RANTES/CCL5 participates in rheumatoid arthritis (RA) pathogenesis by facilitating leukocyte infiltration, however, its other pathological functions are not fully defined in RA. In the present study, we evaluated the effect of RANTES/CCL5 on tissue degrading enzymes matrix metalloproteinase-1 (MMP-1) and MMP-13 expression and its contribution to the progressive joint damage by RA synovial fibroblasts (RASFs). Our results showed that RANTES/CCL5 dose-dependently induced MMP-1 and MMP-13 expression in monolayers and three-dimentional (3D) micromass of human RASFs, which correlated with an increase in collagenase activity. This activation by RANTES/CCL5 was observed in RASF, but not in osteoarthritis SFs (OASFs). Evaluation of the signaling events showed that RANTES/CCL5 selectively activated PKCδ, JNK, and ERK proteins to induce MMP expression in human RASFs. Pretreatment with a functional antagonist (Met-RANTES) or heparinase III (an enzyme that selectively digests heparan sulfate proteoglycans, HSPGs) completely abrogated RANTES/CCL5-induced MMP-1 and MMP-13 expression. Interestingly, the inhibition of RANTES/CCL5 using small interfering RNA (siRNA) approach reduced the ability of IL-1β to induce MMP-1 and MMP-13 expression, asserting its mediatory role in tissue remodeling. In the inhibitor study, only the selective inhibition of HSPGs or PKCδ, ERK, and JNK markedly inhibited RANTES/CCL5-induced MMP-1 and MMP-13 production. Circular dichroism (CD) spectroscopy results demonstrated the degradation of collagen triple-helical structure upon exposure to the conditioned media from RANTES/CCL5 stimulated RASFs, which was reverted by a broad-spectrum MMP inhibitor (GM6001). These findings suggest that RANTES/CCL5 not only up-regulates MMP-1 and MMP-13 expression by utilizing HSPGs and/or PKCδ-JNK/ERK pathways, but also mediates IL-1β-induced MMP-1 and MMP-13 expression.

TNF-α is a major cytokine implicated in rheumatoid arthritis (RA), and its expression is regulated at the transcriptional and posttranscriptional levels. However, the impact of changes in microRNA expression on posttranslational processes involved in TNF-α signaling networks is not well defined in RA. In this study, we evaluated the effect of miR-17, a member of the miR-17–92 cluster, on the TNF-α signaling pathway in human RA synovial fibroblasts (SFs). We demonstrated that miR-17 expression was significantly low in RA serum, SFs, and synovial tissues, as well as in the serum and joints of adjuvant-induced arthritis rats. RNA-sequencing analysis showed modulation of 664 genes by pre–miR-17 in human RA SFs. Ingenuity pathway analysis of RNA-sequencing data identified the ubiquitin proteasome system in the TNF-α signaling pathway as a primary target of miR-17. Western blot analysis confirmed the reduction in TRAF2, cIAP1, cIAP2, USP2, and PSMD13 expression by miR-17 in TNF-α–stimulated RA SFs. Immunoprecipitation assays showed that miR-17 restoration increased the K48-linked polyubiquitination of TRAF2, cIAP1, and cIAP2 in TNF-α–stimulated RA SFs. Thus, destabilization of TRAF2 by miR-17 reduced the ability of TRAF2 to associate with cIAP2, resulting in the downregulation of TNF-α–induced NF-κBp65, c-Jun, and STAT3 nuclear translocation and the production of IL-6, IL-8, MMP-1, and MMP-13 in human RA SFs. In conclusion, this study provides evidence for the role of miR-17 as a negative regulator of TNF-α signaling by modulating the protein ubiquitin processes in RA SFs.

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 C¹⁷⁴ position, an ATP‐binding site, to inhibit its kinase activity. EGCG pretreatment also inhibited K⁶³‐linked autoubiquitination of TRAF6, a posttranslational modification essential for TAK1 autophosphorylation, by forming a stable H bond at the K¹²⁴ 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 K⁴⁸‐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 K⁴⁸‐linked polyubiquitination.Conclusion: Our findings provide a rationale for targeting TAK1 for the treatment of RA with EGCG.