Collagen regulates transforming growth factor-β receptors of HL-1 cardiomyocytes through activation of stretch and integrin signaling

Yen Yu Lu, Yung Kuo Lin, Yu Hsun Kao, Cheng Chih Chung, Yung Hsin Yeh, Shih Ann Chen, Yi Jen Chen

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Abstract

The extracellular matrix (ECM) and transforming growth factor-β (TGF)-β are important in cardiac fibrosis, however, the effects of the ECM on TGF-β signaling remain to be fully elucidated. The aims of the present study were to evaluate the role of collagen in TGF-β signaling and examine the underlying mechanisms. In the present study, western blot analysis was used to examine TGF-β signaling in HL-1 cells treated with and without (control) type I collagen (10 μg/ml), which was co-administered with either an anti-β1 integrin antibody (10 μg/ml) or a stretch-activated channel inhibitor (gadolinium; 50 μM). Cell proliferation and adhesion assays were used to investigate the roles of integrin, mechanical stretch and mitogen-activated protein kinases (MAPKs) on cell proliferation and adhesion. The type I collagen (10 μg/ml)-treated HL-1 cells were incubated with or without anti-β1 integrin antibody (10 μg/ml), gadolinium (50 μM) or inhibitors of p38 (SB203580; 3 μM), extracellular signal-regulated kinase (ERK; PD98059; 50 μM) and c-Jun N-terminal kinase (JNK; SP600125; 50 μM). Compared with the control cells, the collagen-treated HL-1 cells had lower expression levels of type I and type II TGF-β receptors (TGFβRI and TGFβRII), with an increase in phosphorylated focal adhesion kinase (FAK), p38 and ERK1/2, and a decrease in JNK. Incubation with the anti-β1 integrin antibody reversed the collagen-induced downregulation of the expression of TGFβRII and phosphorylated FAK. Gadolinium downregulated the expression levels of TGFβRI and small mothers against decapentaplegic (Smad)2/3, and decreased the levels of phosphorylated p38, ERK1/2 and JNK. In addition, gadolinium reversed the collagen-induced activation of p38 and ERK1/2. In the presence of gadolinium and anti-β1 integrin antibody, collagen regulated the expression levels of TGFβRI, TGFβRII and Smad2/3, but did not alter the phosphorylation of p38, ERK1/2 or JNK. In addition, collagen increased cell proliferation and adhesion, and this collagen-induced cell proliferation was inhibited by the anti-β1 integrin antibody and ERK inhibitor. Taken together, the data obtained suggested that collagen differentially regulated the expression levels of TGFβRI and TGFβRII, and modulated the phosphorylation of MAPKs through integrin- or stretch-dependent and -independent signaling pathways.

Original languageEnglish
Pages (from-to)3429-3436
Number of pages8
JournalMolecular Medicine Reports
Volume14
Issue number4
DOIs
Publication statusPublished - Oct 1 2016

Fingerprint

Growth Factor Receptors
Transforming Growth Factors
Cardiac Myocytes
Integrins
Collagen
Chemical activation
Gadolinium
Cell proliferation
Cell adhesion
Cell Proliferation
Collagen Type X
Cell Adhesion
Antibodies
Focal Adhesion Protein-Tyrosine Kinases
Phosphorylation
Collagen Type I
Mitogen-Activated Protein Kinases
Extracellular Matrix
Mitogen-Activated Protein Kinase 6
Down-Regulation

Keywords

  • Collagen
  • Integrin
  • Mechanical stretch
  • Mitogen-activated protein kinases
  • Transforming growth factor-β

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Oncology
  • Genetics
  • Cancer Research

