Vascular Endothelial Growth Factor-dependent Down-regulation of Flk-1/KDR Involves Cbl-mediated Ubiquitination

CONSEQUENCES ON NITRIC OXIDE PRODUCTION FROM ENDOTHELIAL CELLS*

Martine Duval {ddagger} §, Sara Bédard-Goulet {ddagger}, Chantal Delisle {ddagger} and Jean-Philippe Gratton {ddagger} § ¶ ||

From the {ddagger}Laboratory of Endothelial Cell Biology, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec H2W 1R7, Canada and the Departments of §Pharmacology and Medicine, Université de Montréal, Montreal, Quebec H3C 3J7, Canada

Received for publication, February 10, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ligand-stimulated degradation of receptor tyrosine kinase (RTK) is an important regulatory step of signal transduction. The vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is responsible for the VEGF-stimulated nitric oxide (NO) production from endothelial cells. Cellular mechanisms mediating the negative regulation of Flk-1 signaling in endothelial cells have not been investigated. Here we show that Flk-1 is rapidly down-regulated following VEGF stimulation of bovine aortic endothelial cells (BAECs). Consequently, VEGF pretreatment of endothelial cells prevents any further stimulation of Flk-1, resulting in decreased NO production from subsequent VEGF challenges. Ubiquitination of RTKs targets them for degradation; we demonstrate that activation of Flk-1 by VEGF leads to its polyubiquitination in BAECs. Furthermore, VEGF stimulation of BAECs or COS-7 cells transiently transfected with Flk-1 results in the phosphorylation of the ubiquitin ligase Cbl, the enhanced association of Cbl with Flk-1, and the relocalization of Cbl to vesicular structures in BAECs. Overexpression of Cbl in COS-7 cells enhances VEGF-induced ubiquitination of Flk-1, whereas a Cbl mutant lacking the ubiquitin ligase RING finger domain, 70Z/3-Cbl, does not. Moreover, expression of Cbl in contrast to 70Z/3-Cbl inhibits the Flk-1-dependent activation of eNOS and, thus, NO release. In BAEC overexpressing Cbl, the degradation of Flk-1 upon VEGF stimulation is accelerated compared with cells transfected with a control vector (green fluorescent protein). Our findings demonstrate that Flk-1 is rapidly down-regulated following sustained VEGF stimulation and identify Cbl as a negative regulator of Flk-1 signaling to eNOS. Cbl thus plays a role in the regulation of VEGF signaling by mediating the stimulated ubiquitination and, consequently, degradation of Flk-1 in endothelial cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vascular endothelial growth factor (VEGF)1 is a multifunctional cytokine essential for vascular development and maintenance (1). VEGF activates specific receptor tyrosine kinases, namely Flt-1 (VEGF receptor 1), Flk-1 (VEGF receptor 2, KDR), and Flt-4 (VEGF receptor 3) (2, 3, 4). VEGF-dependent endothelial cell proliferation, migration, survival, and nitric oxide (NO) release are mediated via the activation of Flk-1 through a plethora of downstream signaling mechanisms (5). These signaling pathways include the classic Ras/Raf/mitogen-activated protein kinase (MAPK) pathway, p38 MAPK, phospholipase C (PLC), and phosphatidylinositol 3-kinase (PI3-kinase)/Akt (6, 7, 8, 9). In particular, concomitant activation of phospholipase C-{gamma} and the PI3-kinase/Akt pathways by VEGF leads to an increase in intracellular calcium and the phosphorylation of Ser1179 of the bovine form of endothelial nitric oxide synthase (eNOS) by Akt (Ser1177 in the human form). This results in a rapid and transient activation of eNOS leading to NO production (10, 11, 12).

Flk-1 follows the classical scheme of receptor tyrosine kinase (RTK) activation (13). Following VEGF-mediated Flk-1 autophosphorylation, several Src homology 2 (SH2)-bearing proteins such as PLC-{gamma}, Shc, Grb2, and Sck are recruited to Flk-1, resulting in the activation of some of the above-mentioned signaling pathways (14, 15, 16, 17). Substantial amounts of data describe the receptorial components necessary for VEGF-mediated activation of Flk-1; however, little is known about factors mediating down-regulation or negative regulation of Flk-1 signaling.

