IQGAP1 Mediates VE-Cadherin–Based Cell–Cell Contacts and VEGF Signaling at Adherence Junctions Linked to Angiogenesis
Objective— Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating reactive oxygen species (ROS) production primarily through the VEGF receptor-2 (VEGFR2). One of the initial responses in established vessels to stimulate angiogenesis is loss of vascular endothelial (VE)-cadherin–based cell–cell adhesions; however, little is known about the underlying mechanisms. IQGAP1 is a novel VEGFR2 binding protein, and it interacts directly with actin, cadherin, and β-catenin, thereby regulating cell motility and morphogenesis.
Methods and Results— Confocal microscopy analysis shows that IQGAP1 colocalizes with VE-cadherin at cell–cell contacts in unstimulated human endothelial cells (ECs). VEGF stimulation reduces staining of IQGAP1 and VE-cadherin at the adherens junction without affecting interaction of these proteins. Knockdown of IQGAP1 using siRNA inhibits localization of VE-cadherin at cell–cell contacts, VEGF-stimulated recruitment of VEGFR2 to the VE-cadherin/β-catenin complex, ROS-dependent tyrosine phosphorylation of VE-cadherin, which is required for loss of cell–cell contacts and capillary tube formation. IQGAP1 expression is increased in a mouse hindlimb ischemia model of angiogenesis.
Conclusions— IQGAP1 is required for establishment of cell–cell contacts in quiescent ECs. To induce angiogenesis, it may function to link VEGFR2 to the VE-cadherin containing adherens junctions, thereby promoting VEGF-stimulated, ROS-dependent tyrosine phosphorylation of VE-cadherin and loss of cell–cell contacts.
- cell–cell adherions
- reactive oxygen species
- vascular endothelial growth factor
Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell (EC) migration and proliferation primarily through the VEGF type2 receptor (VEGFR2, KDR/Flk-1).1 VEGF binding initiates autophosphorylation of VEGFR2, which is followed by activation of diverse key angiogenic enzymes such as MAP kinases and Akt.1 One of the initial responses of quiescent ECs to induce angiogenesis is the loosening of cell–cell contacts, which is followed by migration of ECs to form capillary tube networks that become functional capillaries.2 The molecule primarily responsible for cell–cell adhesions of ECs is the transmembrane homophilic adhesion molecule, vascular endothelial (VE)-cadherin.3 The cytoplasmic domain of VE-cadherin binds to β-catenin, which in turn is linked to the actin cytoskeleton via α-catenin.3 This linkage between VE-cadherin–based adherens junctional complex and the actin cytoskeleton contributes to the strong adhesion. Furthermore, deletion or cytosolic truncation of VE-cadherin impairs remodeling and maturation of the vascular networks, and it inhibits VEGF-stimulated Akt phosphorylation induced by formation of a VEGFR2/VE-cadherin/β-catenin/phosphatidylinositol 3-kinase (PI3 kinase) complex.4 Tyrosine phosphorylation of the VE-cadherin complex is another mechanism that regulates the stability of cell–cell junctions,5–7 which is in part mediated through reactive oxygen species (ROS).8,9 We demonstrated that ROS derived from Rac1-dependent NAD(P)H oxidase play an important role in VEGF signaling and angiogenesis in ECs and in vivo.10,11 Thus, the VE-cadherin–based endothelial adherens junction is a potential site for initial activation of VEGFR2-mediated, ROS-dependent signaling linked to angiogenesis. However, underlying regulatory mechanisms are incompletely understood.
