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Myofibroblasts are pathogenic in pulmonary fibrotic disease due to their exuberant production of matrix rich in collagen that interferes with gas exchange and the ability of these cells to contract and distort the alveolar space. Transforming growth factor-β1 (TGF-β1) is a well-known inducer of myofibroblast differentiation. TGF-β1-induced transformation of fibroblasts to apoptosis-resistant myofibroblasts is adhesion-dependent and focal adhesion kinase (FAK)-mediated. Prostaglandin E2 (PGE2) inhibits this differentiation via E prostanoid receptor 2 (EP2) signaling and cAMP elevation, but whether PGE2 does so by interfering with TGF-β1 signaling is unknown. Thus we examined the effects of PGE2 in the presence and absence of TGF-β1 stimulation on candidate signaling pathways in human lung fibroblasts. We now demonstrate that PGE2 does not interfere with TGF-β1-induced Smad phosphorylation or its translocation to the nucleus. Rather, PGE2 has dramatic effects on cell shape and cytoskeletal architecture and disrupts the formation of appropriate focal adhesions. PGE2 treatment diminishes TGF-β1-induced phosphorylation of paxillin, STAT-3, and FAK and, in turn, limits activation of the protein kinase B (PKB/Akt) pathway. These alterations do not, however, result in increased apoptosis within the first 24 h of treatment. Interestingly, the effects of PGE2 stimulation alone do not always mirror the effects of PGE2 in the presence of TGF-β1, indicating that the context for EP2 signaling is different in the presence of TGF-β1. Taken together, our results demonstrate that PGE2 has the potential to limit TGF-β1-induced myofibroblast differentiation via adhesion-dependent, but Smad-independent, pathways.
- fibrosis
- eicosanoid
- lung
- signal transduction
smooth muscle actin-α (α-SMA) expression (39), stress fiber formation (25), and robust collagen secretion are the distinguishing features of myofibroblasts. The multifunctional cytokine, transforming growth factor-β1 (TGF-β1), is a well-characterized profibrotic mediator (2, 3) that induces transformation of fibroblasts to myofibroblasts both in vitro (7) and in vivo (36). Myofibroblasts display contractile properties intermediate between smooth muscle cells and fibroblasts and appear transiently in the granulation phase of normal wound healing to provide the contractile force required for wound closure (6, 38). Eventual apoptosis of fibroblasts/myofibroblasts is essential for normal repair and resolution of tissue scarring. Myofibroblast differentiation and activation by TGF-β1 is a critical event in the pathogenesis of human fibrotic diseases, since TGF-β1 not only causes the differentiation of fibroblasts to myofibroblasts, but also renders myofibroblasts resistant to apoptosis by activating phosphatidylinositol 3-kinase (PI3K)/Akt signaling (15). In turn, activated Akt induces the prosurvival NF-κB pathway (33, 44). Prolonged persistence of myofibroblasts at the site of injury leads to fibrosis and progressive organ dysfunction of lung, kidney, and other tissues. TGF-β1-induced differentiation of fibroblasts to myofibroblasts is dependent on cell adhesion and integrin signaling involving focal adhesion kinase (FAK) (41). We (21) previously showed that this TGF-β1-mediated differentiation process is inhibited by prostaglandin E2 (PGE2) via E prostanoid receptor 2 (EP2) signaling and cyclic adenosine monophosphate (cAMP) elevation. PGE2 could potentially block any of the well-known signal transduction events that occur as a result of TGF-β1 stimulation in fibroblasts. The best known pathway for TGF-β1 signaling in fibroblasts includes Smad phosphorylation and translocation to the nucleus followed by binding of nuclear Smad complexes to the Smad-binding elements (SBEs) of target genes. The ability of Smads to regulate gene transcription further depends on the presence of transcriptional coactivators such as p300 (1).
TGF-β1 also acts through Smad-independent but adhesion-dependent pathways. FAK is a cytoplasmic nonreceptor protein tyrosine kinase found in focal adhesions of cells with extracellular matrix, and its activity is regulated by ligand binding to cell surface receptors including the integrins (27). The focal adhesion complex is involved in the regulation of cell shape, function, survival, migration, and signaling. The effects of PGE2 on fibroblast cell morphology, adhesion, FAK phosphorylation, and Akt activation remain obscure.
The research presented in this manuscript identifies potential mechanisms whereby PGE2 counteracts TGF-β1 signaling in human lung fibroblasts. Our results indicate for the first time that PGE2 effects are independent of Smad and that PGE2 disrupts the normal formation of TGF-β1-induced focal adhesions and also identify several potential signaling targets for PGE2 downstream from TGF-β1.
Cell culture and reagents.
This study was approved by the University of Michigan Institutional Review Board. All experiments were performed on early passage normal human lung fibroblasts (IMR-90; Institute for Medical Research, Camden, NJ) or primary fibroblasts grown from histologically normal-appearing lung from patients undergoing resection for pulmonary nodules. Adult fibroblasts were the kind gift of Dr. Cory Hogaboam at our institution. IMR-90 cells were maintained in a medium consisting of Dulbecco's modified Eagle's medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (Sigma, St. Louis, MO), 100 U/ml penicillin/streptomycin, and Fungizone (GIBCO, Grand Island, NY). Adult lung fibroblasts were maintained in medium containing 15% serum. Medium was changed every 3 days, and cells were expanded and plated onto 35- and 100-mm dishes. At 85% confluency, cells were growth-arrested by reducing the concentration of fetal calf serum in the medium to 0.01% for 48 h before stimulation with porcine platelet-derived TGF-β1 (2 ng/ml; R&D Systems, Minneapolis, MN) and/or PGE2 (generally at 10 nM unless otherwise indicated; Cayman Chemicals, Ann Arbor, MI). The concentration of PGE2 used in the adult lung fibroblasts was generally 100 nM based on our previous dose response studies (21). Forskolin, a direct activator of adenylyl cyclase used at 100 μM, was from Sigma. Butaprost, an EP2 receptor agonist, was obtained from Cayman Chemicals and used at 5 μM.
Western blotting.
Cells grown on tissue culture plates were washed with phosphate-buffered saline (PBS) and lysed in 0.5 ml of ice-cold RIPA lysis buffer (1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M NaH2PO4, 2 mM EDTA, 0.5 mM NaF) containing 2 mM sodium orthovanadate and 1:100 dilution of protease inhibitor mixture III (Calbiochem). Protein concentrations were determined using the DC protein assay reagents from Bio-Rad (Hercules, CA). SDS-PAGE and Western blot analyses were performed as described previously (14, 21).
Subcellular fractionation.
IMR-90 cells treated with or without PGE2 and/or TGF-β1 for 5 min, 30 min, or 24 h were washed with cold PBS and harvested by mechanical detachment with a rubber policeman in a buffer containing 10 mM Tris·HCl, pH 7.5, 0.32 M sucrose, 3 mM CaCl2, 2 mM magnesium acetate, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.5% Nonidet P-40. Cell suspensions were then homogenized with a Tissue-Tearor (Biospec Products) and centrifuged at 5,000 g for 10 min at 4°C. To isolate nuclei, the pellets were layered over sucrose gradient buffer containing 1.7 M sucrose, 5 mM magnesium acetate, 0.1 mM EDTA, and 10 mM Tris·HCl, pH 8.0, followed by centrifugation at 30,000 g for 45 min at 4°C. The 5,000 g postnuclear supernatant was centrifuged at 105,000 g for 1 h. The pellet was collected as membrane fraction and the supernatant as cytosol. Pellets of nuclei and membrane were suspended in RIPA buffer. Nuclear, membrane, and cytosol fractions were boiled with 6× SDS electrophoresis sample buffer (0.35 M Tris·HCl, pH 6.8, 30% glycerol, 10% SDS, 0.6 M DTT, 0.2% bromophenol blue).