One of the mechanisms by which growth factor responses are negatively regulated is through ligand-stimulated degradation of the receptors. Several RTKs, such as the epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and the colony-stimulating factor-1 (CSF-1), are actively degraded following sustained exposure to their respective ligands. Degradation of RTKs is mainly initiated by the addition of ubiquitin moieties following receptor engagement, which has been show to target them to the lysosomal/proteasomal machinery for degradation (18, 19, 20, 21). Ubiquitination of substrates requires three concerted steps for the conjugation of ubiquitin molecules to a substrate protein. Initially, the ubiquitin-activating enzyme (E1) forms a thiol ester bond with the carboxy-terminal glycine of ubiquitin. Then the ubiquitin molecule is transferred to an ubiquitin-conjugating enzyme (also known as ubiquitin-carrier enzyme or E2), again involving the carboxyl terminus of ubiquitin. The final step involves an ubiquitin ligase (E3) enzyme that complexes with the substrate and catalyzes the transfer from E2 to the {epsilon}-amino group of a lysine residue on the target protein (22).

The multi-adaptor protein Cbl has been shown to possess E3 ligase activity and, thus, has been implicated in the ubiquitination of activated EGFR, PDGFR, and other RTKs (19, 23). The demonstration of a prominent and inducible association of Cbl with the EGFR coupled to the observation that Cbl overexpression induced EGFR and PDGFR ubiquitination confirmed Cbl as a regulatory component of RTK function.

The Flk-1 receptor has been shown to internalize upon VEGF stimulation (24). However, the regulation of VEGF-dependent down-regulation of this important and essential component of vascular integrity has never been investigated. In this study, we demonstrate that sustained VEGF stimulation of endothelial cells results in Flk-1 protein down-regulation. This results in impaired eNOS activation and NO release during subsequent VEGF challenges. Consistent with the VEGF-stimulated degradation of Flk-1, we demonstrate that VEGF stimulates ubiquitination of Flk-1 and that Cbl mediates this effect through its ubiquitin ligase activity. Finally, we also demonstrate that Cbl negatively regulates Flk-1 signaling and VEGF-induced NO release. Our results identify Cbl as a novel component in the negative regulation of VEGF signaling.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Bovine aortic endothelial cells (BAECs) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 2.0 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen), 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were used between passages 5 and 8. Recombinant human VEGF obtained from the Biomedical Research Branch Preclinical Repository of the NCI-National Institutes of Health Frederick Cancer Research and Development Center was used for cell stimulation throughout this study.

Constructs and Transfections—The bovine eNOS (pcDNA3), human Flk-1 (pRK7) and {beta}-galactosidase (pcDNA3) cDNAs were described previously (12, 25). Mouse Cbl (pxM139) and the mutant 70Z-Cbl (pxM139) were obtained from Dr. A. Veillette (Institut de Recherches Cliniques de Montréal), and HA-ubiquitin (pcMV) was obtained from Dr. S. Meloche (Institut de Recherches Cliniques de Montréal). COS-7 cells were transfected in either 6-well plates or 60-mm dishes and transfected using LipofectAMINE 2000 (Invitrogen). BAECs were transfected in 60-mm dishes using FuGENE 6 (Roche Diagnostics) as described previously (26). The pEGFP-N3 expression plasmid (Clontech) was used for the assessment of transfection efficiency as well as for plasmid control in BAEC transfections. Cell stimulations were done 48 h following transfections, and protein expression was confirmed (60 µg of total cell lysate) by SDS-PAGE and Western blot analysis.

Nitric Oxide Release—BAECs were serum starved for 12 h and processed for the measurement of nitrite (), the stable breakdown product of NO in aqueous solution, by NO-specific chemiluminescence as described previously (27). Briefly, samples containing were refluxed in glacial acetic acid containing sodium iodide. Under these conditions, is quantitatively reduced to NO, which was quantified by a chemiluminescence detector after reaction with ozone in a NO analyzer (Sievers, Boulder, CO). Transfected COS-7 cells were also processed for the measurement of nitrite. 48 h after transfection, cells were stimulated with VEGF for 2 h, and NO was quantified as described above.

Antibodies, Immunoprecipitations, and Immunoblotting—Flk-1 (monoclonal antibody), Cbl (polyclonal antibody) and ubiquitin (polyclonal antibody) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-eNOS antibody was purchased from BD Transduction Laboratories. HA-tag, phospho-Akt (Ser473), total Akt, and phospho-eNOS (Ser1179) antibodies were purchased from Cell Signaling, and phospho-tyrosine (4G10) was purchased from Upstate Biotechnology. Serum-starved BAECs (16 h) were washed twice with cold PBS following VEGF stimulation. Total cells lysates were prepared in some cases by directly adding to the dishes boiling SDS sample buffer. For immunoprecipitations, cells were solubilized with a lysis buffer containing 1% Triton X-100, 50 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 20 mM sodium fluoride, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM Pefabloc, and a protease inhibitor mixture (Roche Diagnostics). Insoluble proteins were precipitated by centrifugation at 13,000 rpm for 10 min at 4 °C, and the supernatants were then incubated for 2 h with the primary antibody (2 µg) at 4 °C. Protein A-Sepharose (Sigma) (50 µl of a 50% slurry) was then added and incubated for an additional hour. The immune complexes were precipitated by centrifugation, washed three times with lysis buffer, boiled in SDS sample buffer, and separated by SDS-PAGE onto a nitrocellulose membrane (Hybond ECL, Amersham Biosciences) and Western blotted as described (28). Antibody detection was performed by a chemiluminescence-based detection system (ECL, Amersham Biosciences).