Using a yeast 2-hybrid system, we recently identified IQGAP1 as a novel VEGFR2 binding protein.12 IQGAP1 is a scaffold protein that interacts directly with actin, E-cadherin, β-catenin, active Rac1/Cdc42, calmodulin, and the microtubule plus end binding protein, CLIP-170,13–15 thereby regulating actin cytoskeleton, cell–cell adhesion, cellular motility and morphogenesis. IQGAP1 is a downstream effector of active Rac116,17 and acts as anti-GAP through a GAP-related domain, thereby increasing GTP-bound Rac1.16,18 In mouse fibroblasts, IQGAP1 localizes at sites of cell–cell contact and overexpression of IQGAP1 reduces E-cadherin-mediated cell–cell adhesion via interacting with β-catenin, thereby releasing α-catenin from the cadherin/catenin complex.19 Knockdown of IQGAP1 using siRNA reduces the accumulation of actin filaments, E-cadherin and β-catenin at sites of cell–cell contact in MDCKII cells.20 These results suggest negative and positive roles of IQGAP1 for cell–cell adhesions in epithelial cells. We previously demonstrated that IQGAP1 plays an essential role in VEGF-stimulated ROS production and VEGFR2-mediated EC migration and proliferation.12 However, a specific role of IQGAP1 in VE-cadherin-mediated cell–cell adhesions as well as VEGF-induced loss of cell–cell contacts linked to angiogenesis is unknown.
The present study demonstrates that IQGAP1 colocalizes and forms a complex with VE-cadherin at the site of cell–cell contacts in unstimulated confluent human umbilical vascular endothelial cells (HUVECs). VEGF stimulation reduces the staining of VE-cadherin and IQGAP1 at cell margins without affecting their complex formation. Using IQGAP1 siRNA, we found that IQGAP1 is required for establishment of VE-cadherin-based cell–cell contacts in quiescent ECs. We also suggest that IQGAP1 may function as a scaffold protein to link VEGFR2 to the VE-cadherin/β-catenin complex at the adherens junctions, thereby promoting VEGF-stimulated ROS-dependent tyrosine phosphorylation of VE-cadherin and its downstream Akt phosphorylation, which may contribute to angiogenesis.
Materials and Methods
Antibodies to VEGFR2, IQGAP1, phosphotyrosine, VE-cadherin, α-catenin, β-catenin, Akt, and α-tubulin were from Santa Cruz. Anti-phospho-Akt antibody was from Cell Signaling. Human recombinant VEGF165 was from R&D Systems and BRB Preclinical Repository. Oligofectamine was from Invitrogen Corp. Carboxy-H2–2′,7′-dichlorofluorescein diacetate (DCF-DA) was from Molecular Probes. All other chemicals and reagents were from Sigma.
Cell culture, measurement of intracellular H2O2 levels, confocal immunofluorescence microscopy, synthetic siRNA and its transfection, immunoprecipitation and immunoblotting, tube formation assay in 3-dimensional type I collagen gels, mouse ischemic hindlimb model and histological analysis, and statistical analyses are described in the Material and Methods section in the online data supplement (see http://atvb.ahajournals.org).
Subcellular Localization of IQGAP1 and VE-Cadherin and Their Association in Confluent Monolayers of HUVECs Before and After VEGF Stimulation
To induce angiogenesis, cell–cell contact has to be disrupted by reducing VE-cadherin from the cell–cell adhesion site. We thus examined the subcellular localization of IQGAP1 and VE-cadherin before and after VEGF stimulation in confluent monolayers of HUVECs using confocal microscopy. As shown in Figure 1, in unstimulated ECs, IQGAP1 was mainly found at cell–cell contacts, where it colocalized with VE-cadherin and was diffusely distributed within cytosol. VEGF stimulation for 1 hour reduced the staining of both VE-cadherin and IQGAP1 at cell–cell contacts, consistent with the loss of cell–cell adhesions, whereas it increased the staining of IQGAP1, but not VE-cadherin, at the perinucleus area. Co-immunoprecipitation analysis revealed that IQGAP1 physically associates with VE-cadherin in the basal state. This association was slightly enhanced at 10 minutes after VEGF stimulation, and continued at least for 1 hour (Figure 1B; Figure IA, available online at http://atvb.ahajournals.org). This suggests that IQGAP1 remains in complex with VE-cadherin even when their staining at cell–cell contact is decreased. Western analysis of Triton-soluble and insoluble fractions as well as plasma membrane and cytosolic fractions further confirmed that the protein levels of both IQGAP1 and VE-cadherin remained unchanged during VEGF stimulation (Figure IB and IC).