Sources for the antibodies used in this study were as follows. From Cell Signaling (Danvers, MA): phosphorylated Akt (p-Akt; S-473), Akt rabbit polyclonal, p-paxillin (Y-118) rabbit polyclonal, p-Smad2 (S-465/467), p38 MAP kinase rabbit polyclonal, p-p38 MAP kinase (T-180/Y182) mouse antibody, p-p44/42 MAP kinase rabbit polyclonal, p44/42 rabbit polyclonal, p-JNK rabbit polyclonal, p-p38 MAP kinase rabbit polyclonal, JNK rabbit monoclonal, p38 MAP kinase rabbit polyclonal, p-Y705-STAT-3 rabbit polyclonal, STAT-3 rabbit polyclonal. From Santa Cruz (Santa Cruz, CA): Smad4 mouse monoclonal. From BioSource (Carlsbad, CA): p-FAK (Y-397) rabbit polyclonal, FAK (H-1) mouse monoclonal, lamin A/C mouse monoclonal. From CedarLane (Burlington, NC): rabbit anti-human type I collagen polyclonal. From Sigma: β-actin mouse MAb, paxillin MAb. From Zymed (Carlsbad, CA): Smad3 and Smad2 rabbit polyclonals. From DakoCytomation: α-SMA clone 1A4 mouse monoclonal. From BioSource International (Camarillo, CA): pY397-FAK rabbit polyclonal. From Pierce Biotechnology (Rockford, IL): goat anti-mouse IgG peroxidase conjugate and goat anti-rabbit IgG peroxidase conjugate. pSmad3 rabbit antiserum was a gift from Dr. Ed Leof (Mayo Foundation, Rochester, MN).
Morphological analysis and immunofluorescent staining.
IMR-90 cells or normal adult lung fibroblasts were plated onto 35-mm dishes, grown to 50% confluence, growth-arrested for 48 h, and treated with or without TGF-β1 and/or PGE2 or butaprost from 5 min to 24 h. Cells were washed with ice-cold PBS, fixed in cold methanol for 20 min, and blocked for 1 h in 3% BSA in PBS. Cells were incubated with anti-paxillin MAb (1:50 in 1% BSA/PBS) for 1 h and washed three times with PBS. Indocarbocyanine (Cy3)-conjugated secondary anti-mouse IgG (1:100) and FITC-phalloidin were added to the cells and incubated for 30 min. The cells were washed three times, and their nuclei were stained with ProLong Gold mounting medium containing 4,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA). Images were then captured using an Olympus FV500 laser-scanning confocal microscope with a ×60 water immersion objective. Z-stack analysis confirmed the colocalization of the FITC and Cy3 signals in focal adhesions.
Densitometry measurements.
To determine the intensity of each band on Western blots, blots were scanned, and bands were analyzed for pixel density using the Image-J program available for download at http://rsb.info.nih.gov/ij/download.html.
Statistical analysis.
When three or more groups were analyzed, the data were analyzed by ANOVA with a post hoc Dunnet or Bonferroni examination; P values <0.05 were considered to be statistically significant.
PGE2 is able to limit TGF-β1-induced collagen and α-SMA expression.
Our (15, 21) previous results with IMR-90 cells have demonstrated that 2 ng/ml TGF-β1 induces robust fibroblast-to-myofibroblast transformation within 24 h. In addition, we (Ref. 21 and unpublished observations) have previously demonstrated that a dose response of PGE2 ranging from 10 nM to 100 μM is able to limit TGF-β1-induced expression of both collagen 1 and α-SMA at 24 h. The ability of PGE2 to limit collagen and α-SMA was similar across all concentrations. Thus we chose the lowest effective dose of PGE2 (10 nM) for future experiments unless otherwise indicated. Interestingly, however, PGE2 is able to activate cAMP production in fibroblasts very rapidly (5–15 min; Ref. 21). To determine whether PGE2 was able to inhibit the TGF-β1-induced expression of either collagen 1 or α-SMA at an earlier time point, we cultured IMR-90 human lung fibroblasts in the presence or absence of TGF-β1 ± 10 nM PGE2 for 5 min, 30 min, or 24 h. Figure 1A demonstrates that collagen 1 levels were decreased by PGE2 (within 30 min), whereas the downregulation of α-SMA occurred later (24 h; Fig. 1, A and B). To determine whether PGE2 alone had any effect on the basal expression of α-SMA or collagen 1, we compared the expression of these proteins in IMR-90 cells that had been stimulated with serum-free media alone, PGE2 alone, TGF-β1 alone, or TGF-β1 + PGE2 for 24 h (Fig. 1B). PGE2 was able to inhibit both basal and TGF-β1-induced expression of collagen and α-SMA, although the inhibition was more pronounced in the presence of TGF-β1.
PGE2 did not affect the TGF-β1-stimulated phosphorylation of Smad2/3.
TGF-β1 is known to induce the phosphorylation of the transcription factors Smad2 and Smad3 that are involved in collagen synthesis (10, 29). Hence, we investigated the effect of PGE2 signaling on TGF-β1-induced Smad phosphorylation (Fig. 2A). TGF-β1 induced phosphorylation of Smad2 and Smad3 in 30 min. PGE2 (10 nM) did not have any effect on the phosphorylation of either Smad2 or Smad3 at any time point tested (Fig. 2A). Even doses of PGE2 as high as 50 μM showed no inhibition of Smad phosphorylation (Fig. 2B) despite the fact that we (21) have previously shown these concentrations inhibit α-SMA expression. Thus these data suggest that the inhibitory effects of PGE2 are independent of Smad2/3 phosphorylation.
Translocation of Smad2/3/4 complex to the nucleus by TGF-β1 is not abrogated by PGE2 treatments.
Phosphorylated Smads are known to form Smad2/3/4 heteromeric complexes, which translocate to the nucleus to regulate the transcriptional machinery by binding to Smad-binding enhancer elements (26). Therefore, we determined whether the TGF-β1-induced translocation of Smad complexes in myofibroblasts was blocked by PGE2 treatment. We prepared nuclear and cytosolic fractions from IMR-90 cells treated with TGF-β1 with or without PGE2. There was no decrease in the proportion of Smad2/3/4 in the nucleus or increase in the fraction of Smad2/3/4 found in the cytosol of cells treated with both TGF-β1 and PGE2 compared with cells treated with TGF-β1 only (Fig. 2C). Thus we conclude that PGE2 treatment did not interfere with the translocation of Smad2/3/4 complexes to the nucleus.
TGF-β1 and PGE2 cause sudden and sustained morphological changes.
Differential morphological changes were noted in cells stimulated with TGF-β1 and/or PGE2. Both untreated (control) and TGF-β1-treated cells were flat and spindle-shaped (Fig. 3A). In contrast, addition of 10 nM PGE2 induced a rapid change (within 5 min) in the morphology of cultured lung fibroblasts (Fig. 3A). The spindle-shaped cells narrow and become irregular, and the cells appear shrunken in size. The surface area of cell attachment with fibrillar and focal adhesions also decreases. The vertical height of the cell appears to be increased in the middle compared with the flattened control cells. These effects of PGE2 on fibroblast cell shape were concentration dependent, being even more pronounced at higher concentrations (data not shown). PGE2 in the presence of TGF-β1 induces a morphology that is intermediate between that of cells treated with TGF-β1 alone and PGE2 alone. The adenylyl cyclase activator, forskolin, at 100 μM concentration, was able to mimic the rapid change in cell shape induced by 10 nM PGE2 (data not shown). The rapid morphological changes seen with PGE2 alone were also noted in the presence of TGF-β1. Thus these data suggest that PGE2 stimulation of cAMP induces rapid changes in fibroblast cell shape both in the presence and absence of TGF-β1.
PGE2 alters cytoskeletal structure.
