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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 and matrix metalloproteinase-2 (MMP-2)  expression in vascular smooth muscle cells. (A) Representative  photomicrograph showing SMC morphology on attached type I  collagen gels and on collagen gels that have been released for 2 h  into the culture medium. (B) Autoradiograph (representative of  two different experiments) showing immunoprecipitated TN protein from [35S]methionine/cysteine-labeled SMC lysates harvested from attached and floating type I collagen gels in SFM  with 50 ng/ml EGF at 24 h. Synthesis of two TN-C protein isoforms of apparent molecular masses 220 and 180 kD, is suppressed in cells cultured on floating collagen. (C) Gelatin zymography was used to examine the levels and activity of gelatinases  present in conditioned medium (CM) harvested from SMCs cultured on attached and floating collagen. Fresh SFM with 50 ng/ml  EGF was added for 4 h before its collection at 4, 8, 12, and 24 h.  Equal volumes of concentrated CM were then analyzed by gelatin substrate zymography in nonreducing 10% polyacrylamide  gels containing 0.1% gelatin. A zymogram (representative of  three different experiments) shows that on attached and floating  collagen, SMCs secrete gelatinases with apparent molecular  masses of 68 and 57 kD, and that on floating collagen, the activity  is lower. (D) Densitometry of the 68- and 57-kD gelatinases  shown in C. (E) Western immunoblot for the latent form of  MMP-2 in conditioned medium harvested at 24 h from SMCs cultured on attached and floating collagen shows that expression of  the 72-kD proform of this enzyme (equivalent to the 68-kD species under nonreducing conditions on the zymogram) is suppressed under floating conditions. Bar, 120 μm.
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Figure 1: Tenascin-C and matrix metalloproteinase-2 (MMP-2) expression in vascular smooth muscle cells. (A) Representative photomicrograph showing SMC morphology on attached type I collagen gels and on collagen gels that have been released for 2 h into the culture medium. (B) Autoradiograph (representative of two different experiments) showing immunoprecipitated TN protein from [35S]methionine/cysteine-labeled SMC lysates harvested from attached and floating type I collagen gels in SFM with 50 ng/ml EGF at 24 h. Synthesis of two TN-C protein isoforms of apparent molecular masses 220 and 180 kD, is suppressed in cells cultured on floating collagen. (C) Gelatin zymography was used to examine the levels and activity of gelatinases present in conditioned medium (CM) harvested from SMCs cultured on attached and floating collagen. Fresh SFM with 50 ng/ml EGF was added for 4 h before its collection at 4, 8, 12, and 24 h. Equal volumes of concentrated CM were then analyzed by gelatin substrate zymography in nonreducing 10% polyacrylamide gels containing 0.1% gelatin. A zymogram (representative of three different experiments) shows that on attached and floating collagen, SMCs secrete gelatinases with apparent molecular masses of 68 and 57 kD, and that on floating collagen, the activity is lower. (D) Densitometry of the 68- and 57-kD gelatinases shown in C. (E) Western immunoblot for the latent form of MMP-2 in conditioned medium harvested at 24 h from SMCs cultured on attached and floating collagen shows that expression of the 72-kD proform of this enzyme (equivalent to the 68-kD species under nonreducing conditions on the zymogram) is suppressed under floating conditions. Bar, 120 μm.

Mentions: When chick embryo fibroblasts are cultured on attached type I collagen gels, they express TN-C protein, whereas culture on floating collagen leads to suppression of TN-C expression (Chiquet-Ehrismann et al., 1994). Using this culture method as a model system, we first compared SMC morphology, TN-C production, and MMP activity and expression on attached and floating collagen gels. Within the first few hours of releasing attached collagen gels into the culture medium, SMCs lost their elongated morphology and became rounded (Fig. 1 A). Immunoprecipitation of radiolabeled TN-C protein from SMC conditioned medium after 24 h (Fig. 1 B) showed that synthesis of two TN-C protein isoforms, of apparent molecular masses 220 and 180 kD, was suppressed in SMCs cultured on floating collagen when compared to those cultured on attached collagen gels. No significant differences between the total number of TCA precipitable counts were noted between cells cultured on attached (1.10 × 106 cpm/culture, SEM ± 9.6 × 104, P < 0.05) versus floating collagen (1.13 × 106 cpm/culture, SEM ± 1.59 × 104, P < 0.05), indicating that despite suppression of TN-C protein synthesis, general protein synthesis at this time point was unaffected.


