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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 blocking  αvβ3 integrins on tenascin- C–dependent smooth muscle cell morphology, attachment efficiency, and survival.  (A) Representative phase  contrast photomicrographs  of SMCs plated in SFM on  collagen and TN-C–supplemented collagen gels with  control IgG (15 μg/ml) or  with an anti–αvβ3 integrin antisera (LM609; 15 μg/ml).  IgG and LM609 treatment  had no effect on the stellate  morphology produced by the  collagen substrate. In contrast,  the more elongated SMC morphology observed on control-treated TN-C–enriched substrates was abrogated by  inclusion of LM609, which  prevented cells from spreading, resulting in a rounded  morphology. (B) Effect of  LM609 antisera on SMC attachment to TN-C–supplemented type I collagen gels.  By 6 h after plating, no significant differences in attachment efficiency were noted  between cells plated in SFM  on TN-C–supplemented collagen gels in either the presence of control IgG or  LM609 antisera. (C) Effect  of blocking SMC αvβ3 integrins with LM609 antisera on  EGF-dependent SMC cell  growth on TN-C–supplemented collagen gels. Control and LM609 treatment  produced no differences in  SMC number after culture  for 48 h on collagen gels in SFM with 50 ng/ml EGF. The significant (P < 0.05) increase in SMC growth observed on TN-supplemented  collagen gels in the presence of EGF was attenuated by LM609 antisera. Values represent mean ±SEM from three different experiments. (The asterisk denotes P < 0.05 difference from IgG control level on type I collagen.) Bar, 150 μm.
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Figure 4: Effect of blocking αvβ3 integrins on tenascin- C–dependent smooth muscle cell morphology, attachment efficiency, and survival. (A) Representative phase contrast photomicrographs of SMCs plated in SFM on collagen and TN-C–supplemented collagen gels with control IgG (15 μg/ml) or with an anti–αvβ3 integrin antisera (LM609; 15 μg/ml). IgG and LM609 treatment had no effect on the stellate morphology produced by the collagen substrate. In contrast, the more elongated SMC morphology observed on control-treated TN-C–enriched substrates was abrogated by inclusion of LM609, which prevented cells from spreading, resulting in a rounded morphology. (B) Effect of LM609 antisera on SMC attachment to TN-C–supplemented type I collagen gels. By 6 h after plating, no significant differences in attachment efficiency were noted between cells plated in SFM on TN-C–supplemented collagen gels in either the presence of control IgG or LM609 antisera. (C) Effect of blocking SMC αvβ3 integrins with LM609 antisera on EGF-dependent SMC cell growth on TN-C–supplemented collagen gels. Control and LM609 treatment produced no differences in SMC number after culture for 48 h on collagen gels in SFM with 50 ng/ml EGF. The significant (P < 0.05) increase in SMC growth observed on TN-supplemented collagen gels in the presence of EGF was attenuated by LM609 antisera. Values represent mean ±SEM from three different experiments. (The asterisk denotes P < 0.05 difference from IgG control level on type I collagen.) Bar, 150 μm.