To determine what effect PGE2 stimulation would have over a longer period of time, growth-arrested IMR-90 cells, treated with or without TGF-β1 and/or PGE2 for 24 h, were methanol-fixed and stained with phalloidin for the visualization of actin stress fibers. Immunofluorescence of paxillin and phalloidin was used to visualize focal adhesions (Fig. 3B). Actin stress fibers (green) and focal adhesions (yellow/orange) were well-organized in TGF-β1-treated IMR-90 lung fibroblasts. Note that in TGF-β1-treated cells, the focal adhesions were dispersed along the stress fibers stretching toward opposite poles of the cell. In contrast, actin stress fibers and focal contacts were disorganized with totally distorted cell structure in cells treated with PGE2 or both TGF-β1 and PGE2 simultaneously. Although paxillin staining was evident in the PGE2-treated samples, it was localized near the nucleus and was not found in characteristically dispersed focal adhesions. The changes noted in the TGF-β1 + PGE2 treatment groups were intermediate between those of TGF-β1 alone or PGE2 alone. Of note, treatment of the cells with an EP2 receptor agonist, butaprost alone, or in combination with TGF-β1 mimicked the effects of PGE2 treatment. Thus PGE2 likely acting through EP2 limits cytoskeletal changes normally associated with myofibroblast transformation by TGF-β1.
PGE2 inhibits TGF-β1-induced paxillin phosphorylation.
We next investigated whether the cell shape changes induced by PGE2 stimulation correlated with diminished phosphorylation of paxillin. Paxillin was phosphorylated on Y-118 at baseline in untreated cells, but TGF-β1 significantly enhanced Y-118 phosphorylation of paxillin within 30 min, and this response was maximal at 24 h. PGE2 was able to inhibit paxillin phosphorylation in the presence of TGF-β1 at both the 30-min and 24-h time points (Fig. 4, A and C). Interestingly, however, PGE2 increased the paxillin phosphorylation at shorter time periods when added alone (Fig. 4, B and D). Thus the rapid changes in cytoskeletal architecture that are seen with PGE2 stimulation alone are not due to changes in paxillin phosphorylation per se, but rather diminished paxillin phosphorylation could contribute to the cell shape changes that take place in the presence of TGF-β1 + PGE2. As demonstrated by the immunohistochemistry data in Fig. 3, the organization of paxillin into appropriately distributed focal adhesions on stress fibers is also disrupted in the presence of PGE2.
PGE2 limits TGF-β1-induced phosphorylation of STAT-3.
Because STAT-3 has been shown to associate with paxillin and FAK in focal adhesions (35), we next tested the ability of PGE2 cotreatment to limit STAT-3 phosphorylation. TGF-β1 treatment alone significantly induces STAT-3 phosphorylation within 5 min. However, PGE2 cotreatment was able to block tyrosine phosphorylation of STAT-3 by the 30-min time point (Fig. 5, A and C). Thus diminished phosphorylation of both paxillin and STAT-3 likely contributes to the disruption of cytoskeletal interactions with focal adhesion complexes in the presence of both TGF-β1 and PGE2. Interestingly, when STAT-3 phosphorylation was examined in response to PGE2 alone, PGE2 induced rapid phosphorylation of STAT-3 (5–30 min), but this was diminished by 24 h. These results suggest that the context for PGE2 signaling is different in the presence and absence of TGF-β1.
PGE2 inhibits TGF-β1-stimulated Y-397 FAK autophosphorylation.
We (41) previously demonstrated that the TGF-β1-induced differentiation of fibroblasts to myofibroblasts is dependent on cell adhesion and integrin signaling involving FAK. Since the focal adhesion-cytoskeletal architecture was distorted by PGE2 treatment, we examined the effects of PGE2 on TGF-β1-mediated FAK autophosphorylation (Fig. 6). FAK exhibited basal levels of autophosphorylation that were increased by 24 h with TGF-β1 treatment. PGE2 was able to inhibit FAK autophosphorylation in the presence of TGF-β1 at all time points. PGE2 also decreased the FAK autophosphorylation between 5–30 min when cultured alone and the inhibition was sustained. These data mirror the results seen with paxillin and suggest that signaling cascades induced by PGE2 are different in the presence and absence of TGF-β1. Given that we (41) have previously demonstrated that expression of a dominant negative FAK construct can inhibit myofibroblast differentiation in IMR-90 cells, our current data link the effects of PGE2 to the inhibition of FAK activation. Thus these results suggest that PGE2 may disrupt myofibroblast transformation at least, in part, by limiting FAK signaling.
PGE2 inhibits TGF-β1-stimulated PKB/Akt phosphorylation.
We (15) previously showed that TGF-β1 stimulated activation of the prosurvival PI3K/Akt pathway in human fibroblasts. Treatment of IMR-90 cells with TGF-β1 induced the S-473 phosphorylation of Akt in 5 min, and this phosphorylation increased with the duration of treatment through 24 h (Fig. 7). PGE2 cotreatment inhibited the Akt phosphorylation at 30 min and 24 h. Interestingly, PGE2, when added alone, could inhibit Akt phosphorylation at all time points. Thus the inhibition of Akt signaling by PGE2 is TGF-β1 independent. We next assessed whether these alterations in Akt activation would result in differences in cell survival over the 24-h culture period by examining cell extracts for the presence of cleaved caspase-3 under all conditions. Despite the alterations in Akt induced by PGE2 administration, there were no differences in the expression of cleaved caspase-3 (data not shown) under any conditions up to 24 h.
PGE2 signaling alters the kinetics of MAP kinase activation.
Many signal transduction pathways involve phosphorylation and activation of one or more MAP kinases (9). TGF-β1 stimulation of fibroblasts is known to induce phosphorylation of p38 (12, 15, 20, 40) and both ERK1/2 (12) and JNK (12, 40). Thus we wanted to determine the effect of PGE2 treatment on the phosphorylation of MAP kinases. With respect to ERK1/2 activation, TGF-β1 alone induced phosphorylation of ERK1/2 within 5 min, and the effect was increased at 30 min and sustained through 24 h (Fig. 8, A and B). TGF-β1-induced JNK activation was maximal at 30 min, and this was the time point for maximal inhibition by PGE2 as well (Fig. 8, A and C). The phosphorylation of p38 by TGF-β1 alone was slower, taking 24 h (Fig. 8, A and D), and PGE2 did not inhibit this response at any time point. In all cases, PGE2 coadministration with TGF-β1 altered the kinetics of MAP kinase activation. In the presence of TGF-β1 + PGE2, all three kinases were activated within 5 min to a level higher than that seen with TGF-β1 alone. At later time points, PGE2 inhibited the TGF-β1-induced phosphorylation of ERK1/2 and JNK but not p38. The kinetics of MAP kinase activation in the presence of PGE2 alone also showed rapid activation of all three kinases within 5 min (Fig. 9). The ability of PGE2 alone to limit kinase activation at 24 h, however, was kinase specific.
PGE2 and butaprost disrupt cell shape and focal adhesion formation in adult lung fibroblasts.
To determine whether PGE2 could alter the cellular morphology and formation of focal adhesions in adult lung fibroblasts, we cultured fibroblasts from peripheral adult lung demonstrated to be histologically normal. Adult fibroblasts were growth-arrested for 48 h, treated with or without TGF-β1 and/or PGE2 or butaprost for 24 h, methanol-fixed, and stained with phalloidin for the visualization of actin stress fibers and paxillin. Colocalized immunofluorescence of paxillin and phalloidin was used to identify focal adhesions (Fig. 10). Similar to the results with fetal fibroblasts, TGF-β1 strongly stimulated the formation of stress fibers and dispersed focal adhesions. In contrast, treatment with PGE2 alone or in combination with TGF-β1 distorted cell shape and limited the formation of focal adhesions. Butaprost treatment showed similar changes as PGE2, although in the adult fibroblasts, the effects of butaprost were not as dramatic as the results in fetal fibroblasts. Thus PGE2 is able to limit TGF-β1-induced cytoskeletal changes in both fetal and adult lung fibroblasts.
PGE2 inhibits TGF-β1-induced Y-397 FAK autophosphorylation in adult lung fibroblasts.
