Mechanisms of Integrin–Vascular Endothelial Growth Factor Receptor Cross-Activation in Angiogenesis
The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between β3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and β3 integrin appears to be synergistic, because VEGFR-2 activation induces β3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor–induced β3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent β3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and β3 integrin. Moreover, c-Src mediates growth factor–induced β3 integrin activation, ligand binding, β3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.
- endothelial cell
- β3 integrin signaling
- vascular endothelial growth factor receptor
- extracellular matrix proteins
Angiogenesis, the process of new blood vessel formation from preexisting vasculature, plays critical roles in tissue regeneration, postischemic tissue repair on myocardial infarction and stroke, and in the pathogenesis of cancer, rheumatoid arthritis, and diabetic microvascular disease.1 Angiogenesis is triggered by angiogenic growth factors and their receptors in coordination with extracellular matrix (ECM) receptors known as integrins.2 On integrin engagement, ECM triggers activation of numerous intracellular signaling pathways essential for endothelial cell (EC) survival, proliferation, migration, morphogenesis, and organization of ECs into blood vessels.3 There are several manifestations of a tightly collaborative relationship between integrins and receptors for growth factors.4,5 On ECs, engagement of αvβ3 integrin promotes phosphorylation and activation of vascular endothelial growth factor (VEGF) receptor (VEGFR)-2, thereby augmenting the mitogenic activity of VEGFs.6
Among several integrins on ECs, αvβ3 is the most abundant and influential receptor regulating angiogenesis.7 The upregulation of αvβ3 during angiogenesis suggests that this integrin might play a crucial role during this process. Indeed, antagonists of αvβ3, including blocking monoclonal antibody (LM609) and RGD cyclic peptides, were shown to be efficient inhibitors of neovascularization in several experimental animal models.8–10 Results of preclinical models have justified ongoing clinical trials of humanized monoclonal antibodies and cyclic peptides, which specifically target αvβ3.11 That αvβ3 regulates tumor-induced angiogenesis is clear, although its precise role remains disputed,12–15 indicating the need for conclusive mechanistic studies.
At the structural level, the function of this integrin is regulated by its cytoplasmic domain. Conserved regions present within the β3 integrin (β3) cytoplasmic domain include NPXY and NXXY motifs,16 the tyrosine residues of which can be phosphorylated to regulate interactions with signaling proteins containing SH3 and PTB domains.17 Phosphorylation of β3 modulates several intracellular events, including VAV-1/Rho GTPase activation, actin cytoskeleton reorganization and regulation of the phosphatidylinositol 3-kinase/AKT pathway, which is involved in the regulation of basic cellular functions such as cell spreading and survival.18 Thus, phosphorylation of β3 cytoplasmic domain is critical for αvβ3 integrin–dependent functions because it regulates αvβ3 affinity, avidity, and ligand-binding strength.19–21 Although the responses regulated by β3 phosphorylation are numerous and functionally important, this phenomenon has not been thoroughly studied, leaving critical gaps in our understanding of the molecular mechanisms underlying the process of receptor cross-activation.
Materials and Methods
Reagents and plasmids used in this study, cell culture and transfection, isolation of primary mouse ECs, cell adhesion assays, cell migration assays, tube formation assays, time-lapse video microscopy, in vitro kinase assays, fibrinogen and WOW-1–binding assays, and immunoprecipitation and immunoblotting methods are described in the online data supplement at http://circres.ahajournals.org.
Values were expressed as mean±SD. Probability values were based on the paired t test. Results were considered statistically significant with a probability value less than 0.05.
