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)-β1 expression. It has been shown that Thy-1 expression can be epigenetically modified. Lung fibroblasts (PLFs) were treated with TGF-β1 ± DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-AZA) and analyzed for Thy-1 gene and protein expression, DNMT protein expression, and activity. α-Smooth muscle actin (α-SMA) and collagen type 1 (Col1A1) gene and protein expression was assessed. PLFs were transfected with DNMT1 silencing RNA ± TGF-β1. TGF-β1 inhibited Thy-1 gene and protein expression in PLFs, and cotreatment with 5-AZA ameliorated this effect and appeared to inhibit DNMT1 activation. TGF-β1 induced Thy-1 promoter methylation as assessed by quantitative methyl PCR. Treatment with 5-AZA attenuated TGF-β1-induced Col1A1 gene and protein expression and α-SMA gene expression (but not α-SMA protein expression). Inhibiting DNMT1 with silencing RNA attenuated TGF-β1-induced DNMT activity and its downstream suppression of Thy-1 mRNA and protein expression as well as inhibited α-SMA mRNA and Col1A1 mRNA and protein expression, and showed a decreased trend in Thy-1 promoter methylation. Immunofluorescence for α-SMA suggested that 5-AZA inhibited stress fiber formation. These findings suggest that TGF-β1 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.
Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia.
Wendy A
WA
Neveu
Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia.
Stephen T
ST
Mills
Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia.
Bashar S
BS
Staitieh
Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia vsuebli@emory.edu.
Viranuj
V
Sueblinvong
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D054338:Cell Transdifferentiation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044127:Epigenesis, Genetic / Q000187:drug effects*; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D054990:Idiopathic Pulmonary Fibrosis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008168:Lung / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010641:Phenotype; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D018800:Thy-1 Antigens / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
C558396:Acta2 protein, mouse; D000199:Actins; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D004791:Enzyme Inhibitors; D018800:Thy-1 Antigens; D053773:Transforming Growth Factor beta1; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619381:Dnmt1 protein, mouse
5-aza-2′-deoxycytidine; deoxyribonucleic acid methyltransferase; pulmonary fibrosis; thymocyte differentiation antigen 1; transforming growth factor-β1