Cite this

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title = "Collagen regulates transforming growth factor-β receptors of HL-1 cardiomyocytes through activation of stretch and integrin signaling",
abstract = "The extracellular matrix (ECM) and transforming growth factor-β (TGF)-β are important in cardiac fibrosis, however, the effects of the ECM on TGF-β signaling remain to be fully elucidated. The aims of the present study were to evaluate the role of collagen in TGF-β signaling and examine the underlying mechanisms. In the present study, western blot analysis was used to examine TGF-β signaling in HL-1 cells treated with and without (control) type I collagen (10 μg/ml), which was co-administered with either an anti-β1 integrin antibody (10 μg/ml) or a stretch-activated channel inhibitor (gadolinium; 50 μM). Cell proliferation and adhesion assays were used to investigate the roles of integrin, mechanical stretch and mitogen-activated protein kinases (MAPKs) on cell proliferation and adhesion. The type I collagen (10 μg/ml)-treated HL-1 cells were incubated with or without anti-β1 integrin antibody (10 μg/ml), gadolinium (50 μM) or inhibitors of p38 (SB203580; 3 μM), extracellular signal-regulated kinase (ERK; PD98059; 50 μM) and c-Jun N-terminal kinase (JNK; SP600125; 50 μM). Compared with the control cells, the collagen-treated HL-1 cells had lower expression levels of type I and type II TGF-β receptors (TGFβRI and TGFβRII), with an increase in phosphorylated focal adhesion kinase (FAK), p38 and ERK1/2, and a decrease in JNK. Incubation with the anti-β1 integrin antibody reversed the collagen-induced downregulation of the expression of TGFβRII and phosphorylated FAK. Gadolinium downregulated the expression levels of TGFβRI and small mothers against decapentaplegic (Smad)2/3, and decreased the levels of phosphorylated p38, ERK1/2 and JNK. In addition, gadolinium reversed the collagen-induced activation of p38 and ERK1/2. In the presence of gadolinium and anti-β1 integrin antibody, collagen regulated the expression levels of TGFβRI, TGFβRII and Smad2/3, but did not alter the phosphorylation of p38, ERK1/2 or JNK. In addition, collagen increased cell proliferation and adhesion, and this collagen-induced cell proliferation was inhibited by the anti-β1 integrin antibody and ERK inhibitor. Taken together, the data obtained suggested that collagen differentially regulated the expression levels of TGFβRI and TGFβRII, and modulated the phosphorylation of MAPKs through integrin- or stretch-dependent and -independent signaling pathways.",
keywords = "Collagen, Integrin, Mechanical stretch, Mitogen-activated protein kinases, Transforming growth factor-β",
author = "Lu, {Yen Yu} and Lin, {Yung Kuo} and Kao, {Yu Hsun} and Chung, {Cheng Chih} and Yeh, {Yung Hsin} and Chen, {Shih Ann} and Chen, {Yi Jen}",
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T1 - Collagen regulates transforming growth factor-β receptors of HL-1 cardiomyocytes through activation of stretch and integrin signaling

