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Regulation of tenascin-C, a vascular smooth muscle cell survival factor that interacts with the alpha v beta 3 integrin to promote epidermal growth factor receptor phosphorylation and growth.

Jones PL, Crack J, Rabinovitch M - J. Cell Biol. (1997)

Bottom Line: These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of EGF-Rs, tyrosine-phosphorylated proteins, and increased activation of EGF-Rs after addition of EGF.Cross-linking SMC beta 3 integrins replicates the effect of TN-C on EGF-R clustering and tyrosine phosphorylation.Conversely, suppression of MMPs downregulates TN-C and induces apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Research, Research Institute, The Hospital for Sick Children, University of Toronto, Ontario, Canada.

ABSTRACT
Tenascin-C (TN-C) is induced in pulmonary vascular disease, where it colocalizes with proliferating smooth muscle cells (SMCs) and epidermal growth factor (EGF). Furthermore, cultured SMCs require TN-C for EGF-dependent growth on type I collagen. In this study, we explore the regulation and function of TN-C in SMCs. We show that a matrix metalloproteinase (MMP) inhibitor (GM6001) suppresses SMC TN-C expression on native collagen, whereas denatured collagen promotes TN-C expression in a beta 3 integrin- dependent manner, independent of MMPs. Floating type I collagen gel also suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presence of EGF. Addition of exogenous TN-C to SMCs on floating collagen, or to SMCs treated with GM6001, restores the EGF growth response and "rescues" cells from apoptosis. The mechanism by which TN-C facilitates EGF-dependent survival and growth was then investigated. We show that TN-C interactions with alpha v beta 3 integrins modify SMC shape, and EGF- dependent growth. These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of EGF-Rs, tyrosine-phosphorylated proteins, and increased activation of EGF-Rs after addition of EGF. Cross-linking SMC beta 3 integrins replicates the effect of TN-C on EGF-R clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating beta 3 integrin ligands in type I collagen. In turn, alpha v beta 3 interactions with TN-C alter SMC shape and increase EGF-R clustering and EGF-dependent growth. Conversely, suppression of MMPs downregulates TN-C and induces apoptosis.

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Effect of cross-linking β3 integrins on epidermal growth factor receptor clustering. (A) Representative immunofluorescence  micrographs for β3 integrins and EGF-Rs. Smooth muscle cells cultured on collagen substrates in SFM were preincubated for 60 min  with an anti–β3 integrin antibody (150 μg/ml) and then for 60 min with SFM alone or with an anti-F(ab′)2 IgG (20 μg/ml) to promote  cross-linking. Immunodetection of β3 integrins and EGF-Rs indicates that cross-linking β3 integrins promotes EGF-R clustering. (B) Effect  of cross-linking β3 integrins on ligand-dependent tyrosine phosphorylation of EGF-Rs. Tyrosine phosphorylation of EGF-Rs in response to EGF was examined in SMC cultures treated with anti–β3 integrin antibody alone or in cultures incubated sequentially with an  anti–β3 integrin antibody and anti-F(ab′)2 IgG. Epidermal growth factor receptor immunoprecipitates were analyzed by Western immunoblotting using an antiphosphotyrosine antibody, which indicated that cross-linking β3 integrins potentiates ligand-dependent EGF-R  activation. (C) Densitometry of activated epidermal growth factor Western immunoblots (as shown in B) shows that a significant (*P <  0.05) increase in ligand-dependent tyrosine phosphorylation occurs after β3 integrin cross-linking. Values represent mean ±SEM from  three different experiments. Bar, 25 μm.
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Figure 8: Effect of cross-linking β3 integrins on epidermal growth factor receptor clustering. (A) Representative immunofluorescence micrographs for β3 integrins and EGF-Rs. Smooth muscle cells cultured on collagen substrates in SFM were preincubated for 60 min with an anti–β3 integrin antibody (150 μg/ml) and then for 60 min with SFM alone or with an anti-F(ab′)2 IgG (20 μg/ml) to promote cross-linking. Immunodetection of β3 integrins and EGF-Rs indicates that cross-linking β3 integrins promotes EGF-R clustering. (B) Effect of cross-linking β3 integrins on ligand-dependent tyrosine phosphorylation of EGF-Rs. Tyrosine phosphorylation of EGF-Rs in response to EGF was examined in SMC cultures treated with anti–β3 integrin antibody alone or in cultures incubated sequentially with an anti–β3 integrin antibody and anti-F(ab′)2 IgG. Epidermal growth factor receptor immunoprecipitates were analyzed by Western immunoblotting using an antiphosphotyrosine antibody, which indicated that cross-linking β3 integrins potentiates ligand-dependent EGF-R activation. (C) Densitometry of activated epidermal growth factor Western immunoblots (as shown in B) shows that a significant (*P < 0.05) increase in ligand-dependent tyrosine phosphorylation occurs after β3 integrin cross-linking. Values represent mean ±SEM from three different experiments. Bar, 25 μm.

