Prostaglandin E2 Inhibits α-Smooth Muscle Actin Transcription during Myofibroblast Differentiation via Distinct Mechanisms of Modulation of Serum Response Factor and Myocardin-related Transcription Factor-A*

Background: PGE2 inhibits TGF-β1-induced myofibroblast differentiation, but the mechanism is incompletely understood. Results: PGE2 inhibits α-SMA transcription in human lung fibroblasts by preventing both up-regulation of SRF expression and nuclear translocation of MRTF-A. Conclusion: PGE2 blocks myofibroblast differentiation by targeting two critical determinants of contractile gene expression. Significance: These actions provide a mechanistic basis for therapeutic targeting of lung fibrosis. Differentiation of lung fibroblasts into contractile protein-expressing myofibroblasts by transforming growth factor-β1 (TGF-β1) is a critical event in the pathogenesis of pulmonary fibrosis. Transcription of the contractile protein α-smooth muscle actin (α-SMA) is mediated by the transcription factor serum-response factor (SRF) along with its co-activator, myocardin-related transcription factor-A (MRTF-A). The endogenous lipid mediator prostaglandin E2 (PGE2) exerts anti-fibrotic effects, including the inhibition of myofibroblast differentiation. However, the mechanism by which PGE2 inhibits α-SMA expression is incompletely understood. Here, we show in normal lung fibroblasts that PGE2 reduced the nuclear accumulation of MRTF-A·SRF complexes and consequently inhibited α-SMA promoter activation. It did so both by independently inhibiting SRF gene expression and nuclear import of MRTF-A. We identified that p38 MAPK is critical for TGF-β1-induced SRF gene expression and that PGE2 inhibition of SRF expression is associated with its ability to inhibit p38 activation. Its inhibition of MRTF-A import occurs via activation of cofilin 1 and inactivation of vasodilator-stimulated phosphoprotein. Similar effects of PGE2 on SRF gene expression were observed in fibroblasts from the lungs of patients with idiopathic pulmonary fibrosis. Thus, PGE2 is the first substance described to prevent myofibroblast differentiation by disrupting, via distinct mechanisms, the actions of both SRF and MRTF-A.

Fibrotic diseases affect virtually all vital organs, impair normal organ function, are largely irreversible, and are a leading cause of morbidity and mortality. Idiopathic pulmonary fibrosis (IPF) 2 is the most common of the chronic scarring or interstitial diseases of the lung. Median survival in this disease is only 2.5-3.5 years (1), and the fact that there are no Food and Drug Administration-approved treatments for IPF underscores the need for new insights into its pathogenesis that can be targeted therapeutically. Key features in the pathogenesis of fibrotic disorders such as IPF include expansion of the population of fibroblasts, their differentiation into myofibroblasts that express contractile proteins such as ␣-smooth muscle actin (␣-SMA), and the excessive production of extracellular matrix proteins such as collagen that compose the tissue scar (2,3). Myofibroblasts play a critical role in fibrotic tissue remodeling (4) because of the following: 1) their relative resistance to apoptosis (5,6); 2) their robust capacity for extracellular matrix protein generation (7), and 3) their contribution to tissue contraction and hence stiffness (8,9). Transforming growth factor-␤1 (TGF-␤1) is widely implicated in the pathogenesis of fibrotic diseases (10) and is the best studied inducer of fibroblast differentiation into myofibroblasts.
It is well established that transcription of contractile genes, including ␣-SMA, depends on nuclear complexes of serumresponse factor (SRF) (11) and myocardin-related transcription factor-A (MRTF-A) (12). SRF is a mammalian transcription factor that binds to the consensus sequence CArG box (CC(A/ T) 6 GG) (also known as serum response element or SRE) of smooth muscle cell-specific contractile genes and that is largely localized to the nucleus (13). MRTF-A is a co-activator of SRF that is normally anchored in the cytoplasm by monomeric G-actin (14,15). However, growth factors such as TGF-␤1 activate Rho/ROCK/LIM kinase signaling and subsequent G-actin polymerization to filamentous or F-actin, which frees MRTF-A to translocate into the nucleus (15). MRTF-A associates with SRF within the nucleus, and this complex triggers SRF-dependent ␣-SMA gene expression (14,16).
Prostaglandin E 2 (PGE 2 ) is an endogenous lipid mediator with broad anti-fibrotic actions whose synthesis is dysregulated in fibrotic lung diseases (17). Our group previously established that PGE 2 prevents expression of contractile genes such as ␣-SMA in lung fibroblasts (18,19). Recently, we have also demonstrated that PGE 2 can reverse already established myofibroblast differentiation induced in lung fibroblasts by TGF-␤1 as well as endothelin-1 (20). In this study, we investigated the molecular mechanism(s) by which PGE 2 inhibits TGF-␤1-induced ␣-SMA expression in human lung fibroblasts. We found that PGE 2 prevents TGF-␤1-induced activation of ␣-SMA promoter activity and of nuclear accumulation of SRF⅐MRTF-A complexes. This reflected distinct molecular mechanisms of regulation of SRF and MRTF-A.

