Homeostatic modulation of cell surface KDR and Flt1 expression and expression of the vascular endothelial cell growth factor (VEGF) receptor mRNAs by VEGF

Vascular endothelial cell growth factor (VEGF) is a potent angiogenic factor expressed during embryonic development, during wound healing, and in pathologies dependent on neovascularization, including cancer. Regulation of the receptor tyrosine kinases, KDR and Flt-1, to which VEGF binds on endothelial cells is incompletely understood. Chronic incubation with tumor-conditioned medium or VEGF diminished (125)I-VEGF binding to human umbilical vein endothelial cells, incorporation of (125)I-VEGF into covalent complexes with KDR and Flt1, and immunoreactive KDR in cell lysates. Receptor down-regulation desensitized VEGF activation of mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) and p38 mitogen-activated protein kinase. Preincubation with VEGF or tumor-conditioned medium down-regulated cell surface receptor expression but up-regulated KDR and Flt-1 mRNAs, an effect abrogated by a neutralizing VEGF antibody. Removal of VEGF from the medium led to recovery of (125)I-VEGF binding and resensitization of human umbilical vein endothelial cells. Recovery of receptor expression was inhibited by cycloheximide, indicating that augmented VEGF receptor mRNAs, and not receptor recycling from a cytoplasmic pool, restored responsiveness. As the VEGF receptors promote endothelial cell survival, proliferation, and other events necessary for angiogenesis, the noncoordinate regulation of VEGF receptor proteins and mRNAs suggests that human umbilical vein endothelial cells are protected against inappropriate or prolonged loss of VEGF receptors by a homeostatic mechanism important to endothelial cell function.     

Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation

This study was initiated to identify signaling proteins used by the receptors for vascular endothelial cell growth factor KDR/Flk1, and Flt1. Two-hybrid cloning and immunoprecipitation from human umbilical vein endothelial cells (HUVEC) showed that KDR binds to and promotes the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Neither placental growth factor, which activates Flt1, epidermal growth factor (EGF), or fibroblast growth factor (FGF) induced tyrosine phosphorylation of PLCgamma, indicating that KDR is uniquely important to PLCgamma activation in HUVEC. By signaling through KDR, VEGF promoted the tyrosine phosphorylation of focal adhesion kinase, induced activation of Akt, protein kinase Cepsilon (PKCepsilon), mitogen-activated protein kinase (MAPK), and promoted thymidine incorporation into DNA. VEGF activates PLCgamma, PKCepsilon, and phosphatidylinositol 3-kinase independently of one another. MEK, PLCgamma, and to a lesser extent PKC, are in the pathway through which KDR activates MAPK. PLCgamma or PKC inhibitors did not affect FGF- or EGF-mediated MAPK activation. MAPK/ERK kinase inhibition diminished VEGF-, FGF-, and EGF-promoted thymidine incorporation into DNA. However, blockade of PKC diminished thymidine incorporation into DNA induced by VEGF but not FGF or EGF. Signaling through KDR/Flk1 activates signaling pathways not utilized by other mitogens to induce proliferation of HUVEC.     

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.     

Rapid transactivation of the vascular endothelial growth factor receptor KDR/Flk-1 by the bradykinin B2 receptor contributes to endothelial nitric-oxide synthase activation in cardiac capillary endothelial cells.

Bradykinin (BK) and vascular endothelial growth factor (VEGF)-165 stimulate vasodilatation, microvascular permeability, and angiogenesis via the activation of the B2-type and KDR/Flk-1 receptors. To delineate the signal transduction pathways distal to the receptor activation in microvascular permeability, we compared their effects on two downstream targets, i.e. endothelial nitric-oxide (NO) synthase (eNOS) and F-actin, in primary cultures of cardiac capillary endothelial cells. The two mediators induced a similar cytoskeletal reorganization and both the translocation and activation of eNOS, leading to NO release within the first minutes of cell exposure. At the same time, BK produced the tyrosine phosphorylation and internalization of KDR/Flk-1 as did VEGF itself. This transactivation was blocked by the selective inhibitor of VEGF receptor tyrosine kinase activity but not by inhibitors of epidermal growth factor receptor or protein kinase C activity. The selective inhibitor of VEGF receptor tyrosine kinase activity totally prevented the effects of VEGF but only partially inhibited NO release induced by BK without affecting the concomitant cytoskeletal reorganization. Thus, BK transactivated KDR/Flk-1 through an intrinsic kinase activity of KDR/Flk-1, resulting in a further eNOS activation in endothelial cells. This represents a novel mechanism whereby a G protein-coupled receptor activates a receptor tyrosine kinase to generate biological response.     

Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.

Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor, is an endothelial cell mitogen which stimulates angiogenesis. Here we report that a previously identified receptor tyrosine kinase gene, KDR, encodes a receptor for VEGF. Expression of KDR in CMT-3 (cells which do not contain receptors for VEGF) allows for saturable 125I-VEGF binding with high affinity (KD = 75 pM). Affinity cross-linking of 125I-VEGF to KDR-transfected CMT-3 cells results in specific labeling of two proteins of M(r) = 195 and 235 kDa. The KDR receptor tyrosine kinase shares structural similarities with a recently reported receptor for VEGF, flt, in a manner reminiscent of the similarities between the alpha and beta forms of the PDGF receptors.     

Differential expression of the two VEGF receptors flt and KDR in placenta and vascular endothelial cells

Vascular endothelial growth factor (VEGF) is a newly identified growth and permeability factor with a unique specificity for endothelial cells. Recently the flt-encoded tyrosine kinase was characterized as a receptor for VEGF. A novel tyrosine kinase receptor encoded by the KDR gene was also found to bind VEGF with high affinity when expressed in CMT-3 cells. Screening for flt and KDR expression in a variety of species and tissue-derived endothelial cells demonstrates that flt is predominantly expressed in human placenta and human vascular endothelial cells. Placenta growth factor (PIGF), a growth factor significantly related to VEGF, is coexpressed with flt in placenta and human vascular endothelial cells. KDR is more widely distributed and expressed in all vessel-derived endothelial cells. These data demonstrate that cultured human endothelial cells isolated from different tissues express both VEGF receptors in relative high levels and, additionally, that all investigated nonhuman endothelial cells in culture are also positive for KDR gene expression.     

Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants

Endothelial cells express two related vascular endothelial growth factor (VEGF) receptor tyrosine kinases, KDR (kinase-insert domain containing receptor, or VEGFR-2) and Flt-1 (fms-like tyrosine kinase, or VEGFR-1). Although considerable experimental evidence links KDR activation to endothelial cell mitogenesis, there is still significant uncertainty concerning the role of individual VEGF receptors for other biological effects such as vascular permeability. VEGF mutants that bind to either KDR or Flt-1 with high selectivity were used to determine which of the two receptors serves to mediate different VEGF functions. In addition to mediating mitogenic signaling, selective KDR activation was sufficient for the activation of intracellular signaling pathways implicated in cell migration. KDR stimulation caused tyrosine phosphorylation of both phosphatidylinositol 3-kinase and phospholipase Cgamma in primary endothelial cells and stimulated cell migration. KDR-selective VEGF was also able to induce angiogenesis in the rat cornea to an extent indistinguishable from wild type VEGF. We also demonstrate that KDR, but not Flt-1, stimulation is responsible for the induction of vascular permeability by VEGF.     

Tyrosine residues 951 and 1059 of vascular endothelial growth factor receptor-2 (KDR) are essential for vascular permeability factor/vascular endothelial growth factor-induced endothelium migration and proliferation, respectively.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) exerts its multiple functions by activating two receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR (VEGFR-2), both of which are selectively expressed on primary vascular endothelium. To dissect the respective signaling pathways and biological functions mediated by these receptors in primary endothelial cells with two receptors intact, we, recently developed chimeric receptors (EGDR and EGLT) in which the extracellular domain of the epidermal growth factor receptor was fused to the transmembrane domain and intracellular domain of KDR and Flt-1, respectively. With these fusion receptors, we have shown that KDR is solely responsible for VPF/VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration, whereas Flt-1 showed an inhibitory effect on KDR-mediated proliferation but not migration. To further characterize the VPF/VEGF-stimulated HUVEC proliferation and migration here, we have created several EGDR mutants by site-directed mutagenesis. We show that tyrosine residues 1059 and 951 of KDR are essential for VPF/VEGF-induced HUVEC proliferation and migration, respectively. Furthermore, the mutation of tyrosine 1059 to phenylanaline results in the complete loss of KDR/EGDR-mediated intracellular Ca(2+) mobilization and MAPK phosphorylation, but the mutation of tyrosine 951 to phenylanaline did not affect these events. Our results suggest that KDR mediates different signaling pathways for HUVEC proliferation and migration and, moreover, intracellular Ca(2+) mobilization and MAPK phosphorylation are not essential for VPF/VEGF-induced HUVEC migration.     

