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Enhanced Expression of Integrin αvβ3 Induced by TGF-β Is Required for the Enhancing Effect of Fibroblast Growth Factor 1 (FGF1) in TGF-β-Induced Epithelial-Mesenchymal Transition (EMT) in Mammary Epithelial Cells.

Mori S, Kodaira M, Ito A, Okazaki M, Kawaguchi N, Hamada Y, Takada Y, Matsuura N - PLoS ONE (2015)

Bottom Line: We studied if the enhancing effect of FGF1 on TGF-β1-induced EMT requires enhanced levels of both integrin αvβ3 expression and FGFR1.Antagonists to FGFR suppressed the enhancing effect of FGF1 on EMT.Integrin-binding defective FGF1 mutant did not augment TGF-β1-induced EMT in MCF10A cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathology, Osaka University Graduate School of Medicine, Division of Health Sciences, 1-7 Yamada-oka, Suita-shi, Osaka, 565-0871, Japan.

ABSTRACT
Epithelial-to-mesenchymal transition (EMT) plays a critical role in cancer metastasis, and is regulated by growth factors such as transforming growth factor β (TGF-β) and fibroblast growth factors (FGF) secreted from the stromal and tumor cells. However, the role of growth factors in EMT has not been fully established. Several integrins are upregulated by TGF-β1 during EMT. Integrins are involved in growth factor signaling through integrin-growth factor receptor crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and the interaction was required for FGF1 functions such as cell proliferation and migration. We studied the role of αvβ3 induced by TGF-β on TGF-β-induced EMT. Here, we describe that FGF1 augmented EMT induced by TGF-β1 in MCF10A and MCF12A mammary epithelial cells. TGF-β1 markedly amplified integrin αvβ3 and FGFR1 (but not FGFR2). We studied if the enhancing effect of FGF1 on TGF-β1-induced EMT requires enhanced levels of both integrin αvβ3 expression and FGFR1. Knockdown of β3 suppressed the enhancement by FGF1 of TGF-β1-induced EMT in MCF10A cells. Antagonists to FGFR suppressed the enhancing effect of FGF1 on EMT. Integrin-binding defective FGF1 mutant did not augment TGF-β1-induced EMT in MCF10A cells. These findings suggest that enhanced integrin αvβ3 expression in addition to enhanced FGFR1 expression is critical for FGF1 to augment TGF-β1-induced EMT in mammary epithelial cells.

No MeSH data available.


Related in: MedlinePlus

Integrin αvβ3 and FGFR1 are induced by TGF-β1 in MCF10A cells.A, Starved MCF10A cells were stimulated with or without 5 ng/ml TGF-β1 for 48 h. Cell surface expression of integrins were determined by FACS analysis using indicated antibodies. B, We performed time-course analysis of FGFR1, FGFR2, and integrin αv and β3 expression by Western blotting. Starved MCF10A cells were stimulated with DMSO or TGF-β1 (5 ng/ml) for the indicated time periods. Cell lysates were applied for Western blotting and proved with antibodies shown in figure. GAPDH was used as loading control. C, Dose-dependent effect of TGF-β1 on integrin αvβ3 was assessed by FACS flow cytometry. MCF10A cells were treated with the indicated concentration of TGF-β1. D, Induction of integrin αvβ3 was determined by immunofluorescence microscopy. Cells were stimulated with TGF-β1 (0.5 or 5 ng/ml) for 48 h, and stained with anti-integrin αvβ3 antibody (LM609) with FITC conjugated secondary antibody to visualize (upper). Lower panels indicate representative phase contrast images of TGF-β1 stimulated cells. Scale bar = 20 μm.
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pone.0137486.g002: Integrin αvβ3 and FGFR1 are induced by TGF-β1 in MCF10A cells.A, Starved MCF10A cells were stimulated with or without 5 ng/ml TGF-β1 for 48 h. Cell surface expression of integrins were determined by FACS analysis using indicated antibodies. B, We performed time-course analysis of FGFR1, FGFR2, and integrin αv and β3 expression by Western blotting. Starved MCF10A cells were stimulated with DMSO or TGF-β1 (5 ng/ml) for the indicated time periods. Cell lysates were applied for Western blotting and proved with antibodies shown in figure. GAPDH was used as loading control. C, Dose-dependent effect of TGF-β1 on integrin αvβ3 was assessed by FACS flow cytometry. MCF10A cells were treated with the indicated concentration of TGF-β1. D, Induction of integrin αvβ3 was determined by immunofluorescence microscopy. Cells were stimulated with TGF-β1 (0.5 or 5 ng/ml) for 48 h, and stained with anti-integrin αvβ3 antibody (LM609) with FITC conjugated secondary antibody to visualize (upper). Lower panels indicate representative phase contrast images of TGF-β1 stimulated cells. Scale bar = 20 μm.