EXPERIMENTAL PROCEDURES
Cell Culture and Reagents-CCL-210 fibroblasts (CCD-19Lu), a commercially available primary line of fibroblasts isolated from normal adult human lung, were obtained from ATCC (Manassas, VA). Selected studies were also performed with lung fibroblasts isolated at the University of Michigan, under an IRB-approved protocol described previously (21), from lung tissue determined histologically to be either nonfibrotic or diagnostic of IPF. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT), 100 units/ml penicillin, and 100 g/ml streptomycin (Invitrogen). Recombinant human TGF-␤1 (R&D Systems, Minneapolis, MN), PGE 2 , and butaprost were purchased from Cayman Chemicals (Ann Arbor, MI). PKA RI and PKA RII agonists and forskolin were purchased from Biolog (Hayward, CA). We used PKA RI and RII subunit-specific agonists each at 500 M based on our previous dose-response studies (22). Unless otherwise specified, TGF-␤1 was used at a final concentration of 2 ng/ml, and cells were pretreated for 30 min with or without PGE 2 at a final concentration of 500 nM before addition of TGF-␤1. Antibodies recognizing ␣-SMA, SRF, MRTF-A, GAPDH, Sam 68, and ␣-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for p38 and phospho-p38 were purchased from Cell Signaling Technologies (Beverly, MA). Myristoylated PKA inhibitory peptide 14 -22 (PKI) was from EMD Millipore (Millipore Corp., Billerica, MA). The PKA-specific cAMP analog 6-Bnz-cAMP, PKA RI-selective agonist 8-MA-cAMP, and the PKA-RII-selective agonist 6-MBC-cAMP were purchased from Biolog Life Science Institute (Howard, CA). Fast and Power SYBR Green Master Mix and StepOne real time PCR system were procured from Applied Biosystems (Foster City, CA). ChIP-IT kit was purchased from Active Motif (Carlsbad, CA). The wild-type ␣-SMA promoterluciferase reporter construct (␣-SMApro-Luc) plasmid was a kind gift from Prof. S. H. Phan (Dept. of Pathology, University of Michigan, Ann Arbor, MI). pGL3-Basic, pRL-TK plasmids, and the Dual-Luciferase Reporter Assay System were purchased from Promega (Madison, WI).
Co-immunoprecipitation Assay-For co-immunoprecipitation studies, CCL-210 cells from 10-cm plates were rinsed with ice-cold phosphate-buffered saline (PBS) and then lysed in 500 l of lysis buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM EDTA, and EDTA-free protease inhibitor mixture (Roche Applied Science)). The lysates were incubated on a rocking platform for 2 h at 4°C and cleared by centrifugation at 13,000 ϫ g for 10 min at 4°C. Samples were precleared with 10 l of Magna ChIP TM protein A magnetic beads (Millipore, Billerica, MA) for 1 h at 4°C. Precleared cell lysates were incubated with rabbit anti-SRF polyclonal antibody (G-20, Santa Cruz Biotechnology, Inc.) or normal rabbit polyclonal IgG for 16 h at 4°C and immunoprecipitated with magnetic beads. SRF⅐MRTF-A complexes were detected by Western blotting (WB) with an anti-MRTF-A goat polyclonal antibody (Santa Cruz Biotechnology).
qRT-PCR Analysis of mRNA Expression-Quantitative reverse transcription-PCR (qRT-PCR) analysis of mRNA expression was largely performed as described previously. Briefly, total cellular RNA was extracted and purified using an RNeasy kit from Qiagen (Valencia, CA). cDNA was prepared using the Superscript III First Strand Synthesis SuperMix (Invitrogen), amplified with Fast SYBR Green Master Mix, and analyzed on a StepOne real time PCR system (Applied Biosystems, Carlsbad, CA). Standard curves were generated for each gene using PCRamplified fragments from each target with the primers listed in Table 1.
Quantitative ChIP Assay-Chromatin immunoprecipitation (ChIP) experiments were performed using the EZ-ChIP kit (Millipore Corp., Billerica, MA), with minor modifications. In brief, cells were grown in 10-cm plates and treated with 1% formaldehyde for 10 min at 37°C to cross-link histones to DNA. The reaction was then stopped by the addition of glycine to a final concentration of 0.125 M and incubated for 15 min at room temperature. Fixed cells were rinsed twice with PBS and scraped into 1 ml of cell lysis buffer. The cross-linked chromatin was sonicated for 15 min using a Covaris S2 sonicator (Covaris, Woburn, MA) to shear chromatin fragments to ϳ250 -1,000 bp in length. A portion of sheared chromatin was  ATC ACC AAC TGG GAC GAC AT  CAT ACA TGG CTG GGA CAT TG  60  Srf  CCT ACC AGC TTC ACC CTC AT  GTG AGG TCTGTG CTG CTG TC  60  Mrtf-A  AGC TGC AGA TCC TCA ACC AG  GAG CTG GAG CTG CTA TTG GT  60  Gapdh  CAG CCT CAA GAT CAT CAG CA  ACA GTC TTC TGG GTG GCA GT  60  Mapk14 CTT GCG CAT GCC TAC TTT GC GGT GGC ACA AAG CTG ATG AC 60 reversed at 65°C for 4 h, and cross-linked DNA was purified by the QIAquick PCR purification kit (Qiagen Valencia, CA). The DNA was saved and used as an internal reference control in the subsequent RT-PCRs. The rest of the sonicated chromatin was immunoprecipitated with 4 g of rabbit anti-SRF polyclonal antibody (G-20X, Santa Cruz Biotechnology); immunoprecipitation with 4 g of rabbit IgG isotype antibody was used as a negative control. Immune complexes were recovered with Magna ChIP TM protein A magnetic beads (Millipore, Billerica, MA). Cross-links were reversed as mentioned above, and protein was removed from DNA by treatment with proteinase K. DNA was purified by the QIAquick PCR purification kit. qPCR was performed using Fast SYBR Green PCR master mix to quantify SRF binding to ␣-SMA fragments using the following primers: 5Ј-AGT TTT GTG CTG AGG TCC CTA TAT G-3Ј and 5Ј-TTC CCA AAC AAG GAG CAA AGA-3Ј. Chromatin binding was calculated as the percentage of immunoprecipitated DNA relative to the amount of input. Luciferase Reporter Assays-Cells were grown on 6-well plates and co-transfected at 60% confluence with FuGENE HD (Promega) using 1.0 g of ␣-SMApro-Luc or empty (pGL3-Basic) plasmids together with 0.05 g of a reference promoter driving Renilla luciferase (pRL-TK) to normalize the data. After 24 h of incubation, cells were washed and placed in serum-free medium. After 24 h of serum starvation, cells were treated Ϯ PGE 2 (500 nM) and TGF-␤1 (2 ng/ml) in DMEM, and the incubation was continued for an additional 24 h. Cells were then lysed in 500 l of Passive Lysis Buffer (Promega), and the samples were subjected to a cycle of freeze/thawing and then clarified by centrifugation (12,000 rpm, 5 min at 4°C). Firefly and Renilla luciferase activities were measured by the Dual-Luciferase TM reporter assay system using a GloMax 96 microplate luminometer with dual injectors (Promega) according to the manufacturer's instructions. Results were normalized by dividing the firefly luciferase activity by the Renilla luciferase activity of the same sample. All conditions were assayed in triplicate, and each experiment was repeated at least three times.
