Background/Purpose: In rheumatoid arthritis (RA), the role of interleukin-1β (IL-1β) signaling proteins (IRAK-1/TAK-1/TRAF-6) proximal to IL-1 receptor in mediating proinflammatory response is not completely understood. Using IL-1β-induced IL-6 and IL-8 production in RA synovial fibroblasts (RA-FLS), we examined the role of key signaling molecules critical in mediating its inflammatory response. We also tested if EGCG, a potent anti-inflammatory compound, inhibits IL-1β signaling protein to suppress IL-6 synthesis in RA-FLS. 

Methods:  RA-FLS were treated with IL-1β for different time alone or in the presence of EGCG. Western blotting analysis was utilized to study activation/phosphorylation of different IL-1β signaling proteins. In vitro kinase activity of IRAK-1 was determined using ADAPTA kinase assay. Changes in the ubiquitination patterns of IL-1β-stimulated RA-FLS was studied using immunoprecipitation (IP) assay. Using chemical inhibitors or siRNA for IRAK-1, TAK-1 or TRAF-6, their respective roles were examined in IL-1β-induced IL-6 and IL-8 expression by ELISA and qRT-PCR methods. Therapeutic effect of EGCG on these signaling events was evaluated. 

Results:  IL-1β-induced IL-6 and IL-8 production in RA-FLS was significantly inhibited by EGCG (2.5-20 μM) in a dose-dependent manner (p<0.01; n=5). IL-1β induced rapid degradation of IRAK-1 within 1-5 min followed by the activation of TAK-1 phosphorylation in RA-FLS. Using the chemical inhibitors for TAK-1, IRAK-1, and TRAF-6, our novel findings showed a complete blockade of IL-1β-induced IL-6 and IL-8 production by TAK-1, but only a modest inhibition with IRAK-1 or TRAF-6 inhibitors (p<0.01 for TAK-1; n=4). Confirmatory studies using siRNA method also showed a marked inhibition of IL-6 production by TAK-1 or IRAK-1 siRNA. Although EGCG inhibited in vitro IRAK-1 kinase activity by almost 65%, it did not prevent the IL-1β-induced proteasomal degradation of IRAK-1 in RA-FLS. To our surprise, EGCG selectively inhibited IL-1β-induced TAK-1 phosphorylation in a dose-dependent manner (p<0.05; n=4). Interestingly, we observed that the levels of TRAF-6 remained unchanged upon IL-1β stimulation. IP of RA-FLS cell lysates with global FK2 ubiquitin antibody and further Western blotting analysis showed that IL-1β activated TRAF-6 ubiquitination was not modulated by EGCG, suggesting TAK-1 as a potential therapeutic target in IL-1β signaling.  

Conclusion: Our study provides a novel evidence of an important mediatory role of TAK-1 in IL-1β signaling in RA-FLS and warrants further testing of EGCG or it synthetic analogs as TAK-1 inhibitors for the treatment of RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/epigallocatechin-3-gallate-egcg-suppresses-il-1%ce%b2-induced-il-6-and-il-8-synthesis-by-selectively-inhibiting-tak1-activation-in-human-rheumatoid-arthritis-synovial-fibroblasts/