Type I Collagen Limits VEGFR-2 Signaling by a SHP2 Protein-Tyrosine PhosphataseeCDependent Mechanism 1
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循环研究杂志 2006年第1期
the Institute for Cancer Research and Treatment (S.M., B.B., L.Z., M.A., F.B.), Department of Oncological Sciences (S.M., B.B., L.Z., M.A., F.B.), Department of Medicine and Experimental Oncology (M.P., M.T.R.), University of Torino, Torino, Italy
Netherlands Institute for Developmental Biology (L.T., J.d.H.), Utrecht, The Netherlands
Max Planck Institute for Physiological and Clinical Research (G.B.), D-61231 Bad Nauheim, Germany
University Clinic Carl Gustav Carus (G.B.), Dresden, Germany.
Abstract
During angiogenesis, a combined action between newly secreted extracellular matrix proteins and the repertoire of integrins expressed by endothelial cells contributes in the regulation of their biological functions. Extracellular matrix-engaged integrins influence tyrosine kinase receptors, thus promoting a regulatory cross-talk between adhesive and soluble stimuli. For instance, vitronectin has been reported to positively regulate VEGFR-2. Here, we show that collagen I downregulates VEGF-AeCmediated VEGFR-2 activation. This activity requires the tyrosine phosphatase SHP2, which is recruited to the activated VEGFR-2 when cells are plated on collagen I, but not on vitronectin. Constitutive expression of SHP2C459S mutant inhibits the negative role of collagen I on VEGFR-2 phosphorylation. VEGFR-2 undergoes internalisation, which is associated with dynamin II phosphorylation. Expression of SHP2C459S impairs receptor internalisation suggesting that SHP2-dependent dephosphorylation regulates this process. These findings demonstrate that collagen I in provisional extracellular matrix surrounding nascent capillaries triggers a signaling pathway that negatively regulates angiogenesis.
Key Words: endothelial cell extracellular matrix tyrosine kinase receptor tyrosine phosphatase
Introduction
Angiogenesis takes place during development, tissue growth and repair, and aberrantly in several pathological settings. During angiogenesis, endothelial cells (ECs) modify their genetic program. The final consequences are the assumption of a migratory phenotype, cell cycle activation, and the secretion of proteases and proteins of the extracellular matrix (ECM).1 Changes in ECM physico-chemical features are crucial for EC biological functions.2,3 In the resting vasculature, ECM is mainly composed by collagens, laminin, tenascin, proteoglycans, and perleclan;4 such environment favors celleCcell adhesion, survival, and inhibits cell proliferation and migration.2,3 In contrast, provisional ECM surrounding angiogenic ECs include new proteins such as fibronectin, fibrin, vitronectin, collagen I, and thrombospondin. Furthermore, this ECM contains proteolytic cleavage products of laminin, fibronectin, and collagens generated by metalloproteinases released by activated ECs.2,3 A balance between negative and positive cues triggered by provisional ECM is permissive for neovascularization.2,3 For example, collagen I5 and thrombospondin-16 prevent angiogenesis, whereas fibronectin shows opposite activities.7 Furthermore, the proteolytic of laminin, collagen, and fibronectin generates pro- and antiangiogenic fragments.2,3
Coordinated with these ECM modifications, ECs adapt their repertoire of integrins to allow adhesion to new ECM components and to receive instructive signals from surrounding microenvironment.2,3 In particular, the induction of an angiogenic phenotype is associated with the expression of v3, v5, and 51.8eC10 Actually, vascular endothelial growth factor (VEGF)-A and fibroblast growth factor-2 induce angiogenesis that is, respectively, inhibited by blocking v5 and v3 function.11
VEGF-A represents a rate-limiting step for physiologic and pathologic angiogenesis.12 VEGF-A activates VEGF receptor (R) R-1 and VEGFR-2, the latter being the principal target of the ligand in adult life. Different mechanisms are involved in the fine-tuning of VEGFR-2 signaling:12,13 (1) VEGFR-2 transcription is regulated by soluble molecules and hypoxia; (2) the catalytic activity of VEGFR-2 may be negatively regulated by VEGFR-1, tissue inhibitor of metalloproteases (TIMP)-2 and dopamine; (3) the association of VEGFR-2 with neuropilin-1 is instrumental for increasing its affinity VEGF-A165; and (5) the localization in specific plasma membrane domains is another mechanism to control VEGFR-2 function. For example, in confluent ECs, VEGFR-2 is mainly associated with adherens junctions and mediates survival signals.14 On the contrary, during VEGF-AeCmediated EC migration, VEGFR-2 forms a complex with v3 integrin.15eC17 Finally, caveolae depletion results in an increase of VEGFR-2 phosphorylation but leads to the inhibition of downstream signals and EC motility.18
Here, we demonstrate that fibrillar collagen I is a negative modulator of VEGFR-2 activation via the recruitment of the cytosolic Src homology-2 domain-containing protein tyrosine phosphatase (SHP2)19,20 to the receptor. SHP2 dephosphorylates VEGFR-2 and favors its internalization. These findings extend previous observations on the opposite effects exerted by proteins of ECM on EC functions21,22 and provide new insights into how ECM surrounding nascent vessels could generate regulatory signals that are critical for EC response to VEGF-A.
Materials and Methods
For details on reagents and plasmids, cells, cell motility, and immunoprecipitation and immunoblotting, please see the online data supplement available at http://circres.ahajournals.org.
Tyrosine Phosphatase Assay
Tyrosine phosphatase activity was measured on EC immunoprecipitates anti-SHP1 and anti-SHP2 by HitHunter Fluorescence detection tyrosine phosphatase assay kit (Discover, Fremont, Calif). Alternatively, VEGFR-2 was immunoprecipitated as described from 107 human ECs stimulated with VEGF-A165 (0.23 nmol/L) and beads were washed twice in 50 mmol/L 2-(N-morpholino)ethanesulphonic acid, pH 6.5. VEGFR-2 immunoprecipitate was used as substrate and incubated with the aforementioned immunoprecipitates in 2-(N-morpholino)ethanesulphonic acid buffer for 1 hour at 37°C. At the end of incubation, proteins were denatured, separated by SDS-PAGE, and probed with mAb anti-pY or Ab antieCVEGFR-2.