p38 siRNA and Overexpression Studies-Cells were transiently transfected either with 100 nM siRNA targeting both ␣ and ␤ isoforms of p38 MAPK (CGGCAGGAGCUGAACA-AGAUU) or siRNA negative control (scrambled siRNA) (UUCUCCGAACGUGUCACGUUU) (23), both purchased from Dharmacon (Lafayette, CO). Transfection was accomplished using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. 24 h post-transfection, cells were cultured in serum-free medium for another 24 h followed by TGF-␤1 stimulation for 12 h. Wild-type p38 overexpression construct (pSR␣-3HA-p38) was kindly provided by Dr. Yusen Liu (The Research Institute at Nationwide Children's Hospital, Columbus, OH) (24). Cells were transiently transfected with p38 overexpression plasmid using FuGENE HD (Promega) according to the manufacturer's protocol. In brief, p38 plasmid and transfection reagents were added at a 1:3 DNA/FuGENE HD ratio to the cells. 24 h post-transfection, cells were cultured in serum-free medium for another 24 h before treating cells with or without PGE 2 for 30 min followed by stimulation with TGF-␤1 for 12 h.
Statistical Analyses-Data are presented as means and were analyzed for statistical significance by one-way ANOVA with the Newman-Keuls multiple comparisons test. Error bars represent S.E. 2 Inhibits the TGF-␤1-induced Increase in ␣-SMA Protein and mRNA-As demonstrated previously, CCL-210 normal adult lung fibroblasts treated for 24 h with TGF-␤1 manifested a myofibroblast phenotype as indicated by an increase in ␣-SMA protein, and pretreatment with PGE 2 prevented ␣-SMA protein expression in TGF-␤1-stimulated fibroblasts (Fig. 1A). We found similar effects of PGE 2 on ␣-SMA when added together with TGF-␤1 (data not shown). The increase in ␣-SMA protein elicited by TGF-␤1 is known to occur at the transcriptional level (25). We verified that the increase in ␣-SMA mRNA in TGF-␤1-stimulated fibroblasts is detectable at 6 h, reaches its maximum by 24 h (Fig. 1B), and is abolished by pretreatment with actinomycin D (Fig. 1C). To evaluate the effect of PGE 2 on ␣-SMA mRNA levels, we performed qRT-PCR on cells pretreated with or without PGE 2 followed by stimulation with TGF-␤1 for 6, 12, and 24 h. Indeed, pretreatment with PGE 2 significantly abrogated the TGF-␤1-induced increment in ␣-SMA mRNA expression by ϳ40% at 12 h (Fig. 1D) and ϳ70% at 24 h (Fig. 1E). There was no additional effect of PGE 2 on TGF-␤1-induced ␣-SMA mRNA expression in the presence of actinomycin D (Fig. 1C). These results demonstrate that PGE 2 inhibits TGF-␤1-induced ␣-SMA expression at the mRNA level. PGE 2 Inhibits TGF-␤1-induced ␣-SMA Promoter Activity-To determine whether PGE 2 reduces TGF-␤1-induced ␣-SMA mRNA by inhibiting its transcription, we examined the effects of this prostanoid on ␣-SMA promoter activity by luciferase reporter assay. Cells were co-transfected with the ␣-SMA promoter luciferase (␣-SMA-Luc) and thymidine kinase-Renilla luciferase (TK-RL) constructs. Following transfection, cells were serum-starved for 24 h and treated with or without PGE 2 for 30 min followed by TGF-␤1. TGF-␤1 markedly increased ␣-SMA promoter activity, and this was largely abolished by pretreatment with PGE 2 ( Fig. 2A). TGF-␤1-induced ␣-SMA promoter activation involves the binding of SRF to its SRE (26). We performed quantitative ChIP assay to study SRF binding to the ␣-SMA promoter in the presence and absence of PGE 2 . TGF-␤1 enhanced binding of SRF to the ␣-SMA promoter, and such binding was largely abolished in the presence of PGE 2 (Fig.  2B). Because its association with MRTF-A facilitates SRF binding to the ␣-SMA promoter (27), we asked whether the ability of PGE 2 to decrease SRF binding to the ␣-SMA promoter is associated with a decrease in nuclear SRF⅐MRTF-A complexes. This was evaluated by immunoprecipitating SRF in nuclear lysates and performing WB analysis of these immunoprecipitates for MRTF-A. As expected, SRF⅐MRTF-A complex formation was not demonstrable in control conditions, but it was readily detectable following TGF-␤1 treatment. Pretreatment with PGE 2 abolished the association between SRF and MRTF-A in response to TGF-␤1 (Fig. 2C). Together, these results indicate that PGE 2 negatively regulates TGF-␤1-induced ␣-SMA promoter activation through prevention of SRF⅐MRTF-A association and consequent reduction in SRF binding to the ␣-SMA promoter.

