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Lung fibroblasts from patients with idiopathic pulmonary fibrosis exhibit genome-wide differences in DNA methylation compared to fibroblasts from nonfibrotic lung.

ABSTRACT: Excessive fibroproliferation is a central hallmark of idiopathic pulmonary fibrosis (IPF), a chronic, progressive disorder that results in impaired gas exchange and respiratory failure. Fibroblasts are the key effector cells in IPF, and aberrant expression of multiple genes contributes to their excessive fibroproliferative phenotype. DNA methylation changes are critical to the development of many diseases, but the DNA methylome of IPF fibroblasts has never been characterized. Here, we utilized the HumanMethylation 27 array, which assays the DNA methylation level of 27,568 CpG sites across the genome, to compare the DNA methylation patterns of IPF fibroblasts (n?=?6) with those of nonfibrotic patient controls (n?=?3) and commercially available normal lung fibroblast cell lines (n?=?3). We found that multiple CpG sites across the genome are differentially methylated (as defined by P value less than 0.05 and fold change greater than 2) in IPF fibroblasts compared to fibroblasts from nonfibrotic controls. These methylation differences occurred both in genes recognized to be important in fibroproliferation and extracellular matrix generation, as well as in genes not previously recognized to participate in those processes (including organ morphogenesis and potassium ion channels). We used bisulfite sequencing to independently verify DNA methylation differences in 3 genes (CDKN2B, CARD10, and MGMT); these methylation changes corresponded with differences in gene expression at the mRNA and protein level. These differences in DNA methylation were stable throughout multiple cell passages. DNA methylation differences may thus help to explain a proportion of the differences in gene expression previously observed in studies of IPF fibroblasts. Moreover, significant variability in DNA methylation was observed among individual IPF cell lines, suggesting that differences in DNA methylation may contribute to fibroblast heterogeneity among patients with IPF. These results demonstrate that IPF fibroblasts exhibit global differences in DNA methylation that may contribute to the excessive fibroproliferation associated with this disease.

SUBMITTER: Huang SK

PROVIDER: S-EPMC4162578 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Lung fibroblasts from patients with idiopathic pulmonary fibrosis exhibit genome-wide differences in DNA methylation compared to fibroblasts from nonfibrotic lung.

Huang Steven K SK Scruggs Anne M AM McEachin Richard C RC White Eric S ES Peters-Golden Marc M

PloS one 20140912 9

Excessive fibroproliferation is a central hallmark of idiopathic pulmonary fibrosis (IPF), a chronic, progressive disorder that results in impaired gas exchange and respiratory failure. Fibroblasts are the key effector cells in IPF, and aberrant expression of multiple genes contributes to their excessive fibroproliferative phenotype. DNA methylation changes are critical to the development of many diseases, but the DNA methylome of IPF fibroblasts has never been characterized. Here, we utilized t ...[more]

PMID: 25215577

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Project description:BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease without a cure and new drug strategies are urgently needed. Differences in behavior between diseased and healthy cells are well known and drug response can be different between cells isolated from IPF patients and controls. The macrolide Azithromycin (AZT) has anti-inflammatory and immunomodulatory properties. Recently anti-fibrotic effects have been described. However, the anti-fibrotic effects on primary IPF-fibroblasts (FB) directly compared to control-FB are unknown. We hypothesized that IPF-FB react differently to AZT in terms of anti-fibrotic effects.MethodsPrimary normal human lung and IPF-FB were exposed to TGF-β (5 ng/ml), Azithromycin (50 μM) alone or in combination prior to gene expression analysis. Pro-collagen Iα1 secretion was assessed by ELISA and protein expression by western blot (αSMA, Fibronectin, ATP6V1B2, LC3 AB (II/I), p62, Bcl-xL). Microarray analysis was performed to screen involved genes and pathways after Azithromycin treatment in control-FB. Apoptosis and intraluminal lysosomal pH were analyzed by flow cytometry.ResultsAZT significantly reduced collagen secretion in TGF-β treated IPF-FB compared to TGF-β treatment alone, but not in control-FB. Pro-fibrotic gene expression was similarly reduced after AZT treatment in IPF and control-FB. P62 and LC3II/I western blot revealed impaired autophagic flux after AZT in both control and IPF-FB with significant increase of LC3II/I after AZT in control and IPF-FB, indicating enhanced autophagy inhibition. Early apoptosis was significantly higher in TGF-β treated IPF-FB compared to controls after AZT. Microarray analysis of control-FB treated with AZT revealed impaired lysosomal pathways. The ATPase and lysosomal pH regulator ATP6V0D2 was significantly less increased after additional AZT in IPF-FB compared to controls. Lysosomal function was impaired in both IPF and control FB, but pH was significantly more increased in TGF-β treated IPF-FB.ConclusionWe report different treatment responses after AZT with enhanced anti-fibrotic and pro-apoptotic effects in IPF compared to control-FB. Possibly impaired lysosomal function contributes towards these effects. In summary, different baseline cell phenotype and behavior of IPF and control cells contribute to enhanced anti-fibrotic and pro-apoptotic effects in IPF-FB after AZT treatment and strengthen its role as a new potential anti-fibrotic compound, that should further be evaluated in clinical studies.