IQGAP1 Is Required for Localization of VE-Cadherin at Cell–Cell Contacts
To examine the role of IQGAP1 in localization of VE-cadherin at adherence junctions in confluent monolayers of ECs, HUVECs were transfected with IQGAP1 siRNA. As shown in Figure 2, IQGAP1 siRNA, but not scrambled siRNA, almost completely knocked down IQGAP1 protein without affecting VE-cadherin protein expression. Moreover, IQGAP1 siRNA had no effect on expression of IQGAP2 or β-catenin protein (data not shown), further confirming the specificity of IQGAP1 siRNA. IQGAP1 siRNA markedly reduced VE-cadherin staining at sites of cell–cell contact, resulting in small gaps between adjacent cells in basal and VEGF-stimulated ECs. These results suggest that IQGAP1 is required for proper localization of VE-cadherin at the adherens junctions and for VE-cadherin-mediated cell–cell adhesions in ECs.
IQGAP1 Is Required for VEGF-Induced Association of VEGFR2 With VE-Cadherin/β-Catenin Complex
VEGF stimulation promotes formation of VEGFR2/VE-cadherin/β-catenin complex, but their interaction is not direct.4 We previously demonstrated that VEGF induces direct interaction of VEGFR2 with IQGAP1 in HUVECs. Because IQGAP1 directly binds to E-cadherin and β-catenin,19 we examined whether IQGAP1 is involved in VEGF-induced formation of VEGFR2/VE-cadherin/β-catenin complex in ECs. As shown in Figure 3, VE-cadherin was co-immunoprecipitated with α-catenin and β-catenin in unstimulated confluent HUVECs. VEGF stimulation rapidly promoted recruitment of VEGFR2 to and dissociation of α-catenin from the VE-cadherin/β-catenin complex, which was significantly inhibited by IQGAP1 siRNA. These results suggest that IQGAP1 may function as a scaffold to link VEGFR2 to the adherens junctions through binding to VEGFR2 and VE-cadherin/β-catenin complex, thereby dissociating α-catenin from the adherens junctional complex, and contributing to VEGF-stimulated loss of cell–cell contacts in ECs.
IQGAP1 Is Required for ROS-Dependent Tyrosine Phosphorylation of VE-Cadherin
Because tyrosine phosphorylation of VE-cadherin is required for VEGF-induced dissociation of cell–cell contacts in ECs,6,9,21 we examined whether IQGAP1 is involved in this response. As shown in Figure 4A, VEGF stimulation induced a significant increase in tyrosine phosphorylation of VE-cadherin and IQGAP1 within 5 minutes. These increases were significantly inhibited by IQGAP1 siRNA. Basal phosphorylation of VE-cadherin was rather enhanced in IQGAP1 siRNA-transfected cells, presumably because of the reduction of VE-cadherin–mediated cell–cell adhesions induced by IQGAP1 depletion (Figure 2). Under this condition, IQGAP1 siRNA significantly inhibited VEGF-stimulated Akt phosphorylation (Figure 4B), which is a downstream response of formation of the VEGFR2/VE-cadherin/β-catenin complex.4 These results suggest that IQGAP1-mediated formation of VEGFR2/VE-cadherin/β-catenin complex (Figure 3) may be involved in VEGF-induced tyrosine phosphorylation of VE-cadherin as well as Akt phosphorylation in ECs. Because we found previously that IQGAP1 is involved in VEGF-induced increase in ROS production,12 we examined the role of ROS in phosphorylation of VE-cadherin by VEGF. IQGAP1 siRNA inhibited VEGF-stimulated ROS production (Figure 4C), and VEGF-stimulated tyrosine phosphorylation of VE-cadherin was significantly inhibited by H2O2 scavenger, polyethylene glycol (PEG)-catalase, and thiol antioxidant, NAC (Figure 4D). PEG only had no effects (data not shown). These results suggest that IQGAP1-mediated rapid increase in ROS may participate in the initial activation of VEGF signaling at the VE-cadherin–based adherens junction in ECs.
