Search

Peptidoglycan (PGN), the major component of the cell wall of Gram-positive bacteria, activates the innate immune system of the host and induces the release of cytokines and chemokines. We investigated the signaling pathway involved in IL-6 production stimulated by PGN in rheumatoid arthritis synovial fibroblasts. PGN caused concentration- and time-dependent increases in IL-6 production. PGN-mediated IL-6 production was attenuated by TLR2 small interfering RNA and nucleotide-binding oligomerization domain 2 small interfering RNA. Pretreatment with PI3K inhibitor (Ly294002 and wortmannin), Akt inhibitor, and AP-1 inhibitor (tanshinone IIA) also inhibited the potentiating action of PGN. PGN increased the focal adhesion kinase (FAK), PI3K, and Akt phosphorylation. Stimulation of rheumatoid arthritis synovial fibroblast cells with PGN increased the accumulation of phosphorylated c-Jun in the nucleus, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the IL-6 promoter. PGN mediated an increase in the accumulation of phosphorylated c-Jun in the nucleus, AP-1-luciferase activity, and c-Jun binding to AP-1 element was inhibited by Ly294002, Akt inhibitor, and FAK mutant. Our results suggest that PGN increased IL-6 production in human synovial fibroblasts via the TLR2 receptor/FAK/PI3K/Akt and AP-1 signaling 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.