Substrate specificity of alpha(v)beta(3) integrin-mediated cell migration and phosphatidylinositol 3-kinase/AKT pathway activation

The alpha(v)beta(3) integrin has been shown to bind several ligands, including osteopontin and vitronectin. Its role in modulating cell migration and downstream signaling pathways in response to specific extracellular matrix ligands has been investigated in this study. Highly invasive prostate cancer PC3 cells that constitutively express alpha(v)beta(3) adhere and migrate on osteopontin and vitronectin in an alpha(v)beta(3)-dependent manner. However, exogenous expression of alpha(v)beta(3) in noninvasive prostate cancer LNCaP (beta(3)-LNCaP) cells mediates adhesion and migration on vitronectin but not on osteopontin. Activation of alpha(v)beta(3) by epidermal growth factor stimulation is required to mediate adhesion to osteopontin but is not sufficient to support migration on this substrate. We show that alpha(v)beta(3)-mediated cell migration requires activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB/AKT) pathway since wortmannin, a PI 3-kinase inhibitor, prevents PC3 cell migration on both osteopontin and vitronectin; furthermore, alpha(v)beta(3) engagement by osteopontin and vitronectin activates the PI 3-kinase/AKT pathway. Migration of beta(3)-LNCaP cells on vitronectin also occurs through activation of the PI 3-kinase pathway; however, AKT phosphorylation is not increased upon engagement by osteopontin. Furthermore, phosphorylation of focal adhesion kinase (FAK), known to support cell migration in beta(3)-LNCaP cells, is detected on both substrates. Thus, in PC3 cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin and osteopontin; in beta(3)-LNCaP cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin, whereas adhesion to osteopontin does not support alpha(v)beta(3)-mediated cell migration and PI 3-kinase/AKT pathway activation. We conclude therefore that alpha(v)beta(3) exists in multiple functional states that can bind either selectively vitronectin or both vitronectin and osteopontin and that can differentially activate cell migration and intracellular signaling pathways in a ligand-specific manner.     

Inhibitory role of alpha 6 beta 4-associated erbB-2 and phosphoinositide 3-kinase in keratinocyte haptotactic migration dependent on alpha 3 beta 1 integrin

Keratinocytes and other epithelial cells express two receptors for the basement membrane (BM) extracellular matrix component laminin-5 (Ln-5), integrins alpha 3 beta 1 and alpha 6 beta 4. While alpha 3 beta 1 mediates adhesion, spreading, and migration (Kreidberg, J.A. 2000. Curr. Opin. Cell Biol. 12:548--553), alpha 6 beta 4 is involved in BM anchorage via hemidesmosomes (Borradori, L., and A. Sonnenberg. 1999. J. Invest. Dermatol. 112:411--418). We investigated a possible regulatory interplay between alpha 3 beta 1 and alpha 6 beta 4 in cell motility using HaCaT keratinocytes as a model. We found that alpha 6 beta 4 antibodies inhibit alpha 3 beta 1-mediated migration on Ln-5, but only when migration is haptotactic (i.e., spontaneous or stimulated by alpha 3 beta 1 activation), and not when chemotactic (i.e., triggered by epidermal growth factor receptor). Inhibition of migration by alpha 6 beta 4 depends upon phosphoinositide 3-kinase (PI3-K) since it is abolished by PI3-K blockers and by dominant-negative PI3-K, and constitutively active PI3-K prevents haptotaxis. In HaCaT cells incubated with anti-alpha 6 beta 4 antibodies, activation of PI3-K is mediated by alpha 6 beta 4-associated erbB-2, as indicated by erbB-2 autophosphorylation and erbB-2/p85 PI3-K coprecipitation. Furthermore, dominant-negative erbB-2 abolishes inhibition of haptotaxis by anti-alpha 6 beta 4 antibodies. These results support a model whereby (a) haptotactic cell migration on Ln-5 is regulated by concerted action of alpha 3beta 1 and alpha 6 beta 4 integrins, (b) alpha 6 beta 4-associated erbB-2 and PI3-K negatively affect haptotaxis, and (c) chemotaxis on Ln-5 is not affected by alpha 6 beta 4 antibodies and may require PI3-K activity. This model could be of general relevance to motility of epithelial cells in contact with BM.     

Beta 1 and beta 3 integrins have different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix.