VEGFR-2 Internalization
VEGFR-2 internalization was determined by evaluating the amount of receptor that was resistant to trypsinization23 or by evaluating the ratio between the amount of [125I-VEGF-A internalized and bound to the cell surface.24 The methods are detailed in the online supplementary materials.
Fluorescence Resonance Energy Transfer Analysis
For fluorescence resonance energy transfer (FRET), PAE cells carrying VEGFR-2-CFP and YFP-SHP2 were observed by a Leitz orthoplan upright microscope (Leitz GMBH, Wetzlar, Germany) equipped with an epi-illumination fluorescence detection system and a temperature-controlled specimen holder at 33°C. Measurements were made in a DMEM containing 2% fetal calf serum and 10 mmol/L HEPES (pH 7.4) exactly as previously described.25 Further details are described online.
Results
Effect of Type I Collagen and Vitronectin on VEGF-A165eCInduced EC Chemokinesis
Previous works demonstrated that ECM proteins modulate the autophosphorylation activity of VEGFR-2 and its biological responses.16,17,26 To further support this hypothesis, EC chemokinesis-stimulated by VEGF-A165 was evaluated on cells plated on collagen I or vitronectin by time-lapse video microscopy of individual cells (Figure 1). ECs exhibited a baseline mean migration speed of 17.31±0.09 e蘭/hour on vitronectin and 14.51±0.12 e蘭/hour on collagen I (P<0.001; t test). In presence of VEGF-A165, EC speed increased to 36.09±0.13 e蘭/hour (P<0.0001 versus unstimulated cells) and to 22.25±0.08 e蘭/hour (P<0.0002 versus unstimulated cells) on vitronectin and collagen I, respectively. VEGF-A165 stimulation resulted in higher directional persistence of cell migration27; in contrast unstimulated ECs moved in random directions. The VEGF-A165 effect was more evident in cells plated on vitronectin. Unstimulated cells on collagen I and on vitronectin showed a similar directional persistence (vitronectin: 0.12±0.06; collagen I: 0.10±0.02), whereas VEGF-A165 increased this parameter in a greater extent in cells on vitronectin (0.43±0.05) than on collagen I (0.19±0.04) (P<0.0001) (Figure 1).
To exclude that the observed effects were caused by different levels of VEGFR-2 expression, the high-affinity binding sites of VEGF-A on the surface of EC plated on collagen I or vitronectin were studied. Scatchard analysis indicated that the type of ECM did not influence the number of receptors and their affinity (supplemental Figure I).
Type I Collagen Inhibits VEGFR-2 Phosphorylation by Activating SHP2 Tyrosine Phosphatase
When VEGF-A165 stimulated ECs on vitronectin, VEGFR-2 phosphorylation was markedly higher as compared with cells on collagen I,17 suggesting a modulating role of tyrosine phosphatases.13,28eC30 In particular, both SHP1 and SHP2 have been demonstrated to negatively regulate VEGFR-2.13,28
Therefore, we measured phosphatase activity in SHP1 and SHP2 immunocomplexes from VEGF-A165eCstimulated ECs adherent on native ECM. VEGF-A165 promoted a rapid activation of SHP2 that peaked at 10 minutes and then declined (Figure 2A). SHP-1 activity increased, but in slight manner, without reaching a well-defined peak within 20 minutes. On the basis of this observation and previous data indicating that SHP2 is involved in GAS6 inhibition of VEGFR-2,28 we pointed to this phosphatase. Our hypothesis was further confirmed by the ability of SHP2 immunocomplexes isolated from VEGF-A165eCstimulated ECs to dephosphorylate in vitro phosphorylated VEGFR-2 (Figure 2B; supplemental Figure II). VEGF-A165 rapidly phosphorylated SHP2 in tyrosine residues, an event known to be associated with SHP2 catalytic activity19 (Figure 2C; supplemental Figure II). Both catalytic activity (Figure 2D) and tyrosine phosphorylation (Figure 2E; supplemental Figure II) stimulated by VEGF-A165 were more pronounced in cells on collagen I than on vitronectin.
SHP2 Binds VEGFR-2
Figure 3A shows that SHP2 associated with activated VEGFR-2 immunoprecipitated from ECs on native ECM. To further analyze this interaction, we performed a far Western blot analysis using SHP2-GST fusion protein. Figure 3B and supplemental Figure III show that phosphorylated VEGFR-2 bound SHP2-GST in a time dependent-manner. To follow this interaction in living cells, we analyzed FRET of VEGFR-2-CFP and YFP- SHP2 in PAE stimulated with VEGF-A165. Figure 3C and 3D shows that FRET occurred at the plasma membrane level and disappeared when VEGF-A165 was removed from medium.
Type I Collagen Elicits SHP2 Interaction With VEGFR-2
Next, we investigated whether ECM could modulate the association of SHP2 with VEGFR-2. VEGFR-2 was immunoprecipitated from ECs on collagen I or on vitronectin and the association with SHP2 was analyzed. The amount of associated SHP2 with VEGFR-2 in cells on vitronectin was much reduced compared with cells on collagen I (Figure 3E; supplemental Figure III). In these experiments EC adhesion to ECM substrates was similar, thus excluding the possibility that the observed effect could be caused by differences in cells adhesion (not shown).
