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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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VE-cadherin expression and clustering inhibit VEGFR-2 tyrosine phosphorylation. (A) Confluent VEC- and -positive endothelial cells were stimulated with VEGF (80 ng/ml) for the indicated time intervals. Cell extracts were immunoprecipitated with antibodies to VEGFR-2 (IP αVEGFR-2) and immunoblotted (IB) with antibodies to phosphotyrosine (αphosphoTyr) and VEGFR-2 (αVEGFR-2). A similar experimental procedure was used for B–D. In the representative experiment shown, tyrosine-phosphorylated VEGFR-2 normalized over total VEGFR-2 was 0.5-, five-, and twofold more in VEC- than in VEC-positive cells at time 0, 5, and 30 min, respectively. In 15 independent experiments, the range of increase at 5 min was from two- to sevenfold. (B) Tyrosine phosphorylation of VEGFR-2 in response to VEGF (80 ng/ml for 5 min) in sparse VEC- and -positive endothelial cells was comparable. (C) In HUVEC, phosphorylation of VEGFR-2 in response to VEGF (80 ng/ml for 5 min) was lower in confluent than in sparse cultures (range three- to fivefold lower in four experiments). (D) Addition of antibodies to VE-cadherin (anti-VEC, 100 μg/ml) for 1 h increased receptor phosphorylation by VEGF (80 ng/ml, for 5 min). In response to VEGF, the phosphotyrosine content in VEGFR-2 was higher (from three- to fourfold in three experiments) in cells pretreated with VE-cadherin antibody. The antibody to VEGFR-2 recognized two bands at a molecular mass of ∼200 kD. Only the higher molecular mass band, representing the mature form of the receptor, was phosphorylated in tyrosine, as also described by Takahashi and Shibuya (1997). In the following figures, we therefore show only the heavier band of the doublet, which represents the phosphorylable pool of VEGFR-2.
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fig2: VE-cadherin expression and clustering inhibit VEGFR-2 tyrosine phosphorylation. (A) Confluent VEC- and -positive endothelial cells were stimulated with VEGF (80 ng/ml) for the indicated time intervals. Cell extracts were immunoprecipitated with antibodies to VEGFR-2 (IP αVEGFR-2) and immunoblotted (IB) with antibodies to phosphotyrosine (αphosphoTyr) and VEGFR-2 (αVEGFR-2). A similar experimental procedure was used for B–D. In the representative experiment shown, tyrosine-phosphorylated VEGFR-2 normalized over total VEGFR-2 was 0.5-, five-, and twofold more in VEC- than in VEC-positive cells at time 0, 5, and 30 min, respectively. In 15 independent experiments, the range of increase at 5 min was from two- to sevenfold. (B) Tyrosine phosphorylation of VEGFR-2 in response to VEGF (80 ng/ml for 5 min) in sparse VEC- and -positive endothelial cells was comparable. (C) In HUVEC, phosphorylation of VEGFR-2 in response to VEGF (80 ng/ml for 5 min) was lower in confluent than in sparse cultures (range three- to fivefold lower in four experiments). (D) Addition of antibodies to VE-cadherin (anti-VEC, 100 μg/ml) for 1 h increased receptor phosphorylation by VEGF (80 ng/ml, for 5 min). In response to VEGF, the phosphotyrosine content in VEGFR-2 was higher (from three- to fourfold in three experiments) in cells pretreated with VE-cadherin antibody. The antibody to VEGFR-2 recognized two bands at a molecular mass of ∼200 kD. Only the higher molecular mass band, representing the mature form of the receptor, was phosphorylated in tyrosine, as also described by Takahashi and Shibuya (1997). In the following figures, we therefore show only the heavier band of the doublet, which represents the phosphorylable pool of VEGFR-2.

Mentions: VEGFR-2 plays a major role in the growth factor–induced endothelial cell proliferation (Ferrara, 1999) and p44/42 MAP kinase activation (Takahashi et al., 1999b, 2001). In Fig. 2 A, we show that the presence of VE-cadherin markedly reduced tyrosine phosphorylation of VEGFR-2 after VEGF activation of the cells. We then tested whether the correct clustering of VE-cadherin at junctions, and not only its expression, could affect receptor activation. As shown in Fig. 2 B, sparse VEC-positive cells were able to respond to VEGF like cells. Similarly, when freshly isolated human umbilical vein endothelial cells (HUVEC) were tested, VEGFR-2 phosphorylation was higher in sparse than in confluent cells (Fig. 2 C).


