Background/Purpose:

TAK1 is one of the most important proteins in IL-1β signaling cascade that mediates downstream p38/JNK and NF-κB activation. In the present study, we evaluated the effect of EGCG, a potent anti-inflammatory compound, on TAK1 activation and its downstream consequent effects in human rheumatoid arthritis synovial fibroblasts (RA-FLS).

Methods: Human RA-FLS were treated with IL-1β for 30 minutes alone or in presence of EGCG. Cytoplasmic, nuclear, or whole cell extracts were prepared for Western blotting analysis to study the activation and cellular distribution of IL-1β signaling proteins proximal to IL-1 receptor as well as different phosphorylated forms of TAK1. Immunofluorescence was performed to study TAK1 nuclear translocation. EGCG and IL-1β-stimulated TAK1 interacting protein partners were identified using immunoprecipitation assay. In silico docking studies were performed using EGCG as a ligand and three-dimensional TAK1 structure was generated using ligand and protein preparation wizard of Schrodinger suit 2014.3. The ligand (EGCG) was docked using GLIDE module.

Results: Molecular docking studies on TAK1 structure in silico shows that EGCG occupies the binding pocket at C174 position, an ATP-binding site, which causes a reversible yet stable inhibition in TAK1 phosphorylation at Thr184/187site and kinase activity. Western blotting analysis showed that EGCG facilitates nuclear translocation of TAK1 in a dose dependent manner. Furthermore, we also observed overall reduction in cytoplasmic levels of TAK1 in response to EGCG treatment, which was further confirmed by immunofluorescence staining. Interestingly, TAK1 phosphorylation at Thr184/187 is not at all observed in the nuclear compartment while TAK1 phosphorylated at Ser439 mainly translocate to the nucleus in response to EGCG treatment. Ingenuity Pathway analysis predicted STAT3, ATM, SMAD7, and p300 (histone acetyltransferase) as the potential nuclear partners of TAK1. Further validation studies using immunoprecipitation assays in human RA-FLS confirmed the strongest interaction between TAK1 and p300 in addition to the other identified important nuclear proteins in RA-FLS. In parallel to these findings, EGCG inhibited histone H3K56 acetylation in a dose-dependent manner in RA-FLS. Additionally, these results confirmed that the interaction with p300 was significantly higher in EGCG and IL-1β treated RA-FLS, suggesting regulatory role of TAK1 in chromatin modifications.

Conclusion:

Our study provides a novel mechanism through which EGCG facilitates nuclear translocation of TAK1 and regulates transcriptional machinery involved in RA pathogenesis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/epigallocatechin-3-gallate-egcg-facilitates-tak1-nuclear-translocation-and-its-interaction-with-p300-to-inhibit-histone-acetylation-in-human-ra-synovial-fibroblasts/