Jin [Department of Dermatology, Chonnam National University Medical School, Gwangju, 501-757 (Korea, Republic of)] [Department of Dermatology, Chonnam National University Medical School, Gwangju, 501-757 (Korea, Republic of); Lee, Young; Seo, Young-Joon; Lee, Jeung-Hoon [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of) [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Kim, Chang Deok, E-mail: email@example.com [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of)
Interferon regulatory factor 6 is necessary, but not sufficient, for keratinocyte differentiation.
Regulation of epidermal proliferation and differentiation is critical for maintenance of cutaneous homeostasis. Interferon Regulatory Factor 6 (Irf6)-deficient mice die perinatally and exhibit ectopic proliferation and defective epidermal differentiation. We sought to determine whether these disruptions of epidermal function were cell autonomous, and used embryonic Irf6(-/-) keratinocytes to understand the specific role of Irf6 in keratinocyte proliferation and differentiation. In the absence of Irf6, keratinocytes exhibited a heterogeneous phenotype with the presence of large cells. Irf6(-/-) keratinocytes displayed increased colony-forming efficiency compared with wild-type cells, suggesting that Irf6 represses long-term proliferation. Irf6 was present at low levels in wild-type keratinocytes in culture, and upregulated after induction of differentiation in vitro, along with upregulation of markers of early differentiation. However, Irf6(-/-) keratinocytes did not express markers of terminal differentiation. Overexpression of Irf6 in wild-type keratinocytes was insufficient to induce expression of markers of differentiation under growing conditions. Together, these results indicated that Irf6 is necessary, but not sufficient, for keratinocyte differentiation. Finally, using a transgenic mouse expressing Lac-Z under the regulation of an enhancer element 9.7? kb upstream of the Irf6 start site, we demonstrated that this element contributes to the regulation of Irf6 in the epidermis and keratinocytes in culture. PMID:21918538
Biggs, Leah C; Rhea, Lindsey; Schutte, Brian C; Dunnwald, Martine
Efficient keratinocyte differentiation strictly depends on JNK-induced soluble factors in fibroblasts.
Previous studies demonstrated that fibroblast-derived and JUN-dependent soluble factors have a crucial role on keratinocyte proliferation and differentiation during cutaneous wound healing. Furthermore, mice with a deficiency in Jun N-terminal kinases (JNKs) , JNK1 or JNK2, showed impaired skin development and delayed wound closure. To decipher the role of dermal JNK in keratinocyte behavior during these processes, we used a heterologous coculture model combining primary human keratinocytes and murine fibroblasts. Although cocultured JNK1/JNK2-deficient fibroblasts did not affect keratinocyte proliferation, temporal monitoring of the transcriptome of differentiating keratinocytes revealed that efficient keratinocyte differentiation not only requires the support by fibroblast-derived soluble factors, but is also critically dependent on JNK1 and JNK2 signaling in these cells. Moreover, we showed that the repertoire of fibroblast transcripts encoding secreted proteins is severely disarranged upon loss of JNK under the coculture conditions applied. Finally, our data demonstrate that efficient keratinocyte terminal differentiation requires constant presence of JNK-dependent and fibroblast-derived soluble factors. Taken together, our results imply that mesenchymal JNK has a pivotal role in the paracrine cross talk between dermal fibroblasts and epidermal keratinocytes during wound healing. PMID:24335928
Schumacher, Marion; Schuster, Christian; Rogon, Zbigniew M; Bauer, Tobias; Caushaj, Nevisa; Baars, Sebastian; Szabowski, Sibylle; Bauer, Christine; Schorpp-Kistner, Marina; Hess, Jochen; Holland-Cunz, Stefan; Wagner, Erwin F; Eils, Roland; Angel, Peter; Hartenstein, Bettina
The Effect of Secretory Factors of Adipose-Derived Stem Cells on Human Keratinocytes
The beneficial effects of adipose-derived stem cell conditioned medium (ADSC-CM) on skin regeneration have been reported. Although the mechanism of how ADSC-CM promotes skin regeneration is unclear, ADSC-CM contained various growth factors and it is an excellent raw material for skin treatment. ADSC-CM produced in a hypoxia condition of ADSC�in other words, Advanced Adipose-Derived Stem cell Protein Extract (AAPE)�has great merits for skin regeneration. In this study, human primary keratinocytes (HKs), which play fundamental roles in skin tissue, was used to examine how AAPE affects HK. HK proliferation was significantly higher in the experimental group (1.22 ?g/mL) than in the control group. DNA gene chip demonstrated that AAPE in keratinocytes (p < 0.05) notably affected expression of 290 identified transcripts, which were associated with cell proliferation, cycle and migration. More keratinocyte wound healing and migration was shown in the experimental group (1.22 ?g/mL). AAPE treatment significantly stimulated stress fiber formation, which was linked to the RhoA-ROCK pathway. We identified 48 protein spots in 2-D gel analysis and selected proteins were divided into 64% collagen components and 30% non-collagen components as shown by the MALDI-TOF analysis. Antibody array results contained growth factor/cytokine such as HGF, FGF-1, G-CSF, GM-CSF, IL-6, VEGF, and TGF-?3 differing from that shown by 2-D analysis. Conclusion: AAPE activates HK proliferation and migration. These results highlight the potential of the topical application of AAPE in the treatment of skin regeneration.
Moon, Kyoung Mi; Park, Ye-Hyoung; Lee, Jae Seol; Chae, Yong-Byung; Kim, Moon-Moo; Kim, Dong-Soo; Kim, Byung-Woo; Nam, Soo-Wan; Lee, Jong-Hwan
Omega-3 polyunsaturated fatty acids selectively inhibit growth in neoplastic oral keratinocytes by differentially activating ERK1/2
The long-chain omega-3 polyunsaturated fatty acids (n-3 PUFAs)�eicosapentaenoic acid (EPA) and its metabolite docosahexaenoic acid (DHA)�inhibit cancer formation in vivo, but their mechanism of action is unclear. Extracellular signal-regulated kinase 1/2 (ERK1/2) activation and inhibition have both been associated with the induction of tumour cell apoptosis by n-3 PUFAs. We show here that low doses of EPA, in particular, inhibited the growth of premalignant and malignant keratinocytes more than the growth of normal counterparts by a combination of cell cycle arrest and apoptosis. The growth inhibition of the oral squamous cell carcinoma (SCC) lines, but not normal keratinocytes, by both n-3 PUFAs was associated with epidermal growth factor receptor (EGFR) autophosphorylation, a sustained phosphorylation of ERK1/2 and its downstream target p90RSK but not with phosphorylation of the PI3 kinase target Akt. Inhibition of EGFR with either the EGFR kinase inhibitor AG1478 or an EGFR-blocking antibody inhibited ERK1/2 phosphorylation, and the blocking antibody partially antagonized growth inhibition by EPA but not by DHA. DHA generated more reactive oxygen species and activated more c-jun N-terminal kinase than EPA, potentially explaining its increased toxicity to normal keratinocytes. Our results show that, in part, EPA specifically inhibits SCC growth and development by creating a sustained signalling imbalance to amplify the EGFR/ERK/p90RSK pathway in neoplastic keratinocytes to a supraoptimal level, supporting the chemopreventive potential of EPA, whose toxicity to normal cells might be reduced further by blocking its metabolism to DHA. Furthermore, ERK1/2 phosphorylation may have potential as a biomarker of n-3 PUFA function in vivo.
Parkinson, Eric Kenneth
Ganglioside Modulates Ligand Binding to the Epidermal Growth Factor Receptor
Whereas previous investigations have shown that pharmacologic addition of gangliosides inhibits keratinocyte proliferation by downregulating epidermal growth factor receptor phosphorylation, the underlying biochemical basis and physiologic relevance are unknown. Using Scatchard and displacement plots, we have shown that supplemental purified gangliosides decrease the binding of 125I-labeled epidermal growth factor to keratinocyte-derived SCC12 cells. Conversely, SCC12 cells transfected with sialidase and
Xiaoqi Wang; Zakia Rahman; Ping Sun; Emmanuelle Meuillet; David George; Eric G. Bremer; Abbas Al-Qamari; Amy S. Paller
Human keratinocytes produce the complement inhibitor factor H: synthesis is regulated by interferon-gamma.
Locally synthesized complement is believed to play an important role in host defense and inflammation at organ level. In the epidermis, keratinocytes have so far been shown to synthesize two complement components, C3 and factor B. Here, we studied the synthesis of factor H by human keratinocytes. We also studied the regulation of factor H synthesis in keratinocytes by several cytokines, namely IL-1alpha, IL-2, IL-6, TGF-beta1, TNF-alpha and IFN-gamma. Human keratinocytes expressed factor H mRNA and constitutively released small amounts of factor H protein into the culture medium. This release was strongly upregulated by IFN-gamma but not by other cytokines tested. Western blot analysis revealed that IFN-gamma augments the synthesis of both molecular species, factor H (FH; 155kDa) and factor H-like protein-1 (FHL-1; 45kDa), of factor H. Factor H released in response to IFN-gamma was functionally active. In conclusion, we demonstrate that keratinocytes are capable of synthesizing factor H and that this synthesis is regulated by IFN-gamma. PMID:16310045
Tim�r, Krisztina K; Pasch, Marcel C; van den Bosch, Norbert H A; Jarva, Hanna; Junnikkala, Sami; Meri, Seppo; Bos, Jan D; Asghar, Syed S
Growth and Differentiation of HaCaT Keratinocytes.
HaCaT cells are a spontaneously immortalized, human keratinocyte line that has been widely used for studies of skin biology and differentiation. Under typical culture conditions HaCaT cells have a partially to fully differentiated phenotype due to the high calcium content of both standard media and fetal bovine serum. This chapter describes low-calcium culture conditions for reverting HaCaT cells to the fully basal state followed by subsequent controlled differentiation using calcium induction. PMID:24155234
Wilson, Van G
Hydrogen sulfide impairs keratinocyte cell growth and adhesion inhibiting mitogen-activated protein kinase signaling.
The effects of exogenous hydrogen sulfide (H2S) on normal skin-derived immortalized human keratinocytes have been investigated in detail. We show in vitro that exogenous hydrogen sulfide reduces clonal growth, cell proliferation and cell adhesion of human keratinocytes. H(2)S, in fact, decreases the frequency of the putative keratinocyte stem cell subpopulation in culture, consequently affecting clonal growth, and impairs cell proliferation and adhesion of mature cells. As a mechanistic explanation of these effects, we show at the molecular level that (i) H2S reduces the Raf/MAPK kinase/ERK signaling pathway; (ii) the reduced adhesion of sulfur-treated cells is associated to the downregulation of the expression of beta4, alpha2 and alpha6 integrins that are necessary to promote cell adhesion as well as anti-apoptotic and proliferative signaling in normal keratinocytes. One specific interest of the effects of sulfurs on keratinocytes derives from the potential applications of the results, as sulfur is able to penetrate the skin and a sulfur-rich balneotherapy has been known for long to be effective in the treatment of psoriasis. Thus, the relevance of our findings to the pathophysiology of psoriasis was tested in vivo by treating psoriatic lesions with sulfurs at a concentration comparable to that most commonly found in sulfurous natural springs. In agreement with the in vitro observations, the immunohistochemical analysis of patient biopsies showed a specific downregulation of ERK activation levels, the key molecular event in the sulfur-induced effects on keratinocytes. PMID:19546851
Gobbi, Giuliana; Ricci, Francesca; Malinverno, Chiara; Carubbi, Cecilia; Pambianco, Maurizia; Panfilis, Giuseppe de; Vitale, Marco; Mirandola, Prisco
Small-diameter porous poly (epsilon-caprolactone) films enhance adhesion and growth of human cultured epidermal keratinocyte and dermal fibroblast cells.
Autologous keratinocyte grafts provide clinical benefit by rapidly covering wounded areas, but they are fragile. We therefore developed biocompatible hexagonal-packed porous films with uniform, circular pore sizes to support human keratinocytes and fibroblasts. Cells were cultured on these porous poly (epsilon-calprolactone) films with pore sizes ranging from novel ultra-small 3 microm to 20 microm. These were compared with flat (pore-less) films. Cell growth rates, adhesion, migration, and ultrastructural morphology were examined. Human keratinocytes and fibroblasts attached to all films. Furthermore, small-pore (3-5 microm) films showed the highest levels of cell adhesion and survival and prevented migration into the pores and opposing film surface. Keratinocyte migration over small-pore film surface was inhibited. Keratinocytes optimally attached to 3-microm-pore films due to a combination of greater pore numbers (porosity), a greater circumference of the pore edge per unit surface area, and greater frequency of flat surface areas for attachment, allowing better cell-substrate and cell-cell attachment and growth. The 3-microm pore size allowed cell-cell communication, together with diffusion of soluble nutrients and factors from the culture medium or wound substrate. These characteristics are considered important in developing grafts for use in the treatment of human skin wounds. PMID:17228993
McMillan, James R; Akiyama, Masashi; Tanaka, Masaru; Yamamoto, Sadaki; Goto, Maki; Abe, Riichiro; Sawamura, Daisuke; Shimomura, Masatsugu; Shimizu, Hiroshi
EGF Upregulates, Whereas TGF-? Downregulates, the Hyaluronan Synthases Has2 and Has3 in Organotypic Keratinocyte Cultures: Correlations with Epidermal Proliferation and Differentiation
Hyaluronan, a major extracellular matrix molecule in the vital cell layers of skin epidermis, has been suggested to support proliferation and migration of keratinocytes, during challenges like wounding and inflammation. An organotypic keratinocyte culture originated from continuous rat epidermal keratinocyte cell line was subjected to the proliferative and antiproliferative growth factors epidermal growth factor and transforming growth factor ?, respectively,
Sanna Pasonen-Sepp�nen; Susanna Karvinen; Kari T�rr�nen; Juha M T Hyttinen; Tiina Jokela; Mikko J Lammi; Markku I Tammi; Raija Tammi
Further evidence that acanthosis nigricans maligna is linked to enhanced secretion by the tumour of transforming growth factor alpha
The pathogenesis of cutaneous paraneoplastic syndromes is still under discussion. Since many of these syndromes, including acanthosis nigricans, are proliferative skin disorders it is believed that products secreted by the tumour stimulate the keratinocytes to proliferate. Growth factors like transforming growth factor alpha (TGF-alpha) are known to be highly mitogenic for keratinocytes in vitro. Here we report on a patient
K. Wilgenbus; A. Lentner; R. Kuckelkorn; St. Handt; Ch. Mittermayer
Reciprocal Cytokine-Mediated Cellular Interactions in Mouse Epidermis: Promotion of ? ? T-Cell Growth by IL7 and TNF? and Inhibition of Keratinocyte Growth by ?IFN
A unique subset of ?? T cells, termed dendritic epidermal T cells (DETC), resides in symbiosis with keratinocytes in mouse epidermis. We have shown previously that interleukin 7 (IL-7) which is produced by keratinocytes, promotes growth and prevents apoptosis in DETC. To extend this observation, we examined 12 cytokines, each of which is expressed by epidermal cells at mRNA and\\/or
Hiroyuki Matsue; Paul R Bergstresser; Akira Takashima
Biocompatibility and growth of human keratinocytes and fibroblasts on biosynthesized cellulose-chitosan film.
Bacterial cellulose (BC)-chitosan (BCC) films made via bio-co-polymerization by Acetobacter xylinum were developed and characterized for physical and biological properties. With the incorporation of chitosan MW 3 x 10(4) and 8 x 10(4) into bacterial cellulose, the modified films (BCC-MW 30,000 and BCC-MW 80,000, respectively) became denser, with a smaller average pore size of 13.1-15.3 nm in dry form. The BCC films have no toxicity against L929 mouse fibroblast cells. Tissue compatibility was then evaluated by growth and spreading of human skin keratinocytes and fibroblasts. The results revealed that the growth of human skin keratinocytes and fibroblasts on the BCC films was comparable to that on the BC film; however, improvement of cell adhesion and spreading on the BCC films was observed in human skin keratinocytes. The results of the biological response experiments showed no significant difference between BCC-MW 30,000 and BCC-MW 80,000. PMID:20507705
Kingkaew, Jeerun; Jatupaiboon, Nirun; Sanchavanakit, Neeracha; Pavasant, Prasit; Phisalaphong, Muenduen
Malignant Transformation of Immortalized HaCaT Keratinocytes through Deregulated Nuclear Factor KB Signaling
Previous studies addressing functional aspects of nuclear factor KB (NF-KB) activation in normal and transformed keratinocytes revealed complex and seemingly contradictory roles of this transcription factor in this cell type. In normal skin, NF-KB signaling seems to inhibit squamous cell carcinoma development whereas, in squamous cell carcinoma themselves, deregulated NF-KB expression and\\/or signaling is frequently observed. To further investigate this
Qing Ren; Csaba Kari; Randy Burd; Peter McCue; Adam P. Dicker; Ulrich Rodeck
The 55-kD tumor necrosis factor receptor on human keratinocytes is regulated by tumor necrosis factor-alpha and by ultraviolet B radiation.
In previous studies we showed that cultured human keratinocytes expressed the 55-kD TNF receptor (TNFR) and that its expression the important for TNF alpha-mediated upregulation of intercellular adhesion molecule-1 (ICAM-1) expression on keratinocytes. Because factors that either reduce or enhance TNFR expression are likely to have a major impact on the biological effects of TNF alpha on keratinocytes, these studies were conducted to determine the factors that regulate its expression on keratinocytes. Using reverse transcriptase polymerase chain reaction, human keratinocytes were shown to lack 75-kD TNFR expression, indicating that TNF responsiveness of human keratinocytes critically depended on regulation of 55-kD TNFR expression. Human keratinocyte 55-kD TNFR surface and mRNA expression was found to be regulated in vitro by recombinant human (rh) TNF alpha. Stimulation of keratinocytes with rhTNF alpha initially decreased, but later increased, 55-kD TNFR surface expression. This biphasic modulation of 55-kD TNFR surface expression was associated with concomitant changes in 55-kD TNFR mRNA expression. Ultraviolet B (UVB) radiation, a well-known inducer of synthesis and secretion of TNF alpha by human keratinocytes, was found to mimic TNF alpha-induced modulation of 55-kD TNFR surface and mRNA expression via a TNF alpha-mediated autocrine regulatory mechanism. Production of soluble 55-kD TNFR by human keratinocytes remained unaffected by TNF alpha stimulation or UVB irradiation. These studies provide clear evidence that membrane expression of the human 55-kD TNFR may be regulated in human keratinocytes by the ligand itself: TNF alpha. Since in previous studies UVB irradiation transiently inhibited TNF alpha-induced human keratinocyte ICAM-1 expression, it is proposed that UVB radiation-induced biphasic modulation of human keratinocyte 55-kD TNFR expression may affect the capacity of these cells to respond to TNF alpha. Images
Trefzer, U; Brockhaus, M; Lotscher, H; Parlow, F; Budnik, A; Grewe, M; Christoph, H; Kapp, A; Schopf, E; Luger, T A
Suppression of AP1 Transcription Factor Function in Keratinocyte Suppresses Differentiation
Our previous study shows that inhibiting activator protein one (AP1) transcription factor function in murine epidermis, using dominant-negative c-jun (TAM67), increases cell proliferation and delays differentiation. To understand the mechanism of action, we compare TAM67 impact in mouse epidermis and in cultured normal human keratinocytes. We show that TAM67 localizes in the nucleus where it forms TAM67 homodimers that competitively interact with AP1 transcription factor DNA binding sites to reduce endogenous jun and fos factor binding. Involucrin is a marker of keratinocyte differentiation that is expressed in the suprabasal epidermis and this expression requires AP1 factor interaction at the AP1-5 site in the promoter. TAM67 interacts competitively at this site to reduce involucrin expression. TAM67 also reduces endogenous c-jun, junB and junD mRNA and protein level. Studies with c-jun promoter suggest that this is due to reduced transcription of the c-jun gene. We propose that TAM67 suppresses keratinocyte differentiation by interfering with endogenous AP1 factor binding to regulator elements in differentiation-associated target genes, and by reducing endogenous c-jun factor expression.
Han, Bingshe; Rorke, Ellen A.; Adhikary, Gautam; Chew, Yap Ching; Xu, Wen; Eckert, Richard L.
The skin has long been recognized as a major producer of cytokines, but the keratinocyte as principal epidermal cell has received less attention as potential source and target of cytokines. Nevertheless, keratinocytes produce a plethora of cytokines including interleukin (IL)-1, -6, -7, -8, -10, -12, -15, -18, and -20, and tumor necrosis factor ? (TNF). The production by keratinocytes of
A Gr�ne
Expression of the human papillomavirus type 16 E7 oncoprotein induces an autophagy-related process and sensitizes normal human keratinocytes to cell death in response to growth factor deprivation
Expression of oncogenes, such as the human papillomavirus type 16 (HPV16) E7 oncoprotein, promotes aberrant cell proliferation. In the absence of concurrent mitogenic stimuli, this triggers a cell-intrinsic defense mechanism, the 'trophic sentinel response', which eliminates such aberrant cells. The molecular pathways that elicit this response, however, remain obscure. We set up an experimental system to investigate the trophic sentinel pathway triggered by HPV16 E7 expression in normal human keratinocytes, the natural host cells of HPVs. Keratinocytes expressing HPV16 E7 cultured in E-medium undergo cell death and show increased sub-G1 DNA content when grown to confluence or under conditions of serum deprivation. Moreover, HPV16 E7 expressing human keratinocytes express higher levels of the autophagy marker, LC3-II, which can be abrogated by 3-methyladenine, an autophagy inhibitor. These findings indicate that even under normal culture conditions, HPV16 E7 expression triggers metabolic stress that may result in autophagy, a pathway implicated in carcinogenesis.
Zhou Xiaobo [Infectious Diseases Division, Channing Laboratories, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School (United States); Muenger, Karl [Infectious Diseases Division, Channing Laboratories, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School (United States)], E-mail: firstname.lastname@example.com
Role of Keratinocytes in the Development of Vitiligo
Vitiligo is an acquired depigmentary disorder of the skin that results from the loss of functioning epidermal melanocytes. Most studies on vitiligo have concentrated on the abnormality of melanocytes rather than the abnormality of keratinocytes; however, epidermal melanocytes form a functional and structural unit with neighboring keratinocytes. In fact, direct cell-to cell contact stimulates in vitro proliferation of melanocytes, and growth factors produced by adjacent keratinocytes regulate the proliferation and differentiation of melanocytes. The potential role of keratinocyte-derived cytokines has also been presented. We focused on the structural changes in vitiliginous keratinocytes, which may result in loss of melanocytes, to examine the pathomechanism of vitiligo. The results of a comparison between depigmented and normally pigmented epidermis in patients with vitiligo showed that the keratinocytes in the depigmented epidermis were more vulnerable to apoptosis. Impaired Phosphatidylinositol 3-kinase (PI3K)/serine/threonine protein kinase (Akt) activation followed by reduced nuclear factor-?B activation under increased tumor necrosis factor-? levels was demonstrated as a mechanism for keratinocyte apoptosis. The role of aquaporin 3 in keratinocyte apoptosis was addressed based on the relationship between the PI3K/AKT pathway and the E-cadherin-catenin complex. Apoptotic keratinocytes induced a lower expression of keratinocyte-derived factors, including stem cell factor, in depigmented epidermis, resulting in passive melanocyte death.
Nrf Transcription Factors in Keratinocytes Are Essential for Skin Tumor Prevention but Not for Wound Healing
The Nrf2 transcription factor is a key player in the cellular stress response through its regulation of cytoprotective genes. In this study we determined the role of Nrf2-mediated gene expression in keratinocytes for skin development, wound repair, and skin carcinogenesis. To overcome compensation by the related Nrf1 and Nrf3 proteins, we expressed a dominant-negative Nrf2 mutant (dnNrf2) in the epidermis
U. auf dem Keller; M. Huber; T. A. Beyer; A. Kumin; C. Siemes; S. Braun; P. Bugnon; V. Mitropoulos; D. A. Johnson; J. A. Johnson; D. Hohl; S. Werner
Nrf transcription factors in keratinocytes are essential for skin tumor prevention but not for wound healing.
The Nrf2 transcription factor is a key player in the cellular stress response through its regulation of cytoprotective genes. In this study we determined the role of Nrf2-mediated gene expression in keratinocytes for skin development, wound repair, and skin carcinogenesis. To overcome compensation by the related Nrf1 and Nrf3 proteins, we expressed a dominant-negative Nrf2 mutant (dnNrf2) in the epidermis of transgenic mice. The functionality of the transgene product was verified in vivo using mice doubly transgenic for dnNrf2 and an Nrf2-responsive reporter gene. Surprisingly, no abnormalities of the epidermis were observed in dnNrf2-transgenic mice, and even full-thickness skin wounds healed normally. However, the onset, incidence, and multiplicity of chemically induced skin papillomas were strikingly enhanced, whereas the progression to squamous cell carcinomas was unaltered. We provide evidence that the enhanced tumorigenesis results from reduced basal expression of cytoprotective Nrf target genes, leading to accumulation of oxidative damage and reduced carcinogen detoxification. Our results reveal a crucial role of Nrf-mediated gene expression in keratinocytes in the prevention of skin tumors and suggest that activation of Nrf2 in keratinocytes is a promising strategy to prevent carcinogenesis of this highly exposed organ. PMID:16648473
auf dem Keller, Ulrich; Huber, Marcel; Beyer, Tobias A; K�min, Angelika; Siemes, Christina; Braun, Susanne; Bugnon, Philippe; Mitropoulos, Varvara; Johnson, Delinda A; Johnson, Jeffrey A; Hohl, Daniel; Werner, Sabine
The absence of p21Cip1/WAF1 alters keratinocyte growth and differentiation and promotes ras-tumor progression.
p21Cip1/WAF1 was the first cyclin-dependent kinase (CDK) inhibitor to be identified, as a mediator of p53 in DNA damage-induced growth arrest, cell senescence, and direct CDK regulation. p21 may also play an important role in differentiation-associated growth arrest, as its expression is augmented in many terminally differentiating cells. A general involvement of p21 in growth/differentiation control and tumor suppression has been questioned, as mice lacking p21 undergo a normal development, harbor no gross alterations in any of their organs, and exhibit no increase in spontaneous tumor development. However, a significant imbalance between growth and differentiation could be unmasked under conditions where normal homeostatic mechanisms are impaired. We report here that primary keratinocytes derived from p21 knockout mice, transformed with a ras oncogene, and injected subcutaneously into nude mice exhibit a very aggressive tumorigenic behavior, which is not observed with wild-type control keratinocytes nor with keratinocytes with a disruption of the closely related p27 gene. p21 knockout keratinocytes tested under well-defined in vitro conditions show a significantly increased proliferative potential, which is also observed but to a lesser extent with p27 knockout cells. More profound differences were found in the differentiation behavior of p21 versus p27 knockout keratinocytes, with p21 (but not p27) deficiency causing a drastic down-modulation of differentiation markers linked with the late stages of the keratinocyte terminal differentiation program. Thus, our results reveal a so far undetected role of p21 in tumor suppression, demonstrate that this function is specific as it cannot be attributed to the closely related p27 molecule, and point to an essential involvement of p21 in terminal differentiation control, which may account for its role in tumor suppression. PMID:8957006
Missero, C; Di Cunto, F; Kiyokawa, H; Koff, A; Dotto, G P
UV-B Radiation Induces Macrophage Migration Inhibitory Factor-Mediated Melanogenesis through Activation of Protease-Activated Receptor-2 and Stem Cell Factor in Keratinocytes
UV radiation indirectly regulates melanogenesis in melanocytes through a paracrine regulatory mechanism involving keratinocytes. Protease-activated receptor (PAR)-2 activation induces melanosome transfer by increasing phagocytosis of melanosomes by keratinocytes. This study demonstrated that macrophage migration inhibitory factor (MIF) stimulated PAR-2 expression in human keratinocytes. In addition, we showed that MIF stimulated stem cell factor (SCF) release in keratinocytes; however, MIF had no effect on the release of endothelin-1 or prostaglandin E2 in keratinocytes. In addition, MIF had no direct effect on melanin and tyrosinase synthesis in cultured human melanocytes. The effect of MIF on melanogenesis was also examined using a three-dimensional reconstituted human epidermal culture model, which is a novel, commercially available, cultured human epidermis containing functional melanocytes. Migration inhibitory factor induced an increase in melanin content in the epidermis after a 9-day culture period. Moreover, melanin synthesis induced by UV-B stimulation was significantly down-regulated by anti-MIF antibody treatment. An in vivo study showed that the back skin of MIF transgenic mice had a higher melanin content than that of wild-type mice after 12 weeks of UV-B exposure. Therefore, MIF-mediated melanogenesis occurs mainly through the activation of PAR-2 and SCF expression in keratinocytes after exposure to UV-B radiation.
Enomoto, Akiko; Yoshihisa, Yoko; Yamakoshi, Takako; Ur Rehman, Mati; Norisugi, Osamu; Hara, Hiroshi; Matsunaga, Kenji; Makino, Teruhiko; Nishihira, Jun; Shimizu, Tadamichi
Bovine coronary region keratinocyte colony formation is supported by epidermal-dermal interactions.
Delineating the factors that orchestrate keratinocyte growth and differentiation in the claw is pivotal to understanding the quality of hoof horn production in health and disease. The specific objectives of this investigation were to establish an in vitro culture system for bovine coronary region keratinocytes and dermal fibroblasts, determine the colony-forming capacity of epidermal keratinocytes in the coronary region, and characterize transcriptional changes in specific cytokine, growth factor, and receptor genes during colony formation in coculture. Fibroblasts and keratinocytes from the coronary region of the lateral, hind limb claw were collected, and 5.0 x 10(3) and 7.5 x 10(3) keratinocytes were cultured in the presence or absence of fibroblast monolayers, respectively. The 2 densities of keratinocytes formed 144 +/- 15.8 and 183 +/- 26.9 colonies, respectively, in the presence of dermal fibroblasts, whereas no colonies developed in the absence of dermal fibroblasts. Keratinocytes with the ability to show colony formation comprised 1.09% +/- 0.16 to 1.77% +/- 0.28 of the keratinocyte population isolated from the coronary region. Keratinocyte-fibroblast cocultures developed a time-dependent increased expression of several growth factors, cytokines, and receptors. These findings demonstrated that keratinocytes from the bovine coronary region formed colonies in vitro and that colony formation occurred with an absolute dependence on dermal fibroblasts. Colony growth was associated with increased transcriptional expression of cytokine, growth factor, and receptor expression known to drive keratinocyte colony formation in other species. The results indicate that horn-producing keratinocytes must interact with dermal fibroblasts during normal tissue homeostasis in the bovine claw. PMID:19389949
Mills, J A; Zarlenga, D S; Dyer, R M
Re-epithelialization of human oral keratinocytes in vitro.
Re-epithelialization involves interactions between keratinocytes and the extracellular matrix upon which these cells move. It is hypothesized that keratinocytes are activated when wounded, and the resultant phenotypic change directs re-epithelialization. We have adapted organotypic cultures, in which oral gingival keratinocytes are fully differentiated, to study re-epithelialization following wounding. To elucidate keratinocyte behavior and phenotype during re-epithelialization, we have investigated this process in the presence and absence of the growth factor TGF-beta 1 and have monitored expression of MMP-1 (Type I collagenase) mRNA by in situ hybridization. In addition, we have followed proliferation and migration of wound keratinocytes by genetically marking these cells with a retroviral vector and by measuring their proliferative index. We found that keratinocytes grown without TGF-beta 1 were hyperproliferative in response to wounding, and re-epithelialization was complete by 24 h. However, 2.5 ng/mL TGF-beta 1 induced a transient delay in re-epithelialization, a reduction in proliferation, and fewer clusters of genetically marked cells. Keratinocytes expressed MMP-1 mRNA only when they covered the wounded surface, suggesting that the cells acquire a collagenolytic phenotype during re-epithelialization and that contact with different ECM components may modulate keratinocyte expression of MMP-1. We conclude that the phenotype of oral keratinocytes is altered during re-epithelialization in vitro and that this process is modulated by TGF-beta 1. Re-epithelialization occurs as keratinocytes are activated to move over the wound bed. Understanding the phenotype of wounded keratinocytes may facilitate treatment of chronic oral wounds and periodontal disease. PMID:8675802
Garlick, J A; Parks, W C; Welgus, H G; Taichman, L B
The present invention provides a fibroblast growth factor heparin-binding analog of the formula: ##STR00001## where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, X, Y and Z are as defined, pharmaceutical compositions, coating compositions and medical devices including the fibroblast growth factor heparin-binding analog of the foregoing formula, and methods and uses thereof.
Zamora, Paul O. (Gaithersburg, MD); Pena, Louis A. (Poquott, NY); Lin, Xinhua (Plainview, NY); Takahashi, Kazuyuki (Germantown, MD)
Tanshinone IIA Inhibits Growth of Keratinocytes through Cell Cycle Arrest and Apoptosis: Underlying Treatment Mechanism of Psoriasis
The aim of the present investigation was to elucidate the cellular mechanisms whereby Tanshinone IIA (Tan IIA) leads to cell cycle arrest and apoptosis in vitro in keratinocytes, the target cells in psoriasis. Tan IIA inhibited proliferation of mouse keratinocytes in a dose- and time-dependent manner and induced apoptosis, resulting in S phase arrest accompanied by down-regulation of pCdk2 and cyclin A protein expression. Furthermore, Tan IIA-induced apoptosis and mitochondrial membrane potential changes were also further demonstrated by DNA fragmentation, single-cell gel electrophoresis assay (SCGE), and flow cytometry methods. Apoptosis was partially blocked by the caspase-3 inhibitor Ac-DEVD-CHO. Mitochondrial regulation of apoptosis further downstream was investigated, showing changes in the mitochondrial membrane potential, cytochrome c release into the cytoplasm, and enhanced activation of cleaved caspase-3 and Poly ADP-ribose polymerase (PARP). There was also no translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus in apoptotic keratinocytes, indicating Tan IIA-induced apoptosis occurs mainly through the caspase pathway. Our findings provide the molecular mechanisms by which Tan IIA can be used to treat psoriasis and support the traditional use of Salvia miltiorrhiza Bungee (Labiatae) for psoriasis and related skin diseases.
Li, Fu-Lun; Xu, Rong; Zeng, Qing-chun; Li, Xin; Chen, Jie; Wang, Yi-Fei; Fan, Bin; Geng, Lin; Li, Bin
Growth factors for nanobacteria
NASA Astrophysics Data System (ADS)
Nanobacteria are novel microorganisms recently isolated from fetal bovine serum and blood of cows and humans. These coccoid, gram negative bacteria in alpha-2 subgroup of Proteobacteria grow slowly under mammalian cell culture conditions but not in common media for microbes. Now we have found two different kinds of culture supplement preparations that improve their growth and make them culturable in the classical sense. These are supernatant fractions of conditioned media obtained from 1 - 3 months old nanobacteria cultures and from about a 2 weeks old Bacillus species culture. Both improved multiplication and particle yields and the latter increased their resistance to gentamicin. Nanobacteria cultured with any of the methods shared similar immunological property, structure and protein pattern. The growth supporting factors were heat-stabile and nondialyzable, and dialysis improved the growth promoting action. Nanobacteria formed stony colonies in a bacteriological medium supplemented with the growth factors. This is an implication that nanobacterial growth is influenced by pre-existing bacterial flora.
Ciftcioglu, Neva; Kajander, E. Olavi
Epithelial outgrowths (keratinocytes) from normal human skin in vitro were exposed daily for 30 min to vitamin A alcohol for periods up to 5 weeks. There was a markedly decreased number of keratohyaline granules in treated cultures, indicating an effect on the differentiation process, but there was no evidence for mucous metaplasia. The area of vitamin A-treated outgrowths was greater
Dharam P. Chopra; B. Allen Flaxman
Tumor necrosis factor receptor signaling in keratinocytes triggers interleukin-24-dependent psoriasis-like skin inflammation in mice.
Psoriasis is a common chronic inflammatory skin disease with a prevalence of about 2% in the Caucasian population. Tumor necrosis factor (TNF) plays an essential role in the pathogenesis of psoriasis, but its mechanism of action remains poorly understood. Here we report that the development of psoriasis-like skin inflammation in mice with epidermis-specific inhibition of the transcription factor NF-?B was triggered by TNF receptor 1 (TNFR1)-dependent upregulation of interleukin-24 (IL-24) and activation of signal transducer and activator of transcription�3�(STAT3) signaling in keratinocytes. IL-24 was strongly expressed in human psoriatic epidermis, and pharmacological inhibition of NF-?B increased IL-24 expression in TNF-stimulated human primary keratinocytes, suggesting that this mechanism is relevant for human psoriasis. Therefore, our results expand current views on psoriasis pathogenesis by revealing a new keratinocyte-intrinsic mechanism that links TNFR1, NF-?B, ERK, IL-24, IL-22R1, and STAT3 signaling to disease initiation. PMID:24211183
Kumari, Snehlata; Bonnet, Marion C; Ulvmar, Maria H; Wolk, Kerstin; Karagianni, Niki; Witte, Ellen; Uthoff-Hachenberg, Claudia; Renauld, Jean-Christophe; Kollias, George; Toftgard, Rune; Sabat, Robert; Pasparakis, Manolis; Haase, Ingo
Stathmin Regulates Keratinocyte Proliferation and Migration during Cutaneous Regeneration
Cutaneous regeneration utilizes paracrine feedback mechanisms to fine-tune the regulation of epidermal keratinocyte proliferation and migration. However, it is unknown how fibroblast-derived hepatocyte growth factor (HGF) affects these mutually exclusive processes in distinct cell populations. We here show that HGF stimulates the expression and phosphorylation of the microtubule-destabilizing factor stathmin in primary human keratinocytes. Quantitative single cell- and cell population-based analyses revealed that basal stathmin levels are important for the migratory ability of keratinocytes in vitro; however, its expression is moderately induced in the migration tongue of mouse skin or organotypic multi-layered keratinocyte 3D cultures after full-thickness wounding. In contrast, clearly elevated stathmin expression is detectable in hyperproliferative epidermal areas. In vitro, stathmin silencing significantly reduced keratinocyte proliferation. Automated quantitative and time-resolved analyses in organotypic cocultures demonstrated a high correlation between Stathmin/phospho-Stathmin and Ki67 positivity in epidermal regions with proliferative activity. Thus, activation of stathmin may stimulate keratinocyte proliferation, while basal stathmin levels are sufficient for keratinocyte migration during cutaneous regeneration.
Schmitt, Sabrina; Safferling, Kai; Westphal, Kathi; Hrabowski, Manuel; Muller, Ute; Angel, Peter; Wiechert, Lars; Ehemann, Volker; Muller, Benedikt; Holland-Cunz, Stefan; Stichel, Damian; Harder, Nathalie; Rohr, Karl; Germann, Gunter; Matthaus, Franziska; Schirmacher, Peter; Grabe, Niels; Breuhahn, Kai
Epidermal growth factor receptor distribution in burn wounds. Implications for growth factor-mediated repair.
Epidermal growth factor (EGF) along with several related peptide growth factors has been shown both in vivo and in vitro to accelerate events associated with epidermal wound repair. EGF and transforming growth factor alpha act by binding to a common EGF receptor tyrosine kinase thereby initiating a series of events which ultimately regulate cell proliferation. This study examined the immunohistochemical localization of EGF receptor (EGF-R) in burn wound margins, adjacent proliferating epithelium, and closely associated sweat ducts, sebaceous glands, and hair follicles. Tissue specimens removed during surgical debridement were obtained from full and partial thickness burn wounds in 32 patients with total body surface area burns ranging from 2 to 88%. In the early postburn period (days 2-4), prominent staining for EGF-R was found in undifferentiated, marginal keratinocytes, adjacent proliferating, hypertrophic epithelium, and both marginal and nonmarginal hair follicles, sweat ducts, and sebaceous glands. During the late postburn period (days 5-16), EGF-R was depleted along leading epithelial margins; however, immunoreactive EGF-R remained intensely positive in the hypertrophic epithelium and all skin appendages. Increased detection of immunoreactive EGF-R and the presence of [125I]EGF binding in the hypertrophic epithelium correlated positively with proliferating cell nuclear antigen distributions. Thus, the presence of EGF-R in the appropriate keratinocyte populations suggests a functional role for this receptor during wound repair. Dynamic modulation in EGF receptor distribution during the temporal sequence of repair provides further evidence that an EGF/transforming growth factor alpha/EGF-R-mediated pathway is activated during human wound repair. Images
Wenczak, B A; Lynch, J B; Nanney, L B
Protein kinase C-dependent upregulation of miR-203 induces the differentiation of human keratinocytes.