AU - Lu, Yen Yu

AU - Lin, Yung Kuo

AU - Kao, Yu Hsun

AU - Chung, Cheng Chih

AU - Yeh, Yung Hsin

AU - Chen, Shih Ann

AU - Chen, Yi Jen

PY - 2016/10/1

Y1 - 2016/10/1

N2 - The extracellular matrix (ECM) and transforming growth factor-β (TGF)-β are important in cardiac fibrosis, however, the effects of the ECM on TGF-β signaling remain to be fully elucidated. The aims of the present study were to evaluate the role of collagen in TGF-β signaling and examine the underlying mechanisms. In the present study, western blot analysis was used to examine TGF-β signaling in HL-1 cells treated with and without (control) type I collagen (10 μg/ml), which was co-administered with either an anti-β1 integrin antibody (10 μg/ml) or a stretch-activated channel inhibitor (gadolinium; 50 μM). Cell proliferation and adhesion assays were used to investigate the roles of integrin, mechanical stretch and mitogen-activated protein kinases (MAPKs) on cell proliferation and adhesion. The type I collagen (10 μg/ml)-treated HL-1 cells were incubated with or without anti-β1 integrin antibody (10 μg/ml), gadolinium (50 μM) or inhibitors of p38 (SB203580; 3 μM), extracellular signal-regulated kinase (ERK; PD98059; 50 μM) and c-Jun N-terminal kinase (JNK; SP600125; 50 μM). Compared with the control cells, the collagen-treated HL-1 cells had lower expression levels of type I and type II TGF-β receptors (TGFβRI and TGFβRII), with an increase in phosphorylated focal adhesion kinase (FAK), p38 and ERK1/2, and a decrease in JNK. Incubation with the anti-β1 integrin antibody reversed the collagen-induced downregulation of the expression of TGFβRII and phosphorylated FAK. Gadolinium downregulated the expression levels of TGFβRI and small mothers against decapentaplegic (Smad)2/3, and decreased the levels of phosphorylated p38, ERK1/2 and JNK. In addition, gadolinium reversed the collagen-induced activation of p38 and ERK1/2. In the presence of gadolinium and anti-β1 integrin antibody, collagen regulated the expression levels of TGFβRI, TGFβRII and Smad2/3, but did not alter the phosphorylation of p38, ERK1/2 or JNK. In addition, collagen increased cell proliferation and adhesion, and this collagen-induced cell proliferation was inhibited by the anti-β1 integrin antibody and ERK inhibitor. Taken together, the data obtained suggested that collagen differentially regulated the expression levels of TGFβRI and TGFβRII, and modulated the phosphorylation of MAPKs through integrin- or stretch-dependent and -independent signaling pathways.

AB - The extracellular matrix (ECM) and transforming growth factor-β (TGF)-β are important in cardiac fibrosis, however, the effects of the ECM on TGF-β signaling remain to be fully elucidated. The aims of the present study were to evaluate the role of collagen in TGF-β signaling and examine the underlying mechanisms. In the present study, western blot analysis was used to examine TGF-β signaling in HL-1 cells treated with and without (control) type I collagen (10 μg/ml), which was co-administered with either an anti-β1 integrin antibody (10 μg/ml) or a stretch-activated channel inhibitor (gadolinium; 50 μM). Cell proliferation and adhesion assays were used to investigate the roles of integrin, mechanical stretch and mitogen-activated protein kinases (MAPKs) on cell proliferation and adhesion. The type I collagen (10 μg/ml)-treated HL-1 cells were incubated with or without anti-β1 integrin antibody (10 μg/ml), gadolinium (50 μM) or inhibitors of p38 (SB203580; 3 μM), extracellular signal-regulated kinase (ERK; PD98059; 50 μM) and c-Jun N-terminal kinase (JNK; SP600125; 50 μM). Compared with the control cells, the collagen-treated HL-1 cells had lower expression levels of type I and type II TGF-β receptors (TGFβRI and TGFβRII), with an increase in phosphorylated focal adhesion kinase (FAK), p38 and ERK1/2, and a decrease in JNK. Incubation with the anti-β1 integrin antibody reversed the collagen-induced downregulation of the expression of TGFβRII and phosphorylated FAK. Gadolinium downregulated the expression levels of TGFβRI and small mothers against decapentaplegic (Smad)2/3, and decreased the levels of phosphorylated p38, ERK1/2 and JNK. In addition, gadolinium reversed the collagen-induced activation of p38 and ERK1/2. In the presence of gadolinium and anti-β1 integrin antibody, collagen regulated the expression levels of TGFβRI, TGFβRII and Smad2/3, but did not alter the phosphorylation of p38, ERK1/2 or JNK. In addition, collagen increased cell proliferation and adhesion, and this collagen-induced cell proliferation was inhibited by the anti-β1 integrin antibody and ERK inhibitor. Taken together, the data obtained suggested that collagen differentially regulated the expression levels of TGFβRI and TGFβRII, and modulated the phosphorylation of MAPKs through integrin- or stretch-dependent and -independent signaling pathways.

KW - Collagen

KW - Integrin

KW - Mechanical stretch

KW - Mitogen-activated protein kinases

KW - Transforming growth factor-β

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