title: Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Central Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, 95 Wen Chang Road, Taipei, Taiwan.
authors.forename: Ju-Fang
authors.initials: JF
authors.lastname: Liu
authors.forename: Sheng-Mou
authors.initials: SM
authors.lastname: Hou
authors.forename: Chun-Hao
authors.initials: CH
authors.lastname: Tsai
authors.forename: Chun-Yin
authors.initials: CY
authors.lastname: Huang
authors.forename: Wei-Hung
authors.initials: WH
authors.lastname: Yang
authors.forename: Chih-Hsin
authors.initials: CH
authors.lastname: Tang
mesh_terms: D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology* / Q000502:physiology; D051547:Heme Oxygenase-1 / Q000502:physiology*; D006801:Humans; D044463:Receptor, PAR-1 / Q000502:physiology*; D044462:Receptors, Proteinase-Activated / Q000502:physiology; D018179:Receptors, Thrombin / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D013582:Synovial Fluid / Q000201:enzymology* / Q000502:physiology; D013917:Thrombin / Q000502:physiology*
chemical_list: D044463:Receptor, PAR-1; D044462:Receptors, Proteinase-Activated; D018179:Receptors, Thrombin; C105295:protease-activated receptor 3; D051547:Heme Oxygenase-1; D013917:Thrombin