甲基化 CpG 结合蛋白 2 在硬皮病成纤维细胞中发挥抗纤维化作用。
Methyl-CpG-binding protein 2 mediates antifibrotic effects in scleroderma fibroblasts.
机构信息
Division of Rheumatology, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA.
Department of Dermatology, The Second Xiangya Hospital, Central South University, Changsha, China.
出版信息
Ann Rheum Dis. 2018 Aug;77(8):1208-1218. doi: 10.1136/annrheumdis-2018-213022. Epub 2018 May 14.
OBJECTIVE
Emerging evidence supports a role for epigenetic regulation in the pathogenesis of scleroderma (SSc). We aimed to assess the role of methyl-CpG-binding protein 2 (MeCP2), a key epigenetic regulator, in fibroblast activation and fibrosis in SSc.
METHODS
Dermal fibroblasts were isolated from patients with diffuse cutaneous SSc (dcSSc) and from healthy controls. MeCP2 expression was measured by qPCR and western blot. Myofibroblast differentiation was evaluated by gel contraction assay in vitro. Fibroblast proliferation was analysed by ki67 immunofluorescence staining. A wound healing assay in vitro was used to determine fibroblast migration rates. RNA-seq was performed with and without MeCP2 knockdown in dcSSc to identify MeCP2-regulated genes. The expression of MeCP2 and its targets were modulated by siRNA or plasmid. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) using anti-MeCP2 antibody was performed to assess MeCP2 binding sites within MeCP2-regulated genes.
RESULTS
Elevated expression of MeCP2 was detected in dcSSc fibroblasts compared with normal fibroblasts. Overexpressing MeCP2 in normal fibroblasts suppressed myofibroblast differentiation, fibroblast proliferation and fibroblast migration. RNA-seq in MeCP2-deficient dcSSc fibroblasts identified MeCP2-regulated genes involved in fibrosis, including , and . Plasminogen activator urokinase (PLAU) overexpression in dcSSc fibroblasts reduced myofibroblast differentiation and fibroblast migration, while nidogen-2 (NID2) knockdown promoted myofibroblast differentiation and fibroblast migration. Adenosine deaminase (ADA) depletion in dcSSc fibroblasts inhibited cell migration rates. Taken together, antifibrotic effects of MeCP2 were mediated, at least partly, through modulating PLAU, NID2 and ADA. ChIP-seq further showed that MeCP2 directly binds regulatory sequences in and gene loci.
CONCLUSIONS
This study demonstrates a novel role for MeCP2 in skin fibrosis and identifies MeCP2-regulated genes associated with fibroblast migration, myofibroblast differentiation and extracellular matrix degradation, which can be potentially targeted for therapy in SSc.
目的
越来越多的证据表明表观遗传调控在硬皮病（SSc）发病机制中起作用。我们旨在评估甲基-CpG 结合蛋白 2（MeCP2）作为关键的表观遗传调节剂在 SSc 中纤维母细胞激活和纤维化中的作用。
方法
从弥漫性皮肤硬皮病（dcSSc）患者和健康对照中分离真皮成纤维细胞。通过 qPCR 和 Western blot 测量 MeCP2 表达。通过体外凝胶收缩试验评估肌成纤维细胞分化。通过 ki67 免疫荧光染色分析纤维母细胞增殖。体外划痕愈合试验用于测定纤维母细胞迁移率。用和不用 MeCP2 敲低在 dcSSc 中进行 RNA 测序，以鉴定 MeCP2 调控的基因。用 siRNA 或质粒调节 MeCP2 和其靶基因的表达。用抗 MeCP2 抗体进行染色质免疫沉淀测序（ChIP-seq），以评估 MeCP2 调控基因内的 MeCP2 结合位点。
结果
与正常成纤维细胞相比，dcSSc 成纤维细胞中 MeCP2 的表达升高。在正常成纤维细胞中过表达 MeCP2 抑制肌成纤维细胞分化、纤维母细胞增殖和纤维母细胞迁移。MeCP2 缺陷型 dcSSc 成纤维细胞中的 RNA-seq 鉴定出参与纤维化的 MeCP2 调控基因，包括、和。dcSSc 成纤维细胞中尿激酶型纤溶酶原激活物（PLAU）过表达减少肌成纤维细胞分化和纤维母细胞迁移，而层粘连蛋白 2 受体（NID2）敲低促进肌成纤维细胞分化和纤维母细胞迁移。dcSSc 成纤维细胞中腺苷脱氨酶（ADA）耗竭抑制细胞迁移率。总之，MeCP2 的抗纤维化作用至少部分是通过调节 PLAU、NID2 和 ADA 来介导的。ChIP-seq 进一步显示 MeCP2 直接结合和基因座的调节序列。
结论
这项研究表明 MeCP2 在皮肤纤维化中具有新的作用，并确定了与纤维母细胞迁移、肌成纤维细胞分化和细胞外基质降解相关的 MeCP2 调控基因，这些基因可能成为 SSc 治疗的潜在靶点。
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