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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

FGF1 amplifies TGFβ-1-induced EMT in mammary epithelial cells.Starved MCF10A cells were treated with or without 5 ng/ml TGF-β1 in the absence or presence of 50 ng/ml FGF1 for 48 h. A, Cell lysates were applied for Western blotting with the indicated antibodies. GAPDH and total ERK1/2 were used as a loading control. B, Total RNA was extracted and revers transcribed to cDNA. N-cadherin and vimentin, Snail1 and Snail2 expressions were assessed by real-time RT-PCR. C, Cell lysates were applied for Western blotting with the indicated antibodies. D, Matrigel invasion assay was performed on MCF10A cells following treatment with 5 ng/ml TGF-β1 in the presence or absence of FGF1. E, Conditioned media were applied for gelatin zymography and MMP2 activity were assessed by gelatin digestion in the gel. F, MCF12A cells were starved and treated with or without 5 ng/ml TGF-β1 in the absence or presence of 50 ng/ml FGF1 for 48 h. Western blotting was performed with the indicated antibodies. Data represent the mean ± S.E. (n = 3; *, p < 0.05). Bands intensity was measured by densitometry.
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pone.0137486.g001: FGF1 amplifies TGFβ-1-induced EMT in mammary epithelial cells.Starved MCF10A cells were treated with or without 5 ng/ml TGF-β1 in the absence or presence of 50 ng/ml FGF1 for 48 h. A, Cell lysates were applied for Western blotting with the indicated antibodies. GAPDH and total ERK1/2 were used as a loading control. B, Total RNA was extracted and revers transcribed to cDNA. N-cadherin and vimentin, Snail1 and Snail2 expressions were assessed by real-time RT-PCR. C, Cell lysates were applied for Western blotting with the indicated antibodies. D, Matrigel invasion assay was performed on MCF10A cells following treatment with 5 ng/ml TGF-β1 in the presence or absence of FGF1. E, Conditioned media were applied for gelatin zymography and MMP2 activity were assessed by gelatin digestion in the gel. F, MCF12A cells were starved and treated with or without 5 ng/ml TGF-β1 in the absence or presence of 50 ng/ml FGF1 for 48 h. Western blotting was performed with the indicated antibodies. Data represent the mean ± S.E. (n = 3; *, p < 0.05). Bands intensity was measured by densitometry.

Mentions: We studied the effect of FGF1 on TGF-β1-induced EMT in MCF10A human mammary epithelial cells. Serum-starved MCF10A cells were incubated for 48 h in serum-free medium containing TGF-β1 with or without 50 ng/ml FGF1. EMT markers, N-cadherin, PAI-1 and vimentin levels were upregulated by TGF-β1 in MCF10A cells. Interestingly, combined stimulation of TGF-β1 plus FGF1 enhanced the protein and mRNA levels of these proteins (Fig 1A and 1B).


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)

FGF1 amplifies TGFβ-1-induced EMT in mammary epithelial cells.Starved MCF10A cells were treated with or without 5 ng/ml TGF-β1 in the absence or presence of 50 ng/ml FGF1 for 48 h. A, Cell lysates were applied for Western blotting with the indicated antibodies. GAPDH and total ERK1/2 were used as a loading control. B, Total RNA was extracted and revers transcribed to cDNA. N-cadherin and vimentin, Snail1 and Snail2 expressions were assessed by real-time RT-PCR. C, Cell lysates were applied for Western blotting with the indicated antibodies. D, Matrigel invasion assay was performed on MCF10A cells following treatment with 5 ng/ml TGF-β1 in the presence or absence of FGF1. E, Conditioned media were applied for gelatin zymography and MMP2 activity were assessed by gelatin digestion in the gel. F, MCF12A cells were starved and treated with or without 5 ng/ml TGF-β1 in the absence or presence of 50 ng/ml FGF1 for 48 h. Western blotting was performed with the indicated antibodies. Data represent the mean ± S.E. (n = 3; *, p < 0.05). Bands intensity was measured by densitometry.
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Related In: Results  -  Collection

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pone.0137486.g001: FGF1 amplifies TGFβ-1-induced EMT in mammary epithelial cells.Starved MCF10A cells were treated with or without 5 ng/ml TGF-β1 in the absence or presence of 50 ng/ml FGF1 for 48 h. A, Cell lysates were applied for Western blotting with the indicated antibodies. GAPDH and total ERK1/2 were used as a loading control. B, Total RNA was extracted and revers transcribed to cDNA. N-cadherin and vimentin, Snail1 and Snail2 expressions were assessed by real-time RT-PCR. C, Cell lysates were applied for Western blotting with the indicated antibodies. D, Matrigel invasion assay was performed on MCF10A cells following treatment with 5 ng/ml TGF-β1 in the presence or absence of FGF1. E, Conditioned media were applied for gelatin zymography and MMP2 activity were assessed by gelatin digestion in the gel. F, MCF12A cells were starved and treated with or without 5 ng/ml TGF-β1 in the absence or presence of 50 ng/ml FGF1 for 48 h. Western blotting was performed with the indicated antibodies. Data represent the mean ± S.E. (n = 3; *, p < 0.05). Bands intensity was measured by densitometry.
Mentions: We studied the effect of FGF1 on TGF-β1-induced EMT in MCF10A human mammary epithelial cells. Serum-starved MCF10A cells were incubated for 48 h in serum-free medium containing TGF-β1 with or without 50 ng/ml FGF1. EMT markers, N-cadherin, PAI-1 and vimentin levels were upregulated by TGF-β1 in MCF10A cells. Interestingly, combined stimulation of TGF-β1 plus FGF1 enhanced the protein and mRNA levels of these proteins (Fig 1A and 1B).

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