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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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Confluence and VE-cadherin expression inhibit endothelial proliferation induced by VEGF. (A) Growth curve of endothelial cells expressing (VEC positive) or not expressing (VEC ) VE-cadherin. Cells (seeding 30,000/cm2) were cultured in complete culture medium. At the indicated time point, cells were detached and counted. The standard deviation in three independent experiments was between 5 and 10% of the mean values. (B) Confluent (100,000/cm2) and sparse (20,000/cm2) VEC-positive and VEC- cells were stimulated with VEGF (80 ng/ml) for 24 h. BrdU (30 μM) was added during the last 4 h. A total of 300 nuclei in random fields for each treatment were scored. The mean values of three independent experiments ± SD are shown.
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fig1: Confluence and VE-cadherin expression inhibit endothelial proliferation induced by VEGF. (A) Growth curve of endothelial cells expressing (VEC positive) or not expressing (VEC ) VE-cadherin. Cells (seeding 30,000/cm2) were cultured in complete culture medium. At the indicated time point, cells were detached and counted. The standard deviation in three independent experiments was between 5 and 10% of the mean values. (B) Confluent (100,000/cm2) and sparse (20,000/cm2) VEC-positive and VEC- cells were stimulated with VEGF (80 ng/ml) for 24 h. BrdU (30 μM) was added during the last 4 h. A total of 300 nuclei in random fields for each treatment were scored. The mean values of three independent experiments ± SD are shown.

Mentions: As shown in Fig. 1 A, VEC-positive cells arrested their growth at confluence, whereas VEC-negative cells maintained a sustained growth rate reaching a two to threefold higher density than positive cells. Confluent VEC-positive cells behaved like wild-type endothelium (Caveda et al., 1996; Vinals and Pouyssegur, 1999), showing a markedly lower DNA synthesis upon VEGF stimulation than sparse cells (Fig. 1 B). In contrast, VEC- cells were highly responsive to VEGF both in sparse and confluent conditions. Thus, VE-cadherin substantially reduces the response of confluent, but not sparse, endothelium to VEGF.


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)

Confluence and VE-cadherin expression inhibit endothelial proliferation induced by VEGF. (A) Growth curve of endothelial cells expressing (VEC positive) or not expressing (VEC ) VE-cadherin. Cells (seeding 30,000/cm2) were cultured in complete culture medium. At the indicated time point, cells were detached and counted. The standard deviation in three independent experiments was between 5 and 10% of the mean values. (B) Confluent (100,000/cm2) and sparse (20,000/cm2) VEC-positive and VEC- cells were stimulated with VEGF (80 ng/ml) for 24 h. BrdU (30 μM) was added during the last 4 h. A total of 300 nuclei in random fields for each treatment were scored. The mean values of three independent experiments ± SD are shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Confluence and VE-cadherin expression inhibit endothelial proliferation induced by VEGF. (A) Growth curve of endothelial cells expressing (VEC positive) or not expressing (VEC ) VE-cadherin. Cells (seeding 30,000/cm2) were cultured in complete culture medium. At the indicated time point, cells were detached and counted. The standard deviation in three independent experiments was between 5 and 10% of the mean values. (B) Confluent (100,000/cm2) and sparse (20,000/cm2) VEC-positive and VEC- cells were stimulated with VEGF (80 ng/ml) for 24 h. BrdU (30 μM) was added during the last 4 h. A total of 300 nuclei in random fields for each treatment were scored. The mean values of three independent experiments ± SD are shown.
Mentions: As shown in Fig. 1 A, VEC-positive cells arrested their growth at confluence, whereas VEC-negative cells maintained a sustained growth rate reaching a two to threefold higher density than positive cells. Confluent VEC-positive cells behaved like wild-type endothelium (Caveda et al., 1996; Vinals and Pouyssegur, 1999), showing a markedly lower DNA synthesis upon VEGF stimulation than sparse cells (Fig. 1 B). In contrast, VEC- cells were highly responsive to VEGF both in sparse and confluent conditions. Thus, VE-cadherin substantially reduces the response of confluent, but not sparse, endothelium to VEGF.

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