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. 2014 Jun 13;289(24):17151-62.
doi: 10.1074/jbc.M114.558130. Epub 2014 May 5.

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

Loka R K Penke  1 , Steven K Huang  1 , Eric S White  1 , Marc Peters-Golden  2
Affiliations

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

Loka R K Penke et al. J Biol Chem. .

Abstract

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.

Keywords: Differentiation; Myofibroblast; Prostaglandin; Transcription Regulation; Transforming Growth Factor-{beta} (TGF-{beta}).

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Figures

FIGURE 1.
FIGURE 1.
PGE2 inhibits TGF-β1-induced α-SMA gene expression. A, CCL-210 cells were pretreated ± PGE2 (500 nm) followed by stimulation ± TGF-β1 (2 ng/ml). Cells were harvested at 24 h, and expression of α-SMA protein was determined by WB analysis and normalized for total GAPDH as loading control. A representative blot is shown at top and densitometric analysis is shown at bottom; each bar represents mean values (± S.E.) from three independent experiments. B, cells were stimulated with TGF-β1, and at the indicated times the cells were harvested, and α-SMA mRNA levels were measured by qRT-PCR. Each bar represents mean values (±S.E.) of three independent experiments performed per condition, and the results from representative experiments are shown. C, cells were treated with actinomycin D (5 μg/ml) or PGE2 or both for 30 min followed by TGF-β1 stimulation for an additional 12 h. Cells were harvested, and α-SMA gene expression was assessed by qRT-PCR. Each bar represents mean values (±S.E.) of three independent experiments performed per condition, and the results from representative experiments are shown. D, cells were pretreated ± PGE2 followed by stimulation ± TGF-β1. At the indicated times, cells were harvested, and expression of α-SMA was assessed by qRT-PCR. Each bar represents mean values (±S.E.) of three independent experiments performed per condition, and the results from representative experiments are shown. E, cells were pretreated ± PGE2 and then stimulated ± TGF-β1. Cells were harvested at 24 h, and total RNA was prepared as described under “Experimental Procedures.” α-SMA mRNA was analyzed by qRT-PCR and normalized for GAPDH. Each bar represents mean values (±S.E.) from four independent experiments. The statistical differences were analyzed by one-way ANOVA. *, p < 0.005; **, p < 0.01; ***, p < 0.05. Nonsignificant differences are indicated (NS).
FIGURE 2.
FIGURE 2.
PGE2 inhibits TGF-β1-induced transcriptional activation of the α-SMA promoter complex. A, CCL-210 cells were co-transfected with α-SMA-Luc and TK-RL constructs as described under “Experimental Procedures.” Transfected cells were serum-starved for 48 h, pretreated ± PGE2, and followed by stimulation ± TGF-β1. α-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 ± S.E. from three independent experiments. B, cells were pretreated ± PGE2 followed by stimulation ± TGF-β1. Cells were harvested at 24 h, and ChIP-qPCR was performed with primers specific for the 5′-CArG region (for SRF binding) as described under “Experimental Procedures.” C, cells were pretreated ± PGE2 followed by stimulation ± TGF-β1. 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 (± S.E.) from three independent experiments. A and B, statistical differences were analyzed by one-way ANOVA. *, p < 0.05; **, p < 0.01.
FIGURE 3.
FIGURE 3.
PGE2 inhibits TGF-β1-induced SRF gene up-regulation. A, WB analysis for SRF and MRTF-A proteins from cell lysates isolated from CCL-210 pretreated ± PGE2 followed by stimulation ± TGF-β1. The level of SRF and MRTF-A expression was analyzed by WB analysis. Representative immunoblot is shown (top panel). Bands from three independent experiments were quantified by densitometry and normalized to GAPDH as loading control, and data are shown as mean values (± S.E.) (bottom panel). B, cells were stimulated with TGF-β1 (2 ng/ml). At the indicated times, cells were harvested; RNA was isolated, and SRF gene expression was assessed by qRT-PCR and normalized for GAPDH. C, cells were treated with or without actinomycin D for 1 h followed by TGF-β1 for an additional 12 h, after which RNA was isolated, and SRF gene expression was assessed by qRT-PCR and normalized for GAPDH. D, cells were pretreated ± PGE2 and then treated with TGF-β1; expression of SRF and MRTF-A at the mRNA levels was determined by qRT-PCR and normalized for GAPDH. Each bar represents mean values (± S.E.) of four independent experiments performed per condition. E, cells were pretreated ± PGE2 followed by stimulation ± TGF-β1. At the indicated times, cells were harvested, and expression of SRF was assessed by qRT-PCR. Each bar represents mean values (±S.E.) of three independent experiments performed per condition, and the results from representative experiments are shown. The level of SRF expression was quantified and normalized to GAPDH controls. The statistical differences were analyzed by one-way ANOVA. *, p < 0.005; **, p < 0.01. Nonsignificant differences are indicated (NS).
