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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:617.)
© 2002 American Heart Association, Inc.

Vascular Biology

Vascular Endothelial Growth Factor Induces Shc Association With Vascular Endothelial Cadherin

A Potential Feedback Mechanism to Control Vascular Endothelial Growth Factor Receptor-2 Signaling

Adriana Zanetti; Maria Grazia Lampugnani; Giovanna Balconi; Ferruccio Breviario; Monica Corada; Luisa Lanfrancone; Elisabetta Dejana

From the Mario Negri Institute of Pharmacological Research (A.Z., M.G.L., G.B., F.B., M.C., E.D.), Milan; the FIRC Institute of Molecular Oncology (M.G.L., F.B., M.C., E.D.), Milan; the European Institute of Oncology (L.L.), Milan; and the Dipartimento di Scienze Cliniche e Biologiche (E.D.), Facoltà di Medicina e Chirurgia, Università dell’Insubria, Varese, Italy.

Correspondence to Elisabetta Dejana, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy. E-mail dejana{at}marionegri.it

	Abstract

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Vascular endothelial (VE)-cadherin is endothelium specific, mediates homophilic adhesion, and is clustered at intercellular junctions. VE-cadherin is required for normal development of the vasculature in the embryo and for angiogenesis in the adult. Here, we report that VE-cadherin is associated with VE growth factor (VEGF) receptor-2 (VEGFR-2) on the exposure of endothelial cells to VEGF. The binding parallels receptor phosphorylation on tyrosine residues, which is maximal at 5 minutes and then declines within 30 minutes. Tyrosine phosphorylation of VE-cadherin was maximal at 30 minutes after the addition of the growth factor. At this time point, the protein could be coimmunoprecipitated with the adaptor protein Shc. Pull-down experiments with different Shc domains and mutants of the VE-cadherin cytoplasmic tail have shown that Shc binds to the carboxy-terminal domain of the VE-cadherin tail through its Src homology 2 domain (SH2). We found that Shc phosphorylation lasts longer in endothelial cells carrying a targeted null mutation in the VE-cadherin gene than in VE-cadherin–positive cells. These data suggest that VE-cadherin expression exerts a negative effect on Shc phosphorylation by VEGFR-2. We speculate that VE-cadherin binding to Shc promotes its dephosphorylation through associated phosphatases.

Key Words: endothelium • vascular endothelial cadherin • Shc

	Introduction

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Endothelial cells (ECs) grow as monolayers and are controlled, in their response to growth factors, by the establishment of intercellular contacts. Confluent cells do not proliferate but are able to rapidly start dividing once a wound is performed, and intercellular junctions are disrupted (see reviews^1–5). This observation suggests that molecules at the junctions may be involved in transferring intracellular signals that are able to repress cell proliferation and induce cell cycle withdrawal.

Little is known about membrane proteins involved in contact-induced growth arrest. Cadherins are good candidates for this role.^2,6 These transmembrane proteins are organized in junctional structures called "adherens junctions."^7–12 They promote homophilic cell-to-cell adhesion and, inside the cell, are linked to cytoskeletal and signaling proteins called ß- and -catenin (or plakoglobin) and p120.^7–13 ß-Catenin and plakoglobin mediate the anchoring of cadherins to actin by binding -catenin. When released into the cytoplasm, they are able to translocate to the nucleus and exert transcriptional activity.^7–13

It is well documented that on establishment of intercellular junctions, cells become refractory to growth factor activation.^3–5 An attractive possibility is that cadherins might indirectly signal by interacting with growth factor receptors and their signaling effector proteins. For instance, it has previously been reported that the E-cadherin/ß-catenin complex could bind the endothelial growth factor (EGF) receptor.¹⁴ In ECs, we found that the vascular endothelial (VE)-cadherin/ß catenin complex associates with the VE growth factor (VEGF) receptor (VEGFR)-2 and phosphoinositide-3-OH (PI3)-kinase. In the absence of VE-cadherin, VEGFR-2 was unable to associate and mediate cell survival through the PI3-kinase pathway.¹⁵ It is possible that, similar to integrins, ¹⁶ cadherins regulate growth factor–dependent intracellular signaling by mediating the formation of receptor/effector complexes. In these modules, elements of the signaling cascade would be more susceptible to activation or inactivation by contiguous effectors.

