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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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Down-regulation of DEP-1 increases VEGFR-2 phosphorylation. Transfection of the dominant-negative DEP C/S mutant or DEP siRNA induced higher phosphorylation of VEGFR-2 upon VEGF (80 ng/ml for 5 min) than the respective controls, VEC positive, DEP wild type (DEP wt), and scramble siRNA (scr siRNA), respectively. Data were obtained by immunoprecipitation (IP) with anti–VEGFR-2 antibodies (αVEGFR-2) followed by Western blot (IB) with an antiphosphotyrosine antibody (αphosphoTyr). The graph was obtained by quantifying the gel bands and calculating the ratio of bands labeled with antiphosphotyrosine antibodies over total VEGFR-2. Each column represents the fold increase over unstimulated cells. Data are means ± SD of four experiments. Plus and minus indicate the presence or the absence of the protein indicated on the left.
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fig9: Down-regulation of DEP-1 increases VEGFR-2 phosphorylation. Transfection of the dominant-negative DEP C/S mutant or DEP siRNA induced higher phosphorylation of VEGFR-2 upon VEGF (80 ng/ml for 5 min) than the respective controls, VEC positive, DEP wild type (DEP wt), and scramble siRNA (scr siRNA), respectively. Data were obtained by immunoprecipitation (IP) with anti–VEGFR-2 antibodies (αVEGFR-2) followed by Western blot (IB) with an antiphosphotyrosine antibody (αphosphoTyr). The graph was obtained by quantifying the gel bands and calculating the ratio of bands labeled with antiphosphotyrosine antibodies over total VEGFR-2. Each column represents the fold increase over unstimulated cells. Data are means ± SD of four experiments. Plus and minus indicate the presence or the absence of the protein indicated on the left.

Mentions: We then tested the effect of DEP-1 on VEGFR-2 phosphorylation. As shown in Fig. 9, DEP-1 C/S and siRNA partially restored VEGF-induced receptor phosphorylation in VE-cadherin–positive cells. Wild-type DEP-1 (DEPwt) and the scrambled oligonucleotides slightly, but not significantly, reduced VEGFR-2 activation. We obtained similar results when we measured the phosphorylation of Tyr 996 or 951 of the receptor (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200209019/DC1).


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)

Down-regulation of DEP-1 increases VEGFR-2 phosphorylation. Transfection of the dominant-negative DEP C/S mutant or DEP siRNA induced higher phosphorylation of VEGFR-2 upon VEGF (80 ng/ml for 5 min) than the respective controls, VEC positive, DEP wild type (DEP wt), and scramble siRNA (scr siRNA), respectively. Data were obtained by immunoprecipitation (IP) with anti–VEGFR-2 antibodies (αVEGFR-2) followed by Western blot (IB) with an antiphosphotyrosine antibody (αphosphoTyr). The graph was obtained by quantifying the gel bands and calculating the ratio of bands labeled with antiphosphotyrosine antibodies over total VEGFR-2. Each column represents the fold increase over unstimulated cells. Data are means ± SD of four experiments. Plus and minus indicate the presence or the absence of the protein indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199373&req=5

fig9: Down-regulation of DEP-1 increases VEGFR-2 phosphorylation. Transfection of the dominant-negative DEP C/S mutant or DEP siRNA induced higher phosphorylation of VEGFR-2 upon VEGF (80 ng/ml for 5 min) than the respective controls, VEC positive, DEP wild type (DEP wt), and scramble siRNA (scr siRNA), respectively. Data were obtained by immunoprecipitation (IP) with anti–VEGFR-2 antibodies (αVEGFR-2) followed by Western blot (IB) with an antiphosphotyrosine antibody (αphosphoTyr). The graph was obtained by quantifying the gel bands and calculating the ratio of bands labeled with antiphosphotyrosine antibodies over total VEGFR-2. Each column represents the fold increase over unstimulated cells. Data are means ± SD of four experiments. Plus and minus indicate the presence or the absence of the protein indicated on the left.
Mentions: We then tested the effect of DEP-1 on VEGFR-2 phosphorylation. As shown in Fig. 9, DEP-1 C/S and siRNA partially restored VEGF-induced receptor phosphorylation in VE-cadherin–positive cells. Wild-type DEP-1 (DEPwt) and the scrambled oligonucleotides slightly, but not significantly, reduced VEGFR-2 activation. We obtained similar results when we measured the phosphorylation of Tyr 996 or 951 of the receptor (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200209019/DC1).

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