Terminal differentiation of keratinocytes is a multistep process that requires a coordinated program of gene expression. We aimed to explore the possible involvement of a previously unreported class of non-coding RNA genes, microRNAs (miRNAs) in keratinocyte differentiation by using miRNA expression profiling. Out of 365 miRNAs tested, 7 showed significant change between keratinocytes cultured in low or high calcium concentration. The highest-ranked upregulated gene was miR-203, whose expression was significantly upregulated in response to calcium and other inducers of keratinocyte differentiation such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and vitamin D(3). Differentiation-induced upregulation of miR-203 expression was blocked by treatment with specific inhibitors of protein kinase C (PKC), GF109203X, and Ro31-8220. Moreover, our results showed that the activator protein-1 (AP-1) proteins c-Jun and JunB regulate miR-203 expression in keratinocytes. In contrast to inducers of keratinocyte differentiation, epidermal growth factor and keratinocyte growth factor suppressed miR-203 expression in keratinocytes below the basal level. Overexpression of miR-203 in keratinocytes resulted in enhanced differentiation, whereas inhibition of miR-203 suppressed calcium-induced terminal differentiation as judged by involucrin expression. These results suggest that upregulation of miR-203 in human keratinocytes is required for their differentiation and is dependent on the activation of the PKC/AP-1 pathway. PMID:19759552
Sonkoly, Enik�; Wei, Tianling; Pavez Lori�, Elizabeth; Suzuki, Hiroyuki; Kato, Mitsuyasu; T�rm�, Hans; St�hle, Mona; Pivarcsi, Andor
Fibroblast growth factor-2 (FGF-2) is a member of a large family of proteins that bind heparin and heparan sulfate and modulate the function of a wide range of cell types. FGF-2 stimulates the growth and development of new blood vessels (angiogenesis) that contribute to the pathogenesis of several diseases (i.e. cancer, atherosclerosis), normal wound healing and tissue development. FGF-2 contains
Matthew A Nugent; Renato V Iozzo
Role of Growth Factors, Cytokines, and Their Receptors in the Pathogenesis of Psoriasis
Psoriasis is characterized by epidermal hyperplasia, altered epidermal maturation, and local accumulation of acute and chronic inflammatory cells. Keratinocyte hyperplasia in psoriasis may be explained in part by overproduction of growth factors or cytokines which stimulate epidermal proliferation and by altered metabolism of growth-factor receptors in affected skin. Psoriatic epidermis displays overproduction of TGF-alpha and interleukin-6 (IL-6), factors produced by
James G. Krueger; Jeffrey F. Krane; D. Martin Carter; Alice B. Gottlieb
Effects of nerve growth factor (NGF) and other fibroblast-derived growth factors on immature human mast cells (HMC-1).
We have previously shown that fibroblast and keratinocyte supernatants up-regulate expression of mast cell characteristics in the human immature mast cell line HMC-1. This effect could not be induced in HMC-1 cells by the well-known mast cell growth factor stem cell factor (SCF), probably due to mutations of the SCF receptor c-Kit in these cells. Here we report the effects of several known fibroblast- and keratinocyte-derived growth factors, namely nerve growth factor (NGF), basic fibroblast growth factor, platelet-derived growth factor and transforming growth factor-beta, on mast cell differentiation, using HMC-1 cells as a model. NGF, at 0.1-50 ng/ml concentrations, caused a marked, dose-dependent up-regulation of tryptase, Fc epsilon RI and histamine within 10 days of culture, associated with an enhanced expression of mRNA for Fc epsilon RI and mast cell tryptase. On restriction analysis, only mast cell beta-tryptase, but not alpha-tryptase, could be demonstrated. Furthermore, the high-affinity NGF receptor (TrkA) was found at both the transcriptional and protein levels, while expression of the low-affinity NGF receptor was detectable at the mRNA level only. None of the other growth factors caused a significant alteration of the mast cell markers studied when added to HMC-1 cells at concentrations known to be biologically active in other culture systems. Immature human mast cells are thus induced to assume a more mature phenotype in vitro in response to NGF, most probably via stimulation of the high-affinity NGF receptor expressed on these cells. Besides SCF, NGF should therefore be considered as an additional mast cell growth factor that contributes to human mast cell maturation at tissue sites. Images Figure 2 Figure 3 Figure 4 Figure 6
Welker, P; Grabbe, J; Grutzkau, A; Henz, B M
Hematopoiesis is a complex process that underlines the production of multiple highly specialized cells. The intricate mechanisms involved in this process include both positive and negative feedback by humoral activities, pluripotent stem cell selfrenewal and differentiation, and local interactions between stromal components of the hematopoietic microenvironment and various stem and progenitor cells. A group of hematopoietic growth factors, as well as their genes and chromosomal locations, have been identified. Advances in biochemistry and molecular biology led to the purification, genetic sequencing and molecular cloning of these glycoproteins. They include interleukin-3 (IL-3), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and erythropoietin (EPO). The biologic specificity of these substances is defined by their ability to support proliferation and differentiation of hematopoietic cells in a semisolid clonal assay system. These factors share certain characteristics, including their ability to stimulate the function of mature cells, their overlapping activity affecting progenitor cells of several lineages, and their direct and indirect actions on nonhematopoietic cells. Trials using hematopoietic growth factors demonstrated their remarkable efficacy in a variety of clinical settings. PMID:1708221
Tabbara, I A; Robinson, B E
The oncogenic GLI transcription factors facilitate keratinocyte survival and transformation upon exposure to genotoxic agents.
Ultraviolet B (UVB) light is the principal aetiological factor associated with non-melanoma skin cancer, the most prevalent group of malignancies in the Caucasian population. Exposure to environmental chemicals has also been shown to promote skin carcinogenesis and, as for UVB, this is associated with the acquisition of genomic DNA damage. Cells respond to DNA damage by inducing cell cycle arrest to facilitate DNA repair, although apoptosis will occur if the damage is excessive. Oncogenes may drive carcinogenesis by disrupting the balanced control of cell cycle progression, DNA repair and apoptosis, allowing for the propagation of cells with damaged DNA. The transcription factors GLI1 and GLI2 have been implicated in both the initiation and progression of several cancers, including basal cell carcinoma. Here we show that GLI1 and an active mutant of GLI2 (?NGLI2) promote apoptotic resistance in N/TERT human keratinocytes upon exposure to UVB and the DNA-alkylating chemicals such as methyl methanesulphonate (MMS) and N-ethyl-N-nitrosurea. Compared with control and untreated N/TERT-GLI1 and -GLI2 cells, those that survived genotoxic insult formed significantly more colonies in soft agar and were significantly more invasive when grown in three-dimensional organotypic collagen gel cultures. Indeed, surviving N/TERT-GLI1 and -GLI2 cells expressed higher levels of the epithelial-to-mesenchymal transition markers Snail and vimentin, and a subpopulation of MMS-treated cells displayed an elongated fibroblast-like morphology with decreased levels of E-cadherin. Finally, whereas Bcl2 was strongly increased in N/TERT-GLI2 cells, the level of induction was weak in N/TERT-GLI1 cells, indicating that GLI1 may activate anti-apoptotic mechanisms(s) independently of Bcl2. In summary, our results show that GLI1 and GLI2 facilitate the propagation of cells with damaged DNA, and thus their expression may be naturally higher in cells that form the earliest precursor tumour lesions. PMID:23792444
Harrison, W; Cochrane, B; Neill, G; Philpott, M
Effect of Wnt3a on Keratinocytes Utilizing in Vitro and Bioinformatics Analysis
Wingless-type (Wnt) signaling proteins participate in various cell developmental processes. A suppressive role of Wnt5a on keratinocyte growth has already been observed. However, the role of other Wnt proteins in proliferation and differentiation of keratinocytes remains unknown. Here, we investigated the effects of the Wnt ligand, Wnt3a, on proliferation and differentiation of keratinocytes. Keratinocytes from normal human skin were cultured and treated with recombinant Wnt3a alone or in combination with the inflammatory cytokine, tumor necrosis factor ? (TNF?). Furthermore, using bioinformatics, we analyzed the biochemical parameters, molecular evolution, and protein�protein interaction network for the Wnt family. Application of recombinant Wnt3a showed an anti-proliferative effect on keratinocytes in a dose-dependent manner. After treatment with TNF?, Wnt3a still demonstrated an anti-proliferative effect on human keratinocytes. Exogenous treatment of Wnt3a was unable to alter mRNA expression of differentiation markers of keratinocytes, whereas an altered expression was observed in TNF?-stimulated keratinocytes. In silico phylogenetic, biochemical, and protein�protein interaction analysis showed several close relationships among the family members of the Wnt family. Moreover, a close phylogenetic and biochemical similarity was observed between Wnt3a and Wnt5a. Finally, we proposed a hypothetical mechanism to illustrate how the Wnt3a protein may inhibit the process of proliferation in keratinocytes, which would be useful for future researchers.
Nam, Ju-Suk; Chakraborty, Chiranjib; Sharma, Ashish Ranjan; Her, Young; Bae, Kee-Jeong; Sharma, Garima; Doss, George Priya; Lee, Sang-Soo; Hong, Myung-Sun; Song, Dong-Keun
Dysfunctional ?? T cells contribute to impaired keratinocyte homeostasis in mouse models of obesity
Skin complications and chronic non-healing wounds are common in obesity, metabolic disease and type 2 diabetes. Epidermal ?? T cells normally produce keratinocyte growth factors, participate in wound repair and are necessary for keratinocyte homeostasis. We have determined that in ?? T cell-deficient mice, there are reduced numbers of keratinocytes and the epidermis exhibits a flattened, thinner structure with fewer basal keratinocytes. This is important in obesity, where skin-resident ?? T cells are reduced and rendered dysfunctional. Similar to ?? T cell-deficient mice, keratinocytes are reduced and the epidermal structure is altered in two obese mouse models. Even in regions where ?? T cells are present, there are fewer keratinocytes in obese mice indicating that dysfunctional ?? T cells are unable to regulate keratinocyte homeostasis. The impact of absent or impaired ?? T cells on epidermal structure is exacerbated in obesity as E-cadherin localization and expression is additionally altered. These studies reveal that ?? T cells are unable to regulate keratinocyte homeostasis in obesity and that the obese environment further impairs skin structure by altering cell-cell adhesion. Together, impaired keratinocyte homeostasis and epidermal barrier function through direct and indirect mechanisms results in susceptibility to skin complications, chronic wounds and infection.
Taylor, Kristen R.; Costanzo, Anne E.; Jameson, Julie M.
Growth and differentiation of normal human melanocytes in a TPA-free, cholera toxin-free, low-serum medium and influence of keratinocytes
Melanocyte cultures were obtained from a modification of the keratinocyte culture system MCDB153. Either promelanocytes or mature melanocytes were selected from epidermal cell primary cultures. Pure subcultures of actively dividing melanocytes of both types were grown in a low-serum medium totally deprived of TPA and cholera toxin called melanocyte growth medium (MGM). Early passaged cells from MGM primary cocultures were
P. Donatien; J. E. Surl�ve-Bazeille; A. J. Thody; A. Ta�eb
Protein kinase C ? increases Kruppel-like factor 4 protein, which drives involucrin gene transcription in differentiating keratinocytes.
KLF4 is a member of the Kruppel-like factor family of transcriptional regulators. KLF4 has been shown to be required for normal terminal differentiation of keratinocytes, but the molecular mechanism whereby KLF4 regulates genes associated with the differentiation process has not been studied. In the present study, we explore the impact of KLF4 on expression of involucrin, a gene that is specifically expressed in differentiated keratinocytes. KLF4 overexpression and knockdown studies show that involucrin mRNA and protein level correlates directly with KLF4 level. Moreover, studies of mutant KLF4 proteins indicate that transcriptionally inactive forms do not increase involucrin expression. PKC? is a regulator of keratinocyte differentiation that increases expression of differentiation-associated target genes, including involucrin. Overexpression of KLF4 augments the PKC?-dependent increase in involucrin expression, whereas KLF4 knockdown attenuates this response. The KLF4 induction of human involucrin (hINV) promoter activity is mediated via KLF4 binding to a GC-rich element located in the hINV promoter distal regulatory region, a region of the promoter required for in vivo involucrin expression. Mutation of the GC-rich element, an adjacent AP1 factor binding site, or both sites severely attenuates the response. Moreover, loss of KLF4 in an epidermal equivalent model of differentiation results in loss of hINV expression. These studies suggest that KLF4 is part of a multiprotein complex that interacts that the hINV promoter distal regulatory region to drive differentiation-dependent hINV gene expression in epidermis. PMID:23599428
Chew, Yap Ching; Adhikary, Gautam; Xu, Wen; Wilson, Gerald M; Eckert, Richard L
Reactive Oxygen Species in Tumor Necrosis Factor-?-Activated Primary Human Keratinocytes: Implications for Psoriasis and Inflammatory Skin Disease
The multifunctional cytokine tumor necrosis factor-? (TNF-?) is known to play an important role in inflammatory and immunological responses in human skin. Although it has been documented that reactive oxygen species (ROS) are involved in TNF-?-induced signaling pathways associated with certain inflammatory diseases, their role in TNF-? signaling cascades has not been examined in primary human keratinocytes used as a model of inflammatory skin disease and psoriasis. Employing a series of in vitro and in cellulo approaches, we have demonstrated that in primary human keratinocytes (i) TNF-? rapidly induces ROS generation, I?B degradation, NF-?B p65 nuclear translocation, and ultimately production of inflammatory cytokines; (ii) TNF-?-induced cytokine production is mediated both by the mammalian target of rapamycin signaling pathway via NF-?B activation and by ROS; (iii) TNF-?-dependent NF-?B activation (that is, I?B degradation and NF-?B p65 nuclear translocation) is not mediated by ROS; and (iv) a cell-penetrating derivative of the antioxidant enzyme, catalase, as well as taurine and N-acetyl-cysteine attenuate the TNF-?-induced production of cytokines. These latter results suggest that catalase and perhaps other antioxidants should be considered as part of a more specific and effective therapy for the treatment of inflammatory skin diseases, including psoriasis.
Young, Chen N.; Koepke, Jay I.; Terlecky, Laura J.; Borkin, Michael S.; Boyd, Savoy L.; Terlecky, Stanley R.
A Central Role for Transcription Factor C/EBP-? in Regulating CD1d Gene Expression in Human Keratinocytes1
CD1d is a nonclassical Ag-presenting molecule that presents glycolipid Ags to NKT cells that are involved in immune defense and tumor rejection. It also plays a role in immunoregulatory functions in the epidermis. The mechanisms controlling the expression of CD1d are not well understood. Therefore, we cloned the CD1d gene promoter and characterized its activities in primary human keratinocytes and other cell lines of epithelial origin. We found that a CCAAT box in the CD1d promoter is required for its expression in keratinocytes. We show here that transcription factor C/EBP-? binds to the CCAAT box in the CD1d promoter in vitro and in vivo. Consistent with these observations, deletion of the gene encoding for C/EBP-? caused a loss of CD1d expression. The in vivo regulation of CD1d has significant implications for the pathologic mechanisms of certain immunologic skin diseases in which NKT cells play a role, such as allergic contact dermatitis and psoriasis. Together, these data show a central role for C/EBP-? in regulating CD1d transcription.
Sikder, Hashmat; Zhao, Yuming; Balato, Anna; Chapoval, Andre; Fishelevich, Rita; Gade, Padmaja; Singh, Ishwar S.; Kalvakolanu, Dhananjaya V.; Johnson, Peter F.; Gaspari, Anthony A.
Plant polyphenols differentially modulate inflammatory responses of human keratinocytes by interfering with activation of transcription factors NF{kappa}B and AhR and EGFR-ERK pathway
Molecular mechanisms underlying modulation of inflammatory responses in primary human keratinocytes by plant polyphenols (PPs), namely the glycosylated phenylpropanoid verbascoside, the stilbenoid resveratrol and its glycoside polydatin, and the flavonoid quercetin and its glycoside rutin were evaluated. As non-lethal stimuli, the prototypic ligand for epidermal growth factor receptor (EGFR) transforming growth factor alpha (TGFalpha), the combination of tumor necrosis factor (TNFalpha) and interferon (IFNgamma) (T/I), UVA + UVB irradiation, and bacterial lipopolysaccharide (LPS) were used. We demonstrated differential modulation of inflammatory responses in keratinocytes at signal transduction, gene transcription, and protein synthesis levels as a function of PP chemical structure, the pro-inflammatory trigger used, and PP interaction with intracellular detoxifying systems. The PPs remarkably inhibited constitutive, LPS- and T/I-induced but not TGFalpha-induced ERK phosphorylation. They also suppressed NFkappaB activation by LPS and T/I. Verbascoside and quercetin invariably impaired EGFR phosphorylation and UV-associated aryl hydrocarbon receptor (AhR)-mediated signaling, while rutin, polydatin and resveratrol did not affect EGFR phosphorylation and further activated AhR machinery in UV-exposed keratinocytes. In general, PPs down-regulated gene expression of pro-inflammatory cytokines/enzymes, except significant up-regulation of IL-8 observed under stimulation with TGFalpha. Both spontaneous and T/I-induced release of IL-8 and IP-10 was suppressed, although 50 {mu}M resveratrol and polydatin up-regulated IL-8. At this concentration, resveratrol activated both gene expression and de novo synthesis of IL-8 and AhR-mediated mechanisms were involved. We conclude that PPs differentially modulate the inflammatory response of human keratinocytes through distinct signal transduction pathways, including AhR and EGFR. - Graphical abstract: Display Omitted Highlights: > Effects of plant polyphenols on inflammatory responses in human keratinocytes. > Inflammatory stimuli used: TGFalpha, TNFalpha+IFNgamma, UVA+UVB, and LPS. > Inflammatory pathways connected with NFB, ERK1/2, EGFR, and AhR were investigated. > Plant polyphenols, flavonoids, stilbenoids, and phenylpropanoids, were studied. > Modulation of inflammation depends on phenolic core structure and glycosylation.
Potapovich, Alla I. [Tissue Engineering and Skin Pathophysiology Laboratory, Dermatology Research Institute (IDI IRCCS), Via Monti di Creta 104, Rome 00167 (Italy); Biology Department, Belarus State University, Skorina Prosp. 10, Minsk 220050 (Belarus); Lulli, Daniela; Fidanza, Paolo [Tissue Engineering and Skin Pathophysiology Laboratory, Dermatology Research Institute (IDI IRCCS), Via Monti di Creta 104, Rome 00167 (Italy); Kostyuk, Vladimir A. [Tissue Engineering and Skin Pathophysiology Laboratory, Dermatology Research Institute (IDI IRCCS), Via Monti di Creta 104, Rome 00167 (Italy); Biology Department, Belarus State University, Skorina Prosp. 10, Minsk 220050 (Belarus); De Luca, Chiara; Pastore, Saveria [Tissue Engineering and Skin Pathophysiology Laboratory, Dermatology Research Institute (IDI IRCCS), Via Monti di Creta 104, Rome 00167 (Italy); Korkina, Liudmila G., E-mail: email@example.com [Tissue Engineering and Skin Pathophysiology Laboratory, Dermatology Research Institute (IDI IRCCS), Via Monti di Creta 104, Rome 00167 (Italy)
Functional Characterization of Cultured Keratinocytes after Acute Cutaneous Burn Injury
Background In addition to forming the epithelial barrier against the outside environment keratinocytes are immunologically active cells. In the treatment of severely burned skin, cryoconserved keratinocyte allografts gain in importance. It has been proposed that these allografts accelerate wound healing also due to the expression of a favourable - keratinocyte-derived - cytokine and growth factor milieu. Methods In this study the morphology and cytokine expression profile of keratinocytes from skin after acute burn injury was compared to non-burned skin. Skin samples were obtained from patients after severe burn injury and healthy controls. Cells were cultured and secretion of selected inflammatory mediators was quantified using Bioplex Immunoassays. Immunohistochemistry was performed to analyse further functional and morphologic parameters. Results Histology revealed increased terminal differentiation of keratinocytes (CK10, CK11) in allografts from non-burned skin compared to a higher portion of proliferative cells (CK5, vimentin) in acute burn injury. Increased levels of IL-1?, IL-2, IL-4, IL-10, IFN-? and TNF? could be detected in culture media of burn injury skin cultures. Both culture groups contained large amounts of IL-1RA. IL-6 and GM-CSF were increased during the first 15 days of culture of burned skin compared to control skin. Levels of VEGF, FGF-basic, TGF-� und G-CSF were high in both but not significantly different. Cryoconservation led to a diminished mediator synthesis except for higher levels of intracellular IL-1? and IL-1�. Conclusion Skin allografts from non-burned skin show a different secretion pattern of keratinocyte-derived cytokines and inflammatory mediators compared to keratinocytes after burn injury. As these secreted molecules exert auto- and paracrine effects and subsequently contribute to healing and barrier restoration after acute burn injury therapies affecting this specific cytokine/growth factor micromilieu could be beneficial in burned patients.
Gauglitz, Gerd G.; Zedler, Siegfried; v. Spiegel, Felix; Fuhr, Jasmin; v. Donnersmarck, Guido Henkel; Faist, Eugen
Asymmetric Migration of Human Keratinocytes under Mechanical Stretch and Cocultured Fibroblasts in a Wound Repair Model
Keratinocyte migration during re-epithelization is crucial in wound healing under biochemical and biomechanical microenvironment. However, little is known about the underlying mechanisms whereby mechanical tension and cocultured fibroblasts or keratinocytes modulate the migration of keratinocytes or fibroblasts. Here we applied a tensile device together with a modified transwell assay to determine the lateral and transmembrane migration dynamics of human HaCaT keratinocytes or HF fibroblasts. A novel pattern of asymmetric migration was observed for keratinocytes when they were cocultured with non-contact fibroblasts, i.e., the accumulative distance of HaCaT cells was significantly higher when moving away from HF cells or migrating from down to up cross the membrane than that when moving close to HF cells or when migrating from up to down, whereas HF migration was symmetric. This asymmetric migration was mainly regulated by EGF derived from fibroblasts, but not transforming growth factor ? or ?1 production. Mechanical stretch subjected to fibroblasts fostered keratinocyte asymmetric migration by increasing EGF secretion, while no role of mechanical stretch was found for EGF secretion by keratinocytes. These results provided a new insight into understanding the regulating mechanisms of two- or three-dimensional migration of keratinocytes or fibroblasts along or across dermis and epidermis under biomechanical microenvironment.
Lu, Dongyuan; Liu, Xiaofeng; Gao, Yuxin; Huo, Bo; Kang, Yingyong; Chen, Juan; Sun, Shujin; Chen, Li; Luo, Xiangdong; Long, Mian
Growth hormone, growth factors, and acromegaly
This book contains five sections, each consisting of several papers. The section headings are: Biochemistry and Physiology of GH and Growth Factors, Pathology of Acromegaly, Clinical Endocrinology of Acromegaly, Nonsurgical Therapy of Acromegaly, and Surgical Therapy of Acromegaly.
Ludecke, D.K.; Tolis, G.T.
Arsenic Enhancement of Skin Neoplasia by Chronic Stimulation of Growth Factors
Although numerous epidemiological studies have shown that inorganic arsenicals cause skin cancers and hyperkeratoses in humans, there are currently no established mechanisms for their action or animal models. Previous studies in our laboratory using primary human keratinocyte cultures demonstrated that micromolar concentrations of inorganic arsenite increased cell proliferation via the production of keratinocyte-derived growth factors. As recent reports demonstrate that overexpression of keratinocyte-derived growth factors, such as transforming growth factor (TGF)-?, promote the formation of skin tumors, we hypothesized that similar events may be responsible for those associated with arsenic skin diseases. Thus, the influence of arsenic in humans with arsenic skin disease and on mouse skin tumor development in transgenic mice was studied. After low-dose application of tetradecanoyl phorbol acetate (TPA), a marked increase in the number of skin papillomas occurred in Tg.AC mice, which carry the v-Ha-ras oncogene, that received arsenic in the drinking water as compared with control drinking water, whereas no papillomas developed in arsenic-treated transgenic mice that did not receive TPA or arsenic/TPA-treated wild-type FVB/N mice. Consistent with earlier in vitro findings, increases in granulocyte/macrophage colony-stimulating factor (GM-CSF) and TGF-? mRNA transcripts were found in the epidermis at clinically normal sites within 10 weeks after arsenic treatment. Immunohistochemical staining localized TGF-? overexpression to the hair follicles. Injection of neutralizing antibodies to GM-CSF after TPA application reduced the number of papillomas in Tg.AC mice. Analysis of gene expression in samples of skin lesions obtained from humans chronically exposed to arsenic via their drinking water also showed similar alterations in growth factor expression. Although confirmation will be required in nontransgenic mice, these results suggest that arsenic enhances development of skin neoplasias via the chronic stimulation of keratinocyte-derived growth factors and may be a rare example of a chemical carcinogen that acts as a co-promoter.
Germolec, Dori R.; Spalding, Judson; Yu, Hsin-Su; Chen, G. S.; Simeonova, Petia P.; Humble, Michael C.; Bruccoleri, Alessandra; Boorman, Gary A.; Foley, Julie F.; Yoshida, Takahiko; Luster, Michael I.
Id1 and NF-?B promote the generation of CD133+ and BMI-1+ keratinocytes and the growth of xenograft tumors in mice
Id1 and NF-?B are highly expressed in oral squamous cell carcinoma (OSCC). Whether they have a synergistic role in the carcinogenesis of OSCC is unclear. The current study was designed to demonstrate the synergy of both Id1 and NF-?B in the underlying disease mechanisms of OSCC using in vitro and in vivo animal models. Id1 and NF-?B strengthened the expression of both CD133 and BMI-1 in OSCC cell cultures. CD133+ and BMI-1+ keratinocytes from OSCC tissues and cell cultures initiated the growth of xenograft tumors in SCID/Beige mice. Id1 and NF-?B regulate the expression of CD133 and BMI-1 in an additive or synergistic manner in OSCC, which is associated with the generation of na�ve and self-renewable keratinocytes and initiate the growth of xenograft tumors in vivo.
Interleukin6 promotes human epidermal keratinocyte proliferation and keratin cytoskeleton reorganization in culture
We have studied the effects of interleukin-6 (IL-6) on human epidermal keratinocytes by using serum-free culture conditions that allow the serial transfer, differentiation, and formation of well-organized multilayered epithelia. IL-6 at 2.5�ng\\/ml or higher concentrations promoted keratinocyte proliferation, with an ED50 of about 15�ng\\/ml and a maximum effect at 50�ng\\/ml. IL-6 was 10-fold less potent than epidermal growth factor (EGF)
Miriam Hern�ndez-Quintero; Walid Kuri-Harcuch; Arturo Gonz�lez Robles; Federico Castro-Mu�ozledo
Transforming Growth Factor Beta 3 Is Required for Excisional Wound Repair In Vivo
Wound healing is a complex process that relies on proper levels of cytokines and growth factors to successfully repair the tissue. Of particular interest are the members of the transforming growth factor family. There are three TGF-� isoforms�TGF- � 1, 2, and 3, each isoform showing a unique expression pattern, suggesting that they each play a distinct function during development and repair. Previous studies reported an exclusive role for TGF-� 3 in orofacial development and a potent anti-scarring effect. However, the role of TGF- � 3 in excisional wound healing and keratinocyte migration remains poorly understood. We tested the effect of TGF-� 3 levels on excisional cutaneous wounds in the adult mouse by directly injecting recombinant TGF-� 3 or neutralizing antibody against TGF-� 3 (NAB) in the wounds. Our results demonstrate that TGF-� 3 does not promote epithelialization. However, TGF-� 3 is necessary for wound closure as wounds injected with neutralizing antibody against TGF-� 3 showed increased epidermal volume and proliferation in conjunction with a delay in keratinocyte migration. Wild type keratinocytes treated with NAB and Tgfb3-deficient keratinocytes closed an in vitro scratch wound with no delay, suggesting that our in vivo observations likely result from a paracrine effect.
Le, Mark; Naridze, Rachelle; Morrison, Jasmine; Biggs, Leah C.; Rhea, Lindsey; Schutte, Brian C.; Kaartinen, Vesa; Dunnwald, Martine
Cox2 and ?-Catenin/T-cell Factor Signaling Intestinalize Human Esophageal Keratinocytes When Cultured under Organotypic Conditions12
The incidence of esophageal adenocarcinoma (EAC) is rising in the United States. An important risk factor for EAC is the presence of Barrett esophagus (BE). BE is the replacement of normal squamous esophageal epithelium with a specialized columnar epithelium in response to chronic acid and bile reflux. However, the emergence of BE from squamous keratinocytes has not yet been demonstrated. Our research has focused on this. Wnt and cyclooxygenase 2 (Cox2) are two pathways whose activation has been associated with BE and progression to EAC, but their role has not been tested experimentally. To explore their contribution, we engineered a human esophageal keratinocyte cell line to express either a dominant-active Wnt effector CatCLef or a Cox2 complementary DNA. In a two-dimensional culture environment, Cox2 expression increases cell proliferation and migration, but neither transgene induces known BE markers. In contrast, when these cells were placed into three-dimensional organotypic culture conditions, we observed more profound effects. CatCLef-expressing cells were more proliferative, developed a thicker epithelium, and upregulated Notch signaling and several BE markers including NHE2. Cox2 expression also increased cell proliferation and induced a thicker epithelium. More importantly, we observed cysts form within the epithelium, filled with intestinal mucins including Muc5B and Muc17. This suggests that Cox2 expression in a three-dimensional culture environment induces a lineage of mucin-secreting cells and supports an important causal role for Cox2 in BE pathogenesis. We conclude that in vitro modeling of BE pathogenesis can be improved by enhancing Wnt signaling and Cox2 activity and using three-dimensional organotypic culture conditions.
Kong, Jianping; Crissey, Mary Ann S; Stairs, Douglas B; Sepulveda, Antonia R; Lynch, John P
Tumor necrosis factor ?-mediated induction of interleukin 17C in human keratinocytes is controlled by nuclear factor ?B.
IL-17C is a member of the IL-17 family of cytokines. The expression of IL-17C has been demonstrated to be strongly induced by TNF? in human keratinocytes, and recently the level of IL-17C was found to be increased in the inflammatory skin disease psoriasis. However, little is known about the molecular mechanisms involved in the regulation of IL-17C. Here, we show that pretreatment of cultured human keratinocytes with the inhibitor of ?B kinase 2 inhibitor, SC-514, resulted in a significant reduction in both IL-17C mRNA and protein expression, indicating the significance of this pathway in the regulation of IL-17C. NF-?B binding sites were identified upstream from the IL-17C gene, and by electrophoretic mobility shift assay NF-?B was shown to bind to all three identified binding sites. Moreover, NF-?B binding to these sites was inducible by TNF?. Supershift analysis revealed binding of the NF-?B subunits p65 and p50 to all three NF-?B binding sites. To determine the contribution of NF-?B in IL-17C expression, we conducted luciferase gene reporter experiments and demonstrated that a 3204-bp promoter fragment of IL-17C containing three putative NF-?B binding sites was strongly activated by TNF?. Interestingly, mutations of the three NF-?B binding sites revealed that one specific NF-?B binding site was crucial for the TNF?-mediated IL-17C induction because mutation of this specific site completely abolished TNF?-induced IL-17C promoter activation. We conclude that the activation of NF-?B (p65/p50) is crucial for the TNF?-induced stimulation of IL-17C expression in human keratinocytes. PMID:21628458
Johansen, Claus; Riis, Jette L; Gedebjerg, Anne; Kragballe, Knud; Iversen, Lars
The effect of neurotensin in human keratinocytes--implication on impaired wound healing in diabetes.
Diabetic foot ulcers are an important complication of diabetes mellitus characterized by chronic, non-healing ulcers resulting from poor proliferation and migration of fibroblasts and keratinocytes, thus impairing a correct re-epithelialization of wounded tissues. This healing process can be modulated by neuropeptides released from peripheral nerves; however, little is known regarding the role of neurotensin (NT) as a modulator of human keratinocyte function under hyperglycemic conditions. Therefore, this work is focused on the effect of NT in human keratinocytes, under normal and hyperglycemic conditions at different functional levels, namely NT receptors, cytokine, and growth factor expression, as well as proliferation and migration. Human keratinocyte cells were maintained at either 10/30?mM glucose and treated with or without NT (10?nM). The results show that NT did not affect keratinocyte viability. In addition, NT and all NT receptor expression levels were significantly reduced by hyperglycemia; however, NT treatment stimulated expression of NT and neurotensin receptor 2 (NTR2) while neurotensin receptor 1 (NTR1) and neurotensin receptor 3 (NTR3) expression levels were unchanged. Keratinocyte proliferation was not affected by NT and hyperglycemia, while cell migration was reduced by NT treatment. These results demonstrated that hyperglycemic conditions strongly impaired endogenous NT and NTR2 expression in keratinocytes. Despite the addition of exogenous NT to stimulate the endogenous NT and NTR2 expression, these changes do not translate into functional modifications on keratinocytes, particularly in terms of migration, proliferation, and production of cytokines or growth factors. These results suggest that NT production by keratinocytes may exert a paracrine effect on other skin cells, namely fibroblasts, macrophages, and dendritic cells for correct wound healing. PMID:24198343
Moura, Liane I F; Cruz, Maria T; Carvalho, Eug�nia
Human Keratinocytes That Express hTERT and Also Bypass a p16INK4a-Enforced Mechanism That Limits Life Span Become Immortal yet Retain Normal Growth and Differentiation Characteristics
Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16INK4a cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16INK4a-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT+ keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16INK4a expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16INK4a-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems.
Dickson, Mark A.; Hahn, William C.; Ino, Yasushi; Ronfard, Vincent; Wu, Jenny Y.; Weinberg, Robert A.; Louis, David N.; Li, Frederick P.; Rheinwald, James G.
Chronic CSE Treatment Induces the Growth of Normal Oral Keratinocytes via PDK2 Upregulation, Increased Glycolysis and HIF1alpha Stabilization
BackgroundExposure to cigarette smoke is a major risk factor for head and neck squamous cell carcinoma (HNSCC). We have previously established a chronic cigarette smoke extract (CSE)-treated human oral normal keratinocyte model, demonstrating an elevated frequency of mitochondrial mutations in CSE treated cells. Using this model we further characterized the mechanism by which chronic CSE treatment induces increased cellular proliferation.Methodology\\/Principal
Wenyue Sun; Steven S. Chang; Yumei Fu; Yan Liu; Joseph A. Califano; Robert E. Means
Mouse fibroblast growth factor 10: cDNA cloning, protein characterization, and regulation of mRNA expression
Fibroblast growth factor 7 (FGF-7) or keratinocyte growth factor (KGF), is a potent and specific mitogen for epithelial cells. We have recently identified a novel human FGF-7 homologue, named FGF-10.To study the expression of this new FGF family member and its regulation in wound repair, we cloned the mouse FGF-10 (mFGF-10) cDNA. The encoded protein is 92% identical to human
Hans-Dietmar Beer; Charles Florence; Johanna Dammeier; Linda McGuire; Sabine Werner; D Roxanne Duan
Keratinocyte-derived follistatin regulates epidermal homeostasis and wound repair
Activin is a growth and differentiation factor that controls development and repair of several tissues and organs. Transgenic mice overexpressing activin in the skin were characterized by strongly enhanced wound healing, but also by excessive scarring. In this study, we explored the consequences of targeted activation of activin in the epidermis and hair follicles by generation of mice lacking the activin antagonist follistatin in keratinocytes. We observed enhanced keratinocyte proliferation in the tail epidermis of these animals. After skin injury, an earlier onset of keratinocyte hyperproliferation at the wound edge was observed in the mutant mice, resulting in an enlarged hyperproliferative epithelium. However, granulation tissue formation and scarring were not affected. These results demonstrate that selective activation of activin in the epidermis enhances reepithelialization without affecting the quality of the healed wound.
Antsiferova, Maria; Klatte, Jennifer E; Bodo, Eniko; Paus, Ralf; Jorcano, Jose L; Matzuk, Martin M; Werner, Sabine; Kogel, Heidi
Regulatory Effects of Cell Density on the Binding of Transforming Growth Factor ß, EpidermalGrowth Factor, Platelet-derivedGrowth Factor,and Fibroblast Growth Factor1
The work described in this paper demonstrates that the cellular binding of transforming growth factor ß, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor is reduced as cell density is increased. The reduction in transforming growth factor \\/•>' binding was observed in five different cell lines. Examination of several of the cell lines, under conditions where transforming growth
Angie Rizzino; Peter Kazakoff; Eric Ruff; Charles Kuszynski; John Nebelsick
Growth factors for nanobacteria
Nanobacteria are novel microorganisms recently isolated from fetal bovine serum and blood of cows and humans. These coccoid, gram negative bacteria in alpha-2 subgroup of Proteobacteria grow slowly under mammalian cell culture conditions but not in common media for microbes. Now we have found two different kinds of culture supplement preparations that improve their growth and make them culturable in
Neva Ciftcioglu; E. Olavi Kajander
Peptides from milk protein hydrolysates to improve the growth of human keratinocytes in culture
Milk and colostrum are known to contain constituents having growth promoting activities on various human cell lines. Peptides from milk protein hydrolysates have also been shown to have various nutraceutical and biological properties. The aim of this research was to establish conditions for the in vitro hydrolysis of milk proteins and for the separation and identification of peptides that could
Jean Amiot; Lucie Germain; Sylvie Turgeon; Martine Lemay; Christine Ory-Salam; Fran�ois A. Auger
FGF receptors 1 and 2 are key regulators of keratinocyte migration in vitro and in wounded skin
Summary Efficient wound repair is essential for the maintenance of the integrity of the skin. The repair process is controlled by a variety of growth factors and cytokines, and their abnormal expression or activity can cause healing disorders. Here, we show that wound repair is severely delayed in mice lacking fibroblast growth factor receptors (FGFR) 1 and 2 in keratinocytes. As the underlying mechanism, we identified impaired wound contraction and a delay in re-epithelialization that resulted from impaired keratinocyte migration at the wound edge. Scratch wounding and transwell assays demonstrated that FGFR1/2-deficient keratinocytes had a reduced migration velocity and impaired directional persistence owing to inefficient formation and turnover of focal adhesions. Underlying this defect, we identified a significant reduction in the expression of major focal adhesion components in the absence of FGFR signaling, resulting in a general migratory deficiency. These results identify FGFs as key regulators of keratinocyte migration in wounded skin.
Meyer, Michael; Muller, Anna-Katharina; Yang, Jingxuan; Moik, Daniel; Ponzio, Gilles; Ornitz, David M.; Grose, Richard; Werner, Sabine
KGF Promotes Paracrine Activation of the SCF/c-KIT Axis from Human Keratinocytes to Melanoma Cells1
The paracrine networks of the human melanoma microenvironment are able to influence tumor growth and progression. Among the paracrine growth factors involved in skin homeostasis, the KGF/FGF7 secreted by dermal fibroblasts promotes the epidermal proliferation and differentiation as well as the release from keratinocytes of other paracrine mediators. To evaluate the possible role played by KGF in affecting the behavior of different subtypes of melanoma carrying activating mutations or overexpression of the SCF receptor c-KIT, we used human melanoma cell lines, characterized by different expression levels of c-KIT and opposing responsivity to SCF, and HaCaT keratinocytes. Quantitative real-time reverse transcription-polymerase chain reaction assay and ELISA test on KGF-treated keratinocytes showed enhanced expression and secretion of SCF in response to KGF and dependent on functional KGF receptor. Immunofluorescence microscopy and biochemical analysis showed, in one of the selected melanoma cell models, SCF-dependent c-KIT activation induced by stimulation with the culture supernatants collected from KGF-treated keratinocytes. In keratinocyte-melanoma cocultures stained for the Ki67 proliferation marker, incubation with KGF induced enhanced growth not only of the keratinocytes but also of the melanoma cells, which could be blocked by the c-KIT inhibitor imatinib, demonstrating the establishment of a KGF-induced paracrine signaling network owing to the coexpression of biologically active SCF released from keratinocytes and functional c-KIT on melanoma cells.