To determine whether the disruption of focal adhesion formation in adult fibroblasts correlated with an inhibition of FAK autophosphorylation, adult lung fibroblasts were serum-starved and cultured in the presence or absence of TGF-β1 and/or 100 nM PGE2 for periods of 5 min, 30 min, or 24 h. TGF-β1 was able to stimulate FAK autophosphorylation within 30 min in normal adult fibroblasts, and PGE2 was able to significantly inhibit this activation at the 24-h time point (Fig. 11). Thus PGE2 is able to limit the formation of focal adhesions and the activation of FAK in both fetal and adult fibroblasts.
Fibroblasts and myofibroblasts are essential and transient participants in wound closure and repair, but persistence of myofibroblasts at the site of injury promotes dysregulated repair processes leading to fibrosis. The multifunctional cytokine, TGF-β1, induces the transformation of fibroblasts to apoptosis-resistant myofibroblasts with enhanced extracellular matrix synthetic capacity. Since the signals leading to the disappearance of myofibroblasts by massive apoptosis (8) in the final stages of wound healing are unknown, investigation of signals involved in PGE2 inhibition of TGF-β1-induced myofibroblast transformation may shed light on these events and support new therapeutic strategies for prevention and treatment of fibrotic diseases. This is particularly true for lung fibrosis, since lung fibroblast activation is downregulated by PGE2 (21, 28, 46) and since fibroblasts isolated from fibrotic lung show deficits in PGE2 synthesis (19, 43, 47) and reduced expression of cyclooxygenase-2 (42, 47). Animal models of fibrosis have also been shown to be modulated by PGE2 levels. Fibrosis is worse in situations characterized by underproduction of PGE2 (5, 13) or diminished PGE2 signaling (28). In contrast, situations resulting in enhanced PGE2 production are often protective for fibrosis (32). Thus therapeutic administration of PGE2 or EP2 agonists may improve fibrosis by limiting prosurvival signaling in myofibroblasts and/or by interfering with myofibroblast contraction and extracellular matrix synthesis. We (15) previously demonstrated that adhesion/integrin-dependent autophosphorylation and activation of FAK (41) as well as activation of Akt are required for the induction of myofibroblast differentiation by TGF-β1. This differentiation to myofibroblasts is inhibited by PGE2 via EP2 prostanoid receptor signaling and cAMP elevation (21). Our current research explores the signaling mechanisms that account for the ability of PGE2 to limit myofibroblast transformation induced by TGF-β1. We demonstrate that PGE2 inhibition of myofibroblast transformation is Smad independent. In contrast, PGE2 appears to modulate the adhesion-dependent pathways involved in TGF-β1-induced myofibroblast differentiation including paxillin, STAT-3, FAK, and Akt. These data link the inhibition of myofibroblast differentiation by PGE2 to the inhibition of FAK and corroborate our (41) earlier work demonstrating that interruption of FAK signaling can limit myofibroblast differentiation. Importantly, the current work demonstrates these relationships in both fetal and adult lung fibroblasts.
TGF-β1 signaling in fibroblasts is known to involve phosphorylation of Smad2 and Smad3 followed by association with Smad4. The trimeric Smad2/3/4 complex then translocates to the nucleus to induce transcription via binding to SBE elements in the promoters of target genes. In the case of myofibroblasts, it is interesting to note that PGE2 inhibition of TGF-β1-induced collagen secretion appears to occur earlier than PGE2 inhibition of α-SMA expression. This may reflect the fact that different, but perhaps overlapping, signaling pathways are involved in the regulation of these two genes. Smad2/3/4 does not generally initiate transcription alone but requires transcriptional coactivators such as CBP/p300. These cofactors may induce acetylation of the Smad complex to activate DNA binding (37). The ability of TGF-β1 to activate Smad signaling is adhesion independent (41). Because most TGF-β1-responsive genes are regulated in a Smad-dependent manner, we were surprised to discover that PGE2 inhibition of myofibroblast differentiation was Smad independent. Cotreatment of IMR-90 cells with both TGF-β1 and PGE2 had no effect on the phosphorylation of Smad2 and Smad3 and had no effect on the translocation of Smad2/3/4 to the nucleus compared with treatment with TGF-β1 alone. This was true even at very high concentrations of PGE2. Thus PGE2 did not appear to limit Smad phosphorylation or nuclear translocation. We did not, however, examine the acetylation status of the Smad complexes in our studies. We do not yet know whether PGE2 treatment induces the expression of a transcriptional repressor or sequestration of CBP/p300 or whether PGE2 functions merely by disrupting normal TGF-β1 signaling pathways.
Myofibroblast differentiation by TGF-β1 and the inhibition of that differentiation by PGE2 were accompanied by changes in cell shape, cell adhesion to the matrix (fibrillar and focal adhesions), and cytoskeletal organization. Paxillin is a cytoskeletal protein found predominantly at focal adhesions and in the cytosol. Immunofluorescent staining of paxillin and actin stress fibers demonstrated dramatic alterations in focal adhesion, cytoskeletal structure, and cell shape induced by PGE2 signaling in human fibroblasts. When added simultaneously, PGE2 diminished TGF-β1-mediated Y-118 phosphorylation of paxillin and tyrosine-phosphorylation of STAT-3 at 30 min and 24 h. This was associated with dramatic loss and/or redistributions of focal adhesions within the treated cells. Cell shape changes associated with alterations in cytoskeletal structures and cell adhesions have been reported in human macrophages (34), murine Y1 adrenal cells (11), and rat vascular smooth muscle cells (31) by elevation in cAMP and in human aortic smooth muscle cells by prostacyclin analogs and PGE2 (4). Microinjection of the catalytic subunit of PKA into living fibroblasts or the treatment of these cells with dibutyryl cAMP caused reversible alterations in cell morphology and complete loss of actin microfilament bundles (23). We (21) previously demonstrated that PGE2 stimulation induces rapid cAMP elevations in IMR-90 cells. We have confirmed that PGE2 stimulation alone activates the cAMP-responsive transcription factor CREB within 5 min in both IMR-90 cells and primary adult fibroblasts (data not shown and Ref. 16). PGE2 decreases the Y-397 autophosphorylation of FAK when added in the absence of TGF-β1 between 5–30 min in IMR-90 cells, and this inhibition is sustained. FAK inhibition in adult lung fibroblasts treated with PGE2 in the presence or absence of TGF-β1 occurs by 24 h. Thus cAMP elevations are not sufficient to induce FAK activation in lung fibroblasts. Interestingly, PGE2 is able to rapidly phosphorylate STAT-3 and paxillin when added alone. In contrast, PGE2 signaling inhibits STAT-3 and paxillin activation in the presence of TGF-β1. These results clearly demonstrate that PGE2 signaling differs in the presence and absence of TGF-β1. Although we do not yet fully understand why the context for PGE2 signaling is different in the presence and absence of TGF-β1, it may, in part, reflect differential cell-cell signaling related to adherens junctions. Our unpublished observations suggest that TGF-β1 treatment diminishes expression of the adherens junction protein, zonula occludens (ZO)-1α, whereas PGE2 has no influence on this protein when added alone or in combination with TGF-β1. Thus differences such as this may contribute to the differential cellular context for PGE2 signaling in the presence and absence of TGF-β1. It is also possible that the distribution of transcriptional coactivators may also be different in the presence and absence of PGE2. For instance, PGE2-mediated signaling phosphorylates CREB and activates MAP kinases much faster than TGF-β1-mediated signaling can activate Smads. As such, it is likely that in the presence of PGE2, available transcription factors may be preferentially sequestered for CREB-mediated or activator protein-1-dependent transcription as opposed to Smad-mediated transcription. Similarly, the later inhibition of MAP kinase signaling by PGE2 may prevent Smad-mediated transcription at the 24-h time point. Additionally, Rho/Rac/Cdc42-dependent signaling pathways need to be investigated. These questions will be the focus of our future work.
