Cytokines modulate integrin alpha(v)beta(3)-mediated human endothelial cell adhesion and calcium signaling.

Angiogenesis is a complex process regulated by the interactions of endothelial cells with cytokines and the adhesive protein matrix. The cytokines basic fibroblast growth factor (bFGF) and tumor necrosis factor-alpha (TNF-alpha) are two of the modulators of angiogenesis. One mechanism by which these cytokines induce their effects may be through the regulation of integrin adhesion receptor activity, in particular, alpha(v)beta(3). In this study, we examined the ability of these angiogenic factors to modulate the adhesion of human umbilical vein endothelial cells (HUVECs) to immobilized disintegrins (i.e., rhodostomin and arietin), which are specific in antagonizing integrin alpha(v)beta(3) in cells. As these disintegrins were immobilized as substrates, they acted as agonists to induce HUVEC adhesion in a dose- and alpha(v)beta(3)-dependent manner. In addition, adhesion also triggered a sustained increase of intracellular free calcium. Furthermore, bFGF-primed HUVECs potentiated, but TNF-alpha primed cells attenuated, about 50% adhesion events and calcium signaling triggered by immobilized disintegrin compared to naive cells, respectively. The mechanisms of modulating alpha(v)beta(3)-dependent HUVEC adhesion by cytokines may be related to changes of integrin alpha(v)beta(3) conformation, as demonstrating the antagonistic effect of Mn(2+) on decreased adhesion by TNF-alpha pretreatment, and confirmed with flow cytometric analysis probed by anti-LIBS1 mAb. However, cytokine pretreatment did not alter the expression of this integrin on the cell surface, as determined by flow cytometry. Phosphoinositide-3 kinase may be one of the signaling molecules involved in the enhanced adhesion of bFGF-primed cells.     

Regulation of integrin alpha vbeta 3-mediated endothelial cell migration and angiogenesis by integrin alpha5beta1 and protein kinase A

Recent studies indicate that angiogenesis depends, in part, on ligation of integrin alpha(5)beta(1) by fibronectin. Evidence is now provided that integrin alpha(5)beta(1) regulates the function of integrin alpha(v)beta(3) on endothelial cells during their migration in vitro or angiogenesis in vivo. Secretion of fibronectin by endothelial cells leads to the ligation of integrin alpha(5)beta(1), which potentiates alpha(v)beta(3)-mediated migration on vitronectin without influencing alpha(v)beta(3)-mediated cell adhesion. Endothelial cell attachment to vitronectin suppresses protein kinase A (PKA) activity, while addition of soluble anti-alpha(5)beta(1) restores this activity. Moreover, agents that activate intracellular PKA, such as forskolin, dibutyryl cAMP or alpha(5)beta(1) antagonists, suppress endothelial cell migration on vitronectin in vitro or angiogenesis in vivo. In contrast, inhibitors of PKA reverse the anti-migratory or anti-angiogenic effects mediated by alpha(5)beta(1) antagonists. Therefore, alpha(v)beta(3)-mediated endothelial cell migration and angiogenesis can be regulated by PKA activity, which depends on the ligation state of integrin alpha(5)beta(1).     

Thymidine phosphorylase and 2-deoxyribose stimulate human endothelial cell migration by specific activation of the integrins alpha 5 beta 1 and alpha V beta 3