β3 Integrin Tyrosine Phosphorylation Is Required for Maximum Tyrosine Phosphorylation of VEGFR-2
As a first step to examine the relationship between integrin ligation, β3 phosphorylation and VEGFR-2 activation, we monitored phosphorylation of β3 at Tyr747 and Tyr759 in ECs plated on the αvβ3 ligand vitronectin, the α2β1 ligand collagen, or the α6β1/α6β4 ligand laminin. As a control, the ECs were maintained in suspension in the presence or absence of VEGF stimulation. As shown in Figure 1A, vitronectin, but not laminin or collagen, was able to induce β3 phosphorylation, which was augmented on VEGF treatment. At the same time, phosphorylation of β3 was minimal in cells in suspension or plated on laminin or collagen despite stimulation with VEGF (Figure 1A). Human umbilical vein ECs (HUVECs) also exhibited differential adhesion to various integrin ligands (Figure IIA and IIB in the online data supplement). Analysis of VEGFR-2 tyrosine phosphorylation in the same set of samples revealed that basal level VEGFR-2 activation can be triggered by αvβ3 ligation induced by vitronectin but does not occur in cells plated on collagen or laminin. Parallel analysis of β1 phosphorylation status showed no significant difference with VEGF stimulation (supplemental Figure IA). Thus, whereas VEGF stimulation promotes phosphorylation of αvβ3, ligation of αvβ3 also stimulates VEGFR-2 phosphorylation, and activation, demonstrating a mutual relationship between VEGFR-2 and αvβ3.
β3 Integrin Tyrosine Phosphorylation Is Complementary to VEGF-Induced Tyrosine Phosphorylation of VEGFR-2
αvβ3 is expressed on proliferating ECs during angiogenesis and vascular remodeling and the blockade of αvβ3 suppresses angiogenesis in several in vivo models.13–16 Therefore, we assessed whether the blockade of αvβ3 affected tyrosine phosphorylation of the β3 subunit. Accordingly, HUVECs grown on gelatin-coated plates were incubated with anti-αv, anti-β3, anti-β1, and anti-β5 blocking antibodies and induced with VEGF for 5 minutes at 37°C. Cell lysates were analyzed for phosphorylation of β3 at Tyr747 and Tyr759. Figure 1B shows that both anti-αv and anti-β3 blocking antibodies inhibited VEGF-induced phosphorylation of β3 at both residues. Simultaneously, control IgG, anti-β1, or anti-β5 blocking antibodies had no substantial effects on VEGF-induced phosphorylation of β3.
To further examine the consequences of integrin blocking antibodies, cell lysates were analyzed for tyrosine phosphorylation of VEGFR-2. Results indicated that only anti-αv and anti-β3 function blocking antibodies suppressed VEGF-induced phosphorylation of VEGFR-2, whereas anti-β1 or anti-β5 blocking antibodies had no effect on VEGF-induced activation of VEGFR-2 (Figure 1C). Furthermore, treatment of HUVECs grown on gelatin-coated plates with VEGFR-2 inhibitor significantly reduced VEGF-induced β3 phosphorylation (Figure 1D). Taken together, these results demonstrate the cross-activation between the 2 receptors: αvβ3 ligation controls not only tyrosine phosphorylation of β3 but also of VEGFR-2, and VEGF stimulation promotes not only VEGFR-2 but also β3 tyrosine phosphorylation.
As VEGFR-2 activation and signaling seemed to be tightly associated with integrins, we extended our examination to the interaction of VEGFR-2 with other EC integrins. Results indicate that VEGF induced interaction of VEGFR-2 with β3 (Figure 1E). A small fraction of VEGFR-2 was also found to interact with β1 but not with β5 (supplemental Figure IB). Thus, of the 3 classes of integrins expressed on ECs, only αvβ3 forms a complex with VEGFR-2 after VEGF treatment.
VEGF Differentially Induces Interaction of Src Family Tyrosine Kinases With β3 Integrin in Endothelial Cells
The presence of Src family tyrosine kinases (SFKs) is critical for the phosphorylation of several intracellular signaling molecules following integrin-mediated cell adhesion and cell spreading.22 Therefore, we sought to analyze the degree and range of SFKs interactions with β3 and VEGFR-2 on VEGF stimulation. In resting ECs, only small amounts of c-Src associated with both VEGFR-2 and β3. VEGF stimulation dramatically enhanced interaction of c-Src with VEGFR-2 and β3. No basal level or VEGF-induced interaction was found between Fyn and β3 or VEGFR-2. A moderate amount of Yes was always associated with β3 and VEGFR-2, even after VEGF induction (Figure 2A and 2B). Therefore, we conclude that c-Src is the major tyrosine kinase associated with β3 following stimulation of cells with growth factor, possibly the kinase responsible for phosphorylation of β3 cytoplasmic tyrosines. To confirm these observations, kinetic analyses of β3 and c-Src tyrosine phosphorylation were performed. VEGF induced phosphorylation of β3 at 2.5 minutes, and it remained phosphorylated up to 30 minutes. Maximal β3 phosphorylation was observed between 5 and 15 minutes. VEGF also induced a similar activation phosphorylation of c-Src in ECs (Figure 2C), leading us to consider that activated c-Src might be responsible for β3 phosphorylation, which is crucial for activation of β3 integrin–dependent cellular signaling and EC functions.
