Snail and Slug are Key Mediators of TGF-beta-induced Epithelial-to-Mesenchymal Transition in Lung Fibrosis

Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by an increase of interstitial fibroblasts and excessive extracellular matrix deposition, in part mediated through enhanced TGF-β signaling. Epithelial-to-mesenchymal transition (EMT), the reversible phenotypic switching of epithelial to fibroblast-like cells, has recently been proposed as a causative factor in tissue fibrosis, but its precise role in lung fibrosis remains to be resolved. Methods: We analyzed whether EMT occurs in human A549 and mouse primary alveolar epithelial cells (AEC), whether TGF-β is a key inducer of EMT in these cells, and whether EMT can be demonstrated in bleomycin-induced lung fibrosis in vivo. Results: We demonstrate by immunofluorescence costaining of E-cadherin and smooth muscle actin (SMA; an epithelial and mesenchymal marker, respectively) that EMT is induced in A549 and primary AEC by TGF-β (up to 68% SMA-positive cells after 48 hrs). This coincided with decreased expression of E-cadherin, ZO-1, and occludin, as assessed by RT-PCR. In contrast, vimentin, snail, and slug were rapidly induced by TGF-β. Increased snail expression was due to direct binding of Smad3 to the snail promoter, as analyzed by chromatin immunoprecipitation. Using the bleomycin-induced model of lung fibrosis, we furthermore demonstrate that EMT also occurs in vivo, as assessed by SMA-positive epithelial cells, and increases in snail and slug expression, as early as 7 days after bleomycin challenge. Conclusions: Our results thus demonstrate that EMT 1) is induced by TGF-β in vitro, 2) is controlled by the transcription factors snail and slug, and 3) is found in a relevant animal model of lung fibrosis. Reversal and/or inhibition of EMT may therefore be a valid therapeutic option in lung fibrosis.