Thymidine phosphorylase is an angiogenic factor that is frequently overexpressed in solid tumors, in rheumatoid arthritis, and in response to inflammatory cytokines. Our previous studies showed that cells expressing thymidine phosphorylase stimulated endothelial cell migration in vitro. This was a consequence of the intracellular metabolism of thymidine by thymidine phosphorylase and subsequent extracellular release of 2-deoxyribose. The mechanisms by which 2-deoxyribose might mediate thymidine phosphorylase-induced cell migration in vitro, however, are obscure. Here we show that both thymidine phosphorylase and 2-deoxyribose stimulated the formation of focal adhesions and the tyrosine 397 phosphorylation of focal adhesion kinase in human umbilical vein endothelial cells. Although similar actions occurred upon treatment with the angiogenic factor vascular endothelial growth factor (VEGF), thymidine phosphorylase differed from VEGF in that its effect on endothelial cell migration was blocked by antibodies to either integrin alpha 5 beta 1 or alpha v beta 3, whereas VEGF-induced endothelial cell migration was only blocked by the alpha v beta 3 antibody. Further, thymidine phosphorylase and 2-deoxyribose, but not VEGF, increased the association of both focal adhesion kinase and the focal adhesion-associated protein vinculin with integrin alpha 5 beta 1 and, in intact cells, increased the co-localization of focal adhesion kinase with alpha 5 beta 1. Thymidine phosphorylase and 2-deoxyribose-induced focal adhesion kinase phosphorylation was blocked by the antibodies to alpha 5 beta 1 and alpha v beta 3, directly linking the migration and signaling components of thymidine phosphorylase and 2-deoxyribose action. Cell surface expression of alpha 5 beta 1 was also increased by thymidine phosphorylase and 2-deoxyribose. These experiments are the first to demonstrate a direct effect of thymidine phosphorylase and 2-deoxyribose on signaling pathways associated with endothelial cell migration.     

Involvement of integrin alpha(v)beta(3) and cell adhesion molecule L1 in transendothelial migration of melanoma cells

Tumor metastasis involves many stage-specific adhesive interactions. The expression of several cell adhesion molecules, notably the integrin alpha(v)beta(3), has been associated with the metastatic potential of tumor cells. In this study, we used a novel in vitro assay to examine the role of alpha(v)beta(3) in the transmigration of melanoma cells through a monolayer of human lung microvascular endothelial cells. Confocal microscopy revealed the presence of the integrin alpha(v)beta(3) on melanoma membrane protrusions and pseudopods penetrating the endothelial junction. alpha(v)beta(3) was also enriched in heterotypic contacts between endothelial cells and melanoma cells. Transendothelial migration of melanoma cells was inhibited by either a cyclic Arg-Gly-Asp peptide or the anti-alpha(v)beta(3) monoclonal antibody LM609. Although both platelet endothelial cell adhesion molecule-1 and L1 are known to bind integrin alpha(v)beta(3), only L1 serves as a potential ligand for alpha(v)beta(3) during melanoma transendothelial migration. Also, polyclonal antibodies against L1 partially inhibited the transendothelial migration of melanoma cells. However, addition of both L1 and alpha(v)beta(3) antibodies did not show additive effects, suggesting that they are components of the same adhesion system. Together, the data suggest that interactions between the integrin alpha(v)beta(3) on melanoma cells and L1 on endothelial cells play an important role in the transendothelial migration of melanoma cells.     

Regulation of cell-cell interactions by phosphatidic acid phosphatase 2b/VCIP

We identified vascular endothelial growth factor and type I collagen inducible protein (VCIP), also known as phosphatidic acid phosphatase 2b (PAP2b), in a functional assay of angiogenesis. VCIP/PAP2b exhibits an Arg-Gly-Asp (RGD) cell adhesion sequence. Immunoprecipitation and fluorescence-activated cell sorting analyses demonstrated that VCIP-RGD is exposed to the outside of the cell surface. Retroviral transduction of VCIP induced cell aggregation/cell- cell interactions, modestly increased p120 catenin expression and promoted activation of the Fak, Akt and GSK3beta protein kinases. Furthermore, expression of recombinant VCIP promoted adhesion, spreading and tyrosine phosphorylation of Fak, Shc, Cas and paxillin in endothelial cells. GST-VCIP-RGD, but not GST-VCIP-RGE, specifically interacted with a subset of integrins, and these interactions were effectively blocked by anti-alpha(v)beta(3) and anti-alpha(5)beta(1) integrin antibodies, and by PAP2b/VCIP-derived peptides. Interestingly, PAP2b/VCIP is expressed in close proximity to vascular endothelial growth factor, von Willebrand factor and alpha(v)beta(3) integrin in tumor vasculatures. These findings demonstrate an unexpected function of PAP2b/VCIP, and represent an important step towards understanding the molecular mechanisms by which PAP2b/VCIP-induced cell-cell interactions regulate specific intracellular signaling pathways.     

