Background/Purpose:  Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that causes progressive joint destruction. In spite of the modern medications, including biologic reagents, it is still hard to cure RA completely. 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 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 important regulators in gene transcription. Trimethylation of lysine 4 at histone H3 (H3K4me3) is an active histone marker whereas trimethylation of lysine 27 at histone H3 (H3K27me3) is a repressive histone marker. We have hypothesized that epigenetic dysregulation might induce RASF activation. The purpose of this study is to clarify whether histone modifications are associated with MMPs gene activation in RASFs and whether IL-6 regulates MMPs expression in RASFs.

Methods:  We compared MMPs gene expression by quantitative RT-PCR and histone lysine 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 osteoarthritis (OA) SFs as a control. Chromatin structures in the MMP promoters were evaluated by 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. 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, phosphorylation of STAT3 by immunoblotting, and binding of STAT3 to the MMP promoters after IL-6 stimulation by ChIP assay in RASFs and OASFs.

Results:  MMP-1, 3, 9 and 13 genes were actively transcribed in RASFs. The profiles of histone lysine methylation (H3K4me3 and H3K27me3) and the result of MNase assay indicated that chromatin structures were open in the MMP-1, 3, 9 and 13 promoters in RASFs. The depletion of WDR5 reduced the levels of H3K4me3 as well as the MMP-1, 3, 9 and 13 gene expression. Interestingly, IL-6 and sIL-6Rα significantly increased the expression of MMP-1, 3 and 13, but not MMP-9, in RASFs. Although the expression levels of gp130 as well as IL-6Rα were comparable and STAT3 was similarly phosphorylated after IL-6 stimulation in RASFs and OASFs, STAT3 bound to the MMP-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 MMP-1, 3 and 13 gene activation after IL-6 stimulation in RASFs.

Conclusion:  Altered profiles of histone lysine methylation and binding of STAT3 to the promoters differentially regulate constitutive and IL-6-induced MMPs gene activation in RASFs and possibly arthritogenic properties of RASFs.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/histone-lysine-methylation-and-stat3-differentially-regulate-constitutive-and-il-6-induced-mmps-gene-activation-in-rheumatoid-arthritis-synovial-fibroblasts/