| Summary: | 碩士 === 長庚大學 === 天然藥物研究所 === 97 === Rheumatoid arthritis (RA) is a complex systemic disease. Several factors have been implicated to trigger the mechanisms for the pathogenesis of RA. Although these factors concerning the increased inflammatory responses are well known, but the intracellular signaling pathways implicated in RA are not completely recognized. COX-2 is implicated in the pathogenesis of rheumatoid arthritis (RA) and has previously been shown to be induced by TNF-alpha. However, the molecular mechanisms underlying TNF-alpha-induced cyclooxygenase-2 (COX-2) expression in human synoviocytes (HS) which contributes to inflammatory responses in RA were remain unclear. Here, we reported that TNF-alpha-induced COX-2 expression in human synoviocytes (HS), via TNF receptor-activated MAPKs, transactivation of Src, EGFR, PDGFR, PI3K/Akt, and protein kinase C (PKC), transcription factor NF-kappaB and AP-1 signaling pathways. Transfection with siRNA of p42, p38, JNK2, Src, EGFR, AKT and p65 abolished the COX-2 expression induced by TNF-alpha. Accordingly, TNF-alpha-induced activation of COX-2 promoter was also attenuated by pretreatment with inhibitors of MAPKs (U0126, SB202190, and SP600125), transactivation (PP1, AG1478, AG1296 and LY294002), PKCs (GF109203, Ro318220, Gö6976 and Rottlerin), NF-kappaB (Bay117082) and AP-1 (Tanshinone). Consistently, TNF-alpha-stimulated phosphorylation of p42/p44 MAPK, p38 MAPK, JNK, Src, EGFR and Akt was also reduced by pretreatment with U0126, SB202190, SP600125, PP1, AG1478 and LY294002, respectively. Furthermore, TNF-alpha-stimulated NF-kB translocation into nucleus in HS determined by immunofluorescence staining. Previous study shows that COX-2 promoter has an AP-1 binding region. We reported that TNF-alpha-induced mRNA of c-Jun and c-Fos (subunits of AP-1) expression in HS. In addition, TNF-alpha-induced phosphorylation of c-Jun was attenuated by the inhibitors of MAPKs, transactivation and PKCs. MAPKs, transactivation and PKCs pathways regulated AP-1 promoter activities were revealed by promoter assays. Moreover, TNF-alpha-induced PGE2 generation on HS via COX-2 expression was decreased by pretreatment with inhibitors of MAPKs, transactivation and PKCs, Bay11-7082, GR343 and Tanshinone. These results suggest that TNF-alpha induces COX-2 expression in HS via TNF receptor, Src, EGFR, PDGFR, PI3K/Akt, PKCs, MAPKs, transcription factor p65 and AP-1 pathways. Particularly, TNF-alpha-induced COX-2 expression via NF-kB pathway and was not mediated through MAPKs, RTKs and PKCs. These results will provide a new insight into the mechanisms of TNF-alpha in RA. Increased understanding of signal transduction mechanisms underlying COX-2 gene regulation will create opportunities for the development of anti-inflammation therapeutic strategies.
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