The integrin complex alpha v beta 3 participates in the adhesion of microvascular endothelial cells to fibronectin.

Fibronectin is a major adhesive glycoprotein of the vascular basement membrane. Since fibronectin is also found in the interstitium, it may be important not only for attachment but also for endothelial cell migration during neovascularization. We have analyzed how human dermal microvascular endothelial cells use their diverse set of integrin receptors to interact with this ligand. Immunofluorescent staining with specific antibodies identified both beta 1 and beta 3 integrin receptor complexes in focal adhesion plaques on cells adhering to immobilized fibronectin. Adhesion assays with blocking monoclonal antibodies implicated both beta 1 and beta 3 complexes, specifically alpha 5 beta 1 and alpha v beta 3, in the initial adhesion of cells to fibronectin. Finally, ligand affinity chromatography of extracts of surface radiolabeled cells established that both alpha 5 beta 1 and alpha v beta 3 could bind to the 110-kDa cell-binding fragment of fibronectin. An additional receptor complex composed of an alpha v subunit and a beta 5-like subunit was also detected. These results provide evidence that microvascular endothelial cells use multiple integrin receptors, from several beta families, to attach to fibronectin surfaces.     

Proteolytically processed soluble tumor endothelial marker (TEM) 5 mediates endothelial cell survival during angiogenesis by linking integrin alpha(v)beta3 to glycosaminoglycans

Tumor endothelial marker (TEM) 5 is a member of the adhesion family of G-protein-coupled receptors and up-regulated in endothelial cells during tumor and physiologic angiogenesis. Here, we report that TEM5 is expressed on the surface of endothelial cells. A soluble TEM5 (sTEM5) fragment is shed by endothelial cells during capillary-like network formation and upon growth factor stimulation. We found that sTEM5 binds to several glycosaminoglycans. Furthermore, sequence analysis and functional and biochemical studies revealed that sTEM5 contains a cryptic RGD-binding site for integrin alpha(v)beta3. Matrix metalloprotease 9-processed, but not full-length, sTEM5 mediated endothelial cell adhesion by direct interaction with integrin alpha(v)beta3. Adhesion to proteolytically processed sTEM5 (ppsTEM5) or glycosaminoglycan-bound ppsTEM5 promoted survival of growth factor deprived endothelial cells. ppsTEM5-mediated cell survival was inhibited by a function blocking integrin alpha(v)beta3 antibody. Based on our results we conclude that sTEM5 is shed by endothelial cells during angiogenesis and binds to glycosaminoglycans present on extracellular matrix and cell surface proteoglycans. Further proteolytic processing of sTEM5 leads to exposure of its RGD motif mediating endothelial cell survival by linking integrin alpha(v)beta3 to glycosaminoglycans.     

CYR61 stimulates human skin fibroblast migration through Integrin alpha vbeta 5 and enhances mitogenesis through integrin alpha vbeta 3, independent of its carboxyl-terminal domain

CYR61, an angiogenic factor and a member of the CCN protein family, is an extracellular matrix-associated, heparin-binding protein that mediates cell adhesion, promotes cell migration, and enhances growth factor-stimulated cell proliferation. CYR61 induces angiogenesis and promotes tumor growth in vivo and is expressed in dermal fibroblasts during cutaneous wound healing. It has been demonstrated recently that adhesion of primary skin fibroblasts to CYR61 is mediated through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans, resulting in adhesive signaling and up-regulation of matrix metalloproteinases 1 and 3. CYR61 is composed of four discrete structural domains that bear sequence similarities to the insulin-like growth factor-binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and a carboxyl-terminal (CT) domain that resembles cysteine knots found in some growth factors. In this study, we show that a CYR61 mutant (CYR61DeltaCT) that has the CT domain deleted is unable to support adhesion of primary human skin fibroblasts but is still able to stimulate chemotaxis and enhance basic fibroblast growth factor-induced mitogenesis similar to wild type. In addition, fibroblast migration to CYR61 is mediated through integrin alpha(v)beta(5) but not integrins alpha(6)beta(1) or alpha(v)beta(3). Furthermore, we show that CYR61 binds directly to purified integrin alpha(v)beta(5) in vitro. By contrast, CYR61 enhancement of basic fibroblast growth factor-induced DNA synthesis is mediated through integrin alpha(v)beta(3), a known receptor for CYR61 that mediates CYR61-dependent cell adhesion and chemotaxis in vascular endothelial cells. Thus, CYR61 promotes primary human fibroblast adhesion, migration, and mitogenesis through integrins alpha(6)beta(1), alpha(v)beta(5), and alpha(v)beta(3), respectively. Together, these findings establish CYR61 as a novel ligand for integrin alpha(v)beta(5) and show that CYR61 interacts with distinct integrins to mediate disparate activities in a cell type-specific manner.     