Adhesion and Growth Factor–Induced β3 Integrin Tyrosine Phosphorylation Is Mediated Through c-Src
The cytoplasmic tyrosine motifs of β3 integrin known to regulate FAK phosphorylation, integrin-dependent actin cytoskeletal reorganization, the ability of integrins to localize to focal contacts, and cellular adhesion and spreading.16 Therefore, we examined the role of c-Src in adhesion- and growth factor–induced phosphorylation of β3 cytoplasmic tyrosine motifs. Accordingly, HUVECs were either kept in suspension (Figure 3A, lane 1) or plated on vitronectin (lane 2) and then treated with VEGF (lane 3) or SU6656, a specific c-Src inhibitor (lane 4). Cell lysates were analyzed for β3 integrin phosphorylation. As anticipated, in comparison with HUVECs kept in suspension, cells plated on vitronectin showed high levels of β3 phosphorylation at Tyr747 and Tyr759, which were further augmented by VEGF. Treatment of adherent ECs with SU6656 significantly reduced β3 phosphorylation. Furthermore, SU6656 also reduced basal adhesion-induced β3 tyrosine phosphorylation in these cells (supplemental Figure VIIB). As an independent approach, HUVECs were transfected with wild-type (WT), dominant negative (DN), and catalytically active (CA) forms of Src. Cells transfected with DN-Src showed severe impairment in adhesion as well as VEGF-induced β3 phosphorylation (lanes 5 and 6). In contrast, expression of CA-Src dramatically enhanced adhesion as well as VEGF-induced phosphorylation of β3 on both Tyr747 and Tyr759 (lanes 9 and 10). Transfection of HUVECs with WT c-Src did not significantly alter adhesion or VEGF-induced β3 phosphorylation (lanes 7 and 8).
To further substantiate the specific role of c-Src in β3 phosphorylation, we used cellular systems in which c-Src expression is highly regulated.22 As shown in Figure 3B, no β3 phosphorylation was observed in any of these cell types when either plated on uncoated plastic surfaces or kept in suspension (lanes 2 and 3). Attachment to vitronectin stimulated high levels of β3 phosphorylation in Src++ and SYF+c-Src cells but not in SYF cells (lane 4). Cells plated on laminin or collagen showed very low β3 phosphorylation (lanes 5 and 6). On growth factor stimulation, β3 phosphorylation was observed only in Src++ and SYF+ c-Src cells, not in SYF-cells (supplemental Figure IXA). Thus, c-Src controls cell adhesion as well as growth factor-induced β3 tyrosine phosphorylation.
c-Src Directly Phosphorylates Cytoplasmic Tyrosine Motifs of β3 Integrin
To examine whether c-Src can directly mediate β3 tyrosine phosphorylation, c-Src was immunoprecipitated from VEGF-stimulated HUVECs. Immunocomplexes were incubated with purified full-length β3 cytoplasmic domain, and [γ-32P] ATP incorporation was monitored. As shown in Figure 3C, the immunoprecipitated c-Src can phosphorylate β3 cytoplasmic domain (lane 2). The inhibitor SU6656 blocked this process, confirming the specificity of the reaction (lane 3). Recombinant protein tyrosine phosphatase also prevented phosphorylation, indicating that it is a tyrosine substrate that is being phosphorylated (lane 4). Recombinant purified c-Src also phosphorylated β3 whereas none was observed without the substrate, demonstrating specificity (lanes 1 and 5). Together, these results clearly show that c-Src can directly phosphorylate β3 cytoplasmic tyrosines.
