title: Inhibition of DNA Methylation and Methyl-CpG-Binding Protein 2 Suppresses RPE Transdifferentiation: Relevance to Proliferative Vitreoretinopathy.
abstract: OBJECTIVE
  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: Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States 2Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, Unit.
authors: Shikun
authors: S
authors: He
authors: Doheny Eye Institute, Los Angeles, California, United States.
authors: Ernesto
authors: E
authors: Barron
authors: Doheny Eye Institute, Los Angeles, California, United States.
authors: Keijiro
authors: K
authors: Ishikawa
authors: Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States.
authors: Hossein
authors: H
authors: Nazari Khanamiri
authors: Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States.
authors: Chris
authors: C
authors: Spee
authors: Doheny Eye Institute, Los Angeles, California, United States.
authors: Peng
authors: P
authors: Zhou
authors: Doheny Eye Institute, Los Angeles, California, United States.
authors: Satoru
authors: S
authors: Kase
authors: Doheny Eye Institute, Los Angeles, California, United States.
authors: Zhuoshi
authors: Z
authors: Wang
authors: Department of Preventive Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States.
authors: Laurie Diane
authors: LD
authors: Dustin
authors: Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States 2Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, Unit.
authors: David R
authors: DR
authors: Hinton
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015153:Blotting, Western; D002465:Cell Movement; D054338:Cell Transdifferentiation; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics*; D019175:DNA Methylation / Q000187:drug effects*; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D051783:Methyl-CpG-Binding Protein 2 / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*; D010766:Phosphorylation; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D018630:Vitreoretinopathy, Proliferative / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D051783:Methyl-CpG-Binding Protein 2; D000077209:Decitabine; D004247:DNA; D015254:DNA Modification Methylases; D001374:Azacitidine