Mentions: Tenascin-C has the capacity to bind and interact with multiple cell surface receptors, including the αvβ3 integrin (Prieto et al., 1993; Sriramarao et al., 1993). To determine whether this integrin mediates TN-C/EGF–dependent SMC growth, we evaluated the effect of a functional-blocking anti-αvβ3 integrin monoclonal antibody (LM609) on cell morphology and proliferation of SMC cultured on collagen and TN-C–enriched substrates in the presence of EGF. While LM609 had no effect on the stellate SMC morphology observed on collagen, the more elongated SMC morphology induced by TN-C was attenuated by this antibody. Instead, SMCs failed to spread and remained rounded (Fig. 4 A). LM609 had no significant effect on SMC attachment to TN-C–enriched substrates (Fig. 4 B) or on cell number after culture of SMC on collagen in the presence of EGF (Fig. 4 C). In contrast, TN-C–dependent growth in response to EGF was inhibited by LM609 (Fig. 4 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)

Effect of blocking  αvβ3 integrins on tenascin- C–dependent smooth muscle cell morphology, attachment efficiency, and survival.  (A) Representative phase  contrast photomicrographs  of SMCs plated in SFM on  collagen and TN-C–supplemented collagen gels with  control IgG (15 μg/ml) or  with an anti–αvβ3 integrin antisera (LM609; 15 μg/ml).  IgG and LM609 treatment  had no effect on the stellate  morphology produced by the  collagen substrate. In contrast,  the more elongated SMC morphology observed on control-treated TN-C–enriched substrates was abrogated by  inclusion of LM609, which  prevented cells from spreading, resulting in a rounded  morphology. (B) Effect of  LM609 antisera on SMC attachment to TN-C–supplemented type I collagen gels.  By 6 h after plating, no significant differences in attachment efficiency were noted  between cells plated in SFM  on TN-C–supplemented collagen gels in either the presence of control IgG or  LM609 antisera. (C) Effect  of blocking SMC αvβ3 integrins with LM609 antisera on  EGF-dependent SMC cell  growth on TN-C–supplemented collagen gels. Control and LM609 treatment  produced no differences in  SMC number after culture  for 48 h on collagen gels in SFM with 50 ng/ml EGF. The significant (P < 0.05) increase in SMC growth observed on TN-supplemented  collagen gels in the presence of EGF was attenuated by LM609 antisera. Values represent mean ±SEM from three different experiments. (The asterisk denotes P < 0.05 difference from IgG control level on type I collagen.) Bar, 150 μm.
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Figure 4: Effect of blocking αvβ3 integrins on tenascin- C–dependent smooth muscle cell morphology, attachment efficiency, and survival. (A) Representative phase contrast photomicrographs of SMCs plated in SFM on collagen and TN-C–supplemented collagen gels with control IgG (15 μg/ml) or with an anti–αvβ3 integrin antisera (LM609; 15 μg/ml). IgG and LM609 treatment had no effect on the stellate morphology produced by the collagen substrate. In contrast, the more elongated SMC morphology observed on control-treated TN-C–enriched substrates was abrogated by inclusion of LM609, which prevented cells from spreading, resulting in a rounded morphology. (B) Effect of LM609 antisera on SMC attachment to TN-C–supplemented type I collagen gels. By 6 h after plating, no significant differences in attachment efficiency were noted between cells plated in SFM on TN-C–supplemented collagen gels in either the presence of control IgG or LM609 antisera. (C) Effect of blocking SMC αvβ3 integrins with LM609 antisera on EGF-dependent SMC cell growth on TN-C–supplemented collagen gels. Control and LM609 treatment produced no differences in SMC number after culture for 48 h on collagen gels in SFM with 50 ng/ml EGF. The significant (P < 0.05) increase in SMC growth observed on TN-supplemented collagen gels in the presence of EGF was attenuated by LM609 antisera. Values represent mean ±SEM from three different experiments. (The asterisk denotes P < 0.05 difference from IgG control level on type I collagen.) Bar, 150 μm.
Mentions: Tenascin-C has the capacity to bind and interact with multiple cell surface receptors, including the αvβ3 integrin (Prieto et al., 1993; Sriramarao et al., 1993). To determine whether this integrin mediates TN-C/EGF–dependent SMC growth, we evaluated the effect of a functional-blocking anti-αvβ3 integrin monoclonal antibody (LM609) on cell morphology and proliferation of SMC cultured on collagen and TN-C–enriched substrates in the presence of EGF. While LM609 had no effect on the stellate SMC morphology observed on collagen, the more elongated SMC morphology induced by TN-C was attenuated by this antibody. Instead, SMCs failed to spread and remained rounded (Fig. 4 A). LM609 had no significant effect on SMC attachment to TN-C–enriched substrates (Fig. 4 B) or on cell number after culture of SMC on collagen in the presence of EGF (Fig. 4 C). In contrast, TN-C–dependent growth in response to EGF was inhibited by LM609 (Fig. 4 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