Mentions: To address this question, we first studied which integrins are induced by TGF-β1 in MCF10A cells. FACS analysis revealed that integrin αvβ3 was the only integrin induced by TGF-β1 among tested integrins (Fig 2A). We next determined the levels of integrin αvβ3 expressions by Western blotting. TGF-β1 quickly and strongly induced integrin αvβ3 (Fig 2B). We also studied the time-course of TGF- β signaling. Interestingly, 5 ng/ml TGF-β1 induced both canonical Smad pathway and non-canonical MAP kinase pathway. Smad2 phosphorylation peaked at 3 h and gradually decreased until 48 h, while phosphorylated ERK peaked at 48 h after TGF-β1 stimulation (Fig 2B). Integrin αvβ3 expression on the cell surface was assessed by flow cytometer using anti-integrin αvβ3 antibody (LM609). Integrin αvβ3 was induced by TGF-β1 dose-dependently in MCF10A cells (Fig 2C). Similar results were obtained by immuno-staining analysis. Integrin αvβ3 was localized to plasma membrane, and the level of integrin αvβ3 was much increased by TGF-β1 (Fig 2D). To clarify the morphological changes of the EMT by the TGF, we included phase contrast images in Fig 2D lower panel. These results suggest that TGF-β1 markedly induces αvβ3 in MCF10A cells.


Enhanced Expression of Integrin αvβ3 Induced by TGF-β Is Required for the Enhancing Effect of Fibroblast Growth Factor 1 (FGF1) in TGF-β-Induced Epithelial-Mesenchymal Transition (EMT) in Mammary Epithelial Cells.

Mori S, Kodaira M, Ito A, Okazaki M, Kawaguchi N, Hamada Y, Takada Y, Matsuura N - PLoS ONE (2015)

Integrin αvβ3 and FGFR1 are induced by TGF-β1 in MCF10A cells.A, Starved MCF10A cells were stimulated with or without 5 ng/ml TGF-β1 for 48 h. Cell surface expression of integrins were determined by FACS analysis using indicated antibodies. B, We performed time-course analysis of FGFR1, FGFR2, and integrin αv and β3 expression by Western blotting. Starved MCF10A cells were stimulated with DMSO or TGF-β1 (5 ng/ml) for the indicated time periods. Cell lysates were applied for Western blotting and proved with antibodies shown in figure. GAPDH was used as loading control. C, Dose-dependent effect of TGF-β1 on integrin αvβ3 was assessed by FACS flow cytometry. MCF10A cells were treated with the indicated concentration of TGF-β1. D, Induction of integrin αvβ3 was determined by immunofluorescence microscopy. Cells were stimulated with TGF-β1 (0.5 or 5 ng/ml) for 48 h, and stained with anti-integrin αvβ3 antibody (LM609) with FITC conjugated secondary antibody to visualize (upper). Lower panels indicate representative phase contrast images of TGF-β1 stimulated cells. Scale bar = 20 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4559424&req=5