To further investigate phosphorylation of Tyr747 and/or Tyr759, kinase assays were performed using as substrate peptides derived from these 2 distinct β3 sites (Figure 3D). SU6656, which blocks c-Src autophosphorylation, was used to demonstrate specificity. In the absence of integrin substrate, a significant amount of c-Src autophosphorylation was observed, indicating that the immunoprecipitated c-Src complex is active. In the presence of Tyr747/Tyr759 β3 cytoplasmic peptide, a dramatic increase in [γ-32P] ATP incorporation was observed compared with control peptides devoid of tyrosine motifs. Similar results were observed using purified recombinant c-Src protein. As anticipated, [γ-32P] ATP incorporation was minimal in the presence of purified recombinant protein tyrosine phosphatase, demonstrating the role of c-Src in phosphorylation of tyrosine residues. Together, these results clearly indicate that c-Src directly phosphorylates both tyrosine motifs (Tyr747 and Tyr759) of the cytoplasmic tail of β3. Furthermore, VEGF-stimulated Akt phosphorylation was maximum in HUVECs grown on vitronectin compared with other integrin ligands (supplemental Figure VIA and VIB). However, the phosphatidylinositol 3-kinase/Akt pathway was not involved in VEGF-induced β3 tyrosine phosphorylation (supplemental Figure VIC).
c-Src–Mediated β3 Integrin Tyrosine Phosphorylation Is Critical for VEGF- Induced VEGFR-2–β3 Integrin Macromolecular Complex Formation
Our previous results indicate that VEGF-induced VEGFR-2 tyrosine phosphorylation was maximal in cells plated on only αvβ3 ligand vitronectin and that VEGFR-2 shows maximum interaction with β3 in ECs. Therefore, we sought to examine the role of Src-mediated β3 phosphorylation in β3 interaction with VEGFR-2. Accordingly, c-Src activity was modified by overexpression of WT, DN, or CA forms of Src or by treatment of cells with Src and VEGFR-2 inhibitors; interaction between VEGFR-2 and β3 was then assessed. In unstimulated cells, no interaction between β3 and VEGFR-2 was observed (Figure 4A and 4B, lane 1), whereas VEGF induced strong interaction between VEGFR-2 and β3 (lane 2). Treatment of cells with pharmacological inhibitors of c-Src and VEGFR-2 known to inhibit β3 phosphorylation prevented VEGF-stimulated interaction between β3 and VEGFR-2 (lanes 3 and 4). Likewise, transfection with DN-Src reduced, whereas CA-Src enhanced, interaction between the 2 receptors (lanes 6 and 7). Furthermore, c-Src also partially regulated VEGFR-2 phosphorylation in ECs (supplemental Figure VIIA). These results indicate that the c-Src–mediated phosphorylation of β3 integrin essentially regulates interaction between β3 integrin and VEGFR-2 in ECs.
To further analyze the roles of β3 cytoplasmic tyrosine motifs in β3 integrin/VEGFR-2 interaction, we used lung ECs derived from β3 knock-in mice in which Tyr747 and Tyr759 were mutated to phenylalanine (DiYF). Supplemental Figure IIC shows that VEGF induced phosphorylation of β3 at Tyr747 and Tyr759 in WT but not in DiYF EC. To further assess the role of c-Src–mediated β3 phosphorylation, WT and DiYF ECs were transfected with DN or CA forms of Src and then interactions between VEGFR-2 and β3 were examined. VEGF-induced interaction between β3 and VEGFR-2 was observed only in WT cells (Figure 4C, lane 3), whereas in unstimulated cells, no interaction was found (lane 2). Transfection with DN-Src diminished, whereas CA-Src enhanced, the interaction between β3 and VEGFR-2 only in WT cells (lanes 4 and 5). Transfection with CA-Src also enhanced the interaction between VEGFR-2 and β3 in the absence of VEGF (supplemental Figure VIIIA). Under all of these conditions, no interactions were found between β3 integrin and VEGFR-2 in DiYF cells. These results clearly demonstrate that c-Src–mediated tyrosine phosphorylation of β3 integrin controls VEGF-induced β3 integrin and VEGFR-2 interaction in ECs.