Extracellular matrix receptors on ductus arteriosus smooth muscle cells (SMC) must enable the cells to migrate through both interstitial and basement membrane matrices to form intimal mounds during postnatal ductus closure. We examined the role of beta 1 and beta 3 integrin receptors on SMC adhesion and migration. Using a new assay to measure cell migration, we found that lamb ductus arteriosus SMC attach to and migrate over surfaces coated with fibronectin (FN), laminin (LN), vitronectin (VN), and collagens I (I) and IV (IV). Blocking antibodies, specific to different integrin complexes, showed that SMC adhesion to FN, LN, I, and IV depended exclusively on functioning beta 1 integrins with little, if any, contribution by the alpha V beta 3 integrin; on the other hand, cell migration over these substrates depended to a large extent on the alpha V beta 3 receptor. Immunofluorescent staining demonstrated that during the early phase of SMC migration, the beta 1 integrins organized rapidly into focal plaques that, with time, gradually covered the cell's basal surface; on the other hand, the beta 3 receptor remained concentrated at all times at the cell's margins. Ligand affinity chromatography and immunoprecipitation techniques identified a unique series of beta 1 integrins binding to each matrix component: FN (alpha 5 beta 1, alpha 3 beta 1, alpha V beta 1), LN (alpha 1 beta 1, alpha 7 beta 1), VN (alpha V beta 1), I (alpha 1 beta 1, alpha 2 beta 1), and IV (alpha 1 beta 1). In contrast, the beta 3 integrin, alpha V beta 3, bound to all the substrates tested: FN, LN, VN, I, and IV. The results indicate that beta 1 and beta 3 integrins may play different roles in attachment and migration as SMC move through the vascular extracellular matrix to produce obliteration of the ductus arteriosus lumen.     

Ligand-dependent activation of integrin alpha vbeta 3

The ability of leukocytes to self-regulate adhesion during transendothelial and extravascular migration is fundamental to the performance of immune surveillance in complex extracellular matrices. Leukocyte adhesion is regulated through the modulation of integrin receptors such as alpha(v)beta(3). In this study, we examined the activation of alpha(v)beta(3) resulting from attachment to vitronectin or fibronectin. In K562 cells stably expressing transfected alpha(v)beta(3), adhesion to vitronectin required tyrosine phosphorylation of the beta(3) subunit and activation of phosphoinositide 3-kinase and protein kinase C. In contrast, adhesion to fibronectin proceeded without beta(3)-tyrosine phosphorylation or the activities of phosphoinositide 3-kinase or protein kinase C. Firm adhesion to both ligands and actin stress fiber formation required both Syk and Rho activity, suggesting that each ligand employs unique signaling pathways to achieve an active integrin complex, likely merging at a common RhoGEF such as Vav. Distinct signaling by a single integrin species interacting with different ligands permits initiation of additional cellular processes specific to the current task and provides an explanation for what has been described as promiscuous ligand specificity among integrins.     

The CK2 phosphorylation of vitronectin. Promotion of cell adhesion via the alpha(v)beta 3-phosphatidylinositol 3-kinase pathway

Phosphorylation of vitronectin (Vn) by casein kinase II was previously shown to occur at Thr50 and Thr57 and to augment a major physiological function of vitronectin-cell adhesion and spreading. Here we show that this phosphorylation increases cell adhesion via the alpha(v)beta3 (not via the alpha(v)beta5 integrin), suggesting that alpha(v)beta3 differs from alpha(v)beta5 in its biorecognition profile. Although both the phospho (CK2-PVn) and non-phospho (Vn) analogs of vitronectin (simulated by mutants Vn(T50E,T57E), and Vn(T50A,T57A), respectively) trigger the alpha(v)beta3 as well as the alpha(v)beta5 integrins, and equally activate the ERK pathway, these two forms are different in their activation of the focal adhesion kinase/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) pathway. Specifically, we show (i) that, upon exposure of cells to Vn/CK2-PVn, their PKB activation depends on the availability of the alpha(v)beta3 integrin on their surface; (ii) that upon adhesion of the beta3-transfected cells onto the CK2-PVn, the extent of PKB activation coincides with the enhanced adhesion of these cells, and (iii) that both the PKB activation and the elevation in the adhesion of these cells is PI3K-dependent. The occurrence of a cell surface receptor that specifically distinguishes between a phosphorylated and a non-phosphorylated analog of Vn, together with the fact that it preferentially activates a distinct intra-cellular signaling pathway, suggest that extra-cellular CK2 phosphorylation may play an important role in the regulation of cell adhesion and migration.     

