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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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Regulation of tenascin-C protein expression by matrix  metalloproteinases and β3 integrins. (A) Immunoprecipitation of  TN-C protein from [35S]methionine/cysteine-labeled SMCs cultured on native type I collagen gels in serum-free control medium  with 50 ng/ml EGF and 0.4% DMSO, or in control medium supplemented with 1 or 2 μM of GM6001, an MMP inhibitor. Inhibition of MMP activity with GM6001 leads to a marked decrease in  TN-C protein synthesis. Positions of molecular mass standards in  kD are indicated on the left. (B) Western immunoblotting for  TN-C protein from SMCs cultured in SFM with 50 ng/ml EGF on  native and heat-denatured type I collagen gels. A Western immunoblot shows that expression of two TN-C isoforms of apparent  molecular masses 220 and 180 kD is increased in cells cultured on  denatured collagen. (C) Western immunoblot of TN-C protein  from SMCs cultured on heat-denatured type I collagen gels in  control medium supplemented with either 0.4% DMSO or 2 μM  of GM6001. Inhibition of MMP activity with GM6001 had no effect on TN-C protein expression. (D) Effect of β3 integrin blockade  on TN-C protein expression on heat-denatured collagen. Immunoprecipitation of TN-C protein from [35S]methionine/cysteine- labeled SMCs cultured on native type I collagen gels in the presence of control IgG or with anti-β3 antibody shows that blocking  β3 integrins inhibits TN-C protein expression on native type I collagen. (E) Effect of β3 integrin blockade on TN-C protein expression on heat-denatured collagen. Western immunoblot analysis  of lysates derived from cells cultured on heat-denatured collagen  in the presence of control IgG, or with anti-β3 integrin antibody  shows that blocking β3 integrins inhibits TN-C protein expression  on denatured collagen. Arrows indicate the presence of the 220-kD  (upper band) and 180-kD (lower band) TN-C isoforms.
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Figure 2: Regulation of tenascin-C protein expression by matrix metalloproteinases and β3 integrins. (A) Immunoprecipitation of TN-C protein from [35S]methionine/cysteine-labeled SMCs cultured on native type I collagen gels in serum-free control medium with 50 ng/ml EGF and 0.4% DMSO, or in control medium supplemented with 1 or 2 μM of GM6001, an MMP inhibitor. Inhibition of MMP activity with GM6001 leads to a marked decrease in TN-C protein synthesis. Positions of molecular mass standards in kD are indicated on the left. (B) Western immunoblotting for TN-C protein from SMCs cultured in SFM with 50 ng/ml EGF on native and heat-denatured type I collagen gels. A Western immunoblot shows that expression of two TN-C isoforms of apparent molecular masses 220 and 180 kD is increased in cells cultured on denatured collagen. (C) Western immunoblot of TN-C protein from SMCs cultured on heat-denatured type I collagen gels in control medium supplemented with either 0.4% DMSO or 2 μM of GM6001. Inhibition of MMP activity with GM6001 had no effect on TN-C protein expression. (D) Effect of β3 integrin blockade on TN-C protein expression on heat-denatured collagen. Immunoprecipitation of TN-C protein from [35S]methionine/cysteine- labeled SMCs cultured on native type I collagen gels in the presence of control IgG or with anti-β3 antibody shows that blocking β3 integrins inhibits TN-C protein expression on native type I collagen. (E) Effect of β3 integrin blockade on TN-C protein expression on heat-denatured collagen. Western immunoblot analysis of lysates derived from cells cultured on heat-denatured collagen in the presence of control IgG, or with anti-β3 integrin antibody shows that blocking β3 integrins inhibits TN-C protein expression on denatured collagen. Arrows indicate the presence of the 220-kD (upper band) and 180-kD (lower band) TN-C isoforms.

Mentions: To first establish whether MMPs regulate TN-C expression, we cultured SMCs on attached type I collagen gels in the presence of a specific MMP inhibitor, GM6001 (Galardy, 1993; Strauss et al., 1996). Immunoprecipitation of TN-C from radiolabeled cell lysates showed that inhibition of MMP activity results in suppression of TN-C protein synthesis (Fig. 2 A). In addition, the ability of this inhibitor to suppress TN-C protein synthesis is not due to cytotoxicity since there were no significant differences between the total number of TCA precipitable counts in radiolabeled control (3.18 × 106 cpm/culture, SEM ± 2.12 × 105, P < 0.05) and GM6001-treated cultures (3.75 × 106 cpm/culture, SEM ± 1.63 × 105, P < 0.05) at the time point examined.