The integrin alpha5beta1 regulates chondrocyte hypertrophic differentiation induced by GTP-bound transglutaminase 2.

Soluble GTP-bound transglutaminase 2 (TG2) induces hypertrophic differentiation in chondrocyte cultures in a beta1 integrin-dependent fashion. beta1 integrin subfamily consists of 12 heterodimers with 12 different alpha subunits and a beta1 subunit. To identify the specific integrin heterodimer(s) responsible for this process, we specifically blocked individual beta1 integrins on the CH-8 immortalized human chondrocytes during hypertrophic differentiation. Blockade of alpha5beta1 inhibited matrix metalloproteinase 13 (MMP-13), type X collagen expression, alkaline phosphatase activity and matrix calcification by 30-50% associated with weak effects of anti-alpha3beta1 and -alpha4beta1. Anti-alpha1beta1, -alpha2beta1 and -alpha6beta1 had no effect. To examine whether the dominant effect of integrin alpha5beta1 was due to a direct interaction with TG2, we incubated the chondrocytic cells on plates coated with GTP-bound TG2. The immobilized GTP-bound TG2 induced hypertrophic differentiation to the same extent as the soluble GTP-bound TG2, which was also inhibited by anti-alpha5beta1. CH-8 cells grown on plates coated with GTP-bound TG2 demonstrated adherence associated with focal adhesion kinase phosphorylation. These properties were inhibited by anti-alpha5beta1. Furthermore, engagement of alpha5beta1 on CH-8 cells via anti-alpha5beta1 antibody did, in fact, induce differentiation. Although CH-8 cells adhered to GTP-free TG2 via integrin alpha5beta1, the cells failed to undergo hypertrophic differentiation. Thus, integrin alpha5beta1 is critical for the chondrocyte hypertrophic differentiation induced by GTP-bound TG2, and this induction is ligand dependent.     

Extracellular fragment of brain-specific angiogenesis inhibitor 1 suppresses endothelial cell proliferation by blocking alphavbeta5 integrin

Brain-specific angiogenesis inhibitor 1 (BAI1) is a transmembrane protein with anti-angiogenic activity. The mechanisms underlying BAI1 activity are unknown. In this study, we found that overexpression of BAI1 increased cell death in human umbilical vein endothelial cells (HUVECs) and, to a lesser degree, in SHSY5Y and U343 cells. Conditioned medium from BAI1-transfected U343 cells inhibited proliferation of HUVECs, and this effect was neutralized by addition of anti-BAI1 serum. The conditioned medium contained four cleavage products of the BAI1 extracellular domain. BAI1's middle extracellular region containing five thrombospondin type 1 repeats (BAI1-TSR) was sufficient for BAI1's antiproliferative effect on HUVECs. BAI1's action on HUVECs was blocked by anti-alpha(v) integrin, but not by anti-CD36 antibody treatment. Introduction of alpha(v)beta(5) integrin into HEK293 cells rendered them susceptible to cell death by BAI1, and BAI1-TSR bound with alpha(v)beta(5) integrin, but not to alpha(v)beta(3) integrin in brain tissue. Fluorescent BAI1-TSR colocalized with alpha(v)beta(5) integrin in HUVECs. Together, our results indicate that BAI1 has antiproliferative action on surrounding endothelial cells by blocking alpha(v)beta(5) integrin, and its active region is BAI1-TSR. BAI1-TSR could be valuable for regulating brain angiogenesis.     

