Involvement of up-regulated Necl-5/Tage4/PVR/CD155 in the loss of contact inhibition in transformed NIH3T3 cells

Normal cells show contact inhibition of cell movement and proliferation, but this is lost following transformation. We found that Necl-5, originally identified as a poliovirus receptor and up-regulated in many cancer cells, enhances growth factor-induced cell movement and proliferation. We showed that when cells contact other cells, Necl-5 interacts in trans with nectin-3 and is removed by endocytosis from the cell surface, resulting in a reduction of cell movement and proliferation. We show here that up-regulation of the gene encoding Necl-5 by the oncogene V12-Ki-Ras causes enhanced cell movement and proliferation. Upon cell-cell contact, de novo synthesis of Necl-5 exceeds the rate of Necl-5 endocytosis, eventually resulting in a net increase in the amount of Necl-5 at the cell surface. In addition, expression of the gene encoding nectin-3 is markedly reduced in transformed cells. Thus, up-regulation of Necl-5 following transformation contributes to the loss of contact inhibition in transformed cells.     

Inhibition of cell movement and proliferation by cell-cell contact-induced interaction of Necl-5 with nectin-3

Immunoglobulin-like Necl-5/Tage4/poliovirus receptor (PVR)/CD155, originally identified as the PVR, has been shown to be up-regulated in cancer cells and to enhance growth factor-induced cell movement and proliferation. In addition, Necl-5 heterophilically trans-interacts with nectin-3, a cell-cell adhesion molecule known to form adherens junctions in cooperation with cadherin. We show here that Necl-5 was down-regulated from cell surface upon cell-cell contacts in NIH3T3 cells. This down-regulation of Necl-5 was initiated by its interaction with nectin-3 and was mainly mediated by clathrin-dependent endocytosis. Then, the down-regulation of Necl-5 induced in this way reduced movement and proliferation of NIH3T3 cells. These results indicate that the down-regulation of Necl-5 induced by its interaction with nectin-3 upon cell-cell contacts may be at least one mechanism underlying contact inhibition of cell movement and proliferation.     

Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin,beta-catenin,and the phosphatase DEP-1/CD148

Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in beta-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. beta-Catenin-null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density-enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin-beta-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell-cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin-null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.     

Enhancement of serum- and platelet-derived growth factor-induced cell proliferation by Necl-5/Tage4/poliovirus receptor/CD155 through the Ras-Raf-MEK-ERK signaling

Necl-5/Tage4/poliovirus receptor/CD155 has been shown to be the poliovirus receptor and to be up-regulated in rodent and human carcinoma. We have found previously that mouse Necl-5 regulates cell motility. We show here that mouse Necl-5 is furthermore involved in the regulation of cell proliferation. Studies using a specific antibody against Necl-5 and a dominant negative mutant of Necl-5 revealed that Necl-5 enhanced the serum-induced proliferation of NIH3T3, Swiss3T3, and mouse embryonic fibroblast cells. Necl-5 enhanced the serum-induced activation of the Ras-Raf-MEK-ERK signaling, up-regulated cyclins D2 and E, and down-regulated p27(Kip1), eventually shortening the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 similarly enhanced the platelet-derived growth factor-induced activation of the Ras-Raf-MEK-ERK signaling and shortened the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 acted downstream of the platelet-derived growth factor receptor and upstream of Ras. Moreover, up-regulated Necl-5 was involved at least partly in the enhanced proliferation of transformed cells including NIH3T3 cells transformed by an oncogenic Ras or v-Src. These results indicate that Necl-5 plays roles not only in cell motility but also in cell proliferation.     