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)

Regulation of tenascin-C protein expression by matrix  metalloproteinases and β3 integrins. (A) Immunoprecipitation of  TN-C protein from [35S]methionine/cysteine-labeled SMCs cultured on native type I collagen gels in serum-free control medium  with 50 ng/ml EGF and 0.4% DMSO, or in control medium supplemented with 1 or 2 μM of GM6001, an MMP inhibitor. Inhibition of MMP activity with GM6001 leads to a marked decrease in  TN-C protein synthesis. Positions of molecular mass standards in  kD are indicated on the left. (B) Western immunoblotting for  TN-C protein from SMCs cultured in SFM with 50 ng/ml EGF on  native and heat-denatured type I collagen gels. A Western immunoblot shows that expression of two TN-C isoforms of apparent  molecular masses 220 and 180 kD is increased in cells cultured on  denatured collagen. (C) Western immunoblot of TN-C protein  from SMCs cultured on heat-denatured type I collagen gels in  control medium supplemented with either 0.4% DMSO or 2 μM  of GM6001. Inhibition of MMP activity with GM6001 had no effect on TN-C protein expression. (D) Effect of β3 integrin blockade  on TN-C protein expression on heat-denatured collagen. Immunoprecipitation of TN-C protein from [35S]methionine/cysteine- labeled SMCs cultured on native type I collagen gels in the presence of control IgG or with anti-β3 antibody shows that blocking  β3 integrins inhibits TN-C protein expression on native type I collagen. (E) Effect of β3 integrin blockade on TN-C protein expression on heat-denatured collagen. Western immunoblot analysis  of lysates derived from cells cultured on heat-denatured collagen  in the presence of control IgG, or with anti-β3 integrin antibody  shows that blocking β3 integrins inhibits TN-C protein expression  on denatured collagen. Arrows indicate the presence of the 220-kD  (upper band) and 180-kD (lower band) TN-C isoforms.
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Related In: Results  -  Collection

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Figure 2: Regulation of tenascin-C protein expression by matrix metalloproteinases and β3 integrins. (A) Immunoprecipitation of TN-C protein from [35S]methionine/cysteine-labeled SMCs cultured on native type I collagen gels in serum-free control medium with 50 ng/ml EGF and 0.4% DMSO, or in control medium supplemented with 1 or 2 μM of GM6001, an MMP inhibitor. Inhibition of MMP activity with GM6001 leads to a marked decrease in TN-C protein synthesis. Positions of molecular mass standards in kD are indicated on the left. (B) Western immunoblotting for TN-C protein from SMCs cultured in SFM with 50 ng/ml EGF on native and heat-denatured type I collagen gels. A Western immunoblot shows that expression of two TN-C isoforms of apparent molecular masses 220 and 180 kD is increased in cells cultured on denatured collagen. (C) Western immunoblot of TN-C protein from SMCs cultured on heat-denatured type I collagen gels in control medium supplemented with either 0.4% DMSO or 2 μM of GM6001. Inhibition of MMP activity with GM6001 had no effect on TN-C protein expression. (D) Effect of β3 integrin blockade on TN-C protein expression on heat-denatured collagen. Immunoprecipitation of TN-C protein from [35S]methionine/cysteine- labeled SMCs cultured on native type I collagen gels in the presence of control IgG or with anti-β3 antibody shows that blocking β3 integrins inhibits TN-C protein expression on native type I collagen. (E) Effect of β3 integrin blockade on TN-C protein expression on heat-denatured collagen. Western immunoblot analysis of lysates derived from cells cultured on heat-denatured collagen in the presence of control IgG, or with anti-β3 integrin antibody shows that blocking β3 integrins inhibits TN-C protein expression on denatured collagen. Arrows indicate the presence of the 220-kD (upper band) and 180-kD (lower band) TN-C isoforms.
Mentions: To first establish whether MMPs regulate TN-C expression, we cultured SMCs on attached type I collagen gels in the presence of a specific MMP inhibitor, GM6001 (Galardy, 1993; Strauss et al., 1996). Immunoprecipitation of TN-C from radiolabeled cell lysates showed that inhibition of MMP activity results in suppression of TN-C protein synthesis (Fig. 2 A). In addition, the ability of this inhibitor to suppress TN-C protein synthesis is not due to cytotoxicity since there were no significant differences between the total number of TCA precipitable counts in radiolabeled control (3.18 × 106 cpm/culture, SEM ± 2.12 × 105, P < 0.05) and GM6001-treated cultures (3.75 × 106 cpm/culture, SEM ± 1.63 × 105, P < 0.05) at the time point examined.

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