Adhesion-generated signaling and Y-397 phosphorylation of FAK is a prerequisite for TGF-β1-induced fibroblast to myofibroblast transformation (41). The present investigations demonstrate that inhibition of Y-397 phosphorylation of FAK by PGE2 in the presence of TGF-β1 is associated with decreased myofibroblast differentiation and changes in focal adhesion distribution, cytoskeletal changes, and fibroblast shape in both fetal and adult fibroblasts. Hypophosphorylation of FAK and disassembly of actin stress fibers and focal adhesions by prostacyclin analogs and PGE2 has been reported recently in human aortic smooth muscle cells (4). Thus PGE2 signaling is likely dictated by both cell type and the nature of the costimulation. Our data support the concept that FAK regulates cytoskeletal fluidity, focal adhesions, and cell movement (27). These data are consistent with a role for adhesion signaling in survival, growth, and apoptosis of myofibroblasts (18, 22, 24, 45) and the fact that FAK promotes the organization of fibronectin matrix and fibrillar adhesions (17). These data are further supported by the observation that the stellate morphology induced by cAMP in rat vascular smooth muscle cells is associated with loss of actin stress fibers and focal adhesions (31). We have previously shown the fibronectin accumulation and the increased expression of α4-, α5-, and β1-integrins are associated with TGF-β1-induced myofibroblast differentiation (41). The effects of PGE2 on these and other matrix components remain to be investigated.
Myofibroblast differentiation is associated with Akt phosphorylation events. TGF-β1 induced S-473 Akt phosphorylation in human lung fibroblasts at all time points tested. However, this phosphorylation was inhibited by PGE2 within 30 min. This could occur via inhibition of PI3K, activation of PTEN (46), or both. These data suggest that PGE2 is able to limit the prosurvival pathways that are normally active in myofibroblasts. This would imply that PGE2 treatment would render myofibroblasts susceptible to apoptosis as would be expected in normal wound resolution. Certainly, the cytoskeletal changes noted in the presence of PGE2 suggest that the cells may be susceptible to anoikis. However, when we tested IMR-90 cells cultured with TGF-β1 in the presence or absence of PGE2 for 24 h for evidence of alterations in apoptosis, Western blot analysis did not reveal any increase in cleaved caspase-3 expression in the presence of PGE2 during the first 24 h of culture. Thus, if it occurs, induction of apoptosis by PGE2 may require longer exposure times. In fact, our previous studies (15) have suggested inhibition of the Akt pathway induces apoptosis in TGF-β1-treated IMR-90 cells only after 5 days. Another possibility is that PGE2 may serve to limit myofibroblast differentiation and function without diminishing cell survival.
As mentioned earlier, the cellular context for PGE2 signaling is likely different in the presence and absence of TGF-β1. It is likely that cAMP signaling is compartmentalized within the cell. Thus changes in cell shape may serve to alter the proximity of effectors and targets at the subcellular level. Since many of the effects brought about by PGE2 occur within minutes (rapid activation of MAP kinases or STAT-3), the fibroblast context, its interactions with the environment, and its metabolic state are altered dramatically. Thus the conditions required for TGF-β1 to bring about the differentiation of fibroblasts to myofibroblasts no longer exist in the presence of PGE2. The temporal onset of phosphorylation of MAP kinases by PGE2 or TGF-β1 is strikingly different. PGE2 is able to phosphorylate all members of the MAP kinase family within 5 min, whereas the effect of TGF-β1 is delayed. It is possible that the MAP kinases activated by PGE2 may influence pathways that are distinct from pathways stimulated by the activation of MAP kinases with TGF-β1. These investigations point to the importance of stimulus-specific dynamic activation of MAP kinases with respect to time of onset and subcellular localization (30).
Our research has determined that PGE2 signaling inhibits adhesion-dependent signaling (paxillin, STAT-3, FAK, and Akt) necessary for fibroblast-to-myofibroblast transformation to occur. PGE2 signaling does not prevent the TGF-β1-induced activation or translocation of Smad2/3/4 to the nucleus. Additionally, PGE2 coadministration alters the kinetics of MAP kinase activation. Although the inhibitory effects of PGE2 may be mediated by any or all of these actions on TGF-β1 signaling, we think it likely that inhibition of FAK activation is critical. We (41) have previously shown that FAK inhibition can limit TGF-β1-induced differentiation of fetal fibroblasts to myofibroblasts. Our current data link the actions of PGE2 to inhibition of FAK in both fetal and adult lung fibroblasts. Further characterizations of the inhibitory signaling pathways induced by PGE2 may identify potential new targets for therapeutic manipulation.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-071586 (B. B. Moore), P50-HL-56402 (B. B. Moore and M. Peters-Golden), HL-06767 (V. J. Thannickal), P50-HL-074024 (V. J. Thannickal), and K08-HL-070990 (E. S. White).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society


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Sat, Apr 22, 2017
Luray, VA
This event is produced by Vacation Races and is the ? race of the year in the National Park Half Marathon Series.
Our Shenandoah is a scenic road race near Shenandoah National Park. We'll start at Hawksbill Recreation Park (205 Pool Dr, Stanley, VA 22851) and end at Lake Arrowhead Recreation Park (265 Lake Arrowhead Rd, Luray, VA 22835).

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2323 Randall Rd
Carpentersville, IL 60110
Dr. Taran Walia can improve the entire health of your mouth with a non-removable bridge. That will keep your surrounding teeth in position and help maintain the structure of your face and jaw.
Come visit Prairie Meadows Dental in the Carpentersville, IL area. See what we can do to improve your appearance and oral health!
You're unique, and you deserve quality, individualized care. Dr. Taran Walia believes your dental experience should blend the best of the personal and the professional. At Prairie Meadows Dental in the Carpentersville, Illinois area, we take the time to get to know you and understand your dental needs. Dr. Walia and her staff are dedicated to providing you with top-quality dental care designed exclusively for you.
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Prairie Meadows Dental is pleased to offer long-term financing for our qualified Carpentersville area dental patients. We realize that some dental treatments are easier to pay off over time. We want to help make it possible for you to have the smile you've always wanted.
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Dental Arts of Orland
10737 Winterset Dr
Orland Park, IL, 60467-1106
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Meyer Dental Group
73 N Broadway St
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James D. Rohan D.D.S. PC
30 N Michigan Ave
Chicago, IL, 60602-3402
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Roselle Dental Care
603 E Irving Park Rd
Roselle, IL, 60172-2302
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Everyone, even your dentist, knows that when we age, we see certain changes in our face (for example, wrinkled skin, less skin tone, shrunken appearance). The soft tissue in the lower one-third of the face is supported by the teeth and jawbone, and gives support to your smile. As we age, we lose support to our smile and we begin to appear older.
Anatomically, the face is divided into thirds: the upper, middle and lower one-third. The space between your nose and your chin is referred to as the lower one-third. The teeth support the vertical height of your lower face, and more specifically the back teeth support your lower face.
Most dentists agree that minimal and gradual wearing away of the top enamel of the teeth is considered normal during the lifespan of a patient. However, excessive wear on the top surfaces of the teeth can result in abscessed teeth, an irregular bite, decreased chewing capacity and esthetic disharmony. Patients with these types of problems often require extensive restorative cosmetic dentistry treatment.
Although the prevalence of tooth wear, or attrition, is not known, it is thought to be very common in adults over the age of 40. The wearing of the top surfaces of the teeth is most often attributed to attrition, which is the wearing away of one tooth surface by another tooth surface. Attrition is the result of bruxism, or the involuntary grinding of the teeth against each other.
Attrition can be the result of one or a combination of problems such as:
Depending on the severity of the tooth wear, teeth may be broken, shortened and unattractive. Having worn teeth can result in jaw joint pain (TMJ), a decreased ability to chew and a sunken appearance to the lower face. All of these results can make a person appear more wrinkled and older.
Generally, the worn teeth will have to have new fillings placed or redone. When severe wear occurs in the mouth, a dental crown or multiple crowns may be the only solution.