Several growth factor receptors signal through tyrosine phosphorylation of Shc proteins. Phosphorylated Shc is able to mediate the coupling of Grb2-Sos to Ras, which in turn leads to the activation of mitogen-activated protein kinase and cell proliferation.^17,18 VEGF mitogenic activity seems to be largely mediated by VEGFR-2 and, at least in part, by mitogen-activated protein kinase activation. Pharmacological inhibition of this pathway leads to the inhibition of VEGF-induced EC proliferation.¹⁹ In the embryo, ShcA is primarily expressed in the cardiovascular system. Targeted null mutation of the corresponding gene prevents a normal development of the heart and the vascular system. Analysis of ShcA-null cells showed that the protein plays a role in sensitizing the cells to growth factor activation and in regulating the cytoskeletal organization.²⁰

In the present study, we report that VE-cadherin binds Shc on activation of the cells with VEGF. The binding occurs at later times than does VEGFR-2 phosphorylation and parallels VE-cadherin phosphorylation. Shc phosphorylation in response to VEGF lasts longer in VE-cadherin–null cells than in control cells. We propose that VE-cadherin modulates VEGFR-2 interaction with Shc by reducing Shc phosphorylation.

	Methods

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Cell and Culture Conditions
Human ECs isolated from umbilical veins (HUVECs) were routinely cultured in medium 199 supplemented with 20% newborn calf serum (GIBCO-BRL Life Technologies Ltd), 50 µg/mL EC growth supplement (ECGS), and 100 µg/mL heparin (Sigma Chemical Co), as previously described.²¹

Mouse ECs with a homozygous-null mutation of the VE-cadherin gene (VE-cadherin null) were generated from embryonic stem cells induced to differentiate to ECs.^15,22–24 The homogeneous endothelial nature of the cultures was detected by Western blot and immunofluorescence microscopy with antibodies to endothelial markers, as previously described.^15,22

A retroviral vector (PINCO) was used to express wild-type and mutant forms of human VE-cadherin in null ECs.²⁵ Deletion mutants of VE-cadherin are indicated as -p120 (deleted from amino acid [aa] 621 to aa 702, which corresponds to the p120-binding region)²⁶ and -ß-cat (deleted from aa 702 to aa 784, which corresponds to the ß-catenin–binding region).²⁷ Mutant and wild-type molecules were cloned into the PINCO vector and then transfected in amphotropic Phoenix packaging cells.²⁵ Culture supernatant containing high-titer viral particles was used to infect VE-cadherin–null ECs as previously described.²⁸ Efficiency of infection was measured as the percentage of green fluorescent protein–positive cells. VE-cadherin expression in targets cells was confirmed by immunofluorescence microscopy and fluorescence-activated cell sorting analysis. To avoid clonal selection heterogeneity, cells were then sorted. Cells were cultured in DMEM with 20% FCS (GIBCO), ECGS, and heparin on tissue culture plastic (Falcon Becton Dickinson Labware) that had been coated with gelatin (Sigma).

Antibodies
The following antibodies were used: monoclonal anti-human VE-cadherin antibody (BV9),^29–31 monoclonal anti–P-tyrosine (anti–P-tyr) antibody (PY20, horseradish peroxidase–conjugated), and rabbit polyclonal anti-Shc antibody, which were purchased from Transduction Laboratories; rabbit polyclonal anti–VEGFR-2 antibody from Santa Cruz Biotechnology, Inc; and rabbit anti-mouse IgG from DAKO A/S.

Immunoprecipitation and Western Blot Analysis
Cells (2 to 5x10⁴/cm²) were cultured for 3 days in culture medium without ECGS and heparin. Cell monolayers were then washed once with MCDB131 (GIBCO) and cultured with 1% BSA (Sigma) for 24 hours (HUVECs) or 48 hours (mouse EC lines) before the experiments.