Snake venom vascular endothelial growth factors (VEGFs) exhibit potent activity through their specific recognition of KDR (VEGF receptor 2)

Vascular endothelial growth factor (VEGF165) exhibits multiple effects via the activation of two distinct endothelial receptor tyrosine kinases: Flt-1 (fms-like tyrosine kinase-1) and KDR (kinase insert domain-containing receptor). KDR shows strong ligand-dependent tyrosine phosphorylation in comparison with Flt-1 and mainly mediates the mitogenic, angiogenic, and permeability-enhancing effects of VEGF165. Here we show the isolation of two VEGFs from viper venoms and the characterization of their unique biological properties. Snake venom VEGFs strongly stimulated proliferation of vascular endothelial cells in vitro. Interestingly, the maximum activities were almost twice that of VEGF165. They also induced strong hypotension on rat arterial blood pressure compared with VEGF165 in vivo. A receptor binding assay revealed that snake venom VEGFs bound to KDR-IgG with high affinity (Kd = approximately 0.1 nm) as well as to VEGF165 but did not interact with Flt-1, Flt-4, or neuropilin-1 at all. Our data clearly indicate that snake venom VEGFs act through the specific activation of KDR and show potent effects. Snake venom VEGFs are a highly specific ligand to KDR and form a new group of the VEGF family.     

Stretch induces upregulation of key tyrosine kinase receptors in microvascular endothelial cells

We previously demonstrated that cyclic stretch of cardiac myocytes activates paracrine signaling via vascular endothelial growth factor (VEGF) leading to angiogenesis. The present study tested the hypothesis that cyclic stretch upregulates tyrosine kinase receptors in rat coronary microvascular endothelial cells (RCMEC) and human umbilical vein endothelial cells (HUVEC). VEGF receptor-2 (Flk-1) protein levels increased in HUVEC and RCMEC in a time-dependent manner, but the increase occurred much earlier in RCMEC than in HUVEC. The enhancement of Flk-1 protein level was not inhibited by addition of VEGF neutralizing antibodies, indicating that VEGF is not involved in stretch-induced Flk-1 expression. VEGF receptor-1 (Flt-1) protein and mRNA were not changed by stretch. However, Tie-2 and Tie-1 protein levels increased in RCMEC. Angiopoietin-1 and -2, the ligands for Tie-2, increased in cardiac myocytes subjected to cyclic stretch but were not affected by stretch in endothelial cells (EC). Stretch or incubation of RCMEC with VEGF increased cell proliferation moderately, whereas stretch + VEGF had an additive effect on proliferation. Mechanical stretch induces upregulation of the key tyrosine kinase receptors Flk-1, Tie-2, and Tie-1 in vascular EC, which underlies the increase in sensitivity of EC to growth factors and, therefore, facilitates angiogenesis. These in vitro findings support the concept that stretch of cardiac myocytes and EC plays a key role in coronary angiogenesis.     

A fusion fragment from Flt-1 and KDR,acted as VEGF decoy receptor and exhibited anti-tumor function

Angiogenesis is important in tumor development. Vascular endothelial growth factor (VEGF) is involved in this process. In this report, we constructed a recombinant protein (called FK) by fusing the second immunoglobulin-like (Ig-like) domain of a human fms-like tyrosine kinase (Flt-1) with the third Ig-like domain of human kinase insert domain-containing receptor (KDR). FK bound to VEGF₁₆₅ in a dose-dependent manner with a disocciation constant (Kd) of 2.7 pM. In addition, FK specifically inhibited the proliferation of human microvascular endothelial cell (HMEC) and human umbilical vein endothelial Cell (HUVEC) stimulated by VEGF₁₆₅. Subsequent studies also demonstrate that FK efficaciously suppresses growth of a variety of tumors, which could make FK a potential drug candidate in anti-tumor therapy.     