Belleudi, Francesca; Cardinali, Giorgia; Kovacs, Daniela; Picardo, Mauro; Torrisi, Maria Rosaria
Alternative Proteolytic Processing of Hepatocyte Growth Factor during Wound Repair
Wound healing is a crucial regenerative process in all organisms. We examined expression, integrity, and function of the proteins in the hepatocyte growth factor (HGF)/c-Met signaling pathway in normally healing and non-healing human skin wounds. Whereas in normally healing wounds phosphorylation of c-Met was most prominent in keratinocytes and dermal cells, in non-healing wounds phosphorylation of c-Met was barely detectable, suggesting reduced c-Met activation. In wound exudates obtained from non-healing, but not from healing wounds, HGF protein was a target of substantial proteolytic processing that was different from the classical activation by known serine proteases. Western blot analysis and protease inhibitor studies revealed that HGF is a target of neutrophil elastase and plasma kallikrein during skin repair. Proteolytic processing of HGF by each of these proteases significantly attenuated keratinocyte proliferation, wound closure capacity in vitro, and c-Met signal transduction. Our findings reveal a novel pathway of HGF processing during skin repair. Conditions in which proteases are imbalanced and tend toward increased proteolytic activity, as in chronic non-healing wounds, might therefore compromise HGF activity due to the inactivation of the HGF protein and/or the generation of HGF fragments that ultimately mediate a dominant negative effect and limit c-Met activation.
Buchstein, Nils; Hoffmann, Daniel; Smola, Hans; Lang, Sabina; Paulsson, Mats; Niemann, Catherin; Krieg, Thomas; Eming, Sabine A.
Barium promotes anchorage-independent growth and invasion of human HaCaT keratinocytes via activation of c-SRC kinase.
Explosive increases in skin cancers have been reported in more than 36 million patients with arsenicosis caused by drinking arsenic-polluted well water. This study and previous studies showed high levels of barium as well as arsenic in the well water. However, there have been no reports showing a correlation between barium and cancer. In this study, we examined whether barium (BaCl(2)) may independently have cancer-related effects on human precancerous keratinocytes (HaCaT). Barium (5-50 �M) biologically promoted anchorage-independent growth and invasion of HaCaT cells in vitro. Barium (5 �M) biochemically enhanced activities of c-SRC, FAK, ERK and MT1-MMP molecules, which regulate anchorage-independent growth and/or invasion. A SRC kinase specific inhibitor, protein phosphatase 2 (PP2), blocked barium-mediated promotion of anchorage-independent growth and invasion with decreased c-SRC kinase activity. Barium (2.5-5 �M) also promoted anchorage-independent growth and invasion of fibroblasts (NIH3T3) and immortalized nontumorigenic melanocytes (melan-a), but not transformed cutaneous squamous cell carcinoma (HSC5 and A431) and malignant melanoma (Mel-ret) cells, with activation of c-SRC kinase. Taken together, our biological and biochemical findings newly suggest that the levels of barium shown in drinking well water independently has the cancer-promoting effects on precancerous keratinocytes, fibroblast and melanocytes in vitro. PMID:22022425
Thang, Nguyen Dinh; Yajima, Ichiro; Kumasaka, Mayuko Y; Ohnuma, Shoko; Yanagishita, Takeshi; Hayashi, Rumiko; Shekhar, Hossain U; Watanabe, Daisuke; Kato, Masashi
Interstitial fibrosis and growth factors.
Interstitial pulmonary fibrosis (IPF) is scarring of the lung caused by a variety of inhaled agents including mineral particles, organic dusts, and oxidant gases. The disease afflicts millions of individuals worldwide, and there are no effective therapeutic approaches. A major reason for this lack of useful treatments is that few of the molecular mechanisms of disease have been defined sufficiently to design appropriate targets for therapy. Our laboratory has focused on the molecular mechanisms through which three selected peptide growth factors could play a role in the development of IPF. Hundreds of growth factors and cytokines could be involved in the complex disease process. We are studying platelet-derived growth factor because it is the most potent mesenchymal cell mitogen yet described, transforming growth factor beta because it is a powerful inducer of extracellular matrix (scar tissue) components by mesenchymal cells, and tumor necrosis factor alpha because it is a pleiotropic cytokine that we and others have shown is essential for the development of IPF in animal models. This review describes some of the evidence from studies in humans, in animal models, and in vitro, that supports the growth factor hypothesis. The use of modern molecular and transgenic technologies could elucidate those targets that will allow effective therapeutic approaches. Images Figure 1 Figure 2
Lasky, J A; Brody, A R
Growth factors in synaptic function
Synapses are increasingly recognized as key structures that malfunction in disorders like schizophrenia, mental retardation, and neurodegenerative diseases. The importance and complexity of the synapse has fuelled research into the molecular mechanisms underlying synaptogenesis, synaptic transmission, and plasticity. In this regard, neurotrophic factors such as netrin, Wnt, transforming growth factor-? (TGF-?), tumor necrosis factor-? (TNF-?), and others have gained prominence for their ability to regulate synaptic function. Several of these factors were first implicated in neuroprotection, neuronal growth, and axon guidance. However, their roles in synaptic development and function have become increasingly clear, and the downstream signaling pathways employed by these factors have begun to be elucidated. In this review, we will address the role of these factors and their downstream effectors in synaptic function in vivo and in cultured neurons.
Poon, Vivian Y.; Choi, Sojoong; Park, Mikyoung
Gingko biloba extract reduces VEGF and CXCL-8\\/IL8 levels in keratinocytes with cumulative effect with epigallocatechin-3-gallate
In skin inflammation, vascular endothelial growth factor (VEGF) and CXCL-8\\/IL-8 play an important role and are produced by\\u000a activated keratinocytes. Extracts from Ginkgo biloba leaves (GBE), widely used in phytotherapy, have been reported to exert\\u000a antioxidant and anti-inflammatory properties in the skin. We therefore evaluated the effects of GBE on the release of VEGF\\u000a and CXCL8\\/IL-8 by normal human keratinocytes
Sandra Trompezinski; Marl�ne Bonneville; Ingrid Pernet; Alain Denis; Daniel Schmitt; Jacqueline Viac
Tumor Necrosis Factor-? from Macrophages Enhances LPS-Induced Clara Cell Expression of Keratinocyte-Derived Chemokine
Tumor necrosis factor (TNF)-? is a cytokine produced by alveolar macrophages in response to LPS in the lung. Clara cells are bronchiolar epithelial cells that produce a variety of proinflammatory cytokines in response to LPS but not to TNF-?. In this study, we examined whether TNF-? affects Clara cell cytokine production in the setting of LPS stimulation. Using a transformed murine Clara cell line (C22), we observed that both LPS and TNF-? induced production of keratinocyte-derived chemokine (KC) and monocyte chemoattractant protein (MCP)-1. We also found that simultaneous LPS and TNF-? stimulation is synergistic for KC production, but additive for MCP-1 production. By using a Transwell coculture system of RAW264.7 macrophages and Clara cells isolated from C57Bl/6 mice, we found that macrophages produce a soluble factor that enhances Clara cell KC production in response to LPS. Cocultures of Clara cells from mice deficient in TNF-? receptors with RAW264.7 macrophages demonstrated that the effect of macrophages on Clara cells is mediated primarily via TNF-?. To determine whether these findings occur in vivo, we treated wild-type and TNF receptor�deficient mice intratracheally with LPS and examined the expression of KC. LPS-treated, TNF receptor�deficient mice showed much less KC mRNA in airway epithelial cells compared with wild-type mice. In contrast, a similar number of KC-expressing cells was seen in the lung periphery. Thus, upregulation of KC by Clara cells in the setting of LPS stimulation is largely dependent on TNF-? originating from alveolar macrophages. These findings shed light on macrophage�Clara cell interactions in regulating the pulmonary inflammatory response to LPS.
Elizur, Arnon; Adair-Kirk, Tracy L.; Kelley, Diane G.; Griffin, Gail L.; deMello, Daphne E.; Senior, Robert M.
Platelet-Derived Growth Factor-BB Controls Epithelial Tumor Phenotype by Differential Growth Factor Regulation in Stromal Cells
Platelet-derived growth factor (PDGF) stimulates tumor growth and progression by affecting tumor and stromal cells. In the HaCaT skin carcinogenesis model, transfection of immortal nontumorigenic and PDGF-receptor-negative HaCaT keratinocytes with PDGF-B induced formation of benign tumors. Here, we present potential mechanisms underlying this tumorigenic conversion. In vivo, persistent PDGF-B expression induced enhanced tumor cell proliferation but only transiently stimulated stromal cell proliferation and angiogenesis. In vitro and in vivo studies identified fibroblasts as PDGF target cells essential for mediating transient angiogenesis and persistent epithelial hyperproliferation. In fibroblast cultures, long-term PDGF-BB treatment caused an initial up-regulation of vascular endothelial growth factor (VEGF)-A, followed by a drastic VEGF down-regulation and myofibroblast differentiation. Accordingly, in HaCaT/PDGF-B transplants, initially enhanced VEGF expression by stromal fibroblasts was subsequently reduced, followed by down-regulation of angiogenesis, myofibroblast accumulation, and vessel maturation. The PDGF-induced, persistently increased expression of the hepatocyte growth factor by fibroblasts in vitro and in vivo was most probably responsible for enhanced epithelial cell proliferation and benign tumor formation. Thus, by paracrine stimulation of the stroma, PDGF-BB induced epithelial hyperproliferation, thereby promoting tumorigenicity, whereas the time-limited activation of the stroma followed by stromal maturation provides a possible explanation for the benign tumor phenotype.
Lederle, Wiltrud; Stark, Hans-Jurgen; Skobe, Mihaela; Fusenig, Norbert E.; Mueller, Margareta M.
Production of interleukin-8 by human dermal fibroblasts and keratinocytes in response to interleukin-1 or tumour necrosis factor.
Cultured normal human fibroblasts were stimulated to produce neutrophil-activating protein/interleukin-8 (IL-8) in response to IL-1 alpha (0.1-1000 U/ml) or tumour necrosis factor (TNF) alpha (0.1-1000 U/ml). Induction of mRNA for IL-8 in fibroblasts was rapid (within 30 min) and maximal responses were obtained with either 100 U/ml IL-1 alpha or 100 U/ml TNF alpha. Expression of mRNA for IL-8 was accompanied by the production of high levels of neutrophil chemotactic activity. IL-1 alpha (1000 U/ml), but not TNF alpha, induced mRNA for IL-8 in cultured normal human keratinocytes. The relevance of production of IL-8 by these cell types was evaluated further by comparing the local inflammatory effects of IL-1 alpha, TNF alpha and IL-8. Intradermal injection of either recombinant IL-8, IL-1 alpha or TNF alpha lead to a similar in vivo effect, i.e. dose-dependent accumulation of lymphocytes and polymorphonuclear leucocytes at sites of injection. The in vivo attraction of neutrophils and lymphocytes to the site of injection by TNF or IL-1 (which is not chemotactic for neutrophils or lymphocytes in vitro), may be partly mediated by locally produced IL-8. Thus, IL-8 may be a vital participant in the cascade of interacting cytokines that is induced by tissue injury and immunologically induced inflammation. Images Figure 1 Figure 2 Figure 3 Figure 4
Larsen, C G; Anderson, A O; Oppenheim, J J; Matsushima, K
Transcription factor Nrf2 activation by inorganic arsenic in cultured keratinocytes: involvement of hydrogen peroxide
Inorganic arsenic is a well-documented human carcinogen that targets the skin. The induction of oxidative stress, as shown with arsenic, may have a bearing on the carcinogenic mechanism of this metalloid. The transcription factor Nrf2 is a key player in the regulation of genes encoding for many antioxidative response enzymes. Thus, the effect of inorganic arsenic (as sodium arsenite) on
Jingbo Pi; Wei Qu; Jeffrey M Reece; Yoshito Kumagai; Michael P Waalkes
EGF upregulates, whereas TGF-beta downregulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: correlations with epidermal proliferation and differentiation.
Hyaluronan, a major extracellular matrix molecule in the vital cell layers of skin epidermis, has been suggested to support proliferation and migration of keratinocytes, during challenges like wounding and inflammation. An organotypic keratinocyte culture originated from continuous rat epidermal keratinocyte cell line was subjected to the proliferative and antiproliferative growth factors epidermal growth factor and transforming growth factor beta, respectively, to study their influence on hyaluronan synthesis and epidermal morphology. Epidermal growth factor induced a 4-fold increase of epidermal hyaluronan concentration. This was associated with upregulation of the hyaluronan synthases Has2 and Has3, and the hyaluronan receptor CD44. 5-Bromo-2'-deoxyuridine labeling, basal cell height, and the thickness of vital epidermis were increased, reflecting the hyperplastic effects of epidermal growth factor. The expression of keratin 10 and the maturation of filaggrin were inhibited, and epidermal permeability barrier became less efficient, indicating compromised terminal differentiation by epidermal growth factor. In contrast, transforming growth factor beta reduced the content of hyaluronan and the mRNA of Has2 and Has3. At the same time, transforming growth factor beta suppressed keratinocyte proliferation and epidermal thickness, but retained intact differentiation. The results suggest that epidermal hyaluronan synthesis, controlled by epidermal growth factor and transforming growth factor beta through changes in the expression of Has2 and Has3, correlates with epidermal proliferation, thickness, and differentiation. PMID:12787132
Pasonen-Sepp�nen, Sanna; Karvinen, Susanna; T�rr�nen, Kari; Hyttinen, Juha M T; Jokela, Tiina; Lammi, Mikko J; Tammi, Markku I; Tammi, Raija
The zinc finger transcription factor Egr1 is upregulated in arsenite-treated human keratinocytes
Arsenite is a human carcinogen that may induce cancer in skin, liver, kidney, bladder or lung. Arsenite executes its toxic effects by the induction of signaling cascades. In particular, the activation of the stress-induced protein kinase c-Jun N-terminal protein kinase and p38 and the phosphorylation and activation of the transcription factor c-Jun have been linked to the biological effects of
Alia Al-Sarraj; Gerald Thiel
Vitamin D receptor expression is linked to cell cycle control in normal human keratinocytes.
To improve our understanding of the cutaneous vitamin D system, we studied vitamin D receptor (VDR) gene regulation in cultured human keratinocytes. Because VDR and its ligand 1 alpha,25-dihydroxyvitamin D(3) have been implicated in epidermal growth control, we investigated VDR expression as related to cellular proliferation by using different cell cycle synchronization protocols. Keratinocytes, deprived of growth factors, were forced into quiescence and a concomitant loss of VDR expression was observed. Mitogenic stimulation of these G(0) cells however quickly upregulated VDR levels several hours ahead the G(1)-S transition point. Growth arrest at the G(1)-S border by mimosine treatment or at the metaphase by nocodazole also downregulated VDR levels but a restoration of VDR expression was again quickly achieved after reentering the cell cycle. These findings indicate that VDR expression in keratinocytes is restricted to actively cycling cells, but not limited to one particular phase of the cell cycle. PMID:11112422
Segaert, S; Degreef, H; Bouillon, R
Fetal Fibroblasts and Keratinocytes with Immunosuppressive Properties for Allogeneic Cell-Based Wound Therapy
Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.
Zuliani, Thomas; Saiagh, Soraya; Knol, Anne-Chantal; Esbelin, Julie; Dreno, Brigitte
Cytokine release and cytotoxicity in human keratinocytes and fibroblasts induced by phenols and sodium dodecyl sulfate.
Phenolic compounds used in pharmaceutical and industrial products can cause irritant contact dermatitis. We studied the effects of resorcinol, phenol, 3,5-xylenol, chloroxylenol, and 4-hexyl-resorcinol on normal human epidermal keratinocytes and dermal fibroblasts for cytotoxicity and cytokine release, determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide methodology and enzyme-linked immunosorbent assay, respectively. An inverse correlation between phenol concentrations causing a 50% reduction in keratinocyte and fibroblast viability at 24 h and their octanol water-partition coefficients (i.e., hydrophobicity) was observed. 3,5-xylenol, chloroxylenol, hexyl-resorcinol, and sodium dodecyl sulfate, but not resorcinol or phenol, induced release of interleukin-1alpha from keratinocytes at cytotoxic concentrations. Variable release of tumor necrosis factor-alpha and interleukin-8 from keratinocytes occurred only at toxic threshold concentrations of the phenols or sodium dodecyl sulfate. Subtoxic concentrations of phenols or sodium dodecyl sulfate did not induce cytokine release from keratinocytes. Neither the phenols nor sodium dodecyl sulfate induced release of the chemokines interleukin-8, growth-related oncogene-alpha or monocyte chemotactic protein-1 from fibroblasts. Conditioned media from keratinocytes treated with cytotoxic concentrations of 3,5-xylenol, chloroxylenol, hexyl-resorcinol, or sodium dodecyl sulfate stimulated further release of the chemokines from fibroblasts above that obtained with control media. Rabbit anti-interleukin-1alpha serum inhibited keratinocyte-conditioned media induction of chemokine release. We have shown a structure-cytotoxicity relationship for a series of phenols as well as an association of interleukin-1alpha release with a cytotoxic effect. We demonstrated a cytokine cascade amplification step by the actions of stimulated keratinocyte media on cultured dermal fibroblasts, identifying interleukin-1alpha as the principal initiator of chemokine synthesis. PMID:10951249
Newby, C S; Barr, R M; Greaves, M W; Mallet, A I
The Epidermal Growth Factor Receptor Increases Cytokine Production and Cutaneous Inflammation in Response to Ultraviolet Irradiation
The epidermal growth factor receptor (EGFR) is activated in cutaneous keratinocytes upon ultraviolet (UV) exposure and has been implicated in ultraviolet-(UV-)induced inflammation and skin tumorigenesis. Egfr mutant mice and EGFR inhibitors were used to investigate the hypothesis that EGFR activation augments inflammation following UV irradiation. Topical treatment of mouse skin with the EGFR inhibitor AG1478 before UV exposure suppressed UV-induced erythema, edema, mast cell infiltration, and neutrophil infiltration. Genetic ablation of Egfr and EGFR inhibition by AG1478 also suppressed the increase in the proinflammatory cytokines tumor necrosis factor ? (TNF-?), interleukin-1?, KC (murine IL-8), and cyclooxygenase-2 (COX-2) after UV exposure of cultured keratinocytes. Finally, genetic ablation of inhibition of EGFR in cultured keratinocytes decreased p38 activation after UV, while inhibition of p38 kinase reduced COX-2 expression after UV. These data demonstrate that EGFR regulates multiple aspects of UV-induced inflammation and suggest activation of p38 kinase leading to increased COX-2 and cytokine expression as one mechanism through which it acts.
El-Abaseri, Taghrid Bahig; Repertinger, Susan K.; Hansen, Laura A.
Heparin-binding ligands mediate autocrine epidermal growth factor receptor activation In skin organ culture.
Exogenous EGF and TGF-alpha accelerate wound healing, but treatment effects are often modest. Using short-term human skin organ culture, we found that autocrine EGF receptor activation could account for this observation. Amphiregulin and heparin-binding EGF-like growth factor (HB-EGF) transcripts were rapidly and markedly induced, whereas EGF and TGF-alpha mRNAs were undetectable or only slightly increased. Vascular permeability factor and keratin 6 transcripts were also strongly induced, albeit with a >/= 3 h delay relative to HB-EGF and amphiregulin. All four transcripts were upregulated in actual healing skin wounds, HB-EGF and keratin 6 being the most prominent. The highly EGF receptor-specific tyrosine kinase inhibitor PD153035 strongly inhibited induction of all four transcripts in organ culture, as well as release of immunoreactive HB-EGF into the medium. These effects were confirmed using the anti-EGF receptor mAb 225 IgG. Neither PD153035 nor 225 IgG was toxic to keratinocytes, as judged by calcein-AM uptake. PD153035 completely abrogated the proliferative phase of keratinocyte outgrowth in skin explant cultures, whereas it had no effect on the antecedent migratory phase. Based on these results, we conclude that EGF receptor activation by highly inducible, keratinocyte-derived heparin-binding ligands is an important mechanism for amplification and transmission of the cutaneous wound healing signal.
Stoll, S; Garner, W; Elder, J
The trk family of receptors mediates nerve growth factor and neurotrophin-3 effects in melanocytes.
We have recently shown that (a) human melanocytes express the p75 nerve growth factor (NGF) receptor in vitro; (b) that melanocyte dendricity and migration, among other behaviors, are regulated at least in part by NGF; and (c) that cultured human epidermal keratinocytes produce NGF. We now report that melanocyte stimulation with phorbol 12-tetra decanoate 13-acetate (TPA), previously reported to induce p75 NGF receptor, also induces trk in melanocytes, and TPA effect is further potentiated by the presence of keratinocytes in culture. Moreover, trk in melanocytes becomes phosphorylated within minutes after NGF stimulation. As well, cultures of dermal fibroblasts express neurotrophin-3 (NT-3) mRNA; NT-3 mRNA levels in cultured fibroblasts are modulated by mitogenic stimulation, UV irradiation, and exposure to melanocyte-conditioned medium. Moreover, melanocytes constitutively express low levels of trk-C, and its expression is downregulated after TPA stimulation. NT-3 supplementation to cultured melanocytes maintained in Medium 199 alone prevents cell death. These combined data suggest that melanocyte behavior in human skin may be influenced by neurotrophic factors, possibly of keratinocyte and fibroblast origin, which act through high affinity receptors. Images
Yaar, M; Eller, M S; DiBenedetto, P; Reenstra, W R; Zhai, S; McQuaid, T; Archambault, M; Gilchrest, B A
Fibroblast growth factor receptor inhibitors.
Fibroblast growth factor receptors (FGFRs) play an important role in embryonic development, angiogenesis, wound healing, cell proliferation and differentiation. The fibroblast growth factor receptor (FGFR) isoforms have been under intense scrutiny for effective anticancer drug candidates. The fibroblast growth factor (FGF) and its receptor (FGFR) provide another pathway that seems critical to monitoring angiogenesis. Recent findings suggest that FGFR mediates signaling, regulates the PKM2 activity, and plays a crucial role in cancer metabolism. The current review also covers the recent findings on the role of FGFR1 in cancer metabolism. This paper reviews the progress, mechanism, and binding modes of recently known kinase inhibitors such as PD173074, SU series and other inhibitors still under clinical development. Some of the structural classes that will be highlighted in this review include Pyrido[2,3-d]pyrimidines, Indolin- 2-one, Pyrrolo[2,1-f][1,2,4]triazine, Pyrido[2,3-d]pyrimidin-7(8H)-one, and 1,6- Naphthyridin-2(1H)-ones. PMID:23016864
Kumar, Suneel B V S; Narasu, Lakshmi; Gundla, Rambabu; Dayam, Raveendra; J A R P, Sarma
IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes
While the pro-differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms remain poorly understood. We report here that interferon regulatory factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is induced in differentiation through a Notch-dependent mechanism and is a primary Notch target in keratinocytes and keratinocyte-derived SCC cells. Increased IRF6 expression contributes to the impact of Notch activation on growth/differentiation-related genes, while it is not required for induction of �canonical' Notch targets like p21WAF1/Cip1, Hes1 and Hey1. Down-modulation of IRF6 counteracts differentiation of primary human keratinocytes in vitro and in vivo, promoting ras-induced tumour formation. The clinical relevance of these findings is illustrated by the strikingly opposite pattern of expression of Notch1 and IRF6 versus epidermal growth factor receptor in a cohort of clinical SCCs, as a function of their grade of differentiation. Thus, IRF6 is a primary Notch target in keratinocytes, which contributes to the role of this pathway in differentiation and tumour suppression.
Restivo, Gaetana; Nguyen, Bach-Cuc; Dziunycz, Piotr; Ristorcelli, Elodie; Ryan, Russell J H; Ozuysal, Ozden Yalcin; Di Piazza, Matteo; Radtke, Freddy; Dixon, Michael J; Hofbauer, Gunther F L; Lefort, Karine; Dotto, G Paolo
Functional analysis of the nuclear localization signal of the POU transcription factor Skn?1a in epidermal keratinocytes.
POU domain proteins are a family of critical regulators of development and differentiation due to their transcriptional activity in the nucleus. Skn?1a, a member of the POU domain protein family, appears to be expressed predominantly in epidermal keratinocytes and is thought to play a critical role in keratinocyte differentiation and proliferation. In this study, we examined the mechanisms involved in the nuclear localization of Skn?1a. We transiently expressed enhanced green fluorescent protein�(EGFP) reporter constructs encoding EGFP fusions with Skn?1a deletion and mutation proteins in normal human epidermal keratinocytes�(NHEKs). The experiments clearly demonstrated that Skn?1a contained a functional nuclear localization signal�(NLS) domain, and that the smallest domain necessary for Skn?1a nuclear transport was the GRKRKKR sequence located within amino acids�279?285. Previous studies have shown that the phosphorylation of specific amino acids neighboring the NLS may regulate nuclear transport and that the amino acid residues threonine�(Thr) and serine�(Ser) have the potential to undergo phosphorylation. We examined whether the amino acids Thr286 and Ser287, which reside adjacent to the NLS at the carboxy?terminal side, play a role in Skn?1a nuclear localization. For this purpose, we generated three EGFP?Skn?1a mutation constructs, in which Thr286, Ser287, or both Thr286 and Ser287 residues were replaced with alanine, respectively. The results showed that the Thr286 and Ser287 residues were involved in the regulation of nuclear localization as�well�as epidermal differentiation. These results suggested that the epidermal differentiation signaling pathway, involving kinase and phosphatase activation, may regulate the NLS activity of Skn?1a in keratinocytes. Collectively, these data contribute to understanding the mechanisms of nuclear translocation of POU domain proteins and epidermal differentiation. PMID:24954220
Moritsugu, Ryuta; Tamai, Katsuto; Nakano, Hajime; Aizu, Takayuki; Nakajima, Koji; Yamazaki, Takehiro; Sawamura, Daisuke
Growth factor regulation of growth factor production by multiple gene transfer to chondrocytes
Of the many classes of molecules regulated by growth factors, growth factors themselves are not well investigated. We tested the hypothesis that combinations of endogenous growth factors interactively regulate the production of other growth factors. Growth factors have therapeutic potential for articular cartilage repair, and gene transfer is a promising approach to growth factor delivery. We tested the hypothesis using adult bovine articular chondrocytes treated with combinations of cDNAs encoding insulin-like growth factor I, bone morphogenetic protein-2 and protein-7, transforming growth factor ?1, and fibroblast growth factor 2. We found that these growth factor transgenes regulated each other�s growth factor production. This regulation ranged from stimulation to inhibition. Regulation by multiple transgenes was not predictable from the regulatory actions of the individual transgenes. Such interactions may be important for the selection of growth factor genes for cell-based therapies, including articular cartilage repair.
?-Adrenergic Receptor Activation Inhibits Keratinocyte Migration via a Cyclic Adenosine Monophosphate-independent Mechanism
There is increasing evidence that G-protein-coupled receptors cross-talk with growth factor receptor-mediated signal transduction in a variety of cell types. We have investigated mechanisms by which the activation of ?-adrenergic receptors, classically GTP-binding proteins coupled receptors, influence the migration of cultured human keratinocytes. We found that iso-proterenol, a ?-adrenergic receptor-selective agonist, inhibited cell migration stimulated by either epidermal growth factor,
Jin Chen; Brian B Hoffman; R. Rivkah Isseroff
3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals.
Stable protein microcrystals called polyhedra are produced by certain insect viruses. Cytokines, such as fibroblast growth factors (FGFs), can be immobilized within polyhedra. Here, we investigated three-dimensional (3D) co-cultures of keratinocytes and melanocytes on collagen gel containing FGF-2 and FGF-7 polyhedra. Melanocytes were observed to reside at the base of the 3D cell culture and melanin was also typically observed in the lower layer. The 3D cell culture model with FGF-2 and FGF-7 polyhedra was a useful in vitro model of the epidermis due to effective melanogenesis, proliferation and differentiation of keratinocytes. FGF-7 polyhedra showed a potent cytoprotective effect when keratinocytes were treated with menadione, which is a generator of reactive oxygen species. The cytoprotective effect was activated by the inositol triphosphate kinase-Akt pathway leading to upregulation of the antioxidant enzymes superoxide dismutase and peroxiredoxin 6. PMID:25063093
Shimabukuro, Junji; Yamaoka, Ayako; Murata, Ken-Ichi; Kotani, Eiji; Hirano, Tomoko; Nakajima, Yumiko; Matsumoto, Goichi; Mori, Hajime
Keratinocytes as initiators of inflammation.
Environmental stimuli responsible for inducing cutaneous inflammation include contact allergens and ultraviolet light. We postulate that these diverse stimuli trigger a cutaneous inflammatory response by directly inducing epidermal keratinocytes to elaborate specific pro-inflammatory cytokines and adhesion molecules. The consequences are activation of dermal microvascular endothelial cells and selective accumulation of specific mononuclear cells in the dermis and epidermis. Thus, keratinocytes may act as "signal transducers", capable of converting exogenous stimuli into the production of cytokines, adhesion molecules, and chemotactic factors (acting in an autocrine and paracrine fashion) responsible for initiation of "antigen-independent" cutaneous inflammation. The initiation phase may facilitate or promote an amplification phase with additional production of tumour-necrosis factor alpha and interferon gamma via an "antigen-dependent" pathway, and keratinocyte/T cell/antigen-presenting dendritic cellular associations. The direct activation of keratinocytes, with their ability to produce the complete repertoire of pro-inflammatory cytokines, can profoundly influence endogenous and recruited immunocompetent cells, thereby providing the critical trigger responsible for the swift and clinically dramatic alterations that occur following contact between the epidermis and a host of "noxious" agents. PMID:1670850
Barker, J N; Mitra, R S; Griffiths, C E; Dixit, V M; Nickoloff, B J
Sustained release of growth factors.
With the identification, characterization and cloning of specific growth factors, recombinant proteins are now widely used in the clinic. The use of recombinant hematopoietic growth factors has, for example, allowed the clinical manipulation of the hematopoietic system. Recombinant human granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are now widely used to mobilize hematopoietic stem cells (HSC) thereby providing a source of HSC for autologous or allogeneic transplantation, in addition to treating congenic, iatrogenic and disease-related neutropenia. However, one disadvantage associated with the use of most recombinant molecules is their rapid clearance. Clearance mechanisms include glomerular filtration, receptor binding and/or enzymatic degradation. Because of the rapid clearance, of recombinant molecules they require repeated administration to achieve biological efficacy. Initially, continuous infusion (CI) was used to address this pharmacological deficiency. CI has the advantage of delivering drugs in a controlled manner and is particularly appropriate when it is important to maintain constant plasma drug concentrations. However, the requirement for continuous venous access and the use of ambulatory pumps limits its use. Thus other approaches have been developed to improve the pharmacokinetic and pharmacodynamic properties of recombinant proteins in vivo. These have included the addition of polyethylene glycol (PEG) to the recombinant molecules (PEGylation) and the use of sustained release delivery matrices and liposomes. One goal of these approaches is to achieve clinical efficacy with significantly fewer, possibly single injections, thereby increasing patient compliance. In addition to improving the pharmacokinetic and pharmacodynamic profile of recombinant molecules, sustained release may also increase the biological activity of the molecules. PMID:12494898
Robinson, Simon N; Talmadge, James E
Human papillomavirus causes an angiogenic switch in keratinocytes which is sufficient to alter endothelial cell behavior
One of the requirements for tumor growth is the ability to recruit a blood supply, a process known as angiogenesis. Angiogenesis begins early in the progression of cervical disease from mild to severe dysplasia and on to invasive cancer. We have previously reported that expression of human papillomavirus type 16 E6 and E7 (HPV16 E6E7) proteins in primary foreskin keratinocytes (HFKs) decreases expression of two inhibitors and increases expression of two angiogenic inducers [Toussaint-Smith, E., Donner, D.B., Roman, A., 2004. Expression of human papillomavirus type 16 E6 and E7 oncoproteins in primary foreskin keratinocytes is sufficient to alter the expression of angiogenic factors. Oncogene 23, 2988-2995]. Here we report that HPV-induced early changes in the keratinocyte phenotype are sufficient to alter endothelial cell behavior both in vitro and in vivo. Conditioned media from HPV16 E6E7 expressing HFKs as well as from human cervical keratinocytes containing the intact HPV16 were able to stimulate proliferation and migration of human microvascular endothelial cells. In addition, introduction of the conditioned media into immunocompetent mice using a Matrigel plug model resulted in a clear angiogenic response. These novel data support the hypothesis that HPV proteins contribute not only to the uncontrolled keratinocyte growth seen following HPV infection but also to the angiogenic response needed for tumor formation.
Chen, W. [Department of Microbiology and Immunology and the Walther Oncology Center, Indiana University School of Medicine and the Walther Cancer Institute, Indianapolis, 635 Barnhill Drive, Indianapolis, IN 46202-5120 (United States); Li, F.; Mead, L.; White, H. [Department of Pediatrics, Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Walker, J. [Department of Microbiology and Immunology and the Walther Oncology Center, Indiana University School of Medicine and the Walther Cancer Institute, Indianapolis, 635 Barnhill Drive, Indianapolis, IN 46202-5120 (United States); Ingram, D.A. [Department of Pediatrics, Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Roman, A. [Department of Microbiology and Immunology and the Walther Oncology Center, Indiana University School of Medicine and the Walther Cancer Institute, Indianapolis, 635 Barnhill Drive, Indianapolis, IN 46202-5120 (United States)], E-mail: firstname.lastname@example.com
Cellular signaling by fibroblast growth factor receptors
The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. Unlike other growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce
V. P. Eswarakumar; I. Lax; J. Schlessinger
Palmitic Acid Induces Production of Proinflammatory Cytokines Interleukin-6, Interleukin-1?, and Tumor Necrosis Factor-? via a NF-?B-Dependent Mechanism in HaCaT Keratinocytes
To investigate whether palmitic acid can be responsible for the induction of inflammatory processes, HaCaT keratinocytes were treated with palmitic acid at pathophysiologically relevant concentrations. Secretion levels of interleukin-6 (IL-6), tumor necrosis factor-? (TNF-?), interleukin-1? (IL-1?), NF-?B nuclear translocation, NF-?B activation, Stat3 phosphorylation, and peroxisome proliferator-activated receptor alpha (PPAR?) mRNA and protein levels, as well as the cell proliferation ability were measured at the end of the treatment and after 24 hours of recovery. Pyrrolidine dithiocarbamate (PDTC, a selective chemical inhibitor of NF-?B) and goat anti-human IL-6 polyclonal neutralizing antibody were used to inhibit NF-?B activation and IL-6 production, respectively. Our results showed that palmitic acid induced an upregulation of IL-6, TNF-?, IL-1? secretions, accompanied by NF-?B nuclear translocation and activation. Moreover, the effect of palmitic acid was accompanied by PPAR? activation and Stat3 phosphorylation. Palmitic acid-induced IL-6, TNF-?, IL-1? productions were attenuated by NF-?B inhibitor PDTC. Palmitic acid was administered in amounts able to elicit significant hyperproliferation and can be attenuated by IL-6 blockage. These data demonstrate for the first time that palmitic acid can stimulate IL-6, TNF-?, IL-1? productions in HaCaT keratinocytes and cell proliferation, thereby potentially contributing to acne inflammation and pilosebaceous duct hyperkeratinization.
Zhou, Bing-rong; Zhang, Jia-an; Zhang, Qian; Xu, Yang; Wu, Di; Yin, Zhi-qiang; Luo, Dan
Protein Kinase C (PKC) ? Suppresses Keratinocyte Proliferation by Increasing p21Cip1 Level by a KLF4 Transcription Factor-dependent Mechanism*
PKC? increases keratinocyte differentiation and suppresses keratinocyte proliferation and survival. However, the mechanism of proliferation suppression is not well understood. The present studies show that PKC? overexpression increases p21Cip1 mRNA and protein level and promoter activity and that treatment with dominant-negative PKC?, PKC?-siRNA, or rottlerin inhibits promoter activation. Analysis of the p21Cip1 promoter upstream regulatory region reveals three DNA segments that mediate PKC?-dependent promoter activation. The PKC? response element most proximal to the transcription start site encodes six GC-rich DNA elements. Mutation of these sites results in a loss of PKC?-dependent promoter activation. Gel mobility supershift and chromatin immunoprecipitation reveal that these DNA elements bind the Kruppel-like transcription factor KLF4. PKC? increases KLF4 mRNA and protein level and KLF4 binding to the GC-rich elements in the p21Cip1 proximal promoter. In addition, KLF4-siRNA inhibits PKC?-dependent p21Cip1 promoter activity. PKC? increases KLF4 expression leading to enhanced KLF4 interaction with the GC-rich elements in the p21Cip1 promoter to activate transcription.
Chew, Yap Ching; Adhikary, Gautam; Wilson, Gerald M.; Reece, E. Albert; Eckert, Richard L.
Growth Regulation of Skin Cells by Epidermal Cell-Derived Factors: Implications for Wound Healing
Epidermal cell-derived factors (EDF), present in extracts and supernatant fluids of cultured epidermal cells, were found to stimulate the proliferation of keratinocytes but to inhibit fibroblasts. In vitro, the effect of EDF on epiermal cells resulted in an increased number of rapidly proliferating colonies composed mainly of basal keratinocytes. Control cultures grown in the absence of EDF had a high
M. Eisinger; S. Sadan; I. A. Silver; R. B. Flick
Paracrine loop of keratinocyte proliferation and directed neuritic outgrowth in a neuroepithelial coculture.
In the absence of skin innervation, wound healing is delayed and chronic nonhealing wounds may occur. Keratinocytes produce neurotrophic factors, such as nerve growth factor (NGF), which has been suggested to attract primary cutaneous afferent axons and exert mitogenic effects on keratinocytes. The present study was performed to examine the interaction of primary human keratinocytes (hKTs) and rat cutaneous primary afferent dorsal root ganglion (DRG) neurons with regard to neuritic outgrowth and keratinocyte proliferation. Neuritic outgrowth was assessed with neurofilament immunostaining where cell bodies and fine neuritic processes were identified. Neuritic outgrowth of neurons alone in culture is spatially random and radial. Neurites in cocultures of DRG neurons insinuated between the hKTs and grew to "clumps" of hKTs within the cultures. Immunostaining with anti-NGF antibody indicates that hKTs expressed the neurotrophin NGF. Proliferation of keratinocytes was significantly enhanced in coculture with DRG and hKT, and NGF levels were increased as compared to DRG or hKT culture alone. These results indicate a dynamic interaction between DRG neurons and hKTs whereby the DRG neurons issue neurites in association with hKTs and the hKTs up-regulate NGF and increase their proliferation rate. These findings support the hypothesis that nerve-skin interactions play a significant role in wound healing. PMID:23328123
Radtke, Christine; Rennekampff, Hans-Oliver; Reimers, Kerstin; Vogt, Peter M; Kocsis, Jeffery D
Genetically Modified Keratinocytes Transplanted to Wounds Reconstitute the Epidermis
Normal and retrovirally transfected keratinocyte suspensions expressing either the beta-galactosidase gene or the human growth hormone (hGH) gene were transplanted into chamber-enclosed skin full-thickness wounds of Yorkshire pigs. Immunostaining of sequential skin biopsies obtained for 4 weeks after transplantation showed survival of the transplanted keratinocytes as well as expression of beta-galactosidase. Transfected keratinocytes were first seen in the neodermal portions
Peter M. Vogt; Simon Thompson; Christoph Andree; Paul Liu; Karl Breuing; Dimitrios Hatzis; Henry Brown; Richard C. Mulligan; Elof Eriksson
RIP2: A novel player in the regulation of keratinocyte proliferation and cutaneous wound repair?