The cells were stimulated at 37°C with recombinant human VEGF (PeproTech Inc) at a concentration of 80 ng/mL for different times. HUVECs were treated with a combination of vanadate (100 µmol/L, Sigma) and hydrogen peroxide (200 µmol/L, Fluka Chemie GmbH) to produce pervanadate, a potent inhibitor of P-tyr phosphatases, for the last 7 minutes before extraction.³² Cell layers were washed twice with cold PBS with Ca²⁺ and Mg²⁺ (GIBCO) supplemented with vanadate (300 µmol/L) and hydrogen peroxide (600 µmol/L) and were scraped on ice for 20 minutes in lysis buffer (50 mmol/L Tris and 150 mmol/L NaCl, pH 7.4, containing 1% Triton X-100, 1% NP-40, 1 mmol/L phenylmethylsulfonyl fluoride, 2 mmol/L Ca²⁺, 15 µg/mL leupeptin, 71 µg/mL phenanthroline, and 20 U/mL aprotinin; Sigma) containing 300 µmol/L vanadate and 600 µmol/L hydrogen peroxide. Then, insoluble material was removed by centrifugation at 14 000 rpm for 5 minutes. Protein content was measured according to the BCA method (Pierce), and samples (each containing 900 µg protein) were precleared with protein A or protein G (Amersham Pharmacia Biotech) for 1 hour at 4°C. Supernatants were collected and immunoprecipitated with protein G or protein A (previously conjugated with the appropriate antibody) for 1.5 hours at 4°C under continuous mixing, followed by 5 washings. Proteins were separated by SDS-PAGE under reducing conditions and analyzed in immunoblots with specific antibodies.

Binding Experiments
Glutathione S-transferase (GST), GST–Src homology 2 (SH2), GST-phosphotyrosine binding domain, and GST–collagen homologous region 1 fusion proteins corresponding to different Shc domains were expressed and purified by using standard techniques, as previously described.^17,33 The cells were lysed as described above, and lysates were incubated with the fusion proteins bound to glutathione Sepharose 4B beads (Amersham Pharmacia Biotech). For each reaction, 4 µg of either GST or GST fusion proteins was incubated for 1.5 hours at 4°C with 700 µg of the appropriate cell lysate. Protein complexes were washed 5 times in ice-cold lysis buffer, eluted by boiling in Laemmli sample buffer under reducing conditions, and analyzed by immunoblot.

	Results

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VE-Cadherin Is Associated With VEGFR-2 and Shc on Stimulation With VEGF
We have previously published that VE-cadherin can associate with VEGFR-2.¹⁵ In the present study, we show that this phenomenon is regulated by VEGF stimulation of the receptor. As reported in Figure 1a, on the addition of VEGF to the cells, VE-cadherin could be coimmunoprecipitated by anti–VEGFR-2 antibodies (Figure 1a). This effect paralleled receptor phosphorylation on tyrosine, reaching its maximum at 5 minutes and declining within 30 minutes.

View larger version (37K): [in this window] [in a new window]   Figure 1. Interaction of VE-cadherin with VEGFR-2 and Shc. a, Association of VEGFR-2 with VE-cadherin on cell activation with VEGF. HUVEC extracts were immunoprecipitated with VEGFR-2 antibodies and immunoblotted with antibodies to VEGFR-2, P-tyr (PY), or VE-cadherin. Cells were starved for 24 hours before addition of VEGF (80 ng/mL). The apparent molecular weights are reported on the right. One typical experiment is reported of 5 performed. b, Association of Shc with VE-cadherin on cell activation with VEGF. HUVEC extracts were immunoprecipitated with either anti-Shc or anti–VE-cadherin antibodies and then immunoblotted with anti–VE-cadherin or anti–PY antibodies, respectively. Cells were stimulated with VEGF as described above. One typical experiment is shown of 4 performed.