Kinase insert domain receptor (KDR) extracellular immunoglobulin-like domains 4-7 contain structural features that block receptor dimerization and vascular endothelial growth factor-induced signaling

The vascular endothelial growth factor (VEGF) receptor tyrosine kinase subtype kinase insert domain receptor (KDR) contains seven extracellular Ig-like domains, of which the three most amino-terminal contain the necessary structural features required for VEGF binding. To clarify the functional role of KDR Ig-like domains 4-7, we compared VEGF-induced signaling in human embryonic kidney and porcine aortic endothelial cells expressing native versus mutant receptor proteins in which Ig-like domains 4-7, 4-6, or 7 had been deleted. Western blotting using an anti-receptor antibody indicated equivalent expression levels for each of the recombinant proteins. As expected, VEGF treatment robustly augmented native receptor autophosphorylation. In contrast, receptor autophosphorylation, as well as downstream signaling events, were VEGF-independent for cells expressing mutant receptors. (125)I-VEGF(165) bound with equal or better affinity to mutant versus native receptor, although the number of radioligand binding sites was significantly reduced because a significant percentage of mutant, but not native, receptors were localized to the cell interior. As was the case for native KDR, (125)I-VEGF(165) binding to the mutant receptors was dependent upon cell surface heparan sulfate proteoglycans, and (125)I-VEGF(121) bound with an affinity equal to that of (125)I-VEGF(165) to the native and mutant receptors. It is concluded that KDR Ig-like domains 4-7 contain structural features that inhibit receptor signaling by a mechanism that is independent of neuropilin-1 and heparan sulfate proteoglycans. We speculate that this provides a cellular mechanism for blocking unwanted signaling events in the absence of VEGF.     

Vascular endothelial growth factor governs endothelial nitric-oxide synthase expression via a KDR/Flk-1 receptor and a protein kinase C signaling pathway.

The mechanism by which vascular endothelial growth factor (VEGF) regulates endothelial nitric-oxide synthase (eNOS) expression is presently unclear. Here we report that VEGF treatment of bovine adrenal cortex endothelial cells resulted in a 5-fold increase in both eNOS protein and activity. Endothelial NOS expression was maximal following 2 days of constant VEGF exposure (500 pM) and declined to base-line levels by day 5. The elevated eNOS protein level was sustained over the time course if VEGF was co-incubated with L-N(G)-nitroarginine methyl ester, a competitive eNOS inhibitor. Addition of S-nitroso-N-acetylpenicillamine, a nitric oxide donor, prevented VEGF-induced eNOS up-regulation. These data suggest that nitric oxide participates in a negative feedback mechanism regulating eNOS expression. Various approaches were used to investigate the role of the two high affinity VEGF receptors in eNOS up-regulation. A KDR receptor-selective mutant increased eNOS expression, whereas an Flt-1 receptor-selective mutant did not. Furthermore, VEGF treatment increased eNOS expression in a KDR but not in an Flt-1 receptor-transfected porcine aorta endothelial cell line. SU1498, a selective inhibitor of the KDR receptor tyrosine kinase, blocked eNOS up-regulation, thus providing further evidence that the KDR receptor signals for eNOS up-regulation. Finally, treatment of adrenal cortex endothelial cells with VEGF or phorbol ester resulted in protein kinase C activation and elevated eNOS expression, whereas inhibition of protein kinase C with isoform-specific inhibitors abolished VEGF-induced eNOS up-regulation. Taken together, these data demonstrate that VEGF increases eNOS expression via activation of the KDR receptor tyrosine kinase and a downstream protein kinase C signaling pathway.     