We could recently demonstrate an important role of receptor interacting protein 4 (RIP4) in the regulation of keratinocyte differentiation. Now, we analyzed a potential role of the RIP4 homolog RIP2 in keratinocytes. Specifically, we demonstrate here that rip2 expression is induced by scratch-wounding and after the induction of differentiation in these cells. Furthermore, serum growth factors and cytokines can induce rip2, with TNF-{alpha}-dependent induction being dependent on p38 MAPK. In addition, we demonstrate that scratch-induced upregulation of rip2 expression is completely blocked by the steroid dexamethasone. Since we also show that RIP2 is an important player in the regulation of keratinocyte proliferation, these data suggest that inhibition of rip2 upregulation after wounding might contribute to the reduced and delayed wound re-epithelialization phenotype seen in glucocorticoid-treated patients.
Adams, Stephanie; Valchanova, Ralitsa S. [Charite-University Medicine Berlin, Institute of Physiology, Arnimallee 22, D-14195 Berlin (Germany)] [Charite-University Medicine Berlin, Institute of Physiology, Arnimallee 22, D-14195 Berlin (Germany); Munz, Barbara, E-mail: email@example.com [Charite-University Medicine Berlin, Institute of Physiology, Arnimallee 22, D-14195 Berlin (Germany)] [Charite-University Medicine Berlin, Institute of Physiology, Arnimallee 22, D-14195 Berlin (Germany)
Growth Factor Mediated Signaling in Pancreatic Pathogenesis
Functionally, the pancreas consists of two types of tissues: exocrine and endocrine. Exocrine pancreatic disorders mainly involve acute and chronic pancreatitis. Acute pancreatitis typically is benign, while chronic pancreatitis is considered a risk factor for developing pancreatic cancer. Pancreatic carcinoma is the fourth leading cause of cancer related deaths worldwide. Most pancreatic cancers develop in the exocrine tissues. Endocrine pancreatic tumors are more uncommon, and typically are less aggressive than exocrine tumors. However, the endocrine pancreatic disorder, diabetes, is a dominant cause of morbidity and mortality. Importantly, different growth factors and their receptors play critical roles in pancreatic pathogenesis. Hence, an improved understanding of how various growth factors affect pancreatitis and pancreatic carcinoma is necessary to determine appropriate treatment. This chapter describes the role of different growth factors such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor (TGF) in various pancreatic pathophysiologies. Finally, the crosstalk between different growth factor axes and their respective signaling mechanisms, which are involved in pancreatitis and pancreatic carcinoma, are also discussed.
Nandy, Debashis; Mukhopadhyay, Debabrata
Growth factors and antimicrobial factors of bovine colostrum
Colostrum is the first natural food produced by female mammals during the first 24�36h directly after giving birth. Chemically, colostrum is a very complex fluid rich in nutrients, antibodies and growth factors. In cows the antibodies provide passive immunity to the new born calf, whereas the growth factors especially stimulate the growth of the gut. The other antimicrobial components of
R. Pakkanen; J. Aalto
Double-Stranded RNA-Exposed Human Keratinocytes Promote Th1 Responses by Inducing a Type1 Polarized Phenotype in Dendritic Cells: Role of Keratinocyte-Derived Tumor Necrosis Factor ?, Type I Interferons, and Interleukin18
Dendritic cells play a key role in establishing the class of immune response against invading pathogens. Upon engagement with double-stranded RNA, a major bioactive constituent of many virus types, immature dendritic cells develop into type 1 immunostimulatory dendritic cells that promote Th1 responses. Immature dendritic cells reside in the epithelia and are in close contact with keratinocytes. We studied to
M Cristina Lebre; Jeanine C Antons; Pawel Kalinski; Joost H N Schuitemaker; Toni M M van Capel; Martien L Kapsenberg; Esther C de Jong
Antithrombin Regulates Matriptase Activity Involved in Plasmin Generation, Syndecan Shedding, and HGF Activation in Keratinocytes
Matriptase, a membrane-associated serine protease, plays an essential role in epidermal barrier function through activation of the glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin. The matriptase-prostasin proteolytic cascade is tightly regulated by hepatocyte growth factor activator inhibitor (HAI)-1 such that matriptase autoactivation and prostasin activation occur simultaneously and are followed immediately by the inhibition of both enzymes by HAI-1. However, the mechanisms whereby matriptase acts on extracellular substrates remain elusive. Here we report that some active matriptase can escape HAI-1 inhibition by being rapidly shed from the cell surface. In the pericellular environment, shed active matriptase is able to activate hepatocyte growth factor (HGF), accelerate plasminogen activation, and shed syndecan 1. The amount of active matriptase shed is inversely correlated with the amount of antithrombin (AT) bound to the surface of the keratinocytes. Binding of AT to the surface of keratinocytes is dependent on a functional heparin binding site, Lys-125, and that the N-glycosylation site Asn-135 be unglycosylated. This suggests that ?-AT, and not ?-AT, is responsible for regulation of pericellular matriptase activity in keratinocytes. Keratinocytes appear to rely on AT to regulate the level of pericellular active matriptase much more than breast and prostate epithelial cells in which AT regulation of matriptase activity occurs at much lower levels than keratinocytes. These results suggest that keratinocytes employ two distinct serine protease inhibitors to control the activation and processing of two different sets of matriptase substrates leading to different biological events: 1) HAI-1 for prostasin activation/inhibition, and 2) AT for the pericellular proteolysis involved in HGF activation, accelerating plasminogen activation, and shedding of syndecans.
Chen, Ya-Wen; Xu, Zhenghong; Baksh, Adrienne N. H.; Wang, Jehng-Kang; Chen, Chiu-Yuan; Swanson, Richard; Olson, Steve T.; Kataoka, Hiroaki; Johnson, Michael D.; Lin, Chen-Yong
Cell-impermeant pyridinium derivatives of psoralens as inhibitors of keratinocyte growth 1 1 Abbreviations: BQ, 1,4-benzoquinone; DEPC, diethyl pyrocarbonate; DMEM, Dulbecco�s Modified Eagle Medium; EGF, epidermal growth factor; FADU, fluorescent analysis of DNA unwinding; 4?-Pyr-H 2TMP, 4?-pyridinium-4?,5?-dihydro-4,4?,8-trimethylpsoralen; 5?-Pyr-H 2TMP, 5?-pyridinium-4?,5?-dihydro-4,5?,8-trimethylpsoralen; H 2TMP, 4?,5?-dihydro-4,5?,8-trimethylpsoralen; IFN-?, interferon-?; NOS2, inducible nitric oxide synthase; LB, Luria-Bertani broth; PUVA, psoralen plus UVA light; RT-PCR, reverse transcription-polymerase chain reaction; TdR, thymidine; TMP, 4,5?,8-trimethylpsoralen; and UVA light, ultraviolet light in the range of 320�400 nm
Psoralens such as 8-methoxypsoralen and 4,5?,8-trimethylpsoralen (TMP) are used in photochemotherapy for the treatment of a variety of epidermal proliferative diseases. Sequential treatments of the skin with psoralens plus ultraviolet light in the range of 320�400 nm (UVA light), referred to as PUVA therapy, results in the suppression of abnormal keratinocyte growth. With the recognition that the psoralens are phototoxic
Thomas M. Mariano; Anna M. Vetrano; Shannon L. Gentile; Diane E. Heck; Marilyn S. Whittemore; Christophe D. Guillon; Ivan Jabin; Robert D. Rapp; Ned D. Heindel; Jeffrey D. Laskin
Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts.
Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes. PMID:24371457
Ramos-Jerz, Maria Del R; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M
Stimulation of In Vivo Angiogenesis by In Situ Crosslinked, Dual Growth Factor-loaded, Glycosaminoglycan Hydrogels
As part of a study of elicited angiogenesis, hyaluronan (HA)-based hydrogels crosslinked by polyethylene glycol diacrylate (PEGDA) were loaded with combinations of the cytokine growth factors vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), keratinocyte growth factor (KGF) and platelet derived growth factor (PDGF). GF release in vivo was controlled by covalent incorporation of thiol-modified heparin into thiolated HA hydrogels, which were injected into the ear pinnae of mice and allowed to crosslink in situ. GF release in vivo was controlled by covalent incorporation of thiol-modified heparin in the gels. The ears were harvested at 7 or 14 days post implantation, and vascularization evaluated via a Neovascularization Index (NI). The study demonstrates that in situ gelling implants produced no gross inflammation, redness or swelling, and an improved tolerance compared to HA-based dry film implants. All treatments showed significantly more vascularization than either contralateral ears or ears receiving a sham surgery. The maximum response was observed after 14 days in the ears receiving 0.3% Hp, gelatin-containing gels loaded with VEGF+KGF (NI = 3.91). The study revealed injected growth factor-loaded HA-based hydrogels can successfully produce localized controllable vascularization, while minimizing tissue necrosis, polymorphonuclear leukocytes and inflammation. The ability to target and controllably release growth factors can prove a useful tool in specific diseased tissue/organ angiogenesis.
Elia, Roberto; Fuegy, Peter W.; VanDelden, Aaron; Firpo, Matthew A.; Prestwich, Glenn D.; Peattie, Robert A.
Factor Migration: Trade Theory and Growth Centers.
National Technical Information Service (NTIS)
The paper examines the growth center and polarization within the framework of international trade theory, particularly as it relates to factor movements and, in turn, to regional growth possibilities. International trade theory suggests that all countries...
C. W. Hultman
Design and Synthesis of Binding Growth Factors
Growth factors play important roles in tissue regeneration. However, because of their instability and diffusible nature, improvements in their performance would be desirable for therapeutic applications. Conferring binding affinities would be one way to improve their applicability. Here we review techniques for conjugating growth factors to polypeptides with particular affinities. Conjugation has been designed at the level of gene fusion and of polypeptide ligation. We summarize and discuss the designs and applications of binding growth factors prepared by such conjugation approaches.
Tada, Seiichi; Kitajima, Takashi; Ito, Yoshihiro
Production of functional active human growth factors in insects used as living biofactories.
Growth factors (GFs) are naturally signalling proteins, which bind to specific receptors on the cell surface. Numerous families of GFs have already been identified and remarkable progresses have been made in understanding the pathways that these proteins use to activate/regulate the complex signalling network involved in cell proliferation or wound healing processes. The bottleneck for a wider clinical and commercial application of these factors relay on their scalable cost-efficient production as bioactive molecules. The present work describes the capacity of Trichoplusia ni insect larvae used as living bioreactors in combination with the baculovirus vector expression system to produce three fully functional human GFs, the human epidermal growth factor (huEGF), the human fibroblast growth factor 2 (huFGF2) and the human keratinocyte growth factor 1 (huKGF1). The expression levels obtained per g of insect biomass were of 9.1, 2.6 and 3mg for huEGF, huFGF2 and huKGF1, respectively. Attempts to increase the productivity of the insect/baculovirus system we have used different modifications to optimize their production. Additionally, recombinant proteins were expressed fused to different tags to facilitate their purification. Interestingly, the expression of huKGF1 was significantly improved when expressed fused to the fragment crystallizable region (Fc) of the human antibody IgG. The insect-derived recombinant GFs were finally characterized in terms of biological activity in keratinocytes and fibroblasts. The present work opens the possibility of a cost-efficient and scalable production of these highly valuable molecules in a system that favours its wide use in therapeutic or cosmetic applications. PMID:24915129
Dudognon, Benoit; Romero-Santacreu, Lorena; G�mez-Sebasti�n, Silvia; Hidalgo, Ana B; L�pez-Vidal, Javier; Bellido, Mar�a L; Mu�oz, Eduardo; Escribano, Jos� M
Growth factors, nutrient signaling, and cardiovascular aging
Growth factors regulated by specific macronutrients have been shown to promote aging and accelerate mortality in the great majority of the organisms studied. In particular, the enzymes activated by growth hormone (GH), insulin and insulin-like growth factor 1 (IGF-I) in mammals and their orthologs in simple model organisms represent perhaps the best-understood proteins involved in the aging process. Dietary restriction (DR), which reduces the level of IGF-I and of other growth factors, has been associated with protection from diabetes, cancer, and cardiovascular diseases and deficiencies in GH signaling and IGF-I are strongly associated with protection from cancer and diabetes in both mice and humans, but their role in cardiac function and cardiovascular diseases is controversial. Here we review the link between growth factors, cardiac function and heart disease with focus on the cardioprotective and sensitizing effect of growth factors in both model organisms and humans.
Fontana, Luigi; Vinciguerra, Manlio; Longo, Valter D.
Insulin like growth factors axis and growth disorders
The growth hormone-insulin like growth factor (GH-IGF) axis plays a crucial role in the regulation of growth. Initially considered\\u000a to be a mediator of growth hormone actions, IGF axis has been established as an independent endocrine system with wide array\\u000a of actions. Recent advances have led to tremendous increase in the clinical utility of the IGF axis. IGF-based investigations\\u000a (IGF1
Anurag Bajpai I; P. S. N. Menon
Growth factor transgenes interactively regulate articular chondrocytes.
Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin-like growth factor I (IGF-I), fibroblast growth factor-2 (FGF-2), transforming growth factor beta1 (TGF-?1), bone morphogenetic protein-2 (BMP-2), and bone morphogenetic protien-7 (BMP-7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF-I and FGF-2 maximized cell proliferation. The three-transgene group encoding IGF-I, BMP-2, and BMP-7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell-based articular cartilage repair. PMID:23097312
Shi, Shuiliang; Mercer, Scott; Eckert, George J; Trippel, Stephen B
Developmental and Hormonal Regulation of Keratinocyte Growth Factor Expression and Action in the Ovarian Follicle
The developing ovarian follicle is one of the most rapidly prolifer- ating normal tissues in vivo. Mesenchymal-epithelial cell interactions between theca cells and granulosa cells are essential for this follicular expansion. Ovarian hormones (i.e. estrogen and LH) may promote follicular development by regulating the local production of mesen- chymal inducer proteins that mediate theca cell-granulosa cell inter- actions. Recently, theca
Activated keratinocytes in the epidermis of hypertrophic scars.
The etiology of hypertrophic scarring, a pathological end point of wound healing, is unknown. The scars most commonly occur when epithelialization has been delayed during, for example, the healing of deep dermal burn wounds. Hypertrophic scars are conventionally described as a dermal pathology in which the epidermis has only a passive role. In this study, the expression of keratin intermediate filament proteins and filaggrin has been investigated in the epidermis of hypertrophic scars and site-matched controls from the same patients. Hypertrophic scar epidermis was found to express the hyperproliferative keratins K6 and K16 in interfollicular epidermis in association with K17 and precocious expression of filaggrin. K16 mRNA was localized by in situ hybridization using a highly specific cRNA probe. In contrast to the immunohistochemical location of K16 protein, the K16 mRNA was found to be expressed in the basal cell layer of normal skin. In hypertrophic scars the mRNA distribution corroborated the abnormal K16 protein distribution. These results suggest the keratinocytes in hypertrophic scar epidermis have entered an alternative differentiation pathway and are expressing an activated phenotype. Activated keratinocytes are a feature of the early stages of wound healing producing growth factors that influence fibroblasts, endothelial cells, and the inflammatory response. We propose that cellular mechanisms in the pathogenesis of hypertrophic scarring are more complex than isolated dermal phenomena. The persistence of activated keratinocytes in hypertrophic scar epidermis implicates abnormal epidermal-mesenchymal interactions. Images Figure 1 Figure 3
Machesney, M.; Tidman, N.; Waseem, A.; Kirby, L.; Leigh, I.
Growth factor involvement in tension-induced skeletal muscle growth
NASA Technical Reports Server (NTRS)
Long-term manned space travel will require a better understanding of skeletal muscle atrophy which results from microgravity. Astronaut strength and dexterity must be maintained for normal mission operations and for emergency situations. Although exercise in space slows the rate of muscle loss, it does not prevent it. A biochemical understanding of how gravity/tension/exercise help to maintain muscle size by altering protein synthesis and/or degradation rate should ultimately allow pharmacological intervention to prevent muscle atrophy in microgravity. The overall objective is to examine some of the basic biochemical processes involved in tension-induced muscle growth. With an experimental in vitro system, the role of exogenous and endogenous muscle growth factors in mechanically stimulated muscle growth are examined. Differentiated avian skeletal myofibers can be 'exercised' in tissue culture using a newly developed dynamic mechanical cell stimulator device which simulates different muscle activity patterns. Patterns of mechanical activity which significantly affect muscle growth and metabolic characteristics were found. Both exogenous and endogenous growth factors are essential for tension-induced muscle growth. Exogenous growth factors found in serum, such as insulin, insulin-like growth factors, and steroids, are important regulators of muscle protein turnover rates and mechanically-induced muscle growth. Endogenous growth factors are synthesized and released into the culture medium when muscle cells are mechanically stimulated. At least one family of mechanically induced endogenous factors, the prostaglandins, help to regulate the rates of protein turnover in muscle cells. Endogenously synthesized IGF-1 is another. The interaction of muscle mechanical activity and these growth factors in the regulation of muscle protein turnover rates with our in vitro model system is studied.
Vandenburgh, Herman H.
Vascular Endothelial Growth Factor B, a Novel Growth Factor for Endothelial Cells
We have isolated and characterized a novel growth factor for endothelial cells, vascular endothelial growth factor B (VEGF-B), with structural similarities to vascular endothelial growth factor (VEGF) and placenta growth factor. VEGF-B was particularly abundant in heart and skeletal muscle and was coexpressed with VEGF in these and other tissues. VEGF-B formed cell-surface-associated disulfide-linked homodimers and heterodimerized with VEGF when
Birgitta Olofsson; Katri Pajusola; Arja Kaipainen; Gabriel von Euler; Vladimir Joukov; Olli Saksela; Arto Orpana; Ralf F. Pettersson; Kari Alitalo; Ulf Eriksson
Growth Factors in Alzheimer�s Disease
Growth factors have the potential to potently influence neuronal survival and function. Their broad efficacy against a variety\\u000a of pathogenic insults likely arises because growth factors influence final common mechanisms that mediate cell atrophy and\\u000a death, including apoptosis-related mechanisms (bcl-2, bax, and the caspases) as well as genes important for cell function\\u000a (ERK\\/MAP kinase, CREB, others). As such, the growth
A. H. Nagahara; M. H. Tuszynski
Transforming growth factor ? and epidermal growth factor expression in experimental murine polycystic kidney disease
Cystic change in polycystic kidney disease (PKD) is associated with epithelial hyperplasia, altered fluid and electrolyte transport, and de-differentiation of renal tubular epithelium. The role of polypeptide growth factors as potential modulators of cystic change remains an area of controversy. In this study, the expression of epidermal growth factor (EGF) and transforming growth factor-a (TGFa) were assessed by immunohistochemistry and
Malcolm R. Ogborn; Sanjay Sareen
Growth factors from genes to clinical application
The last decade has witnessed an explosion in the identification of growth factors and their receptors. This has been greatly facilitated by recombinant DNA technology, which has provided the tools not only to identify these proteins at the gene level but also to produce recombinant proteins for evaluating their biological activities. With the help of such techniques, we are moving toward an understanding of the biosynthesis of growth factors and their receptors, structure-function relationships, as well as mechanisms for intracellular signal transmission. The possibility of modifying these factors has opened new fields of clinical application. In this paper, four major areas of growth factor research are presented: the characterization of growth factor genes and their protein products, growth factor receptors and signal transduction by the receptors to mediate biological action, the biological actions of the various growth factors, and the role of growth factors in health and disease and their possible clinical application. Some of the topics covered include: structure of the IGFs and their variants; isoforms of PDGF receptor types; tyrosine kinase activation; structure of G-proteins in biological membranes; possible therapeutic application of NGF in the treatment of Parkinson's and Alzheimer's diseases; PDGF's possible role in the development of several fibroproliferative diseases and its therapeutic application in wound healing; and the possible use of angiogenic inhibitors in tumor treatment.
Sara, V.R. (Dept. of Pathology, Karolinska Hospital, Stockholm (SE)); Hall, K.; Low, H. (Dept. of Endocrinology, Karolinska Hospital, Stockholm (SE))
Id2 gene-targeted crosstalk between Wnt and retinoid signaling regulates proliferation in human keratinocytes
We investigated the effect of all-trans-retinoic acid (atRA) on proliferation in several human skin cell lines and found that antiproliferative potency of atRA correlated with the endogenous activity of canonical Wnt signaling. In HaCaT keratinocytes, we found that atRA significantly suppressed the expression of Id2, a member of the inhibitor of differentiation family of transcription factors that regulate cell growth
A Memezawa; I Takada; K Takeyama; M Igarashi; S Ito; S Aiba; S Kato; A P Kouzmenko
\\u000a In autism, disruption of normal neurobiological mechanisms is found, but it is not known which specific developmentally important\\u000a molecules might be involved in this disorder. Increased cerebral volume or brain weight is found across studies in autism.\\u000a Pathological brain growth and premature developmental arrest are suggested to be restricted to the first years of life. We\\u000a found a correlation between
Raili Riikonen
Growth factor involvement in tension-induced skeletal muscle growth
NASA Technical Reports Server (NTRS)
Muscle tissue culture techniques were developed to grow skeletal myofibers which differentiate into more adult-like myofibers. Mechanical simulation studies of these muscle cells in a newly developed mechanical cell simulator can now be performed to study growth processes in skeletal muscle. Conditions in the mechanical cell simulator were defined where mechanical activity can either prevent muscle wasting or stimulate muscle growth. The role of endogenous and exogenous growth factors in tension-induced muscle growth is being investigated under the defined conditions of tissue culture.
Vandenburgh, H. H.
Antiplatelet-derived growth factor (PDGF) activity in the saliva of ixodid ticks is linked with their long mouthparts.
The saliva of blood-feeding arthropods modulates their vertebrate hosts' haemostatic, inflammatory and immune responses to facilitate blood feeding. In a previous study, we showed that salivary gland products from ixodid tick species also manipulate the wound-healing response by targeting at least four different mammalian growth factors: transforming growth factor ?1, hepatocyte growth factor, fibroblast growth factor 2 and platelet-derived growth factor (PDGF). In addition, species that showed PDGF-binding activity also inhibited cell proliferation in vitro and induced changes in cell morphology accompanied by disruption of the actin cytoskeleton. Here, we show a correlation between the length of the tick hypostome, the sclerotized feeding tube of the mouthparts inserted into the host's skin and anti-PDGF activity. This apparent link between hypostome length, and hence the potential depth of skin damage, and PDGF-binding activity was not apparent for the other growth factors or for other cytokines important in wound healing (keratinocyte growth factor, interleukin 6 and stromal cell-derived factor 1). However, PDGF-binding activity was no longer correlated with anti-cell activities, indicating that an additional as yet unidentified activity in tick saliva may affect cellular changes in wound repair. PMID:24102426
Slov�k, M; �tibr�niov�, I; Hajnick�, V; Nuttall, P A
P2Y2 receptor inhibits EGF-induced MAPK pathway to stabilise keratinocyte hemidesmosomes.
?6?4 integrin is the main component of hemidesmosomes (HD) that stably anchor the epithelium to the underlying basement membrane. Epithelial cell migration requires HD remodelling, which can be promoted by epidermal growth factor (EGF). We previously showed that extracellular nucleotides inhibit growth factor-induced keratinocyte migration. Here, we investigate the effect of extracellular nucleotides on ?6?4 integrin localisation in HD during EGF-induced cell migration. Using a combination of pharmacological inhibition and gene silencing approaches, we found that UTP activates the P2Y2 purinergic receptor and G?q protein to inhibit EGF/ERK1/2-induced cell migration in keratinocytes. Using a keratinocyte cell line expressing an inducible form of the Raf kinase, we show that UTP inhibits the EGF-induced ERK1/2 pathway activation downstream of Raf. Moreover, we established that ERK1/2 activation by EGF leads to the mobilisation of ?6?4 integrin from HD. Importantly, activation of P2Y2R and G?q by UTP promotes HD formation and protects these structures from EGF-triggered dissolution as revealed by confocal analysis of the distribution of ?6?4 integrin, plectin, BPAG1, BPAG2 and CD151 in keratinocytes. Finally, we demonstrated that the activation of p90RSK, downstream of ERK1/2, is sufficient to promote EGF-mediated HD dismantling and that UTP does not stabilise HD in cells expressing an activated form of p90RSK. Our data underline an unexpected role of P2Y2R and G?q in the inhibition of the ERK1/2 signalling pathway and in the modulation of hemidesmosome dynamics and keratinocyte migration. PMID:22718344
Faure, Emilie; Garrouste, Fran�oise; Parat, Fabrice; Monferran, Sylvie; Leloup, Ludovic; Pommier, Gilbert; Kovacic, Herv�; Lehmann, Maxime
Virus transcript levels and cell growth rates after naturally occurring HPV16 integration events in basal cervical keratinocytes.
Cervical carcinogenesis is characterized by a clonal selection process in which the high-risk human papillomavirus (HRHPV) genome usually changes from the extra-chromosomal (episomal) state seen in productive infections to DNA that is integrated into host chromosomes. However, it is not clear whether all HRHPV integration events provide cells with a selective growth advantage compared with the episome-containing cells from which they originate. It is also unclear whether selection of cells containing a particular integrant from a mixed population simply reflects the highest levels of virus oncogene expression or has additional determinants. These early events in cervical carcinogenesis cannot readily be addressed by cross-sectional studies of clinical samples. We used the W12 model system to generate a panel of cervical squamous cell clones that were derived from an identical background under non-competitive conditions and differed only by the genomic site of HPV16 integration. Compared with the 'baseline' episome-containing cells from which they were isolated, only 9/17 clones (53%) showed significantly greater growth rates and only 7/17 (41%) showed significantly greater expression of the major virus oncogenes E7/E6. There were significant variations in levels of HPV16 transcription per DNA template, changes that were associated with histone modifications in the integrated virus chromatin. Cell growth rates showed only weak and non-significant associations with protein and mRNA levels for E7, E6, and the mean E7/E6 values. We conclude that HPV16 integration in basal cervical cells does not necessarily lead to increased levels of virus oncogenes, or to a competitive growth advantage, when compared with the initiating episome-containing cells. � 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. PMID:24752734
Scarpini, Cinzia G; Groves, Ian J; Pett, Mark R; Ward, Dawn; Coleman, Nicholas
Growth factors, tumor promoters, and cancer genes
This book contains over 30 selections. Some of the titles are: Growth-regulated genes and human leukemias; Tyrosyl and phosphatidylinositol kinases of human erythrocyte membranes; Growth factors, oncogenes, and multistage carcinogenesis; Tumorigenic transformation of human teratocarcinoma cells by activated ras oncogene but not the homologous photo-oncogene; and Genes that cooperate with tumor promoters in transformation.
Colburn, N.H.; Moses, H.L.; Stanbridge, E.J.
Transforming growth factor ?s and wound healing
The Transforming Growth Factor ? superfamily (TGF?) is one of the most complex groups of cytokines with widespread effects on many aspects of growth and development. The TGF? isoforms and other family members, e.g. Activins and BMPs, have diverse effects in similar physiological situations. TGF? is involved in the wound healing process. The three mammalian isoforms (TGF?1, 2 and 3)
Sharon O'Kane; Mark W. J. Ferguson
Prevention of disc degeneration with growth factors
Clinically, a large number of patients have persistent low back pain attributable to intervertebral disc (IVD) degeneration.\\u000a After the concept of biologically regenerating the degenerated IVD using growth factor injection was first proposed in early\\u000a 1990, the advancement of molecular technology to produce recombinant proteins, including growth factors, on an industrial\\u000a scale accelerated research in this field. The purpose of
Koichi Masuda; Howard S. An
Autocrine Growth Factor Signaling in Motility
The objective of this chapter is to summarize our current understanding of the role of growth factor autocrine signaling in\\u000a cell motility. The Epidermal Growth Factor Receptor (EGFR) was chosen as a central example system for motivating autocrine\\u000a operation as important in both normal physiological processes as well as pathological conditions such as cancer. We provide\\u000a specific evidence from research
Elizabeth J. Joslin; Douglas A. Lauffenburger
Controlled Delivery of Heparin-Binding EGF-Like Growth Factor Yields Fast and Comprehensive Wound Healing
Wound healing is a dynamic process that relies on coordinated signaling molecules to succeed. Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is proven to accelerate healing, however precise control over its application is necessary to reduce side effects and achieve desired therapeutic benefit. To achieve effective growth factor delivery we designed a bioactive heparin-based coacervate. In vitro, HB-EGF released from the coacervate delivery system displayed enhanced bioactivity and promoted human keratinocyte migration while preserving cell proliferative capability. In a mouse excisional full-thickness wound model, controlled release of HB-EGF within the wound significantly accelerated wound closure more effectively than an equal dosage of free HB-EGF. Healing was induced by rapid re-epithelialization, granulation tissue formation, and accompanied by angiogenesis. Consistent with in vitro results, wounds treated with HB-EGF coacervate exhibited enhanced migration of keratinocytes with retained proliferative potential, forming a confluent layer for regained barrier function within 7 days. Collectively, these results suggest that coacervate-based controlled release of HB-EGF may serve as a new therapy to accelerate healing of cutaneous wounds.
Johnson, Noah Ray; Wang, Yadong
Solar ultraviolet-induced erythema in human skin and nuclear factor-kappa-B-dependent gene expression in keratinocytes are modulated by a French maritime pine bark extract.
The procyanidin-rich French maritime pine bark extract Pycnogenol (PBE) has been investigated for its effect in protecting human skin against solar UV-simulated light-induced erythema. Twenty-one volunteers were given an oral supplementation of Pycnogenol: 1.10 mg/kg body weight (b. wt.)/d for the first 4 weeks and 1.66 mg/kg b. wt./d for the next 4 weeks. The minimal erythema dose (MED) was measured twice before supplementation (baseline MED), once after the first 4 weeks of supplementation, and a last time at the end of the study. The UVR dose necessary to achieve 1 MED was significantly increased during PBE supplementation. Since the activation of the pro-inflammatory and redox-regulated transcription factor NF-kappaB is thought to play a major role in UVR-induced erythema, the effect of PBE was also investigated in the human keratinocyte cell line HaCaT. PBE, added to the cell culture medium, inhibited UVR-induced NF-kappaB-dependent gene expression in a concentration-dependent manner. However, NF-kappaB-DNA-binding activity was not prevented, suggesting that PBE affects the transactivation capacity of NF-kappaB. These data indicate that oral supplementation of PBE reduces erythema in the skin. Inhibition of NF-kappaB-dependent gene expression by PBE possibly contributes to the observed increase in MED. PMID:11163532
Saliou, C; Rimbach, G; Moini, H; McLaughlin, L; Hosseini, S; Lee, J; Watson, R R; Packer, L
Calcium-Regulated Differentiation of Normal Human Epidermal Keratinocytes in Chemically Defined Clonal Culture and Serum-Free Serial Culture
An improved serum-free culture system has been developed for normal human epidermal keratinocytes (HK), Short-term clonal growth and differentiation studies are routinely performed in a defined medium consisting of optimized nutrient medium MCDB 153 supplemented with epidermal growth factor, insulin, hydrocortisone, ethanolamine, and phosphoethanolamine. A small amount of whole bovine pituitary extract (wBPE) is added for initiation of primary cultures,
Steven T. Boyce; Richard G. Ham
Transforming growth factor alpha induces collagen degradation and cell migration in differentiating human epidermal raft cultures.
When cultured on plastic and treated with transforming growth factor alpha (TGF alpha), human keratinocytes exhibit an increase in proliferation at the colony periphery, apparently as a consequence of enhanced cell migration (Barrandon and Green, 1987). To investigate the effects of TGF alpha on a differentiating stratified squamous epithelium and to begin to examine the molecular basis mediating this influence, we cultured human epidermal cells on a gelled lattice of collagen and fibroblasts, floating on the air-liquid interface. Under these conditions, raft cultures differentiate and exhibit morphological and biochemical features of human skin in vivo (Asselineau et al., 1986; Kopan et al., 1987). When 3-wk-old raft cultures were treated with TGF alpha, basal cells showed a marked increase in cell proliferation. At elevated concentrations of TGF alpha, the organization of cells within the artificial tissue changed and islands of basal cells entered the collagen matrix. Biochemical analysis of the response revealed that type I collagenase and gelatinase were induced by keratinocytes within 12 h after TGF alpha treatment. In contrast, invasion of basal cells into the collagen matrix was not significant until 48-72 h post-treatment, suggesting that collagenase and gelatinase production may be a prerequisite to this phenomenon. These results have important implications for the possible role of TGF alpha in squamous cell carcinoma and tumor invasion. Images
Turksen, K; Choi, Y; Fuchs, E
The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes.
The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles.
Straight, S W; Herman, B; McCance, D J
Murine Oligodendroglial Cells Express Nerve Growth Factor
The studies reported here present evidence for the expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) by an oligodendroglial cell line and of NGF by oligodendrocytes in mouse primary culture. An immortalized oligodendroglial cell line (N19) expressing markers for immature oligodendrocytes stimulated PC12 cells to elaborate processes. Polymerase chain reaction analysis with degenerate primers indicated that the
Sujatha Byravan; Lyndon M. Foster; Tommy Phan; A. Neil Verity; Anthony T. Campagnoni
Growth\\/differentiation factor-11: an evolutionary conserved growth factor in vertebrates
Growth and differentiation factor-11 (GDF-11) is a member of the transforming growth factor-? superfamily and is thought to\\u000a be derived together with myostatin (known also as GDF-8) from an ancestral gene. In the present study, we report the isolation\\u000a and characterization of GDF-11 homolog from a marine teleost, the gilthead sea bream Sparus aurata, and show that this growth factor
Bruria Funkenstein; Elena Olekh
Autologous keratinocyte suspension in platelet concentrate accelerates and enhances wound healing - a prospective randomized clinical trial on skin graft donor sites: platelet concentrate and keratinocytes on donor sites
Background Wound healing involves complex mechanisms, which, if properly chaperoned, can enhance patient recovery. The abilities of platelets and keratinocytes may be harnessed in order to stimulate wound healing through the formation of platelet clots, the release of several growth factors and cytokines, and cell proliferation. The aim of the study was to test whether autologous keratinocyte suspensions in platelet concentrate would improve wound healing. The study was conducted at the Lausanne University Hospital, Switzerland in 45 patients, randomized to three different topical treatment groups: standard treatment serving as control, autologous platelet concentrate (PC) and keratinocytes suspended in autologous platelet concentrate (PC?+?K). Split thickness skin graft donor sites were chosen on the anterolateral thighs of patients undergoing plastic surgery for a variety of defects. Wound healing was assessed by the duration and quality of the healing process. Pain intensity was evaluated at day five. Results Healing time was reduced from 13.9?�?0.5 days (mean?�?SEM) in the control group to 7.2?�?0.2 days in the PC group (P?0.01). An addition of keratinocytes in suspension further reduced the healing time to 5.7?�?0.2 days. Pain was reduced in both the PC and PC?+?K groups. Data showed a statistically detectable advantage of using PC?+?K over PC alone (P?0.01). Conclusion The results demonstrate the positive contribution of autologous platelets combined with keratinocytes in stimulating wound healing and reducing pain. This strikingly simple approach could have a significant impact on patient care, especially critically burned victims for whom time is of the essence. Clinical trial registry information Protocol Record Identification Number: 132/03 Registry URL: http://www.clinicaltrials.gov
AHR: Making the Keratinocytes Thick Skinned.
Dysregulated signals from the external environment and/or the internal milieu of the skin can lead to pathological conditions such as psoriasis. Di Meglio et�al. (2014) show that the environment-responsive transcription factor AhR acts in keratinocytes to suppress psoriatic lesions. PMID:24950209
Colonna, Marco
Activated protein C: A regulator of human skin epidermal keratinocyte function
Activated protein C (APC) is a physiological anticoagulant, derived from its precursor protein C (PC). Independent of its anticoagulation, APC possesses strong anti-inflammatory, anti-apoptotic and barrier protective properties which appear to be protective in a number of disorders including chronic wound healing. The epidermis is the outermost skin layer and provides the first line of defence against the external environment. Keratinocytes are the most predominant cells in the epidermis and play a critical role in maintaining epidermal barrier function. PC/APC and its receptor, endothelial protein C receptor (EPCR), once thought to be restricted to the endothelium, are abundantly expressed by skin epidermal keratinocytes. These cells respond to APC by upregulating proliferation, migration and matrix metalloproteinase-2 activity and inhibiting apoptosis/inflammation leading to a wound healing phenotype. APC also increases barrier function of keratinocyte monolayers by promoting the expression of tight junction proteins and re-distributing them to cell-cell contacts. These cytoprotective properties of APC are mediated through EPCR, protease-activated receptors, epidermal growth factor receptor or Tie2. Future preventive and therapeutic uses of APC in skin disorders associated with disruption of barrier function and inflammation look promising. This review will focus on APC�s function in skin epidermis/keratinocytes and its therapeutical potential in skin inflammatory conditions.
McKelvey, Kelly; Jackson, Christopher John; Xue, Meilang
High-Glucose Environment Enhanced Oxidative Stress and Increased Interleukin-8 Secretion From Keratinocytes
Impaired wound healing frequently occurs in patients with diabetes. Interleukin (IL)-8 production by keratinocyte is responsible for recruiting neutrophils during healing. Intense inflammation is associated with diabetic wounds, while reduction of neutrophil infiltration is associated with enhanced healing. We hypothesized that increased neutrophil recruitment by keratinocytes may contribute to the delayed healing of diabetic wounds. Using cultured human keratinocytes and a diabetic rat model, the current study shows that a high-glucose environment enhanced IL-8 production via epidermal growth factor receptor (EGFR)�extracellular signal�regulated kinase (ERK) pathway in a reactive oxygen species (ROS)-dependent manner in keratinocytes. In addition, diabetic rat skin showed enhanced EGFR, ERK, and IL-8 expression compared with control rats. The dermal neutrophil infiltration of the wound, as represented by expression of myeloperoxidase level, was also significantly higher in diabetic rats. Treating diabetic rats with dapsone, an agent known to inhibit neutrophil function, was associated with improved healing. In conclusion, IL-8 production and neutrophil infiltration are increased in a high-glucose environment due to elevated ROS level and contributed to impaired wound healing in diabetic skin. Targeting these dysfunctions may present novel therapeutic approaches.
Lan, Cheng-Che E.; Wu, Ching-Shuang; Huang, Shu-Mei; Wu, I-Hui; Chen, Gwo-Shing
Isolation and propagation of keratinocytes derived from Cashmere goat fetus.
