Regulation of Transforming Growth Factor β-Activated Kinase 1 Activation by Epigallocatechin-3-Gallate in Rheumatoid Arthritis Synovial Fibroblasts Suppression of K63-Linked Autoubiquitination of Tumor Necrosis Factor Receptor-Associated Factor 6

Objective. Transforming growth factor beta-activated kinase 1 (TAK1) is a key MAPKKK family protein in interleukin-1 beta(IL-1 beta), tumor necrosis factor (TNF), and Toll-like receptor signaling. This study was undertaken to examine the posttranslational modification of TAK1 and its therapeutic regulation in rheumatoid arthritis (RA). Methods. The effect of TAK1, IL-1 receptor-associated kinase 1 (IRAK-1), and TNF receptor-associated factor 6 (TRAF6) inhibition was evaluated in IL-1 beta-stimulated human RA synovial fibroblasts (RASFs). Western blotting, immunoprecipitation, and 20S proteasome assay were used to study the ubiquitination process in RASFs. The efficacy of epigallocatechin-3-gallate (EGCG), a potent antiinflammatory molecule, in regulating these processes in RASFs was evaluated. Molecular docking was performed to examine the interaction of EGCG with human TAK1, IRAK-1, and TRAF6. These findings were confirmed using a rat model of adjuvant-induced arthritis (AIA). Results. Inhibition of TAK1, but not IRAK-1 or TRAF6, completely abrogated IL-1 beta-induced IL-6 and IL-8 synthesis in RASFs. EGCG inhibited TAK1 phosphorylation at Thr(184/187) and occupied the C-174 position, an ATP-binding site, to inhibit its kinase activity. EGCG pretreatment also inhibited K-63-linked autoubiquitination of TRAF6, a posttranslational modification essential for TAK1 autophosphorylation, by forming a stable H bond at the K-124 position on TRAF6. Furthermore, EGCG enhanced proteasome-associated deubiquitinase expression to rescue proteins from proteasomal degradation. Western blot analyses of joint homogenates from rats with AIA showed a significant increase in K-48-linked polyubiquitination, TAK1 phosphorylation, and TRAF6 expression when compared to naive rats. Administration of EGCG (50 mg/kg/day) for 10 days ameliorated AIA in rats by reducing TAK1 phosphorylation and K-48-linked polyubiquitination. Conclusion. Our findings provide a rationale for targeting TAK1 for the treatment of RA with EGCG.