title: Hypoxia-Induced Changes in DNA Methylation Alter RASAL1 and TGFβ1 Expression in Human Trabecular Meshwork Cells.
abstract: Fibrosis and a hypoxic environment are associated with the trabecular meshwork (TM) region in the blinding disease glaucoma. Hypoxia has been shown to alter DNA methylation, an epigenetic mechanism involved in regulating gene expression such as the pro-fibrotic transforming growth factor (TGF) β1 and the anti-fibrotic Ras protein activator like 1 (RASAL1). The purpose of this study was to compare DNA methylation levels, and the expression of TGFβ1 and RASAL1 in primary human normal (NTM) with glaucomatous (GTM) cells and in NTM cells under hypoxic conditions.
  
  
  
  Global DNA methylation was assessed by ELISA in cultured age-matched NTM and GTM cells. qPCR was conducted for TGFβ1, collagen 1α1 (COL1A1), and RASAL1 expression. Western immunoblotting was used to determine protein expression. For hypoxia experiments, NTM cells were cultured in a 1%O2, 5%CO2 and 37°C environment. NTM and GTM cells were treated with TGFβ1 (10ng/ml) and the methylation inhibitor 5-azacytidine (5-aza) (0.5μM) respectively to determine their effects on DNA Methyltransferase 1 (DNMT1) and RASAL1 expression.
  
  
  
  We found increased DNA methylation, increased TGFβ1 expression and decreased RASAL1 expression in GTM cells compared to NTM cells. Similar results were obtained in NTM cells under hypoxic conditions. TGFβ1 treatment increased DNMT1 and COL1A1, and decreased RASAL1 expression in NTM cells. 5-aza treatment decreased DNMT1, TGFβ1 and COL1A1 expression, and increased RASAL1 expression in GTM cells.
  
  
  
  TGFβ1 and RASAL1 expression, global DNA methylation, and expression of associated methylation enzymes were altered between NTM and GTM cells. We found that hypoxia in NTM cells induced similar results to the GTM cells. Furthermore, DNA methylation, TGFβ1 and RASAL1 appear to have an interacting relationship that may play a role in driving pro-fibrotic disease progression in the glaucomatous TM.
authors.affiliation: School of Medicine and Medical Science, University College Dublin, Dublin 4, Ireland.
authors.forename: Fiona
authors.identifier: 0000-0001-7563-7258
authors.initials: F
authors.lastname: McDonnell
authors.affiliation: Dept. Ophthalmology, Mater Misericordiae University Hospital, Dublin 7, Ireland.
authors.forename: Mustapha
authors.initials: M
authors.lastname: Irnaten
authors.affiliation: Dept. Cell Biology & Immunology and the North Texas Eye Research Institute, U. North Texas Health Science Center, Ft. Worth, Texas, United States of America.
authors.forename: Abbot F
authors.initials: AF
authors.lastname: Clark
authors.affiliation: School of Medicine and Medical Science, University College Dublin, Dublin 4, Ireland.|Dept. Ophthalmology, Mater Misericordiae University Hospital, Dublin 7, Ireland.
authors.forename: Colm J
authors.initials: CJ
authors.lastname: O'Brien
authors.affiliation: School of Medicine and Medical Science, University College Dublin, Dublin 4, Ireland.|Dept. Ophthalmology, Mater Misericordiae University Hospital, Dublin 7, Ireland.
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Wallace
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D001374:Azacitidine / Q000008:administration & dosage; D002478:Cells, Cultured; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics*; D044127:Epigenesis, Genetic / Q000187:drug effects / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D005260:Female; D020690:GTPase-Activating Proteins / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D005901:Glaucoma / Q000235:genetics; D006801:Humans; D000860:Hypoxia / Q000235:genetics*; D007231:Infant, Newborn; D008297:Male; D014129:Trabecular Meshwork / Q000187:drug effects / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000235:genetics*; D000099222:betaIG-H3 Protein
chemical_list: D001374:Azacitidine; D016326:Extracellular Matrix Proteins; D020690:GTPase-Activating Proteins; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein; C580923:RASAL1 protein, human