Endothelial Cell mTOR Complex-2 Regulates Sprouting Angiogenesis

Tumor neovascularization is targeted by inhibition of vascular endothelial growth factor (VEGF) or the receptor to prevent tumor growth, but drug resistance to angiogenesis inhibition limits clinical efficacy. Inhibition of the phosphoinositide 3 kinase pathway intermediate, mammalian target of rapamycin (mTOR), also inhibits tumor growth and may prevent escape from VEGF receptor inhibitors. mTOR is assembled into two separate multi-molecular complexes, mTORC1 and mTORC2. The direct effect of mTORC2 inhibition on the endothelium and tumor angiogenesis is poorly defined. We used pharmacological inhibitors and RNA interference to determine the function of mTORC2 versus Akt1 and mTORC1 in human endothelial cells (EC). Angiogenic sprouting, EC migration, cytoskeleton re-organization, and signaling events regulating matrix adhesion were studied. Sustained inactivation of mTORC1 activity up-regulated mTORC2-dependent Akt1 activation. In turn, ECs exposed to mTORC1-inhibition were resistant to apoptosis and hyper-responsive to renal cell carcinoma (RCC)-stimulated angiogenesis after relief of the inhibition. Conversely, mTORC1/2 dual inhibition or selective mTORC2 inactivation inhibited angiogenesis in response to RCC cells and VEGF. mTORC2-inactivation decreased EC migration more than Akt1- or mTORC1-inactivation. Mechanistically, mTORC2 inactivation robustly suppressed VEGF-stimulated EC actin polymerization, and inhibited focal adhesion formation and activation of focal adhesion kinase, independent of Akt1. Endothelial mTORC2 regulates angiogenesis, in part by regulation of EC focal adhesion kinase activity, matrix adhesion, and cytoskeletal remodeling, independent of Akt/mTORC1.     

Platelet factor 4 disrupts the intracellular signalling cascade induced by vascular endothelial growth factor by both KDR dependent and independent mechanisms.

The mechanism by which the CXC chemokine platelet factor 4 (PF-4) inhibits endothelial cell proliferation is unclear. The heparin-binding domains of PF-4 have been reported to prevent vascular endothelial growth factor 165 (VEGF(165)) and fibroblast growth factor 2 (FGF2) from interacting with their receptors. However, other studies have suggested that PF-4 acts via heparin-binding independent interactions. Here, we compared the effects of PF-4 on the signalling events involved in the proliferation induced by VEGF(165), which binds heparin, and by VEGF(121), which does not. Activation of the VEGF receptor, KDR, and phospholipase Cgamma (PLCgamma) was unaffected in conditions in which PF-4 inhibited VEGF(121)-induced DNA synthesis. In contrast, VEGF(165)-induced phosphorylation of KDR and PLCgamma was partially inhibited by PF-4. These observations are consistent with PF-4 affecting the binding of VEGF(165), but not that of VEGF(121), to KDR. PF-4 also strongly inhibited the VEGF(165)- and VEGF(121)-induced mitogen-activated protein (MAP) kinase signalling pathways comprising Raf1, MEK1/2 and ERK1/2: for VEGF(165) it interacts directly or upstream from Raf1; for VEGF(121), it acts downstream from PLCgamma. Finally, the mechanism by which PF-4 may inhibit the endothelial cell proliferation induced by both VEGF(121) and VEGF(165), involving disruption of the MAP kinase signalling pathway downstream from KDR did not seem to involve CXCR3B activation.     

Identification and characterization of regulator of G protein signaling 4 (RGS4) as a novel inhibitor of tubulogenesis: RGS4 inhibits mitogen-activated protein kinases and vascular endothelial growth factor signaling

Tubulogenesis by epithelial cells regulates kidney, lung, and mammary development, whereas that by endothelial cells regulates vascular development. Although functionally dissimilar, the processes necessary for tubulation by epithelial and endothelial cells are very similar. We performed microarray analysis to further our understanding of tubulogenesis and observed a robust induction of regulator of G protein signaling 4 (RGS4) mRNA expression solely in tubulating cells, thereby implicating RGS4 as a potential regulator of tubulogenesis. Accordingly, RGS4 overexpression delayed and altered lung epithelial cell tubulation by selectively inhibiting G protein-mediated p38 MAPK activation, and, consequently, by reducing epithelial cell proliferation, migration, and expression of vascular endothelial growth factor (VEGF). The tubulogenic defects imparted by RGS4 in epithelial cells, including its reduction in VEGF expression, were rescued by overexpression of constitutively active MKK6, an activator of p38 MAPK. Similarly, RGS4 overexpression abrogated endothelial cell angiogenic sprouting by inhibiting their synthesis of DNA and invasion through synthetic basement membranes. We further show that RGS4 expression antagonized VEGF stimulation of DNA synthesis and extracellular signal-regulated kinase (ERK)1/ERK2 and p38 MAPK activation as well as ERK1/ERK2 activation stimulated by endothelin-1 and angiotensin II. RGS4 had no effect on the phosphorylation of Smad1 and Smad2 by bone morphogenic protein-7 and transforming growth factor-beta, respectively, indicating that RGS4 selectively inhibits G protein and VEGF signaling in endothelial cells. Finally, we found that RGS4 reduced endothelial cell response to VEGF by decreasing VEGF receptor-2 (KDR) expression. We therefore propose RGS4 as a novel antagonist of epithelial and endothelial cell tubulogenesis that selectively antagonizes intracellular signaling by G proteins and VEGF, thereby inhibiting cell proliferation, migration, and invasion, and VEGF and KDR expression.     