M-CSF induces the stable interaction of cFms with alphaVbeta3 integrin in osteoclasts

The macrophage colony stimulating factor receptor (cFms) and alpha(V)beta(3) integrin are both abundantly expressed and play critical roles in the differentiation, survival and migration of osteoclasts. We have previously demonstrated that cross-talk between cFms- and alpha(V)beta(3)-mediated signaling pathways regulated the cytoskeletal organization required for osteoclast migration. To investigate the nature of interaction between the two receptors, we sequentially used anion-exchange chromatography and immunoprecipitation to purify alpha(V)beta(3)-associated protein complexes. We have demonstrated that cFms stably associated with alpha(V)beta(3) in osteoclasts during adhesion, and that the association was induced by macrophage colony stimulating factor (M-CSF) stimulation. However, the kinetics of association of alpha(V)beta(3) and cFms did not correlate with the kinetics of tyrosine phosphorylation of cFms. Instead, maximally observed alpha(V)beta(3)/cFms association was after the peak of cFms tyrosine phosphorylation and correlated inversely with the total amount of cFms remaining. Furthermore, the complex containing cFms and alpha(V)beta(3) also contained a number of other signaling molecules including Pyk2, p130(Cas) and c-Cbl, known downstream regulators of the integrin-mediated signaling pathways in osteoclasts. In the presence of M-CSF, co-localization of alpha(V)beta(3) integrin and cFms was identified in the podosomal actin ring of the osteoclast during adhesion on glass. Interestingly, co-localization of both receptors was not found in the sealing zone, but in punctate structures associated with adhesion- or transcytosis-like structures in osteoclasts on bone. Taken together, we suggest that the association of alpha(V)beta(3) and cFms could be the result of signaling following tyrosine phosphorylation of cFms. The recruitment of cFms to alpha(V)beta(3) integrin may be an integral part of a larger signaling complex via which both of adhesion- and growth factor receptors coordinately regulate osteoclast adhesion, motility and membrane trafficking.     

Interaction and functional association of protein disulfide isomerase with alphaVbeta3 integrin on endothelial cells

Adhesive properties of endothelial cells are influenced by the thioldisulfide balance. However, the molecular mechanism of this effect is unclear, although recent observations indicate that integrin receptors may be direct targets for redox modulation. The purpose of this study was to examine whether protein disulfide isomerase (PDI) is directly involved in this process. As manganese ions are known to affect the thioldisulfide balance and activate integrins to maximal affinity, we searched for PDI interactions with integrins, particularly with alpha(V)beta(3), in Mn(2+)-treated endothelial cells. By employing confocal microscopy, flow cytometry and coimmunoprecipitation experiments, we showed that exposure of endothelial cells to Mn(2+) resulted in: (a) the appearance of surface protein thiol groups, which can be found in PDI and alpha(V)beta(3), and both proteins colocalizing on the cellular surface; and (b) the formation of the PDI-alpha(V)beta(3) complex, which dissociates upon reduction. In addition, PDI in a complex with alpha(V)beta(3) induces conversion of the integrin to the ligand-competent high-affinity state, as evidenced by increased binding of vitronectin. The membrane-impermeable sulfhydryl blockers 3-N-maleimidylpropionyl biocytin 3-N-maleimidylpropionyl biocytin and p-chloromercuriphenyl sulfonate, as well as the PDI inhibitors bacitracin, MA3 018, and MA3 019, abolished the binding of vitronectin and LM609 to endothelial cells that is activated by Mn(2+). Consistently, LM609 almost completely blocked binding of vitronectin to such cells. The formation of the PDI-alpha(V)beta(3) stoichiometric complex was further demonstrated by surface plasmon resonance analysis, which showed that the initial reversible binding of PDI becomes irreversible in the presence of Mn(2+), probably mediated by disulfide bonds. Thus, we show that Mn(2+) simultaneously modulates the thiol isomerase activity of PDI that is bound to alpha(V)beta(3) and induces its transition to the ligand-competent state, suggesting an alternative mechanism of integrin regulation.     

