Overexpression of the Growth Arrest and DNA Damage-Induced 45α Gene Contributes to Autoimmunity by Promoting DNA Demethylation in Lupus T Cells
Overexpression of the Growth Arrest and DNA Damage-Induced 45α Gene Contributes to Autoimmunity by Promoting DNA Demethylation in Lupus T Cells
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DOI：
10.1002/art.27363
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发表时间：
2010-05-01
影响因子：
--
通讯作者：
Lu, Qianjin
中科院分区：
文献类型：
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作者：
Li, Yaping;Zhao, Ming;Lu, Qianjin
Objective. CD 4 + T细胞中CD 11 a和CD 70调节区的去甲基化有助于自身反应性的发展和自身抗体的过度刺激。由于生长停滞和DNA损伤诱导的45 alpha（GADD 45 alpha）通过去除甲基化标记减少了基因的表观遗传沉默，因此本研究探讨了gadd 45 A基因是否可以通过促进系统性红斑狼疮患者T细胞中的DNA去甲基化来促进自身免疫（SLE）。方法。通过实时逆转录-聚合酶链反应和Western印迹或流式细胞术检测GADD 45 α、CD 11 a和CD 70信使RNA（mRNA）和蛋白水平。使用Methylamp全局DNA甲基化定量试剂盒评价全局DNA甲基化。使用市售试剂盒检测CD 4 + T细胞增殖和自体B细胞IgG抗体。用亚硫酸氢盐测序法测定CD 11 a和CD 70启动子甲基化。SLE患者CD 4 + T细胞中gadd 45 A mRNA表达升高和DNA低甲基化。gadd 45 A mRNA水平与DNA甲基化水平成反比。gadd 45 A与CD 11 a/CD 70 mRNA表达呈正相关。紫外线B照射后CD 4 + T细胞gadd 45 A mRNA表达增加，同时伴随着CD 11 a和CD 70 mRNA水平的增加。此外，gadd 45 A，CD 11 a和CD 70 mRNA的表达增加伴随着增加的自身反应性和过度的B细胞刺激gadd 45 A转染的CD 4 + T细胞。CD 11 a启动子甲基化在转染细胞中也显著降低。转染gadd 45 A小干扰RNA可抑制SLE CD 4 + T细胞的自身反应性，并导致CD 11 a和CD 70启动子区甲基化水平显著升高。这些结果表明，gadd 45 A可能有助于狼疮样自身免疫，促进DNA去甲基化在SLE CD 4 + T细胞。
Objective. Demethylation of CD11a and CD70 regulatory regions in CD4+ T cells contributes to the development of autoreactivity and overstimulation of autoantibodies. Because growth arrest and DNA damage-induced 45 alpha (GADD45 alpha) reduces epigenetic silencing of genes by removing methylation marks, this study examined whether the gadd45A gene could contribute to autoimmunity by promoting DNA demethylation in T cells from patients with systemic lupus erythematosus (SLE).Methods. Levels of GADD45 alpha, CD11a, and CD70 messenger RNA (mRNA) and protein were detected by real-time reverse transcription-polymerase chain reaction and Western blotting or flow cytometry. Global DNA methylation was evaluated using Methylamp global DNA methylation quantification kits. Detection of CD4+ T cell proliferation and autologous B cell IgG antibodies was performed using commercially available kits. CD11a and CD70 promoter methylation was determined with bisulfite sequencing.Results. Elevated gadd45A mRNA expression and global DNA hypomethylation were observed in CD4+ T cells from SLE patients. The levels of gadd45A mRNA were inversely proportional to the levels of DNA methylation. Positive correlations were found between gadd45A and CD11a/CD70 mRNA levels. Expression of gadd45A mRNA was increased in CD4+ T cells following ultraviolet B irradiation, and this was accompanied by increased levels of CD11a and CD70 mRNA. Moreover, increased expression of gadd45A, CD11a, and CD70 mRNA was accompanied by increased autoreactivity and excessive B cell stimulation in gadd45A-transfected CD4+ T cells. CD11a promoter methylation was also significantly reduced in transfected cells. Transfection of gadd45A small interfering RNA inhibited the autoreactivity of SLE CD4+ T cells and led to significant increases in the methylation levels of the CD11a and CD70 promoter regions.Conclusion. These findings indicate that gadd45A may contribute to lupus-like autoimmunity by promoting DNA demethylation in SLE CD4+ T cells.