Ubiquitination Assay—COS-7 cells were transfected with a maximum of 1.6 µg of total DNA encoding for Flk-1, HA-tagged ubiquitin, Cbl, and 70Z-Cbl. Unstimulated and VEGF-stimulated cells were washed twice in cold PBS. Cells were lysed for 30 min in buffer containing 50 mM Tris, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% SDS, 0.1% deoxycholic acid, 1% Nonidet P-40, 125 mM NaCl, 10 mM N-ethylmaleimide, 20 mM sodium fluoride, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM Pefabloc, and a protease inhibitor mixture. Immunoprecipitation and Western blotting were performed as described above.

Immunofluorescence—BAECs were cultured on 0.1% gelatin-coated cover slips. Serum-starved cells were stimulated for 30 min with VEGF (50 ng/ml). Cells were then washed briefly with cold PBS and fixed for 15 min in PBS containing 3% paraformaldehyde. Cells were rinsed with PBS and permeabilized with 0.2% Triton in PBS for 5 min. Fixed cells were blocked with 1% BSA and then incubated for 1 h with the primary antibodies in 0.1% bovine serum albumin in PBS (mouse anti-Flk-1 (1:200) and rabbit anti-Cbl (1:200)). Bound primary antibodies were visualized following 1 h of incubation using Alexa Fluor 488-labeled goat anti-mouse (1:600) and Alexa Fluor 568-labeled goat anti-rabbit (1:600) (Molecular Probes). Mounted cover slips were observed using a Zeiss LSM 510 imaging system.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
VEGF Pretreatment Inhibits Subsequent VEGF Signaling to eNOS—Ligand-dependent down-regulation of receptors has been shown previously for several RTKs; interestingly, VEGF-dependent down-regulation of Flk-1 signaling has not been investigated. To document VEGF-dependent desensitization of Flk-1 receptor signaling in endothelial cells, we studied the effect of VEGF (50 ng/ml) pretreatment on subsequent VEGF (50 ng/ml)-stimulated NO release from bovine aortic endothelial cells (BAEC). Fig. 1A shows that VEGF pretreatment resulted in a time-dependent reduction of VEGF-stimulated NO production. The 30- and 60-min pretreatments completely abolished any subsequent VEGF-stimulated NO production from BAEC (Fig. 1A). To demonstrate the specificity of this VEGF pretreatment, ATP (10 µM) or ionomycin (1 µM) were used as stimuli for NO production (Fig. 1B). A 30-min pretreatment with VEGF (50 ng/ml) did not affect stimulated NO release by either agonist. These results indicate that the reduced responses of BAECs to VEGF are specific to the down-regulation of the Flk-1 receptor.



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FIG. 1.
Inhibition of eNOS activation by VEGF pretreatments of BAEC. A, BAECs were pretreated with VEGF (50 ng/ml) for the indicated times prior to 30 min stimulation with media containing either vehicle (CT) or 50 ng/ml of VEGF. Following the incubation, cell media were processed for the measurement of NO as described under "Experimental Procedures." {dagger}, p < 0.05 compared with CT; *, p < 0.05 compared with time at 0 min. B, BAECs were pretreated with VEGF (50 ng/ml) for 30 min of incubation with media alone (CT) or containing either ATP (10 µM) or ionomycin (1 µM). C, serum-starved BAECs were pretreated with VEGF (50 ng/ml) for 30 min prior to a 10 min of stimulation of VEGF (50 ng/ml). Cells were then lysed, and protein levels were determined by the DC protein assay (Bio-Rad). Cell lysates were separated on SDS-PAGE and immunoblotted using antibodies against phospho-Akt, Akt, phospho-eNOS, eNOS, and Flk-1. A and B are presented as mean ± S.E. (n = 4). Experiments were repeated three times with similar results. wb, Western blot.