IQGAP1 Is Involved in VEGF-Stimulated Tube Formation in Type I Collagen 3-Dimensional Culture of ECs
Loss of cell–cell contacts in confluent monolayers of ECs triggers EC migration to form capillary vascular networks during angiogenesis.2,6 To assess the functional role of IQGAP1 in VEGF-induced angiogenesis in vitro, we examined whether IQGAP1 is involved in capillary tube formation using 3-dimensional culture of HUVECs in type I collagen gels. As shown in Figure 5, at 24 hours after overlaying the second collagen gel containing VEGF on confluent monolayer of HUVECs seeded on top of the collagen-coated wells, capillary tube-like structures were formed in scrambled siRNA-transfected cells. Thus, this model is valuable to study the initial mechanical events of angiogenesis. In contrast, VEGF-induced tube formation was not observed in IQGAP1 siRNA-transfected cells, suggesting that IQGAP1 plays an important role in angiogenesis in vitro.
Induction of IQGAP1 Protein Expression in Mouse Ischemic Hindlimb Model of Angiogenesis
To gain further insight into the role of IQGAP1 in angiogenesis in vivo, we examined the expression of IQGAP1 in a mouse hindlimb ischemia model in which angiogenesis is dependent at least in part on VEGF,22 VEGFR2,23 and NAD(P)H oxidase-derived ROS.11 Figure 6A using LDBF analysis demonstrates that hindlimb blood flow recovery was markedly decreased immediately after femoral artery ligation (day 0) and recovered to the level of that of the nonischemic limb by day 7. Western analysis also shows that IQGAP1 protein expression was significantly increased in the ischemic hindlimb tissues at 7 days after operation (Figure 6B) in association with the increase in VEGF expression (data not shown) compared with that in nonischemic sites. Immunocytochemical analysis of double staining for IQGAP1 and lectin showed that IQGAP1 protein and lectin-positive capillary ECs were dramatically increased and colocalized in the ischemic hindlimbs at 7 days after femoral ligation (Figure 6C). Similarly, VE-cadherin expression was increased in ischemic hindlimbs and partially colocalized with IQGAP1 (Figure III). These data indicate that IQGAP1 may be involved in the process by which new blood vessels are formed in vivo.
Understanding the factors that regulate endothelial cell–cell junctions is important for many pathophysiological processes in which functional vascular integrity is compromised, such as development of neovasculature during angiogenesis and chronic inflammatory disorders. The present study shows that IQGAP1 colocalizes and forms a complex with VE-cadherin at the site of cell–cell contacts in unstimulated confluent HUVECs, and VEGF stimulation reduces their localization at the cell margin without affecting their complex formation. Knockdown of IQGAP1 using siRNA inhibits localization of VE-cadherin at cell–cell contacts as well as the following VEGF-stimulated events: (1) recruitment of VEGF2 to and the dissociation of α-catenin from the VE-cadherin/β-catenin complex; (2) ROS-dependent tyrosine phosphorylation of VE-cadherin, which is required for loss of cell–cell contacts8,9; and (3) capillary tube formation in 3-dimensional collagen gels. We also found that IQGAP1 expression is markedly increased in the mouse hindlimb ischemia model of angiogenesis.
