Inhibition of DNA Methylation and Methyl-CpG-Binding Protein 2 Suppresses RPE Transdifferentiation: Relevance to Proliferative Vitreoretinopathy

• Published in: Investigative ophthalmology & visual science 2015-08, Vol.56 (9), p.5579-5589
• Main Authors: He, Shikun, Barron, Ernesto, Ishikawa, Keijiro, Nazari Khanamiri, Hossein, Spee, Chris, Zhou, Peng, Kase, Satoru, Wang, Zhuoshi, Dustin, Laurie Diane, Hinton, David R
• Format: Article
• Language: English
• Subjects: Azacitidine - analogs & derivatives; Azacitidine - pharmacology; Blotting, Western; Cell Movement; Cell Transdifferentiation; Cells, Cultured; DNA - genetics; DNA Methylation - drug effects; DNA Modification Methylases - antagonists & inhibitors; Enzyme Inhibitors - pharmacology; Epithelial-Mesenchymal Transition - drug effects; Gene Expression Regulation; Humans; Immunohistochemistry; Methyl-CpG-Binding Protein 2 - biosynthesis; Methyl-CpG-Binding Protein 2 - drug effects; Methyl-CpG-Binding Protein 2 - genetics; Phosphorylation; Real-Time Polymerase Chain Reaction; Retinal Cell Biology; Retinal Pigment Epithelium - drug effects; Retinal Pigment Epithelium - metabolism; Retinal Pigment Epithelium - pathology; Vitreoretinopathy, Proliferative - drug therapy; Vitreoretinopathy, Proliferative - genetics; Vitreoretinopathy, Proliferative - metabolism
• ISSN: 1552-5783; 0146-0404; 1552-5783
• DOI: 10.1167/iovs.14-16258

The purpose of this study was to evaluate expression of methyl-CpG-binding protein 2 (MeCP2) in epiretinal membranes from patients with proliferative vitreoretinopathy (PVR) and to investigate effects of inhibition of MeCP2 and DNA methylation on transforming growth factor (TGF)-β-induced retinal pigment epithelial (RPE) cell transdifferentiation. Expression of MeCP2 and its colocalization with cytokeratin and α-smooth muscle actin (α-SMA) in surgically excised PVR membranes was studied using immunohistochemistry. The effects of 5-AZA-2'-deoxycytidine (5-AZA-dC) on human RPE cell migration and viability were evaluated using a modified Boyden chamber assay and the colorimetric 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Expression of RASAL1 mRNA and its promoter region methylation were evaluated by real-time PCR and methylation-specific PCR. Effects of 5-AZA-dC on expression of α-SMA, fibronectin (FN), and TGF-β receptor 2 (TGF-β R2) and Smad2/3 phosphorylation were analyzed by Western blotting. Effect of short interfering RNA (siRNA) knock-down of MeCP2 on expression of α-SMA and FN induced by TGFβ was determined. MeCP2 was abundantly expressed in cells within PVR membranes where it was double labeled with cells positive for cytokeratin and α-SMA. 5-AZA-dC inhibited expression of MeCP2 and suppressed RASAL1 gene methylation while increasing expression of the RASAL1 gene. Treatment with 5-AZA-dC significantly suppressed the expression of α-SMA, FN, TGF-β R2 and phosphorylation of Smad2/3 and inhibited RPE cell migration. TGF-β induced expression of α-SMA, and FN was suppressed by knock-down of MeCP2. MeCP2 and DNA methylation regulate RPE transdifferentiation and may be involved in the pathogenesis of PVR.