 

To further document the VEGF inhibition of NO production, we monitored the impact of a 30-min VEGF pretreatment on Akt phosphorylation at Ser473 and on eNOS phosphorylation at Ser1179 stimulated by a second VEGF stimulation (Fig. 1C). As expected, the 30-min pretreatment with VEGF abolished any further increase in Akt and eNOS phosphorylation following a 10-min VEGF stimulation of cells. Serum (10% fetal bovine serum)-dependent activation of Akt was not affected by VEGF pretreatment (data not shown). Interestingly, in these experiments Flk-1 protein levels were monitored, and VEGF pretreatment significantly down-regulated the expression levels of Flk-1, which could be responsible for the impaired response to further VEGF stimulation.

VEGF-dependent Down-regulation of Flk-1 in Endothelial Cells—To further investigate the VEGF-dependent down-regulation of Flk-1 shown in Fig. 1C, we monitored, by Western blotting on total cell lysates from BAECs, the effect of a VEGF treatment on total Flk-1 protein levels. First, when cells were lysed in 1% Triton X-100-based buffer, VEGF (50 ng/ml) stimulation induced a significant decrease in Flk-1 protein levels (Fig. 2A). Similar results were obtained when cells were lysed directly in SDS sample buffer (2% SDS), indicating that changes in detergent solubility of Flk-1 following stimulation cannot account for the observed down-regulation (Fig. 2B). This reduction, under both lysing conditions, was clearly noticeable after 15 min of stimulation and maximal after 30–60 min (Fig. 2, A and B). Stimulation of BAEC for 30 min with increasing concentrations of VEGF (1–50 ng/ml) showed that this effect was dose-dependent and maximal at 25 ng/ml (Fig. 2C). In all cases, the cellular levels of Akt, used as a control for protein loading, were not affected by VEGF treatment (Fig. 2). These results indicate that VEGF stimulation induces specific degradation of Flk-1, which contributes to the reduced VEGF-dependent eNOS activation following a prior VEGF pretreatment of endothelial cells.



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FIG. 2.
VEGF stimulation of BAEC induces down-regulation of Flk-1. A, serum-starved BAECs were incubated with 50 ng/ml VEGF for the times indicated, and the cells were lysed in 1% Triton X-100 buffer (Tx-100). Protein levels were determined by the DC protein assay (Bio-Rad), and 60 µg of each sample were separated by SDS-PAGE. B, cells were stimulated as in A and lysed directly in 2% SDS sample buffer, and equal volumes of total cell lysates (TCL) were separated by SDS-PAGE. C, cells were incubated for 30 min with different concentrations of VEGF (1–50 ng/ml), and cell lysates were prepared as in A. Western blots (wb) were done using anti-Flk-1 antibodies, and Akt was probed as a control for loading. Panels are representative blots from three other experiments.

 

VEGF Stimulates Ubiquitination of Flk-1—The ubiquitination of cellular proteins targets them for the degradative pathways. We investigated the ability of VEGF to induce the addition of ubiquitin moieties on the activated Flk-1 receptor. BAECs were stimulated with low concentration of VEGF (10 ng/ml), which is sufficient to activate Flk-1 but maintains its degradation to a minimum. Immunoblotting against ubiquitin on the Flk-1 immunoprecipitates showed that Flk-1 is ubiquitinated in response to VEGF treatment of BAECs (Fig. 3A). The anti-ubiquitin immunoblot reveals that the ubiquitination machinery adds multiple and variable numbers of ubiquitin moieties to Flk-1, because the polyubiquitinated form of Flk-1 is detected as a smear (Fig. 3A). Reprobing of the immunoprecipitates with anti-phosphotyrosine antibodies revealed that Flk-1 is rapidly and transiently tyrosine phosphorylated in response to VEGF treatment and that ubiquitination kinetically follows the VEGF-induced autophosphorylation of Flk-1 (Fig. 3A).



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FIG. 3.
VEGF induces ubiquitination of Flk-1 and enhances association with Cbl. A, serum-starved BAECs were stimulated with VEGF for the indicated times. Cells were lysed, and equal amounts of lysates were incubated with anti-Flk-1 antibodies or non-immune serum (NI) for immunoprecipitation (IP). Immunoprecipitates were resolved by SDS-PAGE, transferred, and subjected to Western blot analysis (wb) using anti-ubiquitin (ubi), anti-pTyr (pTyr), and anti-Flk-1 (Flk-1) antibodies. B, following VEGF stimulations, cell lysates were immunoprecipitated using anti-Flk-1, anti-Cbl (Cbl), or non-immune serum as indicated. The immunoprecipitated proteins were resolved by SDS-PAGE, transferred, and subjected to Western blot analysis using anti-Cbl, anti-pTyr, and anti-Flk-1 antibodies. C, COS-7 cells were transfected with Flk-1- and Cbl-expressing vectors as indicated and stimulated for 10 min with VEGF (50 ng/ml). Cell lysates were immunoprecipitated as in A. Immunoprecipitates were immunoblotted with an anti-Cbl antibody to detect the association of Cbl with Flk-1 and control for transfections in total cell lysates (TCL). Anti-Flk-1 antibodies were used as controls for equal transfection and immunoprecipitation. These results are representative of at least three experiments.