Hypoxia-induced mitogenic factor enhances angiogenesis by promoting proliferation and migration of endothelial cells

Our previous studies have indicated that hypoxia-induced mitogenic factor (HIMF) has angiogenic properties in an in vivo matrigel plug model and HIMF upregulates expression of vascular endothelial growth factor (VEGF) in mouse lungs and cultured lung epithelial cells. However, whether HIMF exerts angiogenic effects through modulating endothelial cell function remains unknown. In this study, mouse aortic rings cultured with recombinant HIMF protein resulted in enhanced vascular sprouting and increased endothelial cell spreading as confirmed by Dil-Ac-LDL uptake, von Willebrand factor and CD31 staining. In cultured mouse endothelial cell line SVEC 4-10, HIMF dose-dependently enhanced cell proliferation, in vitro migration and tubulogenesis, which was not attenuated by SU1498, a VEGFR2/Flk-1 receptor tyrosine kinase inhibitor. Moreover, HIMF stimulation resulted in phosphorylation of Akt, p38 and ERK1/2 kinases in SVEC 4-10 cells. Treatment of mouse aortic rings and SVEC 4-10 cells with LY294002, but not SB203580, PD098059 or U0126, abolished HIMF-induced vascular sprouting and angiogenic responses. In addition, transfection of a dominant-negative mutant of phosphatidylinositol 3-kinase (PI-3K), Deltap85, blocked HIMF-induced phosphorylation of Akt, endothelial activation and tubulogenesis. These results indicate that HIMF enhances angiogenesis by promoting proliferation and migration of endothelial cells via activation of the PI-3K/Akt pathways.     

Interaction of nectin-like molecule 2 with integrin alpha6beta4 and inhibition of disassembly of integrin alpha6beta4 from hemidesmosomes

In normal epithelial cells, integrin α(6)β(4) is abundantly expressed and forms hemidesmosomes, which is a cellular structure that mediates cell-extracellular matrix binding. In many types of cancer cells, integrin α(6)β(4) is up-regulated, laminin is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and facilitation of their invasion. We previously showed that the immunoglobulin-like cell adhesion molecule Necl-2 (Nectin-like molecule 2), known as a tumor suppressor, inhibits cancer cell movement by suppressing the ErbB3/ErbB2 signaling. We show here that Necl-2 interacts in cis with integrin α(6)β(4). The binding of Necl-2 with integrin β(4) was mediated by its extracellular region. In human colorectal adenocarcinoma Caco-2 cells, integrin α(6)β(4) was localized at hemidesmosomes. Small interfering RNA-mediated suppression of Necl-2 expression enhanced the phorbol ester-induced disruption of the integrin α(6)β(4) complex at hemidesmosomes, whereas expression of Necl-2 suppressed the disruption of this structure. These results indicate that tumor-suppressive functions of Necl-2 are mediated by the stabilization of the hemidesmosome structure in addition to the inhibition of the ErbB3/ErbB2 signaling.     

Pigment epithelium-derived factor exerts opposite effects on endothelial cells of different phenotypes

The anti-angiogenic activity of pigment epithelium-derived factor (PEDF) has recently been discovered on the basis of its inhibition of ischemia-induced retinal neovascularization in an animal model of retinopathy of the premature. Moreover PEDF inhibits the migration and proliferation of various endothelial cells maintained in culture with FGF(2). Since vascular endothelial growth factor (VEGF) is the main angiogenic factor expressed in hypervascularized retinas, we investigated the functions of PEDF on retinal endothelial cells whose angiogenic phenotype is controlled or not by long term exposure to VEGF as observed in human pathologies such as diabetic retinopathy. Here, we observed that PEDF exerts opposite effects on endothelial cells depending on their phenotype. We determined that when PEDF inhibits endothelial cell growth, it inhibits VEGF-induced MAPK activation. However, in endothelial cells cultured with VEGF, PEDF has a synergistic action on cell proliferation with VEGF, and this corresponds to increased MAPK activation.     

Antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1

Neuropilin-1 (NRP-1), a non-tyrosine kinase receptor of vascular endothelial growth factor-165 (VEGF165), was found expressed on endothelial and some tumor cells. Since its overexpression is correlated with tumor angiogenesis and progression, the targeting of NRP-1 could be a potential anti-cancer strategy. To explore this hypothesis, we identified a peptide inhibiting the VEGF165 binding to NRP-1 and we tested whether it was able to inhibit tumor growth and angiogenesis. To prove the target of peptide action, we assessed its effects on binding of radiolabeled VEGF165 to recombinant receptors and to cultured cells expressing only VEGFR-2 (KDR) or NRP-1. Antiangiogenic activity of the peptide was tested in vitro in tubulogenesis assays and in vivo in nude mice xenotransplanted in fat-pad with breast cancer MDA-MB-231 cells. Tumor volumes, vascularity and proliferation indices were determined. The selected peptide, ATWLPPR, inhibited the VEGF165 binding to NRP-1 but not to tyrosine kinase receptors, VEGFR-1 (flt-1) and KDR; nor did it bind to heparin. It diminished the VEGF-induced human umbilical vein endothelial cell proliferation and tubular formation on Matrigel and in co-culture with fibroblasts. Administration of ATWLPPR to nude mice inhibited the growth of MDA-MB-231 xenografts, and reduced blood vessel density and endothelial cell area but did not alter the proliferation indices of the tumor. In conclusion, ATWLPPR, a previously identified KDR-interacting peptide, was shown to inhibit the VEGF165 interactions with NRP-1 but not with KDR and to decrease the tumor angiogenesis and growth, thus validating, in vivo, NRP-1 as a possible target for antiangiogenic and antitumor agents.     

Downregulation of Fes inhibits VEGF-A-induced chemotaxis and capillary-like morphogenesis by cultured endothelial cells

The aim of this study was to determine whether the downregulation of endogenous Fes by siRNA in cultured endothelial cells affects vascular endothelial growth factor-A (VEGF-A)-induced chemotaxis and capillary-like morphogenesis, which are considered as angiogenic cellular responses in vitro. VEGF-A-treatment induced autophosphorylation of Fes in cultured endothelial cells. LY294002, a phosphoinositide 3-kinase inhibitor, significantly inhibited VEGF-A-induced chemotaxis and capillary-like morphogenesis. Downregulation of Fes attenuated these VEGF-A-induced cellular responses but LY294002 did not produce further inhibition of these responses. Downregulation of Fes neither affected VEGF-A-induced autophosphorylation of VEGF receptor 2 nor mitogen-activated protein kinase activation, but markedly decreased Akt activation. Taken together, our novel results indicate the involvement of Fes in VEGF-A-induced cellular responses by cultured endothelial cells.     

Induction of KDR expression in bovine arterial endothelial cells by thrombin: involvement of nitric oxide.

Thrombin, a multifunctional serine protease, is generated at the site with vascular injuries. It not only participates in the coagulation cascade, but also can induce a lot of events related to cell mitogenesis and migration. In this study, we investigated the effect of thrombin on endothelial cell proliferation induced by vascular endothelial growth factor (VEGF). Thrombin promoted proliferation of cultured bovine carotid endothelial cells in a time- and dose-dependent manner. Moreover, it drastically enhanced the cell growth stimulated by VEGF. This stimulatory effect was reduced by inhibitors of either protein kinase C (PKC) or mitogen-activated protein kinase kinase (MAPKK). Thrombin induced a significant increase in the level of mRNA of the kinase domain-containing receptor (KDR), but not tms-like tyrosine kinase (Flt-1), in a time-dependent manner, which reached the maximum after 24 h of stimulation. This increase coincides well with the KDR protein expression. The luciferase assay showed that thrombin induced an about 7.5-fold increase in the KDR promoter activity compared with the control. This enhanced KDR promoter activity was also abolished by inhibitors of either PKC or MAPKK. The deletion analyses indicated that the region between -115 and -97 (containing Sp1 binding region) within the KDR promoter gene was required for the enhanced KDR expression induced by thrombin and VEGF. Moreover, the nitric oxide synthase (NOS) inhibitor abolished both the accelerated cell proliferation and the increased KDR expression induced by thrombin and VEGF. This inhibition was abrogated by DETA NONOate, a NO donor with long half-life. These findings suggest that thrombin might potentiate the VEGF-induced angiogenic activity through increasing the level of the VEGF receptor KDR, in which production of NO is involved.     