The pattern of enhancement of Src kinase activity on platelet-derived growth factor stimulation of glioblastoma cells is affected by the integrin engaged

Enhanced expression of both integrin alpha v beta 3 and platelet-derived growth factor receptor (PDGFr) has been described in glioblastoma tumors. We therefore explored the possibility that integrin alpha v beta 3 cooperates with PDGFr to promote cell migration in glioblastoma cells, and extended the study to identify the Src family members that are activated on PDGF stimulation. Glioblastoma cells utilize integrins alpha v beta 3 and alpha v beta 5 to mediate vitronectin attachment. We found that physiologic PDGF stimulation (83 pm, 10 min) of vitronectin-adherent cells promoted the specific recruitment of integrin alpha v beta 3-containing focal adhesions to the cell cortex and alpha v beta 3-mediated cell motility. Analysis of PDGFr immunoprecipitates indicated an association of the PDGFr beta with integrin alpha v beta 3, but not integrin alpha v beta 5. Cells plated onto collagen or laminin, which engage different integrins, exhibited significantly less migration on PDGF stimulation, indicating a cooperation of alpha v beta 3 and the PDGFr beta in glioblastoma cells that promotes migration. Further analysis of the cells plated onto vitronectin indicated that PDGF stimulation caused an increase in Src kinase activity, which was associated with integrin alpha v beta 3. In the vitronectin-adherent cells, Lyn was associated preferentially with alpha v beta 3 both in the presence and absence of PDGF stimulation. In contrast, Fyn was associated with both alpha v beta 3 and alpha v beta 5. Moreover, PDGF stimulation increased the activity of Lyn, but not Fyn, in vitronectin-adherent cells, and the activity of Fyn, but not Lyn, in laminin-adherent cells. Using cells attached to mAb anti-alpha v beta 3 or mAb anti-integrin alpha 6, we confirmed the activation of specific members of the Src kinase family with PDGF stimulation. Down-regulation of Lyn expression by siRNA significantly inhibited the cell migration mediated by integrin alpha v beta 3 in PDGF-stimulated cells, demonstrating the PDGFr beta cooperates with integrin alpha v beta 3 in promoting the motility of vitronectin-adherent glioblastoma cells through a Lyn kinase-mediated pathway. Notably, the data indicate that engagement of different integrins alters the identity of the Src family members that are activated on stimulation with PDGF.     

The basement membrane protein laminin-5 acts as a soluble cell motility factor

One of the basement membrane (BM) proteins, laminin-5 (LN5), is known to support efficient cell adhesion and migration through interaction with integrins on the basal plasma membrane. Here, we show that a soluble form of LN5 induced migration of human epithelial cells and carcinoma cells by interacting with integrins on the apical cell surface. Although both LN5 and laminin-10/11 (LN10/11) promoted cell migration when coated onto a plastic surface as insoluble substrata, only LN5 stimulated cell migration in its soluble form on other substrata such as fibronectin (FN), vitronectin (VN) and collagen. Soluble LN5 interacted with integrins alpha3beta1 and alpha6beta1 on the apical cell surface and stimulated cell migration, while the cell morphology was largely dependent on the underlying substratum. Thus, integrin signals from the apical surface and the basal surface synergistically regulated cytoskeletal organization and cell motility. Soluble and insoluble LN5 induced cell motility by activating signal pathways via protein kinase C (PKC), phosphoinositide 3-OH kinase (PI3-K) and MAP kinase. The PKC dependency was more prominent for soluble LN5 than insoluble LN5, and was absent in the stimulation by insoluble LN10/11. In vitro scratch assays with keratinocytes, self-produced soluble LN5 bound to the apical cell surface of migrating cells at the scratched edges, suggesting that soluble LN5 may contribute to cell migration in pathological conditions such as wound healing and tumor invasion.     

Integrin beta 1- and beta 3-mediated endothelial cell migration is triggered through distinct signaling mechanisms

Human umbilical vein endothelial cell attachment, spreading and migration on collagen and vitronectin are mediated by integrins alpha 2 beta 1 and alpha v beta 3, respectively, and these events take place in the absence of cytokines, growth factors, or chemoattractants. Cell attachment and spreading on these ligands occur in the absence of extracellular calcium, as does migration on collagen. In contrast, vitronectin-mediated migration is absolutely dependent on the presence of extracellular calcium. Cell contact with immobilized vitronectin or anti-alpha v beta 3 mAbs promotes a measurable rise in [Ca2+]i which requires an extracellular calcium source, whereas collagen, or anti- alpha 2 beta 1 mAbs fail to promote this signaling event. In fact, vitronectin-mediated migration and the rise in intracellular calcium showed the same dose dependence on extracellular calcium. While vitronectin and collagen differ in their ability to induce a calcium influx both ligands or antibodies to their respective integrins promote an equivalent increase in intracellular pH consistent with activation of the Na/H antiporter an event independent of extracellular calcium. These results support two salient conclusions. Firstly, collagen and vitronectin, through their respective integrins, promote distinct intracellular signaling events. Secondly, the alpha v beta 3 specific influx of calcium is not required for cell spreading yet appears to facilitate cellular migration on vitronectin.     

