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. 2008 Feb;172(2):309-20.
doi: 10.2353/ajpath.2008.070347. Epub 2008 Jan 17.

Factor Xa stimulates proinflammatory and profibrotic responses in fibroblasts via protease-activated receptor-2 activation

Keren Borensztajn  1 , Jurriën Stiekema, Sebastiaan Nijmeijer, Pieter H Reitsma, Maikel P Peppelenbosch, C Arnold Spek
Affiliations

Factor Xa stimulates proinflammatory and profibrotic responses in fibroblasts via protease-activated receptor-2 activation

Keren Borensztajn et al. Am J Pathol. 2008 Feb.

Abstract

Coagulation proteases have been suggested to play a role in the pathogenesis of tissue remodeling and fibrosis. We therefore assessed the proinflammatory and fibroproliferative effects of coagulation protease factor (F)Xa. We show that FXa elicits a signaling response in C2C12 and NIH3T3 fibroblasts. FXa-induced ERK1/2 phosphorylation was dependent on protease-activated receptor (PAR)-2 cleavage because desensitization with a PAR-2 agonist (trypsin) but not a PAR-1 agonist (thrombin) abolished FXa-induced signal transduction and PAR-2 siRNA abolished FXa-induced ERK1/2 phosphorylation. The PAR-2-dependent cellular effects of FXa led to fibroblast proliferation, migration, and differentiation into myofibroblasts, as demonstrated by the expression of alpha-smooth muscle actin and desmin, followed by the secretion of the cytokines monocyte chemotactic protein-1 and interleukin-6 as well as the expression of the fibrogenic proteins transforming growth factor-beta and fibronectin. To assess the relevance of FXa-induced proliferation and cell migration, we examined the effect of FXa in a wound scratch assay. Indeed, FXa facilitated wound healing in a PAR-2- and ERK1/2-dependent manner. Taken together, these results support the notion that, beyond its role in coagulation, FXa-dependent PAR-2 cleavage might play a role in the progression of tissue fibrosis and remodeling.

