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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

Knockdown of DA-Raf abrogates TGF-β1-induced EMT.(A) Suppression of TGF-β1-induced αSMA expression by knockdown of DA-Raf with DAraf siRNAs. RLE cells were transfected with DAraf siRNAs as well as a control siRNA. Twenty-four hours after the transfection, they were treated with 0.5 ng/ml TGF-β1 for 48 h. The levels of DA-Raf, A-Raf, αSMA, and E-cadherin, as well as β-tubulin as a standard, were analyzed by immunoblotting. (B) Suppression of TGF-β1-induced αSMA expression with DAraf siRNAs. RLE cells were transfected with DAraf siRNAs and treated with 0.5 ng/ml TGF-β1. αSMA expression (red) and nuclei (blue) were detected by fluorescence microscopy. Scale bar, 50 μm. (C) The ratio of αSMA-expressing cells in the analysis of (B). The values are means ± SD of 3 independent experiments. **, P < 0.01 by t test. (D) Elevation of the TGF-β1-suppressed Cdh1 (E-cadherin) mRNA level and suppression of the TGF-β1-induced Acta2 (αSMA) mRNA level with DAraf siRNAs. RLE cells were transfected with DAraf siRNAs and treated with TGF-β1 for 48 h. Relative levels of Cdh1 and Acta2 mRNAs normalized to the Actb (β-actin) mRNA level were determined by real-time PCR. The values are means ± SD of 3 independent experiments. **, P < 0.01 by t test. (E) Dose-dependent induction of αSMA expression by TGF-β1 and its suppression by DAraf siRNAs. RLE cells were transfected with DAraf siRNAs and treated with 0.1–5 ng/ml TGF-β1 for 48 h. The intensities of αSMA and β-tubulin bands on immunoblots were analyzed by densitometry. The graph shows the ratio of αSMA to β-tubulin band intensity against TGF-β1 concentration. The values are means ± SD of 3 independent experiments. a.u., arbitrary units.
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pone.0127888.g004: Knockdown of DA-Raf abrogates TGF-β1-induced EMT.(A) Suppression of TGF-β1-induced αSMA expression by knockdown of DA-Raf with DAraf siRNAs. RLE cells were transfected with DAraf siRNAs as well as a control siRNA. Twenty-four hours after the transfection, they were treated with 0.5 ng/ml TGF-β1 for 48 h. The levels of DA-Raf, A-Raf, αSMA, and E-cadherin, as well as β-tubulin as a standard, were analyzed by immunoblotting. (B) Suppression of TGF-β1-induced αSMA expression with DAraf siRNAs. RLE cells were transfected with DAraf siRNAs and treated with 0.5 ng/ml TGF-β1. αSMA expression (red) and nuclei (blue) were detected by fluorescence microscopy. Scale bar, 50 μm. (C) The ratio of αSMA-expressing cells in the analysis of (B). The values are means ± SD of 3 independent experiments. **, P < 0.01 by t test. (D) Elevation of the TGF-β1-suppressed Cdh1 (E-cadherin) mRNA level and suppression of the TGF-β1-induced Acta2 (αSMA) mRNA level with DAraf siRNAs. RLE cells were transfected with DAraf siRNAs and treated with TGF-β1 for 48 h. Relative levels of Cdh1 and Acta2 mRNAs normalized to the Actb (β-actin) mRNA level were determined by real-time PCR. The values are means ± SD of 3 independent experiments. **, P < 0.01 by t test. (E) Dose-dependent induction of αSMA expression by TGF-β1 and its suppression by DAraf siRNAs. RLE cells were transfected with DAraf siRNAs and treated with 0.1–5 ng/ml TGF-β1 for 48 h. The intensities of αSMA and β-tubulin bands on immunoblots were analyzed by densitometry. The graph shows the ratio of αSMA to β-tubulin band intensity against TGF-β1 concentration. The values are means ± SD of 3 independent experiments. a.u., arbitrary units.

