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PLoS One. 2015 May 21;10(5):e0127888. doi: 10.1371/journal.pone.0127888. eCollection 2015.

DA-Raf-Mediated Suppression of the Ras--ERK Pathway Is Essential for TGF-β1-Induced Epithelial-Mesenchymal Transition in Alveolar Epithelial Type 2 Cells.

Author information

1
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.
2
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.
3
Biomedical Research Center, Chiba University, Chuo-ku, Chiba, Japan.
4
Department of Developmental Genetics, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba, Japan.

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.

PMID:
25996975
PMCID:
PMC4440819
DOI:
10.1371/journal.pone.0127888
[Indexed for MEDLINE]
Free PMC Article

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