Karouzakis Emmanuel, Gay Renate E, Michel Beat A, Gay Steffen, Neidhart Michel
Zurich Center of Integrative Human Physiology, University Hospital, Zurich, Switzerland.
Arthritis Rheum. 2009 Dec;60(12):3613-22. doi: 10.1002/art.25018.
Rheumatoid arthritis synovial fibroblasts (RASFs) are phenotypically activated and aggressive. We undertook this study to investigate whether the intrinsic activation of RASFs is due to global genomic hypomethylation, an epigenetic modification.
Global genomic hypomethylation was assessed by immunohistochemistry, flow cytometry, and L1 promoter bisulfite sequencing. The levels of Dnmt1 were determined in synovial tissue and cultured SFs by Western blotting before and after treatment with cytokines and growth factors. Normal SFs were treated for 3 months with a nontoxic dose of the DNA hypomethylation drug 5-azacytidine (5-azaC), and changes in gene expression were revealed using complementary DNA arrays. The phenotypic changes were confirmed by flow cytometry.
In situ and in vitro, RASF DNA had fewer 5-methylcytosine and methylated CG sites upstream of an L1 open-reading frame than did DNA of osteoarthritis SFs, and proliferating RASFs were deficient in Dnmt1. Using 5-azaC, we reproduced the activated phenotype of RASFs in normal SFs. One hundred eighty-six genes were up-regulated>2-fold by hypomethylation, with enhanced protein expression. These included growth factors and receptors, extracellular matrix proteins, adhesion molecules, and matrix-degrading enzymes. The hypomethylating milieu induced irreversible phenotypic changes in normal SFs, which resembled those of the activated phenotype of RASFs.
DNA hypomethylation contributes to the chronicity of RA and could be responsible for the limitation of current therapies.
类风湿性关节炎滑膜成纤维细胞（RASF）在表型上具有活化性且侵袭性强。我们开展本研究以调查RASF的内在活化是否归因于整体基因组低甲基化，这是一种表观遗传修饰。
通过免疫组织化学、流式细胞术和L1启动子亚硫酸氢盐测序评估整体基因组低甲基化。在用细胞因子和生长因子处理之前和之后，通过蛋白质印迹法测定滑膜组织和培养的滑膜成纤维细胞中Dnmt1的水平。用无毒剂量的DNA低甲基化药物5-氮杂胞苷（5-azaC）处理正常滑膜成纤维细胞3个月，使用互补DNA阵列揭示基因表达的变化。通过流式细胞术确认表型变化。
在原位和体外，与骨关节炎滑膜成纤维细胞的DNA相比，RASF DNA在L1开放阅读框上游的5-甲基胞嘧啶和甲基化CG位点较少，并且增殖的RASF缺乏Dnmt1。使用5-azaC，我们在正常滑膜成纤维细胞中重现了RASF的活化表型。186个基因通过低甲基化上调>2倍，蛋白质表达增强。这些包括生长因子和受体、细胞外基质蛋白、黏附分子和基质降解酶。低甲基化环境在正常滑膜成纤维细胞中诱导了不可逆的表型变化，类似于RASF活化表型的变化。
DNA低甲基化导致类风湿性关节炎的慢性化，并可能是当前治疗受限的原因。
