Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by accumulation of activated myofibroblasts and excessive extracellular matrix deposition, in part mediated through enhanced TGF-beta signaling. TGF-beta1 is a potent inducer of epithelial-to-mesenchymal transition (EMT), the reversible phenotypic switching of epithelial to fibroblast-like cells. Recently, EMT has been demonstrated in alveolar epithelial cells (AECs) and has been proposed as a causative factor in lung fibrosis, but its precise mediators and mechanisms in IPF remains to be resolved. During developmental and disease settings, the phenotypic conversion of the epithelium is under tight transcriptional control, however, the transcription factors eliciting EMT in IPF have yet to be identified. Putative roles for SNAI transcription factors as regulators of EMT during development and a wide variety of diseases including cancer and organ fibrosis have been documented.
This study is based on the hypothesis that in AECs, TGF-beta1-induced SNAI transcription factors facilitate the acquisition of new morphology and motility, based on their ability to influence EMT marker gene expression. Thus, the objective of this study was to analyze the molecular mediators of TGF-beta1-induced EMT in vitro, in human A549 and primary mouse AT2 cells, and to assess their contribution to the development of fibrosis in experimental and idiopathic pulmonary fibrosis in vivo.
Immunofluorescent costaining of Tjp1 and a-SMA (an epithelial and mesenchymal marker, respectively) demonstrated TGF-beta1-induced EMT in AECs. Furthermore, in vitro, TGF-beta1 treatment increased the expression and nuclear accumulation of the zinc finger transcription factors SNAI1 (Snail) and SNAI2 (Slug), as assessed by RT-PCR and immunofluorescence. Ectopic expression of SNAI1 and SNAI2 proteins was sufficient to induce EMT in A549 cells, even in the absence of TGF-beta1 stimulation. In contrast, the siRNA-mediated depletion of SNAI1 and SNAI2 attenuated TGF-b1-induced AEC migration and EMT in A549 cells. The detection of EMT in vitro, with an increase in SNAI transcription factors was substantiated in vivo in the bleomycin model of pulmonary fibrosis early in disease. In vivo, SNAI expression was elevated in primary AECs isolated from fibrotic lungs, seven days after bleomycin challenge. An indication of occurrence of EMT with an increase in SNAI transcription factors was also corroborated in IPF patient lungs compared to control lungs. Furthermore, the occurrence of EMT, as well as the involvement of transcriptional control of SNAI factors was clarified in a unilateral ureteral obstruction (UUO) mouse model of renal fibrosis.
This study shows that (1) TGF-beta1-induced EMT in alveolar epithelial cells is accompanied by elevated expression of SNAI transcription factors, (2) EMT in AECs is essentially controlled by SNAI transcription factors, as ectopic expression of SNAI1 and SNAI2 triggers EMT, whereas depletion of these factors abrogates TGF-beta1-induced EMT, (3) increased expression of these zinc finger transcription factors are detected in an experimental model of lung fibrosis, with indication of the occurrence of EMT, (4) SNAI1 and SNAI2 upregulation have important implications for the development of IPF, (5) the detection of SNAI transcription factors early in EMT in a UUO model of renal fibrosis and the inhibition of EMT by leukocyte blocker treatment further emphasizes the significance of SNAI transcription factors in EMT as a causal factor in disease. Thus, reversal and/or inhibition of EMT may present a valid therapeutic option in lung fibrosis.
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