Effect of SHP2C459S Overexpression on VEGFR-2 Activation
To investigate the involvement of SHP2 in VEGFR-2 signaling, ECs were infected with a retroviral vector carrying SHP2C459S dominant-negative mutant31 (Figure 4A), plated on collagen I, and stimulated with VEGF-A165 for different lengths of time. The amount of VEGFR-2 was not modified by the constitutive expression of the transgene (supplemental Figure IV), suggesting that the results obtained were not dependent on a modification of the basal expression of VEGFR-2. The expression of SHP2C459S mutant enhanced VEGFR-2 phosphorylation after 15 minutes of stimulation with VEGF-A165 as compared with ECs infected with vector alone (Figure 4B and 4C).
When EC infected with vector alone (EC-Pinco) were plated on collagen I (Figure 4D and 4E) VEGFR-2 phosphorylation triggered by VEGF-A165 was lower than on vitronectin. The pre-incubation with orthovanadate resulted in increase VEGFR-2 phosphorylation, which was particularly evident in stimulated cells on collagen I. Accordingly, SHP2C459S overexpression abrogated the increased VEGFR-2 phosphorylation caused by orthovanadate. When EC-SHP2C459S were plated on vitronectin, VEGFR-2 phosphorylation was completely unaffected by orthovanadate treatment (Figure 4D and 4E). These data support the concept type I collagen modulates VEGFR-2 through SHP2.
Because the chemokinetic activity of VEGF-A165 was low on EC plated on collagen I (Figure 1), we investigated the rescuing effect of the expression of SHP2C459S. The baseline migration speed on EC-Pinco and EC-SHP2C459S was similar (13.92±0.22 e蘭/hour and 14.21±0.42 e蘭/hour, respectively) and was increased &1.5- to 2-fold by VEGF-A165 (EC-Pinco: 20.33±0.26 e蘭/hour; EC-SHP2C459S: 25.82±0.28 e蘭/hour). Analysis of directional persistence demonstrated that the expression of the mutant did not modify the resting values (EC-Pinco: 0.09±0.04; EC-SHP2C459S: 0.12±0.08), whereas it allowed VEGF-A165 to be more efficient (EC-Pinco: 0.20±0.06; EC-SHP2C459S: 0.32±0.07) (Figure 5). The expression of SHP2C459S did not affect the chemokinetic parameters of unstimulated or stimulated cells plated on vitronectin, which were completely comparable to those shown in Figure 1(data not shown).
SHP2 Regulates VEGFR-2 Internalization
On engagement with its ligands, VEGFR-2 is internalized.18,24,32,33 Recent data suggest that dephosphorylation of tyrosine kinase receptors by protein phosphatases is involved in their downregulation through internalization.34eC36
Therefore, we looked at the internalisation of VEGFR-2 in ECs overexpressing SHP2C459S. To discriminate between the receptor exposed on cell membrane and the internalized fraction, cell surface was treated with trypsin23,35 and VEGFR-2 was immunoprecipitated with an Ab raised against its cytosolic portion. This method allows a direct analysis of receptor endocytosis by means trypsin-directed proteolysis of the membrane-exposed receptor. The internalized VEGFR-2 is in fact the only one retaining the original mass, whereas the receptor molecules that remain on the cell surface are cleaved by trypsin. This assay is an indirect indication of endocytosis, even though it does not offer any indication of the intracellular fate of the receptor.23,35 VEGFR-2 endocytosis was detectable 10 minutes after stimulation and reached the peak at 15 minutes (Figure 6A and 6B). Very little amount of internalized VEGFR-2 was detected in ECs overexpressing SHP2C459S. These results were confirmed by internalisation analysis of [125I]VEGF-A. Expression of SHP2C459S reduced the internalisation of the ligand (Figure 6C).
The GTPase dynamin II is a regulator of both clathrin- and caveolae-dependent endocytosis and its activity is positively regulated by tyrosine phosphorylation.37,38 In ECs dynamin II is a substrate of Src kinase,39 which is activated by VEGF-A.33,40 Furthermore, SHP2 regulates Src by controlling phosphorylation of its inhibitory tyrosine 529.41 If SHP2 contributes to the VEGFR-2 endocytosis, Src and dynamin II should be inhibited in cells carrying SHP2C459S. Indeed, VEGF-A165eCevoked tyrosyl phosphorylation of dynamin II and phosphorylation of Src Tyr-416 were impaired in these cells (Figure 6D and 6E; supplemental Figure V). These data demonstrate that SHP2 stimulation by VEGFR-2 modulates dynamin II activation, probably through Src kinase activation.
SHP2 Binds Directly to Tyrosine 1173 in the VEGFR-2
C-terminal Tyr-1212 and -1173 are the two major VEGF-AeCdependent autophosphorylation sites42 and their removal impairs VEGFR-2 downregulation,43 suggesting that one of these Tyr residues could binds SHP2. VEGFR-2 and its mutants VEGFR-2Y1173F and VEGFR-2Y1212F were immunoprecipitated from transfected PAE cells and the association with SHP2 was analyzed by immunoblot. The VEGF-A165eCdependent tyrosine phosphorylation of the receptor was lower in PAE carrying VEGFR-2Y1173F and VEGFR-2Y1212F mutants than in cells transfected with wild type receptor accordingly to the relevance of these residues in the autophosphorylation event (Figure 7A). Mutation in tyrosine 1173 abrogated the interaction with SHP2, suggesting that this reside is crucial for SHP2 binding to VEGFR-2 (Figure 7B).
