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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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The absence of β-catenin enhances VEGF-induced phosphorylation of VEGFR-2 and cell proliferation. Endothelial cells derived from β-catenin– embryos (β-cat ) did not express β-catenin in comparison with cells obtained from β-catenin–positive (β-cat positive) littermate animals. (A) By immunofluorescence analysis, VE-cadherin was expressed at a comparable level and was concentrated at cell–cell contacts in both β-catenin– and –positive cells. Cell junctions were negative for β-catenin in  cells. Bars: (phase contrast) 100 μm; (VE-cadherin and β-catenin) 20 μm. (B) Immunoprecipitation (IP) of cell extracts with VE-cadherin antibodies followed by Western blot (IB) with anti–VE-cadherin (αVE-cadherin) or β-catenin (αβ-catenin) antibodies showed absence of the last protein in the complex. (C) The absence of β-catenin enhanced the extent and duration of VEGFR-2 phosphorylation in response to VEGF (80 ng/ml). IP with anti–VEGFR-2 and Western blot with antiphosphotyrosine and anti–VEGFR-2 antibodies. (D) VE-cadherin could be coimmunoprecipitated with VEGFR-2 only in β-positive cells after VEGF (80 ng/ml for 5 min). IP with anti–VEGFR-2 and Western blot with anti–VEGFR-2 and anti–VE-cadherin antibodies. (E) Confluent β-cat– endothelial cells incorporated BrdU 2–2.5-fold more than β-cat–positive cells in response to stimulation with VEGF (80 ng/ml for 24 h). Incorporation of BrdU was measured and calculated (mean of three independent experiments ± SD) as in Fig. 1. Two independent pairs of both β-positive and β- endothelial cells obtained from littermate embryos of different mothers have been tested with comparable results.
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fig7: The absence of β-catenin enhances VEGF-induced phosphorylation of VEGFR-2 and cell proliferation. Endothelial cells derived from β-catenin– embryos (β-cat ) did not express β-catenin in comparison with cells obtained from β-catenin–positive (β-cat positive) littermate animals. (A) By immunofluorescence analysis, VE-cadherin was expressed at a comparable level and was concentrated at cell–cell contacts in both β-catenin– and –positive cells. Cell junctions were negative for β-catenin in cells. Bars: (phase contrast) 100 μm; (VE-cadherin and β-catenin) 20 μm. (B) Immunoprecipitation (IP) of cell extracts with VE-cadherin antibodies followed by Western blot (IB) with anti–VE-cadherin (αVE-cadherin) or β-catenin (αβ-catenin) antibodies showed absence of the last protein in the complex. (C) The absence of β-catenin enhanced the extent and duration of VEGFR-2 phosphorylation in response to VEGF (80 ng/ml). IP with anti–VEGFR-2 and Western blot with antiphosphotyrosine and anti–VEGFR-2 antibodies. (D) VE-cadherin could be coimmunoprecipitated with VEGFR-2 only in β-positive cells after VEGF (80 ng/ml for 5 min). IP with anti–VEGFR-2 and Western blot with anti–VEGFR-2 and anti–VE-cadherin antibodies. (E) Confluent β-cat– endothelial cells incorporated BrdU 2–2.5-fold more than β-cat–positive cells in response to stimulation with VEGF (80 ng/ml for 24 h). Incorporation of BrdU was measured and calculated (mean of three independent experiments ± SD) as in Fig. 1. Two independent pairs of both β-positive and β- endothelial cells obtained from littermate embryos of different mothers have been tested with comparable results.