Receptor-selective variants of human vascular endothelial growth factor. Generation and characterization

Vascular endothelial growth factor (VEGF) is a pleiotropic factor that exerts a multitude of biological effects through its interaction with two receptor tyrosine kinases, fms-like tyrosine kinase (Flt-1) or VEGF receptor 1 and kinase insert domain-containing receptor (KDR) or VEGF receptor 2. Whereas it is commonly accepted that KDR is responsible for the proliferative activities of VEGF, considerable controversy and uncertainty exist about the role of the individual receptors in eliciting many of the other effects. Based on a comprehensive mutational analysis of the receptor-binding site of VEGF, an Flt-1-selective variant was created containing four substitutions from the wild-type protein. This variant bound with wild-type affinity to Flt-1, was at least 470-fold reduced in binding to KDR, and had no activity in cell-based assays measuring autophosphorylation of KDR or proliferation of primary human vascular endothelial cells. Using a competitive phage display strategy, two KDR-selective variants were discovered with three and four changes from wild-type, respectively. Both variants had approximately wild-type affinity for KDR, were about 2000-fold reduced in binding to Flt-1, and showed activity comparable with the wild-type protein in KDR autophosphorylation and endothelial cell proliferation assays. These variants will serve as useful reagents in elucidating the roles of Flt-1 and KDR.     

Lipocortin V may function as a signaling protein for vascular endothelial growth factor receptor-2/Flk-1.

Binding of vascular endothelial growth factor (VEGF) to its receptor, VEGFR-2 (Flk-1/KDR), induces dimerization and activation of the tyrosine kinase domain of the receptor, resulting in autophosphorylation of cytoplasmic tyrosine residues used as docking sites for signaling proteins that relay the signals for cell proliferation, migration, and permeability enhancement. We explored the VEGF/receptor signaling pathway by performing a two-hybrid screen of a rat lung cDNA library in yeast using the intracellular domain of rat VEGFR-2 as bait. Two clones encoding lipocortin V were isolated. Subsequent studies with the yeast two-hybrid assay showed that the complete intracellular domain of VEGFR-2 was required for the interaction. Co-immunoprecipitation of translated proteins confirmed the interaction between the VEGF receptor and lipocortin V. VEGF induced a rapid tyrosine phosphorylation of lipocortin V in human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with antisense oligodeoxyribonucleotide (ODN) for lipocortin V significantly inhibited VEGF-induced cell proliferation, which was accompanied by a decrease in protein synthesis and tyrosine phosphorylation of lipocortin V. Our results indicate that lipocortin V may function as a signaling protein for VEGFR-2 by directly interacting with the intracellular domain of the receptor and appears to be involved in regulation of vascular endothelial cell proliferation mediated by VEGFR-2.     

Requirement of protein kinase D tyrosine phosphorylation for VEGF-A165-induced angiogenesis through its interaction and regulation of phospholipase Cgamma phosphorylation

Vascular endothelial cell growth factor-A(165) (VEGF-A(165)) is critical for angiogenesis. Although protein kinase C-mediated protein kinase D(PKD)activation was implicated in the response, the detailed mechanism remains unclear. In this study, we found that VEGF-A(165)-stimulated tyrosine phosphorylation of PKD and the dominant negative mutant of PKD, PKD(Y463F), inhibited VEGF-A(165)-induced human umbilical vein endothelial cell (HUVEC) proliferation. In addition, PKD(S738A/S742A) overexpression inhibited VEGF-induced HUVEC migration. Furthermore, knockdown of PKD by its specific small interfering RNA inhibited VEGF-induced HUVEC proliferation and migration. Moreover transfection of PKD(Y463F), PKD(S738A/S742A), or PKD-small interfering RNA blocked VEGF-induced angiogenesis in vivo. Our signaling experiments show that KDR not Flt-1 mediated PKD tyrosine phosphorylation and KDR tyrosine residues 951 and 1059 were required for VEGF-A(165)-stimulated PKD serine and tyrosine phosphorylation, respectively. Whereas G protein Gbetagamma subunits were required for both PKD serine phosphorylation and tyrosine phosphorylation, intracellular Ca(2+) mobilization was required for VEGF-A(165)-stimulated PKD tyrosine phosphorylation and phospholipase C (PLC) activity was required for PKD serine phosphorylation. Surprisingly, the PLC inhibitor did not inhibit PKD tyrosine phosphorylation. Instead, PKD tyrosine 463 was required for VEGF-A(165)-stimulated PLCgamma tyrosine phosphorylation. Moreover, PKD interacted with PLCgamma even in unstimulated cells, and PKD tyrosine 463 phosphorylation was not required for this interaction. Together, we demonstrate that PKD interacts with PLCgamma and becomes tyrosine phosphorylated upon VEGF stimulation, leading to PLCgamma activation and angiogenic response of VEGF-A(165).     