Discussion
Our study establishes a role for collagen I, a protein released within the provisional ECM by angiogenic ECs, as a negative regulator of VEGFR-2 through activation of tyrosine phosphatase SHP2. This concept parallels the observation that 11 integrin engaged by collagen activates the T-cell protein tyrosine phosphatase function that inhibits epidermal growth factor receptor signaling.44
We demonstrated that EC adhesion to collagen I reduces VEGF-A165eCinduced VEGFR-2 autophosphorylation by recruiting SHP2 to the phosphorylated tyrosine 1173. This observation explains our previous data showing that EC proliferation stimulated by VEGF-A165 is lower on collagen I than on vitronectin.17 Moreover, we provide evidences that: (1) EC motility induced by VEGF-A165 is enhanced on vitronectin as compared with collagen I; (2) SHP2 activity and tyrosine residue phosphorylation are activated by VEGF-A when ECs adhere on native ECM or on vitronectin; however, these effects are greatly increased when EC adhere on collagen I; (3) constitutive expression of SHP2C459S blocks the inhibition of VEGFR-2 phosphorylation observed in cells adhering to collagen I; (4) VEGFR-2 is a substrate of SHP2; (5) in living cells VEGFR-2 closely interacts with SHP2 in proximity of plasma membrane of ECs; and (6) VEGF-A165 dependent association of VEGFR-2 with SHP2 occurs when ECs are plated on collagen I, but not on vitronectin.
Downregulation of tyrosine kinase receptors by phosphotyrosine phosphatases could be considered a simpler and faster way to modify their behavior and functions as compared with clathrin-mediated or caveolae-dependent endocytosis or to proteasome-mediated degradation.45 SHP2 has been generally considered a positive downstream signal activated by membrane receptors.46 However, emerging evidences suggest that it may positively or negatively regulate signaling pathways depending on the specific type of signaling network. For example, SHP2 may act as a negative regulator of platelet-derived growth factor receptor by inducing dephosphorylation of the receptor itself or of its cognate substrates.47eC49 However, SHP2 is also implicated in positive regulation of this receptor through regulation of Ras or focal adhesion kinase.50,51 Similarly, SHP2 exerts both negative and positive influences on T-cell receptor52,53 and JAKs/STATs pathway.54,55 Finally, SHP2 exclusively acts as negative regulator in angiotensin II receptor A1-mediated signals.56
Here, we do not investigate the precise mechanism by which collagen I favors SHP2-mediated VEGFR-2 dephosphorylation. Recently, we have demonstrated that SHP2 is involved in the negative control of VEGFR-2 triggered by GAS6-dependent Axl stimulation.28 Furthermore, our observation parallels the results that VEGFR-2 may be negatively regulated by TIMP-2 through SHP1.13 In ECs stimulated by TIMP-2, SHP1 shifts from 31 integrin to VEGFR-2, which is dephosphorylated.
It has been reported that in vascular smooth muscle cells 3 engagement by vitronectin results in tyrosine phosphorylation of its cytosolic domain and recruitment of SHP2, which modulate insulin growth factor I receptor.57 Thus, we hypothesize a protective role on VEGFR-2 signaling by vitronectin-engaged v3, which recruits SHP2 and preserves the receptor from phosphatase activity. In contrast, EC adhesion on collagen I, which does not depend on v3 integrin, could allow SHP2 interacting with VEGFR-2. Preliminary evidences demonstrate that a phosphatase activity is associated to 3 in ECs plated on vitronectin and stimulated by VEGF-A165. However, 1 immunocomplexes do not contain any phosphatase activity both in cells plated on collagen I and vitronectin (S. Mitola, unpublished).
Then we showed that SHP2 plays a relevant role in VEGFR-2 internalisation. VEGFR-2 undergoes endocytosis by a Cbl-dependent ubiquitination process58 or through a mechanism caveolin-1eCdependent.18 Here, we extend these observations by showing overexpression of SHP2C459S dramatically decreases VEGFR-2 internalization, suggesting that dephosphorylation is involved in regulating its trafficking.
VEGF-A165 induced tyrosyl phosphorylation of dynamin II, a GTPase regulator of vesicle fission required for the control of both clathrin- and caveolae-mediated endocytosis.38 Previous studies showed that Src-dependent dynamin II phosphorylation is required for endocytosis.39,59,60 Furthermore, SHP2 activates Src kinase by inhibiting the recruitment of C-terminal Src kinase, which exerts its inhibitory role by phosphorylating Tyr-529 of Src.41 Here, we show that overexpression of SHP2C459S significantly reduced the effect of VEGF-A165 on dynamin II phosphorylation and Src activation. These data suggest that SHP2 activation dephosphorylates VEGFR-2 and concomitantly activates Src kinase, which triggers a dynamin-dependent receptor internalization. SHP2 binds Tyr-1173 of stimulated VEGFR-2. This residue seems important in internalisation process as demonstrated by studying truncated receptors lacking Tyr-1173 and Tyr-1212.61 This process occurs in ECs growing on native ECM, as demonstrated by the data showing that this phosphatase is also activated by VEGF-A165 in ECs adherent to this ECM (Figures 2A, 2B, and 3) as well as in cells on collagen I. Here, we did not address the behavior of the internalized VEGFR-2 in EC adhering on collagen I. VEGFR-2 could either be accumulated in the endosomal compartment and degraded or recycled to the membrane, thus allowing a different degree of EC activation or a modified spatial regulation of the signal. It is known that tyrosine kinase receptors may be translocated to the nucleus.62 Notably, it has been recently reported that VEGF-A through VEGFR-2 may be transferred to the nucleus of migrating ECs.32 We may speculate that collagen I mediated internalization of VEGFR-2 results in attenuation of VEGFR-2eCmediated signals or in activation of undefined EC responses and that a modulation of SHP2-dependent mechanism of internalization by ECM characterizes specific steps of angiogenesis.
Acknowledgments
This study was supported by Associazione Italiana per la Ricerca sul Cancro, Istituto Superiore di Sanite? (AIDS and Stem Cells National Projects), Ministero dell’ Universite?e della Ricerca (MIUR) (60% and PRIN 2004 projects), Fondi incentivazione della Ricerca di Base (RBNE0184P, RBNE01MAWA, RBNE01T8C8), Ministero della Salute (Ricerca Finalizzata 2002 and 2003 to M.A. and F.B.), Regione Piemonte, and European Community (LSHM-CT-2003-503251) (http:://www.engv.org). We thank Letizia Lanzetti, Luca Primo, and Guido Serini for their suggestions, and Vincenzo Bagnardi for statistical analysis.
