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Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, beta-catenin, and the phosphatase DEP-1/CD148.

Grazia Lampugnani M, Zanetti A, Corada M, Takahashi T, Balconi G, Breviario F, Orsenigo F, Cattelino A, Kemler R, Daniel TO, Dejana E - J. Cell Biol. (2003)

Bottom Line: Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation.A dominant-negative mutant of high cell density-enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation.In sparse cells or in VE-cadherin- cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.

View Article: PubMed Central - PubMed

Affiliation: FIRC Institute of Molecular Oncology, 20139 Milan, Italy.

ABSTRACT
Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in beta-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. beta-Catenin- endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density-enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin-beta-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell-cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin- cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.

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VEGFR-2 association and dephosphorylation requires the β-catenin binding domain of VE-cadherin. VEC- cells were transfected with VE-cadherin wild type or truncated mutants lacking β-catenin (Δ-βcat) or p120 (Δ-p120) binding domains. Intra, intracellular region; extra, extracellular region (C). (A) After stimulation with VEGF (80 ng/ml) for 5 and 30 min, cell extracts were immunoprecipitated (IP) with antibodies to VEGFR-2 (αVEGFR-2) and immunoblotted (IB) with antibodies to phosphotyrosine (αphosphoTyr), VEGFR-2 (αVEGFR-2), and VE-cadherin (αVEC). Wild-type (molecular mass, ∼120 kD) and Δ-p120 VE-cadherin (molecular mass, ∼100 kD) were coimmunoprecipitated with VEGFR-2 (A, lower panel). Receptor phosphorylation was significantly reduced in VEC-positive and Δ-p120, but not in Δ-βcat, in comparison with VEC- cells. The quantification of receptor phosphorylation data from three experiments ± SD is shown in A on the right. The values represent the ratio between the phosphorylated and total amount of VEGFR-2 and are normalized to the ratio calculated in untreated VEC-positive cells. The peak of VEGFR-2 phosphorylation at 5 min is similar in VEC- and Δ-βcat, but lower in Δ-p120. At longer stimulation (30 min), the level of phosphorylation of VEGFR-2 in Δ-p120 was comparable to VEC-positive cells. Incubation of VEC-positive cell extract with nonimmune (NI) rabbit immunoglobulin (matched with VEGFR-2 antibody used for IP) did not precipitate bands corresponding to either VEGFR-2 or VE-cadherin, last lane from the left (IP NI). (B) VE-cadherin mutants modulate endothelial growth induced by VEGF. VEC- and Δ-βcat had comparable effects and were the most permissive mutations in terms of cell proliferation (>160% increase over VEC-positive cells). Mutations that affected binding of p120 (Δ-p120) allowed cell proliferation, but to a lower extent (60% increase over stimulation of VEC-positive cells). Proliferation was measured as BrdU incorporation as described in the legend to Fig. 1. Mean ± SD of three independent experiments, each in duplicate, is shown.
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fig6: VEGFR-2 association and dephosphorylation requires the β-catenin binding domain of VE-cadherin. VEC- cells were transfected with VE-cadherin wild type or truncated mutants lacking β-catenin (Δ-βcat) or p120 (Δ-p120) binding domains. Intra, intracellular region; extra, extracellular region (C). (A) After stimulation with VEGF (80 ng/ml) for 5 and 30 min, cell extracts were immunoprecipitated (IP) with antibodies to VEGFR-2 (αVEGFR-2) and immunoblotted (IB) with antibodies to phosphotyrosine (αphosphoTyr), VEGFR-2 (αVEGFR-2), and VE-cadherin (αVEC). Wild-type (molecular mass, ∼120 kD) and Δ-p120 VE-cadherin (molecular mass, ∼100 kD) were coimmunoprecipitated with VEGFR-2 (A, lower panel). Receptor phosphorylation was significantly reduced in VEC-positive and Δ-p120, but not in Δ-βcat, in comparison with VEC- cells. The quantification of receptor phosphorylation data from three experiments ± SD is shown in A on the right. The values represent the ratio between the phosphorylated and total amount of VEGFR-2 and are normalized to the ratio calculated in untreated VEC-positive cells. The peak of VEGFR-2 phosphorylation at 5 min is similar in VEC- and Δ-βcat, but lower in Δ-p120. At longer stimulation (30 min), the level of phosphorylation of VEGFR-2 in Δ-p120 was comparable to VEC-positive cells. Incubation of VEC-positive cell extract with nonimmune (NI) rabbit immunoglobulin (matched with VEGFR-2 antibody used for IP) did not precipitate bands corresponding to either VEGFR-2 or VE-cadherin, last lane from the left (IP NI). (B) VE-cadherin mutants modulate endothelial growth induced by VEGF. VEC- and Δ-βcat had comparable effects and were the most permissive mutations in terms of cell proliferation (>160% increase over VEC-positive cells). Mutations that affected binding of p120 (Δ-p120) allowed cell proliferation, but to a lower extent (60% increase over stimulation of VEC-positive cells). Proliferation was measured as BrdU incorporation as described in the legend to Fig. 1. Mean ± SD of three independent experiments, each in duplicate, is shown.