VEGF-induced vascular permeability is mediated by FAK

Endothelial cells (ECs) form cell-cell adhesive junctional structures maintaining vascular integrity. This barrier is dynamically regulated by vascular endothelial growth factor (VEGF) receptor signaling. We created an inducible knockin mouse model to study the contribution of the integrin-associated focal adhesion tyrosine kinase (FAK) signaling on vascular function. Here we show that genetic or pharmacological FAK inhibition in ECs prevents VEGF-stimulated permeability downstream of VEGF receptor or Src tyrosine kinase activation in vivo. VEGF promotes tension-independent FAK activation, rapid FAK localization to cell-cell junctions, binding of the FAK FERM domain to the vascular endothelial cadherin (VE-cadherin) cytoplasmic tail, and direct FAK phosphorylation of β-catenin at tyrosine-142 (Y142) facilitating VE-cadherin-β-catenin dissociation and EC junctional breakdown. Kinase inhibited FAK is in a closed conformation that prevents VE-cadherin association and limits VEGF-stimulated β-catenin Y142 phosphorylation. Our studies establish a role for FAK as an essential signaling switch within ECs regulating adherens junction dynamics.     

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.     

Vasoactive intestinal peptide increases VEGF expression to promote proliferation of brain vascular endothelial cells via the cAMP/PKA pathway after ischemic insult in vitro

Vasoactive intestinal peptide (VIP) enhances angiogenesis in rats with focal cerebral ischemia. In the present study, we investigated the molecular mechanism of the proangiogenic action of VIP using an in vitro ischemic model, in which rat brain microvascular endothelial cells (RBMECs) are subjected to oxygen and glucose deprivation (OGD). Western blotting and immunocytochemistry were carried out to examine the expression of VIP receptors and vascular endothelial growth factor (VEGF) in cultured RBMECs. The cell proliferation was assessed by the MTT assay. Cyclic adenosine monophosphate (cAMP) and VEGF levels were measured by using the enzyme-linked immunosorbent assay. The cultured RBMECs expressed VPAC1, VPAC2 and PAC1 receptors. Treatment with VIP significantly promoted the proliferation of RBMECs and increased OGD-induced expression of VEGF, and this effect was antagonized by the VPAC receptor antagonist VIP6-28 and VEGF antibody. VIP significantly increased contents of cAMP in RBMECs and VEGF in the culture medium. The VIP-induced VEGF production was blocked by H89, a protein kinase A (PKA) inhibitor. These data suggest that treatment with VIP promotes VEGF-mediated endothelial cell proliferation after ischemic insult in vitro, and this effect appears to be initiated by the VPAC receptors leading to activation of the cAMP/PKA pathway.     

cAMP-dependent protein kinase inhibits the mitogenic action of vascular endothelial growth factor and fibroblast growth factor in capillary endothelial cells by blocking Raf activation

Proliferation of endothelial cells is regulated by angiogenic and antiangiogenic factors whose actions are mediated by complex interactions of multiple signaling pathways. Both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) stimulate cell proliferation and activate the mitogen-activated protein kinase (MAPK) cascade in bovine brain capillary endothelial (BBE) cells. We have extended these findings to show that both mitogens activate MAPK via stimulation of Raf-1. Activation of Raf/MAPK is inhibited by increasing intracellular cAMP levels pharmacologically or via stimulation of endogenously expressed β-adrenergic receptors. Both VEGF- and bFGF-induced Raf-1 activity are blocked in the presence of forskolin or 8-bromo-cAMP by 80%. The actions of increased cAMP appear to be mediated by cAMP-dependent protein kinase (PKA), since treatment with H-89, a the specific inhibitor of PKA, reversed the inhibitory effect of elevated cAMP levels on mitogen-induced cell proliferation and Raf/MAPK activation. Moreover, elevations in cAMP/PKA activity inhibit mitogen-induced cell proliferation. These findings demonstrate, in cultured endothelial cells, that the cAMP/PKA signaling pathway is potentially an important physiological inhibitor of mitogen activation of the MAPK cascade and cell proliferation. J. Cell. Biochem. 67:353–366, 1997. © 1997 Wiley-Liss, Inc.     