Yes, tooth wear can be prevented, but only if you make regular visits to the dentist. If detected early enough, your dentist may prescribe a plastic night guard to protect your teeth, much like an athletic mouth guard.
By Benjamin O. Watkins, III, DDS
Hollywood's most fortunate faces often rely on the skills of their cosmetic dentist in ways that may surprise you.
Dustin Hoffman and Meryl Streep have perfectly nice teeth on their own. But, to meet the challenges of demanding roles in films, both used cosmetic dental appliances (dentures) to change the looks of their natural teeth.
Since women have smaller, rounder teeth, Hoffman, (who played a woman in "Tootsie") used a partial denture to make his teeth look more feminine.
To suggest the brutality of life in a concentration camp, Meryl Streep's teeth and mouth were altered for her role as Sophie Zawistowska, the poignant heroine of "Sophie's Choice."
More commonly, actors see their cosmetic dentists long before they get in front of the camera. In an image-conscious industry, it's no secret the camera is a harsh critic. And a mouth with missing teeth, or a mouthful of stained or broken teeth never helped an actor's image - or the image of a banker, doctor or secretary for that matter.
Few of us face a camera at work every day. But we all face an audience and it's the same audience that goes to the movies. The movies tell us successful people look good, failures don't. Ugly teeth can tell the tale.
Hollywood uses dentistry to make villains out of heroes - you too can use cosmetic dentistry to get a "winning" smile. Restorative dentistry is available to anyone who wants to look better.
Techniques such as dental veneers and tooth bonding have joined the traditional process of dental caps as ways to improve smiles. Orthodontic braces aren't just for children anymore, they're also available to adults who want straight teeth. In some cases, you can opt for teeth bleaching, or have your teeth lengthened or sculpted. If you think you would benefit from these treatments, ask your cosmetic dentist. Not all the new techniques will work for everyone, but he or she will be glad to discuss the best plan for your smile.
With today's new cosmetic dental techniques, you can cast yourself successfully in a competitive world with an award-winning smile.


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We live in a moment of remarkable change and opportunity. Data and technology are transforming industries, society and even the workplace -- by creating professions that did not exist before the emergence of data, cloud, social and mobile. IBM Global Business Services is a leader in this transformation and just the place to launch your career or stat a new one.IBM is now hiring for our Rocket Center, West Virginia location (near Cumberland, Maryland) and has immediate opportunities for forward thinking SAP Security Architects with a passion for growth and innovation. Join our team and utilize leading-edge technology to support projects for our Public Sector.This position will be responsible for building and maintaining SAP user roles across the SAP landscape and providing authorization support to project and business managers. This person will:
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Updated by CasinosAvenue on 08/03/2016See Infos & Directions Voir les jeux
0 reviews > Donner son avis
Share your opinion on this place, we offer you a nice card deck!
Join us for our amazing Cocktail of The Week, available daily at our TanziBar and Sports Bar from 3pm - 3am. Every week this summer we'll bring you a new and delicious cocktail from just…
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At the foot of the Gate complex which houses many bars, cafes, restaurants, and a cinema, the casino is near Haymarket and close to the main shopping area in Newcastle.
The gaming establishment counts 20 slot machines and 70 electronic terminals, with various types and forms of games.
It also h…
Newcastle is located North-East of England in the Tyne and Wear County.
Nearly 400,000 habitants live in Newcastle.
The city provides activities for all, between architecture, nature and history.
In Newcastle you will find many historical moments, as some medieval castles, including the 14th centu…
There are about 20 slots machines of the latest generation, and a good number of electronic terminals in Aspers Newcastle's casino.
|Machine types
|Minimum bet
|Maximum bet
|Traditional reel
|Video reel
|Video poker
Classic games have about twenty tables where you can play in the casino.
|Minimum bet
|Maximum bet
|American Roulette
|Three Card Poker
|Punto Banco
|Wheel of Fortune
|Double Zero Roulette
|Casino War
|Electronic Punto Banco
|Electronic Roulette
The poker room boasts the stunning number of 200 permanent seats, and the poker games selection is wide. Cash games are launched daily from 2pm, blinds ranges from £0,25 / £0,25 to £1 / £2.
|Buy-in min
|Texas Hold'em
|£0,25+ / £0,25+
|Buy In
|Texas Hold'em
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From October 13 to 17, the Monte-Carlo casino held the "One Drop Extravaganza". This poker festival is rather special in its …
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Lead investigator Hugh S. Taylor, M.D., associate professor in the Department of Obstetrics, Gynecology & Reproductive Sciences (Ob/Gyn) at Yale, said the study shows that BPA changes the expression of key developmental genes that form the uterus. Taylor explained that if pregnant women are exposed to the estrogen-like properties found in BPA, it may impact female reproductive tract development and the future fertility of female fetuses the mother is carrying.
The study was conducted on pregnant female mice by administering a range of doses of BPA on days 9-16 of their pregnancies. The aim was to see what interaction BPA would have with the HOXA10 gene, which is necessary for uterine development.
Taylor and co-author Caroline C. Smith of the Department of Epidemiology and Public Health at Yale School of Medicine, found that BPA does, in fact, alter the expression of the HOXA 10 gene, implying that exposure to the popular plastics component may lead to infertility in humans.
"The net effect is concerning," said Taylor. "We are all exposed to multiple estrogen-like chemicals in industrial products, food and pollutants."
Karen N. Peart | EurekAlert!
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Researchers from the Institute for Quantum Computing (IQC) at the University of Waterloo led the development of a new extensible wiring technique capable of controlling superconducting quantum bits, representing a significant step towards to the realization of a scalable quantum computer.
"The quantum socket is a wiring method that uses three-dimensional wires based on spring-loaded pins to address individual qubits," said Jeremy Béjanin, a PhD...
In a paper in Scientific Reports, a research team at Worcester Polytechnic Institute describes a novel light-activated phenomenon that could become the basis for applications as diverse as microscopic robotic grippers and more efficient solar cells.
A research team at Worcester Polytechnic Institute (WPI) has developed a revolutionary, light-activated semiconductor nanocomposite material that can be used...
By forcefully embedding two silicon atoms in a diamond matrix, Sandia researchers have demonstrated for the first time on a single chip all the components needed to create a quantum bridge to link quantum computers together.
"People have already built small quantum computers," says Sandia researcher Ryan Camacho. "Maybe the first useful one won't be a single giant quantum computer...
COMPAMED has become the leading international marketplace for suppliers of medical manufacturing. The trade fair, which takes place every November and is co-located to MEDICA in Dusseldorf, has been steadily growing over the past years and shows that medical technology remains a rapidly growing market.
In 2016, the joint pavilion by the IVAM Microtechnology Network, the Product Market "High-tech for Medical Devices", will be located in Hall 8a again and will...
'Ferroelectric' materials can switch between different states of electrical polarization in response to an external electric field. This flexibility means they show promise for many applications, for example in electronic devices and computer memory. Current ferroelectric materials are highly valued for their thermal and chemical stability and rapid electro-mechanical responses, but creating a material that is scalable down to the tiny sizes needed for technologies like silicon-based semiconductors (Si-based CMOS) has proven challenging.
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Western Mountaineering (WM) was started in 1970 by two Californian mountaineers, whose emphasis on the finest raw materials, meticulous design and unsurpassed workmanship built the foundation of the company. WM have a vast range of down sleeping bags offering at least 3 bag lengths, 2 different zip options, 2 width fittings on key models and a choice of shell fabrics to suit different conditions.
WM down is the very best available and is ethically sourced in Europe. Each batch is tested in Switzerland to ensure it surpasses the EU900 fill power rating, before shipping to the factory in San Jose.
The Apache GWS is a great lightweight winter or alpine bag. The Gore Windstopper (GWS) outer, with DWR treatment, provides an enhanced weather protection where the bag is likely to come into contact with snow or wet equipment/tents.
