Thrombin-induced IL-6 production in human synovial fibroblasts is mediated by PAR1, phospholipase C, protein kinase C alpha, c-Src, NF-kappa B and p300 pathway.
Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis and proinflammatory processes. Abnormalities in these processes are primary features of rheumatoid arthritis (RA) in synovial tissues. We investigated the signaling pathway involved in IL-6 production caused by thrombin in synovial fibroblasts. Thrombin caused concentration- and time-dependent increases in IL-6 production. By using pharmacological inhibitors or activators or genetic inhibition by the protease activated receptor (PAR), siRNA revealed that the PAR1 receptor but not other PAR receptors is involved in thrombin-mediated up-regulation of IL-6. Thrombin-mediated IL-6 production was attenuated by thrombin inhibitor (PPACK), phospholipase C inhibitor (U73122), protein kinase C alpha inhibitor (Ro320432), Src inhibitor (PP2), NF-kappaB inhibitor (PDTC), I kappa B protease inhibitor (TPCK), or NF-kappaB inhibitor peptide. Stimulation of synovial fibroblasts with thrombin activated I kappa B kinase alpha/beta (IKK alpha/beta), I kappa B alpha phosphorylation, I kappa B alpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Thrombin-mediated an increase of IKK alpha/beta activity, kappaB-luciferase activity and p65 and p50 binding to the NF-kappaB element was inhibited by PPACK, U73122, Ro320432 and PP2. The binding of p65 and p50 to the NF-kappaB elements, as well as the recruitment of p300 and the enhancement of p50 acetylation on the IL-6 promoter was enhanced by thrombin. Our results suggest that thrombin increased IL-6 production in synovial fibroblasts via the PAR1 receptor/PI-PLC/PKC alpha/c-Src/NF-kappaB and p300 signaling pathway.
Department of Orthopaedics, Taichung Veterans General Hospital, Taichung, Taiwan; Department of Pharmacology, College of Medicine, China Medical University, Taichung, Taiwan.
Yung-Cheng
YC
Chiu
Yi-Chin
YC
Fong
Chih-Ho
CH
Lai
Chien-Hui
CH
Hung
Horng-Chaung
HC
Hsu
Tu-Sheng
TS
Lee
Rong-Sen
RS
Yang
Wen-Mei
WM
Fu
Chih-Hsin
CH
Tang
D002478:Cells, Cultured; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000378:metabolism; D015398:Signal Transduction; D013583:Synovial Membrane / Q000166:cytology; D013917:Thrombin / Q000502:physiology*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015854:Up-Regulation; D050880:p300-CBP Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors
D015850:Interleukin-6; D016328:NF-kappa B; D044463:Receptor, PAR-1; D050880:p300-CBP Transcription Factors; D016392:Proto-Oncogene Proteins pp60(c-src); D051571:Protein Kinase C-alpha; D010738:Type C Phospholipases; D013917:Thrombin; D010975:Platelet Aggregation Inhibitors