Mentions: To determine whether clustering of β3 integrins on SMC surfaces, in the absence of exogenous TN-C, promotes EGF-R clustering and ligand-dependent phosphorylation of EGF-Rs, anti–β3 integrin antibody cross-linking studies were performed. Smooth muscle cells cultured on type I collagen gels were treated with either a combination of anti–β3 integrin and secondary goat anti–mouse IgG F(ab′)2 antibodies or with primary mouse monoclonal anti–β3 integrin antibody alone. Immunofluorescent microscopy revealed extensive β3 integrin clusters on SMC surfaces treated with primary and secondary antibodies, whereas a more diffuse pattern of β3 integrins was apparent in cultures treated with primary antibody alone (Fig. 8 A). Immunofluorescent microscopy with an anti–EGF-R antibody demonstrated that cross-linking the β3 integrin receptor leads to clustering of EGF-Rs, which appeared diffuse in SMCs treated with primary antibody alone (Fig. 8 A).


Regulation of tenascin-C, a vascular smooth muscle cell survival factor that interacts with the alpha v beta 3 integrin to promote epidermal growth factor receptor phosphorylation and growth.

Jones PL, Crack J, Rabinovitch M - J. Cell Biol. (1997)

Effect of cross-linking β3 integrins on epidermal growth factor receptor clustering. (A) Representative immunofluorescence  micrographs for β3 integrins and EGF-Rs. Smooth muscle cells cultured on collagen substrates in SFM were preincubated for 60 min  with an anti–β3 integrin antibody (150 μg/ml) and then for 60 min with SFM alone or with an anti-F(ab′)2 IgG (20 μg/ml) to promote  cross-linking. Immunodetection of β3 integrins and EGF-Rs indicates that cross-linking β3 integrins promotes EGF-R clustering. (B) Effect  of cross-linking β3 integrins on ligand-dependent tyrosine phosphorylation of EGF-Rs. Tyrosine phosphorylation of EGF-Rs in response to EGF was examined in SMC cultures treated with anti–β3 integrin antibody alone or in cultures incubated sequentially with an  anti–β3 integrin antibody and anti-F(ab′)2 IgG. Epidermal growth factor receptor immunoprecipitates were analyzed by Western immunoblotting using an antiphosphotyrosine antibody, which indicated that cross-linking β3 integrins potentiates ligand-dependent EGF-R  activation. (C) Densitometry of activated epidermal growth factor Western immunoblots (as shown in B) shows that a significant (*P <  0.05) increase in ligand-dependent tyrosine phosphorylation occurs after β3 integrin cross-linking. Values represent mean ±SEM from  three different experiments. Bar, 25 μm.
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Related In: Results  -  Collection

