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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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Expression of tyrosine phosphatase DEP-1 in VEC-positive and -negative cells and effect of transfection of wild-type and DEP-1 C/S constructs and DEP-1 RNAi. In VEC-positive endothelial cells (third lane), DEP-1 was 80–90% higher than in VEC  (fourth lane). In VEC-positive cells, transfection of wild-type (wt, first lane) or point-mutated (C/S, second lane) DEP-1 constructs resulted in increased expression of the protein (50–60% more than in control VEC positive). siRNA directed against DEP-1 (DEP siRNA) resulted in 35–40% inhibition of DEP expression (fifth lane). Scramble oligonucleotides (scr siRNA), used as control (100 nM as DEP siRNA oligonucleotide), were ineffective. Plus and minus indicate the presence or the absence of the protein indicated on the left. The columns report the mean of four experiments ± SD.
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fig8: Expression of tyrosine phosphatase DEP-1 in VEC-positive and -negative cells and effect of transfection of wild-type and DEP-1 C/S constructs and DEP-1 RNAi. In VEC-positive endothelial cells (third lane), DEP-1 was 80–90% higher than in VEC (fourth lane). In VEC-positive cells, transfection of wild-type (wt, first lane) or point-mutated (C/S, second lane) DEP-1 constructs resulted in increased expression of the protein (50–60% more than in control VEC positive). siRNA directed against DEP-1 (DEP siRNA) resulted in 35–40% inhibition of DEP expression (fifth lane). Scramble oligonucleotides (scr siRNA), used as control (100 nM as DEP siRNA oligonucleotide), were ineffective. Plus and minus indicate the presence or the absence of the protein indicated on the left. The columns report the mean of four experiments ± SD.

Mentions: As reported in Fig. 8, expression of VE-cadherin increased DEP-1 protein levels by 80–90% by Western blot analysis. Transfection of VE-cadherin–positive cells with wild-type and mutant DEP-1 constructs resulted in increased levels of the corresponding proteins by Western blot (∼50% in comparison with VEC positive). siRNA directed against DEP-1 reduced DEP-1 production by ∼40% in VE-cadherin–positive cells, whereas the scramble oligonucleotides did not have a significant effect.


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)

Expression of tyrosine phosphatase DEP-1 in VEC-positive and -negative cells and effect of transfection of wild-type and DEP-1 C/S constructs and DEP-1 RNAi. In VEC-positive endothelial cells (third lane), DEP-1 was 80–90% higher than in VEC  (fourth lane). In VEC-positive cells, transfection of wild-type (wt, first lane) or point-mutated (C/S, second lane) DEP-1 constructs resulted in increased expression of the protein (50–60% more than in control VEC positive). siRNA directed against DEP-1 (DEP siRNA) resulted in 35–40% inhibition of DEP expression (fifth lane). Scramble oligonucleotides (scr siRNA), used as control (100 nM as DEP siRNA oligonucleotide), were ineffective. Plus and minus indicate the presence or the absence of the protein indicated on the left. The columns report the mean of four experiments ± SD.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Expression of tyrosine phosphatase DEP-1 in VEC-positive and -negative cells and effect of transfection of wild-type and DEP-1 C/S constructs and DEP-1 RNAi. In VEC-positive endothelial cells (third lane), DEP-1 was 80–90% higher than in VEC (fourth lane). In VEC-positive cells, transfection of wild-type (wt, first lane) or point-mutated (C/S, second lane) DEP-1 constructs resulted in increased expression of the protein (50–60% more than in control VEC positive). siRNA directed against DEP-1 (DEP siRNA) resulted in 35–40% inhibition of DEP expression (fifth lane). Scramble oligonucleotides (scr siRNA), used as control (100 nM as DEP siRNA oligonucleotide), were ineffective. Plus and minus indicate the presence or the absence of the protein indicated on the left. The columns report the mean of four experiments ± SD.
Mentions: As reported in Fig. 8, expression of VE-cadherin increased DEP-1 protein levels by 80–90% by Western blot analysis. Transfection of VE-cadherin–positive cells with wild-type and mutant DEP-1 constructs resulted in increased levels of the corresponding proteins by Western blot (∼50% in comparison with VEC positive). siRNA directed against DEP-1 reduced DEP-1 production by ∼40% in VE-cadherin–positive cells, whereas the scramble oligonucleotides did not have a significant effect.

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