Mentions: Although FGF2-induced strong and sustained activation of the Ras—ERK pathway hindered EMT, TGF-β1-induced weak and transient activation of the Ras—ERK pathway did not affect EMT (see Fig 1A–1E). Thus, regulatory mechanisms of the weak and transient activation of the Ras—ERK pathway induced by TGF-β1 need to be elucidated. Accordingly, we addressed the question of whether DA-Raf, an intrinsic antagonist of the Ras—ERK pathway [28, 29], negatively regulates the TGF-β1-induced Ras—ERK pathway to a level that does not suppress EMT. RLE cells highly expressed DA-Raf regardless of treatment with TGF-β1 (Fig 4A). To determine whether DA-Raf participates in EMT, we knocked down DA-Raf expression by RNA interference (RNAi) using two distinct small interfering RNAs (siRNAs) of DAraf. Both these siRNAs target the 3’ untranslated region of DAraf mRNA because this is the only region not included in Araf mRNA, which is generated by alternative splicing of common Araf/DAraf pre-mRNA [28]. Transfection of either of these two siRNAs in RLE cells strikingly reduced the level of endogenous DA-Raf protein, whereas neither affected the A-Raf protein level (Fig 4A). Thus, these siRNAs specifically knocked down DA-Raf expression without showing off-target effects on A-Raf expression. The knockdown of DA-Raf strongly suppressed TGF-β1-induced elevation of αSMA expression to a level comparable to that of the control cells without TGF-β1 treatment (Fig 4A). In addition, immunofluorescent staining of αSMA showed that the introduction of either of these two siRNAs strikingly reduced the number of αSMA-expressing cells induced by TGF-β1 treatment (Fig 4B and 4C). Since EMT in RLE cells is accompanied by a decrease in epithelial cell marker E-cadherin as well as an increase in myofibroblast marker αSMA [27], we also analyzed the effect of DA-Raf knockdown on E-cadherin expression. TGF-β1 treatment reduced the E-cadherin protein level, whereas the level was hardly affected in DAraf siRNA-introduced cells (Fig 4A).


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)

Knockdown of DA-Raf abrogates TGF-β1-induced EMT.(A) Suppression of TGF-β1-induced αSMA expression by knockdown of DA-Raf with DAraf siRNAs. RLE cells were transfected with DAraf siRNAs as well as a control siRNA. Twenty-four hours after the transfection, they were treated with 0.5 ng/ml TGF-β1 for 48 h. The levels of DA-Raf, A-Raf, αSMA, and E-cadherin, as well as β-tubulin as a standard, were analyzed by immunoblotting. (B) Suppression of TGF-β1-induced αSMA expression with DAraf siRNAs. RLE cells were transfected with DAraf siRNAs and treated with 0.5 ng/ml TGF-β1. αSMA expression (red) and nuclei (blue) were detected by fluorescence microscopy. Scale bar, 50 μm. (C) The ratio of αSMA-expressing cells in the analysis of (B). The values are means ± SD of 3 independent experiments. **, P < 0.01 by t test. (D) Elevation of the TGF-β1-suppressed Cdh1 (E-cadherin) mRNA level and suppression of the TGF-β1-induced Acta2 (αSMA) mRNA level with DAraf siRNAs. RLE cells were transfected with DAraf siRNAs and treated with TGF-β1 for 48 h. Relative levels of Cdh1 and Acta2 mRNAs normalized to the Actb (β-actin) mRNA level were determined by real-time PCR. The values are means ± SD of 3 independent experiments. **, P < 0.01 by t test. (E) Dose-dependent induction of αSMA expression by TGF-β1 and its suppression by DAraf siRNAs. RLE cells were transfected with DAraf siRNAs and treated with 0.1–5 ng/ml TGF-β1 for 48 h. The intensities of αSMA and β-tubulin bands on immunoblots were analyzed by densitometry. The graph shows the ratio of αSMA to β-tubulin band intensity against TGF-β1 concentration. The values are means ± SD of 3 independent experiments. a.u., arbitrary units.
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Related In: Results  -  Collection