c-Src Is Critical for Growth Factor–Induced β3 Integrin Activation and Ligand Binding
An intrinsic property of integrins is an increase in soluble ligand binding in response to stimulation, a process called integrin activation. Our results indicate that c-Src directly phosphorylates β3 on cytoplasmic tyrosines, which might affect integrin functional activity. To address this issue, HUVECs were transfected with WT, DN, and CA forms of c-Src and then stimulated with VEGF. Subsequently, αvβ3 activation was assessed by WOW-1 binding as described under Materials and Methods. As anticipated, VEGF induced a 6-fold increase in WOW-1 binding (Figure 4D). DN-Src reduced αvβ3 activation triggered by VEGF by at least 2-fold. In contrast, CA-Src promoted WOW-1 binding to unstimulated as well as to VEGF-stimulated cells (Figure 4D). Similar results were observed using fibrinogen as a soluble ligand for αvβ3 (Figure 4E). Furthermore, activation and ligand binding were also assessed using Src++, SYF, and SYF+Src cells. Basic fibroblast growth factor stimulation resulted in a dramatic increase in integrin activation in Src++ and SYF+Src but not in SYF cells, as measured by WOW-1 or fibrinogen binding (supplemental Figure IXB and IXD). No differences in β3 expression levels were found between these 3 cell lines (supplemental Figure IXC). Thus, c-Src and c-Src–dependent β3 cytoplasmic tyrosine phosphorylation are essential for VEGF-induced αvβ3 activation and ligand binding to activated integrin, both crucial steps in integrin signaling.
c-Src Is Required for αvβ3 Integrin–Dependent Cellular Adhesion to Distinct Ligand
To examine the role of c-Src in αvβ3-dependent cell adhesion to ECM ligands, we used Src++, SYF, and SYF+Src cell systems. Src++ and SYF+Src cells displayed the highest levels of adhesion to vitronectin, which is primarily recognized by αvβ3. SYF cells showed at least 3-fold lower adhesion on vitronectin compared with Src++ or SYF+Src cells (Figure 5A and 5B), although no differences in cell adhesion to collagen or laminin were observed. Adhesion of HUVECs on vitronectin was assessed on expression of WT, DN, and CA forms of Src or on treatment with inhibitor SU6656 (Figure 5C). CA-Src promoted αvβ3-dependent cell adhesion, whereas DN-Src and Src inhibitor caused impairment of adhesion to vitronectin. These results indicate that c-Src plays a crucial role in αvβ3-dependent cell adhesion to ECM ligands.
Our results also indicated that Src phosphorylates β3 on cytoplasmic tyrosines, which may be required for αvβ3-dependent ECM reorganization. To evaluate this, WT and DiYF mouse lung microvascular ECs were incubated on vitronectin or bone sialoprotein, αvβ3-specific ligands. DiYF ECs showed significantly impaired adhesion to vitronectin as well as bone sialoprotein (Figure 5D). No significant differences in adhesion were found between WT and DiYF ECs incubated on BSA-coated plates. These results clearly show that β3 cytoplasmic tyrosines and c-Src–mediated phosphorylation of these residues are essential for αvβ3-dependent cellular adhesion to distinct ECM ligands.
