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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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VE-cadherin expression and clustering reduce the extent of p44/42 MAP kinase phosphorylation in response to VEGF. (A) Confluent and sparse VEC- and VEC-positive endothelial cells were stimulated with VEGF (80 ng/ml for 10 min), and phospho p44/42 MAP kinase and total MAP kinase were assayed by Western blot with specific antibodies. The columns represent the ratio between phosphorylated and total values as fold increase over the ratio calculated in sparse unstimulated VEC . VEGF-stimulated phosphorylation of MAP kinases was reduced at confluence only in VEC-positive cells. The mean ± SD of three independent experiments is reported. In a total of 12 experiments, the increase of p44/42 MAP kinase phosphorylation in VEC- cells ranged from three- to sixfold over VEC-positive cells. (B) In confluent HUVEC, phosphorylation of p44/42 MAP kinase in response to VEGF (80 ng/ml for 10 min) was reduced about threefold in comparison with sparse cells. Column values are as in A.
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fig3: VE-cadherin expression and clustering reduce the extent of p44/42 MAP kinase phosphorylation in response to VEGF. (A) Confluent and sparse VEC- and VEC-positive endothelial cells were stimulated with VEGF (80 ng/ml for 10 min), and phospho p44/42 MAP kinase and total MAP kinase were assayed by Western blot with specific antibodies. The columns represent the ratio between phosphorylated and total values as fold increase over the ratio calculated in sparse unstimulated VEC . VEGF-stimulated phosphorylation of MAP kinases was reduced at confluence only in VEC-positive cells. The mean ± SD of three independent experiments is reported. In a total of 12 experiments, the increase of p44/42 MAP kinase phosphorylation in VEC- cells ranged from three- to sixfold over VEC-positive cells. (B) In confluent HUVEC, phosphorylation of p44/42 MAP kinase in response to VEGF (80 ng/ml for 10 min) was reduced about threefold in comparison with sparse cells. Column values are as in A.

Mentions: Activation of MAP kinases is an important event in endothelial cell proliferation induced by growth factors (Vinals and Pouyssegur, 1999). As reported in Fig. 3 A and Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200209019/DC1), in confluent but not in sparse cells, VE-cadherin expression reduced the peak and duration of MAP kinase phosphorylation induced by VEGF. Consistently, upon VEGF stimulation, sparse HUVEC presented higher MAP kinase activation than confluent cells (Fig. 3 B).


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)

VE-cadherin expression and clustering reduce the extent of p44/42 MAP kinase phosphorylation in response to VEGF. (A) Confluent and sparse VEC- and VEC-positive endothelial cells were stimulated with VEGF (80 ng/ml for 10 min), and phospho p44/42 MAP kinase and total MAP kinase were assayed by Western blot with specific antibodies. The columns represent the ratio between phosphorylated and total values as fold increase over the ratio calculated in sparse unstimulated VEC . VEGF-stimulated phosphorylation of MAP kinases was reduced at confluence only in VEC-positive cells. The mean ± SD of three independent experiments is reported. In a total of 12 experiments, the increase of p44/42 MAP kinase phosphorylation in VEC- cells ranged from three- to sixfold over VEC-positive cells. (B) In confluent HUVEC, phosphorylation of p44/42 MAP kinase in response to VEGF (80 ng/ml for 10 min) was reduced about threefold in comparison with sparse cells. Column values are as in A.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199373&req=5

fig3: VE-cadherin expression and clustering reduce the extent of p44/42 MAP kinase phosphorylation in response to VEGF. (A) Confluent and sparse VEC- and VEC-positive endothelial cells were stimulated with VEGF (80 ng/ml for 10 min), and phospho p44/42 MAP kinase and total MAP kinase were assayed by Western blot with specific antibodies. The columns represent the ratio between phosphorylated and total values as fold increase over the ratio calculated in sparse unstimulated VEC . VEGF-stimulated phosphorylation of MAP kinases was reduced at confluence only in VEC-positive cells. The mean ± SD of three independent experiments is reported. In a total of 12 experiments, the increase of p44/42 MAP kinase phosphorylation in VEC- cells ranged from three- to sixfold over VEC-positive cells. (B) In confluent HUVEC, phosphorylation of p44/42 MAP kinase in response to VEGF (80 ng/ml for 10 min) was reduced about threefold in comparison with sparse cells. Column values are as in A.
Mentions: Activation of MAP kinases is an important event in endothelial cell proliferation induced by growth factors (Vinals and Pouyssegur, 1999). As reported in Fig. 3 A and Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200209019/DC1), in confluent but not in sparse cells, VE-cadherin expression reduced the peak and duration of MAP kinase phosphorylation induced by VEGF. Consistently, upon VEGF stimulation, sparse HUVEC presented higher MAP kinase activation than confluent cells (Fig. 3 B).

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