Puromycin insensitive leucyl-specific aminopeptidase (PILSAP) is involved in the activation of endothelial integrins

We previously reported that mouse orthologue of puromycin insensitive leucyl-specific aminopeptidase (mPILSAP) played an important role in angiogenesis by regulating the proliferation and migration of endothelial cells (ECs) (Miyashita et al., 2002. Blood 99:3241-3249). Here, we examined the mechanism as to how mPILSAP regulates the migration of ECs. Cell adhesion through integrins plays a crucial role in cell migration, and ECs use at least type-1 collagen receptor integrin alpha2beta1, fibronectin receptor alpha5beta1, and vitronectin receptors alphavbeta3 and alphavbeta5. mPILSAP antisense oligodeoxynucleotide (AS-ODN) or leucinethiol (LT), a leucyl-aminopeptidase inhibitor, did not affect the attachment but did significantly inhibit the spreading of cells of the murine endothelial cell line MSS31 when they were plated on vitronectin-, fibronectin-, or type-1 collagen, although they did not affect the expression of integrin alpha2, alpha5, alphav, beta1, beta3, and beta5 subunits on the cell surface. AS-ODN and LT also inhibited the tyrosine phosphorylation of FAK when cells were plated on vitronectin, fibronectin, or type-1 collagen. This inhibition of cell spreading and of tyrosine phosphorylation of FAK could be negated by Mg(2+). These results suggest that mPILSAP is involved in the activation of endothelial integrins.     

Targeting of alpha(v) integrins interferes with FAK activation and smooth muscle cell migration and invasion

Aberrant migration of smooth muscle cells (SMCs) is a key feature of restenosis. Since extracellular matrix proteins and their receptors of the integrin family play a critical role in this process, it is instrumental to understand their contribution to cell migration and invasive motility of SMC on the molecular level. Therefore, we investigated the role of alpha(v)-containing integrins expressed by primary human coronary artery smooth muscle cells (hCASMCs) in vitronectin (VN)-initiated signaling events and cell migration. In hCASMC plated on VN, alpha(v)-containing integrins were localized at focal adhesion sites. Haptotactic stimulation through VN led to a dose-dependent increase in cell migration and concomitantly to enhanced tyrosine phosphorylation of focal adhesion kinase. Both events were completely blocked by a specific inhibitor of integrin alpha(v). Additionally, the integrin alpha(v) inhibitor abolished PDGF-BB-stimulated chemotactic migration. Confocal microscopy confirmed the increased tyrosine phosphorylation at VN-initiated focal contact sites in hCASMC, that was abolished upon alpha(v) inhibition. In vitro invasion of hCASMC was severely compromised in the presence of the integrin alpha(v) inhibitor paralleled by decreased levels of secreted matrix metalloprotease 2 (MMP-2). Together, integrin alpha(v) inhibition abrogates tyrosine phosphorylation at focal adhesion sites and diminishes MMP-2 secretion leading to reduced migration and invasion of hCASMCs.     

Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase

Herein we report that, despite the similarity of Rac2 to Rac1 (92% amino acid identity), macrophages derived from Rac2-/- mice, which continue to express Rac1, display a marked defect in alphavbeta3/alphavbeta5 and alpha4beta1 integrin-directed migration measured on vitronectin and fibronectin fragments (FN-H296), respectively. In contrast, mouse embryo fibroblasts derived from the Rac2 knockout mice utilize Rac1 for migration via alphavbeta3/alphavbeta5 and alpha4beta1. The genetic reconstitution of bone marrow-derived macrophages (BMM) with Rac2 restores the integrin-dependent migration of Rac2-deficient macrophages on vitronectin (VN) and FN-H296. The levels of GTP-Rac2 generated upon specific integrin engagement in wild type macrophages parallels the phenotypic defect observed in Rac2-deficient macrophages; i.e. FN-H296, alpha4beta1 > VN, alphavbeta3/alphavbeta5 > FN-CH271, alpha5beta1 > intact FN. In a COS7 cell system, the expression of Syk kinase alone is sufficient to convert the alpha4beta1 migration response to Rac2 dependence. Therefore, we present the first evidence that the alpha4beta1 receptor in blood cells has evolved a Syk-Rac2 signaling axis to transmit signals required for integrin-directed migration suggesting that Syk kinase in part encodes myeloid Rac2 specificity in vivo.     