Mentions: As reported in Fig. 7 (A and B), β-catenin was absent in these mutants whereas VE-cadherin expression was comparable. β-Catenin– cells retained the endothelial cell markers tested (including VEGFR-2, Tie2, and CD34) and correctly organized junctions (as detected by the localization of VE-cadherin, ZO-1, occludin, PECAM, and JAM) (Fig. 7 A; unpublished data). β-Catenin– cells presented higher density at confluence and elongated morphology in comparison with the positive cells (Fig. 7 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)

The absence of β-catenin enhances VEGF-induced phosphorylation of VEGFR-2 and cell proliferation. Endothelial cells derived from β-catenin– embryos (β-cat ) did not express β-catenin in comparison with cells obtained from β-catenin–positive (β-cat positive) littermate animals. (A) By immunofluorescence analysis, VE-cadherin was expressed at a comparable level and was concentrated at cell–cell contacts in both β-catenin– and –positive cells. Cell junctions were negative for β-catenin in  cells. Bars: (phase contrast) 100 μm; (VE-cadherin and β-catenin) 20 μm. (B) Immunoprecipitation (IP) of cell extracts with VE-cadherin antibodies followed by Western blot (IB) with anti–VE-cadherin (αVE-cadherin) or β-catenin (αβ-catenin) antibodies showed absence of the last protein in the complex. (C) The absence of β-catenin enhanced the extent and duration of VEGFR-2 phosphorylation in response to VEGF (80 ng/ml). IP with anti–VEGFR-2 and Western blot with antiphosphotyrosine and anti–VEGFR-2 antibodies. (D) VE-cadherin could be coimmunoprecipitated with VEGFR-2 only in β-positive cells after VEGF (80 ng/ml for 5 min). IP with anti–VEGFR-2 and Western blot with anti–VEGFR-2 and anti–VE-cadherin antibodies. (E) Confluent β-cat– endothelial cells incorporated BrdU 2–2.5-fold more than β-cat–positive cells in response to stimulation with VEGF (80 ng/ml for 24 h). Incorporation of BrdU was measured and calculated (mean of three independent experiments ± SD) as in Fig. 1. Two independent pairs of both β-positive and β- endothelial cells obtained from littermate embryos of different mothers have been tested with comparable results.
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fig7: The absence of β-catenin enhances VEGF-induced phosphorylation of VEGFR-2 and cell proliferation. Endothelial cells derived from β-catenin– embryos (β-cat ) did not express β-catenin in comparison with cells obtained from β-catenin–positive (β-cat positive) littermate animals. (A) By immunofluorescence analysis, VE-cadherin was expressed at a comparable level and was concentrated at cell–cell contacts in both β-catenin– and –positive cells. Cell junctions were negative for β-catenin in cells. Bars: (phase contrast) 100 μm; (VE-cadherin and β-catenin) 20 μm. (B) Immunoprecipitation (IP) of cell extracts with VE-cadherin antibodies followed by Western blot (IB) with anti–VE-cadherin (αVE-cadherin) or β-catenin (αβ-catenin) antibodies showed absence of the last protein in the complex. (C) The absence of β-catenin enhanced the extent and duration of VEGFR-2 phosphorylation in response to VEGF (80 ng/ml). IP with anti–VEGFR-2 and Western blot with antiphosphotyrosine and anti–VEGFR-2 antibodies. (D) VE-cadherin could be coimmunoprecipitated with VEGFR-2 only in β-positive cells after VEGF (80 ng/ml for 5 min). IP with anti–VEGFR-2 and Western blot with anti–VEGFR-2 and anti–VE-cadherin antibodies. (E) Confluent β-cat– endothelial cells incorporated BrdU 2–2.5-fold more than β-cat–positive cells in response to stimulation with VEGF (80 ng/ml for 24 h). Incorporation of BrdU was measured and calculated (mean of three independent experiments ± SD) as in Fig. 1. Two independent pairs of both β-positive and β- endothelial cells obtained from littermate embryos of different mothers have been tested with comparable results.
Mentions: As reported in Fig. 7 (A and B), β-catenin was absent in these mutants whereas VE-cadherin expression was comparable. β-Catenin– cells retained the endothelial cell markers tested (including VEGFR-2, Tie2, and CD34) and correctly organized junctions (as detected by the localization of VE-cadherin, ZO-1, occludin, PECAM, and JAM) (Fig. 7 A; unpublished data). β-Catenin– cells presented higher density at confluence and elongated morphology in comparison with the positive cells (Fig. 7 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