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

Abstract

Background/Purpose Transforming growth factor β activated kinase 1 (TAK1) is a key MAPKKK family protein in interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), or toll-like receptor signaling. We examined the posttranslational modification of TAK1 and its therapeutic regulation in rheumatoid arthritis (RA). Methods The effect of TAK1, IRAK-1, or TRAF6 inhibition was evaluated in IL-1β-induced human RA synovial fibroblasts (RA-FLS). Western blotting, immunoprecipitation, and 20S proteasome assay were used to study the ubiquitination process in RA-FLS. The efficacy of epigallocatechin-3-gallate (EGCG), a potent anti-inflammatory molecule, in regulating these processes in RA-FLS was evaluated. Molecular docking was performed to examine the interaction of EGCG with human TAK1, IRAK-1, and TRAF6. These findings were validated using a rat adjuvant-induced arthritis (AIA) model. Results Inhibition of TAK1, not IRAK-1 or TRAF6, completely abrogates IL-1β-induced IL-6 and IL-8 synthesis in RA-FLS. EGCG inhibits TAK1 phosphorylation at Thr184/187 and occupies the C174 position, an ATP-binding site, to inhibit its kinase activity. EGCG pretreatment also inhibits K63-autoubiquitination of TRAF6, a post-translational modification essential for TAK1 autophosphorylation, by forming stable hydrogen-bond at the K124 position on TRAF6. Furthermore, EGCG enhances associated deubiquitinase expression to rescue proteins from proteasomal degradation. Western blot analyses on the joint homogenates from rat AIA show a significant increase in K48-linked polyubiquitination, TAK1 phosphorylation, and TRAF6 expression when compared to the naïve group. Administration of EGCG (50 mg/kg/day) for 10 days ameliorates AIA by reducing TAK1 phosphorylation and K48 polyubiquitination. Conclusions This study provides rationale for targeting TAK1 for RA treatment by EGCG.

Critical role of IL‐1α in IL‐1β‐induced inflammatory responses: cooperation with NF‐κBp65 in transcriptional regulation

“…Our initial observations with PDTC suggested that NF-kB is involved in IL-1a expression, which is already known in fibroblast biology (33). We further confirmed this by inhibiting upstream activation of TAK1, a known activator of NF-kBp65 via IL-1b canonical signaling (14). In addition to NF-kBp65, IL-1a has been reported to bind chromatin in mouse macrophages and to associate with p300, both of which were observed in our study (34,35).…”

“…These 2 complexes were subjected to the 100 ns molecular dynamics (MD) simulation. The methodology of MD has been adopted from our previous studies (14). The frames in the trajectory were recorded at 20 ps intervals to study the conformational variations on IL-1a and IL-1b ligand binding.…”

“…6A). Because recent studies have shown that IRAK phosphorylation is a rapid event occurring within minutes of IL-1R activation (14,20), looking at the phosphorylation status at early time points might give us some insights into the differences in the kinetics of IL-1a and IL-1b binding to IL-1R. RASFs stimulated for 5, 15, and 30 min with IL-1a or IL-1b showed marked differences in their capabilities for inducing key IL-1 signaling proteins.…”

MicroRNA-17 Suppresses TNF-α Signaling by Interfering with TRAF2 and cIAP2 Association in Rheumatoid Arthritis Synovial Fibroblasts

“…The inhibition of p-c-Jun and NF-κB transcription factors also caused a significant reduction in TNF-α-induced MMP-1 and MMP-13 production in RASFs. We have recently shown that a similar reduction in TRAF6 and TAK1 association by epigallocatechin-3gallate (EGCG) inhibited IL-1β signaling and downstream mediators of inflammation in RASFs (47). It is worth noting that both MMP-1 and MMP-13 have no 3’UTR binding regions for miR-17, which further suggest this to be an indirect regulatory effect on tissue remodeling mediated by RASFs Our results showed that miR-17 targets ASK1 3’UTR to regulate ASK1 expression, which may result in the reduction of p38 and JNK phosphorylation in RASFs.…”

Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1‐mediated Noxa expression and proteasomal degradation of Mcl‐1

“…Some of these proteins, including TRAIL, TGF-b, and PI3K/Akt, have been identified to play role in the pharmacological effects of UA (40,41). Important relevance to RA is the fact that inhibition of these pathways has been shown to reduce inflammation and tissue remodeling in RA (1,19,42). Administration of UA has been shown to reduce significantly the symptoms of arthritis in preclinical models (43,44).…”