Thrombin-induced IL-6 production in human synovial fibroblasts is mediated by PAR1, phospholipase C, protein kinase Cα, c-Src, NF-kappaB 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 PAR I 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-kappa B inhibitor (PDTC), I kappa B protease inhibitor (TPCK), or NF-kappa B inhibitor peptide. Stimulation of synovial fibroblasts with thrombin activated I kappa B kinase alpha/beta (IKK alpha/beta), I kappa B alpha phosphorylation, IKB alpha degradation, p65 phosphorylation at Ser 276, p65 and p50 translocation from the cytosol to the nucleus, and kappa B-luciferase activity. Thrombi n-mediated an increase of IKK alpha/beta activity, kappa B-luciferase activity and p65 and p50 binding to the NF-kappa B element was inhibited by PPACK, U73122, Ro320432 and PP2. The binding of p65 and p50 to the NF-kappa B 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/Pl-PLC/PKCa/c-Src/NF-KB and p300 signaling pathway. (c) 2007 Elsevier Ltd. All rights reserved.