PGE 2 Inhibits SRF but Not MRTF-A Gene Expression-We
next sought to understand the mechanism(s) by which PGE 2 interferes with TGF-␤1-induced SRF⅐MRTF-A nuclear com-plex formation. We initially evaluated the effects of TGF-␤1 and PGE 2 on SRF and MRTF-A protein expression in CCL-210 cells. TGF-␤1 induced a significant increase in SRF protein, as  and TK-RL constructs as described under "Experimental Procedures." Transfected cells were serum-starved for 48 h, pretreated Ϯ PGE 2 , and followed by stimulation Ϯ TGF-␤1. ␣-SMA promoter activity was determined by luciferase activity. Renilla was used as a control for transfection efficiency and was used to normalize raw luciferase values. Results were presented in terms of fold change, and the values represent the mean of Ϯ S.E. from three independent experiments. B, cells were pretreated Ϯ PGE 2 followed by stimulation Ϯ TGF-␤1. Cells were harvested at 24 h, and ChIP-qPCR was performed with primers specific for the 5Ј-CArG region (for SRF binding) as described under "Experimental Procedures." C, cells were pretreated Ϯ PGE 2 followed by stimulation Ϯ TGF-␤1. Cells were harvested at 24 h, and endogenous SRF was immunoprecipitated (IP) with rabbit anti-SRF antibody. WB was performed on the eluate and probed with an anti-MRTF-A goat polyclonal antibody as described under "Experimental Procedures." Experiments were repeated independently at least three times, and the results from representative experiments are shown. Each bar represents mean values (Ϯ S.E.) from three independent experiments. A and B, statistical differences were analyzed by one-way ANOVA. *, p Ͻ 0.05; **, p Ͻ 0.01. has previously been reported (28). PGE 2 substantially abrogated this induction of SRF in response to TGF-␤1 and also reduced SRF levels in the absence of TGF-␤1 (Fig. 3A). By contrast, neither TGF-␤1 nor PGE 2 had any effect on MRTF-A protein expression. Whether the recognized increase in SRF protein induced by TGF-␤1 is due to an increase in SRF gene transcription has never previously been determined. Using qRT-PCR, SRF mRNA levels were observed to increase in response to TGF-␤1 at 3 h and reached maximum levels at 12 h (Fig. 3B). Pretreatment of fibroblasts with actinomycin D abolished TGF-␤1-induced SRF gene expression (Fig. 3C), confirming that TGF-␤1 up-regulates SRF at the transcriptional level. Importantly, PGE 2 prevented this induction of SRF mRNA by TGF-␤1 (Fig. 3, D and E). As was also observed for MRTF-A protein, neither TGF-␤1 nor PGE 2 had any effect on MRTF-A mRNA. These data therefore identify inhibition of TGF-␤1induced SRF gene expression as one mechanism by which PGE 2 diminishes SRF⅐MRTF-A complexes in the nucleus and prevents ␣-SMA promoter activity. PGE 2 Inhibits TGF-␤1-induced SRF Expression in Fibroblasts from Nonfibrotic Controls and IPF Patients-We sought to confirm the inhibitory effects of PGE 2 on SRF mRNA expression in cells isolated at our institution from both IPF patients (n ϭ 5) and nonfibrotic control patients (n ϭ 4) undergoing surgical resection for other reasons. Data for each individual control cell line are presented in Fig. 4A, and data for each IPF cell line are presented in Fig. 4B. Basal levels of SRF were significantly greater in IPF than in control fibroblasts (Fig. 4C). As observed in CCL-210 cells (Fig. 3, D and E), SRF was up-regulated by TGF-␤1 in fibroblasts from both IPF and nonfibrotic controls, but the mean increment was significantly greater in IPF than control cells (Fig. 4D). As also observed in CCL-210 cells, the enhancement of SRF was attenuated by pretreatment with PGE 2 in both IPF and control fibroblasts, but the degree of inhibition was significantly less in IPF than control cells (Fig.  4E), consistent with our previous finding of relative PGE 2 resistance in IPF fibroblasts (21). PGE 2 Inhibits TGF-␤1-induced SRF Gene Expression via an EP2/cAMP/PKA Pathway-We next investigated the receptor and signaling intermediates by which PGE 2 inhibits TGF-␤1induced SRF gene expression. PGE 2 can act by ligation of any of four distinct G-protein-coupled receptors, termed E prostanoid receptors type 1-4 (EP1-4), each of which is coupled to distinct signaling pathways. We have previously found that the G␣ s -coupled receptor EP2 is the most abundantly expressed in human lung fibroblasts and, via increases in cAMP, is largely responsible for most of the anti-fibrotic actions of PGE 2 , including its ability to inhibit myofibroblast differentiation (29). We therefore hypothesized that this same pathway was responsible for SRF inhibition by PGE 2 . Indeed, the direct adenylyl cyclase activator forskolin has been shown to inhibit SRF protein expression during myofibroblast differentiation (28). Treatment of CCL-210 cells with either forskolin or butaprost (a selective agonist for the EP2 receptor) mimicked the ability of PGE 2 to inhibit TGF-␤1-induced SRF gene expression (Fig. 5A), indicating that EP2/cAMP signaling is capable of this effect. Protein kinase A (PKA) is the classically recognized downstream effector of cAMP actions, and we have shown it to mediate some but not all of the inhibitory actions of PGE 2 on fibroblasts (22). To assess the involvement of PKA in the ability of PGE 2 to abrogate SRF expression, cells were pretreated for 1 h with the cell-permeable PKA inhibitory peptide PKI(14 -22)-amide, followed by PGE 2 treatment for 30 min and then TGF-␤1 for 24 h. The ability of PGE 2 to prevent TGF-␤1-induced SRF protein expression was entirely abolished by PKI, suggesting the critical role of PKA in SRF inhibition (Fig. 5B). cAMP binding is mediated by the regulatory subunit of PKA, which exists in two distinct isoforms (RI and RII) that can mediate distinct signaling responses. To determine the specific PKA R subunit isoform involved in SRF inhibition, cells were pretreated for 30 min with a cAMP analog, which binds to both R isoforms, or with analogs specific for PKA RI or RII, prior to addition of TGF-␤1. Although the pan-PKA agonist caused a modest reduction in TGF-␤1-induced SRF protein, the RI agonist had no effect, whereas the RII agonist had a marked inhibitory effect (Fig. 5C). Together, these data suggest that PGE 2 inhibits SRF expression via an EP2/cAMP/type II PKA pathway.