β3 Integrin Cytoplasmic Tyrosines Are Required for Directional Migration of Endothelial Cells
EC motility is the defining feature of angiogenesis, required for the organization of proliferating ECs into vessel-like structures. To assess the role of Src-mediated β3 tyrosine phosphorylation in αvβ3-dependent EC migration, HUVECs transfected with various forms (DN and CA) of Src were evaluated in migration assays using VEGF as an agonist and vitronectin as a substrate. DN-Src significantly reduced EC migration in response to VEGF, whereas CA-Src dramatically increased both basal and VEGF-induced EC migration (Figure 6A). Pharmacological inhibitors of c-Src and VEGFR-2 also reduced EC migration triggered by VEGF (supplemental Figure VIIIB). To further evaluate the c-Src–mediated β3 phosphorylation in αvβ3-dependent EC migration, WT and DiYF mouse lung microvascular ECs were transfected with WT, DN, and CA forms of c-Src and stimulated with VEGF (Figure 6B). VEGF stimulation induced increases in WT EC migration by 2.5-fold in WT-Src and 3.5-fold in CA-Src cells. DN-Src significantly tempered the VEGF-induced increase in migration in only WT ECs. Surprisingly, c-Src activity modulation did not result in any significant differences in migration of DiYF ECs. These results clearly indicate that c-Src mediates signaling through tyrosine phosphorylation of β3 integrin. Lack of these tyrosine residues in the cytoplasmic domain of β3 severely impaired c-Src–mediated αvβ3 integrin–dependent EC migration.
To examine the role of β3 cytoplasmic tyrosine motifs in directional migration of ECs, WT and DiYF ECs were cultured on collagen-, laminin-, or vitronectin-coated plates. Wounds were created, and VEGF-A165-stimulated cell migration was monitored by time-lapse video microscopy. Cell paths were recorded and are presented in Figure 6C. WT and DiYF ECs plated on laminin exhibited similar migration speeds, 53.2±4.8 and 47.2±4.2 μm/h, respectively. On collagen, WT and DiYF ECs also showed similar migration rates, of 43.2±3.5 and 44.5±3.3 μm/h, respectively. On vitronectin, WT and DiYF ECs showed relatively slow but similar migration at 30.8±3.2 and 28.3±2.7 μm/h, respectively. However, when random movement was distinguished from directed migration, the results became quite different. Despite the high speed of migration, the average distance of directed migration from site of origin was relatively low for WT cells on laminin and collagen, 71±6.2 and 92±7.2 μm respectively (Figure 6C). No differences between WT and DiYF cells were found. In contrast, WT ECs showed maximum directed migration when plated on vitronectin (229±6.6 μm). Importantly, directed migration by DiYF cells was dramatically impaired (77±5.4 μm versus 229±6.6 μm for WT). WT ECs plated on αvβ3 ligand vitronectin following induction with VEGF-A165 resulted in higher directional persistence of cell migration (supplemental Figure IVA). Simultaneously, DiYF ECs plated on vitronectin and induced with VEGF showed random movement with no distinct pattern. Furthermore, ECs stimulated with VEGF grown on vitronectin also exhibited greater rates of proliferation without significant differences in rates of apoptosis (supplemental Figures IIIA, IIIB, IVB, VA, VB). These results clearly demonstrate that β3 integrin cytoplasmic tyrosine motifs are required for persistent and directional migration of ECs during the process of angiogenesis.
β3 Integrin Cytoplasmic Tyrosine Phosphorylation Is Crucial for Organization of the Angiogenic Program in Endothelial Cells
To further evaluate the functional significance of c-Src–dependent β3 phosphorylation, the ability of ECs to organize into precapillary tube-like structures was tested. Accordingly, WT and DiYF mouse microvascular ECs were transfected with the various activation forms of Src. These cells were seeded on Matrigel-coated plates and allowed to organize into precapillary tube-like structures. Overexpression of DN-Src significantly reduced, whereas the CA form of Src dramatically enhanced, VEGF-induced tube formation on Matrigel in only WT cells (Figure 6D and 6E). Both VEGF and the varied forms of Src failed to modify the degree of tube formation in DiYF ECs. From all of these results, we conclude that c-Src–dependent β3 integrin cytoplasmic tyrosine phosphorylation is essential for αvβ3-dependent EC migration as well as precapillary endothelial tube formation on extracellular matrix substrates. Thus, β3 cytoplasmic tyrosine motifs are crucial for initiation of the angiogenic program in ECs and ultimately regulate the processes of angiogenesis.