Mentions: In previous work (Carmeliet et al., 1999), we found that VE-cadherin can be coimmunoprecipitated with VEGFR-2. We investigated whether this interaction is required for inhibition of VEGFR-2 phosphorylation. To this end, we analyzed VEC- endothelial cells transfected with different mutants of VE-cadherin cytoplasmic tail. As described previously in detail (Lampugnani et al., 2002), mutant proteins are expressed at levels comparable to the wild type and are correctly clustered to cell–cell contacts. As reported in Fig. 6 (A and C), when the region responsible for binding to β-catenin was truncated (from aa 703 to 784, Δ-βcat), VEGFR-2 could not associate with VE-cadherin. In contrast, when the region responsible for p120 association was deleted (from aa 621 to 702, Δ-p120), the receptor was still coimmunoprecipitated with VE-cadherin. Compared with cells, expression of Δ-βcat did not change VEGFR-2 phosphorylation, whereas transfection of Δ-p120 reduced this parameter, although not as effectively as VE-cadherin wild type (Fig. 6 A).


Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, beta-catenin, and the phosphatase DEP-1/CD148.

Grazia Lampugnani M, Zanetti A, Corada M, Takahashi T, Balconi G, Breviario F, Orsenigo F, Cattelino A, Kemler R, Daniel TO, Dejana E - J. Cell Biol. (2003)

