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

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.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.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.Osteoarthritis (OA) is a degenerative disease characterized by a slow progressive degeneration of articular cartilage, variable secondary synovial inflammation, and osteophyte formation [1,2]. The exact etiology of OA is not well understood. In response to macrophage-derived proinflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor-α (TNF)-α, OA synovial fibroblasts (OASFs) produce chemokines that promote inflammation, neovascularization, and cartilage degradation through activation of matrix-degrading enzymes such as matrix metalloproteinases (MMPs) [3].Heme oxygenase (HO)-1 is the key enzyme responsible for the degradation of heme to carbon monoxide, fr