title: Endothelial glucocorticoid receptor promoter methylation according to dexamethasone sensitivity.
abstract: We have previously shown that in vitro sensitivity to dexamethasone (DEX) stimulation in human endothelial cells is positively regulated by the glucocorticoid receptor (NR3C1, GR). The present study determined the role of differential GR transcriptional regulation in glucocorticoid sensitivity. We studied 25 human umbilical vein endothelial cells (HUVECs) that had been previously characterized as DEX-sensitive (n=15), or resistant (n=10). Real-time PCR analysis of GR 5'UTR mRNA isoforms showed that all HUVECs expressed isoforms 1B, 1C, 1D, 1F, and 1H, and isoforms 1B and 1C were predominantly expressed. DEX-resistant cells expressed higher basal levels of the 5'UTR mRNA isoforms 1C and 1D, but lower levels of the 5'UTR mRNA isoform 1F than DEX-sensitive cells. DEX treatment significantly decreased GRα and GR-1C mRNA isoform expression in DEX-resistant cells only. Reporter luciferase assays indicated that differential GR mRNA isoform expression was not due to differential promoter usage between DEX-sensitive and DEX-resistant cells. Analysis of promoter methylation, however, showed that DEX-sensitive cells have higher methylation levels of promoter 1D and lower methylation levels of promoter 1F than DEX-resistant cells. Treatment with 5-aza-2-deoxycytidine abolished the differential 5'UTR mRNA isoform expression between DEX-sensitive and DEX-resistant cells. Finally, both GRα overexpression and 5-aza-2-deoxycytidine treatment eliminated the differences between sensitivity groups to DEX-mediated downregulation of endothelial nitric oxide synthase (NOS3), and upregulation of plasminogen activator inhibitor 1 (SERPINE1). In sum, human endothelial GR 5'UTR mRNA expression is regulated by promoter methylation with DEX-sensitive and DEX-resistant cells having different GR promoter methylation patterns.
authors.affiliation: Divisions of Pharmacology and Physiology Department of Basic Sciences, School of Medicine, Center for Perinatal Biology, Medical Center, Loma Linda University, Room A572, 11234 Anderson Street, Loma Linda, CA 92350, USA ematagreenwood@llu.edu.
authors.forename: Eugenia
authors.initials: E
authors.lastname: Mata-Greenwood
authors.affiliation: Divisions of Pharmacology and Physiology Department of Basic Sciences, School of Medicine, Center for Perinatal Biology, Medical Center, Loma Linda University, Room A572, 11234 Anderson Street, Loma Linda, CA 92350, USA.
authors.forename: P Naomi
authors.initials: PN
authors.lastname: Jackson
authors.affiliation: Divisions of Pharmacology and Physiology Department of Basic Sciences, School of Medicine, Center for Perinatal Biology, Medical Center, Loma Linda University, Room A572, 11234 Anderson Street, Loma Linda, CA 92350, USA.
authors.forename: William J
authors.initials: WJ
authors.lastname: Pearce
authors.affiliation: Divisions of Pharmacology and Physiology Department of Basic Sciences, School of Medicine, Center for Perinatal Biology, Medical Center, Loma Linda University, Room A572, 11234 Anderson Street, Loma Linda, CA 92350, USA.
authors.forename: Lubo
authors.initials: L
authors.lastname: Zhang
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002478:Cells, Cultured; D019175:DNA Methylation / Q000235:genetics*; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D000077209:Decitabine; D003907:Dexamethasone / Q000494:pharmacology*; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis*; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D020121:5' Untranslated Regions; D005938:Glucocorticoids; C497713:NR3C1 protein, human; D017395:Plasminogen Activator Inhibitor 1; D020033:Protein Isoforms; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; C051321:SERPINE1 protein, human; D000077209:Decitabine; D003907:Dexamethasone; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D015254:DNA Modification Methylases; D001374:Azacitidine
keywords: 5′UTR mRNA isoform; endothelium; glucocorticoids; methylation