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Netherlands Institute for Developmental Biology (L.T., J.d.H.), Utrecht, The Netherlands
Max Planck Institute for Physiological and Clinical Research (G.B.), D-61231 Bad Nauheim, Germany
University Clinic Carl Gustav Carus (G.B.), Dresden, Germany.
Abstract
During angiogenesis, a combined action between newly secreted extracellular matrix proteins and the repertoire of integrins expressed by endothelial cells contributes in the regulation of their biological functions. Extracellular matrix-engaged integrins influence tyrosine kinase receptors, thus promoting a regulatory cross-talk between adhesive and soluble stimuli. For instance, vitronectin has been reported to positively regulate VEGFR-2. Here, we show that collagen I downregulates VEGF-AeCmediated VEGFR-2 activation. This activity requires the tyrosine phosphatase SHP2, which is recruited to the activated VEGFR-2 when cells are plated on collagen I, but not on vitronectin. Constitutive expression of SHP2C459S mutant inhibits the negative role of collagen I on VEGFR-2 phosphorylation. VEGFR-2 undergoes internalisation, which is associated with dynamin II phosphorylation. Expression of SHP2C459S impairs receptor internalisation suggesting that SHP2-dependent dephosphorylation regulates this process. These findings demonstrate that collagen I in provisional extracellular matrix surrounding nascent capillaries triggers a signaling pathway that negatively regulates angiogenesis.
Key Words: endothelial cell extracellular matrix tyrosine kinase receptor tyrosine phosphatase
Introduction
Angiogenesis takes place during development, tissue growth and repair, and aberrantly in several pathological settings. During angiogenesis, endothelial cells (ECs) modify their genetic program. The final consequences are the assumption of a migratory phenotype, cell cycle activation, and the secretion of proteases and proteins of the extracellular matrix (ECM).1 Changes in ECM physico-chemical features are crucial for EC biological functions.2,3 In the resting vasculature, ECM is mainly composed by collagens, laminin, tenascin, proteoglycans, and perleclan;4 such environment favors celleCcell adhesion, survival, and inhibits cell proliferation and migration.2,3 In contrast, provisional ECM surrounding angiogenic ECs include new proteins such as fibronectin, fibrin, vitronectin, collagen I, and thrombospondin. Furthermore, this ECM contains proteolytic cleavage products of laminin, fibronectin, and collagens generated by metalloproteinases released by activated ECs.2,3 A balance between negative and positive cues triggered by provisional ECM is permissive for neovascularization.2,3 For example, collagen I5 and thrombospondin-16 prevent angiogenesis, whereas fibronectin shows opposite activities.7 Furthermore, the proteolytic of laminin, collagen, and fibronectin generates pro- and antiangiogenic fragments.2,3
Coordinated with these ECM modifications, ECs adapt their repertoire of integrins to allow adhesion to new ECM components and to receive instructive signals from surrounding microenvironment.2,3 In particular, the induction of an angiogenic phenotype is associated with the expression of v3, v5, and 51.8eC10 Actually, vascular endothelial growth factor (VEGF)-A and fibroblast growth factor-2 induce angiogenesis that is, respectively, inhibited by blocking v5 and v3 function.11
VEGF-A represents a rate-limiting step for physiologic and pathologic angiogenesis.12 VEGF-A activates VEGF receptor (R) R-1 and VEGFR-2, the latter being the principal target of the ligand in adult life. Different mechanisms are involved in the fine-tuning of VEGFR-2 signaling:12,13 (1) VEGFR-2 transcription is regulated by soluble molecules and hypoxia; (2) the catalytic activity of VEGFR-2 may be negatively regulated by VEGFR-1, tissue inhibitor of metalloproteases (TIMP)-2 and dopamine; (3) the association of VEGFR-2 with neuropilin-1 is instrumental for increasing its affinity VEGF-A165; and (5) the localization in specific plasma membrane domains is another mechanism to control VEGFR-2 function. For example, in confluent ECs, VEGFR-2 is mainly associated with adherens junctions and mediates survival signals.14 On the contrary, during VEGF-AeCmediated EC migration, VEGFR-2 forms a complex with v3 integrin.15eC17 Finally, caveolae depletion results in an increase of VEGFR-2 phosphorylation but leads to the inhibition of downstream signals and EC motility.18
Here, we demonstrate that fibrillar collagen I is a negative modulator of VEGFR-2 activation via the recruitment of the cytosolic Src homology-2 domain-containing protein tyrosine phosphatase (SHP2)19,20 to the receptor. SHP2 dephosphorylates VEGFR-2 and favors its internalization. These findings extend previous observations on the opposite effects exerted by proteins of ECM on EC functions21,22 and provide new insights into how ECM surrounding nascent vessels could generate regulatory signals that are critical for EC response to VEGF-A.
Materials and Methods
For details on reagents and plasmids, cells, cell motility, and immunoprecipitation and immunoblotting, please see the online data supplement available at http://circres.ahajournals.org.
Tyrosine Phosphatase Assay
Tyrosine phosphatase activity was measured on EC immunoprecipitates anti-SHP1 and anti-SHP2 by HitHunter Fluorescence detection tyrosine phosphatase assay kit (Discover, Fremont, Calif). Alternatively, VEGFR-2 was immunoprecipitated as described from 107 human ECs stimulated with VEGF-A165 (0.23 nmol/L) and beads were washed twice in 50 mmol/L 2-(N-morpholino)ethanesulphonic acid, pH 6.5. VEGFR-2 immunoprecipitate was used as substrate and incubated with the aforementioned immunoprecipitates in 2-(N-morpholino)ethanesulphonic acid buffer for 1 hour at 37°C. At the end of incubation, proteins were denatured, separated by SDS-PAGE, and probed with mAb anti-pY or Ab antieCVEGFR-2.
