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.

Figure 1. Methylation differences between IPF fibroblasts and two groups of nonfibrotic control cells.

Levels of DNA methylation were analyzed using the HumanMethylation27 array in 6 IPF fibroblast lines, 3 patient-derived nonfibrotic controls, and 3 commercially available nonfibrotic cell lines (CCL190, CCL204, and CCL210). A) The number of differentially methylated CpG loci between IPF and patient-derived controls, between IPF and commercial cell line controls, and the overlap of these differences are shown. B) Fraction of differentially methylated CpG loci that are within and outside of CpG islands.

Figure 2. Methylation levels of CDKN2B, CARD10, and MGMT by bisulfite sequencing in IPF and nonfibrotic control fibroblasts.

The DNA methylation levels of various CpG sites within the CDKN2B (A), CARD10 (B), and MGMT (C) genes were analyzed by bisulfite pyrosequencing in fibroblasts from IPF (n = 6) and nonfibrotic control patients (n = 3). The hashtag indicates the CpG site that was assayed and identified to be differentially methylated by the array. Illustrated are the location of the CpG sites analyzed (based on NCBI Build 36.1) and position relative to the gene location, theoretical CpG islands, and MeDIP-Seq data from UCSC Genome Browser. *P<0.05.

Figure 3. Expression of CDKN2B, CARD10, and MGMT in IPF and nonfibrotic control fibroblasts.

Expression of (A) CDKN2B mRNA (n = 3 nonfibrotic, n = 5 IPF), (B) CDKN2B protein, (C) MGMT protein, and (D) CARD10 protein were assayed in IPF and nonfibrotic control fibroblasts. (E) IPF cells were treated with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza) at the indicated concentrations, and expression of CDKN2B, CARD10, and MGMT were assayed by immunoblot relative to α-tubulin and normalized to untreated control (n = 3). (F) The methylation of the CARD10 gene promoter was assayed in IPF cells after 72 h treatment with 5-aza or vehicle control (n = 2). *P<0.05.

Figure 4. Silencing of CDKN2B and cell proliferation.

CCL210 fibroblasts were treated with either control siRNA or siRNA targeted against CDKN2B. A) Levels of CDKN2B mRNA was assayed by RT-PCR (n = 3). B) Cell proliferation was measured by the CyQuant assay. Shown are the mean data from 6 replicates of a representative experiment.

Figure 5. Network analysis of differentially methylated genes in potassium ion binding gene ontology (GO) concept.

The GO category “potassium ion binding” was identified as an overrepresented concept in our dataset. The 18 differentially methylated genes with annotations in this category were analyzed by STITCH network analysis (http://stitch.embl.de) with their interrelationship shown. Protein-protein interactions are shown in blue, protein-chemical interactions are shown in green, and interactions between chemicals are shown in red.

Figure 6. Variability in DNA methylation of IPF cells.

A) Heirarchical cluster analysis was performed in each cell line studied, which also includes 3 separate samples of IMR-90 cells, a primary fetal fibroblast cell line. The mean methylation levels of the upstream CARD10 promoter (B) and the methylation levels of the individual CpG sites in the MGMT promoter (C) were compared among each individual IPF cell line and nonfibrotic cell lines.

Figure 7. Stability of DNA methylation differences with cell passage.

Three different IPF cell lines (A, B, and C) were assessed at passages 5, 6, and 7, and the DNA methylation for each cell line and each passage was compared. A) Shown are the methylation level of CpG sites 8, 9, and 10 in the upstream segment of the CARD10 promoter for IPF cell lines A and B at serial passage. B) Shown are the methylation levels of CpG sites 7–14 of the MGMT promoter for IPF cell lines A–C at serial passage.