 

VEGF Stimulates Cbl Activation and Association to Flk-1— The ubiquitin ligase Cbl has been shown to mediate ligand-dependent down-regulation of several RTKs. We therefore investigated the ability of VEGF to activate Cbl and promote its association to Flk-1 upon activation. As seen in Fig. 3B, the activation of Flk-1 resulted in a rapid and transient phosphorylation (maximal at 5 min) of Flk-1 (Fig. 3B, top panels). Secondly, the BAEC lysates were immunoprecipitated with antibodies against Cbl, and the tyrosine phosphorylation levels of Cbl and its association with Flk-1 were monitored. Immunoblotting analysis of the Cbl immunoprecipitates confirmed that VEGF stimulation of BAEC led to a rapid and sustained phosphorylation of Cbl on tyrosine residues (Fig. 3B,middle panels). Furthermore, blotting with anti-Flk-1 antibodies revealed that VEGF stimulation of BAEC increased the presence of Flk-1 in the Cbl immunoprecipitates (Fig. 3B, lower panel).

To confirm the VEGF-dependent association of Cbl with Flk-1, we transiently cotransfected COS-7 cells with plasmids coding for Flk-1 and either {beta}-Gal or Cbl (Fig. 3C). Overexpression of Cbl in COS-7 cells resulted in increased detection of Cbl associated with the transfected Flk-1 receptor (Fig. 3C, middle panel; compare lanes 1 and 2). Moreover, in Cbl-overexpressed COS-7 cells, VEGF stimulation resulted in enhanced association of Cbl with Flk-1 (Fig. 3C, middle panel; compare lanes 2 and 3). Cbl was strongly detected only when Flk-1 immunoprecipitates were performed on COS-7 cells overexpressing both Flk-1 and Cbl, indicating that the co-immunoprecipitation was specific for the presence of Flk-1 (Fig. 3C, middle panel; compare lanes 2 and 4). Overall, these results suggest that stimulation of Flk-1 activates Cbl and also promotes its recruitment to the activated Flk-1 receptor.

Cbl Mediates Stimulus-dependent Ubiquitination of Flk-1— To demonstrate that Cbl has the capacity to mediate ubiquitination of Flk-1, we monitored the impact of Cbl overexpression on Flk-1-stimulated ubiquitination. We thus transfected COS-7 cells with Flk-1 and HA-tagged ubiquitin in the presence or absence of Cbl or the mutant form 70Z/3-Cbl. 70Z/3-Cbl has been shown to be an oncogenic form of Cbl that lacks part of the ubiquitin ligase RING domain ({triangleup}366–382) (21). The addition of HA-ubiquitin on Flk-1 was monitored in transfected COS-7 cell lysates immunoprecipitated for Flk-1. VEGF stimulation of Cbl-overexpressing COS-7 cells resulted in increased detection of HA-ubiquitin in the Flk-1 immunoprecipitates (Fig. 4A; top panel). The anti-HA-immunoblot is again detected as a smear rather than a distinct band as Flk-1 is polyubiquitinated in response to VEGF (Fig. 4A, anti-HA immunoblot). By contrast, overexpression of the mutant 70Z/3-Cbl did not enhance VEGF-induced ubiquitination of the transfected Flk-1 (Fig. 4B, top panel). Moreover, the addition of HA-ubiquitin in our assay was specific for Flk-1, because no HA was detected in Flk-1 immunoprecipitates from cells transfected with HA-ubiquitin and Cbl only (Fig. 4B, lane 7). These findings demonstrate that Cbl can mediate Flk-1 ubiquitination and that the RING finger motif of Cbl is necessary for this effect.



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FIG. 4.
Cbl mediates VEGF-dependent ubiquitination of Flk-1. COS-7 cells were transfected with Flk-1, HA-ubiquitin (HA-ubi), and Cbl (panel A) or with Flk-1, HA-ubiquitin, Cbl, and 70Z/3-Cbl (panel B) expression constructs as indicated. Cells were stimulated with VEGF (50 ng/ml) for the indicated times. Cells were lysed as described under "Experimental Procedures" and incubated with monoclonal Flk-1 antibodies for immunoprecipitation. Complexes were resolved by 7% SDS-PAGE gel electrophoresis. Immunoprecipitates were immunoblotted with anti-HA antibodies to detect Flk-1 ubiquitination. Anti-Flk-1 antibodies were used to control for equal transfection and immunoprecipitation (IP), and anti-Cbl antibodies were used to document Cbl and 70Z/3-Cbl transfections in total cell lysates (TCL). These experiments are representative of at least three others. wb, Western blot.