A novel peptide isolated from a phage display library inhibits tumor growth and metastasis by blocking the binding of vascular endothelial growth factor to its kinase domain receptor

Vascular endothelial growth factor (VEGF), one of the most important angiogenic factors, plays an essential role in both physiological and pathological angiogenesis. The VEGF receptor KDR/Flk-1 (a kinase domain receptor) mediates various biological activities of VEGF related to proliferation, differentiation, and migration of endothelial cells. Here we present a novel peptide designated K237-(HTMYYHHYQHHL), which was isolated from a phage-displayed peptide library, binding to KDR with high affinity and specificity. By interfering with the VEGF-KDR interaction, the peptide K237 inhibited proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF(165) in a dose-dependent and cell type-specific manner. The peptide also exerted an anti-angiogenesis activity in vivo as revealed using the chick embryo chorioallantoic membrane angiogenesis assay. Moreover, the peptide K237 significantly inhibited the growth of solid tumors implanted beneath the breasts and their metastases to lungs in severe combined immunodeficient mice. Taken together, these findings suggest that the peptide K237 can functionally disrupt the interaction between VEGF and the KDR receptor and cause potent biological effects that include the inhibition of angiogenesis and tumor growth. As a consequence, this peptide (and its future derivatives) may have use as a potential cancer therapy.     

LRRC4 inhibits glioblastoma cell proliferation, migration, and angiogenesis by downregulating pleiotropic cytokine expression and responses

Leucine-rich repeat C4 (LRRC4) has been shown to inhibit glioma cell proliferation, however, little is known about the mechanism(s) underlying the action of LRRC4. Here, we show that two glioblstoma U251 cell clones stably expressing LRRC4 were established. LRRC4 expression significantly inhibited the expression of some cytokines and their receptors determined by microarray and Western blot assays, and dramatically reduced cytokine-induced AP-1, NF-kB, and CyclinD1 activation in glioma cells. Furthermore, LRRC4 expression in glioma cells significantly downregulated spontaneous and cytokine-induced expression of K-RAS and phosphorylation of c-Raf, ERK, AKT, NF-kBp65, p70S6K, and PKC, suggesting that LRRC4 inhibited receptor tyrosine kinase (RTK) signaling pathways. Moreover, treatment with bFGF, IGF1, or IGF2 stimulated LRRC4(-/-), but not the LRRC4(+), glioma cell proliferation, indicating that LRRC4 mitigated cytokine-stimulated proliferation in glioma cells. In addition, treatment of LRRC4(-/-) glioma cells with EGF, IGF2, or PDGF promoted long distance mobilization, but induced little migration in LRRC4(+) glioma cells, suggesting that LRRC4 retarded cytokine-promoted glioma cell migration in vitro. Finally, human vessel endothelial cells (ECV304) treated with VEGF grew, aligned and formed hollow tube-like structures in vitro. In contrast, LRRC4(+) ECV304 treated with VEGF failed to form vessel-tube structures. Collectively, LRRC4 expression inhibited the expression of some growth factors, cytokines and their receptors, and the capacity of glioma cells responding to cytokine stimulation, leading to inhibition of glioma cell proliferation. Conceivably, induction of LRRC4 expression may provide new intervention for human glioma in the clinic.     

Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments

Receptor endocytosis is a fundamental step in controlling the magnitude, duration, and nature of cell signaling events. Confluent endothelial cells are contact inhibited in their growth and respond poorly to the proliferative signals of vascular endothelial growth factor (VEGF). In a previous study, we found that the association of vascular endothelial cadherin (VEC) with VEGF receptor (VEGFR) type 2 contributes to density-dependent growth inhibition (Lampugnani, G.M., A. Zanetti, M. Corada, T. Takahashi, G. Balconi, F. Breviario, F. Orsenigo, A. Cattelino, R. Kemler, T.O. Daniel, and E. Dejana. 2003. J. Cell Biol. 161:793-804). In the present study, we describe the mechanism through which VEC reduces VEGFR-2 signaling. We found that VEGF induces the clathrin-dependent internalization of VEGFR-2. When VEC is absent or not engaged at junctions, VEGFR-2 is internalized more rapidly and remains in endosomal compartments for a longer time. Internalization does not terminate its signaling; instead, the internalized receptor is phosphorylated, codistributes with active phospholipase C-gamma, and activates p44/42 mitogen-activated protein kinase phosphorylation and cell proliferation. Inhibition of VEGFR-2 internalization reestablishes the contact inhibition of cell growth, whereas silencing the junction-associated density-enhanced phosphatase-1/CD148 phosphatase restores VEGFR-2 internalization and signaling. Thus, VEC limits cell proliferation by retaining VEGFR-2 at the membrane and preventing its internalization into signaling compartments.