Heterotrimeric G alpha q/G alpha 11 proteins function upstream of vascular endothelial growth factor (VEGF) receptor-2 (KDR) phosphorylation in vascular permeability factor/VEGF signaling

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) functions by activating two receptor-tyrosine kinases, Flt-1 (VEGF receptor (VEGFR)-1) and KDR (VEGFR-2), both of which are selectively expressed on primary vascular endothelium. KDR is responsible for VPF/VEGF-stimulated endothelial cell proliferation and migration, whereas Flt-1 down-modulates KDR-mediated endothelial cell proliferation. Our most recent works show that pertussis toxin-sensitive G proteins and Gbetagamma subunits are required for Flt-1-mediated down-regulation of human umbilical vein endothelial cell (HUVEC) proliferation and that Gq/11 proteins are required for KDR-mediated RhoA activation and HUVEC migration. In this study, we demonstrate that Gq/11 proteins are also required for VPF/VEGF-stimulated HUVEC proliferation. Our results further indicate that Gq/11 proteins specifically mediate KDR signaling such as intracellular Ca2+ mobilization rather than Flt-1-induced CDC42 activation and that a Gq/11 antisense oligonucleotide completely inhibits MAPK phosphorylation induced by KDR but has no effect on Flt-1-induced MAPK activation. More importantly, we demonstrate that Gq/11 proteins interact with KDR in vivo, and the interaction of Gq/11 proteins with KDR does not require KDR tyrosine phosphorylation. Surprisingly, the Gq/11 antisense oligonucleotide completely inhibits VPF/VEGF-stimulated KDR phosphorylation. Expression of a constitutively active mutant of G11 but not Gq can cause phosphorylation of KDR and MAPK. In addition, a Gbetagamma minigene, hbetaARK1(495), inhibits VPF/VEGF-stimulated HUVEC proliferation, MAPK phosphorylation, and intracellular Ca2+ mobilization but has no effect on KDR phosphorylation. Taken together, this study demonstrates that Gq/11 proteins mediate KDR tyrosine phosphorylation and KDR-mediated HUVEC proliferation through interaction with KDR.     

Transactivation of vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is involved in sphingosine 1-phosphate-stimulated phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS)

Sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) elicit numerous biological responses including cell survival, growth, migration, and differentiation in endothelial cells mediated by the endothelial differentiation gene, a family of G-protein-coupled receptors, and fetal liver kinase-1/kinase-insert domain-containing receptor (Flk-1/KDR), one of VEGF receptors, respectively. Recently, it was reported that S1P or VEGF treatment of endothelial cells leads to phosphorylation at Ser-1179 in bovine endothelial nitric oxide synthase (eNOS), and this phosphorylation is critical for eNOS activation. S1P stimulation of eNOS phosphorylation was shown to involve G(i) protein, phosphoinositide 3-kinase, and Akt. VEGF also activates eNOS through Flk-1/KDR, phosphoinositide 3-kinase, and Akt, which suggested that S1P and VEGF may share upstream signaling mediators. We now report that S1P treatment of bovine aortic endothelial cells acutely increases the tyrosine phosphorylation of Flk-1/KDR, similar to VEGF treatment. S1P-mediated phosphorylation of Flk-1/KDR, Akt, and eNOS were all inhibited by VEGF receptor tyrosine kinase inhibitors and by antisense Flk-1/KDR oligonucleotides. Our study suggests that S1P activation of eNOS involves G(i), calcium, and Src family kinase-dependent transactivation of Flk-1/KDR. These data are the first to establish a critical role of Flk-1/KDR in S1P-stimulated eNOS phosphorylation and activation.