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

Abstract

PURPOSE:

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.

METHODS:

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.

RESULTS:

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.

CONCLUSIONS:

MeCP2 and DNA methylation regulate RPE transdifferentiation and may be involved in the pathogenesis of PVR.

• Authors: Shikun He, Ernesto Barron, Keijiro Ishikawa, Hossein Nazari Khanamiri, Chris Spee, Peng Zhou, Satoru Kase, Zhuoshi Wang, Laurie Diane Dustin, David R Hinton
• Journal: Investigative ophthalmology & visual science (Invest Ophthalmol Vis Sci) Vol. 56 Issue 9 Pg. 5579-89 (Aug 2015) ISSN: 1552-5783 [Electronic] United States
• PMID: 26305530 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Enzyme Inhibitors
• Methyl-CpG-Binding Protein 2
• Decitabine
• DNA
• DNA Modification Methylases
• Azacitidine

Topics

• Azacitidine (analogs & derivatives, pharmacology)
• Blotting, Western
• Cell Movement
• Cell Transdifferentiation
• Cells, Cultured
• DNA (genetics)
• DNA Methylation (drug effects)
• DNA Modification Methylases (antagonists & inhibitors)
• Decitabine
• Enzyme Inhibitors (pharmacology)
• Epithelial-Mesenchymal Transition (drug effects)
• Gene Expression Regulation
• Humans
• Immunohistochemistry
• Methyl-CpG-Binding Protein 2 (biosynthesis, drug effects, genetics)
• Phosphorylation
• Real-Time Polymerase Chain Reaction
• Retinal Pigment Epithelium (drug effects, metabolism, pathology)
• Vitreoretinopathy, Proliferative (drug therapy, genetics, metabolism)