We previously demonstrated that IQGAP1 plays an essential role in both VEGF-induced and wound injury-induced EC migration.12,24 One of the initial responses to stimulate EC migration is the loosening of stable cell–cell contacts between ECs, and the molecule primarily responsible for cell–cell adhesions of ECs is the VE-cadherin. Recent studies reveal that IQGAP1 regulates E cadherin-mediated cell–cell adhesion both positively and negatively in epithelial cells.15 However, its role in VE-cadherin-mediated cell–cell adhesion in ECs is unknown. Using confocal microscopy and co-immunoprecipitation assays, here we show that IQGAP1 colocalizes and associates with VE-cadherin at the sites of cell–cell contacts in confluent monolayers of ECs. Of note, IQGAP1, but not VE-cadherin, is also found in the cytosol in unstimulated HUVECs (Figure 1 and Figure I). VEGF stimulation reduces IQGAP1 and VE-cadherin staining at the adherens junction, whereas it increased the staining of IQGAP1, but not VE-cadherin, at the perinucleus area without changing their complex formation and protein expression. Thus, it is likely that perinuclear IQGAP1 protein which is increased after VEGF stimulation may be translocated mainly from the cytosol where VE-cadherin–unbound IQGAP1 localizes in basal state. Similar results are obtained for β-catenin (unpublished observation). Our findings are consistent with the previous reports that both VE-cadherin and β-catenin are dissociated from adherens junctions as a complex without changing their protein expression in HUVEC monolayers in response to VEGF, thrombin, H2O2, and fluid shear stress.5,25–27 This may be because of the possibility that VE-cadherin staining using confocal microscopy in Triton X-permeabilized and fixed cultured confluent ECs reflects the disruption of cell–cell adhesion, which may reduce the accessibility to the VE-cadherin antibody presumably caused by conformational change and/or phosphorylation of VE-cadherin induced by VEGF stimulation. Because Western analysis was performed in denatured condition, these factors might not be reflected. Moreover, knockdown of IQGAP1 using siRNA inhibits localization of VE-cadherin at cell–cell contacts but causes its mislocalization to the perinucleus area before and after VEGF stimulation, thereby reducing cell–cell adhesions. Consistent with our result, Noritake et al20 reported that IQGAP1 siRNA reduces the accumulation of E-cadherin and β-catenin at cell–cell contacts in MDCKII cells. These results suggest that IQGAP1 is required for proper localization of VE-cadherin at cell–cell contacts, and for establishment of VE-cadherin-mediated cell–cell adhesions in ECs.
It has been shown that VEGFR2 associates with VE-cadherin/β-catenin complex after VEGF stimulation to activate VE-cadherin–dependent signaling including Akt in ECs.4 However, interaction between VEGFR2 and VE-cadherin/β-catenin is not direct. In the present study, we show that IQGAP1 siRNA inhibits VEGF-induced recruitment of VEGFR2 to as well as dissociation of α-catenin from the VE-cadherin/β-catenin complex. Because IQGAP1 directly binds to activated VEGFR212 as well as to E-cadherin and β-catenin,19 these results suggest that IQGAP1 may function to link VEGFR2 to the adherens junctions through binding to VE-cadherin/β-catenin complex, thereby dissociating α-catenin from the adherens junctional complex, which in turn results in loss of cell–cell adhesions. Given that IQGAP1 siRNA inhibits VEGF-stimulated activation of Akt, and that disrupting stable cell–cell contacts is required to stimulate EC migration, it is conceivable that IQGAP1-dependent formation of the VEGFR2/VE-cadherin/β-catenin complex at the adherens junction is necessary for downstream activation of VEGFR2-mediated signaling linked to angiogenesis. It is important to characterize the interacting domains of IQGAP1 with its binding partners in future study.
