Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways
- Equal contributors
1 Central Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, 95 Wen Chang Road, Taipei, Taiwan
2 Department of Orthopedic Surgery, Shin-Kong Wo Ho-Su Memorial Hospital, 95 Wen Chang Road, Taipei, Taiwan
3 Department of Orthopaedic Surgery, China Medical University Hospital, 91 Hsueh-Shih Road, Taichung, Taiwan
4 School of Medicine and Graduate Institute of Clinical Medical Science, China Medical University, 91 Hsueh-Shih Road, Taichung, Taiwan
5 Department of Orthopaedic Surgery, China Medical University Beigang Hospital, 123 Hsin Te Road, Yun-Lin County, Taiwan
6 Department of Orthopedic Surgery, Taichung Hospital, Department of Health, 1 San Min Road, Taichung, Taiwan
7 Graduate Institute of Biotechnology, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan
8 School of Chinese Medicine, China Medical University, 91 Hsueh-Shih Road, Taichung, Taiwan
9 Department of Pharmacology, School of Medicine, China Medical University, 91 Hsueh-Shih Road, Taichung, Taiwan
10 Graduate Institute of Basic Medical Science, China Medical University, 91 Hsueh-Shih Road, Taichung, Taiwan
Arthritis Research & Therapy 2012, 14:R91 doi:10.1186/ar3815Published: 27 April 2012
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).
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.