To analyze the role of VE-cadherin in VEGF signaling, we investigated whether it could associate with elements of the VEGFR-2 signaling pathway. As shown in Figure 1b, anti-Shc antibodies could coimmunoprecipitate VE-cadherin. The association was induced by VEGF activation of ECs and paralleled VEGF-induced VE-cadherin phosphorylation. In contrast, Shc phosphorylation on tyrosine was maximal at 5 minutes and declined to basal values within 30 minutes. This finding suggests that tyrosine phosphorylation of the VE-cadherin tail, but not of Shc, is required for their interaction.

SH2 Domain of Shc Binds to VEGFR-2 and VE-Cadherin
To further analyze Shc interaction with VE-cadherin, we performed binding experiments with GST fragments spanning different domains of Shc (see Methods for details). As reported in Figure 2, only the GST-SH2 domain of Shc was able to bind VE-cadherin, whereas GST-PTB and GST-CH1 were unable to do so. The same region of Shc was also responsible for binding to VEGFR-2 (Figure 2).

View larger version (33K): [in this window] [in a new window]   Figure 2. Binding of GST-SH2 Shc to VE-cadherin and VEGFR-2. Wild-type VE-cadherin–expressing murine ECs (see also Figure 3) were lysed after 5 minutes (for VEGFR-2 association) or 30 minutes (for VE-cadherin association) of VEGF activation and incubated with GST or GST fusion proteins immobilized on glutathione Sepharose beads. GST fusion proteins correspond to the SH2, PTB, and CH1 domains of the Shc gene, as previously reported.33 The bound proteins were washed, separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with either anti–VEGFR-2 or anti–VE-cadherin antibodies, as described in Methods. One typical experiment is reported of 3 performed.

View larger version (26K): [in this window] [in a new window]   Figure 3. Association of GST-SH2 Shc with the ß-catenin–binding domain of the VE-cadherin cytoplasmic tail. VE-cadherin mutants were starved for 48 hours and then activated with VEGF (80 ng/mL) for 30 minutes. Cells were lysed, and the lysates were incubated with GST-SH2 fusion protein immobilized on glutathione Sepharose 4B beads, as described above. The bound proteins were analyzed by Western blot with VE-cadherin antibody. VE-cadherin–null ECs were transfected with the wild-type VE-cadherin (WT) or with deletion mutants of the protein lacking either the ß-catenin–binding (-ß-cat) or the p120-binding (-p120) domain, as specified in Methods and schematically reported in the lower part of the figure. One typical experiment is reported of 3 performed.

Shc Binds to ß-Catenin–Binding Region of VE-Cadherin
To identify the domain of the VE-cadherin tail responsible for Shc binding, we used VE-cadherin–null cell lines expressing either the wild-type or 2 different VE-cadherin mutants, which lack either the -ß-cat– or -p120–binding region (Figure 3). Wild-type cadherin and mutant VE-cadherin were expressed at the cell surface at comparable levels and were correctly organized at intercellular junctions (M.G. Lampugnani, A. Zanetti, F. Breviario, G. Balconi, F. Orenigo, M. Corada, R. Spagnudo, M. Beston, V. Braga, E. Dejana, unpublished data, 2002). As shown in Figure 3, GST-SH2 Shc was able to precipitate wild-type VE-cadherin and the -p120 mutant. In contrast, only very small amounts of VE-cadherin -ß-cat could be precipitated by the GST-SH2 Shc fragment. These data indicate that the Shc binding site is contained in the region of VE-cadherin spanning aa 702 to aa 784.

VE-Cadherin Is Required for Shc Dephosphorylation
The data reported above indicate that Shc is able to form a complex with VE-cadherin in a VEGF-dependent manner. To understand the biological meaning of this association, we compared the effect of VEGF on Shc phosphorylation in the presence or in the absence of VE-cadherin. To this purpose, we compared VE-cadherin–null ECs¹⁵ with the same line infected with wild-type VE-cadherin (see above). VE-cadherin–null and –positive cells express comparable amounts of all known endothelial markers, such as platelet and endothelial cell adhesion molecule, junctional adhesion molecule, occludin, zonula occludens 1, endoglin, S-Endo 1, and VEGFR-2¹⁵ (M.G. Lampugnani, A. Zanetti, F. Breviario, G. Balconi, F. Orenigo, M. Corada, R. Spagnudo, M. Beston, V. Braga, E. Dejana, unpublished data, 2002) and only differ in the expression of VE-cadherin. Figure 4 shows that VE-cadherin expression influences the kinetics of Shc phosphorylation. As expected and reported above for HUVECs, on VEGF activation, VE-cadherin–positive cells showed increased Shc phosphorylation at 5 minutes, which then declined within 30 minutes. In VE-cadherin–null cells, Shc phosphorylation was increased at 5 minutes after activation with the growth factor but declined more slowly and remained high up to 60 minutes after cell activation. These data suggest that the physiological dephosphorylation of Shc is less effective in the presence of VE-cadherin.