VEGFR-2 Internalization
VEGFR-2 internalization was determined by evaluating the amount of receptor that was resistant to trypsinization23 or by evaluating the ratio between the amount of [125I-VEGF-A internalized and bound to the cell surface.24 The methods are detailed in the online supplementary materials.
Fluorescence Resonance Energy Transfer Analysis
For fluorescence resonance energy transfer (FRET), PAE cells carrying VEGFR-2-CFP and YFP-SHP2 were observed by a Leitz orthoplan upright microscope (Leitz GMBH, Wetzlar, Germany) equipped with an epi-illumination fluorescence detection system and a temperature-controlled specimen holder at 33°C. Measurements were made in a DMEM containing 2% fetal calf serum and 10 mmol/L HEPES (pH 7.4) exactly as previously described.25 Further details are described online.
Results
Effect of Type I Collagen and Vitronectin on VEGF-A165eCInduced EC Chemokinesis
Previous works demonstrated that ECM proteins modulate the autophosphorylation activity of VEGFR-2 and its biological responses.16,17,26 To further support this hypothesis, EC chemokinesis-stimulated by VEGF-A165 was evaluated on cells plated on collagen I or vitronectin by time-lapse video microscopy of individual cells (Figure 1). ECs exhibited a baseline mean migration speed of 17.31±0.09 e蘭/hour on vitronectin and 14.51±0.12 e蘭/hour on collagen I (P<0.001; t test). In presence of VEGF-A165, EC speed increased to 36.09±0.13 e蘭/hour (P<0.0001 versus unstimulated cells) and to 22.25±0.08 e蘭/hour (P<0.0002 versus unstimulated cells) on vitronectin and collagen I, respectively. VEGF-A165 stimulation resulted in higher directional persistence of cell migration27; in contrast unstimulated ECs moved in random directions. The VEGF-A165 effect was more evident in cells plated on vitronectin. Unstimulated cells on collagen I and on vitronectin showed a similar directional persistence (vitronectin: 0.12±0.06; collagen I: 0.10±0.02), whereas VEGF-A165 increased this parameter in a greater extent in cells on vitronectin (0.43±0.05) than on collagen I (0.19±0.04) (P<0.0001) (Figure 1).
To exclude that the observed effects were caused by different levels of VEGFR-2 expression, the high-affinity binding sites of VEGF-A on the surface of EC plated on collagen I or vitronectin were studied. Scatchard analysis indicated that the type of ECM did not influence the number of receptors and their affinity (supplemental Figure I).
Type I Collagen Inhibits VEGFR-2 Phosphorylation by Activating SHP2 Tyrosine Phosphatase
When VEGF-A165 stimulated ECs on vitronectin, VEGFR-2 phosphorylation was markedly higher as compared with cells on collagen I,17 suggesting a modulating role of tyrosine phosphatases.13,28eC30 In particular, both SHP1 and SHP2 have been demonstrated to negatively regulate VEGFR-2.13,28
Therefore, we measured phosphatase activity in SHP1 and SHP2 immunocomplexes from VEGF-A165eCstimulated ECs adherent on native ECM. VEGF-A165 promoted a rapid activation of SHP2 that peaked at 10 minutes and then declined (Figure 2A). SHP-1 activity increased, but in slight manner, without reaching a well-defined peak within 20 minutes. On the basis of this observation and previous data indicating that SHP2 is involved in GAS6 inhibition of VEGFR-2,28 we pointed to this phosphatase. Our hypothesis was further confirmed by the ability of SHP2 immunocomplexes isolated from VEGF-A165eCstimulated ECs to dephosphorylate in vitro phosphorylated VEGFR-2 (Figure 2B; supplemental Figure II). VEGF-A165 rapidly phosphorylated SHP2 in tyrosine residues, an event known to be associated with SHP2 catalytic activity19 (Figure 2C; supplemental Figure II). Both catalytic activity (Figure 2D) and tyrosine phosphorylation (Figure 2E; supplemental Figure II) stimulated by VEGF-A165 were more pronounced in cells on collagen I than on vitronectin.
SHP2 Binds VEGFR-2
Figure 3A shows that SHP2 associated with activated VEGFR-2 immunoprecipitated from ECs on native ECM. To further analyze this interaction, we performed a far Western blot analysis using SHP2-GST fusion protein. Figure 3B and supplemental Figure III show that phosphorylated VEGFR-2 bound SHP2-GST in a time dependent-manner. To follow this interaction in living cells, we analyzed FRET of VEGFR-2-CFP and YFP- SHP2 in PAE stimulated with VEGF-A165. Figure 3C and 3D shows that FRET occurred at the plasma membrane level and disappeared when VEGF-A165 was removed from medium.
Type I Collagen Elicits SHP2 Interaction With VEGFR-2
Next, we investigated whether ECM could modulate the association of SHP2 with VEGFR-2. VEGFR-2 was immunoprecipitated from ECs on collagen I or on vitronectin and the association with SHP2 was analyzed. The amount of associated SHP2 with VEGFR-2 in cells on vitronectin was much reduced compared with cells on collagen I (Figure 3E; supplemental Figure III). In these experiments EC adhesion to ECM substrates was similar, thus excluding the possibility that the observed effect could be caused by differences in cells adhesion (not shown).
Effect of SHP2C459S Overexpression on VEGFR-2 Activation
To investigate the involvement of SHP2 in VEGFR-2 signaling, ECs were infected with a retroviral vector carrying SHP2C459S dominant-negative mutant31 (Figure 4A), plated on collagen I, and stimulated with VEGF-A165 for different lengths of time. The amount of VEGFR-2 was not modified by the constitutive expression of the transgene (supplemental Figure IV), suggesting that the results obtained were not dependent on a modification of the basal expression of VEGFR-2. The expression of SHP2C459S mutant enhanced VEGFR-2 phosphorylation after 15 minutes of stimulation with VEGF-A165 as compared with ECs infected with vector alone (Figure 4B and 4C).