pone.0137486.g002: Integrin αvβ3 and FGFR1 are induced by TGF-β1 in MCF10A cells.A, Starved MCF10A cells were stimulated with or without 5 ng/ml TGF-β1 for 48 h. Cell surface expression of integrins were determined by FACS analysis using indicated antibodies. B, We performed time-course analysis of FGFR1, FGFR2, and integrin αv and β3 expression by Western blotting. Starved MCF10A cells were stimulated with DMSO or TGF-β1 (5 ng/ml) for the indicated time periods. Cell lysates were applied for Western blotting and proved with antibodies shown in figure. GAPDH was used as loading control. C, Dose-dependent effect of TGF-β1 on integrin αvβ3 was assessed by FACS flow cytometry. MCF10A cells were treated with the indicated concentration of TGF-β1. D, Induction of integrin αvβ3 was determined by immunofluorescence microscopy. Cells were stimulated with TGF-β1 (0.5 or 5 ng/ml) for 48 h, and stained with anti-integrin αvβ3 antibody (LM609) with FITC conjugated secondary antibody to visualize (upper). Lower panels indicate representative phase contrast images of TGF-β1 stimulated cells. Scale bar = 20 μm.
Mentions: To address this question, we first studied which integrins are induced by TGF-β1 in MCF10A cells. FACS analysis revealed that integrin αvβ3 was the only integrin induced by TGF-β1 among tested integrins (Fig 2A). We next determined the levels of integrin αvβ3 expressions by Western blotting. TGF-β1 quickly and strongly induced integrin αvβ3 (Fig 2B). We also studied the time-course of TGF- β signaling. Interestingly, 5 ng/ml TGF-β1 induced both canonical Smad pathway and non-canonical MAP kinase pathway. Smad2 phosphorylation peaked at 3 h and gradually decreased until 48 h, while phosphorylated ERK peaked at 48 h after TGF-β1 stimulation (Fig 2B). Integrin αvβ3 expression on the cell surface was assessed by flow cytometer using anti-integrin αvβ3 antibody (LM609). Integrin αvβ3 was induced by TGF-β1 dose-dependently in MCF10A cells (Fig 2C). Similar results were obtained by immuno-staining analysis. Integrin αvβ3 was localized to plasma membrane, and the level of integrin αvβ3 was much increased by TGF-β1 (Fig 2D). To clarify the morphological changes of the EMT by the TGF, we included phase contrast images in Fig 2D lower panel. These results suggest that TGF-β1 markedly induces αvβ3 in MCF10A cells.

Bottom Line: We studied if the enhancing effect of FGF1 on TGF-β1-induced EMT requires enhanced levels of both integrin αvβ3 expression and FGFR1.Antagonists to FGFR suppressed the enhancing effect of FGF1 on EMT.Integrin-binding defective FGF1 mutant did not augment TGF-β1-induced EMT in MCF10A cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathology, Osaka University Graduate School of Medicine, Division of Health Sciences, 1-7 Yamada-oka, Suita-shi, Osaka, 565-0871, Japan.

ABSTRACT
Epithelial-to-mesenchymal transition (EMT) plays a critical role in cancer metastasis, and is regulated by growth factors such as transforming growth factor β (TGF-β) and fibroblast growth factors (FGF) secreted from the stromal and tumor cells. However, the role of growth factors in EMT has not been fully established. Several integrins are upregulated by TGF-β1 during EMT. Integrins are involved in growth factor signaling through integrin-growth factor receptor crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and the interaction was required for FGF1 functions such as cell proliferation and migration. We studied the role of αvβ3 induced by TGF-β on TGF-β-induced EMT. Here, we describe that FGF1 augmented EMT induced by TGF-β1 in MCF10A and MCF12A mammary epithelial cells. TGF-β1 markedly amplified integrin αvβ3 and FGFR1 (but not FGFR2). We studied if the enhancing effect of FGF1 on TGF-β1-induced EMT requires enhanced levels of both integrin αvβ3 expression and FGFR1. Knockdown of β3 suppressed the enhancement by FGF1 of TGF-β1-induced EMT in MCF10A cells. Antagonists to FGFR suppressed the enhancing effect of FGF1 on EMT. Integrin-binding defective FGF1 mutant did not augment TGF-β1-induced EMT in MCF10A cells. These findings suggest that enhanced integrin αvβ3 expression in addition to enhanced FGFR1 expression is critical for FGF1 to augment TGF-β1-induced EMT in mammary epithelial cells.

No MeSH data available.


Related in: MedlinePlus