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.