Inhibition of binding of fibronectin to matrix assembly sites by anti- integrin (alpha 5 beta 1) antibodies

Fibroblasts have cell surface sites that mediate assembly of plasma and cellular fibronectin into the extracellular matrix. Cell adhesion to fibronectin can be mediated by the interaction of an integrin (alpha 5 beta 1) with the Arg-Gly-Asp-Ser (RGDS)-containing cell adhesion region of fibronectin. We have attempted to elucidate the role of the alpha 5 beta 1 fibronectin receptor in assembly of fibronectin in matrices. Rat monoclonal antibody mAb 13, which recognizes the integrin beta 1 subunit, completely blocked binding and matrix assembly of 125I-fibronectin as well as binding of the 125I-70-kD amino-terminal fragment of fibronectin (70 kD) to fibroblast cell layers. Fab fragments of the anti-beta 1 antibody were also inhibitory. Antibody mAb 16, which recognizes the integrin alpha 5 subunit, partially blocked binding of 125I-fibronectin and 125I-70-kD. When cell layers were coincubated with fluoresceinated fibronectin and either anti-beta 1 or anti-alpha 5, anti-beta 1 was a more effective inhibitor than anti-alpha 5 of binding of labeled fibronectin to the cell layer. Inhibition of 125I-fibronectin binding by anti-beta 1 IgG occurred within 20 min. Inhibition of 125I-fibronectin binding by anti-beta 1 Fab fragments or IgG could not be overcome with increasing concentrations of fibronectin, suggesting that anti-beta 1 and exogenous fibronectin may not compete for the same binding site. No beta 1-containing integrin bound to immobilized 70 kD. These data indicate that the beta 1 subunit plays an important role in binding and assembly of exogenous fibronectin, perhaps by participation in the organization, regeneration, or cycling of the assembly site rather than by a direct interaction with fibronectin.     

Activation-dependent adhesion of human platelets to Cyr61 and Fisp12/mouse connective tissue growth factor is mediated through integrin alpha(IIb)beta(3).

Cyr61 and connective tissue growth factor (CTGF), members of a newly identified family of extracellular matrix-associated signaling molecules, are found to mediate cell adhesion, promote cell migration and enhance growth factor-induced cell proliferation in vitro, and induce angiogenesis in vivo. We previously showed that vascular endothelial cell adhesion and migration to Cyr61 and Fisp12 (mouse CTGF) are mediated through integrin alpha(v)beta(3). Both Cyr61 and Fisp12/mCTGF are present in normal blood vessel walls, and it has been demonstrated that CTGF is overexpressed in advanced atherosclerotic lesions. In the present study, we examined whether Cyr61 and Fisp12/mCTGF could serve as substrates for platelet adhesion. Agonist (ADP, thrombin, or U46619)-stimulated but not resting platelets adhered to both Cyr61 and Fisp12/mCTGF, and this process was completely inhibited by prostaglandin I(2), which prevents platelet activation. The specificity of Cyr61- and Fisp12/mCTGF-mediated platelet adhesion was demonstrated by specific inhibition of this process with polyclonal anti-Cyr61 and anti-Fisp12/mCTGF antibodies, respectively. The adhesion of ADP-activated platelets to both proteins was divalent cation-dependent and was blocked by RGDS, HHLGGAKQAGDV, or echistatin, but not by RGES. Furthermore, this process was specifically inhibited by the monoclonal antibody AP-2 (anti-alpha(IIb)beta(3)), but not by LM609 (anti-alpha(v)beta(3)), indicating that the interaction is mediated through integrin alpha(IIb)beta(3). In a solid phase binding assay, activated alpha(IIb)beta(3), purified by RGD affinity chromatography, bound to immobilized Cyr61 and Fisp12/mCTGF in a dose-dependent and RGD-inhibitable manner. In contrast, unactivated alpha(IIb)beta(3) failed to bind to either protein. Collectively, these findings identify Cyr61 and Fisp12/mCTGF as two novel activation-dependent adhesive ligands for the integrin alpha(IIb)beta(3) on human platelets, and implicate a functional role for these proteins in hemostasis and thrombosis.     

Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2

Interaction between integrin alphavbeta3 and extracellular matrix is crucial for endothelial cells sprouting from capillaries and for angiogenesis. Furthermore, integrin-mediated outside-in signals co-operate with growth factor receptors to promote cell proliferation and motility. To determine a potential regulation of angiogenic inducer receptors by the integrin system, we investigated the interaction between alphavbeta3 integrin and tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR-2) in human endothelial cells. We report that tyrosine-phosphorylated VEGFR-2 co-immunoprecipitated with beta3 integrin subunit, but not with beta1 or beta5, from cells stimulated with VEGF-A165. VEGFR-2 phosphorylation and mitogenicity induced by VEGF-A165 were enhanced in cells plated on the alphavbeta3 ligand, vitronectin, compared with cells plated on the alpha5beta1 ligand, fibronectin or the alpha2beta1 ligand, collagen. BV4 anti-beta3 integrin mAb, which does not interfere with endothelial cell adhesion to vitronectin, reduced (i) the tyrosine phosphorylation of VEGFR-2; (ii) the activation of downstream transductor phosphoinositide 3-OH kinase; and (iii) biological effects triggered by VEGF-A165. These results indicate a new role for alphavbeta3 integrin in the activation of an in vitro angiogenic program in endothelial cells. Besides being the most important survival system for nascent vessels by regulating cell adhesion to matrix, alphavbeta3 integrin participates in the full activation of VEGFR-2 triggered by VEGF-A, which is an important angiogenic inducer in tumors, inflammation and tissue regeneration.     

The CXC-chemokine CXCL4 interacts with integrins implicated in angiogenesis

The human CXC-chemokine CXCL4 is a potent inhibitor of tumor-induced angiogenesis. Considering that CXCL4 is sequestered in platelet alpha-granules and released following platelet activation in the vicinity of vessel wall injury, we tested the hypothesis that CXCL4 might function as a ligand for integrins. Integrins are a family of adhesion receptors that play a crucial role in angiogenesis by regulating early angiogenic processes, such as endothelial cell adhesion and migration. Here, we show that CXCL4 interacts with alphavbeta3 on the surface of alphavbeta3-CHO. More importantly, human umbilical vein endothelial cells adhere to immobilized CXCL4 through alphavbeta3 integrin, and also through other integrins, such as alphavbeta5 and alpha5beta1. We further demonstrate that CXCL4-integrin interaction is of functional significance in vitro, since immobilized CXCL4 supported endothelial cell spreading and migration in an integrin-dependent manner. Soluble CXCL4, in turn, inhibits integrin-dependent endothelial cell adhesion and migration. As a whole, our study identifies integrins as novel receptors for CXCL4 that may contribute to its antiangiogenic effect.     

A co-clustering model involving alpha5beta1 integrin for the biological effects of GPI-anchored human carcinoembryonic antigen (CEA)

CEA functions as an intercellular adhesion molecule and is up-regulated in a wide variety of human cancers, including colon, breast and lung. Its over-expression inhibits cellular differentiation, blocks cell polarization, distorts tissue architecture, and inhibits anoikis of many different cell types. Here we report results concerning the molecular mechanism involved in these biological effects, where relatively rapid molecular changes not requiring alterations in gene expression were emphasized. Confocal microscopy experiments showed that antibody-mediated clustering of a deletion mutant of CEA (DeltaNCEA), normally incapable of self binding and clustering, led to the co-localization of integrin alpha5beta1 with patches of DeltaNCEA on the cell surface. Activation of alpha5, as defined by an anti-alpha5 mAb-sensitive increase in cell adhesion to immobilized fibronectin, and an increased binding of soluble fibronectin to cells, was also observed. This was accompanied by the recruitment of integrin-linked kinase (ILK), protein kinase B (PKB/Akt), and the mitogen-activated protein kinase (MAPK) to membrane microdomains and the phosphorylation of Akt and MAPK. Inhibition of PI3-K and ILK, but not MAPK, prevented the alpha5beta1 integrin activation. Conversely, anti-alpha5 antibody inhibited the PI3-K-mediated activation of Akt, implying the involvement of outside-in and inside-out signaling in integrin activation. Therefore we propose that CEA-mediated signaling involves clustering of CEA and co-clustering and activation of the alpha5beta1 and associated specific signaling elements on the internal surfaces of membrane microdomains. These changes may represent a molecular mechanism for the biological effects of CEA.     

