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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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Matrix metalloproteinases and tenascin-C act as vascular smooth muscle cell survival factors. (A) Smooth muscle cells  (2 × 104 cells per dish) plated on type I collagen were maintained  in serum-free control medium with 0.4% DMSO and 50 ng/ml  EGF, or in control medium supplemented with 1 or 2 μM of  GM6001 and EGF. GM6001 treatment resulted in a significant  decline in SMC numbers by 48 h, whereas addition of exogenous  human TN-C protein (15 μg/ml) to attached collagen substrates  inhibited this effect. (B) Effect of exogenous TN-C on SMC numbers on floating collagen gels. A significant decline in SMC numbers is apparent on floating compared to attached collagen gels  (P < 0.05), whereas addition of TN-C suppresses this effect. Values shown in C and D represent mean ±SEM derived from three  experiments. The asterisk denotes a P < 0.05 difference from cell  numbers recorded on collagen gels in EGF-containing medium.  (C) Effect of TN-C on SMC apoptosis on floating collagen gels.  Smooth muscle cells plated on attached type I collagen gels (2 ×  104 cells per dish) were maintained in SFM with EGF (50 ng/ml)  for 48 h or were floated in the same medium, either with or without addition of exogenous human TN-C (15 mg/ml). Genomic  DNA was isolated from each culture and 10 μg per sample was  analyzed on 1% agarose gels. DNA fragments comprised of  ∼180-bp multimers, which are indicative of apoptosis, were apparent on floating collagen gels. In contrast, no evidence of DNA  fragmentation was observed on either attached or TN-C–supplemented floating collagen. Positions of a standardized DNA ladder in kb are indicated on the right.
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Figure 3: Matrix metalloproteinases and tenascin-C act as vascular smooth muscle cell survival factors. (A) Smooth muscle cells (2 × 104 cells per dish) plated on type I collagen were maintained in serum-free control medium with 0.4% DMSO and 50 ng/ml EGF, or in control medium supplemented with 1 or 2 μM of GM6001 and EGF. GM6001 treatment resulted in a significant decline in SMC numbers by 48 h, whereas addition of exogenous human TN-C protein (15 μg/ml) to attached collagen substrates inhibited this effect. (B) Effect of exogenous TN-C on SMC numbers on floating collagen gels. A significant decline in SMC numbers is apparent on floating compared to attached collagen gels (P < 0.05), whereas addition of TN-C suppresses this effect. Values shown in C and D represent mean ±SEM derived from three experiments. The asterisk denotes a P < 0.05 difference from cell numbers recorded on collagen gels in EGF-containing medium. (C) Effect of TN-C on SMC apoptosis on floating collagen gels. Smooth muscle cells plated on attached type I collagen gels (2 × 104 cells per dish) were maintained in SFM with EGF (50 ng/ml) for 48 h or were floated in the same medium, either with or without addition of exogenous human TN-C (15 mg/ml). Genomic DNA was isolated from each culture and 10 μg per sample was analyzed on 1% agarose gels. DNA fragments comprised of ∼180-bp multimers, which are indicative of apoptosis, were apparent on floating collagen gels. In contrast, no evidence of DNA fragmentation was observed on either attached or TN-C–supplemented floating collagen. Positions of a standardized DNA ladder in kb are indicated on the right.

Mentions: Since MMPs regulate SMC TN-C protein synthesis on native type I collagen, and exogenous TN-C cooperates with EGF to facilitate SMC growth on this substrate (Jones and Rabinovitch, 1996), we next determined the functional consequences of suppressing MMPs on EGF-dependent SMC morphology, growth, and survival, in the presence and absence of exogenously added TN-C. Inhibition of MMPs with GM6001 in SMCs cultured on attached collagen resulted in cellular rounding (data not shown) and a significant decline in SMC number, whereas inclusion of exogenous TN-C in the collagen gels prevented this decrease (Fig. 3 A) and restored their characteristic elongated morphology (data not shown). Similarly, on floating collagen, in which MMPs and TN-C expression are suppressed, SMC survival is diminished (Fig. 3 B) via apoptosis as determined by oligonucleosomal DNA fragmentation assays (Fig. 3 C). In contrast, SMC numbers were unaffected (Fig. 3 B), and apoptosis was suppressed in cells cultured on floating collagen gels supplemented with exogenous TN-C protein (Fig. 3 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)