On the standard 6' length bag, 510 g (19 oz) of EU900+ down provides 6� (15 cm) of loft giving a -4�C Comfort Rating and a -10�C Limit Rating. Yet the weight is only 1175g (2lb 6 oz).
The efficient cut allows the bag to heat quickly, and is complimented with 3D sewn full down collar and an oversized zip draft tube, both of which are capped with GWS fabric.
The Apache GWS is 59� (150 cm) wide across the shoulder and 38� (97 cm) at the foot. It is available in 5'6�, 6' and 6' 6� lengths, and left (standard) and right zip options.
There is also a wider fitting version called Badger GWS, providing an extra 7� of width.
Apache GWS: £440
Badger GWS: £500
More information and a list of UK stockists here.
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Look: Donald Trump's Ridiculously Condescending Comment to A Black Voter at the Second Debate
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According to entertainment blogger Necole Bitchie, Cole recently announced via Twitter, that former Def Jam president and former Warner Music Group EVP Kevin Liles would be representing her. Liles, an idustry vet who played a big role in the success of Def Jam Records, is credited with turning Trey Songz into a star most recently.
Back in January, Coles let go of long time manager Manny Halley.
"Would like for u guys to know that Manny Halley is no longer my management," Cole tweeted. Someone said if the people around u won't change, change the people around you."
Cole's last album, Calling All Hearts, was plagued by poor sales.
(Photo: Ivan Nikolov/WENN.com)
Plus, the former reality star staged a gross photo op with Bill Clinton's alleged mistresses.
Thanks to the power of social media.


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second half notes
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This 12 page Class Notes was uploaded by Ashley Sintetas on Monday February 16, 2015. The Class Notes belongs to Hwc205 at Kansas taught by Barnard in Fall. Since its upload, it has received 101 views. For similar materials see western civ 2 in Arts and Humanities at Kansas.
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Date Created: 02/16/15
Organic Chemistry test 2 3664 36 Application of solubility Soap soap molecules 2 distinct parts 1 a hydrophilic portion made of ions called polar head 2 a hydrophobic carbon chain of nonpolar CC and CH bonds called the nonpolar tail Dissolving soap in water forms micelles spherical droplets with ionic heads on the surface and nonpolar tails packed together in the interior Soap mixed with water nonpolar hydrocarbon tails dissolve dirt in interior of the micelle Polar head of soap remains on surface of micelle to interact with water Micelles are water soluble they separate from our clothes and are washed down the drain with water 37 Applicationthe cell membrane cell basic unit of life aqueous medium inside the cell separated from water outside by cell membrane 1 barrier to passage of ions water and other molecules in and out of cell 2 selectively permeable nutrients in waste out Phospholipids in membranepolar head and two non polar tails when mixed with water lipid bilayer with ionic heads out tails inpolar heads interact with polar h20 and nonpolar tails head close by VDW interactions Transport across a cell membrane Some non polar molecules like 02 are small enough to enter and exit the cell by diffusion onophores molecules that help some ions transport across cell membrane organic molecules that complex cations hydrophobic exterior soluble in nonpolar interior of membrane central cavity w A 02 atoms whose lone pairs complex with a given atom some antibiotics act as ionophores Nonactin and valinomycin Synthetic ionophores Crown ethers cyclic ethers w A 02 atoms that bind specific cations depending size of cavity ionophore binds to ion on one side of membrane in its polar interior move across membrane bc hydrophobic exterior interacts with hydrophobic tails of phospholipid releases ion on other side helps cell function 38 Functional groups and reactivity functional groups create reactive sites in molecules electron rich sites react with electron poor sites all FGs heteroatom TE bond or both make electron deficient or electrophilic sites and electron rich or nucleophilic sites in a molecule Determine reactivity 1 find functional group determine resulting sites it creates a an electronegative heteroatom NO or X makes C atom electrophilic b lone pair on heteroatom makes it basic and nucleophilic c TE bonds make nucleophilic sites more easily broken than 6 bonds 2 electorn deficient C atom reacts with nucleophile 3 electorn rich C atom reacts with electrophile 39 Biomolecules biomolecules 0 compounds found in biological systems small molecules 4 groups Simple sugars Amino acids Lipids and Nucleotides Van Der Waals Caused by interaction of temp dipoles larger surface area stronger forces larger more polarizable atoms stronger forces DipoleDipole interaction of permanent dipoles Hydrogen bonding h to N O or F Ionon charge attraction of two ions Compounds w comparable weight Increasing strength of intermolecular increasing boiling points and Increasing mp Compounds w similar FGs Increasing surface area increasing bp Increasing polarizability increasing bp More symmetrical compound increasing mp Types of water soluble compounds Ionic compounds Organic compounds w less than 5 carbons and 0 or n atom for h bonding for a compound w one functional group Types of compounds soluble in organic solvents 0 compounds any size or F6 Chapter 4 Alkanes organic compounds no functional group less reactive than other 0 compounds Lipids group of biomolecules similar to alkanes composed of nonpolar CC and CH 6 bonds 41 Alkanes aliphatic hydrocarbons w CC and CH o Acyclic Alkanes CnH2n2 saturated hydrocarbons max number of hydrogen atoms per carbons Cycloalkanes carbons joined in one or more rinds CnH2n Butane and isobutene Butane straight chain alkane sobutene branched chain alkane somers 2 different compounds w same same molecular formula Constitutional isomers differ in the way atoms are connected to each other C atoms in alkanes and 0 compounds classified by of other C directly bonded to them Primary bonded to one other C atom secondary 2 tertiary 3 quaternary 4 Hs are classified depending on type of C atom they are bonded Primary H is on a C bonded to another C atom Secondary H is on a C bonded to 2 other C atoms Tertiary H is on a C bonded to 3 other C atoms CH2 group methylene group compounds that differ by only a CH2 group called homologous series All alkanes end in ane 42 cycloalkanes carbon atoms arranged in a ring add prefix cyclo to name of acyclic alkane having same number of Cs 43 intro to naming many drugs have 3 names 1 Systematic follows accepted rules of naming and indicates compounds chemical structure 2 Generic official internationally approved name for the drug 3 Trade assigned by company that makes it usually catchy easy to remember Ex 1 242 methylpropyl phenyl propanoic acid 2 ibuprofen 3 motrin advil 44 naming alkanes 1 parent name longest number of Cs in a molecule 2 suffix which functional group is present 3 prefix identity location and number of substituants attached to carbon chain prefix pa rent suffix carbon substituents bonded to a long C chain alkyl groups formed by removing one H from an alkane to name change the ane ending of parent alkane to yl R general C group bonded to F6 any alkyl group Acyclic alkane naming 4 steps find parent carbon chain and add suffix ane for alkane number the atoms in the c chain name and number the substituents combine substituent names and numbers parent suffix PWP 45 naming cycloalkanes add prefix cyclo before parent prefix cyclo parent suffix 46 common names isopentane dodecahedrane all names end in ane alkane 47 Fossil Fuels natural gas made of methane w lower amounts of ethane propane and butane burn in presence of 02 and release E petroleum mixture of compounds hydrocarbone w 140 c atoms refining distilling crude petro separate into usable fuel for heat each fuel has diff comp of hydrocarbons 48 physical properties of alkanes only nonpolar CH and C2 bonds only weak VDW forces not water soluble 49 conformations of acyclic alkanes Ethane stereochemistry 3d structure of molecules rotation around single bonds CC sigma bonds conformations different shapes w rotation physical properties of alkanes alkanes have low bps compared to more polar compounds of similar size increasing imf increasing bp bp increases as of cs increases bc of increased sa increase sa increase bp bp of isomers decrease w branching bf of decreased surface area alkanes low mp compared to more polar compounds increasing strength of IMF increasing mp mp increases as of cs increase bc of increased sa increased symmetry increase mp alkanes soluble in o solvents alkanes are insoluble in water eclipsed conformation CH bonds on one carbon behind CH bonds of another Staggered conformation CH bonds on one C bisect HCH bond angle on adjacent C Rotate atoms on 1 c by 60 degrees converts one to another How to draw neuman projection on back of page Staggered conformations are more stable lower in Energy than eclipsed conformations energy difference bw staggered and eclipsed caed torsional energy eclipsing induces torsional strain an increase in E caused by eclipsing interactions 410 