The study was conducted to isolate epidermal keratinocytes from Cashmere goat fetus with the aim to develop suitable conditions for keratinocyte cultivation and propagation. The methods developed for keratinocyte culture include (i) use of a feeder-layer of mitotically inactivated fibroblasts obtained from goat and mouse fetal skin, (ii) use of a substrate such as collagen IV, or (iii) without use of any substrate. Epidermal cell removal was established by enzymatically separating keratinocytes from 12 to 16 weeks aged fetal skin tissues treated with 0.125% trypsin solution overnight at 4 degrees C. The cells were maintained in all culture conditions with serum containing medium. Keratinocyte multiplication and proliferation were comparable in different culture conditions and the improved cellular attachment and growth have been obtained in cultures on feeder layers. Colony forming keratinocytes on feeder layer were heterogeneous in their growth potential. In feeder free conditions, high cellular density was required at plating for sub-cultivation as their poor attachment in culture dishes. This study reports the comparative efficacy of different culture conditions for keratinocyte isolation and in vitro propagation originating from Cashmere goat fetus. PMID:17881027
Islam, M S; Zhou, H M
Placenta Growth Factor in Diabetic Wound Healing
Reduced microcirculation and diminished expression of growth factors contribute to wound healing impairment in diabetes. Placenta growth factor (PlGF), an angiogenic mediator promoting pathophysiological neovascularization, is expressed during cutaneous wound healing and improves wound closure by enhancing angiogenesis. By using streptozotocin-induced diabetic mice, we here demonstrate that PlGF induction is strongly reduced in diabetic wounds. Diabetic transgenic mice overexpressing PlGF in the skin displayed accelerated wound closure compared with diabetic wild-type littermates. Moreover, diabetic wound treatment with an adenovirus vector expressing the human PlGF gene (AdCMV.PlGF) significantly accelerated the healing process compared with wounds treated with a control vector. The analysis of treated wounds showed that PlGF gene transfer improved granulation tissue formation, maturation, and vascularization, as well as monocytes/macrophages local recruitment. Platelet-derived growth factor, fibroblast growth factor-2, and vascular endothelial growth factor mRNA levels were increased in AdCMV.PlGF-treated wounds, possibly enhancing PlGF-mediated effects. Finally, PlGF treatment stimulated cultured dermal fibroblast migration, pointing to a direct role of PlGF in accelerating granulation tissue maturation. In conclusion, our data indicate that reduced PlGF expression contributes to impaired wound healing in diabetes and that PlGF gene transfer to diabetic wounds exerts therapeutic activity by promoting different aspects of the repair process.
Cianfarani, Francesca; Zambruno, Giovanna; Brogelli, Laura; Sera, Francesco; Lacal, Pedro Miguel; Pesce, Maurizio; Capogrossi, Maurizio C.; Failla, Cristina Maria; Napolitano, Monica; Odorisio, Teresa
Transforming Growth Factor-?1Antisense Modulates the Expression of Hepatocyte Growth Factor\\/Scatter Factor in Keloid Fibroblast Cell Culture
Abnormal wound healing processes can result in hypertrophic scars and keloids. Transforming growth factor-?1 (TGF-?1) and\\u000a hepatocyte growth factor\\/scatter factor (HGF\\/SF) are biphasic growth factor cytokines in physiologic and pathophysiologic\\u000a conditions. Findings have shown TGF-?1 to be pivotal in the formation of keloid tissue. Therefore, neutralizing antibodies\\u000a may allow wound healing without keloid formation. As reported, TGF-?1 is antagonized by
R. Naim; A. Naumann; J. Barnes; A. Sauter; K. Hormann; D. Merkel; W. Aust; T. Braun; M. Bloching
Sinclair, N. A. (Washington State University, Pullman) and J. L. Stokes. Factors which control maximal growth of bacteria. J. Bacteriol. 83:1147�1154. 1962.�In a chemically defined medium containing 1% glucose and 0.1% (NH4)2SO4, both of these compounds are virtually exhausted by the growth of Pseudomonas fluorescens. If these carbon, energy, and nitrogen sources are added back to the culture filtrate, maximal growth to the level of the original culture is obtained. This process can be repeated several times with the same results. Eventually, however, the supply of minerals in the culture limits growth. When the nutrient levels are raised to 3% glucose and 0.3% (NH4)2SO4, lack of oxygen and low pH limit growth before the supply of nutrients is exhausted. There is no evidence that specific autoinhibitory substances are produced either in chemically defined or complex nitrogenous media or that physical crowding of the cells limits growth. The results with Escherichia coli are similar to those with P. fluorescens. However, after a few growth cycles aerobically and after only one growth cycle anaerobically, inhibitory substances, probably organic acids, accumulate and limit growth.
Sinclair, N. A.; Stokes, J. L.
Peptide growth factors: clinical and therapeutic strategies.
The literature contains many accounts of studies in which tumour growth has been accelerated by administration of a particular mitogen and the response then inhibited by co-administration of the corresponding antagonist. Much effort has been focused on the development of cytokine or growth factor antagonists. Like most other cancer therapies, biological therapies will undoubtedly have undesirable toxicities because the proteins they target may not be unique to malignant cells. We reviewed the clinical and therapeutic potential of growth factor agonists and antagonists in some non urologic and urologic diseases. In a recent report we demonstrated that both androgen and antiandrogen treatments enhance the proliferation rate of the hormone-dependent prostate cancer cell line LNCaP, expressing a mutated androgen receptor. Simultaneous treatment with 1 nM R1881 and 100 nM OH-Flutamide, completely counteracted the androgen-induced increase of Epidermal Growth Factor (EGF) levels. Moreover we found that Testosterone, DHT and EGF are mainly concentrated in the periurethral zone in human BPH and long term treatment with Finasteride and with Flutamide modify the distribution and concentration of these factors. Some authors analyzed whether and addition of aurin tricarboxylic acid (ATA) can reduce the growth rate of basic FGF-dependent cells in a manner similar to suramin. PMID:9228827
Di Silverio, F; Sciarra, A; Di Nicola, S; Di Chiro, C
Epidermal growth factor receptors in idiopathic and virally induced skin diseases.
The altered distribution of epidermal growth factor receptors (EGF-R) in hyperproliferative skin lesions such as psoriasis vulgaris, seborrheic keratoses, acanthosis nigricans, ichthyosis, and others implies aberrant control of growth/proliferation by epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and other growth factors/cytokines. Whether overexpression of EGF-R: 1) correlates with epidermal proliferation, 2) serves as a hallmark of specific dermatoses, or 3) is due to modulation by multiple growth factors remains unclear. To correlate distributions of EGF-R with in vivo proliferative status, two benign diseases of unknown etiology, seborrheic keratoses and acrochordons (skin tags), were examined using EGF-R immunolocalization and 125I-EGF binding techniques. Lesions documented as growing by clinical criteria or 5-bromodeoxyuridine incorporation (a measure of cell proliferation) were compared to nongrowing lesions of the same type. To correlate distributions of EGF-R to specific dermatoses, skin diseases of viral etiology (verruca vulgaris and molluscum contagiosum) were also probed by EGF-R immunolocalization and 125I-EGF binding. Elevated immunostaining for EGF-R and 125I-EGF binding sites were associated with actively growing seborrheic keratoses and skin tags whereas normal patterns of immunostaining and 125I-EGF binding were seen in nongrowing seborrheic keratoses and skin tags. Viral diseases showed unique patterns. No EGF-R were detected in verruca vulgaris. Molluscum contagiosum lesions showed intense EGF-R in basal keratinocytes and no EGF-R in virally infected cells. Thus elevations in EGF-R show a positive in vivo correlation with proliferation in at least two differing benign diseases of the epidermis. The decreased levels of EGF-R in virally infected lesions suggests that EGF-R may show unique patterns for specific dermatoses and are not universally elevated in benign hyperproliferative skin disorders. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7
Nanney, L. B.; Ellis, D. L.; Levine, J.; King, L. E.
Review of epidermal growth factor receptor biology
The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that constitutes one of four members of the erbB family of tyrosine kinase receptors. Binding of EGFR to its cognate ligands leads to autophosphorylation of receptor tyrosine kinase and subsequent activation of signal transduction pathways that are involved in regulating cellular proliferation, differentiation, and survival. Although present in normal cells,
Roy S Herbst
Epidermal growth factor receptors: a functional perspective.
The epidermal growth factor receptors (EGFRs) belong to the ErbB family of receptor tyrosine kinases (TKs) involved in the proliferation of normal and malignant cells. EGFR has attracted considerable attention as a target for cancer therapy. This article considers various functional roles of EGFR-based systems that are relevant for the early detection and staging of cancers overexpressing EGFR. PMID:22074479
Mitrasinovic, Petar M
Growth factor interactions in bone regeneration
Bone regeneration is a complex process regulated by a large number of bioactive molecules. Many growth factors and cytokines involved in the natural process of bone healing have been identified and tested as potential therapeutic candidates to enhance the regeneration process. Although many of these studies show an enhancement of the bone regeneration process by a single drug therapy, in
D. H. R. Kempen; L. B. Creemers; J. Alblas; L. Lu; A. J. Verbout; M. J. Yaszemski; W. J. A. Dhert
Platelet-Derived Growth Factor-B Normalizes Micromorphology and Vessel Function in Vascular Endothelial Growth Factor-A-Induced Squamous Cell Carcinomas
Vascular endothelial growth factor (VEGF), which is a key regulator of angiogenesis, often induces formation of immature vessels with increased permeability and reduced vessel functionality. Here, we demonstrate that de novo expression of murine (m)VEGF-164 induces malignant and invasive tumor growth of HaCaT keratinocytes. However, the mVEGF-164-induced tumors are ulcerated with a disorganized epithelium that is interrupted by lacunae with limited basement membrane and endothelial cell coverage. Vessel maturation is strongly impaired. Tumor and vessel micromorphology are markedly improved by the combined expression of human platelet-derived growth factor (hPDGF)-B and mVEGF-164. Although tumor size and malignancy are comparable with either mVEGF-164 alone or combined human PDGF-B and mVEGF-164 expression, combined hPDGF-B and mVEGF-164 expression leads to a more solid and compact tumor tissue with a mature functional tumor vasculature and a higher microvessel density, as demonstrated histologically and by dynamic contrast-enhanced magnetic resonance imaging. Treatment of the hPDGF-B- and mVEGF-164-expressing tumors with imatinib mesylate to block PDGF-B signaling reverses this effect. In addition, tumor cell invasion of mVEGF-164 transfectants and mVEGF-164 plus hPDGF-B transfectants in vivo is associated with a marked induction of tumor-derived matrix metalloproteinase-1 and stromal matrix metalloproteinase-9 and -13, as was confirmed in three-dimensional organotypic co-cultures with fibroblasts in vitro. These data clearly demonstrate the need for a concerted action of different growth factors in the establishment of solid tumors with functional vasculature and emphasize the need for a multifactorial therapy.
Lederle, Wiltrud; Linde, Nina; Heusel, Julia; Bzyl, Jessica; Woenne, Eva C.; Zwick, Stefan; Skobe, Mihaela; Kiessling, Fabian; Fusenig, Norbert E.; Mueller, Margareta M.
Differentiated Keratinocyte-Releasable Stratifin (14-3-3 Sigma) Stimulates MMP-1 Expression in Dermal Fibroblasts
Through the use of a keratinocyte\\/fibroblast co-culture system, we have recently identified a potent keratinocyte-derived anti-fibrogenic factor (KDAF) for dermal fibroblasts. A sequential chromatography of the active fractions of keratinocyte-conditioned medium (KCM) and peptide mapping of the candidate proteins identified KDAF as being the keratinocyte-releasable 14-3-3 sigma (14-3-3?) protein, which is also known as stratifin. In this study, we hypothesize
Aziz Ghahary; Yvonne Marcoux; Feridoun Karimi-Busheri; Yunyaun Li; Edward E. Tredget; Ruhangiz T. Kilani; Eugene Lam; Michael Weinfeld
SEOM clinical guidelines for myeloid growth factors.
Neutropenia induced by chemotherapy (CT) is an infection risk factor associated to greater morbidity/mortality and dose-limiting toxicity that on many occasions requires a reduction of the dose of cytostatics or a delay in the administration of treatment. This may have a negative effect on the patient's quality of life and even diminish the efficacy of the treatment, especially when the intention is to cure or prolong survival. Management of treatment or prophylaxis of grade 3-4 neutropenia and febrile neutropenia with myeloid growth factors (CSF) varies very much in clinical practice, both in the time of starting treatment and the types of patients it is given to. The need to generalise and facilitate practice based on clinical evidence has led the Spanish Society of Medical Oncology (SEOM) to prepare clinical practice guidelines on the use of myeloid growth factors. PMID:22721792
Mu�oz Langa, Jos�; Gasc�n, Pere; de Castro, Javier
Nerve growth factor promotes human hemopoietic colony growth and differentiation
Nerve growth factor (NGF) is a neurotropic polypeptide necessary for the survival and growth of some central neurons, as well as sensory afferent and sympathetic neurons. Much is now known of the structural and functional characteristics of NGF, whose gene has recently been clones. Since it is synthesized in largest amounts by the male mouse submandibular gland, its role exclusively in nerve growth is questionable. These experiments indicate that NGF causes a significant stimulation of granulocyte colonies grown from human peripheral blood in standard hemopoietic methylcellulose assays. Further, NGF appears to act in a relatively selective fashion to induce the differentiation of eosinophils and basophils/mast cells. Depletion experiments show that the NGF effect may be T-cell dependent and that NGF augments the colony-stimulating effect of supernatants from the leukemic T-cell (Mo) line. The hemopoietic activity of NGF is blocked by {sup 125}I-polyclonal and monoclonal antibodies to NGF. The authors conclude that NGF may indirectly act as a local growth factor in tissues other than those of the nervous system by causing T cells to synthesize or secrete molecules with colony-stimulating activity. In view of the synthesis of NGF in tissue injury, the involvement of basophils/mast cells and eosinophils in allergic and other inflammatory processes, and the association of mast cells with fibrosis and tissue repair, they postulate that NGF plays an important biological role in a variety of repair processes.
Matsuda, H.; Coughlin, M.D.; Bienenstock, J.; Denburg, J.A. (McMaster Univ. Health Sciences Center, Hamilton, Ontario (Canada))
PCB153 reduces telomerase activity and telomere length in immortalized human skin keratinocytes (HaCaT) but not in human foreskin keratinocytes (NFK)
Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are characterized by long term-persistence in the environment, bioaccumulation, and biomagnification in the food chain. Exposure to PCBs may cause various diseases, affecting many cellular processes. Deregulation of the telomerase and the telomere complex leads to several biological disorders. We investigated the hypothesis that PCB153 modulates telomerase activity, telomeres and reactive oxygen species resulting in the deregulation of cell growth. Exponentially growing immortal human skin keratinocytes (HaCaT) and normal human foreskin keratinocytes (NFK) were incubated with PCB153 for 48 and 24 days, respectively, and telomerase activity, telomere length, superoxide level, cell growth, and cell cycle distribution were determined. In HaCaT cells exposure to PCB153 significantly reduced telomerase activity, telomere length, cell growth and increased intracellular superoxide levels from day 6 to day 48, suggesting that superoxide may be one of the factors regulating telomerase activity, telomere length and cell growth compared to untreated control cells. Results with NFK cells showed no shortening of telomere length but reduced cell growth and increased superoxide levels in PCB153-treated cells compared to untreated controls. As expected, basal levels of telomerase activity were almost undetectable, which made a quantitative comparison of treated and control groups impossible. The significant down regulation of telomerase activity and reduction of telomere length by PCB153 in HaCaT cells suggest that any cell type with significant telomerase activity, like stem cells, may be at risk of premature telomere shortening with potential adverse health effects for the affected organism. -- Highlights: ? Human immortal (HaCaT) and primary (NFK) keratinocytes were exposed to PCB153. ? PCB153 significantly reduced telomerase activity and telomere length in HaCaT. ? No effect on telomere length and telomerase activity was found in NFK. ? Increased intracellular superoxide levels and reduced cell growth was seen in both. ? PCB153 may damage telomerase expressing cells like stem cells.
Senthilkumar, P.K. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States)] [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Robertson, L.W. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States) [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA (United States); Ludewig, G., E-mail: firstname.lastname@example.com [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA (United States)
Altered (/sup 125/I)epidermal growth factor binding and receptor distribution in psoriasis
Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that (/sup 125/I)EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers.
Nanney, L.B.; Stoscheck, C.M.; Magid, M.; King, L.E. Jr.
Pulsed electric current induces the differentiation of human keratinocytes.
Although normal human keratinocytes are known to migrate toward the cathode in a direct current (DC) electric field, other effects of the electric stimulation on keratinocyte activities are still unclear. We have investigated the keratinocyte differentiation under monodirectional pulsed electric stimulation which reduces the electrothermal and electrochemical hazards of a DC application. When cultured keratinocytes were exposed to the electric field of 3 V (ca. 100 mV/mm) or 5 V (ca. 166 mV/mm) at a frequency of 4,800 Hz for 5 min a day for 5 days, cell growth under the 5-V stimulation was significantly suppressed as compared with the control culture. Expression of mRNAs encoding keratinocyte differentiation markers such as keratin 10, involucrin, transglutaminase 1, and filaggrin was significantly increased in response to the 5-V stimulation, while the 3-V stimulation induced no significant change. After the 5-V stimulation, enhanced immunofluorescent stainings of involucrin and filaggrin were observed. These results indicate that monodirectional pulsed electric stimulation induces the keratinocyte differentiation with growth arrest. PMID:23564189
Arai, Koji Y; Nakamura, Yohei; Hachiya, Yuko; Tsuchiya, Hiroyuki; Akimoto, Ryuji; Hosoki, Katsu; Kamiya, Shohei; Ichikawa, Hideyuki; Nishiyama, Toshio
Tumor necrosis factor-alpha promotes tumor growth by inducing vascular endothelial growth factor.
Tumor necrosis factor (TNF)-? has been proved as an adjuvant therapy for tumor by FDA. However, the effect of chronic TNF-? expression for tumor is still controversial. In this study, we investigated the effect of low-dose TNF-? on tumor growth. We confirmed that low-dose TNF-? promoted angiogenesis of tumor in vivo, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1?, the transcription factor of VEGF, were both upregulated. Our results suggested that low-dose TNF-? was a powerful activator of angiogenesis in tumor and HIF-1?-VEGF pathway seemed to be the most important molecular mechanism. PMID:21740086
Jing, Yingying; Ma, Nannan; Fan, Tingting; Wang, Chenyang; Bu, Xinxin; Jiang, Guocheng; Li, Rong; Gao, Lu; Li, Ding; Wu, Mengchao; Wei, Lixin
The E5 Oncoprotein of Human Papillomavirus Type 16 Inhibits the Acidification of Endosomes in Human Keratinocytes
Thehumanpapillomavirustype16E5oncoproteinpossessesmitogenicactivitythatactssynergisticallywith epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observa- tions is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component ofthevacuolarproton-ATPase,sinceanimalandhumanpapillomavirusE5proteinsbindthissubunitprotein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of
Fibroblast Growth Factor Receptor and Platelet-Derived Growth Factor Receptor Abnormalities in Eosinophilic Myeloproliferative Disorders
Rearrangements of the genes encoding the fibroblast growth factor receptor 1 (FGFR1) and platelet-derived growth factor receptors (PDGFR) ? or ? receptor tyrosine kinases are found in a rare but important subset of patients with atypical myeloproliferative disorders that are usually but not always associated with eosinophilia. Chromosomal translocations or other rearrangements at 8p11�12, 4q12 or 5q31�33 give rise to
Nicholas C. P. Cross; Andreas Reiter
Scatter Factor\\/Hepatocyte Growth Factor Expression Enhances Human Glioblastoma Tumorigenicity and Growth
We have shown previously that the multifunctional cytokine scatter factor\\/hepatocyte growth factor (SF\\/HGF) is elevated in human malignant gliomas. In this study we investigated how human SF\\/HGF expression affects the malignancy of the U373 human glioblastoma cell linein vivoandin vitro.Human SF\\/HGF gene transfer increased U373 glioblastoma tumorigenicity by ?20-fold and enhanced the growth rate of intracerebral U373 xenografts by 3-
John Laterra; Eliot Rosen; Myeong Nam; Srikanth Ranganathan; Kevin Fielding; Peter Johnston
Epidermal growth factor receptor signaling in tissue
Abstract: A peptide purified from the salivary gland of a mouse was shown few years ago to accelerate incisor eruption and eyelid opening in newborn mice, and was named epidermal growth factor (EGF). The members of this family of peptide growth factors had been identified in numerous physiological and pathological contexts. EGF binds to a cell surface EGF receptor, which induces a biochemical modification (phosphorylation) of the receptor's cytoplasmic tail. There is a growing consensus in the research community that, in addition to cellular and molecular studies, the dynamics of the EGFR network and its operation must be examined in tissues. A key challenge is to integrate the existing molecular and cellular information into a system-level description of the EGFR network at the tissue and organism level. In this paper, the two examples of EGFR signaling in tissues are described, and the recent efforts to model EGFR autocrine loops, which is a predominant mode of EGFR activation in vivo, are summarized.
Shvartsman, Stanislav; Wiley, H. S.; Lauffenburger, Douglas A.
The linear C-terminal regions of epidermal growth factor (EGF) and transforming growth factor-alpha bind to different epitopes on the human EGF receptor.
Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) bind with similar affinities in a competitive fashion to the human EGF receptor, and basically induce similar mitogenic responses. In spite of the fact that EGF and TGFalpha are structurally alike, it is still not clear if the two growth factors bind the receptor in an identical manner. The observation that the 13A9 antibody blocks binding of TGFalpha, but not that of EGF, to the human EGF receptor [Winkler, O'Connor, Winget and Fendly (1989) Biochemistry 28, 6373-6378] suggests that their binding characteristics are not identical. In the present study we have made use of a set of EGF/TGFalpha chimaeric molecules to show that the 13A9 antibody blocks receptor binding of ligands with TGFalpha sequences, but not of ligands with EGF sequences, in their C-terminal linear regions. Using HaCaT human keratinocyte cells in culture, it was determined that ligands that are able to bind the EGF receptor in the presence of 13A9 are also able to induce calcium release from intracellular stores in these cells, indicating that these ligands have the ability to activate the EGF receptor in the presence of the antibody. From these data it is concluded that the flexible C-terminal linear domains of EGF and TGFalpha bind to separate sequences on the EGF receptor, such that the binding domain of TGFalpha, but not that of EGF, overlaps with the binding epitope of the 13A9 antibody.
Lenferink, A E; De Roos, A D; Van Vugt, M J; Van de Poll, M L; Van Zoelen, E J
Growth Factors and Tension-Induced Skeletal Muscle Growth
NASA Technical Reports Server (NTRS)
The project investigated biochemical mechanisms to enhance skeletal muscle growth, and developed a computer based mechanical cell stimulator system. The biochemicals investigated in this study were insulin/(Insulin like Growth Factor) IGF-1 and Steroids. In order to analyze which growth factors are essential for stretch-induced muscle growth in vitro, we developed a defined, serum-free medium in which the differentiated, cultured avian muscle fibers could be maintained for extended periods of time. The defined medium (muscle maintenance medium, MM medium) maintains the nitrogen balance of the myofibers for 3 to 7 days, based on myofiber diameter measurements and myosin heavy chain content. Insulin and IGF-1, but not IGF-2, induced pronounced myofiber hypertrophy when added to this medium. In 5 to 7 days, muscle fiber diameters increase by 71 % to 98% compared to untreated controls. Mechanical stimulation of the avian muscle fibers in MM medium increased the sensitivity of the cells to insulin and IGF-1, based on a leftward shift of the insulin dose/response curve for protein synthesis rates. (54). We developed a ligand binding assay for IGF-1 binding proteins and found that the avian skeletal muscle cultures produced three major species of 31, 36 and 43 kD molecular weight (54) Stretch of the myofibers was found to have no significant effect on the efflux of IGF-1 binding proteins, but addition of exogenous collagen stimulated IGF-1 binding protein production 1.5 to 5 fold. Steroid hormones have a profound effect on muscle protein turnover rates in vivo, with the stress-related glucocorticoids inducing rapid skeletal muscle atrophy while androgenic steroids induce skeletal muscle growth. Exercise in humans and animals reduces the catabolic effects of glucocorticoids and may enhance the anabolic effects of androgenic steroids on skeletal muscle. In our continuing work on the involvement of exogenrus growth factors in stretch-induced avian skeletal muscle growth, we have performed experiments to determine whether mechanical stimulation of cultured avian muscle cells alters their response to anabolic steroids or glucocorticoids. In static cultures, testosterone had no effect on muscle cell growth, but 5alpha-dihydrotestosterone and the synthetic steroid stanozolol increased cell growth by up to 18% and 30%, respectively, after a three day exposure. We completed development of a new IBM-based mechanical cell stimulator system to provide greater flexibility in operating and monitoring our experiments. Our previous long term studies on myofiber growth were designed around a perfusion system of our own design. We have recently changed to performing these studies using a modified CELLCO cartridge bioreactor system Z since it has been certified as the ground-based model for the Shuttle's Space Tissue Loss (STL) F= Cell Culture Module. The current goals of this aspect of the project are three fold: 1) to design a Z cell culture system for studying avian skeletal myofiber atrophy on the Shuttle and Space Station; 0 2) to expand the use of bioreactors to cells which do not grow in either suspension or attached to the hollow fibers; and 3) to combine the bioreactor system with our computerized mechanical cell stimulator to have a better in vitro model to study tension/gravity/stretch regulation of skeletal muscle size. Preliminary studies also reported on involved : (1) how release of tension can induce rapid atrophy of tissues cultured avian skeletal muscle cells, and (2) a mechanism to transfer and maintain avian skeletal muscle organoids in modified cartridges in the Space Tissue Loss Module.
Vandenburgh, Herman H.
Signaling via Vascular Endothelial Growth Factor Receptors
Angiogenesis, or development of blood vessels from preexisting vasculature, has important functions under both normal and pathophysiological conditions. Vascular endothelial growth factor receptors 1�3, also known as flt-1, KDR, and flt-4, are endothelial cell-specific receptor tyrosine kinases which serve as key mediators of the angiogenic responses. The review focuses on the signaling pathways that are initiated from these receptors and
Tatiana V. Petrova; Taija Makinen; Kari Alitalo
Inhibition of ultraviolet B-mediated activation of nuclear factor ?B in normal human epidermal keratinocytes by green tea Constituent (-)-epigallocatechin-3-gallate
Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, possesses significant anti-inflammatory and cancer chemopreventive properties. Studies have shown the photochemopreventive effects of green tea and EGCG in cell culture, animal models, and human skin. The molecular mechanism(s) of photochemopreventive effects of EGCG are incompletely understood. We recently showed that EGCG treatment of the normal human epidermal keratinocytes (NHEK) inhibits ultraviolet
Farrukh Afaq; Vaqar M Adhami; Nihal Ahmad; Hasan Mukhtar; H Mukhtar
Milk Epidermal Growth Factor and Gut Protection
Maternal milk is a complex fluid with multifunctional roles within the developing gastrointestinal tract. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) are members of the family of EGF-related peptides. Biological actions of these growth factors are mediated via interaction with the EGF-receptor (EGF-R). In the early postnatal period, breast milk is the major source of EGF for the developing intestinal mucosa. HB-EGF is also detected in breast milk, but in concentrations 2 to 3 times lower than EGF. Under normal physiological conditions, the intestinal epithelium undergoes a continuing process of cell proliferation, differentiation and maturation. EGF plays an important role in these processes. In pathophysiologic situations, EGF contributes to epithelial protection from injury and post-injury mucosal repair. Necrotizing enterocolitis (NEC) is a devastating disease affecting prematurely born infants. The pathogenesis of NEC is not known and there is no effective treatment for this disease. In an experimental NEC model, oral administration of a physiological dose of EGF significantly reduces the incidence and severity of NEC. HB-EGF provides similar protection against NEC, but only when pharmacological doses are used. Further studies are necessary before EGF can be introduced as an efficient therapeutic approach of intestinal injury.
Dvorak, Bohuslav
Computerized Microassay of Keratinocyte Cell-Plastic Attachment and Proliferation for Assessing Net Stimulatory, Inhibitory and Toxic Effects of Compounds on Nonimmortalized Cell Lines
Testing of pharmacological agents that affect growth of epidermal keratinocytes (EK) requires a standardized assay. We have developed an assay measuring net effects of stimulatory (e.g. growth factors), inhibitory (e.g. methotrexate) or toxic (e.g. Triton X-100) compounds. The amount of crystal violet staining viable EK attached to the wells of standard 96-well microplates is measured in situ using an ELISA
Sergei A. Grando; Raul Cabrera; Bruce S. Hostager; Paul L. Bigliardi; John S. Blake; Michael J. Herron; Mark V. Dahl; Robert D. Nelson
Transforming Growth Factor fi and Cell Cycle Regulation
In many cases tumor cells develop when normal progenitor cells lose control of signaling pathways that regulate responses to soluble growth factors. These signaling pathways consist of the molecular machinery that regulates cell cycle progression primarily during the first gap phase (G1)of the cell cycle. Whereasgrowth factors such as epidermal growth factor and platelet-derived growth factor stimulate these signaling pathways
Mark G. Alexandrow; Harold L. Moses
Impaired keratinocyte function on matrix metalloproteinase-1 (MMP-1) damaged collagen
Healing of superficial skin wounds depends on the proliferation and migration of keratinocytes at the wound margin. When human epidermal keratinocytes were incubated on polymerized type I collagen, they rapidly attached and spread. The cells underwent a proliferative response and, over the subsequent 6-day period, covered the collagen surface with a monolayer of cells. When keratinocytes were plated on collagen that had been fragmented by exposure to matrix metalloproteinase-1 (MMP-1, collagenase-1), the cells attached as readily as to intact collagen but spread more slowly and less completely. Growth was reduced by approximately 50%. Instead of covering the collagen surface, the keratinocytes remained localized to the site of attachment. Keratinocytes on fragmented collagen expressed a more differentiated phenotype as indicated by a higher level of surface E-cadherin. Based on these findings, we suggest that damage to the underlying collagenous matrix may impede efficient keratinocyte function and retard wound closure.
Perone, Patricia; Deming, Monica O'Brien; Warner, Roscoe L.; Aslam, Muhammad N.; Bhagavathula, Narasimharao; Dame, Michael K.; Voorhees, John J.
Insulin-like growth factor 1: common mediator of multiple enterotrophic hormones and growth factors
Purpose of review To summarize recent evidence that IGF1 mediates growth effects of multiple trophic factors and discuss clinical relevance. Recent findings Recent reviews and original reports indicate benefits of growth hormone (GH) and long-acting glucagon-like peptide 2 (GLP2) analogues in short bowel syndrome and Crohn�s disease. This review highlights evidence that biomarkers of sustained small intestinal growth or mucosal healing and evaluation of intestinal epithelial stem cell biomarkers may improve clinical measures of intestinal growth or response to trophic hormones. Compelling evidence that IGF1 mediates growth effects of GH and GLP2 on intestine or linear growth in preclinical models of resection or Crohn�s disease is presented, along with a concept that these hormones or IGF1 may enhance sustained growth if given early after bowel resection. Evidence that SOCS protein induction by GH or GLP2 in normal or inflamed intestine, may limit IGF1-induced growth, but protect against risk of dysplasia or fibrosis is reviewed. Whether IGF1 receptor mediates IGF1 action and potential roles of insulin receptors are addressed. Summary IGF1 has a central role in mediating trophic hormone action in small intestine. Better understanding of benefits and risks of IGF1, receptors that mediate IGF1 action, and factors that limit undesirable growth are needed.
Bortvedt, Sarah F.; Lund, P. Kay
Resistance of human squamous carcinoma cells to transforming growth factor beta 1 is a recessive trait.
Because most human squamous carcinoma cell lines of the aerodigestive and genital tracts are refractory to the antiproliferative action of transforming growth factor beta 1 (TGF beta 1) in vitro, we have begun to identify the causes for resistance of squamous carcinoma cell lines to TGF beta 1 by using somatic cell genetics. Two stable hybrid cell lines (FaDu-HKc.1 and FaDu-HKc.2) were obtained by fusing a TGF beta 1-resistant human squamous carcinoma cell line, FaDu-HygR, with a human papilloma virus 16-immortalized, TGF beta 1-sensitive, human foreskin keratinocyte cell line, HKc-neoR. Whereas TGF beta 1 did not inhibit DNA synthesis in parental FaDu-HygR cells, it reduced DNA synthetic activity of HKc-neoR, FaDu-HKc.1, and FaDu-HKc.2 cells by 75-85% (IC50, 2-5 pM). Although squamous carcinoma cells express lower than normal levels of TGF beta 1 type II receptors on their cell surface, TGF beta 1 type II receptor mRNA was detected in all four cell lines. Recessive genes involved in TGF beta 1 signaling may be localized to the distal portion of chromosome 18q, as this was the sole chromosomal region of homozygous deletion in parental FaDu-HygR cells. Furthermore, our previous observation that mutant p53 decreases sensitivity of keratinocytes to TGF beta 1 was supported by the finding that the level of the mutant p53 protein expressed by the hybrid cell lines was greatly reduced. In summary, TGF beta 1 resistance of FaDu cells appears to be recessive and is presumably due to the loss of one or more post-receptor elements of the signaling pathway. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 Fig. 6
Reiss, M; Munoz-Antonia, T; Cowan, J M; Wilkins, P C; Zhou, Z L; Vellucci, V F
Epidermal growth factor modulates fetal thymocyte growth and differentiation.
In the present study, we used the fetal organ culture (FTOC) technique in order to study a putative effect of epidermal growth factor (EGF) on the thymus ontogeny. Functional EGF receptors and more recently the EGF molecule itself, respectively, on the membrane of epithelial components of thymic stroma and on a few thymocytes in adult thymus, had been reported in the literature. We could observe a dose-dependent decrease in cellularity and a progressive retention of thymocytes in the double-negative (CD4-/CD8-) stage of differentiation when exogenous EGF was added. Epidermal growth factor interfered with both fetal stroma growth and thymocyte development at a precise moment, that is, in the passage from double-negative to the double-positive (CD4+/CD8+) stage. After a 7-day FTOC in the presence of EGF, most cells recovered were Thy-1.2+, c-kit+, TSA1-/int, CD3-, and one of CD44high/CD25int, CD44-/CD25int, or CD44/CD25-. Some developed into gammadeltaTCR+ cells with a mature (CD3+) phenotype, but not into alphabetaTCR+ thymocytes. It seems that EGF addition makes the cultures "nonpermissible" for alphabetaTCR+ thymocyte generation. We report here the presence of a high Mr "EGF-like" molecule on the membrane of fetal thymocytes, which role in the observed effects is under investigation. Further biochemical characterization of this molecule is still required, because its presence was only evidenced on the basis of its antigenicity. PMID:9851357
Freitas, C S; Dalmau, S R; Kovary, K; Savino, W
Differentiation-Induced Enhancement of the Ability of Cultured Human Keratinocytes to Suppress Oxidative Stress
Human keratinocytes in culture were harvested at different stages of differentiation. Both the level of antioxidants and the response of cells to oxidative stress were measured as a function of growth and differentiation. As the keratinocyte cultures became confluent and began to differentiate, the cellular levels of glutathione, glutathione peroxidase, glutathione S transferase, and glucose-6-phosphate dehydrogenase increased. This higher level
Donald A. Vessey; Kyung-Hee Lee; Thomas D. Boyer
Epidermal growth factor receptor: mechanisms of activation and signalling
The epidermal growth factor (EGF) receptor (EGFR) is one of four homologous transmembrane proteins that mediate the actions of a family of growth factors including EGF, transforming growth factor-?, and the neuregulins. We review the structure and function of the EGFR, from ligand binding to the initiation of intracellular signalling pathways that lead to changes in the biochemical state of
Robert N. Jorissen; Francesca Walker; Normand Pouliot; Thomas P. J. Garrett; Colin W. Ward; Antony W. Burgessa
Growth factors VEGF and TGF-?1 in peritoneal dialysis
The morphologic alterations in the kidney and the retina that can be present in patients with diabetic microangiopathy are mediated by growth factors. Vascular endothelial growth factor (VEGF) is a mediator of neoangiogenesis in diabetic retinopathy. Transforming growth factor-? (TGF-?) is involved in the extracellular matrix proliferation in diabetic nephropathy. The aim of the present study was to investigate the
Machteld M Zweers; Dirk R de Waart; Watske Smit; Dirk G Struijk; Raymond T Krediet
Effects of recombinant basic fibroblast growth factor, insulin-like growth factor-II and transforming growth factor- ? 1 on dog dental pulp cells in vivo
The effects of recombinant basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-II and transforming growth factor (TGF)-?1 on dental pulp cells were investigated by light and transmission electron microscopy after their implantation for 1 and 3 weeks at central sites of mechanically exposed pulps in dog molar and canine teeth. The implants were Millipore filters that have been soaked
D. Tziafas; A. Alvanou; S. Papadimitriou; J. Gasic; A. Komnenou
Reduced Expression of Fibroblast Growth Factor Receptor 2IIIb in Hepatocellular Carcinoma Induces a More Aggressive Growth
Fibroblast growth factor receptor 2 isoform b (FGFR2-IIIb) is highly expressed in hepatocytes and plays an important role in liver homeostasis and regeneration. Here, we analyzed the expression and function of FGFR2-IIIb in hepatocellular carcinoma (HCC). FGFR2-IIIb expression in HCC tissues and cell lines was lower than in primary human hepatocytes and nontumorous tissue. FGFR2-IIIb-negative HCCs showed a significantly higher Ki-67 labeling index, and loss of FGFR2-IIIb expression correlated significantly with vascular invasion and more advanced tumor stages. A decrease in FGFR-2IIIb expression in HCC cell lines was not related to promoter hypermethylation. However, PCR analysis indicated that chromosomal deletion at 10q accounted for the loss of FGFR2 expression in a subset of HCC cells. FGFR2-IIIb re-expression in stable transfected HCC cell lines induced a higher basal apoptosis rate and a significantly reduced proliferation and migratory potential in vitro. In nude mice, FGFR2-IIIb re-expressing HCC cells grew significantly slower, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed higher apoptosis rates. The antitumorigenic effects of FGFR2-IIIb expression in HCC cells were not affected by keratinocyte growth factor or an inhibitor of FGFR-phosphorylation, indicating that they are independent of tyrosine kinase activation. In conclusion, our data indicate that FGFR2-IIIb inhibits tumorigenicity of HCC cells. Identification of the molecular mechanisms promoting regeneration in normal tissue while suppressing malignancy may lead to novel therapeutic targets of this highly aggressive tumor.
Amann, Thomas; Bataille, Frauke; Spruss, Thilo; Dettmer, Katja; Wild, Peter; Liedtke, Christian; Muhlbauer, Marcus; Kiefer, Paul; Oefner, Peter J.; Trautwein, Christian; Bosserhoff, Anja-Katrin; Hellerbrand, Claus
Self-assembling elastin-like peptides growth factor chimeric nanoparticles for the treatment of chronic wounds
Chronic wounds are associated with poor epidermal and dermal remodeling. Previous work has shown the efficacy of keratinocyte growth factor (KGF) in reepithelialization and elastin in dermal wound healing. Here we demonstrate the fabrication of a fusion protein comprising of elastin-like peptides and KGF. This fusion protein retains the performance characteristics of KGF and elastin as evidenced by its enhancement of keratinocyte and fibroblast proliferation. It also preserved the characteristic elastin-like peptides inverse phase transitioning allowing the recombinant protein to be expressed in bacterial hosts (such as Escherichia coli) and purified rapidly and easily using inverse temperature cycling. The fusion protein self-assembled into nanoparticles at physiological temperatures. When applied to full thickness, wounds in Leprdb diabetic mice these particles enhanced reepithelialization and granulation, by 2- and 3-fold respectively, when compared to the controls. The data strongly suggests that these self-assembled nanoparticles may be beneficial in the treatment of chronic wounds resulting from diabetes or other underlying circulatory conditions.
Koria, Piyush; Yagi, Hiroshi; Kitagawa, Yuko; Megeed, Zaki; Nahmias, Yaakov; Sheridan, Robert; Yarmush, Martin L.