Identification of a novel integrin alpha 6 beta 1 binding site in the angiogenic inducer CCN1 (CYR61)

The angiogenic inducer CCN1 (cysteine-rich 61, CYR61), a secreted matricellular protein of the CCN family, is a ligand of multiple integrins, including alpha 6 beta 1. Previous studies have shown that CCN1 interaction with integrin alpha 6 beta 1 mediates adhesion of fibroblasts, endothelial cells, and smooth muscle cells, as well as migration of smooth muscle cells. Recently, we have reported that CCN1-induced tubule formation of unactivated endothelial cells is also mediated through integrin alpha 6 beta 1. In this study, we demonstrate that human skin fibroblasts adhere specifically to the T1 sequence (GQKCIVQTTSWSQCSKS) within domain III of CCN1, and this process is blocked by anti-alpha 6 and anti-beta 1 monoclonal antibodies. Alanine substitution mutagenesis of the T1 sequence further defines the sequence TTSWSQCSKS as the critical determinant for mediating alpha 6 beta 1-dependent adhesion. Soluble T1 peptide specifically inhibits fibroblast adhesion to CCN1 in a dose-dependent manner. Furthermore, T1 also inhibits cell adhesion to other alpha 6 beta 1 ligands, including CCN2 (CTGF), CCN3 (NOV), and laminin, but not to ligands of other integrins. In addition, T1 specifically inhibits alpha 6 beta 1-dependent tubule formation of unactivated endothelial cells in a CCN1-containing collagen gel matrix. To confirm that T1 binds integrin alpha 6 beta 1 directly, we perform affinity chromatography and show that integrin alpha 6 beta 1 is isolated from an octylglucoside extract of fibroblasts on T1-coupled Affi-gel. Taken together, these findings define the T1 sequence in CCN1 as a novel binding motif for integrin alpha 6 beta 1, providing the basis for the development of peptide mimetics to examine the functional role of alpha 6 beta 1 in angiogenesis.     

Sphingosine 1-phosphate-induced endothelial cell migration requires the expression of EDG-1 and EDG-3 receptors and Rho-dependent activation of alpha vbeta3- and beta1-containing integrins

Sphingosine 1-phosphate (SPP), a platelet-derived bioactive lysophospholipid, is a regulator of angiogenesis. However, molecular mechanisms involved in SPP-induced angiogenic responses are not fully defined. Here we report the molecular mechanisms involved in SPP-induced human umbilical vein endothelial cell (HUVEC) adhesion and migration. SPP-induced HUVEC migration is potently inhibited by antisense phosphothioate oligonucleotides against EDG-1 as well as EDG-3 receptors. In addition, C3 exotoxin blocked SPP-induced cell attachment, spreading and migration on fibronectin-, vitronectin- and Matrigel-coated surfaces, suggesting that endothelial differentiation gene receptor signaling via the Rho pathway is critical for SPP-induced cell migration. Indeed, SPP induced Rho activation in an adherence-independent manner, whereas Rac activation was dispensible for cell attachment and focal contact formation. Interestingly, both EDG-1 and -3 receptors were required for Rho activation. Since integrins are critical for cell adhesion, migration, and angiogenesis, we examined the effects of blocking antibodies against alpha(v)beta(3), beta(1), or beta(3) integrins. SPP induced Rho-dependent integrin clustering into focal contact sites, which was essential for cell adhesion, spreading and migration. Blockage of alpha(v)beta(3)- or beta(1)-containing integrins inhibited SPP-induced HUVEC migration. Together our results suggest that endothelial differentiation gene receptor-mediated Rho signaling is required for the activation of integrin alpha(v)beta(3) as well as beta(1)-containing integrins, leading to the formation of initial focal contacts and endothelial cell migration.     

