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. 2008 Apr 29;105(17):6386-91.
doi: 10.1073/pnas.0801490105. Epub 2008 Apr 23.

The cyclic AMP effector Epac integrates pro- and anti-fibrotic signals

Utako Yokoyama  1 , Hemal H Patel, N Chin Lai, Nakon Aroonsakool, David M Roth, Paul A Insel
Affiliations

The cyclic AMP effector Epac integrates pro- and anti-fibrotic signals

Utako Yokoyama et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A. 2008 Jun 10;105(23):8160

Abstract

Scar formation occurs during the late stages of the inflammatory response but, when excessive, produces fibrosis that can lead to functional and structural damage of tissues. Here, we show that the profibrogenic agonist, transforming growth factor beta1, transcriptionally decreases expression of Exchange protein activated by cAMP 1 (Epac1) in fibroblasts/fibroblast-like cells from multiple tissues (i.e., cardiac, lung, and skin fibroblasts and hepatic stellate cells). Overexpression of Epac1 inhibits transforming growth factor beta1-induced collagen synthesis, indicating that a decrease of Epac1 expression appears to be necessary for the fibrogenic phenotype, an idea supported by evidence that Epac1 expression in cardiac fibroblasts is inhibited after myocardial infarction. Epac and protein kinase A, a second mediator of cAMP action, have opposite effects on migration but both inhibit synthesis of collagen and DNA by fibroblasts. Epac is preferentially activated by low concentrations of cAMP and stimulates migration via the small G protein Rap1 but inhibits collagen synthesis in a Rap1-independent manner. The regulation of Epac expression and activation thus appear to be critical for the integration of pro- and anti-fibrotic signals and for the regulation of fibroblast function.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Profibrogenic agents attenuate Epac1 expression. (A) Expression of Epac1 and Epac2 protein in rat cardiac fibroblasts (passage 1) grown for 48 h in serum-free media and examined by immunoblot analysis. Freshly isolated rat brain tissue was also blotted. GAPDH was used to calibrate protein loading. (B) Time-dependent changes in the mRNA expression of Epac1, Epac2, and GAPDH in cardiac fibroblasts treated with or without 10 ng/ml TGFβ1. Transcripts from quantitative RT-PCR were analyzed by 2% agarose gel electrophoresis and ethidium bromide staining and were detected as clear bands of the expected length. No template control (NTC) had no amplification. (C) Quantitative RT-PCR standardized to GAPDH was used to examine time-dependent changes in the mRNA expression of Epac1 and Epac2 in cardiac fibroblasts treated with 10 ng/ml TGFβ1 or 100 nM angiotensin II. Data are the fold-increase vs. control for each time-course. n = 4–6; ***, P < 0.001 compared with control for each time-course. (D) Densitometry of the blots for Epac1 and Epac2 protein normalized to GAPDH protein expression. Cardiac fibroblasts were incubated with TGFβ1 (10 ng/ml) for 16–24 h after 48 h serum-starvation. n = 4; **, P < 0.01. (E) Cardiac fibroblasts, serum-starved for 48 h, were incubated with or without TGFβ1 (10 ng/ml) for 24 h and then with Me-cAMP (50 μM) for 15min. Cells were lysed and assayed for Rap1 activation. Precipitates (Top) and total cell lysates (Middle) were analyzed by immunoblotting with an anti-Rap1 antibody. (F and G) Expression of Epac1 mRNA in cardiac fibroblasts treated with actinomycin D (5 μg/ml), TGFβ1 (10 ng/ml) or angiotensin II (ATII, 100 nM), using quantitative RT-PCR standardized to GAPDH. n = 4–6. (H–K) Time-dependent (6 h and 24 h) changes in the mRNA expression of Epac1 standardized to GAPDH in rat lung fibroblasts, rat skin fibroblasts, mouse hepatic stellate cells (HSC), and human hepatic stellate cells (hTERT-HSC) treated with 10 ng/ml TGFβ1. Data are the fold- increase vs. control for each time point. n = 6; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with control for each time point.
Fig. 2.
Fig. 2.
Effect of Epac on collagen synthesis, migration and morphology in cardiac fibroblasts. (A–C) Fibroblasts were infected with Adv.GFP or Adv.Epac1 in serum-free media for 24 h and then with or without TGFβ1 (10 ng/ml) for 24 h. Cells were assayed by using real-time RT-PCR to quantify collagen Iα1, Iα2, and IIIα1; the data are normalized to GAPDH. n = 4. *, P < 0.05; ***, P < 0.001. (D) Fibroblast migration was examined by using a modified Boyden chamber method in the absence (CTRL) or presence of Me-cAMP, an Epac-activator, or Phe-cAMP, a PKA-activator. Data are shown as the fold-increase relative to control. n = 6–8. **, P < 0.01; ***, P < 0.001 compared with control (CTRL). (E) Immunocytochemistry was performed by using cardiac fibroblasts (passage 1) grown for 48 h in serum-free media and then for 1 h in serum-free media alone (CTRL) or