When EC infected with vector alone (EC-Pinco) were plated on collagen I (Figure 4D and 4E) VEGFR-2 phosphorylation triggered by VEGF-A165 was lower than on vitronectin. The pre-incubation with orthovanadate resulted in increase VEGFR-2 phosphorylation, which was particularly evident in stimulated cells on collagen I. Accordingly, SHP2C459S overexpression abrogated the increased VEGFR-2 phosphorylation caused by orthovanadate. When EC-SHP2C459S were plated on vitronectin, VEGFR-2 phosphorylation was completely unaffected by orthovanadate treatment (Figure 4D and 4E). These data support the concept type I collagen modulates VEGFR-2 through SHP2.
Because the chemokinetic activity of VEGF-A165 was low on EC plated on collagen I (Figure 1), we investigated the rescuing effect of the expression of SHP2C459S. The baseline migration speed on EC-Pinco and EC-SHP2C459S was similar (13.92±0.22 e蘭/hour and 14.21±0.42 e蘭/hour, respectively) and was increased &1.5- to 2-fold by VEGF-A165 (EC-Pinco: 20.33±0.26 e蘭/hour; EC-SHP2C459S: 25.82±0.28 e蘭/hour). Analysis of directional persistence demonstrated that the expression of the mutant did not modify the resting values (EC-Pinco: 0.09±0.04; EC-SHP2C459S: 0.12±0.08), whereas it allowed VEGF-A165 to be more efficient (EC-Pinco: 0.20±0.06; EC-SHP2C459S: 0.32±0.07) (Figure 5). The expression of SHP2C459S did not affect the chemokinetic parameters of unstimulated or stimulated cells plated on vitronectin, which were completely comparable to those shown in Figure 1(data not shown).
SHP2 Regulates VEGFR-2 Internalization
On engagement with its ligands, VEGFR-2 is internalized.18,24,32,33 Recent data suggest that dephosphorylation of tyrosine kinase receptors by protein phosphatases is involved in their downregulation through internalization.34eC36
Therefore, we looked at the internalisation of VEGFR-2 in ECs overexpressing SHP2C459S. To discriminate between the receptor exposed on cell membrane and the internalized fraction, cell surface was treated with trypsin23,35 and VEGFR-2 was immunoprecipitated with an Ab raised against its cytosolic portion. This method allows a direct analysis of receptor endocytosis by means trypsin-directed proteolysis of the membrane-exposed receptor. The internalized VEGFR-2 is in fact the only one retaining the original mass, whereas the receptor molecules that remain on the cell surface are cleaved by trypsin. This assay is an indirect indication of endocytosis, even though it does not offer any indication of the intracellular fate of the receptor.23,35 VEGFR-2 endocytosis was detectable 10 minutes after stimulation and reached the peak at 15 minutes (Figure 6A and 6B). Very little amount of internalized VEGFR-2 was detected in ECs overexpressing SHP2C459S. These results were confirmed by internalisation analysis of [125I]VEGF-A. Expression of SHP2C459S reduced the internalisation of the ligand (Figure 6C).
The GTPase dynamin II is a regulator of both clathrin- and caveolae-dependent endocytosis and its activity is positively regulated by tyrosine phosphorylation.37,38 In ECs dynamin II is a substrate of Src kinase,39 which is activated by VEGF-A.33,40 Furthermore, SHP2 regulates Src by controlling phosphorylation of its inhibitory tyrosine 529.41 If SHP2 contributes to the VEGFR-2 endocytosis, Src and dynamin II should be inhibited in cells carrying SHP2C459S. Indeed, VEGF-A165eCevoked tyrosyl phosphorylation of dynamin II and phosphorylation of Src Tyr-416 were impaired in these cells (Figure 6D and 6E; supplemental Figure V). These data demonstrate that SHP2 stimulation by VEGFR-2 modulates dynamin II activation, probably through Src kinase activation.
SHP2 Binds Directly to Tyrosine 1173 in the VEGFR-2
C-terminal Tyr-1212 and -1173 are the two major VEGF-AeCdependent autophosphorylation sites42 and their removal impairs VEGFR-2 downregulation,43 suggesting that one of these Tyr residues could binds SHP2. VEGFR-2 and its mutants VEGFR-2Y1173F and VEGFR-2Y1212F were immunoprecipitated from transfected PAE cells and the association with SHP2 was analyzed by immunoblot. The VEGF-A165eCdependent tyrosine phosphorylation of the receptor was lower in PAE carrying VEGFR-2Y1173F and VEGFR-2Y1212F mutants than in cells transfected with wild type receptor accordingly to the relevance of these residues in the autophosphorylation event (Figure 7A). Mutation in tyrosine 1173 abrogated the interaction with SHP2, suggesting that this reside is crucial for SHP2 binding to VEGFR-2 (Figure 7B).
Discussion
Our study establishes a role for collagen I, a protein released within the provisional ECM by angiogenic ECs, as a negative regulator of VEGFR-2 through activation of tyrosine phosphatase SHP2. This concept parallels the observation that 11 integrin engaged by collagen activates the T-cell protein tyrosine phosphatase function that inhibits epidermal growth factor receptor signaling.44
We demonstrated that EC adhesion to collagen I reduces VEGF-A165eCinduced VEGFR-2 autophosphorylation by recruiting SHP2 to the phosphorylated tyrosine 1173. This observation explains our previous data showing that EC proliferation stimulated by VEGF-A165 is lower on collagen I than on vitronectin.17 Moreover, we provide evidences that: (1) EC motility induced by VEGF-A165 is enhanced on vitronectin as compared with collagen I; (2) SHP2 activity and tyrosine residue phosphorylation are activated by VEGF-A when ECs adhere on native ECM or on vitronectin; however, these effects are greatly increased when EC adhere on collagen I; (3) constitutive expression of SHP2C459S blocks the inhibition of VEGFR-2 phosphorylation observed in cells adhering to collagen I; (4) VEGFR-2 is a substrate of SHP2; (5) in living cells VEGFR-2 closely interacts with SHP2 in proximity of plasma membrane of ECs; and (6) VEGF-A165 dependent association of VEGFR-2 with SHP2 occurs when ECs are plated on collagen I, but not on vitronectin.