View larger version (24K): [in this window] [in a new window]   Figure 4. Effect VE-cadherin expression on Shc phosphorylation. VE-cadherin–positive and –null cells were extracted after incubation with VEGF for different lengths of time. Cell extracts were then immunoprecipitated with anti-Shc antibodies, and the bound proteins were analyzed by Western blot with anti-PY antibody. One typical experiment is reported of 7 performed.

	Discussion

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In the present study, we report the following: (1) VEGF stimulation induces Shc association with VE-cadherin. This interaction requires the SH2 domain of Shc and the carboxy-terminal domain of the VE-cadherin tail. (2) Shc interaction with VE-cadherin occurs when VE-cadherin is phosphorylated on tyrosine and detached from VEGFR-2. (3) Shc phosphorylation induced by VEGF lasts longer in the absence of VE-cadherin than in its presence.

It has been demonstrated that N-cadherin can bind Shc.³⁴ The binding is direct, is dependent on tyrosine phosphorylation of N-cadherin, and is mediated by its carboxy-terminal tail. The data reported in the present study confirm and extend these observations to VE-cadherin. Using different mutants of the protein, we could demonstrate that Shc is associated with a region contained in the last 82 residues of the tail.

A novel observation contained in the present work is that this association is VEGF dependent. As schematically reported in Figure 5, we found that at early time points (5 minutes) after VEGF activation, VE-cadherin links VEGFR-2 and that this event is followed by tyrosine phosphorylation of VE-cadherin within 30 minutes. VE-cadherin phosphorylation parallels its dissociation from the receptor and association with Shc. Therefore, the action of VEGF is different from what has been reported with EGF, which did not change the Shc association with N-cadherin in a physiological medium.³⁴ This apparent discrepancy may be due to the fact the authors used tumoral and immortalized cell lines that, even in absence of the growth factor, presented phosphorylated N-cadherin and a constitutive binding of Shc to it. We used freshly isolated cells in which VE-cadherin phosphorylation and Shc binding were almost undetectable under resting conditions and could, therefore, be increased by activation with VEGF.

View larger version (31K): [in this window] [in a new window]   Figure 5. Schematic representation of the sequence of events that follows VEGFR-2 activation of endothelial cells. For step 1, at early times (5 minutes) after the addition of VEGF, VEGFR-2 and Shc are phosphorylated on tyrosine. VE-cadherin is associated with VEGFR-2. For step 2, at later times (30 minutes) of activation, VE-cadherin is phosphorylated on tyrosine and binds Shc, which is dephosphorylated. We hypothesize that a VE-cadherin–associated phosphatase induces Shc dephosphorylation.

Xu and Carpenter³⁵ found that Tyr851 and Tyr883 in the N-cadherin tail are targets of Src phosphorylation and are required for optimal Shc binding. In the absence of activated Src, Shc association with N-cadherin does not occur. Tyr851 and Tyr883 conform to the consensus recognition sequence of the Shc SH2 domain (PY-hydrophobic, X-hydrophobic). In VE-cadherin, the tyrosines in positions Tyr725 and Tyr757 are conserved and conform to the consensus sequence for binding the Shc SH2 domain. The SH2 domain is responsible for Shc binding to VEGFR-2, suggesting that there may be competition between VEGFR-2 and VE-cadherin for Shc association.