Growth factor regulation of insulin-like growth factor binding protein secretion by cultured human granulosa-luteal cells
Objective: To examine the effects of epidermal growth factor (EGF), transforming growth factor-? (TGF-?), and fibroblast growth factor (FGF) on insulin-like growth factor binding protein (IGFBP) secretion by cultured human granulosa-luteal cells.Design: Granulosa-luteal cells obtained at the time of oocyte harvest for IVF were cultured in serum-free medium in the presence or absence of EGF, TGF-?, or FGF. Conditioned medium
O. W. Stephanie Yap; Yasmin A. Chandrasekher; Linda C. Giudice
Exogenous Addition of a C-Xylopyranoside Derivative Stimulates Keratinocyte Dermatan Sulfate Synthesis and Promotes Migration
As C-Xyloside has been suggested to be an initiator of glycosaminoglycan (GAG) synthesis, and GAGs such as Dermatan sulfate (DS) are potent enhancers of fibroblast growth factor (FGF) - 10 action, we investigated if a C-Xylopyranoside derivative, (C-?-D-xylopyranoside-2-hydroxy-propane, C-Xyloside), could promote DS production by cultured normal human keratinocytes, how this occurs and if C-Xyloside could also stimulate FGF-dependent cell migration and proliferation. C-Xyloside-treated keratinocytes greatly increased secretion of total sulfated GAGs. Majority of the induced GAG was chondroitin sulfate/dermatan sulfate (CS/DS) of which the major secreted GAG was DS. Cells lacking xylosyltransferase enzymatic activity demonstrated that C-Xyloside was able to stimulate GAG synthesis without addition to core proteins. Consistent with the observed increase in DS, keratinocytes treated with C-Xyloside showed enhanced migration in response to FGF-10 and secreted into their culture media GAGs that promoted FGF-10-dependent cellular proliferation. These results indicate that C-Xyloside may enhance epithelial repair by serving as an initiator of DS synthesis.
Muto, Jun; Naidu, Nandita Natasha; Yamasaki, Kenshi; Pineau, Nathalie
Epidermal Expression of Neuropilin 1 Protects Murine keratinocytes from UVB-induced apoptosis
Background Neuropilin 1 (NRP1) is expressed on several cell types including neurons and endothelial cells, where it functions as an important regulator in development and during angiogenesis. As a cell surface receptor, NRP1 is able to bind to members of the VEGF family of growth factors and to secreted class 3 semaphorins. Neuropilin 1 is also highly expressed in keratinocytes, but the function of NRP1 in epidermal physiology and pathology is still unclear. Methods and Results To elucidate the role of NRP1 in skin in vivo we generated an epidermis-specific neuropilin 1 knock out mouse model by using the Cre-LoxP-System. Mice were viable and fertile and did not display any obvious skin or hair defects. After challenge with UVB irradiation, we found that deletion of epidermal NRP1 leads to increased rates of apoptosis both in vitro and in vivo. NRP1-deficient primary keratinocytes cultured in vitro showed significantly higher rates of apoptosis 24 hours after UVB. Likewise, there is a significant increase of active caspase 3 positive cells in the epidermis of Keratin 14-Cre-NRP1 (?/?) mice 24 hours after UVB irradiation. By Western Blot analysis we could show that NRP1 influences the cytosolic levels of Bcl-2, a pro-survival member of the Bcl-2 family. After UVB irradiation the amounts of Bcl-2 decrease in both protein extracts from murine epidermis and in NRP1-deficient keratinocytes in vitro, whereas wild type cells retain their Bcl-2 levels. Likewise, levels of phospho-Erk and Rac1 were lower in NRP1-knock out keratinocytes, whereas levels of pro-apoptotic p53 were higher. Conclusion NRP1 expression in keratinocytes is dispensable for normal skin development. Upon UVB challenge, NRP1 contributes to the prevention of keratinocyte apoptosis. This pro-survival function of NRP1 is accompanied by the maintenance of high levels of the antiapoptotic regulator Bcl-2 and by lower levels of pro-apoptotic p53.
Riese, Anna; Eilert, Yvonne; Meyer, Yvonne; Arin, Meral; Baron, Jens M.; Eming, Sabine; Krieg, Thomas; Kurschat, Peter
Comparison of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis in SCID mice.
Therapeutic angiogenesis is a promising approach to treat patients with cardiovascular disease, and will likely be critical to engineering large tissues. Many growth factors have been found to play significant roles in angiogenesis, and vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the most extensively investigated angiogenic factors to date. However, the appropriate dose to obtain a desired response and the effectiveness of each factor, relative to the other, in promoting angiogenesis at a specific site in the body remains unclear. We have used alginate hydrogels as localized delivery vehicles for VEGF and bFGF, and compared the ability of these factors to promote new blood vessel formation in the subcutaneous tissue of severe combined immunodeficient (SCID) mice. We have found that the thickness of a granulation tissue layer formed around the gel and the number of blood vessels in the layer increased with the dose of VEGF in the gel, but the density of new blood vessels remained relatively constant. Sustained and localized delivery of bFGF from the gels, while similarly leading to an increase in the density of blood vessels in the granulation tissue, did not lead to as high of a blood vessel density as VEGF. The results of this study support previous studies demonstrating the utility of both VEGF and bFGF in promoting angiogenesis, and suggest VEGF is more appropriate for creating a dense bed of new blood vessels in this model. PMID:12618022
Lee, Kuen Yong; Peters, Martin C; Mooney, David J
Healing of Burn Wounds in Transgenic Mice Overexpressing Transforming Growth Factor-?1 in the Epidermis
Transforming growth factor-? (TGF-?) isoforms are multifunctional cytokines that play an important role in wound healing. Transgenic mice overexpressing TGF-? in the skin under control of epidermal-specific promoters have provided models to study the effects of increased TGF-? on epidermal cell growth and cutaneous wound repair. To date, most of these studies used transgenic mice that overexpress active TGF-? in the skin by modulating the latency-associated-peptide to prevent its association with active TGF-?. The present study is the first to use transgenic mice that overexpress the natural form of latent TGF-?1 in the epidermis, driven by the keratin 14 gene promoter to investigate the effects of locally elevated TGF-?1 on the healing of partial-thickness burn wounds made on the back of the mice using a CO2 laser. Using this model, we demonstrated activation of latent TGF-? after wounding and determined the phenotypes of burn wound healing. We found that introduction of the latent TGF-?1 gene into keratinocytes markedly increases the release and activation of TGF-? after burn injury. Elevated local TGF-? significantly inhibited wound re-epithelialization in heterozygous (42% closed versus 92% in controls, P < 0.05) and homozygous (25% versus 92%, P < 0.01) animals at day 12 after wounding. Interestingly, expression of type I collagen mRNA and hydroxyproline significantly increased in the wounds of transgenic mice, probably as a result of a paracrine effect of the transgene.
Yang, Liju; Chan, Teddy; Demare, Jack; Iwashina, Takashi; Ghahary, Aziz; Scott, Paul G.; Tredget, Edward E.
Growth without growth hormone: evidence for a potent circulating human growth factor.
A potent growth factor was detected in the serum of a child with growth hormone (GH) deficiency and early growth delay whose growth velocity spontaneously increased to supranormal levels despite persistent GH deficiency by both radioimmunoassay (RIA) and radioreceptor assay. Thyroid function, prolactin, insulin response to oral glucose, glucose response to intravenous insulin, and computerised tomography of the head were all normal. Whilst somatomedin-C levels measured by RIA were low or low-normal, in vitro somatomedin bioactivity measured by bioassay was normal, suggesting the presence of a growth factor other than somatomedin-C. By way of confirmation, the patient's serum was incubated with erythroid progenitor cells from peripheral blood of a normal individual and a Laron dwarf. In this system, proliferation of normal erythroid progenitors was almost double that obtained with physiological concentrations of GH or control sera, and Laron erythroid progenitors, which were completely resistant to added GH, also responded strongly to the patient's serum. The patient's growth is therefore independent of GH and other known growth factors. PMID:2868295
Geffner, M E; Lippe, B M; Bersch, N; Van Herle, A; Kaplan, S A; Elders, M J; Golde, D W
A decorin-deficient matrix affects skin chondroitin/dermatan sulfate levels and keratinocyte function.
Decorin is a small leucine-rich proteoglycan harboring a single glycosaminoglycan chain, which, in skin, is mainly composed of dermatan sulfate (DS). Mutant mice with targeted disruption of the decorin gene (Dcn(-/-)) exhibit an abnormal collagen architecture in the dermis and reduced tensile strength, collectively leading to a skin fragility phenotype. Notably, Ehlers-Danlos patients with mutations in enzymes involved in the biosynthesis of DS display a similar phenotype, and recent studies indicate that DS is involved in growth factor binding and signaling. To determine the impact of the loss of DS-decorin in the dermis, we analyzed the glycosaminoglycan content of Dcn(-/-) and wild-type mouse skin. The total amount of chondroitin/dermatan sulfate (CS/DS) was increased in the Dcn(-/-) skin, but was overall less sulfated with a significant reduction in bisulfated ?DiS2,X (X=4 or 6) disaccharide units, due to the reduced expression of uronyl 2-O sulfotransferase (Ust). With increasing age, sulfation declined; however, Dcn(-/-) CS/DS was constantly undersulfated vis-�-vis wild-type. Functionally, we found altered fibroblast growth factor (Fgf)-7 and -2 binding due to changes in the micro-heterogeneity of skin Dcn(-/-) CS/DS. To better delineate the role of decorin, we used a 3D Dcn(-/-) fibroblast cell culture model. We found that the CS/DS extracts of wild-type and Dcn(-/-) fibroblasts were similar to the skin sugars, and this correlated with the lack of uronyl 2-O sulfotransferase in the Dcn(-/-) fibroblasts. Moreover, Ffg7 binding to total CS/DS was attenuated in the Dcn(-/-) samples. Surprisingly, wild-type CS/DS significantly reduced the binding of Fgf7 to keratinocytes in a concentration dependent manner unlike the Dcn(-/-) CS/DS that only affected the binding at higher concentrations. Although binding to cell-surfaces was quite similar at higher concentrations, keratinocyte proliferation was differentially affected. Higher concentration of Dcn(-/-) CS/DS induced proliferation in contrast to wild-type CS/DS. 3D co-cultures of fibroblasts and keratinocytes showed that, unlike Dcn(-/-) CS/DS, wild-type CS/DS promoted differentiation of keratinocytes. Collectively, our results provide novel mechanistic explanations for the reported defects in wound healing in Dcn(-/-) mice and possibly Ehlers-Danlos patients. Moreover, the lack of decorin-derived DS and an altered CS/DS composition differentially influence keratinocyte behavior. PMID:24447999
Nikolovska, Katerina; Renke, Jana K; Jungmann, Oliver; Grobe, Kay; Iozzo, Renato V; Zamfir, Alina D; Seidler, Daniela G
Autologous growth factor injections in chronic tendinopathy.
Reference: de Vos RJ, van Veldhoven PLJ, Moen MH, Weir A, Tol JL. Autologous growth factor injections in chronic tendinopathy: a systematic review. Br Med Bull. 2010;95:63-77. Clinical Question: The authors of this systematic review evaluated the literature to critically consider the effects of growth factors delivered through autologous whole-blood and platelet-rich-plasma (PRP) injections in managing wrist-flexor and -extensor tendinopathies, plantar fasciopathy, and patellar tendinopathy. The primary question was, according to the published literature, is there sufficient evidence to support the use of growth factors delivered through autologous whole-blood and PRP injections for chronic tendinopathy? Data Sources: The authors performed a comprehensive, systematic literature search in October 2009 using PubMed, MEDLINE, EMBASE, CINAHL, and the Cochrane library without time limits. The following key words were used in different combinations: tendinopathy, tendinosis, tendinitis, tendons, tennis elbow, plantar fasciitis, platelet rich plasma, platelet transfusion, and autologous blood or injection. The search was limited to human studies in English. All bibliographies from the initial literature search were also viewed to identify additional relevant studies. Study Selection: Studies were eligible based on the following criteria: (1) Articles were suitable (inclusion criteria) if the participants had been clinically diagnosed as having chronic tendinopathy; (2) the design had to be a prospective clinical study, randomized controlled trial, nonrandomized clinical trial, or prospective case series; (3) a well-described intervention in the form of a growth factor injection with either PRP or autologous whole blood was used; and (4) the outcome was reported in terms of pain or function (or both). Data Extraction: All titles and abstracts were assessed by 2 researchers, and all relevant articles were obtained. Two researchers independently read the full text of each article to determine if it met the inclusion criteria. If opinions differed on suitability, a third reviewer was consulted to reach consensus. The data extracted included number of participants, study design, inclusion criteria, intervention, control group, primary outcome measures (pain using a visual analog or ordinal scale or function), time of follow-up, and outcomes for intervention and control group (percentage improvement) using a standardized data-extraction form. Function was evaluated in 9 of the 11 studies using (1) the Nirschl scale (elbow function) or the modified Mayo score for wrist flexors and extensors, (2) the Victorian Institute of Sports Assessment-Patella score, a validated outcome measure for patellar tendinopathy, or the Tegner score for patellar tendinopathy, and (3) the rearfoot score from the American Orthopaedic Foot and Ankle Scale for plantar fasciopathy. The Physiotherapy Evidence Database (PEDro) scale contains 11 items; items 2-11 receive 1 point each for a yes response. Reliability is sufficient (0.68) for the PEDro scale to be used to assess physiotherapy trials. A score of 6 or higher on the PEDro scale is considered a high-quality study; below 6 is considered a low-quality study. The PEDro score results determined the quality of the randomized controlled trial (RCT), nonrandomized clinical trial, or prospective case series (?6 or <6). A qualitative analysis was used with 5 levels of evidence (strong, moderate, limited, conflicting, or no evidence) to determine recommendations for the use of the intervention. The number of high-quality or low-quality RCT or nonrandomized clinical trial studies with consistent or inconsistent results determined the level of evidence (1-5). Main Results: Using the specific search criteria, the authors identified 418 potential sources. After screening of the title or abstract (or both), they excluded 405 sources, which left 13 studies. After viewing the full text, they excluded 2 additional sources (a case report and a study in which the outcome measure was remission of symptoms and not pain o
Sandrey, Michelle A
Systems Biology of Vascular Endothelial Growth Factors
Several cytokine families have roles in development, maintenance and remodeling of the microcirculation. Of these, the VEGF family is one of the best studied and one of the most complex. Five VEGF ligand genes and five cell surface receptor genes are known in the human, and each of these may be transcribed as multiple splice isoforms to generate an extensive family of proteins, many of which are subject to further proteolytic processing. Using the VEGF family as an example, we describe the current knowledge of growth factor expression, processing and transport in vivo. Experimental studies and computational simulations are being used to measure and predict the activity of these molecules, and we describe avenues of research that seek to fill the remaining gaps in our understanding of VEGF family behavior.
Mac Gabhann, Feilim; Popel, Aleksander S.
Toxicities of the thrombopoietic growth factors
The thrombopoietic growth factors (TGFs) are a novel class of compounds for the treatment of chronic immune thrombocytopenia (ITP). The first of these agents to receive regulatory approval, romiplostim and eltrombopag, have demonstrated impressive efficacy and tolerability in randomized controlled trials and open-label extension studies of several years duration and stand poised to revolutionize the management of ITP. Nonetheless, critical questions regarding the safety of these agents, particularly after long-term administration, remain partially unanswered. The objective of this review is to describe the reported and potential toxicities of the TGFs including bone marrow fibrosis, thrombosis, rebound thrombocytopenia, hematologic malignancy, neutralizing antibody formation, hepatotoxicity, cataract formation, and common adverse events. The incidence and clinical implications of these toxicities as well as strategies for patient safety monitoring are examined.
Cuker, Adam
Matrix control of transforming growth factor-? function.
The cytokine transforming growth factor-beta (TGF-?) has multiple effects in both physiological and pathological conditions. TGF-? is secreted as part of a tripartite complex from which it must be released in order to bind to its receptor. Sequestration of latent TGF-? in the extracellular matrix (ECM) is crucial for proper mobilization of the latent cytokine and its activation. However, contrary to expectation, loss-of-function mutations in genes encoding certain matrix proteins that bind TGF-? yield elevated, rather than decreased, TGF-? levels, posing a 'TGF-? paradox.' In this review, we discuss recent findings concerning the relationship of TGF-?, ECM molecules, and latent TGF-? activation and propose a model to resolve the 'TGF-? paradox.' PMID:22923731
Horiguchi, Masahito; Ota, Mitsuhiko; Rifkin, Daniel B
Matrix control of transforming growth factor-? function
The cytokine transforming growth factor-beta (TGF-?) has multiple effects in both physiological and pathological conditions. TGF-? is secreted as part of a tripartite complex from which it must be released in order to bind to its receptor. Sequestration of latent TGF-? in the extracellular matrix (ECM) is crucial for proper mobilization of the latent cytokine and its activation. However, contrary to expectation, loss-of-function mutations in genes encoding certain matrix proteins that bind TGF-? yield elevated, rather than decreased, TGF-? levels, posing a �TGF-? paradox.� In this review, we discuss recent findings concerning the relationship of TGF-?, ECM molecules, and latent TGF-? activation and propose a model to resolve the �TGF-? paradox.�
Horiguchi, Masahito; Ota, Mitsuhiko; Rifkin, Daniel B.
Growth Factor-Extracellular Matrix Interactions Regulate Wound Repair
Background Wound healing is a process made up of several phases, including hemostasis/inflammation, proliferation, and scar formation/remodeling. An array of growth factors is produced during each of these phases that helps direct the repair process. The Problem Most of the information we have about the biological actions of growth factors are from studies examining a growth factor in isolation. However, growth factors are known to interact with the extracellular matrix (ECM), and these associations influence cell behavior. Details about these interactions within the complex and continuously changing wound environment are not well understood and are likely to be very important during repair. Basic/Clinical Science Advances Several types of growth factor/ECM interactions have been described that could impact wound healing. The ECM can interact directly with growth factors, offering protection from degradation and controlling bioactivity of the growth factor. Portions of the ECM can bind to growth factor receptors, and cell�ECM binding can influence growth factor receptor signaling. Growth factors can also control production and degradation of the ECM; therefore, the relationship between growth factors and ECM is bidirectional. Clinical Care Relevance New information about the relationship between growth factors and ECM could be used to optimize growth factor-based therapies or lead to the development of novel treatment strategies for wound care. Conclusion Growth factor�ECM interactions likely have a strong impact on the rate and quality of healing. A better understanding of the relationship between these classes of molecules and how it can be exploited to enhance healing is needed.
Wilgus, Traci A.
Transient expression of OCT4 is sufficient to allow human keratinocytes to change their differentiation pathway
In this study, we describe a simple system in which human keratinocytes can be redirected to an alternative differentiation pathway. We transiently transfected freshly isolated human skin keratinocytes with the single transcription factor OCT4. Within 2 days these cells displayed expression of endogenous embryonic genes and showed reduced genomic methylation. More importantly, these cells could be specifically converted into neuronal
D Racila; M Winter; M Said; A Tomanek-Chalkley; S Wiechert; R L Eckert; J R Bickenbach; JR Bickenbach
IL-17 and IL-22 mediate IL-20 subfamily cytokine production in cultured keratinocytes via increased IL-22 receptor expression.
IL-20 cytokine subfamily members, including IL-19, IL-20, and IL-24, are highly expressed in psoriatic skin lesions. Here, we demonstrate that psoriasis mediators IL-17 and IL-22 synergistically induce the production of IL-20 subfamily proteins in cultured human keratinocytes. Interestingly, expression of the IL-22 receptor (IL-22R) also increased in epidermal lesions versus normal skin. IL-22R over-expression using an adenoviral vector to mimic psoriatic conditions in cultured keratinocytes significantly enhanced IL-17- and IL-22-induced production of IL-20 subfamily cytokines. Furthermore, IL-17 and IL-22 coordinately enhanced MIP-3alpha, IL-8, and heparin-binding EGF-like growth factor (HB-EGF) production, depending on the amount of IL-22R expression. Additionally, because IL-20 and IL-24 share the IL-22R with IL-22, the function of IL-20 and IL-24 was also increased. IL-20 and IL-24 have effects similar to that of IL-22; IL-24 showed more potent expression than IL-20. A combination of IL-24 and IL-17 increased the production of MIP-3alpha, IL-8, and HB-EGF, as did a combination of IL-22 and IL-17. These data indicate that increased IL-22R expression in epidermal keratinocytes contributes to the pathogenesis of psoriasis through enhancing the coordinated effects of IL-22 and IL-17, inducing the production of the IL-20 subfamily, chemokines, and growth factors. PMID:19731362
Tohyama, Mikiko; Hanakawa, Yasushi; Shirakata, Yuji; Dai, Xjuju; Yang, Lujun; Hirakawa, Satoshi; Tokumaru, Sho; Okazaki, Hidenori; Sayama, Koji; Hashimoto, Koji
The role of the tetraspanin CD151 in primary keratinocyte and fibroblast functions: Implications for wound healing
Previous studies showed that CD151-null mice have a skin wound healing deficit. To gain an understanding of the role of CD151 in re-epithelialisation and dermal contraction, keratinocyte and fibroblast functions were assayed. Primary CD151-null keratinocytes displayed defective migration on Matrigel (a basement membrane equivalent) and laminin-332, the primary adhesion component of basement membranes, but not on collagen-I. Adhesion, spreading and proliferation were also deficient on laminin-332, but not collagen-I. The data suggest that loss of CD151 impairs the function of its primary interaction partners, integrin {alpha}3{beta}1- and/or {alpha}6{beta}4 which bind to laminin-332. Skin fibroblasts also produce CD151 mRNA. CD151-null fibroblasts migrated significantly faster on collagen I than wild type fibroblasts, confirming that they possess functional collagen receptors. However, no significant decrease in the ability of CD151-null fibroblasts to cause contraction in floating collagen gel assays in response to transforming growth factor beta-1 (TGF-{beta}1) or platelet derived growth factor (PDGF-BB) was observed, nor was there an effect on fibroblast adhesion or proliferation on collagen-I. The data implicate CD151 as a facilitator of laminin-332-mediated keratinocyte functions that impact on the re-epithelialisation process intrinsic to wound healing and further suggest a potential novel role for CD151 in fibroblast migration.
Geary, Sean M. [School of Biomedical Sciences, University of Newcastle, New South Wales (Australia); Hunter Medical Research Institute, Newcastle, New South Wales (Australia); Cowin, Allison J. [Child Health Research Institute, North Adelaide, South Australia (Australia); Department of Paediatrics, University of Adelaide, South Australia (Australia); Copeland, Ben; Baleato, Rosa M. [School of Biomedical Sciences, University of Newcastle, New South Wales (Australia); Hunter Medical Research Institute, Newcastle, New South Wales (Australia); Miyazaki, Kaoru [Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Yokohama (Japan); Ashman, Leonie K. [School of Biomedical Sciences, University of Newcastle, New South Wales (Australia); Hunter Medical Research Institute, Newcastle, New South Wales (Australia)], E-mail: firstname.lastname@example.com
Role of platelet-derived growth factor in wound healing: synergistic effects with other growth factors.
Platelet-derived growth factor (PDGF) in vitro stimulates DNA synthesis and chemotaxis of fibroblasts and smooth muscle cells and stimulates collagen, glycosaminoglycan, and collagenase production by fibroblasts. These in vitro properties suggest that PDGF, delivered by platelets to the site of injury in vivo, may play an important role in the initiation of the wound repair process. Studies presented here show that the addition of pure PDGF to a wound site involving the epidermis and dermis has little effect on the morphology or biochemistry of the healing wound. In contrast, the addition of partially purified PDGF resulted in significant dose-dependent increases in the width of the newly synthesized connective tissue and epidermal layers. Autoradiography using [3H]thymidine revealed increased numbers of labeled cells in the new connective tissue and epithelial layers. Furthermore, addition of partially purified PDGF resulted in significant increases in the rate of protein and DNA synthesis and the total content of these components in biopsies taken from the wound site. Similar effects were obtained when insulin-like growth factor I was added in combination with pure PDGF. This combination of factors caused a 2.4-fold increase in the width of the newly formed connective tissue layer and a 95% increase in epidermal thickness compared with controls. Insulin-like growth factor I alone caused no significant morphologic changes. Epidermal growth factor alone or in combination with PDGF resulted in a thickening only of the epidermis. These results indicate that the synergistic actions of other factors with PDGF are important in the modulation of the wound healing process. Images
Lynch, S E; Nixon, J C; Colvin, R B; Antoniades, H N
Insulin-like growth factor binding protein-4 differentially inhibits growth factor-induced angiogenesis.
An in-depth understanding of the molecular and cellular complexity of angiogenesis continues to advance as new stimulators and inhibitors of blood vessel formation are uncovered. Gaining a more complete understanding of the response of blood vessels to both stimulatory and inhibitory molecules will likely contribute to more effective strategies to control pathological angiogenesis. Here, we provide evidence that endothelial cell interactions with structurally altered collagen type IV may suppress the expression of insulin-like growth factor binding protein-4 (IGFBP-4), a well documented inhibitor of the IGF-1/IGF-1R signaling axis. We report for the first time that IGFBP-4 differentially inhibits angiogenesis induced by distinct growth factor signaling pathways as IGFBP-4 inhibited FGF-2- and IGF-1-stimulated angiogenesis but failed to inhibit VEGF-induced angiogenesis. The resistance of VEGF-stimulated angiogenesis to IGFBP-4 inhibition appears to depend on sustained activation of p38 MAPK as blocking its activity restored the anti-angiogenic effects of IGFBP-4 on VEGF-induced blood vessel growth in vivo. These novel findings provide new insight into how blood vessels respond to endogenous inhibitors during angiogenesis stimulated by distinct growth factor signaling pathways. PMID:22134921
Contois, Liangru W; Nugent, Desiree P; Caron, Jennifer M; Cretu, Alexandra; Tweedie, Eric; Akalu, Abebe; Liebes, Leonard; Friesel, Robert; Rosen, Clifford; Vary, Calvin; Brooks, Peter C
Gene Expression of Growth Factors and Growth Factor Receptors for Potential Targeted Therapy of Canine Hepatocellular Carcinoma
ABSTRACT The purpose of this study was to evaluate the gene expression of growth factors and growth factor receptors of primary hepatic masses, including hepatocellular carcinoma (HCC) and nodular hyperplasia (NH), in dogs. Quantitative real-time reverse transcriptase-polymerase chain reaction was performed to measure the expression of 18 genes in 18 HCCs, 10 NHs, 11 surrounding non-cancerous liver tissues and 4 healthy control liver tissues. Platelet-derived growth factor-B (PDGF-B), transforming growth factor-?, epidermal growth factor receptor, epidermal growth factor and hepatocyte growth factor were found to be differentially expressed in HCC compared with NH and the surrounding non-cancerous and healthy control liver tissues. PDGF-B is suggested to have the potential to become a valuable ancillary target for the treatment of canine HCC.
IIDA, Gentoku; ASANO, Kazushi; SEKI, Mamiko; SAKAI, Manabu; KUTARA, Kenji; ISHIGAKI, Kumiko; KAGAWA, Yumiko; YOSHIDA, Orie; TESHIMA, Kenji; EDAMURA, Kazuya; WATARI, Toshihiro
Endorsement of Growth Factors in Experiential Training Groups
ERIC Educational Resources Information Center
The purpose of this study was to identify student growth factors during a semester long Master's level group counseling class. Results indicated that 12 growth factors accounted for 86% of the total number of critical incidents that participants reported as influencing their personal growth and awareness during the group experience. Two other�
Kiweewa, John; Gilbride, Dennis; Luke, Melissa; Seward, Derek
Growth Factors in Development and Diseases of the Exocrine Pancreas
Besides the classical gut hormones, like CCK, which have been extensively studied, several growth factors are now being recognized to influence exocrine pancreatic growth and regeneration. Growth factors are proteins which are secreted in an auto-, para-, or endocrine manner. They are produced either by residential cells or by migratory cells, e.g. in inflamed tissue in extremely variable concentrations. Various
Karlheinz Kiehne; Jan-Michel Otte; Ulrich R. F�lsch; Karl-Heinz Herzig
Keratinocytes Propagated in Serum-Free, Feeder-Free Culture Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin Model
Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification.
Lamb, Rebecca; Ambler, Carrie A.
Keratinocytes propagated in serum-free, feeder-free culture conditions fail to form stratified epidermis in a reconstituted skin model.
Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification. PMID:23326335
Lamb, Rebecca; Ambler, Carrie A
The Role of Macroeconomic Factors in Growth
Using a regression analog of growth accounting, I present cross- sectional and panel regressions showing that growth is negatively associated with inflation, large budget deficits, and distorted foreign exchange markets. Supplementary evidence suggests that the causation runs from macroeconomic policy to growth. The framework makes it possible to identify the channels of these effects: inflation reduces growth by reducing investment
Stanley Fischer
The Expression of Heparin-Binding Epidermal Growth Factor-Like Growth Factor by Regulatory Macrophages
We previously described a population of regulatory macrophages that produced high levels of IL-10 and low levels of IL-12/23. We now describe and characterize the expression of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) by these macrophages. HB-EGF has previously been associated with a number of physiological and pathological conditions, including tumor growth and angiogenesis. The induction of HB-EGF in regulatory macrophages is due to new transcription and not to increased mRNA stability. The transcription factor Sp1 is a major factor in HB-EGF production, and knockdown of Sp1 substantially diminishes HB-EGF production. Sp1 was recruited to three sites within the first 2 kb of the HB-EGF promoter following stimulation, and the site located at �83/�54 was required for HB-EGF promoter activity. These regions of the promoter become more accessible to endonuclease activity following macrophage activation, and this accessibility was contingent on activation of the MAPK, ERK. We show that several experimental manipulations that give rise to regulatory macrophages also result in HB-EGF production. These observations indicate that in addition to the secretion of the anti-inflammatory cytokine IL-10, another novel characteristic of regulatory macrophages is the production of angiogenic HB-EGF.
Edwards, Justin P.; Zhang, Xia; Mosser, David M.
Fibroblast Growth Factors Are Required for Efficient Tumor Angiogenesis1
Although the function of vascular endothelial growth factor in the induction of tumor angiogenesis is well understood, the role of a second group of angiogenic factors, the fibroblast growth factors (FGFs), remains elusive. We used a recombinant adenovirus expressing soluble FGF re- ceptor (AdsFGFR) to interfere with FGF function in tumor angiogenesis. AdsFGFR repressed endothelial cell proliferation in vitro and
Amelia Compagni; Petra Wilgenbus; Maria-Antonietta Impagnatiello; Matt Cotten; Gerhard Christofori
Epidermal Growth Factor Receptor Dynamics Influences Response to Epidermal Growth Factor Receptor Targeted Agents
Analysis of gene expression of cancer cell lines exposed to erlotinib, a small molecule inhibitor of the epidermal growth factor receptor (EGFR), showed a marked increase in EGFR mRNA in resistant cell lines but not in susceptible ones. Because cetuximab induces EGFR down-regulation, we explored the hypothesis that treatment with cetuximab would interfere with erlotinib-induced EGFR up-regulation and result in
Antonio Jimeno; Maria L. Amador; Nadia Bouraoud; Peter Kulesza; Anirban Maitra; Manuel Hidalgo
Growth/differentiation factor-11: an evolutionary conserved growth factor in vertebrates.
Growth and differentiation factor-11 (GDF-11) is a member of the transforming growth factor-? superfamily and is thought to be derived together with myostatin (known also as GDF-8) from an ancestral gene. In the present study, we report the isolation and characterization of GDF-11 homolog from a marine teleost, the gilthead sea bream Sparus aurata, and show that this growth factor is highly conserved throughout vertebrates. Using bioinformatics, we identified GDF-11 in Tetraodon, Takifugu, medaka, and stickleback and found that they are highly conserved at the amino acid sequence as well as gene organization. Moreover, we found conservation of syntenic relationships among vertebrates in the GDF-11 locus. Transcripts for GDF-11 can be found in eggs and early embryos, albeit at low levels, while in post-hatching larvae expression levels are high and decreases as development progresses, suggesting that GDF-11 might have a role during early development of fish as found in tetrapods and zebrafish. Finally, GDF-11 is expressed in various tissues in the adult fish including muscle, brain, and eye. PMID:20694476
Funkenstein, Bruria; Olekh, Elena
Effect of gamma-hydroxybutyrate on keratinocytes proliferation: A preliminary prospective controlled study in severe burn patients
Background: Hypermetabolism and hyposomatotropism related to severe burns lead to impaired wound healing. Growth hormone (GH) boosts wound healing notably following stimulation of the production of insulin-like growth factor-1 (IGF1), a mitogen factor for keratinocytes. Gamma-hydroxybutyrate (GHB) stimulates endogenous GH secretion. Aim: To assess effects of GHB sedation on keratinocytes proliferation (based on immunohistochemical techniques). Design: Monocentric, prospective, controlled trial. Materials and Methods: Patients (aging 18-65 years, burn surface area >30%, expected to be sedated for at least one month) were alternately allocated, at the 5th day following injury, in three groups according to the intravenous GHB dose administered for 21 days: Evening bolus of 50 mg/kg (Group B), continuous infusion at the rate of 10 mg/kg/h (Group C), or absence of GHB (Group P). They all received local standard cares. Immunohistochemistry (Ki67/MIB-1, Ulex europaeus agglutinin-1 and Mac 387 antibodies) was performed at D21 on adjacent unburned skin sample for assessing any keratinocyte activation. Serum IGF1 levels were measured at initiation and completion of the protocol. Statistical Analysis: Categorical variables were compared with Chi-square test. Comparisons of medians were made using Kruskal-Wallis test. Post hoc analyses were performed using Mann-Whitney test with Bonferroni correction for multiple comparisons. A P < 0.05 was considered to be statistically significant. Results: A total of 14 patients completed the study (Group B: n = 5, Group C: n = 5, Group P: n = 4). Continuous administration of GHB was associated with a significant higher Ki67 immunolabeling at D21 (P = 0.049) and with a significant higher increase in the IGF1 concentrations at D21 (P = 0.024). No adverse effects were disclosed. Conclusions: Our preliminary data support a positive effect of GHB on keratinocyte proliferation and are encouraging enough to warrant large prospective studies.
Rousseau, Anne-Francoise; Bargues, Laurent; Bever, Herve Le; Vest, Philippe; Cavalier, Etienne; Ledoux, Didier; Pierard, Gerald E.; Damas, Pierre
Stability of faecal hepatocyte growth factor determination.
In order to evaluate the accuracy and reproducibility of determination of hepatocyte growth factor (HGF) levels in faeces, the stability of HGF in samples processed in different ways was investigated. An ELISA method was used for determination of HGF concentrations. Faeces samples from healthy controls and patients with infectious diarrhoea were studied. It was found that faeces HGF concentration remained stable irrespective of whether samples were freeze-thawed several times, kept for 6, 12 or 24 h at room temperature or refrigerated for 6, 12, 24 or 36 h; the levels of HGF did not change significantly when samples were freeze-dried. Adding protease inhibitor to the faeces samples did not affect the HGF levels. There were no significant differences between HGF levels using phosphate buffered saline (PBS) (pH 7.4) or NaCL as buffer, but it was observed that levels of HGF were significantly lower in the samples that were diluted in distilled water. Although both HGF and albumin through various mechanisms may increase in faeces during infectious diarrhoea, there was no significant correlation between faeces HGF levels and albumin levels, which might indicate local production of HGF in the bowel in response to infection. It is concluded that determination of faeces HGF levels is feasible with a high degree of stability. Increased HGF levels in faeces might represent a local production of HGF during bowel injury and might be of use as a diagnostic and monitoring assay. PMID:15370465
Nayeri, F; Nilsson, I; Brudin, L; Almer, S
Vascular Endothelial Growth Factor in Eye Disease
Collectively, angiogenic ocular conditions represent the leading cause of irreversible vision loss in developed countries. In the U.S., for example, retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration are the principal causes of blindness in the infant, working age and elderly populations, respectively. Evidence suggests that vascular endothelial growth factor (VEGF), a 40 kDa dimeric glycoprotein, promotes angiogenesis in each of these conditions, making it a highly significant therapeutic target. However, VEGF is pleiotropic, affecting a broad spectrum of endothelial, neuronal and glial behaviors, and confounding the validity of anti-VEGF strategies, particularly under chronic disease conditions. In fact, among other functions VEGF can influence cell proliferation, cell migration, proteolysis, cell survival and vessel permeability in a wide variety of biological contexts. This article will describe the roles played by VEGF in the pathogenesis of retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. The potential disadvantages of inhibiting VEGF will be discussed, as will the rationales for targeting other VEGF-related modulators of angiogenesis.
Penn, J.S.; Madan, A.; Caldwell, R.B.; Bartoli, M.; Caldwell, R.W.; Hartnett, M.E.
Vascular endothelial growth factor and ocular neovascularization.
Okamoto et al have developed an extremely useful and interesting model of retinal and subretinal neovascularization. Using molecular techniques, they have developed a transgenic model driven by overexpression of VEGF, a growth factor demonstrated to play an important role in neovascularization in many ocular diseases. They have been able to demonstrate that VEGF overexpression is sufficient to cause intraretinal and subretinal neovascularization. The mouse model is relatively cheap and reliable, does not require any exogenous agent, and has many characteristics of clinical intraocular neovascularization. The new vessels develop in the outer retina and subretinal space and have a characteristic histological appearance. They leak fluorescein on angiography, demonstrating their similarity to human disease and allowing identification and grading of neovascularization in vivo. The model can be used to investigate molecular mechanisms of VEGF-dependent neovascularization, with applications beyond ocular eye disease. The model can also be used to study anti-angiogenic agents that have the potential to treat common blinding diseases such as age-related macular degeneration. Okamoto et al have made a substantial contribution to the angiogenesis field with this work, and one looks forward to future investigations.
Miller, J. W.
Vascular endothelial growth factor in heart failure.
Heart failure is a devastating condition, the progression of which culminates in a mismatch of oxygen supply and demand, with limited options for treatment. Heart failure has several underlying causes including, but not limited to, ischaemic heart disease, valvular dysfunction, and hypertensive heart disease. Dysfunctional blood vessel formation is a major problem in advanced heart failure, regardless of the aetiology. Vascular endothelial growth factor (VEGF) is the cornerstone cytokine involved in the formation of new vessels. A multitude of investigations, at both the preclinical and clinical levels, have garnered valuable information on the potential utility of targeting VEGF as a treatment option for heart failure. However, clinical trials of VEGF gene therapy in patients with coronary artery disease or peripheral artery disease have not, to date, demonstrated clinical benefit. In this Review, we outline the biological characterization of VEGF, and examine the evidence for its potential therapeutic application, including the novel concept of VEGF as adjuvant therapy to stem cell transplantation, in patients with heart failure. PMID:23856679
Taimeh, Ziad; Loughran, John; Birks, Emma J; Bolli, Roberto
Maternal insulin-like growth factor binding protein-1, body mass index, and fetal growth
AIMTo examine the hypothesis that the maternal insulin-like growth factor system may constrain fetal growth.METHODSA prospective observational study of maternal serum insulin-like growth factor binding protein-1 (IGFBP-1) and fetal growth was undertaken in neonates with birthweights below the 5th centile. They had been classified either as having fetal growth restriction (FGR) due to placental dysfunction (increased umbilical artery Doppler pulsatility
Robert P Holmes; J M P Holly; Peter W Soothill
T helper type 1 cytokines and keratinocyte growth factor play a critical role in pseudoepitheliomatous hyperplasia initiation during cutaneous leishmaniasis
Pseudoepitheliomatous hyperplasia (PEH) is an exuberant proliferation of the epidermis. The underlying mechanism(s) that lead\\u000a to PEH have not been completely elucidated. Here, we characterize PEH during the healing stages of cutaneous leishmanial ulcers\\u000a in mice. During experimental cutaneous leishmaniasis (CL) C57BL\\/6 mice produce PEH, and BALB\\/c do not. A series of immunohistochemical\\u000a and immunological studies were performed to identify
Oleg E. Akilov; Michael J. Donovan; Thomas Stepinac; Cristina R. Carter; James P. Whitcomb; Tayyaba Hasan; Mary Ann McDowell
IP10 Inhibits Epidermal Growth Factor-induced Motility by Decreasing Epidermal Growth Factor Receptor-mediated Calpain Activity
During wound healing, fibroblasts are re- cruited from the surrounding tissue to accomplish re- pair. The requisite migration and proliferation of the fibroblasts is promoted by growth factors including those that activate the epidermal growth factor recep- tor (EGFR). Counterstimulatory factors in wound fluid are postulated to limit this response; among these fac- tors is the ELR-negative CXC chemokine, interferon
Hidenori Shiraha; Angela Glading; Kiran Gupta; Alan Wells
Actin filament dynamics impacts keratinocyte stem cell maintenance
Cultured human epidermal keratinocyte stem cells (holoclones) are crucial for regenerative medicine for burns and genetic disorders. In serial culture, holoclones progressively lose their proliferative capacity to become transient amplifying cells with limited growth (paraclones), a phenomenon termed clonal conversion. Although it negatively impacts the culture lifespan and the success of cell transplantation, little is known on the molecular mechanism underlying clonal conversion. Here, we show that holoclones and paraclones differ in their actin filament organization, with actin bundles distributed radially in holoclones and circumferentially in paraclones. Moreover, actin organization sets the stage for a differing response to epidermal growth factor (EGF), since EGF signalling induces a rapid expansion of colony size in holoclones and a significant reduction in paraclones. Furthermore, inhibition of PI3K or Rac1 in holoclones results in the reorganization of actin filaments in a pattern that is similar to that of paraclones. Importantly, continuous Rac1 inhibition in holoclones results in clonal conversion and reduction of growth potential. Together, our data connect loss of stem cells to EGF-induced colony dynamics governed by Rac1.