 

Cbl Overexpression Abrogates Flk-1-dependent Nitric Oxide Production—To investigate the effect of Cbl on VEGF-mediated NO release, we thus transfected COS-7 cells with Flk-1 and eNOS expression vectors and monitored the effect of overexpression of Cbl and 70Z/3-Cbl on VEGF-dependent NO release (Fig. 5A). Nitric oxide accumulation in the tissue culture media, measured as nitrite by specific chemiluminescence, was monitored for 2h in presence of 50 ng/ml of VEGF. As expected, higher levels of NO in the media were detected from COS-7 cells expressing eNOS as compared with {beta}-Gal-expressing cells (data not shown) (12). Coexpression of Flk-1 with eNOS in the presence of VEGF increased NO production significantly when compared with eNOS coexpressed with {beta}-Gal. This indicates that Flk-1 activation has the capacity to stimulate eNOS-dependent NO release in transfected COS-7 cells (Fig. 5A). Interestingly, coexpression of Cbl with Flk-1 and eNOS inhibited the Flk-1-dependent NO release from VEGF-stimulated cells (Fig. 5A, compare lanes 2 and 4). In contrast, Cbl did not exert any inhibiting effects on eNOS alone (compare lanes 1 and 3). This indicates that the effects of Cbl on NO release are specific to Flk-1-dependent stimulations and not on eNOS activity per se. Finally, 70Z/3-Cbl expression, in marked contrast to Cbl, did not affect Flk-1-stimulated NO release. Interestingly, 70Z/3-Cbl showed a tendency (which, however, did not reach significance) to increase NO release induced by stimulation of Flk-1 (compare lanes 2 and 5). These results show that Cbl negatively modulates Flk-1-dependent eNOS activation and that the RING finger motif is essential for the down-regulatory effects of Cbl on Flk-1-mediated NO production.



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FIG. 5.
Cbl overexpression inhibits Flk-1-mediated NO release and enhances Flk-1 degradation. A, COS-7 cells were transfected with Flk-1, eNOS, Cbl, and 70Z/3-Cbl expression vectors as indicated. {beta}-Gal expression vectors were used to balance the amount of transfected DNA throughout. Overnight serum-starved cells were incubated with VEGF (50 ng/ml) for 2 h. Cell media were then processed for the measurement of NO as described under "Experimental Procedures." Cell lysates were separated on SDS-PAGE and immunoblotted. Anti-Flk-1, anti-eNOS, and anti-Cbl antibodies were used as controls for equal transfection. These results are expressed as NO released above the amounts released by cells transfected with eNOS and {beta}-Gal and are representative of at least three experiments. B, BAECs were transfected as detailed under "Experimental Procedures" with a Cbl or GFP expression vector. Overnight serum-starved cells were VEGF-stimulated (25 ng/ml) for the times indicated. BAEC lysates were separated on SDS-PAGE and immunoblotted with anti-Flk-1 antibodies and anti-Cbl for assessment of the transfection levels. Equal protein loading was confirmed by immunoblotting against Akt. The results were repeated three times with essentially identical results. wb, Western blot.

 

Enhanced VEGF-dependent Degradation of Flk-1 in BAEC Overexpressing Cbl—To ascertain the regulatory role of Cbl in endothelial cell responses, we transiently transfected BAEC with expression vectors that code for either Cbl or GFP as a control. Transient transfections in BAEC are relatively inefficient ({approx}30%); however, our results showed that BAEC transfected with Cbl showed a significant overexpression of Cbl when compared with control cells transfected with GFP vector (Fig. 5B). Interestingly, VEGF stimulation of BAEC overexpressing Cbl resulted in a more rapid kinetic of Flk-1 degradation when compared with GFP-transfected cells. Although levels of Flk-1 in unstimulated GFP- or Cbl-transfected BAECs were equivalent, BAECs overexpressing Cbl showed, at 5 and 15 min of VEGF (25 ng/ml) stimulation, a marked decrease in the receptor band when compared with GFP-expressing cells that showed only a slight decrease in band intensity at 15 min of VEGF stimulation. These results confirm a role for Cbl in VEGF-dependent degradation of Flk-1 in endothelial cells.

