TGF-β1 epigenetically modifies Thy-1 expression in primary lung fibroblasts

Idiopathic pulmonary fibrosis is a progressive lung disease that increases in incidence with age. We identified a profibrotic lung phenotype in aging mice characterized by an increase in the number of fibroblasts lacking the expression of thymocyte differentiation antigen 1 (Thy-1) and an increase in transforming growth factor (TGF)-beta 1 expression. It has been shown that Thy-1 expression can be epigenetically modified. Lung fibroblasts (PLFs) were treated with TGF-beta 1 +/- DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-AZA) and analyzed for Thy-1 gene and protein expression, DNMT protein expression, and activity. alpha-Smooth muscle actin (alpha-SMA) and collagen type 1 (Col1A1) gene and protein expression was assessed. PLFs were transfected with DNMT1 silencing RNA +/- TGF-beta 1. TGF-beta 1 inhibited Thy-1 gene and protein expression in PLFs, and cotreatment with 5-AZA ameliorated this effect and appeared to inhibit DNMT1 activation. TGF-beta 1 induced Thy-1 promoter methylation as assessed by quantitative methyl PCR. Treatment with 5-AZA attenuated TGF-beta 1-induced Col1A1 gene and protein expression and alpha-SMA gene expression (but not alpha-SMA protein expression). Inhibiting DNMT1 with silencing RNA attenuated TGF-beta 1-induced DNMT activity and its downstream suppression of Thy-1 mRNA and protein expression as well as inhibited alpha-SMA mRNA and Col1A1 mRNA and protein expression, and showed a decreased trend in Thy-1 promoter methylation. Immunofluorescence for alpha-SMA suggested that 5-AZA inhibited stress fiber formation. These findings suggest that TGF-beta 1 epigenetically regulates lung fibroblast phenotype through methylation of the Thy-1 promoter. Targeted inhibition of DNMT in the right clinical context might prevent fibroblast to myofibroblast transdifferentiation and collagen deposition, which in turn could prevent fibrogenesis in the lung and other organs.