Abstract
Myofibroblasts play critical roles in the development of idiopathic pulmonary fibrosis by depositing components of extracellular matrix. One source of lung myofibroblasts is thought to be alveolar epithelial type 2 cells that undergo epithelial–mesenchymal transition (EMT). Rat RLE-6TN alveolar epithelial type 2 cells treated with transforming growth factor-β1 (TGF-β1) are converted into myofibroblasts through EMT. TGF-β induces both canonical Smad signaling and non-canonical signaling, including the Ras-induced ERK pathway (Raf–MEK–ERK). However, the signaling mechanisms regulating TGF-β1-induced EMT are not fully understood. Here, we show that the Ras–ERK pathway negatively regulates TGF-β1-induced EMT in RLE-6TN cells and that DA-Raf1 (DA-Raf), a splicing isoform of A-Raf and a dominant-negative antagonist of the Ras–ERK pathway, plays an essential role in EMT. Stimulation of the cells with fibroblast growth factor 2 (FGF2), which activated the ERK pathway, prominently suppressed TGF-β1-induced EMT. An inhibitor of MEK, but not an inhibitor of phosphatidylinositol 3-kinase, rescued the TGF-β1-treated cells from the suppression of EMT by FGF2. Overexpression of a constitutively active mutant of a component of the Ras–ERK pathway, i.e., H-Ras, B-Raf, or MEK1, interfered with EMT. Knockdown of DA-Raf expression with siRNAs facilitated the activity of MEK and ERK, which were only weakly and transiently activated by TGF-β1. Although DA-Raf knockdown abrogated TGF-β1-induced EMT, the abrogation of EMT was reversed by the addition of the MEK inhibitor. Furthermore, DA-Raf knockdown impaired the TGF-β1-induced nuclear translocation of Smad2, which mediates the transcription required for EMT. These results imply that intrinsic DA-Raf exerts essential functions for EMT by antagonizing the TGF-β1-induced Ras–ERK pathway in RLE-6TN cells.
Highlights
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible, and usually lethal lung disease characterized by interstitial fibrosis of unknown pathogenesis [1,2,3]
These studies use human cells and tissues from IPF patient lungs; rodent lung models treated with bleomycin or transforming growth factor-β1 (TGF-β1); and rodent primary cultured AEC2s or rat AEC2 cell line RLE-6TN (RLE) cells treated with TGF-β1, endothelin-1 (ET-1), or cultured on fibronectin
Smad signaling is indispensable for TGF-β-induced epithelial—mesenchymal transition (EMT), it has remained controversial whether the Ras—ERK pathway is required for or interferes with EMT
Summary
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible, and usually lethal lung disease characterized by interstitial fibrosis of unknown pathogenesis [1,2,3]. A growing body of evidence indicates that the disease is the result of a fibrotic response driven by abnormally activated alveolar epithelial cells (AECs) These cells produce mediators that induce the formation of myofibroblast foci through the proliferation and activation of resident fibroblasts, attraction of circulating fibrocytes, and stimulation of epithelial—mesenchymal transition (EMT). Several recent studies have supported the notion that myofibroblasts or fibroblasts generated by EMT of type 2 AECs (AEC2s) are, at least in part, responsible for pulmonary fibrosis [4,5,6,7,8] These studies use human cells and tissues from IPF patient lungs; rodent lung models treated with bleomycin or transforming growth factor-β1 (TGF-β1); and rodent primary cultured AEC2s or rat AEC2 cell line RLE-6TN (RLE) cells treated with TGF-β1, endothelin-1 (ET-1), or cultured on fibronectin. EMT by the ET-1 treatment or culture on fibronectin is mediated by the induction of TGF- β 1 signaling
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