VEGF Stimulation of Endothelial Cells Induces Relocalization of Cbl to Vesicular Structures—Finally, we examined cellular localization of Flk-1 and Cbl by immunofluorescence confocal microscopy in fixed and permeabilized BAECs following VEGF stimulation. Fig. 6 shows that, in control-unstimulated conditions, Flk-1 staining is prominent in the cytoplasm as punctate membrane-associated structures as well as at the surface of cells. Cbl staining in control conditions showed a light and relatively uniform cytoplasmic staining. In VEGF-stimulated cells, Flk-1 plasma membrane staining disappeared, and the cytoplasmic stain became more uniform. VEGF stimulation of BAEC promoted a marked relocalization of Cbl into vesicular structures, which, in part, colocalized with Flk-1. No staining was observed when cells were incubated with a non-immune serum as well as a secondary antibody alone (data not shown).



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FIG. 6.
Cellular localization of Cbl in BAEC following VEGF stimulation. BAECs were cultured on 0.1% gelatin-coated cover slips. Serum-starved cells were either untreated or treated with 50 ng/ml VEGF for 30 min. Cells were fixed, permeabilized, and incubated with anti-Flk-1 (green) and anti-Cbl (red) primary antibodies. Immunofluorescent staining was performed as described under "Experimental Procedures" and observed by confocal microscopy.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The results presented herein show the following. 1) Flk-1 is rapidly down-regulated following sustained VEGF stimulation of endothelial cells. 2) VEGF stimulation promotes Cbl activation and enhances its association to Flk-1. 3) Cbl is responsible for the VEGF-stimulated ubiquitination and consequently degradation of Flk-1. We thus provide evidence that Cbl-dependent negative regulation of the Flk-1 receptor is a novel mechanism for down-regulation of VEGF-stimulated NO production from endothelial cells. Nitric oxide release from endothelial cells is centrally involved in multiple aspects of VEGF physiological effects. Indeed, as demonstrated in eNOS null mice, NO release has been shown to be essential for VEGF-mediated increases in vascular permeability, angiogenesis, and endothelial cell migration (29, 30, 31). Thus regulation of VEGF cellular signals, including NO production, is tightly controlled.

Our study shows that VEGF pretreatment of BAEC prevents any further activation of the Akt/eNOS signaling pathway from a second VEGF stimulation. Our results demonstrated that VEGF-dependent degradation of Flk-1 is involved in this desensitization of cells to VEGF stimulation. The degradation of Flk-1 actively participates in the regulation of VEGF responses in endothelial cells through the rapid inhibition of VEGF-stimulated eNOS activation and NO release (Figs. 1 and 2). One can also assume that rapid degradation of Flk-1 may also participate in the regulation of other components of VEGF signaling. However, the consequences on long term effects of VEGF such as endothelial cell migration, proliferation, and survival have not yet been investigated.

Ubiquitination of RTKs is known to target them to the lysosomal and proteasomal machineries (21). Indeed, consistent with these findings we demonstrate that, following VEGF stimulation, Flk-1 is multi-ubiquitinated in both BAEC and Flk-1 transfected COS-7 cells (Figs. 3 and 4). Polyubiquitination of cellular proteins, including RTKs, has emerged as a posttranslational modification that controls the cellular levels of proteins that are regulated either constitutively or in response to the cellular environment (22). We now show that polyubiquitination is involved in the regulation of membrane Flk-1 receptor levels.

Our results also demonstrate that Cbl is tyrosine phosphorylated and recruited to Flk-1 following VEGF stimulation of BAEC (Fig. 3). Cbl has initially been identified as a multi-adaptor protein involved in the recruitment of signaling complexes. Tyrosine phosphorylation of Cbl upon stimulation results in its association to the Src homology 2 domain containing proteins such as the p85 subunit of PI3-kinase, the nucleotide exchange factor VAV, and the Src-like kinases Fyn and Lyn (32, 33, 34, 35). Furthermore, Cbl contains a proline-rich domain that is involved in Src homology 3 domain interactions. The demonstration of an involvement of Cbl in the negative regulation of RTKs came from studies of Caenorhabditis elegans that showed that a loss of function of the Cbl homologue Sli-1 compensated for Let-23 (EGF-R) signaling (36, 37). Recent studies revealed that Cbl could function as an ubiquitin ligase that recruits ubiquitin-loaded E2 enzymes to a ligand-activated receptor (38).

