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DA-Raf-Mediated Suppression of the Ras--ERK Pathway Is Essential for TGF-β1-Induced Epithelial-Mesenchymal Transition in Alveolar Epithelial Type 2 Cells.
PLoS One. 2015; 10(5):e0127888.Plos

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+Show Affiliations

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

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

25996975

Citation

Watanabe-Takano, Haruko, et al. "DA-Raf-Mediated Suppression of the Ras--ERK Pathway Is Essential for TGF-β1-Induced Epithelial-Mesenchymal Transition in Alveolar Epithelial Type 2 Cells." PloS One, vol. 10, no. 5, 2015, pp. e0127888.
Watanabe-Takano H, Takano K, Hatano M, et al. DA-Raf-Mediated Suppression of the Ras--ERK Pathway Is Essential for TGF-β1-Induced Epithelial-Mesenchymal Transition in Alveolar Epithelial Type 2 Cells. PLoS One. 2015;10(5):e0127888.
Watanabe-Takano, H., Takano, K., Hatano, M., Tokuhisa, T., & Endo, T. (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. PloS One, 10(5), e0127888. https://doi.org/10.1371/journal.pone.0127888
Watanabe-Takano H, et al. DA-Raf-Mediated Suppression of the Ras--ERK Pathway Is Essential for TGF-β1-Induced Epithelial-Mesenchymal Transition in Alveolar Epithelial Type 2 Cells. PLoS One. 2015;10(5):e0127888. PubMed PMID: 25996975.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - DA-Raf-Mediated Suppression of the Ras--ERK Pathway Is Essential for TGF-β1-Induced Epithelial-Mesenchymal Transition in Alveolar Epithelial Type 2 Cells. AU - Watanabe-Takano,Haruko, AU - Takano,Kazunori, AU - Hatano,Masahiko, AU - Tokuhisa,Takeshi, AU - Endo,Takeshi, Y1 - 2015/05/21/ PY - 2014/12/25/received PY - 2015/04/20/accepted PY - 2015/5/22/entrez PY - 2015/5/23/pubmed PY - 2016/3/5/medline SP - e0127888 EP - e0127888 JF - PloS one JO - PLoS One VL - 10 IS - 5 N2 - 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. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/25996975/DA_Raf_Mediated_Suppression_of_the_Ras__ERK_Pathway_Is_Essential_for_TGF_β1_Induced_Epithelial_Mesenchymal_Transition_in_Alveolar_Epithelial_Type_2_Cells_ L2 - https://dx.plos.org/10.1371/journal.pone.0127888 DB - PRIME DP - Unbound Medicine ER -