Downregulation of tyrosine kinase receptors by phosphotyrosine phosphatases could be considered a simpler and faster way to modify their behavior and functions as compared with clathrin-mediated or caveolae-dependent endocytosis or to proteasome-mediated degradation.45 SHP2 has been generally considered a positive downstream signal activated by membrane receptors.46 However, emerging evidences suggest that it may positively or negatively regulate signaling pathways depending on the specific type of signaling network. For example, SHP2 may act as a negative regulator of platelet-derived growth factor receptor by inducing dephosphorylation of the receptor itself or of its cognate substrates.47eC49 However, SHP2 is also implicated in positive regulation of this receptor through regulation of Ras or focal adhesion kinase.50,51 Similarly, SHP2 exerts both negative and positive influences on T-cell receptor52,53 and JAKs/STATs pathway.54,55 Finally, SHP2 exclusively acts as negative regulator in angiotensin II receptor A1-mediated signals.56
Here, we do not investigate the precise mechanism by which collagen I favors SHP2-mediated VEGFR-2 dephosphorylation. Recently, we have demonstrated that SHP2 is involved in the negative control of VEGFR-2 triggered by GAS6-dependent Axl stimulation.28 Furthermore, our observation parallels the results that VEGFR-2 may be negatively regulated by TIMP-2 through SHP1.13 In ECs stimulated by TIMP-2, SHP1 shifts from 31 integrin to VEGFR-2, which is dephosphorylated.
It has been reported that in vascular smooth muscle cells 3 engagement by vitronectin results in tyrosine phosphorylation of its cytosolic domain and recruitment of SHP2, which modulate insulin growth factor I receptor.57 Thus, we hypothesize a protective role on VEGFR-2 signaling by vitronectin-engaged v3, which recruits SHP2 and preserves the receptor from phosphatase activity. In contrast, EC adhesion on collagen I, which does not depend on v3 integrin, could allow SHP2 interacting with VEGFR-2. Preliminary evidences demonstrate that a phosphatase activity is associated to 3 in ECs plated on vitronectin and stimulated by VEGF-A165. However, 1 immunocomplexes do not contain any phosphatase activity both in cells plated on collagen I and vitronectin (S. Mitola, unpublished).
Then we showed that SHP2 plays a relevant role in VEGFR-2 internalisation. VEGFR-2 undergoes endocytosis by a Cbl-dependent ubiquitination process58 or through a mechanism caveolin-1eCdependent.18 Here, we extend these observations by showing overexpression of SHP2C459S dramatically decreases VEGFR-2 internalization, suggesting that dephosphorylation is involved in regulating its trafficking.
VEGF-A165 induced tyrosyl phosphorylation of dynamin II, a GTPase regulator of vesicle fission required for the control of both clathrin- and caveolae-mediated endocytosis.38 Previous studies showed that Src-dependent dynamin II phosphorylation is required for endocytosis.39,59,60 Furthermore, SHP2 activates Src kinase by inhibiting the recruitment of C-terminal Src kinase, which exerts its inhibitory role by phosphorylating Tyr-529 of Src.41 Here, we show that overexpression of SHP2C459S significantly reduced the effect of VEGF-A165 on dynamin II phosphorylation and Src activation. These data suggest that SHP2 activation dephosphorylates VEGFR-2 and concomitantly activates Src kinase, which triggers a dynamin-dependent receptor internalization. SHP2 binds Tyr-1173 of stimulated VEGFR-2. This residue seems important in internalisation process as demonstrated by studying truncated receptors lacking Tyr-1173 and Tyr-1212.61 This process occurs in ECs growing on native ECM, as demonstrated by the data showing that this phosphatase is also activated by VEGF-A165 in ECs adherent to this ECM (Figures 2A, 2B, and 3) as well as in cells on collagen I. Here, we did not address the behavior of the internalized VEGFR-2 in EC adhering on collagen I. VEGFR-2 could either be accumulated in the endosomal compartment and degraded or recycled to the membrane, thus allowing a different degree of EC activation or a modified spatial regulation of the signal. It is known that tyrosine kinase receptors may be translocated to the nucleus.62 Notably, it has been recently reported that VEGF-A through VEGFR-2 may be transferred to the nucleus of migrating ECs.32 We may speculate that collagen I mediated internalization of VEGFR-2 results in attenuation of VEGFR-2eCmediated signals or in activation of undefined EC responses and that a modulation of SHP2-dependent mechanism of internalization by ECM characterizes specific steps of angiogenesis.
Acknowledgments
This study was supported by Associazione Italiana per la Ricerca sul Cancro, Istituto Superiore di Sanite? (AIDS and Stem Cells National Projects), Ministero dell’ Universite?e della Ricerca (MIUR) (60% and PRIN 2004 projects), Fondi incentivazione della Ricerca di Base (RBNE0184P, RBNE01MAWA, RBNE01T8C8), Ministero della Salute (Ricerca Finalizzata 2002 and 2003 to M.A. and F.B.), Regione Piemonte, and European Community (LSHM-CT-2003-503251) (http:://www.engv.org). We thank Letizia Lanzetti, Luca Primo, and Guido Serini for their suggestions, and Vincenzo Bagnardi for statistical analysis.
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