together with Me-cAMP or Phe-cAMP. Cells were stained for Epac1 (green) and nuclear staining of DNA with DAPI (blue). (Scale bar, 30 μm.) (F) Time-dependent Rap1 activation in cardiac fibroblasts. Cells were serum-starved for 48 h, then incubated with Me-cAMP (50 μM) or Phe-cAMP (50 μM) up to 16 h and assayed for Rap1 activation. Precipitates (Upper) and total cell lysates (Lower) were analyzed by immunoblotting with an anti-Rap1 antibody. (G) siRNA-promoted silencing of Rap1 protein expression. Fibroblasts were incubated with Rap1-targeted siRNA for 24 h, lysed and subjected to immunoblot analysis with a Rap1 antibody. (H) Fibroblasts were incubated with Rap1-targeted siRNA for 24 h, treated with Me-cAMP (50 μM) and migration was assayed. n = 4–6; *, P < 0.05. (I) Migration was examined in cardiac fibroblasts treated with Me-cAMP (50 μM) alone, or together with TGFβ1 (10 ng/ml) or angiotensin II (100 nM). n = 4–7, ***, P < 0.001.
Fig. 3.
Fig. 3.
Lower concentrations of cAMP promote cardiac fibroblast migration via Epac. (A–E) Fibroblast migration and cAMP concentrations in response to various agents were measured by a modified Boyden chamber method and RIA, respectively. Cardiac fibroblasts were grown for 48 h in serum-free media and then incubated for 16 h to assess migration or stimulated for 3–10 min for assay of cAMP concentrations after treatment with the indicated concentrations of each drug. The bar graph and the left y axis indicate migration, and the line graph and the right y axis indicate cellular cAMP concentrations. Values represent mean ± SEM of at least three independent experiments. Data are the fold-increase relative to control. n = 6–8. *, P < 0.05; **, P < 0.01 compared with control (CTRL). (F) The relation between migration and cAMP production by cAMP-elevating drugs. (G) The effect of Epac1-targeted siRNA (si-A) on adrenomedullin-induced migration was examined as in B. n = 4–8; *, P < 0.05. (H) Effect of the PKA inhibitor (PKI) Wiptide on adrenomedullin-induced migration. Cardiac fibroblasts were incubated with 10 μM PKI 20 min before assessment of migration. n = 4–7; *, P < 0.05.
Fig. 4.
Fig. 4.
Epac and PKA activation inhibit collagen I and III expression and DNA synthesis of cardiac fibroblasts. (A–C) Cardiac fibroblasts were incubated in serum-free media for 48 h and then with either Me-cAMP (50 μM) or Phe-cAMP (50 μM) for 24 h. Cells were assayed by using real-time RT-PCR to quantify collagens Iα1, Iα2, and IIIα1; the data are normalized to GAPDH. n = 4. **, P < 0.01; ***, P < 0.001 compared with CTRL. (D) Cells were serum-starved for 48 h and then incubated with either Me-cAMP (50 μM) or Phe-cAMP (50 μM) in presence of [3H]thymidine for 24 h and processed to quantify DNA synthesis. n = 6. ***, P < 0.01 compared with CTRL. (E) Cells were incubated with Rap1-targeted siRNA for 24 h and then stimulated with Me-cAMP for 24 h. Quantitative RT-PCR was used to analyze collagen type Iα1 mRNA expression. n = 4–6. *, P < 0.05. (F) Cells were incubated with Rap1-targeted siRNA for 24 h, then for 24 h with Me-cAMP and [3H]thymidine. n = 4. ***, P < 0.001.
Fig. 5.
Fig. 5.
Epac1 expression is decreased after myocardial infarction in vivo. (A) Sections of cardiac intraventricular septa from rats subjected to myocardial infarction (MI) or sham operation (sham) were stained for TGFβ1 (red), Epac1 (green) and nuclei with DAPI (blue). The images of the border zone that abuts on areas of infarction are shown. (Scale bar, 100 μm.) (B) Sections of intraventricular septa of rats subjected to myocardial infarction (MI) or sham operation (sham) were stained for αSMA (red), Epac1 (green), and DNA with DAPI (blue). The images of the border zone of infarction are shown. (Scale bar, 100 μm.) (C) Cardiac fibroblasts isolated from intraventricular septa of postmyocardial infarction and sham-operated rats were cultured for 16 h to eliminate other cell types. RNA was isolated and subjected to quantitative RT-PCR. GAPDH was used as standard. n = 4–5. **, P = 0.0085 compared with the sham-operated group. (D) Expression of Epac1 protein. (Upper) Representative images of immunoblots. (Lower) Densitometry of the blots for Epac1 protein normalized to GAPDH protein expression is shown in the graph. n = 3, **, P = 0.0367. (E) Model for the role of Epac and PKA in fibroblasts after injury. After injury, levels of cAMP-elevating hormones increase, activating cognate GPCR. The enhanced synthesis of cAMP by adenylyl cyclase activation stimulates Epac and PKA. Epac promotes fibroblast migration, whereas PKA inhibits migration and fibroblast-to-myofibroblast transformation. Both Epac and PKA inhibit collagen synthesis and fibroblast proliferation. Profibrotic stimuli inhibit Epac expression and stimulate collagen synthesis.
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