Regulation of Transforming Growth Factor ß-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.

OBJECTIVE: Transforming growth factor ß-activated kinase 1 (TAK1) is a key MAPKKK family protein in interleukin-1ß (IL-1ß), 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-1ß-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-1ß-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. CONCLUSION: Our findings provide a rationale for targeting TAK1 for the treatment of RA with EGCG.

Role of ADAM-17, p38 MAPK, cathepsins, and the proteasome pathway in the synthesis and shedding of fractalkine/CX3 CL1 in rheumatoid arthritis.

OBJECTIVE: To evaluate the mechanism of fractalkine (FKN)/CX3 CL1 synthesis and shedding in rheumatoid arthritis synovial fibroblasts (RASFs) and in rat adjuvant-induced arthritis (AIA). METHODS: The effect of tumor necrosis factor α (TNFα) and/or interferon-γ (IFNγ) on FKN synthesis and shedding in human RASFs was determined over time by immunostaining, quantitative reverse transcription-polymerase chain reaction, and Western blotting. The role of protease enzymes and signaling pathways was evaluated using chemical inhibitors and small interfering RNA (siRNA). The activity of 20S proteasome in the lysates and the DNA binding of NF-κB/p65 in the nuclear fractions were evaluated. The in vivo relevance of these findings was examined in rat AIA. RESULTS: In RASFs, stimulation with the combination of TNFα and IFNγ induced cellular expression of FKN within 24 hours. Activation of ADAM-17, but not ADAM-10, partly mediated the proteolytic shedding and release of soluble FKN (sFKN) following TNFα/IFNγ stimulation for 24-72 hours. Compared with control siRNA, ADAM-17 siRNA markedly inhibited TNFα/IFNγ-induced sFKN production (by ∼33%). TNFα/IFNγ-induced sFKN release was markedly suppressed by inhibitors of ADAM-17, p38 MAPK, proteasome, or cathepsin inhibitor but not by inhibitors of caspase 3 or calpain. TNFα/IFNγ-induced proteasome activity also correlated with rapid degradation of IκBα and p38 MAPK phosphorylation. In vivo findings showed increased FKN expression in the joints of rats with AIA, which correlated with increased expression of ADAM-17 and phospho-p38 MAPK. CONCLUSION: Our results provide new understanding of the role of ADAM-17, p38 MAPK, cathepsins, and the proteasome pathway in FKN expression and shedding. Regulating these pathways may suppress FKN-mediated inflammation and tissue destruction.

Potential benefits of green tea polyphenol EGCG in the prevention and treatment of vascular inflammation in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease of the joints in which systemic overproduction of pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) may accelerate cardiovascular (CV) complications. Synovial inflammation in RA spreads systemically and transforms silently into chronic inflammation manifested by increased cytokine release and abnormally high levels of acute reactive proteins (ARPs) such as C-reactive protein (CRP), suggesting inflammation as a connecting link between RA and CV dysfunction. While the treatment to improve CV function in RA patients is being validated, it is timely to propose and test two-pronged therapies that ameliorate arthritis concomitant to improving CV functions. In this review, we summarized the pre-clinical and clinical studies validating the cardiovascular and anti-rheumatic activities of epigallocatechin-3 gallate (EGCG), a potent anti-inflammatory molecule found in green tea. The review also draws many parallels that have emerged between the paradigm of cytokine-driven inflammation in the pathogenesis of RA and its CV complications. Finally, based on extensive clinical evidence of the 'synovial inflammation-systemic inflammation' link and the benefits of EGCG in regulating these two pathologies via common driving factors, authors put forward an argument that EGCG may be tested for its potential CV benefit along with anti-rheumatic activity in animal models of human RA.

Largazole, a class I histone deacetylase inhibitor, enhances TNF-α-induced ICAM-1 and VCAM-1 expression in rheumatoid arthritis synovial fibroblasts.

In the present study, we evaluated the effect of largazole (LAR), a marine-derived class I HDAC inhibitor, on tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinase-2 (MMP-2) activity. LAR (1-5 µM) had no adverse effect on the viability of RA synovial fibroblasts. Among the different class I HDACs screened, LAR (0.5-5 µM) inhibited the constitutive expression of HDAC1 (0-30%). Surprisingly, LAR increased class II HDAC [HDAC6] by ~220% with a concomitant decrease in HDAC5 [30-58%] expression in RA synovial fibroblasts. SAHA (5 µM), a pan-HDAC inhibitor, also induced HDAC6 expression in RA synovial fibroblasts. Pretreatment of RA synovial fibroblasts with LAR further enhanced TNF-α-induced ICAM-1 and VCAM-1 expression. However, LAR inhibited TNF-α-induced MMP-2 activity in RA synovial fibroblasts by 35% when compared to the TNF-α-treated group. Further, the addition of HDAC6 specific inhibitor Tubastatin A with LAR suppressed TNF-α+LAR-induced ICAM-1 and VCAM-1 expression and completely blocked MMP-2 activity, suggesting a role of HDAC6 in LAR-induced ICAM-1 and VCAM-1 expression. LAR also enhanced TNF-α-induced phospho-p38 and phospho-AKT expression, but inhibited the expression of phospho-JNK and nuclear translocation of NF-κBp65 in RA synovial fibroblasts. These results suggest that LAR activates p38 and Akt pathways and influences class II HDACs, in particular HDAC6, to enhance some of the detrimental effects of TNF-α in RA synovial fibroblasts. Understanding the exact role of different HDAC isoenzymes in RA pathogenesis is extremely important in order to develop highly effective HDAC inhibitors for the treatment of RA.