Conformations of Butane staggered conformation with 2 larger groups 180 from each other anti Staggered conformation w 2 larger groups 60 from each other called gauche Steric strain increase in energy from when atoms are forced too close to eachother Gauche conformations are usually higher in energy than anti bc of steric strain Staggered conform At energy minima and eclipsed at energy maxima Unfavorable steric interactions increase energy The energy difference bw the lowest and highest E conformations is called barrier to rotation 411 an intro to cycloalkanes affected by torsional steric and angle strain increase in E when tetrahedral bond angles deviate from optimum 1095 cycloalkanes w more than 3 C atoms in ring are not flat pucker to reduce strain 412 cyclohexane planar cyclohexane ring angle strain bc internal bond angles bw carbon atoms 120 and torsional strain all H s eclipsed chair conformations Axial point up equatorial point out chair flip axial equatorial 413 Substituted cycloalkanes larger substituants are more stable in the equatorial position lower energy boat less stable than chair eclipsing interations bw hs cause torsional strain flagpole hs causes steric strain stereoisomers isomers that differ only in way atoms are oriented in space Cis 2 groups on same side of the ring Trans 2 groups on opposite sides of the ring Whether sub are axial or equatorial depends on relative location of 2 sub 414 oxidation of alkanes combustion oxidation reduction reaction oxidation loss of electrons 0 increase in number of C2 bonds or decrease in number of CH bonds reduction gain of electrons H decrease in number of C2 bonds or increase in number of CH bonds combustion burn in presence of water to form oxygen and coZ and water 415 lipids lipids biomolecules resemble alkanes and other hydrocarbons defined by a physical property soluble in organic solvents and insoluble in water made of nonpolar CH and CC bonds have few polar FGs Chapter 5 51 Starch and Cellulose minute differences in structure can cause different properties polymers in carbohydrate group polymer a large molecule made of repeating smaller units called monomers covalently bonded together starch main carb in seeds and roots of plants glucose simple sugar we use for energy cellulose gives rigidity to tree trunks and plant stems hydrolysis of cellulose glucose humans cant go cellulose glucose both 6 membered ring w oxygen and 3 OH groups joined by an oxygen differ in position of O atom joining rings together in cellulose O atom joins 2 rings w 2 equatorial bonds in starch O atom joins 2 rings w 1 equatorial and 1 axial bond isomers bc different compounds w same molecular formula stereoisomers bc oly 3d arrangment is different 52 Two major classes of isomers 1 constitutional isomers differ in way atoms are connected to another a different IUPAC names b same or different FGS c different physical properties d different chemical properties 2 stereoisomers differ in way they are arranged in space a identical IUPAC names except for prefix b same FGS configuration a particular 3d arrangement 53 Chiral and Achiral Molecules identical or different from mirror image chiral molecule that is not superimposable on its mirror image achiral molecule that is superimposable on its mirror image Usually have plane of symmetrMirror plane that cuts a molecule in 12 12 is reflection of other 12 Test Chirality Draw molecule in 3d Draw its mirror image Try to align all bonds and atoms To superimpose you can rotate but not break bonds PWP Enantiomers are mirror images that are not superimposable Stereogenic center C atom with four different FGS no SC molecule generally not chiral one SC molecule always chiral exists as pair of enantiomers 2 or more SC molecule may or may not be chiral 56 labeling Stereogenic Centers w R or S 1 assign priority to each group bonded to center highest atomic number highest priority a if 2 atoms are the same assign priority based on atomic number of atoms bonded to these atoms 1 atom of higher atomic number determines higher priority b if 2 isotopes are bonded assign priorities in order of decreasing mass number c to assign prority in an atom that has a multiple bond treat atom as equivalent number of singly bonded atoms 2 Orient molecule w lowest priority on dash 3 Find direction of priority group 193 a Clockwise R b CounterClockwiseS 57 Diastereomers for n stereogenic centers max number of stereoisomers is Zn 58 Meso Compounds an achrial compound that has stereogenic center usually have plane of symmetry 59 R and S assignments in compounds w two or more stereogenic centers identical compounds have same R S designations at every SC enantiomers have opposite R S designations distereomers have same R S designation for at least 1 SC and opposite for at least 1 of other SC diastereomers stereoisomers not mirror images of each other Figure 510 and 511 512 physical properties of stereoisomers Chemical and physical properties of 2 enantiomers are identical except in their interaction with chiral substances enantiomers same mp bp solubility except for how they interact w plane polarized light polarity of light rotation clockwise dextrorotatory rotation counterclockwise levorotatory enantiomers rotate plane polarized light in equal extent but in opposite directions reactions cancel an equal amount of 2 enantiomers is called racemic mixture or racemeate optically inactive specific roation equation in book notes diastereomers differ with optical rotation enantiomers can not be separated by distillation diasteriomers and constitutional isomers have different physical properties and can be separated by physical techniques enantiomers when 2 enantiomers react w achiral reagent they react at same rate but w chiral non recemic reagent they react at different rates Two enantiomers have exactly same chemical properties except their reaction with chiral non racemic reagents ex chiral drugs Chapter 6 61 writing equations for organic reactions reagent chemical substance that organic reacts with left side of equation with other reactants sometimes above reaction arror organic starting material on left side solvent and temp of rxn maybe added above or below arrow substitution reaction a reaction in which an atom or group of atoms is replaced by antoher atom or groups of atoms elimination reactions elements of starting material are lost and a pi bond is formed addition reaction reaction in which elements are added to the starting material addition and elimination reacctions are opposite 63 bond making and bond breaking reaction mechanism detailed description of how bonds are broken and formed as a starting material is converted to a product one step reaction called concerted reaction stepwise reaction more than one step starting material unstable intermediate product only 2 ways to break cleave a bond electrons can be devided equally or unequally between 2 atoms in a bond equally dividing es called homolysis or hemolytic clevage unequally dividing es calle heterolysis or heterolytic clevage es usually end up on more electronegative atom both require energy homolysis uncharged reactive intermediates with unpaired es heterolysis charged intermediates movement of single e use curved arrow reactive intermediate with single unpaired electron radical most radicals highly unstable have atom without octet no charge intermediates in a group of reactions called radical reactions heterolysis of C2 bond can generate carbocation or carbanion two electrons to Z and none to C charged carbon intermediate carbocation two electrons to C and non to Z generates charged carbon carbanion carbocations elecrophiles carbanions nucleophiles both intermediates in polar reactions rxns where nucleophilie reacts with elecrophile radicals and carbocations are elecrtrophiles bc they have electron deficient carbon carbanions are nucleophhiles bc they have a carbon and a lone pair two radicals can donate 1 electron to form a 2 e bond two ions w unlike charges can come together w neg chatged ion donating both es to form 2 e bond bond formation releases energy 64 bond dissociation energy energy that is needed to homolytically cleave a covalent bond delta h is enthalpy change or heat of reaction delta h pos energy absorbed endothermic delta h negative energy released exothermic bond dissociation energies always positive and homolysis always endothermic bond formation always releases energy reaction is exothermic the stronger the bind the higher the bond dissociation energy decrease down a column of the periodic table as the valence electrons used in bonding are farther from the nucleus shorter bonds are stronger bonds delta h indicates relative strength of bonds broken and formed in a reaction delta h positive more energy needed to break bonds that is released in forming bonds bonds broken in starting material stronger than bonds formed in product delta h negative more energy is released in forming bonds than is needed to break bondds bonds formed in product are stronger than bonds broken in starting material determine overall delta h of a reaction 1 beginning w balanced equation add bond dissociation energies for all bonds broken in starting materials 2 add bond dissociation energies for all bonds formed in products 3 overall delta h is sum of both these numbers
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