TGF-␤1 Induces SRF Gene Expression in a p38
MAPK-dependent Pathway-As both RhoA and p38 MAPK pathways participate in TGF-␤1-induced ␣-SMA expression (30, 31), we sought to address their roles in its ability to up-regulate SRF expression. Cells were treated with the RhoA inhibitor Y-27632 for 1 h prior to TGF-␤1 addition, and SRF gene expression was determined by qRT-PCR analysis. Although the RhoA inhibitor markedly inhibited TGF-␤1-induced ␣-SMA expression, it caused only a slight inhibition of SRF mRNA (Fig. 5D). By contrast, the p38 MAPK inhibitor SB203580 significantly and substantially attenuated TGF-␤1 induction of SRF gene expression in parallel with its ability to attenuate ␣-SMA expression (Fig.  5E). To further confirm the significance of p38 MAPK in SRF gene expression, we used siRNA targeting both ␣ and ␤ p38 MAPK isoforms (Fig. 5F). Similar to our observations in Fig.  5E, knockdown of p38 MAPK with siRNA impaired TGF-␤1induced SRF gene expression (Fig. 5G). Cells overexpressing p38 MAPK (Fig. 5, H and I) had no significant increase in basal SRF expression, keeping with the fact that this overexpression construct encodes for a kinase that is not constitutively active, although a modest increase with TGF-␤1 stimulations was observed (Fig. 5I). To evaluate the contribution of p38 to SRF mRNA stability, cells were treated with TGF-␤1 for 12 h to induce SRF mRNA, at which point (time 0) they were incubated with actinomycin D (to stop new transcription) in the presence or absence of the p38 inhibitor SB203580 for various times prior to harvest for determination of SRF mRNA. Our results suggest that SRF mRNA  numbers 1-4) were pretreated Ϯ PGE 2 and then stimulated Ϯ TGF-␤1; SRF expression at the mRNA level was determined by qRT-PCR and normalized for GAPDH. B, lung fibroblasts isolated from five IPF patient samples (indicated by numbers [1][2][3][4][5] were pretreated Ϯ PGE 2 and then stimulated Ϯ TGF-␤1. SRF expression at the mRNA level was determined by qRT-PCR and normalized for GAPDH. C, mean (ϮS.E.) basal SRF mRNA levels, normalized for GAPDH, were compared between nonfibrotic and IPF cells by qRT-PCR. D, mean (ϮS.E.) SRF increment with TGF-␤1 was compared between nonfibrotic and IPF cells. E, mean (ϮS.E.) percent of inhibition by PGE 2 of TGF-␤1-induced SRF was compared between nonfibrotic and IPF cells. The statistical differences were analyzed by one-way ANOVA. *, p Ͻ 0.01; **, p Ͻ 0.001. exhibits a steady degradation, and SB203580 causes a modest enhancement of degradation (Fig. 5J). It therefore appears that in fibroblasts p38 likely exerts its major effects by promoting SRF transcription, with a lesser effect on stabilizing the SRF transcripts.

PGE 2 in a cAMP/PKA Pathway Decreases SRF Expression by
Inhibiting TGF-␤1-induced Activation of p38 MAPK-In view of this important role of p38 MAPK in mediating TGF-␤1induced SRF gene expression, we wished to verify that the kinase was activated by TGF-␤1 and to assess the effects of FIGURE 5. PGE 2 in an EP2/cAMP/PKA pathway prevents TGF-␤1-induced SRF gene expression by inhibiting p38 MAPK phosphorylation. A, CCL-210 cells were pretreated with either butaprost (100 nM) or forskolin (10 M) followed by stimulation Ϯ TGF-␤1. Cells were harvested at 12 h, and SRF mRNA was analyzed by qRT-PCR and normalized for GAPDH. B, cells were pretreated Ϯ protein kinase A inhibitor (PKI(14 -22)-amide) (10 M) and then treated with PGE 2 followed by stimulation Ϯ TGF-␤1. Cells were harvested at 24 h, and SRF protein was determined by WB analysis and normalized for total GAPDH as loading control. The results shown are representative of two independent experiments. C, cells were pretreated Ϯ protein kinase A regulatory subunit-specific agonists (RI and RII agonists) and then stimulated with TGF-␤1. Cells were harvested at 24 h, and SRF protein was determined by WB analysis and normalized for total GAPDH as loading control. The results shown are representative of two independent experiments. D, cells were treated Ϯ the Rho kinase inhibitor, Y-27632 (15 M), and then stimulated Ϯ TGF-␤1. mRNA levels of SRF and ␣-SMA were measured by qRT-PCR and normalized for GAPDH. E, cells were treated Ϯ the p38 MAPK inhibitor SB203580 (10 M) and then stimulated Ϯ TGF-␤1 for 12 h. mRNA levels of SRF and ␣-SMA were measured by qRT-PCR and normalized for GAPDH. F, p38 mRNA expression (top panel) and protein levels (bottom panel) were determined in untransfected control (Cont), control siRNA-transfected and p38 siRNA-transfected cells (top panel). G, cells were transfected with either p38 MAPK siRNA (100 nmol) or control scrambled siRNA (100 nmol) for 24 h and then stimulated Ϯ TGF-␤1 for another 12 h. mRNA levels of SRF and ␣-SMA were measured by qRT-PCR and normalized for GAPDH. H, p38 mRNA expression (top panel) and protein levels (bottom panel) were determined in control and pSR␣-3HA-p38 transfected cells. I, cells were transfected Ϯ pSR␣-3HA-p38 plasmid for 24 h and then treated Ϯ PGE 2 followed by stimulation with TGF-␤1 for another 12 h. mRNA levels of SRF were measured by qRT-PCR and normalized for GAPDH. J, cells were stimulated with TGF-␤1 for 12 h and then they were treated Ϯ actinomycin D (Act D) and Ϯ SB203580 Ϯ TGF-␤1. At the indicated times, cells were harvested; RNA was isolated, and SRF gene expression was assessed by qRT-PCR and normalized for GAPDH. K, cells were pretreated Ϯ PGE 2 and then stimulated Ϯ TGF-␤1 for 30 min (top panel). Cells were pretreated Ϯ PKI and then treated Ϯ forskolin followed by Ϯ TGF-␤1 stimulation for additional 30 min (bottom panel). Cells were harvested, and WB was performed for p38 and phospho-p38 proteins. Experiments were repeated independently at least two times, and the results from representative experiments are shown. The statistical differences were analyzed by one-way ANOVA. *, p Ͻ 0.01; **, p Ͻ 0.001; ***, p Ͻ 0.05. PGE 2 on its activation. Phosphorylation of the kinase was utilized as an indicator of its activation state. As expected, TGF-␤1 elicited a strong activation of p38 MAPK within 30 min of its addition. Pretreatment with PGE 2 completely inhibited this TGF-␤1-induced phosphorylation of p38 MAPK (Fig. 5K). We also observed the inhibitory effects of PGE 2 in TGF-␤1-stimulated cells overexpressing p38 MAPK (Fig. 5I), attesting to its robust capacity for inhibition. Similarly, pretreatment with forskolin inhibited TGF-␤1-induced p38 MAPK phosphorylation and PKI-impaired forskolin effects on p38 MAPK phosphorylation (Fig. 5K). Taken together, our data for the first time demonstrate that p38 MAPK is crucial for TGF-␤1-induced SRF expression and strongly suggest that PGE 2 in a cAMP/PKA pathway inhibits SRF gene expression by inhibiting TGF-␤1mediated activation of p38 MAPK. PGE 2 Inhibits TGF-␤1-induced Nuclear Import of MRTF-A-Although our findings strongly implicate a decrease in SRF by PGE 2 (as shown in Fig. 3) as a key factor in the prevention of TGF-␤1-induced ␣-SMA expression, it should be noted that a significant decline in ␣-SMA expression was observed as early as 6 h (Fig. 1D), whereas complete inhibition of SRF was observed only at 24 h (Fig. 3A). This suggests the possible involvement of other inhibitory mechanisms that are operative at time points that precede the ability of PGE 2 to reduce SRF expression. Although we observed no changes in expression of MRTF-A in the presence of TGF-␤1, PGE 2 , or both (Fig. 3, A  and D), this co-activator is known to rapidly shuttle in and out of the nucleus in a RhoA-dependent manner, and TGF-␤1 has been previously reported to promote rapid nuclear accumulation of MRTF-A (32). Based on these considerations, we hypothesized that PGE 2 might also interfere with the nuclear import of MRTF-A. To test this possibility, cells were treated for 24 h with TGF-␤1 in the presence or absence of PGE 2 , and nuclear and cytoplasmic fractions were collected and examined for MRTF-A by WB analysis. The purity of nuclear and cytoplasmic fractions was confirmed by the specific dis-tribution of Sam 68 and ␣-tubulin in nuclear and cytoplasmic fractions, respectively. An increase in nuclear MRTF-A was observed in response to TGF-␤1, and this increase was abrogated in the presence of PGE 2 (Fig. 6A). To evaluate whether this effect of PGE 2 reflected an inhibition of nuclear import or a potentiation of nuclear export of MRTF-A, we examined the effects of a 3-h treatment with the nuclear export inhibitor leptomycin B. Although leptomycin B facilitated nuclear accumulation of MRTF-A in control cells, as expected of a nuclear export inhibitor, it failed to do so in cells treated for 1 h with TGF-␤1 in the presence of PGE 2 (Fig. 6B). These data demonstrate that PGE 2 -mediated inhibition of nuclear accumulation of MRTF-A is not due to facilitation of its nuclear export, but rather it reflects an inhibition of import. MRTF-A nuclear import depends on actin polymerization (14,33). Active (dephosphorylated) cofilin 1 prevents polymerization of G-actin monomers into F-actin and thus prevents MRTF-A nuclear entry. Phosphorylation of cofilin 1 at Ser-3 via a Rho/ROCK/LIM kinase pathway, as activated by TGF-␤1, favors G-actin polymerization and release of MRTF-A, which is thus freed to enter the nucleus (34). We therefore assessed the effect of PGE 2 on cofilin 1 phosphorylation by WB analysis in CCL-210 cells treated for 1 h with TGF-␤1. TGF-␤1 phosphorylated cofilin 1, as expected, and PGE 2 dephosphorylated it (Fig. 6C). Vasodilator-stimulated phosphoprotein (VASP) in its dephosphorylated form stabilizes F-actin and thereby indirectly contributes to ␣-SMA gene expression. In contrast to its effects on cofilin 1, PGE 2 elicited phosphorylation of VASP (indicated by gel shifted band; Fig. 6C, arrow), which would be expected to destabilize F-actin. Collectively these results suggest that PGE 2 inhibits nuclear import of MRTF-A by preventing TGF-␤1-induced actin polymerization, which it accomplishes both by dephosphorylating cofilin 1 and phosphorylating VASP. Nuclear and cytoplasmic fractions were prepared as described under "Experimental Procedures." ␣-Tubulin and Sam 68 were used as markers for cytoplasmic and nuclear lysates, respectively. Using WB, MRTF-A localization was assessed. Bottom panel represents the values obtained from densitometric analyses of nuclear fractions from all samples analyzed. Each bar represents mean values (Ϯ S.E.) of three independent experiments performed per condition. B, cells were incubated Ϯ leptomycin B (LMB) (20 nM) for 3 h. Cells were then pretreated Ϯ PGE 2 followed by stimulation Ϯ TGF-␤1 for 1 h. Nuclear and cytoplasmic fractions were prepared, and MRTF-A localization was assessed by WB. Experiments were repeated independently at least twice, and the blots from a representative experiment are shown. C, cells were pretreated Ϯ PGE 2 followed by stimulation Ϯ TGF-␤1 for 1 h. Then p-cofilin 1 and VASP levels were analyzed by WB. Experiments were repeated independently at least three times, and the results from representative experiments are shown. A, the statistical differences were analyzed by one-way ANOVA. *, p Ͻ 0.05; **, p Ͻ 0.01.

