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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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Tenascin-C potentiates ligand-dependent epidermal  growth factor-receptor tyrosine phosphorylation. (A) Effect of  TN-C on EGF-R protein synthesis. Representative autoradiograph (from two different experiments) showing immunoprecipitation of EGF-R protein from [35S]methionine-labeled SMCs cultured on native type I collagen gels (+/− 15 μg/ml TN-C) in SFM  indicates that TN-C has no effect on the levels of EGF-R protein  synthesis compared to SMCs cultured on collagen alone. (B) Effect of TN-C on tyrosine phosphorylation in SMCs cultured on  collagen and TN-C–supplemented gels in SFM, or after addition  of EGF (50 ng/ml) for 10 and 30 min. A Western immunoblot  (representative of two different studies) was performed on membrane-enriched fractions (15 μg per sample) using an antibody  against tyrosine-phosphorylated proteins. Note that relative to  SMCs cultured on collagen alone, contact with exogenous TN-C  led to an increase in basal levels of tyrosine phosphorylation. In  response to EGF, qualitative and quantitative increases in tyrosine-phosphorylated proteins were observed on both collagen  and TN-C, with changes being more pronounced on TN-C–enriched  gels, including species at 170 and 125 kD. Positions of molecular  mass standards in kD are indicated on the right. The 170-kD species  may represent the EGF receptor, and the 125-kD species the focal adhesion kinase. (C) The 170-kD tyrosine-phosphorylated  protein shown in B represents the EGF-R. Epidermal growth factor receptors were immunoprecipitated from 15 μg of membrane-enriched SMC fractions in cells cultured as in B. Epidermal  growth factor immunoprecipitates were analyzed for evidence of  tyrosine phosphorylation using Western immunoblotting with an  antiphosphotyrosine antibody. After addition of EGF at 10 min,  a transient increase in the levels of tyrosine-phosphorylated  EGF-Rs occurs on collagen and TN-C–supplemented gels, with  greater levels observed on TN-C–supplemented collagen substrates.
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Figure 6: Tenascin-C potentiates ligand-dependent epidermal growth factor-receptor tyrosine phosphorylation. (A) Effect of TN-C on EGF-R protein synthesis. Representative autoradiograph (from two different experiments) showing immunoprecipitation of EGF-R protein from [35S]methionine-labeled SMCs cultured on native type I collagen gels (+/− 15 μg/ml TN-C) in SFM indicates that TN-C has no effect on the levels of EGF-R protein synthesis compared to SMCs cultured on collagen alone. (B) Effect of TN-C on tyrosine phosphorylation in SMCs cultured on collagen and TN-C–supplemented gels in SFM, or after addition of EGF (50 ng/ml) for 10 and 30 min. A Western immunoblot (representative of two different studies) was performed on membrane-enriched fractions (15 μg per sample) using an antibody against tyrosine-phosphorylated proteins. Note that relative to SMCs cultured on collagen alone, contact with exogenous TN-C led to an increase in basal levels of tyrosine phosphorylation. In response to EGF, qualitative and quantitative increases in tyrosine-phosphorylated proteins were observed on both collagen and TN-C, with changes being more pronounced on TN-C–enriched gels, including species at 170 and 125 kD. Positions of molecular mass standards in kD are indicated on the right. The 170-kD species may represent the EGF receptor, and the 125-kD species the focal adhesion kinase. (C) The 170-kD tyrosine-phosphorylated protein shown in B represents the EGF-R. Epidermal growth factor receptors were immunoprecipitated from 15 μg of membrane-enriched SMC fractions in cells cultured as in B. Epidermal growth factor immunoprecipitates were analyzed for evidence of tyrosine phosphorylation using Western immunoblotting with an antiphosphotyrosine antibody. After addition of EGF at 10 min, a transient increase in the levels of tyrosine-phosphorylated EGF-Rs occurs on collagen and TN-C–supplemented gels, with greater levels observed on TN-C–supplemented collagen substrates.

Mentions: Having established that culturing SMCs on exogenous TN-C promotes EGF-R clustering, we next determined the effects of TN-C on EGF-R protein synthesis and tyrosine phosphorylation of this receptor. Immunoprecipitation of radiolabeled whole cell lysates with an anti–EGF-R antibody showed that the levels of receptor protein synthesis were identical on collagen and TN-C–supplemented gels (Fig. 6 A). Consistent with our immunofluorescent studies, Western immunoblotting of membrane-enriched fractions with an antiphosphotyrosine antibody revealed that there was increased tyrosine phosphorylation in the presence of exogenous TN-C (Fig. 6 B). In addition, on both type I collagen and TN-C–supplemented cultures, EGF treatment resulted in a transient increase in protein tyrosine phosphorylation that was enhanced by TN-C. Of particular interest was a 170-kD tyrosine-phosphorylated species that corresponds in molecular mass to the EGF-R. Also noted was an increase in an ∼125-kD species, which may represent focal adhesion kinase (Fig. 6 B). To specifically determine whether TN-C promotes EGF-R activation, EGF-Rs were immunoprecipitated from SMC membrane fractions, and their activation status was assessed using Western immunoblotting with an antiphosphotyrosine antibody. This confirmed that EGF-R phosphorylation occurs by 10 min after addition of EGF, and that this response is enhanced in TN-C–supplemented as opposed to SMC cultures on collagen gels alone (Fig. 6 C).