This study focused on the mechanisms and molecular requirements for the crosstalk and cross-activation between 2 families of cell surface receptors on endothelium: integrins, receptors for extracellular matrix, and tyrosine kinase receptors, represented by VEGFR-2. The major findings in this manuscript are: (1) there is an intimate and coordinated relationship between VEGFR-2 and αvβ3 integrin; (2) adhesion- and growth factor–induced β3 integrin tyrosine phosphorylation is directly mediated by c-Src; (3) c-Src–dependent β3 integrin tyrosine phosphorylation is critical for interaction between VEGFR-2 and β3 integrin; and 4) c-Src is required for growth factor–induced β3 integrin activation, ligand binding, and αvβ3 integrin–dependent cellular adhesion. We have demonstrated that VEGF induces association of its receptor VEGFR-2 with the β3 subunit of αvβ3, but not with β1 or β5 integrins, on ECs. Blocking antibodies against either the αv or β3 subunits independently blocked VEGF-induced phosphorylation of β3 cytoplasmic tyrosines and VEGF-induced VEGFR-2 phosphorylation. We found that phosphorylation of tyrosine within the β3 cytoplasmic domain occurred in response to VEGF and, in turn, was essential for VEGFR-2–β3 integrin association and VEGFR-2 activation and subsequent signaling. Thus, crosstalk between the 2 receptors determines the cellular responses to VEGF as well as to integrin ligation, which, in turn, is regulated by tyrosine phosphorylation events.
We have identified c-Src as a molecule that directly phosphorylates the cytoplasmic tyrosines of β3 in response to VEGF stimulation, enabling it to directly control VEGF-induced and integrin-mediated cellular responses such as cell adhesion and migration. Src, Yes, and Fyn triple mutant cells (SYF) exhibited severely impaired β3 tyrosine phosphorylation in response to growth factors, which was corrected by reexpression of c-Src alone. Potentially, kinases other than c-Src may also contribute to VEGF-induced β3 phosphorylation in other cell lines. In ECs, Src, but neither Yes nor Fyn, was able to interact with VEGFR-2 and β3 in a VEGF-dependent manner. Recent observations have demonstrated that Src is required for VEGF-induced vascular permeability, a response triggered by VEGFR-2 activation.23 Moreover, c-Src is able to modulate blood vessel development in several experimental animal models.24 Here we have demonstrated that growth factor–stimulated ligand binding to αvβ3 on ECs is c-Src dependent. In triple knockout cells (SYF), the lack of Src activity resulted in deficient β3 phosphorylation, which, in turn, led to severe impairment in growth factor–induced ligand binding, a prerequisite for growth factor–modulated cell adhesion, spreading, and migration. Thus, we conclude that c-Src is crucial for integrin activation in response to growth factors and have also demonstrated the intimate association of Src and integrin signaling.25 In fact, several features of the Src knockout phenotype, including osteopetrosis, show a close resemblance to the phenotype of β3-null mice.26
The presence of the β3/Src complex at the leading edges of adherent cells further demonstrates their intimate association in basic cellular processes.27 Furthermore, mouse embryonic fibroblasts containing c-Src (Src++ and SYF+Src) showed a greater tendency to adhere to vitronectin-coated surfaces than to collagen- or laminin-coated surfaces. These results strengthen our conclusion that c-Src is required mainly for vitronectin receptor signaling (αvβ3) rather than collagen or laminin receptor signaling. Interestingly, only Src, not Fyn knockout, mice display impairment of VEGF-induced vascular responses and tumor burden, indicating that Src is the main SFK in the regulation of EC functions.28 Our study also demonstrates that c-Src–dependent β3 cytoplasmic tyrosine phosphorylation is essential for αvβ3 integrin–dependent EC migration as well as precapillary endothelial tube formation on ECM substrates. Thus c-Src, via direct phosphorylation of β3 integrin cytoplasmic tyrosines, controls the functional association between αvβ3 and VEGFR-2, which, in turn, regulates activation of both receptors on ECs. This functional interplay is crucial for EC adhesion, migration, and initiation of angiogenic programming in ECs.
Sources of Funding
We acknowledge financial support from NIH grants HL071625 and HL073311 (to T.V.B.) and American Heart Association Grant 0625271B (to G.H.M.).
Original received January 10, 2007; resubmission received May 8, 2007; revised resubmission received June 26, 2007; accepted July 11, 2007.
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