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 and matrix metalloproteinase-2 (MMP-2)  expression in vascular smooth muscle cells. (A) Representative  photomicrograph showing SMC morphology on attached type I  collagen gels and on collagen gels that have been released for 2 h  into the culture medium. (B) Autoradiograph (representative of  two different experiments) showing immunoprecipitated TN protein from [35S]methionine/cysteine-labeled SMC lysates harvested from attached and floating type I collagen gels in SFM  with 50 ng/ml EGF at 24 h. Synthesis of two TN-C protein isoforms of apparent molecular masses 220 and 180 kD, is suppressed in cells cultured on floating collagen. (C) Gelatin zymography was used to examine the levels and activity of gelatinases  present in conditioned medium (CM) harvested from SMCs cultured on attached and floating collagen. Fresh SFM with 50 ng/ml  EGF was added for 4 h before its collection at 4, 8, 12, and 24 h.  Equal volumes of concentrated CM were then analyzed by gelatin substrate zymography in nonreducing 10% polyacrylamide  gels containing 0.1% gelatin. A zymogram (representative of  three different experiments) shows that on attached and floating  collagen, SMCs secrete gelatinases with apparent molecular  masses of 68 and 57 kD, and that on floating collagen, the activity  is lower. (D) Densitometry of the 68- and 57-kD gelatinases  shown in C. (E) Western immunoblot for the latent form of  MMP-2 in conditioned medium harvested at 24 h from SMCs cultured on attached and floating collagen shows that expression of  the 72-kD proform of this enzyme (equivalent to the 68-kD species under nonreducing conditions on the zymogram) is suppressed under floating conditions. Bar, 120 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139818&req=5

Figure 1: Tenascin-C and matrix metalloproteinase-2 (MMP-2) expression in vascular smooth muscle cells. (A) Representative photomicrograph showing SMC morphology on attached type I collagen gels and on collagen gels that have been released for 2 h into the culture medium. (B) Autoradiograph (representative of two different experiments) showing immunoprecipitated TN protein from [35S]methionine/cysteine-labeled SMC lysates harvested from attached and floating type I collagen gels in SFM with 50 ng/ml EGF at 24 h. Synthesis of two TN-C protein isoforms of apparent molecular masses 220 and 180 kD, is suppressed in cells cultured on floating collagen. (C) Gelatin zymography was used to examine the levels and activity of gelatinases present in conditioned medium (CM) harvested from SMCs cultured on attached and floating collagen. Fresh SFM with 50 ng/ml EGF was added for 4 h before its collection at 4, 8, 12, and 24 h. Equal volumes of concentrated CM were then analyzed by gelatin substrate zymography in nonreducing 10% polyacrylamide gels containing 0.1% gelatin. A zymogram (representative of three different experiments) shows that on attached and floating collagen, SMCs secrete gelatinases with apparent molecular masses of 68 and 57 kD, and that on floating collagen, the activity is lower. (D) Densitometry of the 68- and 57-kD gelatinases shown in C. (E) Western immunoblot for the latent form of MMP-2 in conditioned medium harvested at 24 h from SMCs cultured on attached and floating collagen shows that expression of the 72-kD proform of this enzyme (equivalent to the 68-kD species under nonreducing conditions on the zymogram) is suppressed under floating conditions. Bar, 120 μm.
Mentions: When chick embryo fibroblasts are cultured on attached type I collagen gels, they express TN-C protein, whereas culture on floating collagen leads to suppression of TN-C expression (Chiquet-Ehrismann et al., 1994). Using this culture method as a model system, we first compared SMC morphology, TN-C production, and MMP activity and expression on attached and floating collagen gels. Within the first few hours of releasing attached collagen gels into the culture medium, SMCs lost their elongated morphology and became rounded (Fig. 1 A). Immunoprecipitation of radiolabeled TN-C protein from SMC conditioned medium after 24 h (Fig. 1 B) showed that synthesis of two TN-C protein isoforms, of apparent molecular masses 220 and 180 kD, was suppressed in SMCs cultured on floating collagen when compared to those cultured on attached collagen gels. No significant differences between the total number of TCA precipitable counts were noted between cells cultured on attached (1.10 × 106 cpm/culture, SEM ± 9.6 × 104, P < 0.05) versus floating collagen (1.13 × 106 cpm/culture, SEM ± 1.59 × 104, P < 0.05), indicating that despite suppression of TN-C protein synthesis, general protein synthesis at this time point was unaffected.

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