生长停滞与DNA损伤诱导基因45α的过表达通过促进狼疮T细胞中的DNA去甲基化而导致自身免疫。
Overexpression of the growth arrest and DNA damage-induced 45alpha gene contributes to autoimmunity by promoting DNA demethylation in lupus T cells.
作者信息
Li Yaping, Zhao Ming, Yin Heng, Gao Fei, Wu Xiaoyan, Luo Yongqi, Zhao Sha, Zhang Xiujuan, Su Yuwen, Hu Nan, Long Hai, Richardson Bruce, Lu Qianjin
机构信息
Second Xiangya Hospital and Central South University, Changsha, China.
出版信息
Arthritis Rheum. 2010 May;62(5):1438-47. doi: 10.1002/art.27363.
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 45alpha (GADD45alpha) 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 GADD45alpha, 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.
目的
CD4⁺ T细胞中CD11a和CD70调控区域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损伤诱导基因45α（GADD45α）通过去除甲基化标记减少基因的表观遗传沉默，本研究探讨gadd45A基因是否可通过促进系统性红斑狼疮（SLE）患者T细胞中的DNA去甲基化而导致自身免疫。
方法
通过实时逆转录聚合酶链反应、蛋白质印迹法或流式细胞术检测GADD45α、CD11a和CD70信使核糖核酸（mRNA）及蛋白质水平。使用甲基化酶全球DNA甲基化定量试剂盒评估整体DNA甲基化。使用市售试剂盒检测CD4⁺ 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CD11a和CD70启动子甲基化。
结果
在SLE患者的CD4⁺ T细胞中观察到gadd45A mRNA表达升高和整体DNA低甲基化。gadd45A mRNA水平与DNA甲基化水平呈负相关。gadd45A与CD11a/CD70 mRNA水平之间存在正相关。紫外线B照射后，CD4⁺ T细胞中gadd45A mRNA表达增加，同时CD11a和CD70 mRNA水平升高。此外，gadd45A、CD11a和CD70 mRNA表达增加伴随着gadd45A转染的CD4⁺ T细胞中自身反应性增加和B细胞过度刺激。转染细胞中CD11a启动子甲基化也显著降低。转染gadd45A小干扰RNA可抑制SLE CD4⁺ T细胞的自身反应性，并导致CD11a和CD70启动子区域甲基化水平显著升高。
结论
这些发现表明，gadd45A可能通过促进SLE CD4⁺ T细胞中的DNA去甲基化而导致狼疮样自身免疫。
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