Analysis of the roles of RGD-binding integrins, alpha(4)/alpha(9) integrins, alpha(6) integrins, and CD9 in the interaction of the fertilin beta (ADAM2) disintegrin domain with the mouse egg membrane

Fertilin beta (also known as ADAM2), a mammalian sperm protein that mediates gamete cell adhesion during fertilization, is a member of the ADAM protein family whose members have disintegrin domains with homology to integrin ligands found in snake venoms. Fertilin beta utilizes an ECD sequence within its disintegrin domain to interact with the egg plasma membrane; the Asp is especially critical. Based on what is known about different integrin subfamilies and their ligands, we sought to characterize fertilin beta binding sites on mouse eggs, focusing on integrin subfamilies that recognize short peptide sequences that include an Asp residue: the alpha(5)/alpha(8)/alpha(v)/alpha(IIb) or RGD-binding subfamily (alpha(5)beta(1), alpha(8)beta(1), alpha(V)beta(1), alpha(V)beta(3), alpha(V)beta(5), alpha(V)beta(6), alpha(V)beta(8), and alpha(IIb)beta(3)) and the alpha(4)/alpha(9) subfamily (alpha(4)beta(1), alpha(9)beta(1), and alpha(4)beta(7)). We tested peptide sequences known to perturb interactions mediated by these integrins in two different assays for fertilin beta binding. Peptides with the sequence MLDG, which perturb alpha(4)/alpha(9) integrin-mediated interactions, significantly inhibit fertilin beta binding to eggs, which suggests a role for a member of this integrin subfamily as a fertilin beta receptor. RGD peptides, which perturb alpha(5)/alpha(8)/alpha(v)/alpha(IIb) integrin-mediated interactions, have partial inhibitory activity. The anti-alpha(6) antibody GoH3 has little or no inhibitory activity. An antibody to the integrin-associated tetraspanin protein CD9 inhibits the binding of a multivalent presentation of fertilin beta (immobilized on beads) but not soluble fertilin beta, which we speculate has implications for the role of CD9 in the strengthening of fertilin beta-mediated cell adhesion but not in initial ligand binding.     

Murine lipid phosphate phosphohydrolase-3 acts as a cell-associated integrin ligand

Lipid phosphate phosphohydrolase-3 (LPP3) is a cell surface protein that exhibits ectoenzyme activity. Previously, we identified human LPP3 in a functional assay of angiogenesis and showed that the Arg-Gly-Asp (RGD) motif in the proposed second extracellular domain interacts with a subset of integrins to mediate cell-cell adhesion. In contrast to the RGD domain of human LPP3, murine Lpp3 contains a variant sequence, Arg-Gly-Glu (RGE). Whether the RGE motif of murine Lpp3 mediates cell-cell interaction has not been studied. In this report, we test the hypothesis that the cell adhesion function of the LPP3 protein is conserved across mouse and human. A glutathione S-transferase (GST) fusion protein of the proposed second extracellular loop of the murine Lpp3 sequence (GST-mLpp3-RGE) promoted attachment of cells in a long-term cell adhesion assay. GST-mLpp3-RGE interacted with alpha(5)beta(1) and alpha(v)beta(3) integrins in a solid-phase ELISA, while a mutant control, GST-hLPP3-RAD, did not. Long-term adhesion of endothelial cells to GST-mLpp3-RGE induced phosphorylation of FAK, SHC, and CAS, whereas adhesion to GST-hLPP3-RAD failed to do so. Upon long-term adhesion both the GST-hLPP3-RGD and GST-mLpp3-RGE substrates bound to the alpha(5)beta(1) integrin of FRT-alpha(5)(+) cells, an interaction that was inhibited by an anti-alpha(5) integrin antibody. In addition, a cell aggregation assay showed that the intact mLpp3-RGE protein interacts with alpha(5)beta(1) and alpha(v)beta(3) integrins expressed by adjacent cells, an interaction that can be blocked by GRGDSP peptides and anti-LPP3-RGD antibodies. These data, together with the known importance of integrins in angiogenesis, provide a mechanism for the function of LPP3 in cell-cell interactions in both human and mouse.