Alpha1B-adrenoceptor signaling and cell motility: GTPase function of Gh/transglutaminase 2 inhibits cell migration through interaction with cytoplasmic tail of integrin alpha subunits

A multifunctional enzyme, G(h), is a GTP-binding protein that couples to the alpha(1B)-adrenoreceptor and stimulates phospholipase C-delta1 but also displays transglutaminase 2 (TG2) activity. G(h)/TG2 has been implicated to play a role in cell motility. In this study we have examined which function of G(h)/TG2 is involved in this cellular response and the molecular basis. Treatment of human aortic smooth muscle cell with epinephrine inhibits migration to fibronectin and vitronectin, and the inhibition is blocked by the alpha(1)-adrenoreceptor antagonist prazosin or chloroethylclonidine. Up-regulation or overexpression of G(h)/TG2 in human aortic smooth muscle cells, DDT1-MF2, or human embryonic kidney cells, HEK 293 cells, results in inhibition of the migratory activity, and stimulation of the alpha(1B)-adrenoreceptor with the alpha(1) agonist further augments the inhibition of migration of human aortic smooth muscle cells and DDT1-MF2. G(h)/TG2 is coimmunoprecipitated by an integrin alpha(5) antibody and binds to the cytoplasmic tail peptide of integrins alpha(5), alpha(v), and alpha(IIb) subunits in the presence of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). Mutation of Lys-Arg residues in the GFFKR motif, present in the alpha(5)-tail, significantly reduces the binding of GTPgammaS-G(h)/TG2. Moreover, the motif-containing integrin alpha(5)-tail peptides block G(h)/TG2 coimmunoprecipitation and reverse the inhibition of the migratory activity of HEK 293 cells caused by overexpression G(h)/TG2. These results provide evidence that G(h) function initiates the modulation of cell motility via association of GTP-bound G(h)/TG2 with the GFFKR motif located in integrin alpha subunits.     

The contributions of integrin affinity and integrin-cytoskeletal engagement in endothelial and smooth muscle cell adhesion to vitronectin

The serine proteinase inhibitor, plasminogen activator inhibitor type-1 (PAI-1), binds to the adhesion protein vitronectin with high affinity at a site that is located directly adjacent to the vitronectin RGD integrin binding sequence. The binding of PAI-1 to vitronectin sterically blocks integrin access to this site and completely inhibits the binding of purified integrins to vitronectin; however, its inhibition of endothelial and smooth muscle cell adhesion to vitronectin is at most 50-75%. Because PAI-1 binds vitronectin with approximately 10-100-fold higher affinity than purified integrins, we have analyzed the mechanism whereby these cells are able to overcome this obstacle. Our studies exclude proteolytic removal of PAI-1 from vitronectin as the mechanism, and show instead that cell adhesion in the presence of PAI-1 is dependent on integrin-cytoskeleton engagement. Disrupting endothelial or smooth muscle cell actin polymerization and/or focal adhesion assembly reduces cell adhesion to vitronectin in the presence of PAI-1 to levels similar to that observed for the binding of purified integrins to vitronectin. Furthermore, endothelial cell, but not smooth muscle cell adhesion to vitronectin in the presence of PAI-1 requires both polymerized microtubules and actin, further demonstrating the importance of the cytoskeleton for integrin-mediated adhesion. Finally, we show that cell adhesion in the presence of PAI-1 leads to colocalization of PAI-1 with the integrins alphavbeta3 and alphavbeta5 at the cell-matrix interface.     

Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein.

