title: Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype.
abstract: BACKGROUND
  Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.
  
  
  METHODS
  CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.
  
  
  RESULTS
  Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.
  
  
  CONCLUSIONS
  These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.
authors.affiliation: UCD School of Medicine & Medical Science, UCD Conway Institute, University College Dublin, Dublin 4, Ireland.
authors.forename: Claire M
authors.initials: CM
authors.lastname: Robinson
authors.forename: Roisin
authors.initials: R
authors.lastname: Neary
authors.forename: Ashleigh
authors.initials: A
authors.lastname: Levendale
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Watson
authors.forename: John A
authors.initials: JA
authors.lastname: Baugh
mesh_terms: D000199:Actins / Q000378:metabolism; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D015687:Cell Hypoxia; D002460:Cell Line; D024042:Collagen Type I / Q000378:metabolism; D024061:Collagen Type III / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D008168:Lung / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D058628:Myofibroblasts / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011658:Pulmonary Fibrosis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D018800:Thy-1 Antigens / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D024042:Collagen Type I; D024061:Collagen Type III; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D018800:Thy-1 Antigens; D000077209:Decitabine; D015254:DNA Modification Methylases; D001374:Azacitidine