Project description:BACKGROUND:Idiopathic Pulmonary Fibrosis (IPF) is characterized by profound changes in the lung phenotype including excessive extracellular matrix deposition, myofibroblast foci, alveolar epithelial cell hyperplasia and extensive remodeling. The role of epigenetic changes in determining the lung phenotype in IPF is unknown. In this study we determine whether IPF lungs exhibit an altered global methylation profile. METHODOLOGY/PRINCIPAL FINDINGS:Immunoprecipitated methylated DNA from 12 IPF lungs, 10 lung adenocarcinomas and 10 normal histology lungs was hybridized to Agilent human CpG Islands Microarrays and data analysis was performed using BRB-Array Tools and DAVID Bioinformatics Resources software packages. Array results were validated using the EpiTYPER MassARRAY platform for 3 CpG islands. 625 CpG islands were differentially methylated between IPF and control lungs with an estimated False Discovery Rate less than 5%. The genes associated with the differentially methylated CpG islands are involved in regulation of apoptosis, morphogenesis and cellular biosynthetic processes. The expression of three genes (STK17B, STK3 and HIST1H2AH) with hypomethylated promoters was increased in IPF lungs. Comparison of IPF methylation patterns to lung cancer or control samples, revealed that IPF lungs display an intermediate methylation profile, partly similar to lung cancer and partly similar to control with 402 differentially methylated CpG islands overlapping between IPF and cancer. Despite their similarity to cancer, IPF lungs did not exhibit hypomethylation of long interspersed nuclear element 1 (LINE-1) retrotransposon while lung cancer samples did, suggesting that the global hypomethylation observed in cancer was not typical of IPF. CONCLUSIONS/SIGNIFICANCE:Our results provide evidence that epigenetic changes in IPF are widespread and potentially important. The partial similarity to cancer may signify similar pathogenetic mechanisms while the differences constitute IPF or cancer specific changes. Elucidating the role of these specific changes will potentially allow better understanding of the pathogenesis of IPF.

Project description:BACKGROUND:Idiopathic Pulmonary Fibrosis (IPF) is a debilitating disease characterized by exaggerated extracellular matrix deposition and aggressive lung structural remodeling. Disease pathogenesis is driven by fibroblastic foci formation, consequent on growth factor overexpression and myofibroblast proliferation. We have previously shown that both CTGF overexpression and myofibroblast formation in IPF cell lines are dependent on RhoA signaling. As RhoA-mediated regulation is also involved in cell cycle progression, we hypothesise that this pathway is key to lung fibroblast turnover through modulation of cyclin D1 kinetic expression. METHODS:Cyclin D1 expression was compared in primary IPF patient-derived fibroblasts and equivalent normal control cells. Quantitative real time PCR was employed to examine relative expression levels of cyclin D1 mRNA; protein expression was confirmed by western blotting. Effects of Rho signaling were investigated using transient transfection of constitutively active and dominant negative RhoA constructs as well as pharmacological inhibitors. Cellular proliferation of lung fibroblasts was determined by BrdU incorporation ELISA. To further explore RhoA regulation of cyclin D1 in lung fibroblasts and associated cell cycle progression, an established Rho inhibitor, Simvastatin, was incorporated in our studies. RESULTS:Cyclin D1 expression was upregulated in IPF compared to normal lung fibroblasts under exponential growth conditions (p < 0.05). Serum deprivation inhibited cyclin D1 expression, which was restored following treatment with fibrogenic growth factors (TGF-beta1 and CTGF). RhoA inhibition, using a dominant negative mutant and a pharmacological inhibitor (C3 exotoxin), suppressed levels of cyclin D1 mRNA and protein in IPF fibroblasts, with significant abrogation of cell turnover (p < 0.05). Furthermore, Simvastatin dose-dependently inhibited fibroblast cyclin D1 gene and protein expression, inducing G1 cell cycle arrest. Similar trends were observed in control experiments using normal lung fibroblasts, though exhibited responses were lower in magnitude. CONCLUSION:These findings report for the first time that cyclin D1 expression is deregulated in IPF through a RhoA dependent mechanism that influences lung fibroblast proliferation. This potentially unravels new molecular targets for future anti-IPF strategies; accordingly, Simvastatin inhibition of Rho-mediated cyclin D1 expression in IPF fibroblasts merits further exploitation.

Dataset Information

Lung fibroblasts from patients with idiopathic pulmonary fibrosis exhibit genome-wide differences in DNA methylation compared to fibroblasts from nonfibrotic lung.

Publications

Lung fibroblasts from patients with idiopathic pulmonary fibrosis exhibit genome-wide differences in DNA methylation compared to fibroblasts from nonfibrotic lung.

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