Integrins are a complex family of divalent cation-dependent cell adhesion receptors composed of one alpha and one beta subunit noncovalently bound to one another. A subset of integrins contains the alpha v subunit in association with one of several beta subunits (e.g. beta 3, beta 5, beta 1). We have recently identified a novel integrin beta subunit, beta 6, that is present in a number of epithelial cell lines. Using a polyclonal antibody raised against the carboxyl-terminal peptide of beta 6, we have now identified the integrin heterodimer, alpha v beta 6, on the surface of two human carcinoma cell lines. Using affinity chromatography of lysates from the pancreatic carcinoma cell line, FG-2, we demonstrate that alpha v beta 6 binds to fibronectin, but not to vitronectin or collagen I. In contrast, the alpha v beta 5 integrin, which is also expressed on FG-2 cells, binds exclusively to vitronectin. Immobilized collagen I does not interact with alpha v integrins, but binds beta 1-containing integrins. Both alpha v beta 6 and alpha v beta 5 are eluted from their respective immobilized ligands by a hexa-peptide containing the sequence Arg-Gly-Asp (RGD). RGD is highly effective in the presence of Ca2+, somewhat less effective in Mg2+, and virtually inactive in Mn2+. These results suggest that alpha v beta 6 functions as an RGD-dependent fibronectin receptor in FG-2 carcinoma cells. In agreement with this notion, cell adhesion assays show that FG-2 cell attachment to fibronectin is only partially inhibited by anti-beta 1 integrin antibodies, implying that other fibronectin receptors may be involved. Taken together with recent reports on the vitronectin receptor function of alpha v beta 5, our results suggest that the previously described carcinoma cell integrin, alpha v beta x (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), is a mixture of at least two different receptors: alpha v beta 5, mediating adhesion to vitronectin, and alpha v beta 6, mediating adhesion to fibronectin.     

Antibodies to the vitronectin receptor (integrin alpha V beta 3) inhibit binding and infection of foot-and-mouth disease virus to cultured cells.

The amino acid sequence Arg-Gly-Asp (RGD) is highly conserved on the VP1 proteins of different serotypes and subtypes of foot-and-mouth disease virus (FMDV) and is essential for cell attachment. This sequence is also found in certain extracellular matrix proteins that bind to a family of cell surface receptors called integrins. Within the Picornaviridae family, enterovirus coxsackievirus A9 also has an RGD motif on its VP1 capsid protein and has recently been shown to utilize the vitronectin receptor integrin alpha V beta 3 as a receptor on monkey kidney cells. Competition binding experiments between type A12 FMDV and coxsackievirus A9 using BHK-21 and LLC-MK2 cells revealed shared receptor specificity between these two viruses. Polyclonal anti-serum to the vitronectin receptor and a monoclonal antibody to the alpha V subunit inhibited both FMDV binding and plaque formation, while a monoclonal antibody to the beta 3 subunit inhibited virus binding. In contrast, antibodies to the fibronectin receptor (alpha 5 beta 1) or to the integrin (alpha V beta 5) had no effect on either binding or plaque formation. These data demonstrate that the alpha V beta 3 vitronectin receptor can function as a receptor for FMDV.     

(alpha)v(beta)3 integrins and Pyk2 mediate insulin-like growth factor I activation of Src and mitogen-activated protein kinase in 3T3-L1 cells

IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.     

CCN3 (NOV) is a novel angiogenic regulator of the CCN protein family

CCN3 (NOV) is a matricellular protein of the CCN family, which also includes CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3). During development, CCN3 is expressed widely in derivatives of all three germ layers, and high levels of expression are observed in smooth muscle cells of the arterial vessel wall. Altered expression of CCN3 has been observed in a variety of tumors, including hepatocellular carcinomas, Wilm's tumors, Ewing's sarcomas, gliomas, rhabdomyosarcomas, and adrenocortical carcinomas. To understand its biological functions, we have investigated the activities of purified recombinant CCN3. We show that in endothelial cells, CCN3 supports cell adhesion, induces directed cell migration (chemotaxis), and promotes cell survival. Mechanistically, CCN3 supports human umbilical vein endothelial cell adhesion through multiple cell surface receptors, including integrins alphavbeta3, alpha5beta1, alpha6beta1, and heparan sulfate proteoglycans. In contrast, CCN3-induced cell migration is dependent on integrins alphavbeta3 and alpha5beta1, whereas alpha6beta1 does not play a role in this process. Although CCN3 does not contain a RGD sequence, it binds directly to immobilized integrins alphavbeta3 and alpha5beta1, with half-maximal binding occurring at 10 nm and 50 nm CCN3, respectively. Furthermore, CCN3 induces neovascularization when implanted in rat cornea, demonstrating that it is a novel angiogenic inducer. Together, these findings show that CCN3 is a ligand of integrins alphavbeta3 and alpha5beta1, acts directly upon endothelial cells to stimulate pro-angiogenic activities, and induces angiogenesis in vivo.