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Figure 8: Effect of cross-linking β3 integrins on epidermal growth factor receptor clustering. (A) Representative immunofluorescence micrographs for β3 integrins and EGF-Rs. Smooth muscle cells cultured on collagen substrates in SFM were preincubated for 60 min with an anti–β3 integrin antibody (150 μg/ml) and then for 60 min with SFM alone or with an anti-F(ab′)2 IgG (20 μg/ml) to promote cross-linking. Immunodetection of β3 integrins and EGF-Rs indicates that cross-linking β3 integrins promotes EGF-R clustering. (B) Effect of cross-linking β3 integrins on ligand-dependent tyrosine phosphorylation of EGF-Rs. Tyrosine phosphorylation of EGF-Rs in response to EGF was examined in SMC cultures treated with anti–β3 integrin antibody alone or in cultures incubated sequentially with an anti–β3 integrin antibody and anti-F(ab′)2 IgG. Epidermal growth factor receptor immunoprecipitates were analyzed by Western immunoblotting using an antiphosphotyrosine antibody, which indicated that cross-linking β3 integrins potentiates ligand-dependent EGF-R activation. (C) Densitometry of activated epidermal growth factor Western immunoblots (as shown in B) shows that a significant (*P < 0.05) increase in ligand-dependent tyrosine phosphorylation occurs after β3 integrin cross-linking. Values represent mean ±SEM from three different experiments. Bar, 25 μm.
Mentions: To determine whether clustering of β3 integrins on SMC surfaces, in the absence of exogenous TN-C, promotes EGF-R clustering and ligand-dependent phosphorylation of EGF-Rs, anti–β3 integrin antibody cross-linking studies were performed. Smooth muscle cells cultured on type I collagen gels were treated with either a combination of anti–β3 integrin and secondary goat anti–mouse IgG F(ab′)2 antibodies or with primary mouse monoclonal anti–β3 integrin antibody alone. Immunofluorescent microscopy revealed extensive β3 integrin clusters on SMC surfaces treated with primary and secondary antibodies, whereas a more diffuse pattern of β3 integrins was apparent in cultures treated with primary antibody alone (Fig. 8 A). Immunofluorescent microscopy with an anti–EGF-R antibody demonstrated that cross-linking the β3 integrin receptor leads to clustering of EGF-Rs, which appeared diffuse in SMCs treated with primary antibody alone (Fig. 8 A).

Bottom Line: These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of EGF-Rs, tyrosine-phosphorylated proteins, and increased activation of EGF-Rs after addition of EGF.Cross-linking SMC beta 3 integrins replicates the effect of TN-C on EGF-R clustering and tyrosine phosphorylation.Conversely, suppression of MMPs downregulates TN-C and induces apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Research, Research Institute, The Hospital for Sick Children, University of Toronto, Ontario, Canada.

ABSTRACT
Tenascin-C (TN-C) is induced in pulmonary vascular disease, where it colocalizes with proliferating smooth muscle cells (SMCs) and epidermal growth factor (EGF). Furthermore, cultured SMCs require TN-C for EGF-dependent growth on type I collagen. In this study, we explore the regulation and function of TN-C in SMCs. We show that a matrix metalloproteinase (MMP) inhibitor (GM6001) suppresses SMC TN-C expression on native collagen, whereas denatured collagen promotes TN-C expression in a beta 3 integrin- dependent manner, independent of MMPs. Floating type I collagen gel also suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presence of EGF. Addition of exogenous TN-C to SMCs on floating collagen, or to SMCs treated with GM6001, restores the EGF growth response and "rescues" cells from apoptosis. The mechanism by which TN-C facilitates EGF-dependent survival and growth was then investigated. We show that TN-C interactions with alpha v beta 3 integrins modify SMC shape, and EGF- dependent growth. These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of EGF-Rs, tyrosine-phosphorylated proteins, and increased activation of EGF-Rs after addition of EGF. Cross-linking SMC beta 3 integrins replicates the effect of TN-C on EGF-R clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating beta 3 integrin ligands in type I collagen. In turn, alpha v beta 3 interactions with TN-C alter SMC shape and increase EGF-R clustering and EGF-dependent growth. Conversely, suppression of MMPs downregulates TN-C and induces apoptosis.

Show MeSH
Related in: MedlinePlus