Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways

Abstract

Introduction: Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, and proinflammatory processes. Abnormalities in these processes are primary features of osteoarthritis (OA). Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury. Here, we investigated the intracellular signaling pathways involved in thrombin-induced HO-1 expression in human synovial fibroblasts (SFs).

Methods: Thrombin-mediated HO-1 expression was assessed with quantitative real-time (q)PCR. The mechanisms of action of thrombin in different signaling pathways were studied by using Western blotting. Knockdown of protease-activated receptor (PAR) proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of Nrf2 to the HO-1 promoter. Transient transfection was used to examine HO-1 activity.

Results: Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of thrombin, and expression was higher than in normal SFs. OASFs stimulation with thrombin induced concentration- and time-dependent increases in HO-1 expression. Pharmacologic inhibitors or activators and genetic inhibition by siRNA of protease-activated receptors (PARs) revealed that the PAR1 and PAR3 receptors, but not the PAR4 receptor, are involved in thrombin-mediated upregulation of HO-1. Thrombin-mediated HO-1 expression was attenuated by thrombin inhibitor (PPACK), PKCδ inhibitor (rottlerin), or c-Src inhibitor (PP2). Stimulation of cells with thrombin increased PKCδ, c-Src, and Nrf2 activation.

Conclusion: Our results suggest that the interaction between thrombin and PAR1/PAR3 increases HO-1 expression in human synovial fibroblasts through the PKCδ, c-Src, and Nrf2 signaling pathways.

Figure 1

Thrombin induces HO-1 expression in human synovial fibroblasts. (A) Total protein was extracted from normal synovial fibroblasts (lines 1 and 2) and osteoarthritis synovial fibroblast (OASF) cells (lines 3 to 6), and subjected to Western blot analysis for thrombin. (B; left panel) Human synovial fibroblasts were cultured for 48 hours, and media were collected to measure thrombin. (B; right panel) Synovial fluid was obtained from normal (n = 10) or osteoarthritis patients (n = 15) and examined with ELISA for the expression of thrombin. OASFs (six OA lines) were incubated with various concentrations of thrombin for 24 hours (C, E) or with thrombin (3 U/ml) for 6, 12, or 24 hours (D, F). The HO-1 expression was examined with Western blotting and qPCR. Results are expressed as the mean ± SEM. *P < 0.05 as compared with basal level. #P < 0.05 as compared with the thrombin-treated group.

Figure 2

Involvement of PAR1 and PAR3 receptor in thrombin-induced HO-1 expression. (A) Osteoarthritis synovial fibroblasts (OASFs) (six OA lines) were treated with thrombin (3 U/ml), thrombin plus PPACK (30 nM), SFLLRN-NH₂ (100 μM), TFRGAP-NH₂ (100 μM), and GYPGQV-NH₂ (100 μM) for 24 hours, and HO-1 expression was examined with qPCR. (B) Normal synovial fibroblasts (seven normal lines) were treated with thrombin (3 U/ml), SFLLRN-NH₂ (100 μM), and TFRGAP-NH₂ (100 μM) for 24 hours, and HO-1 expression was examined with qPCR. (C) Cells were transfected with PAR1, PAR3, PAR4, or control siRNA for 24 hours, and PAR receptor level was examined with Western blotting. (D, E) Cells (five OA lines) were transfected with PAR1, PAR3, PAR4, or control siRNA for 24 hours followed by stimulation with thrombin for 24 hours, and HO-1 expression was examined with Western blotting and qPCR. Results are expressed as the mean ± SEM. *P < 0.05 as compared with basal level. #P < 0.05 as compared with thrombin-treated group.

Figure 3

PKCδ is involved in thrombin-induced HO-1 expression in synovial fibroblasts. (A, C) Osteoarthritis synovial fibroblasts (OASFs) (six OA lines) were pretreated for 30 minutes with GF109203X (3 μM), rottlerin (10 μM), and Ro320432 (10 μM) followed by stimulation with thrombin for 24 hours, and HO-1 expression was examined with Western blotting and qPCR. (B) Cells (seven OA lines) were transfected for 24 hours with PKCδ siRNA followed by stimulation with thrombin for 24 hours, and HO-1 expression was examined with qPCR. (C) Cells (six OA lines) were incubated with thrombin for indicated time intervals, and PKCδ phosphorylation was examined with Western blotting. (D, E) Cells (seven OA lines) were incubated with thrombin for 30 minutes or pretreated for 30 minutes with PPACK or transfected 24 hours with PAR1 and PAR3 siRNA, followed by stimulation with thrombin for 30 minutes, and PKCδ kinase activity was determined with the PKCδ kinase kit. Results are expressed as the mean ± SEM. *P < 0.05 as compared with basal level. #P < 0.05 as compared with thrombin-treated group.

Figure 4

c-Src is involved in thrombin-induced HO-1 expression in synovial fibroblasts. (A, B) Osteoarthritis synovial fibroblasts (OASFs) (six OA lines) were pretreated for 30 minutes with PP2 (3 μM) or transfected with c-Src mutant for 24 hours followed by stimulation with thrombin for 24 hours, and the HO-1 expression was examined with qPCR and Western blotting. (C) OASFs (five OA lines) were incubated with thrombin for indicated time intervals, and c-Src phosphorylation was examined with Western blotting. (D) Cells (six OA lines) were pretreated 30 minutes with PPACK or rottlerin, followed by stimulation with thrombin for 30 minutes, and c-Src phosphorylation was examined with Western blotting. Results are expressed as the mean ± SEM. *P < 0.05 as compared with basal level. #P < 0.05 as compared with thrombin-treated group.

Figure 5

Thrombin induced Nrf2 activation in synovial fibroblasts. (A) Osteoarthritis synovial fibroblasts (OASFs; six OA lines) were transfected with Nrf2 siRNA for 24 hours, and HO-1 expression was examined with qPCR. (B) OASFs (six OA lines) were incubated with thrombin for indicated time intervals, and Nrf2 phosphorylation was examined with Western blotting. (C) Cells (five OA lines) were pretreated 30 minutes with PPACK, rottlerin, or PP2 followed by stimulation with thrombin for 30 minutes, and Nrf2 phosphorylation was examined with Western blotting. Results are expressed as the mean ± SEM. *P < 0.05 as compared with basal level. #P < 0.05 as compared with thrombin-treated group.

Figure 6

PAR, PKCδ, and c-Src are involved in thrombin-induced Nrf2 activation. (A) Osteoarthritis synovial fibroblast (OASFs; seven OA lines) were pretreated with GF109203X, rottlerin, and PP2 for 30 minutes followed by stimulation with thrombin for 120 minutes, and ChIP assay was then performed. Chromatin was immunoprecipitated with anti-Nrf2 antibody. One percent of the precipitated chromatin was assayed to verify equal loading (Input). OASFs (eight OA lines) were incubated with thrombin for 24 hours (B) or pretreated with PPACK, GF109203X, rottlerin, and PP2 for 30 minutes (C) or co-transfected with PAR1 siRNA, PAR3 siRNA, PAR4 siRNA, PKCδ siRNA, and c-Src mutant for 24 hours (D) before incubation with thrombin for 24 hours. HO-1 luciferase activity was then assayed. Results are expressed as the mean ± SEM. *P < 0.05 as compared with basal level. #P < 0.05 as compared with thrombin-treated group. (E) Schematic diagram of the signaling pathways involved in thrombin-induced HO-1 expression in OASFs. Thrombin increases HO-1 expression by binding to the PAR1/PAR3 receptor and activating PKCδ and c-Src, which enhances binding of Nrf2 to the ARE site. This results in the transactivation of HO-1 expression.