Horswill Mark A, Narayan Malathi, Warejcka Debra J, Cirillo Lisa A, Twining Sally S
Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Exp Eye Res. 2008 Apr;86(4):586-600. doi: 10.1016/j.exer.2008.01.003. Epub 2008 Jan 12.
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-beta1 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 P1 cells cultured in the presence of serum-free defined medium, FGF-2 and TGF-beta1 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.
Maspin是一种42 kDa的非经典丝氨酸蛋白酶抑制剂（serpin），可控制细胞迁移和侵袭，主要由上皮来源的细胞表达，但在角膜基质角膜细胞中也有表达。在含有胎牛血清（FBS）的条件下培养基质角膜细胞时，到传代两次时maspin下调至几乎检测不到的水平。DNA甲基化是在细胞分化、发育、基因印记和致癌过程中控制基因表达的几种过程之一，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平滑肌成纤维细胞中maspin合成下调的机制。将人供体角膜基质细胞立即置于无血清限定培养基中，或在FBS存在下培养，然后传代至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，或传代至含有TGF-β1的培养基中以诱导平滑肌成纤维细胞表型。这些细胞类型存在于受伤的角膜中。这些细胞用于制备RNA进行半定量或定量RT-PCR，或提取蛋白质进行Western分析。此外，用DNA去甲基化剂5-氮杂-2'-脱氧胞苷（5-Aza-dC）和组蛋白脱乙酰酶抑制剂曲古抑菌素A（TSA）处理P4 FBS培养的成纤维细胞。收获处理和未处理的细胞，使用亚硫酸氢盐测序法检测DNA甲基化。使用染色质免疫沉淀（ChIP）测定法确定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，但在表型分化后，maspin mRNA和蛋白质显著下调。亚硫酸氢盐测序显示，新鲜分离的基质角膜细胞中的maspin启动子是低甲基化的，而在无血清限定培养基、FGF-2和TGF-β1存在下培养的P0基质细胞和P1细胞是高甲基化的。maspin合成的下调也与赖氨酸残基9处的组蛋白H3二甲基化有关。5-Aza-dC处理可使maspin mRNA和蛋白质低水平重新表达，但TSA处理则不能。在5-Aza-dC处理的细胞中添加TSA不会增加maspin的表达。5-Aza-dC处理不会显著改变maspin启动子的去甲基化，但会使组蛋白H3去甲基化。这些结果表明，在角膜基质细胞中，maspin启动子高甲基化和组蛋白甲基化与maspin合成下调同时发生，提示角膜细胞转化为伤口愈合成纤维细胞时基因的调控可能涉及启动子和组蛋白甲基化。
