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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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The role of β3 integrins in tenascin-C–dependent epidermal growth factor receptor clustering and ligand-dependent  receptor activation. (A) Representative immunofluorescence  photomicrographs for EGF-R in SMCs cultured on TN-C (15 μg/ ml) supplemented collagen gels in the presence of control IgG or  with an anti–β3 integrin antibody that prevented the formation of  EGF-R clusters. (B) Effect of blocking β3 integrins on ligand-dependent EGF-R tyrosine phosphorylation in SMCs cultured on  collagen gels supplemented with TN-C. Western immunoblot  (representative of two different studies) of membrane preparations from SMCs treated with control IgG, with anti–β3 integrin  antisera on TN-C–supplemented collagen in SFM alone, or in  SFM with EGF (50 ng/ml) for 10 and 30 min. Epidermal growth  factor receptors were immunoprecipitated from 15 μg of membrane-enriched SMC fractions and were analyzed for evidence of  tyrosine phosphorylation using Western immunoblotting. Note  that the anti–β3 integrin antibody prevents the transient increase  in EGF-R tyrosine phosphorylation observed in TN-treated cultures after addition of EGF at 10 min. Bar, 17 μm.
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Figure 7: The role of β3 integrins in tenascin-C–dependent epidermal growth factor receptor clustering and ligand-dependent receptor activation. (A) Representative immunofluorescence photomicrographs for EGF-R in SMCs cultured on TN-C (15 μg/ ml) supplemented collagen gels in the presence of control IgG or with an anti–β3 integrin antibody that prevented the formation of EGF-R clusters. (B) Effect of blocking β3 integrins on ligand-dependent EGF-R tyrosine phosphorylation in SMCs cultured on collagen gels supplemented with TN-C. Western immunoblot (representative of two different studies) of membrane preparations from SMCs treated with control IgG, with anti–β3 integrin antisera on TN-C–supplemented collagen in SFM alone, or in SFM with EGF (50 ng/ml) for 10 and 30 min. Epidermal growth factor receptors were immunoprecipitated from 15 μg of membrane-enriched SMC fractions and were analyzed for evidence of tyrosine phosphorylation using Western immunoblotting. Note that the anti–β3 integrin antibody prevents the transient increase in EGF-R tyrosine phosphorylation observed in TN-treated cultures after addition of EGF at 10 min. Bar, 17 μm.

Mentions: We next examined the effects of blocking TN-C–β3 integrin SMC interactions on EGF-R clustering and tyrosine phosphorylation. Smooth muscle cells were preincubated with an anti–β3 integrin antibody before plating on TN- C–supplemented collagen gels. As with the αvβ3 functional-blocking antibody experiments, the anti–β3 integrin antibody prevented cell spreading on TN-C (data not shown). We therefore used the anti–β3 integrin antibody in subsequent studies (although similar results would be expected with the αvβ3 antibody, LM609) and showed inhibition of TN-C–dependent EGF-R clustering (Fig. 7 A). Immunoprecipitation of SMC membrane–enriched fractions followed by Western immunoblotting with an antiphosphotyrosine antibody showed that upon addition of EGF, EGF-R activation on TN-C–enriched collagen substrates was abrogated in cells pretreated with the function-blocking anti–β3 integrin antibody (Fig. 7 B).


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)

The role of β3 integrins in tenascin-C–dependent epidermal growth factor receptor clustering and ligand-dependent  receptor activation. (A) Representative immunofluorescence  photomicrographs for EGF-R in SMCs cultured on TN-C (15 μg/ ml) supplemented collagen gels in the presence of control IgG or  with an anti–β3 integrin antibody that prevented the formation of  EGF-R clusters. (B) Effect of blocking β3 integrins on ligand-dependent EGF-R tyrosine phosphorylation in SMCs cultured on  collagen gels supplemented with TN-C. Western immunoblot  (representative of two different studies) of membrane preparations from SMCs treated with control IgG, with anti–β3 integrin  antisera on TN-C–supplemented collagen in SFM alone, or in  SFM with EGF (50 ng/ml) for 10 and 30 min. Epidermal growth  factor receptors were immunoprecipitated from 15 μg of membrane-enriched SMC fractions and were analyzed for evidence of  tyrosine phosphorylation using Western immunoblotting. Note  that the anti–β3 integrin antibody prevents the transient increase  in EGF-R tyrosine phosphorylation observed in TN-treated cultures after addition of EGF at 10 min. Bar, 17 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139818&req=5

Figure 7: The role of β3 integrins in tenascin-C–dependent epidermal growth factor receptor clustering and ligand-dependent receptor activation. (A) Representative immunofluorescence photomicrographs for EGF-R in SMCs cultured on TN-C (15 μg/ ml) supplemented collagen gels in the presence of control IgG or with an anti–β3 integrin antibody that prevented the formation of EGF-R clusters. (B) Effect of blocking β3 integrins on ligand-dependent EGF-R tyrosine phosphorylation in SMCs cultured on collagen gels supplemented with TN-C. Western immunoblot (representative of two different studies) of membrane preparations from SMCs treated with control IgG, with anti–β3 integrin antisera on TN-C–supplemented collagen in SFM alone, or in SFM with EGF (50 ng/ml) for 10 and 30 min. Epidermal growth factor receptors were immunoprecipitated from 15 μg of membrane-enriched SMC fractions and were analyzed for evidence of tyrosine phosphorylation using Western immunoblotting. Note that the anti–β3 integrin antibody prevents the transient increase in EGF-R tyrosine phosphorylation observed in TN-treated cultures after addition of EGF at 10 min. Bar, 17 μm.
Mentions: We next examined the effects of blocking TN-C–β3 integrin SMC interactions on EGF-R clustering and tyrosine phosphorylation. Smooth muscle cells were preincubated with an anti–β3 integrin antibody before plating on TN- C–supplemented collagen gels. As with the αvβ3 functional-blocking antibody experiments, the anti–β3 integrin antibody prevented cell spreading on TN-C (data not shown). We therefore used the anti–β3 integrin antibody in subsequent studies (although similar results would be expected with the αvβ3 antibody, LM609) and showed inhibition of TN-C–dependent EGF-R clustering (Fig. 7 A). Immunoprecipitation of SMC membrane–enriched fractions followed by Western immunoblotting with an antiphosphotyrosine antibody showed that upon addition of EGF, EGF-R activation on TN-C–enriched collagen substrates was abrogated in cells pretreated with the function-blocking anti–β3 integrin antibody (Fig. 7 B).

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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Related in: MedlinePlus