title: DA-Raf-Mediated Suppression of the Ras--ERK Pathway Is Essential for TGF-β1-Induced Epithelial-Mesenchymal Transition in Alveolar Epithelial Type 2 Cells.
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.
authors.affiliation: Department of Biology, Graduate School of Science, Chiba University, Inage-ku, Chiba, Japan; Biomedical Research Center, Chiba University, Chuo-ku, Chiba, Japan; Japan Society for the Promotion of Science (JSPS), Chiyoda-ku, Tokyo, Japan.
authors.forename: Haruko
authors.initials: H
authors.lastname: Watanabe-Takano
authors.affiliation: Department of Biology, Graduate School of Science, Chiba University, Inage-ku, Chiba, Japan; Department of Nanobiology, Graduate School of Advanced Integral Science, Chiba University, Inage-ku, Chiba, Japan.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Takano
authors.affiliation: Biomedical Research Center, Chiba University, Chuo-ku, Chiba, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Hatano
authors.affiliation: Department of Developmental Genetics, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tokuhisa
authors.affiliation: Department of Biology, Graduate School of Science, Chiba University, Inage-ku, Chiba, Japan; Department of Nanobiology, Graduate School of Advanced Integral Science, Chiba University, Inage-ku, Chiba, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Endo
mesh_terms: D056809:Alveolar Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects / Q000235:genetics; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D048491:Proto-Oncogene Proteins A-raf / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D053773:Transforming Growth Factor beta1; D016222:Fibroblast Growth Factor 2; D019869:Phosphatidylinositol 3-Kinases; C521643:DA-Raf1 protein, mouse; D048491:Proto-Oncogene Proteins A-raf; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D018631:ras Proteins