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.
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Authors | Iona C Evans, Josephine L Barnes, Ian M Garner, David R Pearce, Toby M Maher, Xu Shiwen, Elisabetta A Renzoni, Athol U Wells, Christopher P Denton, Geoffrey J Laurent, David J Abraham, Robin J McAnulty |
Journal | Clinical science (London, England : 1979)
(Clin Sci (Lond))
Vol. 130
Issue 8
Pg. 575-86
(Apr 2016)
ISSN: 1470-8736 [Electronic] England |
PMID | 26744410
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Copyright | © 2016 The Author(s). |
Chemical References |
- Enzyme Inhibitors
- Neoplasm Proteins
- RNA, Messenger
- TCIM protein, human
- Cyclooxygenase 2
- PTGS2 protein, human
- DNA Modification Methylases
- Dinoprostone
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Topics |
- Aged
- Binding Sites
- Case-Control Studies
- Cell Proliferation
- Cells, Cultured
- Cyclooxygenase 2
(genetics, metabolism)
- DNA Methylation
(drug effects)
- DNA Modification Methylases
(antagonists & inhibitors, metabolism)
- Dinoprostone
(metabolism)
- Dose-Response Relationship, Drug
- Down-Regulation
- Enzyme Inhibitors
(pharmacology)
- Epigenesis, Genetic
(drug effects)
- Female
- Fibroblasts
(drug effects, enzymology, pathology)
- Gene Expression Regulation, Neoplastic
- Genotype
- Humans
- Lung
(drug effects, enzymology, pathology)
- Male
- Middle Aged
- Neoplasm Proteins
(genetics, metabolism)
- Phenotype
- Promoter Regions, Genetic
- Pulmonary Fibrosis
(enzymology, genetics, pathology)
- RNA Interference
- RNA, Messenger
(genetics, metabolism)
- Scleroderma, Systemic
(enzymology, genetics, pathology)
- Transcription, Genetic
- Transfection
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