Hypermethylation-mediated silencing of p14(ARF) in fibroblasts from idiopathic pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease of unknown etiology. A conspicuous feature is the formation and persistence of fibroblastic/myofibroblastic foci throughout the lung parenchyma. Mechanisms remain unknown, but data indicate that fibroblasts acquire an antiapoptotic phenotype. We hypothesized that transcriptional silencing of proapoptotic genes may be implicated, and accordingly we evaluated the epigenetic regulation of p14(ARF). The expression of p14(ARF) was analyzed by RT-PCR in IPF (n = 8) and normal derived fibroblasts (n = 4) before and after treatment with 5-aza-2'-deoxycytidine (5-aza) and trichostatin A (TSA). p14(ARF) gene promoter methylation was determined by methylation-specific PCR (MS-PCR) and by DNA digestion with endonuclease McrBc, which cleaves 50% of methylated CpG. Apoptosis was evaluated by Annexin-V and nuclear staining. p14(ARF) expression was significantly decreased in four of the eight IPF fibroblasts lines, which was restored after 5-aza treatment. No changes were found with TSA. MS-PCR of bisulfite-treated genomic DNA showed a correlation between the reduced expression of p14(ARF) and the presence of hypermethylated promoter. No amplification was observed in the DNA treated with the McrBc enzyme, corroborating promoter hypermethylation. p14(ARF)-hypermethylated IPF fibroblasts were significantly more resistant to staurosporine-and S-nitrosoglutathione-induced apoptosis compared with normal and nonmethylated IPF fibroblasts (P < 0.01) and showed reduced levels of p53. Resistance to apoptosis was provoked in fibroblasts when p14(ARF) expression was inhibited by siRNA (P < 0.05). These findings demonstrate that many IPF fibroblasts have reduced expression of the proapoptotic p14(ARF) attributable to promoter hypermethylation and indicate that epigenetic mechanisms may underlie their resistance to apoptosis.

Fig. 1.

Methylation of p14^ARF in idiopathic pulmonary fibrosis (IPF) fibroblasts by methylation-specific PCR (MS-PCR) analysis. DNA extracted from normal and IPF fibroblasts before (A) and after (B) treatment with 5-aza was amplified by PCR with primers specific to the unmethylated (U) or the methylated (M) CpG islands of the p14^ARF gene after modification with sodium bisulfite. The expected sizes of PCR products of p14^ARF were 132 bp with U primers and 122 bp with M primers and were identified with commercially available unmethylated and methylated DNA. Methylation products were amplified in four IPF fibroblasts (IPF-5 to 8).

Fig. 2.

Fibroblast p14^ARF promoter amplification. Genomic DNA extracted from IPF fibroblasts was digested with the enzyme McrBc as described in materials and methods. A: p14^ARF promoter was amplified by PCR in undigested DNA (Ctrl-DNA) and DNA cleaved by McrBc (McrBc-DNA) using 4 different amplicons (R1-R4). Absence of amplification product in McrBc-DNA from IPF-5 and IPF-6 indicates methylation of corresponding region. B: methylation status of p14^ARF promoter by relative quantitation of R2 amplicon using real-time-PCR after digestion with McrBc against its undigested cells. White spaces represent a reassembly of noncontiguous gel.

Fig. 3.

Levels of expression of the p14^ARF in normal and IPF fibroblasts. Fibroblasts were cultured alone or in presence of 10 μM of 5-aza-2′-deoxycitidine (5-aza) for 3 days, and mRNA expression was analyzed by real-time RT-PCR. IPF fibroblasts with methylated promoter (IPF-5 to IPF-8) showed a significant reduced expression of p14^ARF at basal level compared with the others fibroblasts (solid bars), and the expression was restored after 5-aza treatment (open bars). Bars represent the means ± SD of copy number of p14^ARF mRNA normalized to 18S rRNA expression of 2 independent experiments made by triplicate. *P < 0.05; **P < 0.01.

Fig. 4.

Increased resistance to apoptosis in p14^ARF hypermethylated IPF fibroblasts. A: fibroblast with negative methylation (N1 and IPF-4) and 1 IPF with positive methylation (IPF-5) were cultured in presence of DMSO (control) or staurosporine (sts) for 6 h, and apoptosis was analyzed by flow cytometry. Representative charts of flow cytometry experiments. Cells negative for 7AAD staining and positive for Annexin-V binding represent apoptotic cells. Right: analysis of apoptotic cells showing the percent of apoptosis obtained in 2 independent experiments. B: fibroblasts grown on coverslips were incubated with DMSO (control) or 1 μM of sts for 6 h and fixed with 2% paraformaldehyde. Cells were stained with Hoechst 33342 (1 μg/ml) for 20 min at room temperature and analyzed by fluorescence microscopy. Left: representative photomicrograph of apoptotic changes in normal (N1) and 1 of IPF fibroblast cell lines with p14^ARF promoter hypermethylation (IPF-5). Arrows indicate cells with fragmented nuclei. Bottom: percentage of cells with apoptotic changes (nuclear fragmentation) of 7 random fields. C: fibroblasts with methylated and nonmethylated p14^ARF promoter were cultured alone or in presence of S-nitrosoglutathione (GSNO, 500 μM) for 24 h, and apoptosis was analyzed by flow cytometry. *P < 0.05; **P < 0.01.

Fig. 5.

Silencing of p14^ARF increases resistance to apoptosis in fibroblasts. Normal fibroblasts were transfected with 5 nM of siRNA for p14^ARF. After 48 h, fibroblasts were exposed to DMSO or 1 μM of sts for 6 h, and apoptosis was analyzed by flow cytometry. A: relative quantitation of gene expression in control and transfected fibroblasts by real time-PCR. B: representative charts of cells negative for 7AAD staining and positive for Annexin-V binding represent apoptotic cells obtained in 1 of 2 independent experiments. The graph represents the percentage of apoptosis obtained in 2 independent experiments. *P < 0.05.

Fig. 6.

Western blot analysis of p53 in IPF fibroblasts. Fibroblasts with methylated (M) and unmethylated (U) p14^ARF promoter (A) and fibroblasts treated with p14^ARF siRNA or nontreated (Ctrl) (B) were plated in 60-mm dishes and cultured in Ham's F-12 medium supplemented with 10% FBS until ∼80% confluence was reached. Cell lysates were analyzed by immunoblotting using anti-human p53 antibody. Detection of GAPDH was used to test equal loading.