Hypermethylation-mediated silencing of p14ARF in fibroblasts from idiopathic pulmonary fibrosis

Cisneros J, Hagood J, Checa M, Ortiz-Quintero B, Negreros M, Herrera I, Ramos C, Pardo A, Selman M. Hypermethylation-mediated silencing of p14(ARF) in fibroblasts from idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 303: L295-L303, 2012. First published June 15, 2012; doi:10.1152/ajplung.00332.2011.-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.