Center of Experimental Rheumatology and Zurich Center of Integrative Human Physiology (ZIHP), University Hospital Zurich, Zurich, Switzerland.
Genes Immun. 2011 Dec;12(8):643-52. doi: 10.1038/gene.2011.45. Epub 2011 Jul 14.
In the search for specific genes regulated by DNA methylation in rheumatoid arthritis (RA), we investigated the expression of CXCL12 in synovial fibroblasts (SFs) and the methylation status of its promoter and determined its contribution to the expression of matrix metalloproteinases (MMPs). DNA was isolated from SFs and methylation was analyzed by bisulfite sequencing and McrBC assay. CXCL12 protein was quantified by enzyme-linked immunosorbent assay before and after treatment with 5-azacytidine. RASFs were transfected with CXCR7-siRNA and stimulated with CXCL12. Expression of MMPs was analyzed by real-time PCR. Basal expression of CXCL12 was higher in RASFs than osteoarthritis (OA) SFs. 5-azacytidine demethylation increased the expression of CXCL12 and reduced the methylation of CpG nucleotides. A lower percentage of CpG methylation was found in the CXCL12 promoter of RASFs compared with OASFs. Overall, we observed a significant correlation in the mRNA expression and the CXCL12 promoter DNA methylation. Stimulation of RASFs with CXCL12 increased the expression of MMPs. CXCR7 but not CXCR4 was expressed and functional in SFs. We show here that RASFs produce more CXCL12 than OASFs due to promoter methylation changes and that stimulation with CXCL12 activates MMPs via CXCR7 in SFs. Thereby we describe an endogenously activated pathway in RASFs, which promotes joint destruction.
在寻找类风湿关节炎 (RA) 中受 DNA 甲基化调控的特定基因时，我们研究了趋化因子 CXCL12 在滑膜成纤维细胞 (SF) 中的表达及其启动子的甲基化状态，并确定其对基质金属蛋白酶 (MMPs) 表达的贡献。从 SF 中分离 DNA，通过亚硫酸氢盐测序和 McrBC 分析检测甲基化。用 5-氮杂胞苷处理前后，通过酶联免疫吸附试验定量 CXCL12 蛋白。用 CXCR7-siRNA 转染 RASF 并用 CXCL12 刺激。通过实时 PCR 分析 MMPs 的表达。RASF 的 CXCL12 基础表达高于骨关节炎 (OA) SF。5-氮杂胞苷去甲基化增加 CXCL12 的表达并减少 CpG 核苷酸的甲基化。与 OASF 相比，RASF 中 CXCL12 启动子的 CpG 甲基化百分比较低。总的来说，我们观察到 mRNA 表达和 CXCL12 启动子 DNA 甲基化之间存在显著相关性。用 CXCL12 刺激 RASF 增加了 MMPs 的表达。CXCR7 而不是 CXCR4 在 SF 中表达并发挥功能。我们在这里表明，由于启动子甲基化变化，RASF 产生的 CXCL12 多于 OASF，并且 CXCL12 刺激通过 SF 中的 CXCR7 激活 MMPs。由此我们描述了 RASF 中一种内源性激活途径，它促进关节破坏。
