Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

Abstract

BackgroundPulmonary 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.MethodsCCD19Lu 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.ResultsSignificant 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.ConclusionThese data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

DNA methylation in fibrosis

“…When the methylation was reversed with 5-aza-2'-deoxycytidine, Thy-1 expression was restored. This provides evidence that methylation alterations can influence myofibroblast differentiation (Robinson et al, 2012). It has also been shown in idiopathic lung fibrosis that there is hypermethylation of the p14 ARF promoter compared to controls, leading to reduced expression.…”

Risk factors for primary Sjögren syndrome-associated interstitial lung disease

“…Studies revealed mechanistic links between aging and lung fibrosis involving telomere attrition, genomic instability, and epigenetic alterations (24)(25)(26). Antihistone antibodies, a type of ANA, are correlated with severe pulmonary fibrosis in systemic sclerosis patients (27).…”

Quantitative Profiling of Chromatome Dynamics Reveals a Novel Role for HP1BP3 in Hypoxia-induced Oncogenesis

“…However, hypoxia can also induce histone modification and chromatin remodeling via hypoxia-inducible factor-independent pathways, indicating that additional mechanisms of epigenetic regulation can shape the cellular response to restricted oxygen supply (9,10), and potentially increase cell survival and promote angiogenesis in hypoxic conditions (11)(12)(13)(14). Hy-poxia-induced changes in the composition of the chromatinassociated proteome (chromatome) are therefore likely to alter gene expression and promote clonal evolution in developing tumors.…”