Our results show that the association of Cbl with Flk-1 is enhanced in response to VEGF stimulation of BAEC and in transfected COS-7 cells. This association of Cbl to activated RTKs is most likely mediated by the N-terminal phosphotyrosine-binding domain of Cbl. However, we cannot exclude that a tyrosine-phosphorylated accessory protein may act as an intermediate in the docking of Cbl to Flk-1. Nonetheless the two proteins need to be in proximity in order for the flanking RING finger motif of Cbl to transfer the ubiquitin group onto the target receptor. Indeed, we demonstrate that overexpression of Cbl strongly enhances ubiquitination and degradation of Flk-1 and that a Cbl mutant lacking the RING finger domain, 70Z/3-Cbl, cannot mediate ligand-dependent Flk-1 ubiquitination (38).

Our report also demonstrates that Cbl regulates VEGF-stimulated NO release from cells. The coexpression of Cbl with Flk-1 and eNOS inhibits VEGF-stimulated NO release specifically. Furthermore, the mutant form of Cbl, 70Z/3-Cbl, failed to inhibit NO release and, interestingly, seemed to have a dominant negative effect on endogenous Cbl function by significantly enhancing NO release as well as inhibiting VEGF-stimulated Flk-1 ubiquitination (Figs. 4 and 5). These results are in line with those showing that oncogenic forms of Cbl, such as v-Cbl and 70Z/3-Cbl, led to enhanced signaling from RTKs and, in some cases, to hyperproliferation and tumorigenesis (39, 40, 41).

The VEGF-stimulated association of Cbl with Flk-1 is rapid, sustained, and consistent within the time frame of Flk-1 degradation. Interestingly, we demonstrate that overexpression of Cbl in BAEC accelerates VEGF-stimulated degradation of Flk-1. This confirms that, in endothelial cells, Cbl mediates Flk-1 degradation and that the levels of expression of Cbl can affect Flk-1 responses by regulating receptor levels following stimulation of cells. In unstimulated conditions, our results indicate that Cbl expression does not, however, alter basal levels of the Flk-1 protein upon stimulation of cells; Cbl influences the rate of its degradation.

The precise subcellular localization where Cbl acts on RTKs is still unclear, as some studies show that receptor ubiquitination might occur in endosomal compartments, and other studies indicate that this occurs at the plasma membrane before the internalization route (23, 42). Moreover, our immunofluorescence data show that, following a 30-min VEGF stimulation of endothelial cells, Cbl is located in vesicular-like structures (Fig. 6). This seems to be in agreement with previous studies reporting that Cbl associates with the EGF receptor and remains receptor-associated throughout endocytosis (42).

Our results show that negative regulation of Flk-1 by Cbl results in impaired NO release and that ubiquitination of Flk-1 through Cbl is involved in the desensitization of the cellular response to rapid VEGF signaling. Cbl has been shown to be activated in response to endothelial cells submitted to fluid shear stress and also participate in the activation of PI3-kinase following shear (43, 44). Knowing that Cbl functions as an adaptor-signaling molecule as well as an E3 ligase, it is tempting to speculate that prolonged shear stress on endothelial cells results in decreased Flk-1 protein expression.

In summary, our report demonstrates that polyubiquitination of the VEGF receptor, Flk-1, by the ubiquitin ligase adaptor protein, Cbl, mediates VEGF-dependent degradation of Flk-1. Importantly, our study provides evidences that Cbl-mediated effects lead to important biological consequences in endothelial cells such as the inhibition of VEGF-mediated NO release and enhanced Flk-1 degradation. Further studies will likely demonstrate the involvement of this novel mode of biological control of Flk-1 signaling in other physiological effects of VEGF.


    FOOTNOTES
 
* This work was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) and an establishment grant from Fonds de la Recherche en Santé du Québec (FRSQ) (to J.-P. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| A scholar of the Canadian Institutes of Health Research (New Investigator Award) and to whom correspondence should be addressed: Inst. de Recherches Cliniques de Montréal, 110 des Pins Ave W., Montreal, Quebec H2W 1R7, Canada. Tel.: 514-987-5610; Fax: 514-987-5610; E-mail: jean-philippe.gratton{at}ircm.qc.ca.

1 The abbreviations used are: VEGF, vascular endothelial growth factor; NO, nitric oxide; PI3-kinase, phosphatidylinositol 3-kinase; eNOS, endothelial NO synthase; RTK, receptor tyrosine kinase; EGFR, epidermal growth factor receptor; PDGFR, platelet-derived growth factor receptor; BAEC, bovine aortic endothelial cells; HA, hemagglutinin A; PBS, phosphate-buffered saline; {beta}-Gal, {beta}-galactosidase; GFP, green fluorescent protein. Back


    ACKNOWLEDGMENTS
 
We are grateful to Ms. Dior Cissé for secretarial assistance, Mr. Christian Charbonneau for assistance with confocal microscopy, Dr. Sylvain Meloche for reagents, and Dr. André Veillette for reagents and critical reading of the manuscript.



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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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