Tyrosine phosphorylation of VE-cadherin is also critical for the loosening of cell–cell contacts in ECs.5–7 We demonstrate here that IQGAP1 siRNA significantly inhibits VEGF-stimulated tyrosine phosphorylation of VE-cadherin, whereas its response in basal state is rather enhanced presumably caused by the reduction of VE-cadherin-mediated cell–cell adhesions. Recently, VEGF-induced or Rac1-induced ROS have been shown to be involved in VE-cadherin tyrosine phosphorylation and loss of cell–cell contacts in ECs.8,9 We previously demonstrated that VEGF induces a rapid increase in ROS production via activation of Rac1-dependent NAD(P)H oxidase in ECs,10 which is mediated through IQGAP1.12 In line with these findings, the present study confirmed that IQGAP1 siRNA inhibits VEGF-stimulated increase in ROS production and that ROS inhibitors block tyrosine phosphorylation of VE-cadherin by VEGF. The mechanisms by which ROS mediate VE-cadherin phosphorylation remain unclear. Accumulating evidence suggests that ROS mediate the oxidation of critical cysteine residues in protein tyrosine phosphatases, thereby deactivating these enzymes, which results in increased tyrosine kinase activity.28–30 Although it is not known which protein tyrosine phosphatases may be involved, it is intriguing to note that SHP-2 has been shown to associate with VE-cadherin complex after thrombin stimulation in ECs31 and that VEGF stimulation promotes SHP-2 binding to IQGAP1/VEGFR2/VE-cadherin/β-catenin complex (authors’ unpublished observations). The precise underlying mechanism requires further investigation. Our present results are consistent with the possibility that VEGF-induced, IQGAP1-mediated formation of VEGFR2/VE-cadherin/β-catenin complex is important for ROS-dependent tyrosine phosphorylation of VE-cadherin at the adherens junction, thereby facilitating loss of cell–cell contacts.
Because loss of cell–cell adhesions is an initial key event for angiogenesis, we assessed the functional role of IQGAP1 in VEGF-induced angiogenesis using in vitro and in vivo models. Using 3-dimensional cultures in type I collagen gels, an in vitro model of angiogenesis,6 we demonstrate that VEGF-stimulated capillary-like tube formation is almost completely blocked in IQGAP1 siRNA-transfected HUVECs. We have shown that IQGAP1 is involved in VEGF-stimulated EC proliferation and migration,12 which may explain in part the mechanisms by which IQGAP1 regulates capillary tube formation in ECs. However, we cannot exclude the possibility that HUVEC data obtained in the present study may not be relevant to microvascular angiogenesis.
Study using mouse hindlimb ischemia model reveals that IQGAP1 is markedly increased in lectin-positive, newly formed capillary ECs and partially colocalizes with VE-cadherin in hindlimb tissues after ischemia. Because IQGAP1 is involved in VEGF-stimulated ROS production, loss of cell–cell contacts, EC migration, and proliferation as well as capillary tube formation in cultured ECs, the functional consequence of upregulation of IQGAP1 in neovasculature is consistent with the possibility that IQGAP1 may play an important role in postnatal angiogenesis, which is dependent on VEGF, VEGFR2, and NAD(P)H oxidase-derived ROS.11,22,23 The definitive role of IQGAP1 in ischemia-induced angiogenesis will require further investigation using IQGAP1−/− mice.
In summary, IQGAP1 plays an important role in establishment of VE-cadherin–based cell–cell contacts in quiescent ECs. It may also function as a scaffold protein to link VEGFR2 to the VE-cadherin/β-catenin complex at the adherens junctions, thereby promoting ROS-dependent tyrosine phosphorylation of VE-cadherin and loss of cell–cell contacts, which may contribute to postnatal angiogenesis. These findings suggest an essential role of IQGAP1 in organization of signaling at endothelial adherens junction and provide novel insight into IQGAP1 as an attractive therapeutic target for modulating development of neovasculature during angiogenesis.
Sources of Funding
This work was supported by National Institutes of Health grant HL077524, an AHA grant-in-aid 0555308B, and an America Heart Association National Scientist Development Grant 0130175N (to M.U.-F.), and America Heart Association Postdoctoral Fellowship 0425460B (to M.Y.-T.).
Original received August 3, 2005; final version accepted May 24, 2006.
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