Matrix metalloproteinases and tenascin-C act as vascular smooth muscle cell survival factors. (A) Smooth muscle cells  (2 × 104 cells per dish) plated on type I collagen were maintained  in serum-free control medium with 0.4% DMSO and 50 ng/ml  EGF, or in control medium supplemented with 1 or 2 μM of  GM6001 and EGF. GM6001 treatment resulted in a significant  decline in SMC numbers by 48 h, whereas addition of exogenous  human TN-C protein (15 μg/ml) to attached collagen substrates  inhibited this effect. (B) Effect of exogenous TN-C on SMC numbers on floating collagen gels. A significant decline in SMC numbers is apparent on floating compared to attached collagen gels  (P < 0.05), whereas addition of TN-C suppresses this effect. Values shown in C and D represent mean ±SEM derived from three  experiments. The asterisk denotes a P < 0.05 difference from cell  numbers recorded on collagen gels in EGF-containing medium.  (C) Effect of TN-C on SMC apoptosis on floating collagen gels.  Smooth muscle cells plated on attached type I collagen gels (2 ×  104 cells per dish) were maintained in SFM with EGF (50 ng/ml)  for 48 h or were floated in the same medium, either with or without addition of exogenous human TN-C (15 mg/ml). Genomic  DNA was isolated from each culture and 10 μg per sample was  analyzed on 1% agarose gels. DNA fragments comprised of  ∼180-bp multimers, which are indicative of apoptosis, were apparent on floating collagen gels. In contrast, no evidence of DNA  fragmentation was observed on either attached or TN-C–supplemented floating collagen. Positions of a standardized DNA ladder in kb are indicated on the right.
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Related In: Results  -  Collection

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Figure 3: Matrix metalloproteinases and tenascin-C act as vascular smooth muscle cell survival factors. (A) Smooth muscle cells (2 × 104 cells per dish) plated on type I collagen were maintained in serum-free control medium with 0.4% DMSO and 50 ng/ml EGF, or in control medium supplemented with 1 or 2 μM of GM6001 and EGF. GM6001 treatment resulted in a significant decline in SMC numbers by 48 h, whereas addition of exogenous human TN-C protein (15 μg/ml) to attached collagen substrates inhibited this effect. (B) Effect of exogenous TN-C on SMC numbers on floating collagen gels. A significant decline in SMC numbers is apparent on floating compared to attached collagen gels (P < 0.05), whereas addition of TN-C suppresses this effect. Values shown in C and D represent mean ±SEM derived from three experiments. The asterisk denotes a P < 0.05 difference from cell numbers recorded on collagen gels in EGF-containing medium. (C) Effect of TN-C on SMC apoptosis on floating collagen gels. Smooth muscle cells plated on attached type I collagen gels (2 × 104 cells per dish) were maintained in SFM with EGF (50 ng/ml) for 48 h or were floated in the same medium, either with or without addition of exogenous human TN-C (15 mg/ml). Genomic DNA was isolated from each culture and 10 μg per sample was analyzed on 1% agarose gels. DNA fragments comprised of ∼180-bp multimers, which are indicative of apoptosis, were apparent on floating collagen gels. In contrast, no evidence of DNA fragmentation was observed on either attached or TN-C–supplemented floating collagen. Positions of a standardized DNA ladder in kb are indicated on the right.
Mentions: Since MMPs regulate SMC TN-C protein synthesis on native type I collagen, and exogenous TN-C cooperates with EGF to facilitate SMC growth on this substrate (Jones and Rabinovitch, 1996), we next determined the functional consequences of suppressing MMPs on EGF-dependent SMC morphology, growth, and survival, in the presence and absence of exogenously added TN-C. Inhibition of MMPs with GM6001 in SMCs cultured on attached collagen resulted in cellular rounding (data not shown) and a significant decline in SMC number, whereas inclusion of exogenous TN-C in the collagen gels prevented this decrease (Fig. 3 A) and restored their characteristic elongated morphology (data not shown). Similarly, on floating collagen, in which MMPs and TN-C expression are suppressed, SMC survival is diminished (Fig. 3 B) via apoptosis as determined by oligonucleosomal DNA fragmentation assays (Fig. 3 C). In contrast, SMC numbers were unaffected (Fig. 3 B), and apoptosis was suppressed in cells cultured on floating collagen gels supplemented with exogenous TN-C protein (Fig. 3 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