Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts
Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts
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DOI：
10.1016/j.exer.2008.01.003
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发表时间：
2008-04-01
影响因子：
3.4
通讯作者：
Twining, Sally S.
中科院分区：
文献类型：
--
作者：
Horswill, Mark A.;Narayan, Malathi;Twining, Sally S.
Maspin是一种控制细胞迁移和侵袭的42 kDa非经典丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂），主要由上皮来源的细胞表达，但也在角膜基质角膜细胞中表达。在FBS存在下培养基质角膜细胞时，maspin在第二代下调至几乎检测不到的水平。DNA甲基化是在细胞分化、发育、遗传印记和致癌过程中控制基因表达的几个过程之一，但尚未在角膜基质细胞中进行研究。本研究的目的是确定maspin启动子的DNA甲基化和组蛋白H3二甲基化是否参与人角膜基质成纤维细胞和肌成纤维细胞中maspin合成下调的机制。立即将人供体角膜基质细胞置于无血清确定成分培养基中或在FBS存在下培养，并转入无血清培养基或含有FBS或FGF-2的培养基中以诱导成纤维细胞表型或用于肌成纤维细胞表型的TGF-β 1。这些细胞类型在受伤的角膜中发现。细胞用于制备用于半定量或定量RT-PCR的RNA或提取用于Western分析的蛋白质。此外，用DNA去甲基化剂5-氮杂-2 '-脱氧胞苷（5-Aza-dC）和组蛋白去乙酰化酶抑制剂曲马斯他丁A（TSA）处理P4 FBS培养的成纤维细胞。收获经处理和未经处理的细胞，并使用亚硫酸氢钠测序测定DNA甲基化。使用ChIP测定法测定P4成纤维细胞中与maspin基因相关的组蛋白H3的甲基化状态。新鲜收获的角膜基质细胞表达maspin，但表型分化后，maspin mRNA和蛋白质显着下调。亚硫酸氢钠测序显示，新鲜分离的基质角膜细胞中的maspin启动子被低甲基化，而在无血清成分培养基、FGF-2和TGF-β 1存在下培养的P0基质细胞和PI细胞均被高甲基化。maspin合成的下调也与组蛋白H3在赖氨酸9处的二甲基化有关。用5-Aza-dC而不是TSA处理，maspin mRNA和蛋白质都以低水平重新表达。向5-Aza-dC处理的细胞中添加TSA不增加maspin表达。用5-Aza-dC处理没有显著改变maspin启动子的去甲基化，但确实使组蛋白H3去甲基化。这些结果表明，maspin启动子高甲基化和组蛋白甲基化与角膜基质细胞中maspin合成的下调一起发生，并表明角膜细胞转化为伤口愈合成纤维细胞后的基因调节可能涉及启动子和组蛋白甲基化。(C)2008爱思唯尔有限公司保留所有权利。
Maspin, a 42 kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, maspin is down-regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the maspin promoter and histone H3 dimethylation is involved in the mechanism of down-regulation of maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-beta 1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for Western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the PI cells cultured in the presence of serum-free defined medium, FGF-2 and TGF-beta 1 were hypermethylated. Down-regulation of maspin synthesis was also associated with histone H3 dimethylation at lysine 9. Both maspin mRNA and protein were re-expressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3. These results show maspin promoter hypermethylation and histone methylation occur with down-regulation of maspin synthesis in corneal stromal cells and suggest regulation of genes upon conversion of keratocytes to wound healing fibroblasts can involve promoter and histone methylation. (C) 2008 Elsevier Ltd. All rights reserved.