We hypothesized that VE-cadherin phosphorylation by VEGF induces recognition and binding by Shc and that this may facilitate Shc dephosphorylation. To verify this hypothesis, we studied the time course of Shc phosphorylation in the presence or absence of VE-cadherin. We found that in the presence of VE-cadherin, Shc phosphorylation on VEGF stimulation returned to control levels within a few minutes, whereas in the absence of the protein, it remained high for up to 60 minutes. Phosphatase activity is increased by cell confluence.^5,36,37 It is possible that the binding of Shc to the VE-cadherin tail facilitates its dephosphorylation by a contiguous phosphatase. Several phosphatases (such as protein tyrosine phosphatase-µ,³⁸ protein tyrosine phosphatase-,³⁹ and SHP2⁴⁰) were found to be associated with cadherin and catenin complexes.

Other authors⁴¹ have found that the addition of VE-cadherin antibodies to porcine ECs overexpressing VEGFR-2 increases receptor phosphorylation. At this stage, we cannot exclude the possibility that VE-cadherin–associated phosphatases may also downregulate receptor phosphorylation and, as a consequence, Shc activation.

It has previously been demonstrated that the concentration of cytoplasmic Shc might be a limiting factor in the EGF receptor signaling pathway.⁴² However, in contrast to EGF, the role of Shc in VEGF signal transduction is still debated. Some authors,^43,44 using primary cultures of sinusoidal rat ECs, reported that Shc and Grb2 were not phosphorylated by VEGF or associated with VEGFR-2. Another report,¹⁹ using porcine ECs overexpressing VEGFR-2, and the present study, using HUVECs and mouse embryonic ECs (see Figure 1b and Figure 4), show that VEGFR-2 activation leads to Shc phosphorylation and association with VEGFR-2. A conceivable explanation for these contrasting reports may be the different origin of the ECs used. Sinusoidal endothelium is a very specialized type of EC, which may follow cell-specific signaling pathways even through the same receptor.

An important question is what is the downstream effect of the Shc association with VE-cadherin. In a previous study, we found that VE-cadherin can associate with PI3-kinase and facilitate VEGFR-2 activation of the enzyme.¹⁵ This process was required for the antiapoptotic activity of VEGF. VE-cadherin–null cells were less effective in activating PI3-kinase and Akt kinase after VEGF induction, and these cells presented a much higher apoptotic index in the absence of serum. An attractive hypothesis is that, on one side, VE-cadherin makes VEGF signaling through PI3-kinase more efficient but, on the other, inhibits VEGF signaling through Shc by facilitating its dephosphorylation (Figure 5). Consistent with this idea is the observation that loss of VE-cadherin expression leads to major alterations in the development of the vascular system in the embryo, with the formation of abnormal vessels with an enlarged lumen and a high apoptotic index of ECs.¹⁵ This phenotype may be due to an unbalanced response of ECs to VEGF.

An alternative possibility is that VE-cadherin binding to Shc contributes to VEGF-induced changes in cytoskeletal organization and permeability. In a previous study,⁴⁵ we observed that the effect of VEGF on endothelial permeability in vitro is not a rapid event but requires at least 2 hours to be significant. VE-cadherin phosphorylation on tyrosine and Shc association occurs within 30 minutes. These events may be followed by cytoskeletal and junctional changes that are compatible with a later permeability increase. ShcA-null embryo fibroblasts present a rounded morphology and a disorganized actin cytoskeleton.⁴¹ It is possible that the association of Shc with VE-cadherin and possibly its withdrawal from the cytosol may contribute to the cytoskeletal changes and the increase in permeability observed after VEGF.⁴⁵

	Acknowledgments

 
This work was supported in part by Associazione Italiana per la Ricerca sul Cancro, Consiglio Nazionale delle Ricerche (CNR grant 97.01299.PF49, CNR Progetto Coordinato-Agenzia 2000), The European Community (QLG1-CT-1999-01036 and QLK3-CT-1999-00020), Ministero Università Ricerche Scientifiche e Tecnologiche (9906317157-003), Telethon-Italy (grant No. E.1254), P.PO, A.S.I., and Ministero della Sanità (ICS 060.2/RF99.72, RF00.73).

Received November 11, 2001; accepted January 7, 2002.

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