DISCUSSION
TGF-␤1 is a potent profibrotic cytokine that plays a critical role in the pathogenesis of fibrotic tissue remodeling. High levels of TGF-␤1 are noted in IPF lung tissue as well as plasma. An action of TGF-␤1 that is central to fibrogenesis is its ability to induce fibroblast differentiation into myofibroblasts. A hallmark of myofibroblasts is their expression of contractile genes such as ␣-SMA that are usually confined to smooth muscle cells. Although this confers upon these cells the capacity to exhibit contractile function, which contributes to tissue stiffness, the myofibroblast program is also associated with elaboration of excessive amounts of extracellular matrix proteins and resistance to apoptosis. PGE 2 is an endogenous substance that inhibits TGF-␤1-induced differentiation of fibroblasts to myofibroblasts, but the mechanisms underlying this action are poorly understood. Here, we have demonstrated that PGE 2 inhibits transcription of the ␣-SMA gene (Fig. 1, D and E). It does so by inhibiting the p38 MAPK-dependent expression of SRF (Fig. 5, E and G), a master transcription factor for contractile genes, and by inhibiting the F-actin-dependent nuclear import of MRTF-A (Fig. 6A), a transcriptional co-activator that partners with SRF. To our knowledge, this represents the first report of a single biological substance that interrupts both of these drivers of myofibroblast differentiation.
Although it was known that TGF-␤1 induces SRF expression at the protein level, our work demonstrates for the first time that it does so via transcriptional up-regulation of the SRF gene. Because ␣-SMA and SRF promoters both contain an SRE sequence, stimulation with TGF-␤1 promotes SRF binding to these SREs and activation of both ␣-SMA and SRF promoters. However, although SRF mRNA levels increased significantly within 6 h of TGF-␤1 addition, ␣-SMA mRNA levels did so only after 12 h. This may be explained by a higher threshold for SRF activation of the ␣-SMA promoter due to its higher content of SRE domains.
Our studies confirm previous findings that Rho/ROCK signaling is crucial for ␣-SMA gene expression. However, we found only a modest effect of Rho/ROCK inhibition on SRF gene expression. This suggests the involvement of other TGF-␤1 signaling pathways in SRF expression. A role for p38 MAPK in TGF-␤1-induced ␣-SMA expression has been reported (31), but we are aware of no reports of a role for p38 in TGF-␤1-induced SRF expression. So, we investigated the role of p38 MAPK using a p38 inhibitor and p38 MAPK siRNA. Our results uncover an important contribution of p38 to TGF-␤1induced up-regulation of SRF mRNA expression. Our findings also demonstrate a modest role for p38 in maintaining SRF mRNA stability, and inhibition of p38 slightly reduced the stability of SRF mRNA. Overall, our data suggest that the predominant role of p38 is in regulating SRF transcription. Although our results strongly suggest p38 as the target for the inhibitory effects of the PGE 2 -cAMP-PKA axis on TGF-␤1-induced SRF gene expression, future work using a constitutively active form of p38 will be necessary to confirm this. Additional work will also be necessary to determine whether PGE 2 -cAMP-PKA inhibits p38 phosphorylation by inhibiting its upstream kinases, activating p38 phosphatases, or both. Although cAMP/ PKA signaling has been reported to have variable effects on p38 activation in various cell types and contexts (35)(36)(37)(38), in our system it was evident that PGE 2 abolishes p38 activation induced by TGF-␤1.
Although increased SRF levels have been detected by immunohistochemistry in myofibroblasts in the bleomycin model of pulmonary fibrosis (39), to our knowledge neither basal expression of SRF nor its regulation has been investigated in fibroblasts from patients with IPF. Although the general patterns of regulation were similar in IPF as control fibroblasts, certain subtle differences were noted. We observed that IPF fibroblasts expressed higher basal levels of SRF mRNA than did nonfibrotic control cells, and SRF levels also increased to a greater degree in IPF cells during myofibroblast differentiation in response to TGF-␤1. It is possible that greater expression of SRF could contribute to the phenotypic activation characteristic of IPF fibroblasts, but this possibility will require direct examination. Although TGF-␤1-induced SRF up-regulation was also attenuated by PGE2 in IPF cells, the extent of inhibition was more modest than in nonfibrotic cells. This too is characteristic of our prior observations in fibrotic fibroblasts from both humans and mice, in which we have found decreased sensitivity to the inhibitory actions of PGE 2 on processes such as proliferation and collagen I expression (21), which reflects both epigenetic down-regulation of EP2 expression (40) as well as impaired PKA activation (22).
