Background Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that causes progressive joint destruction. A line of evidence suggests that synovial fibroblasts (SFs) play an important role in the pathogenesis of RA. RASFs produce matrix metalloproteinases (MMPs) that degrade articular cartilage. Although interleukin-6 (IL-6) is an inflammatory cytokine and is proved to be involved in the pathogenesis of RA, it is unknown whether IL-6 affects MMPs gene transcription in RASFs. Furthermore, recent advances have revealed that epigenetic mechanisms, including histone modifications, are considered to be important regulators in gene transcription. We hypothesized that epigenetic dysregulation might induce RASF activation.
Objectives The aim of this study is to examine whether IL-6 regulates MMPs expression in RASFs and whether histone modifications are associated with the MMPs gene activation in RASFs.
Methods We compared MMPs gene expression by quantitative RT-PCR and histone methylation in the MMP promoters by chromatin immunoprecipitation (ChIP) assay after stimulation with IL-6 and/or soluble IL-6 receptor α (sIL-6Rα) in RASFs and control, osteoarthritis (OA) SFs. Chromatin structures in the MMP promoters were evaluated with micrococcal nuclease (MNase) assay in RASFs and OASFs. We investigated the change in the MMPs gene expression after silencing of WDR5 that is required for generating H3K4me3, an active histone marker. IL-6 signal induces Signal Transducer and Activator of Transcription 3 (STAT3) activation. To elucidate the mechanisms of IL-6-induced MMPs gene activation in RASFs, we investigated cell surface expression of the IL-6 receptor (gp130 and membrane-bound IL-6Rα) by flow cytometry, phospho-STAT3 (p-STAT3) expression by immunoblotting, and binding of STAT3 to the MMPs 1, 3, 9, and 13 promoters after IL-6 stimulation by ChIP assay in RASFs and OASFs.
Results MMPs 1, 3, 9 and 13 genes were actively transcribed in RASFs. The histone methylation profiles (H3K4me3 and H3K27me3) and the result of MNase assay indicated that chromatin structures were open in the MMPs 1, 3, 9 and 13 promoters in RASFs. The depletion of WDR5 reduced the levels of H3K4me3 as well as the MMPs 1, 3, 9 and 13 gene expression. Interestingly, IL-6 and sIL-6Rα significantly increased the expression of MMPs 1, 3 and 13, but not MMP-9, in RASFs. Although the expression levels of gp130 as well as membrane-bound IL-6Rα were comparable and STAT3 was similarly phosphorylated after IL-6 stimulation in RASFs and OASFs, STAT3 bound to the MMPs 1, 3 and 13 promoters, but not the MMP-9 promoter, after stimulation with IL-6 and sIL-6Rα only in RASFs. It was suggested that binding of STAT3 to the promoters resulted in MMPs 1, 3 and 13 gene activation after IL-6 stimulation in RASFs.
Conclusions Our data indicate that altered profiles of histone methylation and binding of STAT3 to the promoters regulate constitutive and IL-6-induced MMPs gene activation in RASFs and possibly arthritogenic properties of RASFs.
Araki Y et al. Arthritis Rheum. Epub. 2015 Dec 29.
Disclosure of Interest None declared