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Show All Figures
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pone.0127888.g004: Knockdown of DA-Raf abrogates TGF-β1-induced EMT.(A) Suppression of TGF-β1-induced αSMA expression by knockdown of DA-Raf with DAraf siRNAs. RLE cells were transfected with DAraf siRNAs as well as a control siRNA. Twenty-four hours after the transfection, they were treated with 0.5 ng/ml TGF-β1 for 48 h. The levels of DA-Raf, A-Raf, αSMA, and E-cadherin, as well as β-tubulin as a standard, were analyzed by immunoblotting. (B) Suppression of TGF-β1-induced αSMA expression with DAraf siRNAs. RLE cells were transfected with DAraf siRNAs and treated with 0.5 ng/ml TGF-β1. αSMA expression (red) and nuclei (blue) were detected by fluorescence microscopy. Scale bar, 50 μm. (C) The ratio of αSMA-expressing cells in the analysis of (B). The values are means ± SD of 3 independent experiments. **, P < 0.01 by t test. (D) Elevation of the TGF-β1-suppressed Cdh1 (E-cadherin) mRNA level and suppression of the TGF-β1-induced Acta2 (αSMA) mRNA level with DAraf siRNAs. RLE cells were transfected with DAraf siRNAs and treated with TGF-β1 for 48 h. Relative levels of Cdh1 and Acta2 mRNAs normalized to the Actb (β-actin) mRNA level were determined by real-time PCR. The values are means ± SD of 3 independent experiments. **, P < 0.01 by t test. (E) Dose-dependent induction of αSMA expression by TGF-β1 and its suppression by DAraf siRNAs. RLE cells were transfected with DAraf siRNAs and treated with 0.1–5 ng/ml TGF-β1 for 48 h. The intensities of αSMA and β-tubulin bands on immunoblots were analyzed by densitometry. The graph shows the ratio of αSMA to β-tubulin band intensity against TGF-β1 concentration. The values are means ± SD of 3 independent experiments. a.u., arbitrary units.
Mentions: Although FGF2-induced strong and sustained activation of the Ras—ERK pathway hindered EMT, TGF-β1-induced weak and transient activation of the Ras—ERK pathway did not affect EMT (see Fig 1A–1E). Thus, regulatory mechanisms of the weak and transient activation of the Ras—ERK pathway induced by TGF-β1 need to be elucidated. Accordingly, we addressed the question of whether DA-Raf, an intrinsic antagonist of the Ras—ERK pathway [28, 29], negatively regulates the TGF-β1-induced Ras—ERK pathway to a level that does not suppress EMT. RLE cells highly expressed DA-Raf regardless of treatment with TGF-β1 (Fig 4A). To determine whether DA-Raf participates in EMT, we knocked down DA-Raf expression by RNA interference (RNAi) using two distinct small interfering RNAs (siRNAs) of DAraf. Both these siRNAs target the 3’ untranslated region of DAraf mRNA because this is the only region not included in Araf mRNA, which is generated by alternative splicing of common Araf/DAraf pre-mRNA [28]. Transfection of either of these two siRNAs in RLE cells strikingly reduced the level of endogenous DA-Raf protein, whereas neither affected the A-Raf protein level (Fig 4A). Thus, these siRNAs specifically knocked down DA-Raf expression without showing off-target effects on A-Raf expression. The knockdown of DA-Raf strongly suppressed TGF-β1-induced elevation of αSMA expression to a level comparable to that of the control cells without TGF-β1 treatment (Fig 4A). In addition, immunofluorescent staining of αSMA showed that the introduction of either of these two siRNAs strikingly reduced the number of αSMA-expressing cells induced by TGF-β1 treatment (Fig 4B and 4C). Since EMT in RLE cells is accompanied by a decrease in epithelial cell marker E-cadherin as well as an increase in myofibroblast marker αSMA [27], we also analyzed the effect of DA-Raf knockdown on E-cadherin expression. TGF-β1 treatment reduced the E-cadherin protein level, whereas the level was hardly affected in DAraf siRNA-introduced cells (Fig 4A).

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