Inhibition of DNA methylation and methyl-CpG-binding protein 2 suppresses RPE transdifferentiation: Relevance to proliferative vitreoretinopathy

Shikun He, Ernesto Barron, Keijiro Ishikawa, Hossein Nazari Khanamiri, Chris Spee, Peng Zhou, Satoru Kase, Zhuoshi Wang, Laurie Diane Dustin, David R. Hinton

Research output: Contribution to journalArticle

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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 a-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 methylationspecific 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.

Original languageEnglish
Pages (from-to)5579-5589
Number of pages11
JournalInvestigative Ophthalmology and Visual Science
Volume56
Issue number9
DOIs
Publication statusPublished - Jan 1 2015
Externally publishedYes

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Methyl-CpG-Binding Protein 2
Proliferative Vitreoretinopathy
Retinal Pigments
DNA Methylation
Transforming Growth Factors
Deoxycytidine
Fibronectins
Epithelial Cells
Keratins
Methylation
Cell Movement
Cell Transdifferentiation
Phosphorylation
Epiretinal Membrane
Membranes
Growth Factor Receptors
Bromides
Genetic Promoter Regions
Small Interfering RNA
Smooth Muscle

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Inhibition of DNA methylation and methyl-CpG-binding protein 2 suppresses RPE transdifferentiation : Relevance to proliferative vitreoretinopathy. / He, Shikun; Barron, Ernesto; Ishikawa, Keijiro; Khanamiri, Hossein Nazari; Spee, Chris; Zhou, Peng; Kase, Satoru; Wang, Zhuoshi; Dustin, Laurie Diane; Hinton, David R.

In: Investigative Ophthalmology and Visual Science, Vol. 56, No. 9, 01.01.2015, p. 5579-5589.

Research output: Contribution to journalArticle

He, Shikun ; Barron, Ernesto ; Ishikawa, Keijiro ; Khanamiri, Hossein Nazari ; Spee, Chris ; Zhou, Peng ; Kase, Satoru ; Wang, Zhuoshi ; Dustin, Laurie Diane ; Hinton, David R. / Inhibition of DNA methylation and methyl-CpG-binding protein 2 suppresses RPE transdifferentiation : Relevance to proliferative vitreoretinopathy. In: Investigative Ophthalmology and Visual Science. 2015 ; Vol. 56, No. 9. pp. 5579-5589.
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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 a-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 methylationspecific 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.",
author = "Shikun He and Ernesto Barron and Keijiro Ishikawa and Khanamiri, {Hossein Nazari} and Chris Spee and Peng Zhou and Satoru Kase and Zhuoshi Wang and Dustin, {Laurie Diane} and Hinton, {David R.}",
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T1 - Inhibition of DNA methylation and methyl-CpG-binding protein 2 suppresses RPE transdifferentiation

T2 - Relevance to proliferative vitreoretinopathy

AU - He, Shikun

AU - Barron, Ernesto

AU - Ishikawa, Keijiro

AU - Khanamiri, Hossein Nazari

AU - Spee, Chris

AU - Zhou, Peng

AU - Kase, Satoru

AU - Wang, Zhuoshi

AU - Dustin, Laurie Diane

AU - Hinton, David R.

PY - 2015/1/1

Y1 - 2015/1/1

N2 - 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 a-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 methylationspecific 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.

AB - 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 a-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 methylationspecific 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.

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