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)

Tenascin-C potentiates ligand-dependent epidermal  growth factor-receptor tyrosine phosphorylation. (A) Effect of  TN-C on EGF-R protein synthesis. Representative autoradiograph (from two different experiments) showing immunoprecipitation of EGF-R protein from [35S]methionine-labeled SMCs cultured on native type I collagen gels (+/− 15 μg/ml TN-C) in SFM  indicates that TN-C has no effect on the levels of EGF-R protein  synthesis compared to SMCs cultured on collagen alone. (B) Effect of TN-C on tyrosine phosphorylation in SMCs cultured on  collagen and TN-C–supplemented gels in SFM, or after addition  of EGF (50 ng/ml) for 10 and 30 min. A Western immunoblot  (representative of two different studies) was performed on membrane-enriched fractions (15 μg per sample) using an antibody  against tyrosine-phosphorylated proteins. Note that relative to  SMCs cultured on collagen alone, contact with exogenous TN-C  led to an increase in basal levels of tyrosine phosphorylation. In  response to EGF, qualitative and quantitative increases in tyrosine-phosphorylated proteins were observed on both collagen  and TN-C, with changes being more pronounced on TN-C–enriched  gels, including species at 170 and 125 kD. Positions of molecular  mass standards in kD are indicated on the right. The 170-kD species  may represent the EGF receptor, and the 125-kD species the focal adhesion kinase. (C) The 170-kD tyrosine-phosphorylated  protein shown in B represents the EGF-R. Epidermal growth factor receptors were immunoprecipitated from 15 μg of membrane-enriched SMC fractions in cells cultured as in B. Epidermal  growth factor immunoprecipitates were analyzed for evidence of  tyrosine phosphorylation using Western immunoblotting with an  antiphosphotyrosine antibody. After addition of EGF at 10 min,  a transient increase in the levels of tyrosine-phosphorylated  EGF-Rs occurs on collagen and TN-C–supplemented gels, with  greater levels observed on TN-C–supplemented collagen substrates.
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Figure 6: Tenascin-C potentiates ligand-dependent epidermal growth factor-receptor tyrosine phosphorylation. (A) Effect of TN-C on EGF-R protein synthesis. Representative autoradiograph (from two different experiments) showing immunoprecipitation of EGF-R protein from [35S]methionine-labeled SMCs cultured on native type I collagen gels (+/− 15 μg/ml TN-C) in SFM indicates that TN-C has no effect on the levels of EGF-R protein synthesis compared to SMCs cultured on collagen alone. (B) Effect of TN-C on tyrosine phosphorylation in SMCs cultured on collagen and TN-C–supplemented gels in SFM, or after addition of EGF (50 ng/ml) for 10 and 30 min. A Western immunoblot (representative of two different studies) was performed on membrane-enriched fractions (15 μg per sample) using an antibody against tyrosine-phosphorylated proteins. Note that relative to SMCs cultured on collagen alone, contact with exogenous TN-C led to an increase in basal levels of tyrosine phosphorylation. In response to EGF, qualitative and quantitative increases in tyrosine-phosphorylated proteins were observed on both collagen and TN-C, with changes being more pronounced on TN-C–enriched gels, including species at 170 and 125 kD. Positions of molecular mass standards in kD are indicated on the right. The 170-kD species may represent the EGF receptor, and the 125-kD species the focal adhesion kinase. (C) The 170-kD tyrosine-phosphorylated protein shown in B represents the EGF-R. Epidermal growth factor receptors were immunoprecipitated from 15 μg of membrane-enriched SMC fractions in cells cultured as in B. Epidermal growth factor immunoprecipitates were analyzed for evidence of tyrosine phosphorylation using Western immunoblotting with an antiphosphotyrosine antibody. After addition of EGF at 10 min, a transient increase in the levels of tyrosine-phosphorylated EGF-Rs occurs on collagen and TN-C–supplemented gels, with greater levels observed on TN-C–supplemented collagen substrates.
Mentions: Having established that culturing SMCs on exogenous TN-C promotes EGF-R clustering, we next determined the effects of TN-C on EGF-R protein synthesis and tyrosine phosphorylation of this receptor. Immunoprecipitation of radiolabeled whole cell lysates with an anti–EGF-R antibody showed that the levels of receptor protein synthesis were identical on collagen and TN-C–supplemented gels (Fig. 6 A). Consistent with our immunofluorescent studies, Western immunoblotting of membrane-enriched fractions with an antiphosphotyrosine antibody revealed that there was increased tyrosine phosphorylation in the presence of exogenous TN-C (Fig. 6 B). In addition, on both type I collagen and TN-C–supplemented cultures, EGF treatment resulted in a transient increase in protein tyrosine phosphorylation that was enhanced by TN-C. Of particular interest was a 170-kD tyrosine-phosphorylated species that corresponds in molecular mass to the EGF-R. Also noted was an increase in an ∼125-kD species, which may represent focal adhesion kinase (Fig. 6 B). To specifically determine whether TN-C promotes EGF-R activation, EGF-Rs were immunoprecipitated from SMC membrane fractions, and their activation status was assessed using Western immunoblotting with an antiphosphotyrosine antibody. This confirmed that EGF-R phosphorylation occurs by 10 min after addition of EGF, and that this response is enhanced in TN-C–supplemented as opposed to SMC cultures on collagen gels alone (Fig. 6 C).

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