FIGURE 4.
FIGURE 4.
PGE2 inhibits TGF-β1-induced SRF expression in fibroblasts isolated from lungs of patients with IPF and from nonfibrotic controls. A, human lung fibroblasts isolated from four nonfibrotic controls (indicated by numbers 1–4) were pretreated ± PGE2 and then stimulated ± TGF-β1; 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 1–5) were pretreated ± PGE2 and then stimulated ± TGF-β1. SRF expression at the mRNA level was determined by qRT-PCR and normalized for GAPDH. C, mean (±S.E.) basal SRF mRNA levels, normalized for GAPDH, were compared between nonfibrotic and IPF cells by qRT-PCR. D, mean (±S.E.) SRF increment with TGF-β1 was compared between nonfibrotic and IPF cells. E, mean (±S.E.) percent of inhibition by PGE2 of TGF-β1-induced SRF was compared between nonfibrotic and IPF cells. The statistical differences were analyzed by one-way ANOVA. *, p < 0.01; **, p < 0.001.
FIGURE 5.
FIGURE 5.
PGE2 in an EP2/cAMP/PKA pathway prevents TGF-β1-induced SRF gene expression by inhibiting p38 MAPK phosphorylation. A, CCL-210 cells were pretreated with either butaprost (100 nm) or forskolin (10 μm) followed by stimulation ± TGF-β1. Cells were harvested at 12 h, and SRF mRNA was analyzed by qRT-PCR and normalized for GAPDH. B, cells were pretreated ± protein kinase A inhibitor (PKI(14–22)-amide) (10 μm) and then treated with PGE2 followed by stimulation ± TGF-β1. 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 ± protein kinase A regulatory subunit-specific agonists (RI and RII agonists) and then stimulated with TGF-β1. 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 ± the Rho kinase inhibitor, Y-27632 (15 μm), and then stimulated ± TGF-β1. mRNA levels of SRF and α-SMA were measured by qRT-PCR and normalized for GAPDH. E, cells were treated ± the p38 MAPK inhibitor SB203580 (10 μm) and then stimulated ± TGF-β1 for 12 h. mRNA levels of SRF and α-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 ± TGF-β1 for another 12 h. mRNA levels of SRF and α-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 pSRα-3HA-p38 transfected cells. I, cells were transfected ± pSRα-3HA-p38 plasmid for 24 h and then treated ± PGE2 followed by stimulation with TGF-β1 for another 12 h. mRNA levels of SRF were measured by qRT-PCR and normalized for GAPDH. J, cells were stimulated with TGF-β1 for 12 h and then they were treated ± actinomycin D (Act D) and ± SB203580 ± TGF-β1. 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 ± PGE2 and then stimulated ± TGF-β1 for 30 min (top panel). Cells were pretreated ± PKI and then treated ± forskolin followed by ± TGF-β1 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 < 0.01; **, p < 0.001; ***, p < 0.05.
FIGURE 6.
FIGURE 6.
PGE2 prevents TGF-β1-induced nuclear import of MRTF-A. A, CCL-210 cells were pretreated ± PGE2 followed by stimulation ± TGF-β1 for 24 h. Nuclear and cytoplasmic fractions were prepared as described under “Experimental Procedures.” α-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 (± S.E.) of three independent experiments performed per condition. B, cells were incubated ± leptomycin B (LMB) (20 nm) for 3 h. Cells were then pretreated ± PGE2 followed by stimulation ± TGF-β1 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 ± PGE2 followed by stimulation ± TGF-β1 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 < 0.05; **, p < 0.01.
FIGURE 7.
FIGURE 7.
Scheme depicting mechanisms of TGF-β1-induced activation of SRF and MRTF-A, α-SMA promoter activation, and inhibition by PGE2. PGE2 inhibits MRTF-A nuclear entry as an early means to repress α-SMA promoter activity. An additional delayed effect of PGE2 is to reduce SRF expression. These two mechanisms allow PGE2 to markedly reduce SRF·MRTF-A complexes within the nucleus and abolish α-SMA promoter activity. These actions of PGE2 result in impaired myofibroblast differentiation in response to the profibrotic factor, TGF-β1.
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