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. 2012 Nov;26(11):4540-7.
doi: 10.1096/fj.12-213496. Epub 2012 Aug 2.

Defining the cellular repertoire of GPCRs identifies a profibrotic role for the most highly expressed receptor, protease-activated receptor 1, in cardiac fibroblasts

Aaron N Snead  1 , Paul A Insel
Affiliations

Defining the cellular repertoire of GPCRs identifies a profibrotic role for the most highly expressed receptor, protease-activated receptor 1, in cardiac fibroblasts

Aaron N Snead et al. FASEB J. 2012 Nov.

Abstract

G-protein-coupled receptors (GPCRs) have many roles in cell regulation and are commonly used as drug targets, but the repertoire of GPCRs expressed by individual cell types has not been defined. Here we use an unbiased approach, GPCR RT-PCR array, to define the expression of nonchemosensory GPCRs by cardiac fibroblasts (CFs) isolated from Rattus norvegicus. CFs were selected because of their importance for cardiac structure and function and their contribution to cardiac fibrosis, which occurs with advanced age, after acute injury (e.g., myocardial infarction), and in disease states (e.g., diabetes mellitus, hypertension). We discovered that adult rat CFs express 190 GPCRs and that activation of protease-activated receptor 1 (PAR1), the most highly expressed receptor, raises the expression of profibrotic markers in rat CFs, resulting in a 60% increase in collagen synthesis and conversion to a profibrogenic myofibroblast phenotype. We use siRNA knockdown of PAR1 (90% decrease in mRNA) to show that the profibrotic effects of thrombin are PAR1-dependent. These findings, which define the expression of GPCRs in CFs, provide a proof of principle of an approach to discover previously unappreciated, functionally relevant GPCRs and reveal a potential role for thrombin and PAR1 in wound repair and pathophysiology of the adult heart.

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Figures

Figure 1.
Figure 1.
Real-time-PCR array expression of nonchemosensory GPCRs in adult rCFs. Histogram of the normalized dC(t) values for all detected GPCRs (152 not detected). Table 2 lists the 12 most abundant GPCRs. Cycle threshold values were normalized relative to 18S rRNA, with a lower dC(t) value indicating higher expression.
Figure 2.
Figure 2.
PAR1 activation alters expression of fibrotic markers in rCFs. mRNA expression of fibrotic markers was determined by RT-PCR in CFs that were initially serum starved for 24 h and then incubated with DMEM (control), thrombin (4.5 nM, A–F), or TRAP6 (20 μM, G–L) for various times up to 24 h. Treatments with thrombin and TRAP6 were staggered so that all samples were collected at the final 24 h time point. Shown are data for PAI-1 (A, G), αSMA (B, H), EPAC1 (C, I), CCN1 (also known as Cyr61; D, J), and TGFβ1 (E, K). Data are summarized in F, L to show the relative magnitude of effects of treatment on each marker. Data are means ± se of ≤4 experiments performed in duplicate. *P ≤ 0.05, **P ≤ 0.01.
Figure 3.
Figure 3.
Effects of thrombin on the protein levels of PAI-1 and αSMA. CFs were treated as described in Fig. 2. Cell lysates were prepared, separated by SDS-PAGE and probed for either PAI-1 (A) or αSMA (B) expression. Densitometry and quantification were performed and normalized to β-tubulin expression. Values represent means ± se of 3 experiments performed in triplicate. **P ≤ 0.01.
Figure 4.
Figure 4.
Effects of thrombin on cell morphology, collagen synthesis, and proliferation in rCFs. A–C) Immunofluorescence microscopy was performed on CFs plated on fibronectin-coated coverslips, serum starved for 24 h, and incubated for 24 h with DMEM (control; A), thrombin (4.5 nM; B), or angiotensin II (1 μM; C). Cells were stained for αSMA (green) and nuclear staining of DNA with DAPI (blue). Scale bars = 30 μm. D) Collagenase-sensitive [3H]proline incorporation was determined for CFs grown for 24 h in serum-free medium and then incubated for 24 h in the presence of [3H]proline (1 μCi/ml) with serum-free medium (control), thrombin (4.5 nM), angiotensin II (Ang II; 1 μM), or FBS (2.5% v/v). E) [3H]thymidine incorporation was determined for cells treated as in D but incubated with [3H]thymidine (1 μCi/ml). Data are means ± se of results from ≤4 experiments performed in triplicate. **P ≤ 0.01.
Figure 5.
Figure 5.
PAR1-dependence of profibrotic effects of thrombin on rCFs. A–C) CFs were serum starved and transfected for 24 h with either control scrambled siRNA (SCR; 5 nM) or PAR1-specific siRNA (PAR1; 5 nM) and then incubated with DMEM (open bars) or thrombin (4.5 nM, solid bars). After 4 h, lysates were collected, and the expression of PAR1 (A), PAI-1 (B), and αSMA (C) was determined by RT-PCR. D) CFs transfected similarly were also incubated (as in Fig. 4D) with [3H]proline and either DMEM (open bars) or thrombin (4.5 nM, solid bars) for 24 h and analyzed for collagenase-sensitive [3H]proline incorporation. Data shown are means ± se of results from ≤3 experiments performed in duplicate. *P ≤ 0.05, ***P ≤ 0.001.
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