title: Regulation of Transforming Growth Factor β-Activated Kinase Activation by Epigallocatechin-3-Gallate in Rheumatoid Arthritis Synovial Fibroblasts: Suppression of K(63) -Linked Autoubiquitination of Tumor Necrosis Factor Receptor-Associated Factor 6.
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 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.
  
  
  CONCLUSIONS
  Our findings provide a rationale for targeting TAK1 for the treatment of RA with EGCG.
authors.affiliation: Washington State University College of Pharmacy, Spokane.
authors.forename: Anil K
authors.initials: AK
authors.lastname: Singh
authors.affiliation: Washington State University College of Pharmacy, Spokane.
authors.forename: Sadiq
authors.initials: S
authors.lastname: Umar
authors.affiliation: University of Toledo College of Pharmacy and Pharmaceutical Sciences, Toledo, Ohio.
authors.forename: Sharayah
authors.initials: S
authors.lastname: Riegsecker
authors.affiliation: National Institute of Pharmaceutical Education and Research, Hajipur, India.
authors.forename: Mukesh
authors.initials: M
authors.lastname: Chourasia
authors.affiliation: Washington State University College of Pharmacy, Spokane.
authors.forename: Salahuddin
authors.initials: S
authors.lastname: Ahmed
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D001169:Arthritis, Experimental / Q000378:metabolism*; D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D015153:Blotting, Western; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D004195:Disease Models, Animal; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D053592:Interleukin-1 Receptor-Associated Kinases / Q000187:drug effects / Q000378:metabolism; D053583:Interleukin-1beta / Q000494:pharmacology*; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000187:drug effects / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000187:drug effects* / Q000378:metabolism; D062105:Molecular Docking Simulation; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D011917:Rats, Inbred Lew; D013583:Synovial Membrane / Q000166:cytology; D048029:TNF Receptor-Associated Factor 6 / Q000187:drug effects* / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects*; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D000893:Anti-Inflammatory Agents; D002392:Catechin; D053592:Interleukin-1 Receptor-Associated Kinases; D053583:Interleukin-1beta; D015850:Interleukin-6; D016209:Interleukin-8; D008239:Lysine; D020930:MAP Kinase Kinase Kinases; D046988:Proteasome Endopeptidase Complex; D048029:TNF Receptor-Associated Factor 6; D000099151:MAP Kinase Kinase Kinase 7; C045651:epigallocatechin gallate; C507768:IRAK1 protein, human