Nanba, Daisuke; Toki, Fujio; Matsushita, Natsuki; Matsushita, Sachi; Higashiyama, Shigeki; Barrandon, Yann
Angiotensin II?Induced Transactivation of Epidermal Growth Factor Receptor Regulates Fibronectin and Transforming Growth Factor-b Synthesis via
Abstract�The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of a growth factor, and recent evidence indicates transactivation of epidermal growth factor receptor (EGF-R) by G protein? coupled receptors. Here, we report the involvement of EGF-R in Ang II?induced synthesis of fibronectin and transforming growth factor-b (TGF-b )i n cardiac fibroblasts. Ang II stimulated fibronectin mRNA levels
Yasutaka Moriguchi; Hiroaki Matsubara; Yasukiyo Mori; Satoshi Murasawa; Hiroya Masaki; Katsuya Maruyama; Yoshiaki Tsutsumi; Yasunobu Shibasaki; Yoko Tanaka; Takuma Nakajima; Kinichiro Oda; Toshiji Iwasaka
Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3
Epidermal growth factor receptor (EGFR) is frequently overexpressed in esophageal carcinoma and its precursor lesions. To gain insights into how EGFR overexpression affects cellular functions in primary human esophageal cells, we performed gene expression profiling and identified insulin-like growth factor-binding protein (IGFBP)-3 as the most up- regulated gene. IGFBP-3 regulates cell proliferation through both insulin- like growth factor-dependent and independent
Munenori Takaoka; Hideki Harada; Claudia D. Andl; Kenji Oyama; Yoshio Naomoto; Kelly L. Dempsey; Andres J. Klein-Szanto; Wafik S. El-Deiry; Adda Grimberg; Hiroshi Nakagawa
Growth factor receptors as therapeutic targets: strategies to inhibit the insulin-like growth factor I receptor
Neoplastic transformation is often related to abnormal activation of growth factor receptors and their signaling pathways. The concept of targeting specific tumorigenic receptors and\\/or signaling molecules has been validated by the development and successful clinical application of drugs acting against the epidermal growth factor receptor 2 (HER2\\/neu, Erb2), the epidermal growth factor receptor 1 (EGFR, HER1), the Brc-Abl kinase, the
Eva Surmacz
Spatiotemporal control over growth factor signaling for therapeutic neovascularization ?
Many of the qualitative roles of growth factors involved in neovascularization have been delineated, but it is unclear yet from an engineering perspective how to use these factors as therapies. We propose that an approach that integrates quantitative spatiotemporal measurements of growth factor signaling using 3-D in vitro and in vivo models, mathematic modeling of factor tissue distribution, and new delivery technologies may provide an opportunity to engineer neovascularization on demand.
Cao, Lan; Mooney, David J.
Vascular endothelial growth factor in tuberculous meningitis.
Vascular endothelial growth factor (VEGF) is linked to brain edema and infarction, but there is paucity of studies correlating VEGF level with magnetic resonance imaging (MRI) changes in tuberculous meningitis (TBM). The aim of this study was to measure serum VEGF level in TBM and correlate it with clinical, laboratory, and MRI findings. Forty patients with TBM underwent cranial evaluation, cranial MRI, and MR angiography (MRA). Presence of exudates, hydrocephalous, infarction, tuberculoma, and MRA abnormalities was noted. Serum VEGF level was measured by enzyme-linked immunosorbent assay and compared in patients and controls. The VEGF level was also correlated with clinical, MRI, and MRA findings. The median age of the patients was 26.5 years. There was a trend towards higher serum VEGF level in TBM patients (100.7 � 110.6 pg/ml) compared to the controls (60.6 � 20.3 pg/ml). There was also a trend towards higher VEGF level in patients with shorter duration of illness (127.5 � 152.4 pg/ml vs 76.5 � 40.9 pg/ml), MRI evidence of infarction (131.4 � 150.7 pg/ml vs. 73.0 � 41.4 pg/ml), and paradoxical response (122.3 � 157.6 pg/ml vs. 88.8 � 50.8 pg/ml). Five patients died, and death was not related to VEGF level. It can be concluded that serum VEGF level in TBM patients is insignificantly higher in those with shorter duration of illness and infarction. PMID:23098361
Misra, Usha K; Kalita, Jayantee; Singh, Avadhesh P; Prasad, Sreeram
Fibroblast growth factor 19 entry into brain
Background Fibroblast growth factor (FGF)-19, an endocrine FGF protein mainly produced by the ileum, stimulates metabolic activity and alleviates obesity. FGF19 modulates metabolism after either intravenous or intracerebroventricular injection, and its receptor FGFR4 is present in the hypothalamus. This led to the question whether blood-borne FGF19 crosses the blood-brain barrier (BBB) to exert its metabolic effects. Methods We determined the pharmacokinetics of FGF19 permeation from blood to brain in comparison with its distribution in peripheral organs. Multiple-time regression analysis after intravenous bolus injection, in-situ brain perfusion, and HPLC assays were performed. Results FGF19 was relatively stable in blood and in the brain compartment. Significant influx was seen in the presence of excess unlabeled FGF19 in blood. This coincided with a slower decline of 125I-FGF19 in blood which suggested there was decreased clearance or peripheral tissue uptake. In support of an altered pattern of peripheral processing of 125I-FGF19 by excess unlabeled FGF19, the high influx to liver was significantly attenuated, whereas the minimal renal uptake was linearly accelerated. In the present setting, we did not detect a saturable transport of FGF19 across the BBB, as the entry rate of 125I-FGF19 was not altered by excess unlabeled FGF19 or its mouse homologue FGF15 during in-situ brain perfusion. Conclusion FGF19 remained stable in the blood and brain compartments for up to 10�min. Its influx to the brain was non-linear, non-saturable, and affected by its blood concentration and distribution in peripheral organs. Liver showed a robust and specific uptake of FGF19 that could be inhibited by the presence of excess unlabeled FGF19, whereas kidney clearance was dose-dependent.
Integrin control of the transforming growth factor-? pathway in glioblastoma.
Transforming growth factor-? is a central mediator of the malignant phenotype of glioblastoma, the most common and malignant form of intrinsic brain tumours. Transforming growth factor-? promotes invasiveness and angiogenesis, maintains cancer cell stemness and induces profound immunosuppression in the host. Integrins regulate cellular adhesion and transmit signals important for cell survival, proliferation, differentiation and motility, and may be involved in the activation of transforming growth factor-?. We report that ?v?3, ?v?5 and ?v?8 integrins are broadly expressed not only in glioblastoma blood vessels but also in tumour cells. Exposure to ?v, ?3 or ?5 neutralizing antibodies, RNA interference-mediated integrin gene silencing or pharmacological integrin inhibition using the cyclic RGD peptide EMD 121974 (cilengitide) results in reduced phosphorylation of Smad2 in most glioma cell lines, including glioma-initiating cell lines and reduced transforming growth factor-?-mediated reporter gene activity, coinciding with reduced transforming growth factor-? protein levels in the supernatant. Time course experiments indicated that the loss of transforming growth factor-? bioactivity due to integrin inhibition likely results from two distinct mechanisms: an early effect on activation of preformed inactive protein, and second, major effect on transforming growth factor-? gene transcription as confirmed by decreased activity of the transforming growth factor-? gene promoter and decreased transforming growth factor-?(1) and transforming growth factor-?(2) messenger RNA expression levels. In vivo, EMD 121974 (cilengitide), which is currently in late clinical development as an antiangiogenic agent in newly diagnosed glioblastoma, was a weak antagonist of pSmad2 phosphorylation. These results validate integrin inhibition as a promising strategy not only to inhibit angiogenesis, but also to block transforming growth factor-?-controlled features of malignancy including invasiveness, stemness and immunosuppression in human glioblastoma. PMID:23378223
Roth, Patrick; Silginer, Manuela; Goodman, Simon L; Hasenbach, Kathy; Thies, Svenja; Maurer, Gabriele; Schraml, Peter; Tabatabai, Ghazaleh; Moch, Holger; Tritschler, Isabel; Weller, Michael
Adsorption and release properties of growth factors from biodegradable implants.
The present investigation was performed to study the adsorption behavior of growth factors and their release characteristics from biodegradable implants in an in vitro study. We investigated the stability of growth factors administered on various scaffolds. We used porous tricalcium phosphate ceramics (alpha-TCP), a neutralized glass-ceramics (GB9N), a composite (polylactid/-glycolid/GB9N), and solvent dehydrated human bone as carriers. Block shaped scaffolds (sized: 7 x 7 x 10 mm) were loaded with 5 microg of either bone morphogenetic protein (rxBMP-4), basic fibroblast growth factor (rh-bFGF), or vascular endothelial growth factor (rh-VEGF) solved in 150 microL PBS. The growth factors were labeled with Iodine125 (I-125) for detecting the adsorbed and released amount of growth factors by counting the samples for total I-125 activity. We observed that the adsorption of these growth factors seems to depend on two different parameters: first on the nature of the tested material, and second on the growth factors on their own. The release kinetics of the growth factors from the biodegradable implants can be described as a two phase process-a very rapid release during the first hours by an elution of not adsorbed protein, followed by a specific release, which depends upon the chemical/physical interaction of the material and the growth factor used. Analyzing the eluted proteins on SDS-PAGEs rh-VEGF was degraded into a smaller fragment with a size of around 15 kDa, while rxBMP-4 and rh-bFGF showed a complete degradation into fragments smaller than 3 kDa after more than 3 days. Although this in vitro study suggests that biodegradable implants might be successfully used as carriers for osteogenic growth factors, the different release kinetics as well as the alteration of their molecular structure including loss of biological activity should be considered. PMID:11774299
Ziegler, J; Mayr-Wohlfart, U; Kessler, S; Breitig, D; G�nther, K-P
Induction of Hair Growth by Insulin-Like Growth Factor-1 in 1,763 MHz Radiofrequency-Irradiated Hair Follicle Cells
Radiofrequency (RF) radiation does not transfer high energy to break the covalent bonds of macromolecules, but these low energy stimuli might be sufficient to induce molecular responses in a specific manner. We monitored the effect of 1,763 MHz RF radiation on cultured human dermal papilla cells (hDPCs) by evaluating changes in the expression of cytokines related to hair growth. The expression of insulin-like growth factor-1 (IGF-1) mRNA in hDPCs was significantly induced upon RF radiation at the specific absorption rate of 10 W/kg, which resulted in increased expression of B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL-2) and cyclin D1 (CCND1) proteins and increased phosphorylation of MAPK1 protein. Exposure to 10 W/kg RF radiation 1 h per day for 7 days significantly enhanced hair shaft elongation in ex vivo hair organ cultures. In RF-exposed follicular matrix keratinocytes in the hair bulb, the expression of Ki-67 was increased, while the signal for terminal deoxynucleotidyl transferase dUTP nick end labeling was reduced. From these results, we suggest that 1,763 MHz RF exposure stimulates hair growth in vitro through the induction of IGF-1 in hDPCs.
Jo, Seong Jin; Cho, A-Ri; Jeon, Soon-Ik; Choi, Hyung-Do; Kim, Kyu Han; Park, Gun-Sik; Pack, Jeong-Ki; Kwon, Oh Sang; Park, Woong-Yang
Activated extracellular signal-regulated kinases: association with epidermal growth factor receptor/transforming growth factor alpha expression in head and neck squamous carcinoma and inhibition by anti-epidermal growth factor receptor treatments.
The expression of the activated mitogen-activated kinases/extracellular signal-regulated kinases (ERKs) ERK1 and ERK2 was characterized in 101 humanhead and neck squamous carcinoma specimens. Activated ERK1/2were detected at different levels in the majority of these tumors, as assayed by immunostaining with an antibody specific for the dually phosphorylated and activated ERK1 and ERK2. ERK1/2 activation levels were higher in tumors with advanced regional lymph node metastasis (P = 0.048) and in relapsed tumors (P = 0.021). The expression of epidermal growth factor (EGF) receptor (P = 0.037), transforming growth factor alpha (TGF-alpha; P < 0.001), and HER2 (P = 0.066; positive trend) correlated with activation of ERK1/2. In a multivariate analysis, both TGF-alpha (P < 0.0001) and HER2 (P = 0.045) were independently correlated with ERK1/2 activation. In turn, activation of ERK1/2 was associated with a higher Ki-67 proliferative index (P = 0.002). In EGF receptor-dependent model cells (A431 and DiFi), a specific EGF receptor tyrosine kinase inhibitor ("Iressa"; ZD1839) and a chimeric anti-EGF receptor antibody ("Cetuximab"; C225) inhibited ERK 1/2 activation at concentrations that inhibited autocrine cell proliferation. In patients on treatment with C225, the activation of ERK1/2 in skin, an EGF receptor-dependent tissue, was lower compared with control skin. Parallel changes were seen in keratinocyte Ki67 proliferation indexes in skin from C225-treated patients. Taken together, these studies provide support for a role of activation of ERK1/2 in head and neck squamous carcinoma and a correlation with EGF receptor/TGF-alpha expression. The inhibition of ERK1/2 activation in vitro and in vivo by compounds targeting the EGF receptor points to the interest of ERK1/2 as potential surrogate markers of EGF-receptor signaling in clinical therapeutic studies. PMID:11522647
Albanell, J; Codony-Servat, J; Rojo, F; Del Campo, J M; Sauleda, S; Anido, J; Raspall, G; Giralt, J; Rosell�, J; Nicholson, R I; Mendelsohn, J; Baselga, J
Basic fibroblast growth factor promotes epidermal growth factor responsiveness and survival of mesencephalic neural precursor cells.
Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) induce proliferation of neural precursor cells from several central nervous system regions in vitro. We have previously described two neural precursor cell populations from 13.5 days postcoitium (dpc) mesencephalon, one forming colonies in response to EGF, present in the ventral mesencephalon, and other forming colonies in response to EGF + bFGF, mainly present in the dorsal mesencephalon. In the present work, we show that 13.5 dpc dorsal mesencephalic cells required bFGF only for 1 h to form colonies in response to EGF alone, indicating that these two growth factors act in sequence rather than simultaneously. Absence of bFGF at the beginning of the culture gave rise to very few colonies, even after the addition of EGF + bFGF, suggesting that cells responsive to bFGF were very labile in the primary culture condition. This result is in contrast with cells pretreated with bFGF, which could survive for up to 5 days in the absence of bFGF or EGF, and then were capable of efficiently forming colonies in response to EGF. Basic FGF was also able to support survival of EGF-responsive neural precursors from both ventral and dorsal mesencephalon. The population requiring bFGF to form colonies in response to EGF was identified at different developmental stages (11.5-15.5 dpc), with higher contribution to the total number of neural precursors cells detected (EGF-responsive plus bFGF-responsive) at early stages and in the dorsal region. We show that the differentiation effect of bFGF resulted in the appearance of the mRNA coding for the EGF receptor. Our data suggest that bFGF-responsive neural precursors are the source of EGF-responsive neural precursors. PMID:10398068
Santa-Olalla, J; Covarrubias, L
Platelet-derived growth factor receptor regulates salivary gland morphogenesis via fibroblast growth factor expression.
A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is beta-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor alpha is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRalpha, and PDGFRbeta were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme. PMID:18559345
Yamamoto, Shinya; Fukumoto, Emiko; Yoshizaki, Keigo; Iwamoto, Tsutomu; Yamada, Aya; Tanaka, Kojiro; Suzuki, Hiroharu; Aizawa, Shizuko; Arakaki, Makiko; Yuasa, Kenji; Oka, Kyoko; Chai, Yang; Nonaka, Kazuaki; Fukumoto, Satoshi
Possible role of transforming growth factor-?1 and vascular endothelial growth factor in Fabry disease nephropathy
Fabry disease is a lysosomal storage disorder (LSD) caused by deficiency of ?-galactosidase A (?-gal A), resulting in deposition of globotriaosylceramide (Gb3; also known as ceramide trihexoside) in the vascular endothelium of many organs. A gradual accumulation of Gb3 leads to cardiovascular, cerebrovascular and renal dysfunction. Endothelial cell dysfunction leads to renal complications, one of the main symptoms of Fabry disease. However, the pathological mechanisms by which endothelial dysfunction occurs in Fabry disease are poorly characterized. The purpose of this study was to investigate whether the expression of transforming growth factor-?1 (TGF-?1) and vascular endothelial growth factor (VEGF) is associated with the renal pathogenesis of Fabry disease. We found that the protein expression levels of renal thrombospondin-1 (TSP-1), TGF-?1 and VEGF were higher in the kidneys from Fabry mice compared to wild-type mice. The expression levels of VEGF receptor 2 (VEGFR2), fibroblast growth factor-2 (FGF-2) and phospho-p38 (P-p38) were also higher in the kidneys from Fabry mice compared with wild-type mice. Activities of cysteine aspartic acid protease (caspase)-6 and caspase-9 were higher in kidneys from Fabry than from the wild-type mice. These results suggest that overexpression of TGF-?1 and VEGF in the Fabry mouse kidney might contribute to Fabry disease nephropathy by inducing apoptosis. To test whether Gb3 accumulation can induce apoptosis, we incubated bovine aortic endothelial cells with Gb3 and found increased expression of TGF-?1, VEGFR2, VEGF, FGF-2 and P-p38. The combination of increased expression of TGF-?1 and VEGF caused by Gb3 accumulation may allow upregulation of FGF-2, VEGFR2 and P-p38 expression, and these changes may be associated with Fabry disease nephropathy by inducing apoptosis.
Possible role of transforming growth factor-?1 and vascular endothelial growth factor in Fabry disease nephropathy.
Fabry disease is a lysosomal storage disorder (LSD) caused by deficiency of ?-galactosidase�A (?-gal A), resulting in deposition of globotriaosylceramide (Gb3; also known as ceramide trihexoside) in the vascular endothelium of many organs. A gradual accumulation of Gb3 leads to cardiovascular, cerebrovascular and renal dysfunction. Endothelial cell dysfunction leads to renal complications, one of the main symptoms of Fabry disease. However, the pathological mechanisms by which endothelial dysfunction occurs in Fabry disease are poorly characterized. The purpose of this study was to investigate whether the expression of transforming growth factor-?1 (TGF-?1) and vascular endothelial growth factor (VEGF) is associated with the renal pathogenesis of Fabry disease. We found that the protein expression levels of renal thrombospondin-1 (TSP-1), TGF-?1 and VEGF were higher in the kidneys from Fabry mice compared to wild-type mice. The expression levels of VEGF receptor�2 (VEGFR2), fibroblast growth factor-2 (FGF-2) and phospho-p38 (P-p38) were also higher in the kidneys from Fabry mice compared with wild-type mice. Activities of cysteine aspartic acid protease (caspase)-6 and caspase-9 were higher in kidneys from Fabry than from the wild-type mice. These results suggest that overexpression of TGF-?1 and VEGF in the Fabry mouse kidney might contribute to Fabry disease nephropathy by inducing apoptosis. To test whether Gb3 accumulation can induce apoptosis, we incubated bovine aortic endothelial cells with Gb3 and found increased expression of TGF-?1, VEGFR2, VEGF, FGF-2 and P-p38. The combination of increased expression of TGF-?1 and VEGF caused by Gb3 accumulation may allow upregulation of FGF-2, VEGFR2 and P-p38 expression, and these changes may be associated with Fabry disease nephropathy by inducing apoptosis. PMID:23007467
Lee, Mi Hee; Choi, Eun Nam; Jeon, Yeo Jin; Jung, Sung-Chul
Insulin-Like Growth Factor (IGF) family and prostate cancer
There is abundant in vitro, animal and epidemiologic evidence to suggest that the Insulin-Like Growth Factor (IGF) family is a multi-component network of molecules which is involved in the regulation of both physiological and pathological growth processes in prostate. The IGF family plays a key role in cellular metabolism, differentiation, proliferation, transformation and apoptosis, during normal development and malignant growth.
C. Gennigens; C. Menetrier-Caux; J. P. Droz
Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cytoprotective agent in several organ systems but its roles in liver fibrosis are unclear. We studied the roles of HB-EGF in experimental liver fibrosis in mice and during hepatic stellate cell (HSC) activation. Thioacetamide (TAA; 100mg/kg) was administered by intra-peritoneal injection three times a week for 4 weeks to wild-type HB-EGF+/+ or HB-EGF-null (HB-EGF?/?) male mice. Livers were examined for histology and expression of key fibrotic markers. Primary cultured HSC isolated from untreated HB-EGF+/+ or HB-EGF?/? mice were examined for fibrotic markers and/or cell migration either during culture-induced activation or after exogenous HB-EGF (100 ng/ml) treatment. TAA induced liver fibrosis in both HB-EGF+/+ and HB-EGF?/? mice. Hepatic HB-EGF expression was decreased in TAA-treated HB-EGF+/+ mice by 37.6% (p < 0.05) as compared to animals receiving saline alone. HB-EGF?/? mice treated with TAA showed increased hepatic ?-smooth muscle actin-positive cells and collagen deposition, and, as compared to HB-EGF+/+ mice, TAA-stimulated hepatic mRNA levels in HB-EGF?/? mice were, respectively, 2.1-, 1.7-, 1.8-, 2.2-, 1.2-, or 3.3-fold greater for ?-smooth muscle actin, ?1 chain of collagen I or III (COL1A1 or COL3A1), transforming growth factor-?1, connective tissue growth factor, or tissue inhibitor of metalloproteinase-1 (p < 0.05). HB-EGF expression was detectable in primary cultured HSC from HB-EGF+/+ mice. Both endogenous and exogenous HB-EGF inhibited HSC activation in primary culture, and HB-EGF enhanced HSC migration. These findings suggest that HB-EGF gene knockout in mice increases susceptibility to chronic TAA-induced hepatic fibrosis and that HB-EGF expression or action is associated with suppression of fibrogenic pathways in HSC.
Huang, Guangcun; Besner, Gail E.; Brigstock, David R.
Redundancy of myostatin and growth\\/differentiation factor 11 function
BACKGROUND: Myostatin (Mstn) and growth\\/differentiation factor 11 (Gdf11) are highly related transforming growth factor ? (TGF?) family members that play important roles in regulating embryonic development and adult tissue homeostasis. Despite their high degree of sequence identity, targeted mutations in these genes result in non-overlapping phenotypes affecting distinct biological processes. Loss of Mstn in mice causes a doubling of skeletal
Alexandra C McPherron; Thanh V Huynh; Se-Jin Lee
Control of Growth Factor Networks by Heparan Sulfate Proteoglycans
Growth factor binding to transmembrane protein receptors is generally understood to initiate cell signaling. Receptor binding of heparin-binding growth factors (HB-GFs), such as fibroblast growth factor-2 (FGF-2), is regulated by interactions with heparan sulfate proteoglycans. While there is some specificity for binding to heparan sulfate, overlap in sites for different growth factors may allow for cross regulation. Here we demonstrate, using experiments and computer simulations, that the HB-GFs FGF-2 and heparin-binding EGF-like growth factor (HB-EGF) can cross regulate receptor binding of the other despite having unique receptors. The ability of HSPG to stabilize HB-GF receptor binding is critical for competing growth factors to modulate receptor binding with both enhanced and reduced binding possible depending on this stabilization process. HSPG density and affinity for HB-GF are also critical factors for HB-GF cross regulation. Simulations further reveal that HB-GF can regulate receptor binding of non-HB-GFs such as EGF even when the two proteins share no binding sites when other HB-GF are present within the network. Proliferation studies demonstrate potentiation of HB-EGF-induced growth by FGF-2 indicating that competition networks can alter biological response. Exogenous manipulation of cellular responses to growth factors in complex living systems will require understanding the HSPG-controlled network.
Forsten-Williams, Kimberly; Chu, Chia Lin; Fannon, Michael; Buczek-Thomas, Jo Ann; Nugent, Matthew A.
Molecular cloning and expression of human hepatocyte growth factor
HEPATOCYTE growth factor (HGF) is the most potent mitogen for mature parenchyma! hepatocytes in primary culture, and seems to be a hepatotrophic factor that acts as a trigger for liver regeneration after partial hepatectomy and liver injury. The partial purification and characterization of HGF have been reported1-4. We have demonstrated that pure HGF from rat platelets is a new growth
Toshikazu Nakamura; Tsutomu Nishizawa; Mitchio Hagiya; Tatsuya Seki; Manabu Shimonishi; Atsushi Sugimura; Kosuke Tashiro; Shin Shimizu
In vivo cooperation between Bcl-xL and the phosphoinositide 3-kinase-Akt signaling pathway for the protection of epidermal keratinocytes from apoptosis.
To investigate the function of Bcl-xL in the skin, we established keratinocyte-specific Bcl-x gene-targeted mice under the keratin 5 promoter (K5). K5.Bcl-xL-/- mice were viable, devoid of alteration in the development of skin or appendages. However, they harbored spontaneous apoptotic keratinocytes in the epidermis. Bcl-xL-deficient keratinocytes cultured in vitro readily underwent apoptosis in the absence of growth factors, but the addition of HGF or EGF resulted in restoration of cell survival, which was reversed by treatment with wortmannin, an inhibitor of phosphoinositide-3 kinase (PI3K). Topical treatment of K5.Bcl-xL-/- mice with wortmannin sensitized the skin for apoptosis induced by UV (UV) B, although wild-type epidermis was only marginally affected by this treatment, suggesting that the resistance to UVB largely depended on PI3K-Akt signaling in Bcl-xL-deficient mice but not in wild-type mice. Furthermore, UVB irradiation resulted in redistribution of phosphorylated Akt from the basal layer to the suprabasal layer, indicating that Akt could spatially cooperate with Bcl-xL upon UVB exposure in the upper epidermis where Bcl-xL is normally localized. These results suggest that Bcl-xL and the PI3K-Akt pathway form a cooperative, intercompensatory axis for the protection of epidermal keratinocytes from apoptosis in vivo. PMID:12665473
Umeda, Jiro; Sano, Shigetoshi; Kogawa, Kazuhiko; Motoyama, Noboru; Yoshikawa, Kunihiko; Itami, Satoshi; Kondoh, Gen; Watanabe, Takeshi; Takeda, Junji
A unique growth factor in patients with acromegaloidism.
Acromegaloidism is a syndrome characterized by features of acromegaly without biochemical evidence of excessive GH or somatomedin production. We searched for a growth factor in the serum of patients with this syndrome. Growth-promoting activity was measured by determining the stimulatory effect of whole and fractionated serum on colony formation by human erythroid progenitors in vitro. Sera from five subjects with acromegaloidism gave a mean (+/- SEM) stimulated colony growth of 211 +/- 4.0 colonies, in contrast to normal sera which yielded a mean colony growth of 100 +/- 11.0 (n = 9; P less than 0.001). When serum was chromatographed on a Sephadex G-200 column, the maximal stimulation of colony growth was found in the fractions coinciding with the descending slope of the second protein peak. Based on gel filtration chromatography, the estimated molecular weight was 70,000 daltons. Epidermal growth factor, nerve growth factor, fibroblast growth factor, and platelet-derived growth factor resulted in no substantial stimulation of colony growth under the conditions used. Although the erythroid progenitor cells of a Laron dwarf were unresponsive to 200 ng/ml human GH, they were clearly stimulated by serum from a patient with acromegaloidism. The present study describes the presence of a heretofore unidentified growth factor in the serum of subjects with acromegaloidism. This factor also stimulated the erythroid precursor cells of a Laron dwarf whose cells were unresponsive to GH. The physiological role of this growth factor in normal man as well as its pathogenic role in subjects with acromegaloidism remain to be established. PMID:6863475
Ashcraft, M W; Hartzband, P I; Van Herle, A J; Bersch, N; Golde, D W
Extracellular matrix and growth factors in branching morphogenesis
NASA Technical Reports Server (NTRS)
The unifying hypothesis of the NSCORT in gravitational biology postulates that the ECM and growth factors are key interrelated components of a macromolecular regulatory system. The ECM is known to be important in growth and branching morphogenesis of embryonic organs. Growth factors have been detected in the developing embryo, and often the pattern of localization is associated with areas undergoing epithelial-mesenchymal interactions. Causal relationships between these components may be of fundamental importance in control of branching morphogenesis.
Hardman, P.; Spooner, B. S.
Role of fibroblast growth factor receptor signaling in kidney development
Fibroblast growth factor receptors (Fgfrs) are expressed throughout the developing kidney. Several early studies have shown\\u000a that exogenous fibroblast growth factors (Fgfs) affect growth and maturation of the metanephric mesenchyme (MM) and ureteric\\u000a bud (UB). Transgenic mice that over-express a dominant negative receptor isoform develop renal aplasia\\/severe dysplasia, confirming\\u000a the importance of Fgfrs in renal development. Furthermore, global deletion of
Carlton M. Bates
Human Epidermal Growth Factor: Isolation and Chemical and Biological Properties
A polypeptide hormone has been isolated from human urine, human epidermal growth factor. It was assayed by its ability to compete with 125I-labeled mouse-derived epidermal growth factor in binding to human foreskin fibroblasts. The biological effects of the human polypeptide are similar to those previously described for the mouse hormone. These include the stimulation of the growth in vitro of
Stanley Cohen; Graham Carpenter
Insulin-Like Growth Factor I Stimulates Dendritic Growth in Primary Somatosensory Cortex
The temporal and spatial distributions of several growth factors suggest roles in the regulation of neuronal differentiation in the neocortex. Among such growth factors, the insulin-like growth factors (IGF-I and -II) are of particular interest because they are available to neurons from multiple sources under independent control. IGF-I is produced by many neurons throughout the brain and also by cells
Mary M. Niblock; Brunso Bechtold Jk
Effect of 1,25-dihydroxyvitamin D 3 on human keratinocytes grown under different culture conditions
Summary��1,25-Dihydroxyvitamin D3 (1,25-(OH)2-D3) is known to decrease the proliferation and increase the differentiation of different cell types including human keratinocytes.\\u000a The growth and differentiation of keratinocytes in the presence of 1,25-(OH)2-D3 using serum-free media formulations has been described previously. This investigation extends these studies to describe various\\u000a culture conditions with human foreskin keratinocytes to determine the optimal antiproliferative activity of
John A. McLane; Marion Katz; Nana Abdelkader
TITLE: TERATOGENIC RESPONSES ARE MODULATED IN MICE LACKING EXPRESSION OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF). AUTHORS (ALL): Abbott, Barbara D.1; Best, Deborah S.1; Narotsky, Michael G.1. SPONSOR NAME: None INSTITUTIONS (ALL): 1. Repro Tox ...
Transgenic Studies with a Keratin Promoter-Driven Growth Hormone Transgene: Prospects for Gene Therapy
NASA Astrophysics Data System (ADS)
Keratinocytes are potentially appealing vehicles for the delivery of secreted gene products because they can be transferred to human skin by the relatively simple procedure of grafting. Adult human keratinocytes can be efficiently propagated in culture with sufficient proliferative capacity to produce enough epidermis to cover the body surface of an average adult. However, the feasibility of delivering secreted proteins through skin grafting rests upon (i) the strength of the promoter in keratinocytes and (ii) the efficiency of protein transport through the basement membrane of the stratified epithelium and into the bloodstream. In this paper, we use transgenic technology to demonstrate that the activity of the human keratin 14 promoter remains high in adult skin and that keratinocyte-derived human growth hormone (hGH) can be produced, secreted, and transported to the bloodstream of mice with efficiency that is sufficient to exceed by an order of magnitude the circulating hGH concentration in growing children. Transgenic skin grafts from these adults continue to produce and secrete hGH stably, at ? 1/10 physiological levels in the bloodstream of nontransgenic recipient mice. These studies underscore the utility of the keratin 14 promoter for expressing foreign transgenes in keratinocytes and demonstrate that keratinocytes can be used as effective vehicles for transporting factors to the bloodstream and for eliciting metabolic changes. These findings have important implications for considering the keratinocyte as a possible vehicle for gene therapy.
Wang, Xiaoming; Zinkel, Sandra; Polonsky, Kenneth; Fuchs, Elaine
Vascular endothelial growth factor serum concentrations in ovarian cancer
Objective: To determine whether serum vascular endothelial growth factor is an independent prognostic factor in ovarian cancer patients.Methods: We measured vascular endothelial growth factor in pretreatment serum samples of 60 women with International Federation of Gynecology and Obstetrics stages I to IV epithelial ovarian cancer, using an enzyme-linked immunosorbent assay. The results were correlated to clinical data.Results: The median vascular
Clemens Tempfer; Andreas Obermair; Lukas Hefler; Guenther Haeusler; Gerald Gitsch; Christian Kainz
Early changes in protein synthesis induced by basic fibroblast growth factor, nerve growth factor, and epidermal growth factor in PC12 pheochromocytoma cells.
Nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) stimulate neuronal differentiation, whereas epidermal growth factor (EGF) promotes only mitogenic responses in PC12 pheochromocytoma cells. The early changes in protein synthesis induced by bFGF, NGF, and EGF in these cells have been determined by two-dimensional PAGE of [35S]methionine-labeled proteins and computerized image analysis. The rate of synthesis of only 29 proteins (out of approximately 1500 identified) was found to be modulated during the first several hours of growth factor stimulation. Individually, 12 were affected by EGF, 23 were affected by bFGF, and 20 were affected by NGF. Eight of these were regulated by all three growth factors, while 10 proteins were commonly induced by bFGF and NGF, in accordance with the essentially identical morphological responses induced by these two factors. In addition, the effects of bFGF and NGF were about equally divided between increases and decreases in the rate of synthesis of individual proteins, whereas EGF caused significantly more positive (increased) responses. All proteins modulated by NGF or FGF alone were negative in their response and those induced by only EGF were positive. Of particular interest, the rate of synthesis of two proteins of 55 kDa and pI 5.45 and 5.50 was dramatically and transiently induced during the first 2 hr of bFGF and NGF treatment and was not affected by EGF. This study indicates that all three factors elicit early increases and decreases in the synthesis of a quite limited number of proteins and provides molecular evidence for the specificity of a differentiative vs. a proliferative growth factor-induced signaling pathway in these cells. Images
Hondermarck, H; McLaughlin, C S; Patterson, S D; Bradshaw, R A
Insulin-like growth factor-I and cancer risk.
Growth factor pathways are fundamental in normal tissue regulation and development. In many tissues, factors that function in normal growth and development also have important regulatory roles in transformed malignant cells. The insulin-like growth factor (IGF) system is implicated in the regulation of the malignant phenotype by its effects on proliferation, differentiation, and apoptosis. IGF-I has also been linked to malignant transformation. The role of the IGF-I in cancer has been recognized in both experimental and clinical settings, suggesting that the enhancement of growth factor pathways potentially could increase the risk for cancer development. In this paper, the role of IGF-I signaling in tumor regulation, and the impact of IGF-I modulation using growth hormone replacement therapy are discussed. PMID:15231294
Ibrahim, Yasir H; Yee, Douglas
Transcription factor control of growth rate dependent genes in Saccharomyces cerevisiae: A three factor design
BACKGROUND: Characterization of cellular growth is central to understanding living systems. Here, we applied a three-factor design to study the relationship between specific growth rate and genome-wide gene expression in 36 steady-state chemostat cultures of Saccharomyces cerevisiae. The three factors we considered were specific growth rate, nutrient limitation, and oxygen availability. RESULTS: We identified 268 growth rate dependent genes, independent
Alessandro Fazio; Michael C. Jewett; Pascale Daran-Lapujade; Roberta Mustacchi; Renata Usaite; Jack T. Pronk; Christopher T. Workman; Jens Nielsen
Characteristic Cytokines Generated by Keratinocytes and Mononuclear Infiltrates in Oral Lichen Planus
Cytokine production was investigated in oral keratinocytes and tissue-infiltrated mononuclear cells (TIMC) obtained from patients with oral lichen planus (OLP). The numbers of cells producing interleukine (IL) -1?, IL-4, IL-6, granulocyte colony-stimulating factor, and tumor necrosis factor-? per 104 cells in keratinocytes from patients with OLP were determined by enzyme-linked immunospot assay. These levels were two to threefold greater than
Tetsuya Yamamoto; Tokio Osaki
Keratinocytes synthesize and activate cortisol.
The bioavailability of circulating and/or endogenous hydrocortisone (cortisol) in epidermal cells is a key determinant in inflammatory disease and chronic wounds. It is not known, however, whether epidermal cells can regulate tissue cortisol and whether they are capable of producing endogenous glucocorticoids. In the present study, we show by microarray analysis that epidermal cells express mRNAs to all the major enzymes involved in the metabolic chain from cholesterol to cortisol, including cytocrome P450 chain, 11?-hydroxysteroid dehydrogenases (HSD11Bs), adrenocorticotropic hormone (ACTH) receptor (MC2R), and glucocorticoid receptor. The two enzymes mediating activation/deactivation of cortisone to cortisol, namely HSD11B1 and HSD11B2, were expressed at the protein level in cultured keratinocytes as well as human skin samples, as shown by Western blotting and immunohistochemistry, respectively. In functional assays, we show that keratinocytes are not only able to activate cortisone to cortisol in a HSD11B-dependent manner but also silencing of either HSD11B1 or HSD11B2 specifically modulates the bioavailability of the inactive glucocorticoid and the active steroid, respectively. A further key observation was that keratinocytes responded to stimulation with ACTH by a significant increase in the de novo synthesis of cortisol. Taken together, we provide evidence for a novel non-adrenal steroideal system in human keratinocytes. PMID:21344493
Cirillo, Nicola; Prime, Stephen S
DNA repair in cultured keratinocytes
Most of our understanding of DNA repair mechanisms in human cells has come from the study of these processes in cultured fibroblasts. The unique properties of keratinocytes and their pattern of terminal differentiation led us to a comparative examination of their DNA repair properties. The relative repair capabilities of the basal cells and the differentiated epidermal keratinocytes as well as possible correlations of DNA repair capacity with respect to age of the donor have been examined. In addition, since portions of human skin are chronically exposed to sunlight, the repair response to ultraviolet (UV) irradiation (254 nm) when the cells are conditioned by chronic low-level UV irradiation has been assessed. The comparative studies of DNA repair in keratinocytes from infant and aged donors have revealed no significant age-related differences for repair of UV-induced damage to DNA. Sublethal UV conditioning of cells from infant skin had no appreciable effect on either the repair or normal replication response to higher, challenge doses of UVL. However, such conditioning resulted in attenuated repair in keratinocytes from adult skin after UV doses above 25 J/m2. In addition, a surprising enhancement in replication was seen in conditioned cells from adult following challenge UV doses.
Liu, S.C.; Parsons, S.; Hanawalt, P.C.
Involvement of growth factor receptors in the mammalian UVC response.
Irradiation of HeLa cells with short-wavelength ultraviolet light (UVC) induces the modification and activation of the preexisting transcription factors c-Fos-c-Jun (AP-1) and TCF/Elk-1, as well as the protein synthesis independent transcriptional activation of the c-fos and c-jun genes. This response to UVC is mediated via obligatory cytoplasmic signal transduction, involving Ras and Raf, Src, and MAP kinases. The UVC response is inhibited by prior down-modulation of growth factor receptor signaling upon growth factor prestimulation, by suramin (an inhibitor of receptor activation) or by expression of a dominant negative epidermal growth factor (EGF) receptor mutant. These data suggest the involvement of several growth factor receptors in the UVC response. Indeed, UVC induces the suramin-inhibitable immediate tyrosine phosphorylation of the EGF receptor. PMID:7923365
Sachsenmaier, C; Radler-Pohl, A; Zinck, R; Nordheim, A; Herrlich, P; Rahmsdorf, H J
A tumor growth inhibitory factor and a tumor growth promoting factor isolated from unfertilized ova of shad (Alosa sapidissima).