VEGFR-2 association and dephosphorylation requires the β-catenin binding domain of VE-cadherin. VEC- cells were transfected with VE-cadherin wild type or truncated mutants lacking β-catenin (Δ-βcat) or p120 (Δ-p120) binding domains. Intra, intracellular region; extra, extracellular region (C). (A) After stimulation with VEGF (80 ng/ml) for 5 and 30 min, cell extracts were immunoprecipitated (IP) with antibodies to VEGFR-2 (αVEGFR-2) and immunoblotted (IB) with antibodies to phosphotyrosine (αphosphoTyr), VEGFR-2 (αVEGFR-2), and VE-cadherin (αVEC). Wild-type (molecular mass, ∼120 kD) and Δ-p120 VE-cadherin (molecular mass, ∼100 kD) were coimmunoprecipitated with VEGFR-2 (A, lower panel). Receptor phosphorylation was significantly reduced in VEC-positive and Δ-p120, but not in Δ-βcat, in comparison with VEC- cells. The quantification of receptor phosphorylation data from three experiments ± SD is shown in A on the right. The values represent the ratio between the phosphorylated and total amount of VEGFR-2 and are normalized to the ratio calculated in untreated VEC-positive cells. The peak of VEGFR-2 phosphorylation at 5 min is similar in VEC- and Δ-βcat, but lower in Δ-p120. At longer stimulation (30 min), the level of phosphorylation of VEGFR-2 in Δ-p120 was comparable to VEC-positive cells. Incubation of VEC-positive cell extract with nonimmune (NI) rabbit immunoglobulin (matched with VEGFR-2 antibody used for IP) did not precipitate bands corresponding to either VEGFR-2 or VE-cadherin, last lane from the left (IP NI). (B) VE-cadherin mutants modulate endothelial growth induced by VEGF. VEC- and Δ-βcat had comparable effects and were the most permissive mutations in terms of cell proliferation (>160% increase over VEC-positive cells). Mutations that affected binding of p120 (Δ-p120) allowed cell proliferation, but to a lower extent (60% increase over stimulation of VEC-positive cells). Proliferation was measured as BrdU incorporation as described in the legend to Fig. 1. Mean ± SD of three independent experiments, each in duplicate, is shown.
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fig6: VEGFR-2 association and dephosphorylation requires the β-catenin binding domain of VE-cadherin. VEC- cells were transfected with VE-cadherin wild type or truncated mutants lacking β-catenin (Δ-βcat) or p120 (Δ-p120) binding domains. Intra, intracellular region; extra, extracellular region (C). (A) After stimulation with VEGF (80 ng/ml) for 5 and 30 min, cell extracts were immunoprecipitated (IP) with antibodies to VEGFR-2 (αVEGFR-2) and immunoblotted (IB) with antibodies to phosphotyrosine (αphosphoTyr), VEGFR-2 (αVEGFR-2), and VE-cadherin (αVEC). Wild-type (molecular mass, ∼120 kD) and Δ-p120 VE-cadherin (molecular mass, ∼100 kD) were coimmunoprecipitated with VEGFR-2 (A, lower panel). Receptor phosphorylation was significantly reduced in VEC-positive and Δ-p120, but not in Δ-βcat, in comparison with VEC- cells. The quantification of receptor phosphorylation data from three experiments ± SD is shown in A on the right. The values represent the ratio between the phosphorylated and total amount of VEGFR-2 and are normalized to the ratio calculated in untreated VEC-positive cells. The peak of VEGFR-2 phosphorylation at 5 min is similar in VEC- and Δ-βcat, but lower in Δ-p120. At longer stimulation (30 min), the level of phosphorylation of VEGFR-2 in Δ-p120 was comparable to VEC-positive cells. Incubation of VEC-positive cell extract with nonimmune (NI) rabbit immunoglobulin (matched with VEGFR-2 antibody used for IP) did not precipitate bands corresponding to either VEGFR-2 or VE-cadherin, last lane from the left (IP NI). (B) VE-cadherin mutants modulate endothelial growth induced by VEGF. VEC- and Δ-βcat had comparable effects and were the most permissive mutations in terms of cell proliferation (>160% increase over VEC-positive cells). Mutations that affected binding of p120 (Δ-p120) allowed cell proliferation, but to a lower extent (60% increase over stimulation of VEC-positive cells). Proliferation was measured as BrdU incorporation as described in the legend to Fig. 1. Mean ± SD of three independent experiments, each in duplicate, is shown.
Mentions: In previous work (Carmeliet et al., 1999), we found that VE-cadherin can be coimmunoprecipitated with VEGFR-2. We investigated whether this interaction is required for inhibition of VEGFR-2 phosphorylation. To this end, we analyzed VEC- endothelial cells transfected with different mutants of VE-cadherin cytoplasmic tail. As described previously in detail (Lampugnani et al., 2002), mutant proteins are expressed at levels comparable to the wild type and are correctly clustered to cell–cell contacts. As reported in Fig. 6 (A and C), when the region responsible for binding to β-catenin was truncated (from aa 703 to 784, Δ-βcat), VEGFR-2 could not associate with VE-cadherin. In contrast, when the region responsible for p120 association was deleted (from aa 621 to 702, Δ-p120), the receptor was still coimmunoprecipitated with VE-cadherin. Compared with cells, expression of Δ-βcat did not change VEGFR-2 phosphorylation, whereas transfection of Δ-p120 reduced this parameter, although not as effectively as VE-cadherin wild type (Fig. 6 A).

Bottom Line: Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation.A dominant-negative mutant of high cell density-enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation.In sparse cells or in VE-cadherin- cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.

View Article: PubMed Central - PubMed

Affiliation: FIRC Institute of Molecular Oncology, 20139 Milan, Italy.

ABSTRACT
Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in beta-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. beta-Catenin- endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density-enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin-beta-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell-cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin- cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.

Show MeSH
Related in: MedlinePlus