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)

VE-cadherin expression and clustering inhibit VEGFR-2 tyrosine phosphorylation. (A) Confluent VEC- and -positive endothelial cells were stimulated with VEGF (80 ng/ml) for the indicated time intervals. Cell extracts were immunoprecipitated with antibodies to VEGFR-2 (IP αVEGFR-2) and immunoblotted (IB) with antibodies to phosphotyrosine (αphosphoTyr) and VEGFR-2 (αVEGFR-2). A similar experimental procedure was used for B–D. In the representative experiment shown, tyrosine-phosphorylated VEGFR-2 normalized over total VEGFR-2 was 0.5-, five-, and twofold more in VEC- than in VEC-positive cells at time 0, 5, and 30 min, respectively. In 15 independent experiments, the range of increase at 5 min was from two- to sevenfold. (B) Tyrosine phosphorylation of VEGFR-2 in response to VEGF (80 ng/ml for 5 min) in sparse VEC- and -positive endothelial cells was comparable. (C) In HUVEC, phosphorylation of VEGFR-2 in response to VEGF (80 ng/ml for 5 min) was lower in confluent than in sparse cultures (range three- to fivefold lower in four experiments). (D) Addition of antibodies to VE-cadherin (anti-VEC, 100 μg/ml) for 1 h increased receptor phosphorylation by VEGF (80 ng/ml, for 5 min). In response to VEGF, the phosphotyrosine content in VEGFR-2 was higher (from three- to fourfold in three experiments) in cells pretreated with VE-cadherin antibody. The antibody to VEGFR-2 recognized two bands at a molecular mass of ∼200 kD. Only the higher molecular mass band, representing the mature form of the receptor, was phosphorylated in tyrosine, as also described by Takahashi and Shibuya (1997). In the following figures, we therefore show only the heavier band of the doublet, which represents the phosphorylable pool of VEGFR-2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199373&req=5

fig2: VE-cadherin expression and clustering inhibit VEGFR-2 tyrosine phosphorylation. (A) Confluent VEC- and -positive endothelial cells were stimulated with VEGF (80 ng/ml) for the indicated time intervals. Cell extracts were immunoprecipitated with antibodies to VEGFR-2 (IP αVEGFR-2) and immunoblotted (IB) with antibodies to phosphotyrosine (αphosphoTyr) and VEGFR-2 (αVEGFR-2). A similar experimental procedure was used for B–D. In the representative experiment shown, tyrosine-phosphorylated VEGFR-2 normalized over total VEGFR-2 was 0.5-, five-, and twofold more in VEC- than in VEC-positive cells at time 0, 5, and 30 min, respectively. In 15 independent experiments, the range of increase at 5 min was from two- to sevenfold. (B) Tyrosine phosphorylation of VEGFR-2 in response to VEGF (80 ng/ml for 5 min) in sparse VEC- and -positive endothelial cells was comparable. (C) In HUVEC, phosphorylation of VEGFR-2 in response to VEGF (80 ng/ml for 5 min) was lower in confluent than in sparse cultures (range three- to fivefold lower in four experiments). (D) Addition of antibodies to VE-cadherin (anti-VEC, 100 μg/ml) for 1 h increased receptor phosphorylation by VEGF (80 ng/ml, for 5 min). In response to VEGF, the phosphotyrosine content in VEGFR-2 was higher (from three- to fourfold in three experiments) in cells pretreated with VE-cadherin antibody. The antibody to VEGFR-2 recognized two bands at a molecular mass of ∼200 kD. Only the higher molecular mass band, representing the mature form of the receptor, was phosphorylated in tyrosine, as also described by Takahashi and Shibuya (1997). In the following figures, we therefore show only the heavier band of the doublet, which represents the phosphorylable pool of VEGFR-2.
Mentions: VEGFR-2 plays a major role in the growth factor–induced endothelial cell proliferation (Ferrara, 1999) and p44/42 MAP kinase activation (Takahashi et al., 1999b, 2001). In Fig. 2 A, we show that the presence of VE-cadherin markedly reduced tyrosine phosphorylation of VEGFR-2 after VEGF activation of the cells. We then tested whether the correct clustering of VE-cadherin at junctions, and not only its expression, could affect receptor activation. As shown in Fig. 2 B, sparse VEC-positive cells were able to respond to VEGF like cells. Similarly, when freshly isolated human umbilical vein endothelial cells (HUVEC) were tested, VEGFR-2 phosphorylation was higher in sparse than in confluent cells (Fig. 2 C).

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