In the present study, a cytostatic tumor growth inhibitory peptide and a tumor growth promoting peptide with molecular weights of 20,000-30,000 Da have been identified in the supernatant fraction of unfertilized ova from Shad. The factors can be separated by gel chromatography, thus indicating that the factors are individual molecules. Both of the factors are nondialyzable, heat stable, and resistant to trypsin digestion and periodate oxidation. PMID:2930539
Sheid, B; Prat, J C; Gaetjens, E
ENOD40 encodes a peptide growth factor
Rhizobium bacteria induce the formation of nodules on the roots of leguminous plants. The nodules create the right biological niche for the rhizobia to carry out biological nitrogen fixation by which atmospheric nitrogen is reduced to ammonia. The nodule is a new organ that provides the plant with a nitrogen source for its growth and development. The formation of a
Sande van de G. P. C. M
Covalent binding of BP-metabolites to DNA of cultured human hair follicle keratinocytes
Primary cultures of human hair follicle keratinocytes were established by using a basement membrane-like growth substrate, the bovine eye lens capsule. A method was adapted for the isolation of 3H-benzo(a)pyrene (BP)-modified DNA from the cellular outgrowth of only one hair follicle (approximately 2�105 cells). In a routine procedure hair follicle keratinocytes were incubated with 0.5 �M 3H-BP for 24 h.
M. W. A. C. Hukkelhoven; A. M. Bronkhorst; A. J. M. Vermorken
Immunoglobulins, growth factors and growth hormone in bovine colostrum and the effects of processing
In colostrum collected 0�80h postpartum the contents of immunoglobulins (Igs), transforming growth factor beta-2 (TGF-?2), insulin-like growth factor-1 (IGF-1) and growth hormone (GH) were analysed. Colostrum initially contained 90mgmL?1 IgG1, 2.8mgmL?1 IgG2, 1.6mgmL?1 IgA, 4.5mgmL?1 IgM, and these concentrations declined by 92%, 87%, 93% and 84%, respectively, in the samples collected later. Of the growth factors, colostrum initially contained 289�310ngmL?1
Lidia Elfstrand; Helena Lindmark-M�nsson; Marie Paulsson; Lena Nyberg; Bj�rn �kesson
Targeting the Insulin Growth Factor Receptor 1
Synopsis The IGF axis is a tightly controlled endocrine system that regulates cell growth and development, known to have an important function in cancer biology. IGF1 and IGF2 can promote cancer growth in a GH-independent manner both through paracrine and autocrine secretion and can also confer resistance to chemotherapy and radiation. Many alterations of this system have been found in neoplasias, including increased expression of ligands and receptors, loss of heterozigosity of the IGF2 locus and increased IGF1R gene copy number. The IGF1 network is an attractive candidate for targeted therapy, including receptor blockade with monoclonal antibodies and small molecule inhibitors of receptor downstream signaling. This article reviews the role of the IGF axis in the initiation and progression of cancer, and describes the recent advances in IGF inhibition as a therapeutic tool.
Arnaldez, Fernanda I.; Helman, Lee J.
Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice.
The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/"metabolizing acquired dioxin-induced skin hamartomas" (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis. PMID:24503019
Sch�fer, Matthias; Willrodt, Ann-Helen; Kurinna, Svitlana; Link, Andrea S; Farwanah, Hany; Geusau, Alexandra; Gruber, Florian; Sorg, Olivier; Huebner, Aaron J; Roop, Dennis R; Sandhoff, Konrad; Saurat, Jean-Hilaire; Tschachler, Erwin; Schneider, Marlon R; Langbein, Lutz; Bloch, Wilhelm; Beer, Hans-Dietmar; Werner, Sabine
Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice
The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/�metabolizing acquired dioxin-induced skin hamartomas� (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis.
Schafer, Matthias; Willrodt, Ann-Helen; Kurinna, Svitlana; Link, Andrea S; Farwanah, Hany; Geusau, Alexandra; Gruber, Florian; Sorg, Olivier; Huebner, Aaron J; Roop, Dennis R; Sandhoff, Konrad; Saurat, Jean-Hilaire; Tschachler, Erwin; Schneider, Marlon R; Langbein, Lutz; Bloch, Wilhelm; Beer, Hans-Dietmar; Werner, Sabine
Release kinetics of platelet-derived and plasma-derived growth factors from autologous plasma rich in growth factors.
Many studies have evaluated the biological effects of platelet rich plasma reporting the final outcomes on cell and tissues. However, few studies have dealt with the kinetics of growth factor delivery by plasma rich in growth factors. Venous blood was obtained from three healthy volunteers and processed with PRGF-Endoret technology to prepare autologous plasma rich in growth factors. The gel-like fibrin scaffolds were then incubated in triplicate, in a cell culture medium to monitor the release of PDGF-AB, VEGF, HGF and IGF-I during 8 days of incubation. A leukocyte-platelet rich plasma was prepared employing the same technology and the concentrations of growth factors and interleukin-1? were determined after 24h of incubation. After each period, the medium was collected, fibrin clot was destroyed and the supernatants were stored at -80�C until analysis. The growth factor delivery is diffusion controlled with a rapid initial release by 30% of the bioactive content after 1h of incubation and a steady state release when almost 70% of the growth factor content has been delivered. Autologous fibrin matrix retained almost 30% of the amount of the growth factors after 8 days of incubation. The addition of leukocytes to the formula of platelet rich plasma did not increase the concentration of the growth factors, while it drastically increased the presence of pro-inflammatory IL-1?. Further studies employing an in vitro inflammatory model would be interesting to study the difference in growth factors and pro-inflammatory cytokines between leukocyte-free and leukocyte-rich platelet rich plasma. PMID:23722041
Anitua, Eduardo; Zalduendo, Mari Mar; Alkhraisat, Mohammad Hamdan; Orive, Gorka
Body size regulation and insulin-like growth factor signaling.
How animals achieve their specific body size is a fundamental, but still largely unresolved, biological question. Over the past decades, studies on the insect model system have provided some important insights into the process of body size determination and highlighted the importance of insulin/insulin-like growth factor signaling. Fat body, the Drosophila counterpart of liver and adipose tissue, senses nutrient availability and controls larval growth rate by modulating peripheral insulin signaling. Similarly, insulin-like growth factor I produced from liver and muscle promotes postnatal body growth in mammals. Organismal growth is tightly coupled with the process of sexual maturation wherein the sex steroid hormone attenuates body growth. This review summarizes some important findings from Drosophila and mammalian studies that shed light on the general mechanism of animal size determination. PMID:23508807
Hyun, Seogang
Role of VEGF Receptors in Normal and Psoriatic Human Keratinocytes: Evidence from Irradiation with Different UV Sources
Vascular endothelial growth factor (VEGF) promotes angiogenesis and plays important roles both in physiological and pathological conditions. VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and are originally considered specific to endothelial cells. We previously reported that VEGFRs were also constitutively expressed in normal human keratinocytes and overexpressed in psoriatic epidermis. In addition, UVB can activate VEGFRs in normal keratinocytes, and the activated VEGFR-2 signaling is involved in the pro-survival mechanism. Here, we show that VEGFRs were also upregulated and activated by UVA in normal human keratinocytes via PKC, and interestingly, both the activated VEGFR-1 and VEGFR-2 protected against UVA-induced cell death. As VEGFRs were over-expressed in psoriatic epidermis, we further investigated whether narrowband UVB (NB-UVB) phototherapy or topical halomethasone monohydrate 0.05% cream could affect their expression. Surprisingly, the over-expressed VEGFRs in psoriatic epidermis were significantly attenuated by both treatments. During NB-UVB therapy, VEGFRs declined first in the basal, and then gradually in the upper psoriatic epidermis. VEGFRs were activated in psoriatic epidermis, their activation was enhanced by NB-UVB, but turned undetectable after whole therapy. This process was quite different from that by halomethasone, in which VEGFRs and phospho-VEGFRs decreased in a gradual, homogeneous manner. Our findings further suggest that UV-induced activation of VEGFRs serves as a pro-survival signal for keratinocytes. In addition, VEGFRs may be involved in the pathological process of psoriasis, and UV phototherapy is effective for psoriasis by directly modulating the expression of VEGFRs.
Zhu, Jian-Wei; Wu, Xian-Jie; Lu, Zhong-Fa; Luo, Dan; Cai, Sui-Qing; Zheng, Min
Growth Factor Receptor-Directed Therapy in Human Breast Cancer.
National Technical Information Service (NTIS)
Overexpression of HER-2 growth factor receptor in human breast cancer is associated with poor prognosis and disease progression. We have targeted these receptor pathways for therapeutic intervention, using a humanized monoclonal antibody to HER-2 receptor...
R. J. Pietras
Transforming Growth Factor-B Receptors in Humans.
National Technical Information Service (NTIS)
Transforming Growth Factor-Beta (TGF-Beta) is the most potent known inhibitor of cell cycle progression of normal mammary epithelial cells. In general, advanced breast cancers are refractory to TGF-Beta-mediated growth inhibition. The TGF-Beta type I and ...
M. Reiss
Epidermal growth factor receptor inhibition strategies in oncology
Molecular targeting strategies for cancer therapy are distinct from conventional chemotherapy and radiotherapy in their potential to provide increased tumor specificity. One particular molecular target of high promise in oncology is the epidermal growth factor receptor (EGFR). The EGFR is overexpressed, dysregulated or mutated in many epithelial malignancies, and EGFR activation appears important in tumor growth and progression. Advances in
P M Harari
A family of autocrine growth factors in Mycobacterium tuberculosis
Summary Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micro- coccus luteus . Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show
Galina V. Mukamolova; Obolbek A. Turapov; Danielle I. Young; Arseny S. Kaprelyants; Douglas B. Kell; Michael Young
Expression of transforming growth factor-?1 during diabetic renal hypertrophy
Expression of transforming growth factor-?1 during diabetic renal hypertrophy. Experimental type I diabetes mellitus is characterized by an early increase in kidney weight and glomerular volume, but changes in gene expression accompanying diabetic renal growth have not been fully elucidated. In the current study, total RNA was extracted from renal cortex and isolated glomeruli of streptozotocin-induced diabetic rats 24 hours,
Stuart J Shankland; James W Scholey; Hao Ly; Kerri Thai
This study identifies some of the critical factors that affect tree and shrub growth on reclaimed sanitary landfill sites and determines which woody species are adaptable to the adverse growth conditions of such sites. Trees planted at the Edgeboro Landfill, East Brunswick, New J...
Insulin and insulin-like growth factor signalling in neoplasia
Insulin and insulin-like growth factors (IGFs) are well known as key regulators of energy metabolism and growth. There is now considerable evidence that these hormones and the signal transduction networks they regulate have important roles in neoplasia. Epidermiological, clinical and laboratory research methods are being used to investigate novel cancer prevention and treatment strategies related to insulin and IGF signalling.
Michael Pollak
Epidermal growth factor receptor (EGFR) signaling in cancer
The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTK). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Evidence suggests that the EGFR is involved in the pathogenesis and progression of different carcinoma types. The EGFR and EGF-like peptides are often over-expressed in human carcinomas, and
Nicola Normanno; Antonella De Luca; Caterina Bianco; Luigi Strizzi; Mario Mancino; Monica R. Maiello; Adele Carotenuto; Gianfranco De Feo; Francesco Caponigro; David S. Salomon
Growth Differentiation Factor15 in Idiopathic Pulmonary Arterial Hypertension
Rationale: Growth-differentiation factor (GDF)-15 is a stress-respon- sive, transforming growth factor-b-related cytokine. Circulating levels of GDF-15 provide independent prognostic information in patients with acute pulmonary embolism and chronic left-sided heart failure. Objectives: To assess the prognostic value of GDF-15 in idiopathic pul- monary arterial hypertension. Methods: GDF-15 levels were determined in 76 treatment-naive patients at the time of baseline right
Nils Nickel; Tibor Kempf; Heike Tapken; J�rn Tongers; Florian Laenger; Ulrich Lehmann; Heiko Golpon; Karen Olsson; Martin R. Wilkins; J. Simon; R. Gibbs; Marius M. Hoeper; Kai C. Wollert
Expression of vascular endothelial growth factor in renal cell carcinomas
Vascular endothelial growth factor (VEGF) is an angiogenic factor that may be involved in tumor growth and metastasis. Only\\u000a a few data concerning the role of VEGF in renal cell carcinomas (RCCs) are available, and no studies have yet evaluated its\\u000a prognostic value. The aim of the present study was to assess VEGF expression in a large series of renal
V. Paradis; N. Ben Lagha; L. Zeimoura; P. Blanchet; P. Eschwege; N. Ba; G. Beno�t; A. Jardin; P. Bedossa
Vascular Endothelial Growth Factor C Induces Angiogenesis in vivo
Vascular endothelial growth factor C (VEGF-C) recently has been described to be a relatively specific growth factor for the lymphatic vascular system. Here we report that ectopic application of recombinant VEGF-C also has potent angiogenic effects in vivo. VEGF-C is sufficiently potent to stimulate neovascularization from limbal vessels in the mouse cornea. Similar to VEGF, the angiogenic response of corneas
Yihai Cao; Philip Linden; Jacob Farnebo; Renhai Cao; Anna Eriksson; Vijay Kumar; Jian-Hua Qi; Lena Claesson-Welsh; Kari Alitalo
Tpl2 knockout keratinocytes have increased biomarkers for invasion and metastasis.
Skin cancer is the most common form of cancer in the USA, with an estimated two million cases diagnosed annually. Tumor progression locus 2 (Tpl2), also known as MAP3K8, is a serine/threonine protein kinase in the mitogen-activated protein kinase signal transduction cascade. Tpl2 was identified by our laboratory as having a tumor suppressor function in skin carcinogenesis, with the absence of this gene contributing to heightened inflammation and increased skin carcinogenesis. In this study, we used gene expression profiling to compare expression levels between Tpl2 (+/+) and Tpl2 (-) (/-) keratinocytes. We identified over 2000 genes as being differentially expressed between genotypes. Functional annotation analysis identified cancer, cell growth/proliferation, cell death, cell development, cell movement and cell signaling as the top biological processes to be differentially regulated between genotypes. Further microarray analysis identified several candidate genes, including Mmp1b, Mmp2, Mmp9 and Mmp13, involved in migration and invasion to be upregulated in Tpl2 (-) (/-) keratinocytes. Moreover, Tpl2 (-/-) keratinocytes had a significant downregulation in the matrix metalloproteinase (MMP) inhibitor Timp3. Real-time PCR validated the upregulation of the MMPs in Tpl2 (-/-) keratinocytes and zymography confirmed that MMP2 and MMP9 activity was higher in conditioned media from Tpl2 (-/-) keratinocytes. Immunohistochemistry confirmed higher MMP9 staining in 12-O-tetradecanoylphorbol-13-acetate-treated skin from Tpl2 (-/-) mice and grafted tumors formed from v-ras(Ha) retrovirus-infected Tpl2 (-/-) keratinocytes. Additionally, Tpl2 (-/-) keratinocytes had significantly higher invasion, malignant conversion rates and increased endothelial cell tube formation when compared with Tpl2 (+/+) keratinocytes. In summary, our studies reveal that keratinocytes from Tpl2 (-/-) mice demonstrate a higher potential to be invasive and metastatic. PMID:24067898
Decicco-Skinner, Kathleen L; Jung, Sarah A; Tabib, Tracy; Gwilliam, J Curtis; Alexander, Hepzibha; Goodheart, Sarah E; Merchant, Anand S; Shan, Mengge; Garber, Caroline; Wiest, Jonathan S
Local Delivery of Growth Factors Using Coated Suture Material
The optimization of healing processes in a wide range of tissues represents a central point for surgical research. One approach is to stimulate healing processes with growth factors. These substances have a short half-life and therefore it seems useful to administer these substances locally rather than systemically. One possible method of local delivery is to incorporate growth factors into a bioabsorbable poly (D, L-lactide) suspension (PDLLA) and coat suture material. The aim of the present study was to establish a procedure for the local delivery of growth factors using coated suture material. Sutures coated with growth factors were tested in an animal model. Anastomoses of the colon were created in a rat model using monofilament sutures. These were either untreated or coated with PDLLA coating alone or coated with PDLLA incorporating insulin�like growth factor-I (IGF-I). The anastomoses were subjected to biomechanical, histological, and immunohistochemical examination. After 3 days the treated groups showed a significantly greater capacity to withstand biomechanical stress than the control groups. This finding was supported by the results of the histomorphometric. The results of the study indicate that it is possible to deliver bioactive growth factors locally using PDLLA coated suture material. Healing processes can thus be stimulated locally without subjecting the whole organism to potentially damaging high systemic doses.
Fuchs, T. F.; Surke, C.; Stange, R.; Quandte, S.; Wildemann, B.; Raschke, M. J.; Schmidmaier, G.
Ultraviolet Irradiation Alters Transforming Growth Factor ?\\/Smad Pathway in Human Skin In Vivo
Solar ultraviolet irradiation damages human skin and causes premature skin aging and skin cancer. As transforming growth factor ? plays an important role in regulating cell growth and extracellular matrix synthesis, we investigated expression of transforming growth factor ? isoforms, transforming growth factor ? receptors, and transforming growth factor ? regulated Smad transcription factors following irradiation with an ultraviolet B
TaiHao Quan; TianYuan He; Sewon Kang; John J. Voorhees; Gary J. Fisher
TNF-alpha stimulates Akt by a distinct aPKC-dependent pathway in premalignant keratinocytes.
Tumor necrosis factor-alpha (TNF-alpha) is an important proinflammatory cytokine involved in the pathogenesis of inflammatory skin diseases and cutaneous squamous cell carcinoma. Some of these effects are mediated by the stimulatory effect of this cytokine on the Akt signalling pathway, which renders keratinocytes less susceptible to proapoptotic stimuli and enhances cell growth. We have recently shown that TNF-alpha-induced Akt activation may promote the early stages of skin cancer. In this work, we demonstrate that in the premalignant keratinocyte cell line HaCaT, TNF-alpha activates Akt, ERK1/2 and p38. The specific peptide blocking the activity of the atypical protein kinase C (aPKC) species zeta and iota/lambda abrogated the effects of TNF-alpha on Akt and ERK1/2 but increased the activation of p38. The TNF-alpha-dependent phosphorylation of Akt-ERK1/2 was slightly decreased by NF kappaB inhibition and in the presence of p38 blockers. Akt/ERK signalling but not p38 activation was abolished in the presence of the iron chelator desferroxamine that blocks formation of hydroxyl ( OH) radicals. Thus, the TNF-alpha signalling in keratinocytes seems to bifurcate into an aPKC-, NFkB- and OH-dependent pathway resulting in the activation of survival and mitogenic pathways mediated by Akt and ERK1/2, and a signalling pathway conveyed by p38 that contributes to Akt activation but is suppressed by aPKC. Our data may be utilized in the development of more selective anti-TNF-alpha therapeutic strategies. PMID:18557926
Faurschou, Annesofie; Gniadecki, Robert
Growth factor action in neural crest cell diversification
At the onset of their migration into the embryo, many neural crest cells are pluripotent in the sense that they have the capacity to generate progeny that consist of more than one cell type. More recently, we have found that there are pluripotent neural crest cell-derived cells even at sites of terminal differentiation. These findings support the notion that cues originating from the microenvironment, at least in part, direct neural crest cell type specification. Based on the rationale that growth factors that are known to support survival of neural crest cell derivatives may have additional functions in progenitor cell development, we have examined the action of pertinent growth factors. Trophic, mitogenic, antiproliferative and differentiation promoting activities were found. Stem cell factor (SCF) is trophic for pluripotent neural crest cells. Contrary to expectation, SCF plus a neurotrophin, rather than SCF alone, is trophic for committed melanogenic cells. Basic fibroblast growth factor (bFGF) is mitogenic both for pluripotent cells and committed melanogenic cells. However, the cells become dependent on another factor for survival. Whereas any neurotrophin tested can rescue bFGF-activated pluripotent neural crest cells, the factor that rescues melanogenic cells remains to be determined. Transforming growth factor ?1 (TGF-?1) is a powerful antimitotic signal for all neural crest cells that overrides the bFGF/neurotrophin proliferative signal. Furthermore, SCF promotes differentiation of neural crest cells into cells of the sensory neuron lineage. Neurotrophin-3 (NT-3) specifically promotes high affinity uptake of norepinephrine by neural crest cells and is thus thought to play a critical role in the differentiation of sympathetic neuroblasts. In summary, our data indicate that neurotrophins and other pertinent growth factors affect survival, proliferation and differentiation of neural crest cells at multiple levels and in different lineages. Moreover, our findings emphasise the importance of the concerted action of combinations of growth factors, rather than of individual factors.
Trafficking of nuclear heparin-binding epidermal growth factor-like growth factor into an epidermal growth factor receptor-dependent autocrine loop in response to oxidative stress.
Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) accumulates in the nucleus in aggressive transitional cell carcinoma (TCC) cells and this histologic feature is a marker of poor prognosis in human bladder cancer tissues. Here we report that HB-EGF can be exported from the nucleus during stimulated processing and secretion of the growth factor. Production of reactive oxygen species (ROS) resulted in mobilization of the HB-EGF precursor, proHB-EGF, from the nucleus of TCCSUP bladder cancer cells to a detergent-resistant membrane compartment, where the growth factor was cleaved by a metalloproteinase-mediated mechanism and shed into the extracellular space. Inhibition of nuclear export suppressed HB-EGF shedding. Production of ROS resulted in EGF receptor (EGFR) and Akt1 phosphorylation in HB-EGF-expressing cells. HB-EGF also stimulated cell proliferation and conferred cytoprotection when cells were challenged with cisplatin. These findings show that the nucleus can serve as an intracellular reservoir for a secreted EGFR ligand and, thus, can contribute to an autocrine loop leading to cell proliferation and protection from apoptotic stimuli. PMID:16166300
Kim, Jayoung; Adam, Rosalyn M; Freeman, Michael R
Comparative studies of erythroid growth factors.
Representative specimens from two classes of Vertebrata Sub-Phyllum, Bufo paracnemis (amphibian) and Gallus domesticus (avian) were made anemic by phenylhydrazine treatment. Appearance of serum factors able to stimulate the proliferation of mammalian erythroid cells was tested. Normal and anemic sera from Gallus domesticus and Bufo paracnemis were fractioned by alcoholic treatment and assayed by the post-hypoxic mice method, showing null uptake of 59Fe. When assayed in semisolid cultures using bone marrow murine cells at different times of incubation (CFU-E and BFU-E colonies), anemia Gallus domesticus serum showed high stimulatory activity, while anemic Bufo paracnemis serum was unable to enhance erythroid proliferation. Gel filtration chromatography of partially purified avian samples on Sephadex G-150 showed three molecular entities responsible for biological activity in vitro, with an apparent molecular weight of 29, 14 and 10 KD respectively. They were submitted to several treatments and then tested for biological activity. All factors were heat stable, sensitive to neuraminidase treatment, while dithiothreitol caused loss of activity on low molecular weight proteins. These results suggest at least under these experimental conditions, the presence of analogous erythroid factors among homeotherms amniotas. PMID:2618752
Juaristi, J A; Aguirre, M V; Guill�n, E R; Alvarez, M A; Brandan, N C
Classical human epidermal keratinocyte cell culture.
It has been more than 30 years since the serial cultivation of human keratinocytes in monolayer culture was first described by Rheinwald and Green. Initially, isolation of primary keratinocytes from disaggregated human skin tissue and subsequent propagation was promoted through use of replication-inactivated murine fibroblast feeder layers. Since then numerous advances have been made to the cultivation of human keratinocytes in both two-dimensional monolayer and three-dimensional organotypic culture. Monolayer culture facilitates keratinocyte proliferation, whereas organotypic culturing techniques promote keratinocyte differentiation using conditions permissive for stratification. The protocols presented here describe traditional culturing methods, providing guidance for isolation and serial cultivation of primary human keratinocytes and dermal fibroblasts, as well as the use of these cells types for generation of stratified skin tissue. PMID:23097107
Rasmussen, Cathy; Thomas-Virnig, Christina; Allen-Hoffmann, B Lynn
Identification and Partial Purification of a Basic Fibroblast Growth Factor-Like Growth Factor Derived from Bovine Colostrum
Bovine colostrum that had been collected up to 6 h postpartum was fractionated by ammonium sulfate precipitation, and various fractions were examined for basic fibroblast growth factor activity. Activity that stimulated cell growth was detected in the cream fraction, which was purified by isoelectric focusing and heparin affinity chromatography. Three peaks were eluted from the heparin affinity column at ap-
T. Hironaka; H. Ohishi; T. Masaki
Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis
In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors\\u000a are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation\\u000a we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results\\u000a demonstrated that EGF and
M. Kassab; Ahmed Abd-Elmaksoud; Mona A. Ali
Connective tissue growth factor mediates transforming growth factor b-induced collagen synthesis: down- regulation by cAMP
Connective tissue growth factor (CTGF) is a cysteine-rich peptide synthesized and secreted by fibroblastic cells after activation with transforming growth factor beta (TGF-b) that acts as a downstream mediator of TGF-b-induced fibroblast proliferation. We performed in vitro and in vivo studies to determine whether CTGF is also essential for TGF-b-induced fibroblast collagen synthesis. In vitro studies with normal rat kidney
Calcium regulation of keratinocyte differentiation
Calcium is the major regulator of keratinocyte differentiation in vivo and in vitro. A calcium gradient within the epidermis promotes the sequential differentiation of keratinocytes as they traverse the different layers of the epidermis to form the permeability barrier of the stratum corneum. Calcium promotes differentiation by both outside�in and inside�out signaling. A number of signaling pathways involved with differentiation are regulated by calcium, including the formation of desmosomes, adherens junctions and tight junctions, which maintain cell�cell adhesion and play an important intracellular signaling role through their activation of various kinases and phospholipases that produce second messengers that regulate intracellular free calcium and PKC activity, critical for the differentiation process. The calcium receptor plays a central role by initiating the intracellular signaling events that drive differentiation in response to extracellular calcium. This review will discuss these mechanisms.
Bikle, Daniel D; Xie, Zhongjian; Tu, Chia-Ling
Dietary Restriction, Growth Factors and Aging: from yeast to humans
Dietary restriction (DR) and reduced growth factor signaling both elevate resistance to oxidative stress, reduce macromolecular damage, and increase lifespan in model organisms. In rodents, both DR and decreased growth factor signaling reduce the incidence of tumors and slow down cognitive decline and aging. DR reduces cancer and cardiovascular disease and mortality in monkeys, and reduces metabolic traits associated with diabetes, cardiovascular disease and cancer in humans. Neoplasias and diabetes are also rare in humans with loss of function mutations in the growth hormone receptor. DR and reduced growth factor signaling may thus slow aging by similar, evolutionarily conserved, mechanisms. We review these conserved anti-aging pathways in model organisms, discuss their link to disease prevention in mammals, and consider the negative side effects that might hinder interventions intended to extend healthy lifespan in humans.
Fontana, Luigi; Partridge, Linda; Longo, Valter D
Regulation of fibroblast growth factor-23 in chronic kidney disease
Background. Fibroblast growth factor-23 (FGF23) is a circulating factor that regulates the renal reabsorption of inorganic phosphate (Pi) and is increased in chronic kidney disease (CKD). The aim of the current investigation was to study the regulation of FGF23 in CKD subjects with various degree of renal function. As such, we analysed the relationship between FGF23, Pi, calcium, parathyriod hormone
Per-Anton Westerberg; Torbjorn Linde; Bjorn Wikstrom; Osten Ljunggren; Mats Stridsberg; Tobias E. Larsson
Epidermal growth factor mediates spermatogonial proliferation in newt testis
The complex processes of spermatogenesis are regulated by various factors. The aim of the current study is to determine the effect of epidermal growth factor (EGF) on spermatogonial proliferation and clarify the mechanism causing the proliferation in newt testis. In the organ culture, EGF stimulated spermatogonial proliferation, but not their differentiation into spermatocytes. cDNA cloning identified 3 members of the
Keisuke Ab�; Ko Eto; Shin-ichi Ab�
Effects of epidermal growth factor on neonatal pancreatic growth in the guinea pig
Summary\\u000a Conclusion��\\u000a EGF and\\/or transforming growth factor-? (TGF-?) are likely to be important in the rapid pancreatic growth that occurs in the\\u000a neonatal guinea pig.\\u000a \\u000a \\u000a \\u000a Background��Rapid pancreatic growth is observed during the neonatal period in the guinea pig. The growth factors that are involved are\\u000a not known but may include members of the EGF family.\\u000a \\u000a \\u000a \\u000a Methods��Mini-osmotic pumps were implanted on
Margery K. Herrington; Corey S. Joekel; Thomas E. Adrian
Hematopoietic growth factor derived from T lymphocytes
US Patent & Trademark Office Database
A T cell-derived colony stimulating factor ("TC-CSF") may be isolated from media conditioned with T lymphocyte cells. TC-CSF stimulates formation of colonies composed of granulocytes, macrophages, megakaryocytes, fibrocytic stromal cells, lymphocytes, and mixed colonies of granulocytes and macrophages. Anion exchange chromatography may be employed in conjunction with gel filtration and rpHPLC to isolate TC-CSF. Human TC-CSF and murine TC-CSF cDNA, MRNA, genomic DNA nucleotide and amino acid sequences, expression products, pharmaceutical formulations and antibody materials are specifically provided.
Endothelin-1 is a transcriptional target of p53 in epidermal keratinocytes and regulates UV induced melanocyte homeostasis
Summary Keratinocytes contribute to melanocyte activity by influencing their microenvironment, in part, through secretion of paracrine factors. Here we discovered that p53 directly regulates Edn1 expression in epidermal keratinocytes and controls UV-induced melanocyte homeostasis. Selective ablation of EDN1 in murine epidermis (EDN1ep?/?) does not alter melanocyte homeostasis in newborn skin but decreases dermal melanocytes in adult skin. Results showed that keratinocytic EDN1 in a non-cell autonomous manner controls melanocyte proliferation, migration, DNA damage and apoptosis after UVB irradiation. Expression of other keratinocyte derived paracrine factors did not compensate for the loss of EDN1. Topical treatment with EDN1 receptor (EDNRB) antagonist BQ788 abrogated UV induced melanocyte activation and recapitulated the phenotype seen in EDN1ep?/? mice. Altogether, present studies establish an essential role of EDN1 in epidermal keratinocytes to mediate UV induced melanocyte homeostasis in vivo.
Hyter, Stephen; Coleman, Daniel J.; Ganguli-Indra, Gitali; Merrill, Gary F.; Ma, Steven; Yanagisawa, Masashi; Indra, Arup K.
Growth factor delivery from genetically modified skin grafts
Summary form only given. Keratinocytes of the epidermis can be cultured in vitro and have been used as part of a skin substitute for the treatment of defects of the skin such as burns and ulcers. Cultured cells are often combined with various biomaterials to create a composite skin graft. Our goal is to enhance the therapeutic effectiveness of these
J. R. Morgan
47 CFR 36.604 - Calculation of the rural growth factor.
Code of Federal Regulations, 2010 CFR
...Calculation of the rural growth factor. 36.604...TELECOMMUNICATIONS COMPANIES 1 Universal Service...Calculation of the rural growth factor. The Rural Growth Factor (RGF...both determined by the company's submissions...
47 CFR 36.604 - Calculation of the rural growth factor.
Code of Federal Regulations, 2010 CFR
...Calculation of the rural growth factor. 36.604...TELECOMMUNICATIONS COMPANIES 1 Universal Service...Calculation of the rural growth factor. The Rural Growth Factor (RGF...both determined by the company's submissions...
Transcriptional profiling of keratinocytes reveals a vitamin D-regulated epidermal differentiation network.
1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] regulates mineral homeostasis and exhibits potent anti-proliferative, prodifferentiative, and immunomodulatory activities. It mediates these effects by binding to the vitamin D receptor (VDR), which belongs to the superfamily of steroid/thyroid hormone nuclear receptors. As a result of keratinocyte differentiation and anti-proliferation activities, 1,25(OH)(2)D(3) and its synthetic analogs are therapeutically effective in psoriasis and show promise for the treatment of actinic keratosis and squamous cell carcinoma. To elucidate the VDR signaling pathway in keratinocytes, we examined the gene expression profile with 1,25(OH)(2)D(3) treatment using oligonucleotide microarrays. Out of the 12,600 genes investigated, 82 were upregulated and 16 were downregulated and many of these were involved in differentiation, proliferation, and immune response. We have identified three vitamin D-responsive chromosomal loci (1p36, 19q13, and 6p25) and show the induction of various class II tumor suppressor/growth-regulatory genes in response to 1,25(OH)(2)D(3). Finally, quantitative differences in gene expression revealed a vitamin D-regulated differentiation network and identified peptidylarginine deiminases, kallikreins, serine proteinase inhibitor family members, Kruppel-like factor 4, and c-fos as vitamin D-responsive genes, whose protein products may play an important role in epidermal differentiation in normal and diseased state. PMID:15816836
Lu, Jianfen; Goldstein, Keith M; Chen, Peining; Huang, Shuguang; Gelbert, Lawrence M; Nagpal, Sunil
Epidermal ADAM17 maintains the skin barrier by regulating EGFR ligand-dependent terminal keratinocyte differentiation
ADAM17 (a disintegrin and metalloproteinase 17) is ubiquitously expressed and cleaves membrane proteins, such as epidermal growth factor receptor (EGFR) ligands, l-selectin, and TNF, from the cell surface, thus regulating responses to tissue injury and inflammation. However, little is currently known about its role in skin homeostasis. We show that mice lacking ADAM17 in keratinocytes (A17?KC) have a normal epidermal barrier and skin architecture at birth but develop pronounced defects in epidermal barrier integrity soon after birth and develop chronic dermatitis as adults. The dysregulated expression of epidermal differentiation proteins becomes evident 2 d after birth, followed by reduced transglutaminase (TGM) activity, transepidermal water loss, up-regulation of the proinflammatory cytokine IL-36?, and inflammatory immune cell infiltration. Activation of the EGFR was strongly reduced in A17?KC skin, and topical treatment of A17?KC mice with recombinant TGF-? significantly improved TGM activity and decreased skin inflammation. Finally, we show that mice lacking the EGFR in keratinocytes (Egfr?KC) closely resembled A17?KC mice. Collectively, these results identify a previously unappreciated critical role of the ADAM17�EGFR signaling axis in maintaining the homeostasis of the postnatal epidermal barrier and suggest that this pathway could represent a good target for treatment of epidermal barrier defects.
Cobzaru, Cristina; Triantafyllopoulou, Antigoni; Loffek, Stefanie; Horiuchi, Keisuke; Threadgill, David W.; Kurz, Thomas; van Rooijen, Nico; Bruckner-Tuderman, Leena
Hypoxia-Mediated Induction of Acidic\\/Basic Fibroblast Growth Factor and Platelet-Derived Growth Factor in Mononuclear Phagocytes Stimulates Growth of Hypoxic Endothelial Cells
Wound repair and tumor vascularization depend upon blood vessel growth into hypoxic tissue. Although hypoxia slows endothelial cell (EC) proliferation and suppresses EC basic fibroblast growth factor (bFGF) expression, we report that macrophages (MPs) exposed to PO_2≈ 12-14 torr (1 torr = 133.3 Pa) synthesize and release in a time-dependent manner platelet-derived growth factor (PDGF) and acidic\\/basic FGFs (a\\/bFGFs), which
Keisuke Kuwabara; Satoshi Ogawa; Masayasu Matsumoto; Shin Koga; Matthias Clauss; David J. Pinsky; Peter Lyn; Jeffrey Leavy; Larry Witte; Jacqueline Joseph-Silverstein; Martha B. Furie; Gabriella Torcia; Federico Cozzolino; Takenobu Kamada; David M. Stern
Tunable dual growth factor delivery from polyelectrolyte multilayer films
A promising strategy to accelerate joint implant integration and reduce recovery time and failure rates is to deliver a combination of certain growth factors to the integration site. There is a need to control the quantity of growth factors delivered at different times during the healing process to maximize efficacy. Polyelectrolyte multilayer (PEM) films, built using the layer-by-layer (LbL) technique, are attractive for releasing controlled amounts of potent growth factors over a sustained period. Here, we present PEM films that sequester physiological amounts of osteogenic rhBMP-2 (recombinant human bone morphogenetic protein - 2) and angiogenic rhVEGF165 (recombinant human vascular endothelial growth factor) in different ratios in a degradable [poly(?-amino ester)/polyanion/growth factor/ polyanion] LbL tetralayer repeat architecture where the biologic load scaled linearly with the number of tetralayers. No burst release of either growth factor was observed as the films degraded. The release of rhBMP-2 was sustained over a period of 2 weeks, while rhVEGF165 eluted from the film over the first 8 days. Both growth factors retained their efficacy, as quantified with relevant in vitro assays. rhBMP-2 initiated a dose dependent differentiation cascade in MC3T3-E1S4 pre-osteoblasts while rhVEGF165 upregulated HUVEC proliferation, and accelerated closure of a scratch in HUVEC cell cultures in a dose dependent manner. In vivo, the mineral density of ectopic bone formed de novo by rhBMP-2/rhVEGF165 PEM films was approximately 33% higher than when only rhBMP-2 was introduced, with a higher trabecular thickness, which would indicate a decrease in the risk of osteoporotic fracture. Bone formed throughout the scaffold when both growth factors were released, which suggests more complete remodeling due to an increased local vascular network. This study demonstrates a promising approach to delivering precise doses of multiple growth factors for a variety of implant applications where control over spatial and temporal release profile of the biologic is desired.
Shah, Nisarg J.; Macdonald, Mara L.; Beben, Yvette M.; Padera, Robert F.; Samuel, Raymond E.; Hammond, Paula T.
Coordinated Epidermal Growth Factor Receptor Pathway Gene Overexpression Predicts Epidermal Growth Factor Receptor Inhibitor Sensitivity in Pancreatic Cancer
The epidermal growth factor receptor (EGFR) inhibitor erlotinib is approved for treatment of pancreatic cancer but the overall activity is minimal, and known predictive factors for EGFR inhibitor efficacy are infrequent in this disease.We tested the hypothesis that global activation of the EGFR pathway is predictive of EGFR inhibitor efficacy.Pancreatic cancer tumors directly xenografted at surgery were treated with the
Antonio Jimeno; Aik Choon Tan; Jordy Coffa; N. V. Rajeshkumar; Peter Kulesza; Belen Rubio-Viqueira; Jenna Wheelhouse; Begona Diosdado; Wells A. Messersmith; Christine Iacobuzio-Donahue; Anirban Maitra; Fred R. Hirsch; Gerrit A. Meijer; Manuel Hidalgo
Vascular endothelial growth factor (VEGF) is an autocrine growth factor for VEGF receptor-positive human tumors
Angiogenesis is required for the progres- sion of tumors from a benign to a malig- nant phenotype and for metastasis. Malig- nant tumor cells secrete factors such as vascular endothelial growth factor (VEGF), which bind to their cognate receptors on endothelial cells to induce angiogenesis. Here it is shown that several tumor types express VEGF receptors (VEGFRs) and that inhibition
Rizwan Masood; Jie Cai; Tong Zheng; D. Lynne Smith; David R. Hinton; Parkash S. Gill
Cytokine and Growth Factor Responses After Radiotherapy for Localized Ependymoma
Purpose: To determine the time course and clinical significance of cytokines and peptide growth factors in pediatric patients with ependymoma treated with postoperative radiotherapy (RT). Methods and Materials: We measured 15 cytokines and growth factors (fibroblast growth factor, e