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

Anil K. Singh, Sadiq Umar, Sharayah Riegsecker, Mukesh Chourasia, Salahuddin Ahmed

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25 Scopus citations

Abstract

Objective 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). 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-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). Results 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. Conclusion Our findings provide a rationale for targeting TAK1 for the treatment of RA with EGCG.

Original languageEnglish (US)
Pages (from-to)347-358
Number of pages12
JournalArthritis and Rheumatology
Volume68
Issue number2
DOIs
StatePublished - Feb 1 2016

Bibliographical note

Funding Information:
Supported by the NIH (grant AR-063104 to Dr. Ahmed), the Arthritis Foundation (Innovative Research Grant to Dr. Ahmed), and Washington State University startup funding. The authors thank the National Disease Research Interchange and the Cooperative Human Tissue Network for providing RA synovial tissue, Dr. David A. Fox (University of Michigan Medical School) for critical review of the manuscript and helpful suggestions, and Ms Maria Beamer for technical support in animal studies.

Publisher Copyright:
© 2015, American College of Rheumatology.

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