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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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Inhibition of p44/42 MAP kinase phosphorylation correlates with reduction of BrdU incorporation in VEC- endothelial cells. (A) Confluent VEC- or -positive cells were treated with PD98059 (100 μM) for 20 min before challenge with VEGF (80 ng/ml for 10 min). VEC-positive cells were transfected with a constitutive active or wild-type MAP kinase kinase (MAPKK) construct or the empty vector. Nuclear incorporation of BrdU was evaluated after 24 h as in the legend to Fig. 1. PD98059 strongly inhibited proliferation of VEC- cells in response to VEGF. Proliferation of VEC-positive cells was very low and was inhibited by the drug. Constitutively active MAPK kinase partially restored BrdU incorporation in response to VEGF in VEC-positive cells. (B) The concentration of PD98059 used in A was able to strongly inhibit phosphorylation of p44/42 MAP kinase after a 10-min stimulation with VEGF (80 ng/ml). Each column represents the fold increase of the ratio between phosphorylated and total values in VEGF-stimulated over the respective unstimulated condition. Data are means ± SD of three independent experiments. The vehicle is DMSO added at the same final concentration (0.1%) as in PD98059-treated cells.
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fig4: Inhibition of p44/42 MAP kinase phosphorylation correlates with reduction of BrdU incorporation in VEC- endothelial cells. (A) Confluent VEC- or -positive cells were treated with PD98059 (100 μM) for 20 min before challenge with VEGF (80 ng/ml for 10 min). VEC-positive cells were transfected with a constitutive active or wild-type MAP kinase kinase (MAPKK) construct or the empty vector. Nuclear incorporation of BrdU was evaluated after 24 h as in the legend to Fig. 1. PD98059 strongly inhibited proliferation of VEC- cells in response to VEGF. Proliferation of VEC-positive cells was very low and was inhibited by the drug. Constitutively active MAPK kinase partially restored BrdU incorporation in response to VEGF in VEC-positive cells. (B) The concentration of PD98059 used in A was able to strongly inhibit phosphorylation of p44/42 MAP kinase after a 10-min stimulation with VEGF (80 ng/ml). Each column represents the fold increase of the ratio between phosphorylated and total values in VEGF-stimulated over the respective unstimulated condition. Data are means ± SD of three independent experiments. The vehicle is DMSO added at the same final concentration (0.1%) as in PD98059-treated cells.

Mentions: Treatment of VEC- and -positive cells with PD98059 significantly reduced p44/42 MAP kinase activation and DNA synthesis after VEGF (Fig. 4, A and B). In addition, transfection of VEC-positive cells with a constitutive active MAP kinase kinase, but not with the wild-type construct or the empty vector, partially but significantly rescued cell proliferation.


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)

Inhibition of p44/42 MAP kinase phosphorylation correlates with reduction of BrdU incorporation in VEC- endothelial cells. (A) Confluent VEC- or -positive cells were treated with PD98059 (100 μM) for 20 min before challenge with VEGF (80 ng/ml for 10 min). VEC-positive cells were transfected with a constitutive active or wild-type MAP kinase kinase (MAPKK) construct or the empty vector. Nuclear incorporation of BrdU was evaluated after 24 h as in the legend to Fig. 1. PD98059 strongly inhibited proliferation of VEC- cells in response to VEGF. Proliferation of VEC-positive cells was very low and was inhibited by the drug. Constitutively active MAPK kinase partially restored BrdU incorporation in response to VEGF in VEC-positive cells. (B) The concentration of PD98059 used in A was able to strongly inhibit phosphorylation of p44/42 MAP kinase after a 10-min stimulation with VEGF (80 ng/ml). Each column represents the fold increase of the ratio between phosphorylated and total values in VEGF-stimulated over the respective unstimulated condition. Data are means ± SD of three independent experiments. The vehicle is DMSO added at the same final concentration (0.1%) as in PD98059-treated cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199373&req=5

fig4: Inhibition of p44/42 MAP kinase phosphorylation correlates with reduction of BrdU incorporation in VEC- endothelial cells. (A) Confluent VEC- or -positive cells were treated with PD98059 (100 μM) for 20 min before challenge with VEGF (80 ng/ml for 10 min). VEC-positive cells were transfected with a constitutive active or wild-type MAP kinase kinase (MAPKK) construct or the empty vector. Nuclear incorporation of BrdU was evaluated after 24 h as in the legend to Fig. 1. PD98059 strongly inhibited proliferation of VEC- cells in response to VEGF. Proliferation of VEC-positive cells was very low and was inhibited by the drug. Constitutively active MAPK kinase partially restored BrdU incorporation in response to VEGF in VEC-positive cells. (B) The concentration of PD98059 used in A was able to strongly inhibit phosphorylation of p44/42 MAP kinase after a 10-min stimulation with VEGF (80 ng/ml). Each column represents the fold increase of the ratio between phosphorylated and total values in VEGF-stimulated over the respective unstimulated condition. Data are means ± SD of three independent experiments. The vehicle is DMSO added at the same final concentration (0.1%) as in PD98059-treated cells.
Mentions: Treatment of VEC- and -positive cells with PD98059 significantly reduced p44/42 MAP kinase activation and DNA synthesis after VEGF (Fig. 4, A and B). In addition, transfection of VEC-positive cells with a constitutive active MAP kinase kinase, but not with the wild-type construct or the empty vector, partially but significantly rescued cell proliferation.

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