Regulation of Transforming Growth Factor β–Activated Kinase Activation by Epigallocatechin-3-Gallate in Rheumatoid Arthritis Synovial Fibroblasts: Suppression of K63-Linked Autoubiquitination of Tumor Necrosis Factor Receptor–Associated Factor 6
Version of Record online: 25 JAN 2016
© 2015, American College of Rheumatology
Arthritis & Rheumatology
Volume 68, Issue 2, pages 347–358, February 2016
How to Cite
Singh, A. K., Umar, S., Riegsecker, S., Chourasia, M. and Ahmed, S. (2016), Regulation of Transforming Growth Factor β–Activated Kinase Activation by Epigallocatechin-3-Gallate in Rheumatoid Arthritis Synovial Fibroblasts: Suppression of K63-Linked Autoubiquitination of Tumor Necrosis Factor Receptor–Associated Factor 6. Arthritis & Rheumatology, 68: 347–358. doi: 10.1002/art.39447
- Issue online: 25 JAN 2016
- Version of Record online: 25 JAN 2016
- Accepted manuscript online: 16 OCT 2015 02:41PM EST
- Manuscript Accepted: 17 SEP 2015
- Manuscript Received: 6 MAR 2015
- NIH. Grant Number: AR-063104
- Arthritis Foundation
- Washington State University
Transforming growth factor β–activated kinase 1 (TAK1) is a key MAPKKK family protein in interleukin-1β (IL-1β), 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).
The effect of TAK1, IL-1 receptor–associated kinase 1 (IRAK-1), and TNF receptor–associated factor 6 (TRAF6) inhibition was evaluated in IL-1β–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).
Inhibition of TAK1, but not IRAK-1 or TRAF6, completely abrogated IL-1β–induced IL-6 and IL-8 synthesis in RASFs. EGCG inhibited TAK1 phosphorylation at Thr184/187 and occupied the C174 position, an ATP-binding site, to inhibit its kinase activity. EGCG pretreatment also inhibited K63-linked autoubiquitination of TRAF6, a posttranslational modification essential for TAK1 autophosphorylation, by forming a stable H bond at the K124 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 K48-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 K48-linked polyubiquitination.
Our findings provide a rationale for targeting TAK1 for the treatment of RA with EGCG.