UCLA

Due to the active inhibition of the adipogenic programming, the default destiny of the developing lung mesenchyme is to acquire a myogenic phenotype. We have previously shown that perinatal nicotine exposure, by down-regulating PPARγ expression, accentuates this property, culminating in myogenic pulmonary phenotype, though the underlying mechanisms remained incompletely understood. We hypothesized that nicotine-induced PPARγ down-regulation is mediated by PPARγ promoter methylation, controlled by DNA methyltransferase 1 (DNMT1) and methyl CpG binding protein 2 (MeCP2), two known key regulators of DNA methylation. Using cultured alveolar interstitial fibroblasts and an in vivo perinatal nicotine exposure rat model, we found that PPARγ promoter methylation is strongly correlated with inhibition of PPARγ expression in the presence of nicotine. Methylation inhibitor 5-aza-2'-deoxycytidine restored the nicotine-induced down-regulation of PPARγ expression and the activation of its downstream myogenic marker fibronectin. With nicotine exposure, a specific region of PPARγ promoter was significantly enriched with antibodies against chromatin repressive markers H3K9me3 and H3K27me3, dose-dependently. Similar data were observed with antibodies against DNA methylation regulatory factors DNMT1 and MeCP2. The knock down of DNMT1 and MeCP2 abolished nicotine-mediated increases in DNMT1 and MeCP2 protein levels, and PPARγ promoter methylation, restoring nicotine-induced down regulation of PPARγ and upregulation of the myogenic protein, fibronectin. The nicotine-induced alterations in DNA methylation modulators DNMT1 and MeCP2, PPARγ promoter methylation, and its down-stream targets, were also validated in perinatally nicotine exposed rat lung tissue. These data provide novel mechanistic insights into nicotine-induced epigenetic silencing of PPARγ that could be exploited to design novel targeted molecular interventions against the smoke exposed lung injury in general and perinatal nicotine exposure induced lung damage in particular.