It is well established that TGF-␤1 stimulation of actin polymerization promotes the translocation of free cytoplasmic MRTF-A into the nucleus, where it forms a complex with SRF to drive transcriptional activation of the ␣-SMA promoter. The importance of adhesion signaling in this process is further evidenced by the previous finding that a FAK inhibitor inhibited ␣-SMA expression (41). An important consequence of adhesion signaling elicited by TGF-␤1 is activation of actin polymerization, which in turn requires alterations in activities of the opposing regulatory proteins, cofilin 1 and VASP. Cofilin 1 is an actin-depolymerization factor that, when activated (dephosphorylated), opposes F-actin formation. Cofilin 1 phosphorylation by the Rho/ROCK/LIM kinase pathway is stimulated by TGF-␤1. VASP has opposing effects on actin dynamics, and its phosphorylation at Ser-239 or Thr-278 leads to VASP inactivation and resulting impaired F-actin formation. Our data demonstrate that PGE 2 leads to both dephosphorylation of phospho-cofilin 1 and phosphorylation of VASP; both of these actions would favor actin depolymerization and thereby disrupt the pro-adhesive actions of TGF-␤1. Although we did not formally investigate the involvement of specific PGE 2 receptors or signaling pathways in mediating these anti-adhesive actions, it is highly likely that EP2 and the cAMP-PKA pathways participate in these effects just as they did in inhibition of SRF expression. First, we have previously reported that PKA mediates phosphorylation of VASP stimulated by PGE 2 in murine lung fibroblasts (22). Second, we have previously shown in macrophages that PGE 2 dephosphorylates cofilin 1 in a cAMP-PKAdependent manner, with dephosphorylation being mediated by the phosphatase and tensin homolog (42). Third, we have shown that PGE 2 activates phosphatase and tensin homolog in both macrophages and lung fibroblasts (42,43). Thus, it seems highly likely that PGE 2 inhibition of TGF-␤1-induced MRTF-A nuclear import involves the depolymerization of actin, which in turn reflects both EP2-and PKA-dependent phosphorylation of VASP as well as dephosphorylation of cofilin 1.
A previous report demonstrating that ␣-SMA expression was increased by overexpression of MRTF-A (27) but not by forced overexpression of SRF (28) suggests that the transcription of this gene is limited by a lack of nuclear MRTF-A relative to that of SRF. This highlights the functional importance of the ability of TGF-␤1 to trigger rapid nuclear translocation of MRTF-A to permit sufficient complex formation with nuclear SRF. The ability of PGE 2 to prevent this early effect of TGF-␤1 therefore represents its first wave of inhibition. Its second wave of inhibition involves its inhibition of SRF gene expression. As a result of both of these temporally distinct mechanisms of inhibition, PGE 2 limits the accumulation of nuclear SRF⅐MRTF-A complexes and thus prevents TGF-␤1-induced ␣-SMA gene expression. PGE 2 has been shown to inhibit not only myofibroblast differentiation but also to inhibit fibroblast migration, proliferation, collagen synthesis, and survival. We and others have demonstrated a deficiency of endogenous PGE 2 production in IPF (44,45), and the lack of this endogenous anti-fibrotic substance has been suggested to facilitate the mesenchymal cell activation that characterizes this disorder. A deficiency of PGE 2 generation has also been described in mesenchymal cells isolated from the lungs in two forms of fibrosis of the conducting airways, namely airway remodeling associated with a murine model of chronic allergen-induced asthma (46) and obliterative bronchiolitis in patients post-lung transplant (47). Culturing lung fibroblasts on stiff matrix, which activates adhesive signaling and is sufficient to trigger myofibroblast differentiation, has likewise been reported to down-regulate PGE 2 synthetic capacity (48). Deficient endogenous PGE 2 synthesis or actions promotes fibrogenesis, as evidenced by studies employing pharmacological interruption of PGE 2 synthesis (46,49) and deletion of cAMP-coupled EP2 (50) and EP4 receptors (51). Taken together, this body of literature suggests that, rather than merely being of interest as an exogenously added substance, the suppressive actions of endogenous PGE 2 are so fundamental that tissue fibrogenesis can only occur when this potent antifibrotic brake is down-regulated. The findings of this study provide new mechanistic insights into the critical anti-fibrotic actions of this prostanoid.
Based on our current and previous findings, we propose a model to explain the mechanisms by which PGE 2 inhibits TGF-␤1-induced ␣-SMA gene expression (Fig. 7). TGF-␤1 signaling promotes rapid MRTF-A nuclear import by activating actin polymerization. TGF-␤1 also promotes SRF gene expression in a p38-dependent manner. Together, these two actions result in the formation of SRF⅐MRTF-A complexes within the nucleus that bind to the ␣-SMA promoter and promote its transcriptional up-regulation. PGE 2 inhibits transcriptional activation of ␣-SMA by interfering with both of these determinants of SRF⅐MRTF-A complex formation. Its earliest effect is to inhibit actin polymerization (via inhibition of VASP and activation of cofilin 1), which serves to prevent nuclear import of MRTF-A. PGE 2 subsequently inhibits p38-dependent SRF gene expression. Both of these actions proceed via EP2 ligation and PKAdependent signaling. It is important to recognize that Rho/ ROCK signaling and p38 MARK activation are conserved pathways downstream from TGF-␤1 as well as other pro-fi- FIGURE 7. Scheme depicting mechanisms of TGF-␤1-induced activation of SRF and MRTF-A, ␣-SMA promoter activation, and inhibition by PGE 2 . PGE 2 inhibits MRTF-A nuclear entry as an early means to repress ␣-SMA promoter activity. An additional delayed effect of PGE 2 is to reduce SRF expression. These two mechanisms allow PGE 2 to markedly reduce SRF⅐MRTF-A complexes within the nucleus and abolish ␣-SMA promoter activity. These actions of PGE 2 result in impaired myofibroblast differentiation in response to the profibrotic factor, TGF-␤1. brotic factors that contribute to the pathogenesis of not only pulmonary fibrosis but also renal, hepatic, and cardiac fibrosis. Thus, our findings regarding the mechanisms by which PGE 2 targets these pathways to inhibit myofibroblast differentiation have pathophysiological and therapeutic relevance for a broad range of fibrotic conditions.