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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.

Watanabe-Takano H, Takano K, Hatano M, Tokuhisa T, Endo T - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Inhibition of MEK but not PI3K recovers TGF-β1-induced and FGF2-suppressed EMT.(A) Recovery of FGF2-suppressed αSMA expression by MEK inhibition but not by PI3K inhibition. RLE cells were pretreated with 10 μM of the MEK inhibitor U0126 or the PI3K inhibitor LY294002 for 30 min. Then they were stimulated with 0.5 ng/ml TGF-β1 along with 100 ng/ml FGF2 for 48 h. αSMA expression (red) and nuclei (blue) were detected as described in Fig 1 legend. Scale bar, 50 μm. (B) The ratio of αSMA-expressing cells in the analysis of (A). The values are means ± SD of 3 independent experiments. **, P < 0.01; #, P > 0.05 (not significant) by t test.
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pone.0127888.g002: Inhibition of MEK but not PI3K recovers TGF-β1-induced and FGF2-suppressed EMT.(A) Recovery of FGF2-suppressed αSMA expression by MEK inhibition but not by PI3K inhibition. RLE cells were pretreated with 10 μM of the MEK inhibitor U0126 or the PI3K inhibitor LY294002 for 30 min. Then they were stimulated with 0.5 ng/ml TGF-β1 along with 100 ng/ml FGF2 for 48 h. αSMA expression (red) and nuclei (blue) were detected as described in Fig 1 legend. Scale bar, 50 μm. (B) The ratio of αSMA-expressing cells in the analysis of (A). The values are means ± SD of 3 independent experiments. **, P < 0.01; #, P > 0.05 (not significant) by t test.

Mentions: Growth factors activate not only the Ras—ERK pathway but also PI3K—Akt signaling [31]. TGF- β also induces both the Ras—ERK pathway and PI3K—Akt signaling as non-Smad signaling [16, 17]. To determine whether only the Ras—ERK pathway or whether both the Ras—ERK pathway and PI3K—Akt signaling are responsible for the FGF2-caused suppression of EMT, we analyzed the effects of inhibitors of these signaling pathways in RLE cells. Treatment of the cells with the MEK inhibitor U0126 recovered the stress fiber-associated αSMA expression that was suppressed by FGF2 stimulation (Fig 2A). U0126 treatment also restored the ratio of αSMA-expressing cells nearly to the ratio induced by TGF- β 1 (Fig 2B). In contrast, treatment with the PI3K inhibitor LY294002 had no detectable effect on FGF2-caused suppression of αSMA expression (Fig 2A and 2B). Consequently, FGF2-induced strong and sustained activation of the Ras—ERK pathway (see Fig 1B) but not PI3K—Akt signaling hinders EMT. We also tried to evaluate the effects of TGF- β 1-induced weak and transient activation of the Ras—ERK pathway and PI3K—Akt signaling on EMT by adding U0126 and LY294002, respectively. However, addition of either inhibitor together with TGF- β 1 treatment resulted in extensive apoptosis. Thus, we could not estimate the effects on these signaling pathways (data not shown, see discussion).


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

Watanabe-Takano H, Takano K, Hatano M, Tokuhisa T, Endo T - PLoS ONE (2015)

Inhibition of MEK but not PI3K recovers TGF-β1-induced and FGF2-suppressed EMT.(A) Recovery of FGF2-suppressed αSMA expression by MEK inhibition but not by PI3K inhibition. RLE cells were pretreated with 10 μM of the MEK inhibitor U0126 or the PI3K inhibitor LY294002 for 30 min. Then they were stimulated with 0.5 ng/ml TGF-β1 along with 100 ng/ml FGF2 for 48 h. αSMA expression (red) and nuclei (blue) were detected as described in Fig 1 legend. Scale bar, 50 μm. (B) The ratio of αSMA-expressing cells in the analysis of (A). The values are means ± SD of 3 independent experiments. **, P < 0.01; #, P > 0.05 (not significant) by t test.
© Copyright Policy
Related In: Results  -  Collection

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pone.0127888.g002: Inhibition of MEK but not PI3K recovers TGF-β1-induced and FGF2-suppressed EMT.(A) Recovery of FGF2-suppressed αSMA expression by MEK inhibition but not by PI3K inhibition. RLE cells were pretreated with 10 μM of the MEK inhibitor U0126 or the PI3K inhibitor LY294002 for 30 min. Then they were stimulated with 0.5 ng/ml TGF-β1 along with 100 ng/ml FGF2 for 48 h. αSMA expression (red) and nuclei (blue) were detected as described in Fig 1 legend. Scale bar, 50 μm. (B) The ratio of αSMA-expressing cells in the analysis of (A). The values are means ± SD of 3 independent experiments. **, P < 0.01; #, P > 0.05 (not significant) by t test.
Mentions: Growth factors activate not only the Ras—ERK pathway but also PI3K—Akt signaling [31]. TGF- β also induces both the Ras—ERK pathway and PI3K—Akt signaling as non-Smad signaling [16, 17]. To determine whether only the Ras—ERK pathway or whether both the Ras—ERK pathway and PI3K—Akt signaling are responsible for the FGF2-caused suppression of EMT, we analyzed the effects of inhibitors of these signaling pathways in RLE cells. Treatment of the cells with the MEK inhibitor U0126 recovered the stress fiber-associated αSMA expression that was suppressed by FGF2 stimulation (Fig 2A). U0126 treatment also restored the ratio of αSMA-expressing cells nearly to the ratio induced by TGF- β 1 (Fig 2B). In contrast, treatment with the PI3K inhibitor LY294002 had no detectable effect on FGF2-caused suppression of αSMA expression (Fig 2A and 2B). Consequently, FGF2-induced strong and sustained activation of the Ras—ERK pathway (see Fig 1B) but not PI3K—Akt signaling hinders EMT. We also tried to evaluate the effects of TGF- β 1-induced weak and transient activation of the Ras—ERK pathway and PI3K—Akt signaling on EMT by adding U0126 and LY294002, respectively. However, addition of either inhibitor together with TGF- β 1 treatment resulted in extensive apoptosis. Thus, we could not estimate the effects on these signaling pathways (data not shown, see discussion).

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus