Hypermethylation‐Mediated Silencing of p14ARF in Fibroblasts from Idiopathic Pulmonary Fibrosis (IPF)

Abstract

IPF is a lethal lung disease characterized by the formation and persistence of fibroblast foci and matrix accumulation. Evidence suggests that fibroblasts acquire an anti‐apoptotic phenotype and we hypothesized that epigenetic silencing of the pro‐apoptotic gene p14ARF may be involved. p14ARF expression was analyzed by RT‐PCR and Western blot in IPF and normal human lung fibroblasts. p14ARF gene promoter methylation was determined by methylation specific PCR (MS‐PCR) and by DNA digestion with endonuclease McrBc. Apoptosis was evaluated by annexin‐V and nuclear staining. p14ARF expression was significantly decreased in 4 of 8 IPF fibroblasts lines, which was restored after 5‐aza‐2′‐deoxycytidine treatment. MS‐PCR of bisulfite‐treated genomic DNA showed a correlation between the reduced expression of p14ARF and the presence of hypermethylated promoter. No amplification was observed in the DNA treated with McrBc confirming promoter hypermethylation. p14ARF hypermethylated IPF fibroblasts were more resistant to staurosporine‐induced apoptosis compared with normal and non‐methylated IPF fibroblasts (p<0.01) and showed reduced levels of p53. Apoptosis resistance was induced in fibroblasts inhibiting p14ARF expression by siRNA (p<0.05). These findings reveal that many IPF fibroblasts have reduced expression of p14ARF indicating that epigenetic mechanisms may cause their resistance to apoptosis.