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Figures

Figure 1
Figure 1
FXa elicits specific phosphorylation of ERK1/2 in fibroblasts via PAR-2 activation. A: Western blot analysis on cell lysates of NIH3T3 (lane 1) and C2C12 (lane 2) cells showing that both cell lines constitutively express PAR-1 and PAR-2. Actin was used as a loading control. B: Serum-starved C2C12 cells (4 hours) treated with PBS (lane 1), thrombin (lane 2), or trypsin (lane 3) for 30 minutes. Western blot analysis of phospho-ERK1/2 indicates the functionality of both PARs. C: Effect of different concentrations of FXa on the phosphorylation of ERK1/2 in C2C12 cells. Cells stimulated for 30 minutes with PBS (lane 1) and FXa (lanes 2 to 5). D: Top: Western blot of C2C12 cells pretreated for 30 minutes with 200 nmol/L TAP (lane 2) or 100 nmol/L hirudin (lane 3) and stimulated for 30 minutes with PBS (lane 1) or 1 U/ml FXa (lanes 2 to 4) indicate that FXa-induced ERK1/2 phosphorylation is specific. Bottom: Densitometric analysis of three independent Western blots (mean ± SEM). E: FXa-induced ERK1/2 phosphorylation is not mediated by PAR-1. Western blots of C2C12 cells incubated for 30 minutes with PBS (lanes 1 to 3) or the anti-PAR-1 antibody ATAP2 (lanes 4 to 6), which were subsequently stimulated for 30 minutes with PBS (lane 1), 1 U/ml thrombin (lanes 2 and 5), or 1 U/ml FXa (lanes 3 and 6). F: FXa-induced ERK1/2 phosphorylation is mediated by PAR-2. Lanes 1 to 16: Western blots of fibroblasts exposed for 150 minutes (desensitization) to thrombin (lanes 1 to 8) or trypsin (lanes 9 to 16), subsequently stimulated with thrombin (lanes 1 to 4 and 13 to 16) or trypsin (lanes 5 to 12) for the indicated time points. Western blots of fibroblasts exposed for 150 minutes to serum-free control medium (lanes 17 to 20), thrombin (lanes 21 to 24), or trypsin (lanes 25 to 28) subsequently stimulated with FXa (1 U/ml) for the indicated time points. B–F: Total ERK1/2 is used as a loading control. G: PAR-2-specific siRNA causes loss of receptor-stimulated ERK1/2 phosphorylation. Left: Western blot of C2C12 cells transfected with control siRNA or PAR-2 siRNA. Right: Western blot of C2C12 cells transfected with control siRNA (lanes 1 to 4) or PAR-2 siRNA (lanes 5 to 8), stimulated with FXa for the indicated time points (minutes). Actin served as a loading control. Results presented are representative of three independent experiments.
Figure 2
Figure 2
FXa induces a profibrotic and proinflammatory phenotype in fibroblasts. A: FXa increases α-SMA expression in NIH3T3 fibroblasts. Lanes 1 and 3: Constitutive expression of α-SMA. Lanes 4 to 8: Cells treated with 1 U/ml FXa. Lanes 9 to 13: Cells treated for 24 hours with various concentrations of FXa. B: FXa increases desmin expression in NIH3T3 fibroblasts. Lanes 1 to 5: Cells treated with 1 U/ml FXa. Lanes 6 to 10: Cells treated for 24 hours with various concentrations of FXa. C: FXa stimulates TGF-β production in C2C12 cells. Lanes 1 to 5: Cells treated with 1 U/ml FXa. Lanes 6 to 10: Cells treated for 24 hours with various concentrations of FXa. D: FXa induces fibronectin production. Lanes 1 to 5: Cells treated with 1 U/ml FXa. Lanes 6 to 10: Cells treated for 24 hours with various concentrations of FXa. A–D: β-Actin served as loading control and results are representative of three independent experiments. E: FXa induces the secretion of MCP-1 and IL-6. IL-10, interferon-γ, TNF-α, IL-6, and MCP-1 expression in supernatants of C2C12 cells stimulated with FXa for 24 hours. Shown is the mean ± SEM (n = 4).
Figure 3
Figure 3
FXa induces NIH3T3 and C2C12 proliferation. A: Cell survival of NIH3T3 and C2C12 cells seeded in medium containing 1% FCS treated with FXa or PBS. B: Proliferation of C2C12 cells stimulated for 24 hours with different FXa concentrations. C: Proliferation of C2C12 cells pretreated with 200 nmol/L TAP or 10 μmol/L U0126 for 2 hours, then stimulated for 24 hours with 1 U/ml FXa. D: Proliferation of C2C12 cells transfected with control or PAR-2 siRNA and incubated for 24 hours with 1 U/ml FXa. Results are shown as mean ± SEM of two independent experiments performed in octuplicate. **P < 0.01; ***P < 0.001.
Figure 4
Figure 4
Effect of FXa signaling on migration and cytoskeletal reorganization of NIH3T3 and C2C12 cells. A: Migration of NIH3T3 and C2C12 cells toward DMEM (control) or DMEM supplemented with FXa. Results represent the mean ± SEM (n = 3). B: Migration of C2C12 cells in the presence of FXa. Results are shown as mean ± SEM and are representative of two independent experiments performed in duplicate. C: FXa (1 U/ml) induced phosphorylation of FAK and Src. β-Actin serves as loading control. The blots are representative of two independent experiments. D: Inhibition of Src abolishes FXa-induced stimulation of cell migration, as demonstrated by pretreatment of cells with PP1. Results represent the number of cells migrated as percentage of the control and are shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. E: Fluorescent microscopy of FXa stimulated C2C12 cells (for 15 minutes) showing that FXa induces phosphorylation of tyrosine residues. Stress fibers are indicated by arrows and the spikes by asterisks. F: Effect of FXa (15-minute incubation) on stress fiber assembly and tyrosine-phosphorylation in the absence or presence of PP1. Red, actin; blue, nucleus; green, phosphotyrosine. Experiments were repeated twice.
Figure 5
Figure 5
FXa facilitates wound healing in a wound scratch assay. A: Wound size of C2C12 fibroblast monolayers after treatment with either PBS (control), FXa (1 U/ml), FCS (10%), FXa/TAP (200 nmol/L), or FXa/PP1 (1 μmol/L) for 18 hours. Shown are photographs of representative microscopic fields. B: Actin staining of fibroblasts treated with PBS (control), FXa (1 U/ml), or FCS (10%) after wound induction. C: Detail of the effect of FXa, in the presence or in the absence of TAP or PP1, on stress fiber formation. D: Quantification of the results depicted in A as described in the Materials and Methods section. E: Involvement of ERK1/2 in FXa-mediated wound healing. Quantification of wound size of fibroblast monolayers after treatment with either PBS (control), FXa (1 U/ml), or FXa/U0126 for 18 hours. F: Wound size of C2C12 cells transfected with control or PAR-2 siRNA, after treatment with either PBS (control) or FXa (1 U/ml) for 18 hours. Results are shown as mean ± SEM of two independent experiments performed in octuplicate. **P < 0.01; ***P < 0.001.
Figure 6
Figure 6
Proposed role of FXa in wound healing and fibrosis. After activation of the coagulation cascade (1), FXa induces fibrosis via thrombin generation (2) or via PAR-2-dependent signaling (3). Signaling leads to fibroblast proliferation (4) and differentiation into myofibroblasts (5) followed by the secretion of MCP-1, IL-6, TGF-β, and fibronectin (6). Ultimately this leads to the accumulation of ECM thereby disrupting the tight balance between production and degradation of ECM, leading to wound healing and fibrosis.
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