title: Epigenetic regulation of cyclooxygenase-2 by methylation of c8orf4 in pulmonary fibrosis.
abstract: Fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) produce low levels of prostaglandin (PG) E2, due to a limited capacity to up-regulate cyclooxygenase-2 (COX-2). This deficiency contributes functionally to the fibroproliferative state, however the mechanisms responsible are incompletely understood. In the present study, we examined whether the reduced level of COX-2 mRNA expression observed in fibrotic lung fibroblasts is regulated epigenetically. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5AZA) restored COX-2 mRNA expression by fibrotic lung fibroblasts dose dependently. Functionally, this resulted in normalization of fibroblast phenotype in terms of PGE2 production, collagen mRNA expression and sensitivity to apoptosis. COX-2 methylation assessed by bisulfite sequencing and methylation microarrays was not different in fibrotic fibroblasts compared with controls. However, further analysis of the methylation array data identified a transcriptional regulator, chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), which is hypermethylated and down-regulated in fibrotic fibroblasts compared with controls. siRNA knockdown of c8orf4 in control fibroblasts down-regulated COX-2 and PGE2 production generating a phenotype similar to that observed in fibrotic lung fibroblasts. Chromatin immunoprecipitation demonstrated that c8orf4 regulates COX-2 expression in lung fibroblasts through binding of the proximal promoter. We conclude that the decreased capacity of fibrotic lung fibroblasts to up-regulate COX-2 expression and COX-2-derived PGE2 synthesis is due to an indirect epigenetic mechanism involving hypermethylation of the transcriptional regulator, c8orf4.
authors.affiliation: UCL Respiratory Centre for Inflammation and Tissue Repair, Rayne Building, University College London, London, WC1E 6JF, U.K.
authors.forename: Iona C
authors.initials: IC
authors.lastname: Evans
authors.affiliation: UCL Respiratory Centre for Inflammation and Tissue Repair, Rayne Building, University College London, London, WC1E 6JF, U.K.
authors.forename: Josephine L
authors.initials: JL
authors.lastname: Barnes
authors.affiliation: UCL Respiratory Centre for Inflammation and Tissue Repair, Rayne Building, University College London, London, WC1E 6JF, U.K.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Garner
authors.affiliation: UCL Respiratory Centre for Inflammation and Tissue Repair, Rayne Building, University College London, London, WC1E 6JF, U.K.
authors.forename: David R
authors.initials: DR
authors.lastname: Pearce
authors.affiliation: UCL Respiratory Centre for Inflammation and Tissue Repair, Rayne Building, University College London, London, WC1E 6JF, U.K. NIHR Biomedical Research Unit & Interstitial Lung Disease Unit, Royal Brompton Hospital, Sydney Street, London, SW3 6NP, U.K.
authors.forename: Toby M
authors.initials: TM
authors.lastname: Maher
authors.affiliation: Centre for Rheumatology and Connective Tissue Diseases, Royal Free Campus, University College London, London, NW3 2PF, U.K.
authors.forename: Xu
authors.initials: X
authors.lastname: Shiwen
authors.affiliation: NIHR Biomedical Research Unit & Interstitial Lung Disease Unit, Royal Brompton Hospital, Sydney Street, London, SW3 6NP, U.K.
authors.forename: Elisabetta A
authors.initials: EA
authors.lastname: Renzoni
authors.affiliation: NIHR Biomedical Research Unit & Interstitial Lung Disease Unit, Royal Brompton Hospital, Sydney Street, London, SW3 6NP, U.K.
authors.forename: Athol U
authors.initials: AU
authors.lastname: Wells
authors.affiliation: Centre for Rheumatology and Connective Tissue Diseases, Royal Free Campus, University College London, London, NW3 2PF, U.K.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Denton
authors.affiliation: Centre for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology, University of Western Australia, Harry Perkins Institute of Medical Research, Crawley, WA 6009, Australia.
authors.forename: Geoffrey J
authors.initials: GJ
authors.lastname: Laurent
authors.affiliation: Centre for Rheumatology and Connective Tissue Diseases, Royal Free Campus, University College London, London, NW3 2PF, U.K.
authors.forename: David J
authors.initials: DJ
authors.lastname: Abraham
authors.affiliation: UCL Respiratory Centre for Inflammation and Tissue Repair, Rayne Building, University College London, London, WC1E 6JF, U.K. r.mcanulty@ucl.ac.uk.
authors.forename: Robin J
authors.initials: RJ
authors.lastname: McAnulty
mesh_terms: D000368:Aged; D001665:Binding Sites; D016022:Case-Control Studies; D049109:Cell Proliferation; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044127:Epigenesis, Genetic / Q000187:drug effects; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D005838:Genotype; D006801:Humans; D008168:Lung / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011658:Pulmonary Fibrosis / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012595:Scleroderma, Systemic / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D012333:RNA, Messenger; C418741:TCIM protein, human; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D015254:DNA Modification Methylases; D015232:Dinoprostone
keywords: DNA methylation; cyclooxygenase-2; fibroblast; idiopathic pulmonary fibrosis; prostaglandin E2; systemic sclerosis