Regulation of tumor cell chemotaxis by type IV collagen is mediated by a Ca(2+)-dependent mechanism requiring CD47 and the integrin alpha(V)beta(3)

Studies from our laboratories demonstrated that synthetic peptides from the non-collagenous (NC-1) domain of the alpha3 (IV) chain of type IV collagen (COL IV) enhanced tumor cell adhesion (Han, J., Ohno, N., Monboisse, J. C., Pasco, S., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395-20401). We have isolated the receptors for the alpha3(IV)185-203 peptide from melanoma and prostate tumor cells and identified them as CD47/integrin-associated protein and the integrin alpha(V)beta(3) (Shahan, T. A., Ziaie, Z., Pasco, S., Fawzi, A., Bellon, G., Monboisse, J. C., and Kefalides, N. A. (1999) Cancer Res. 59, 4584-4590). In the present study we have examined the effect of CD47 and the integrin alpha(V)beta(3) on in vitro tumor cell chemotaxis and Ca(2+)(i) modulation in response to COL IV, from the anterior lens capsule (ALC-COL IV) and peptides from its NC-1 domain. COL IV as well as the alpha3(IV) peptide promoted tumor cell chemotaxis with an immediate increase in intracellular [Ca(2+)]. Treating tumor cells with CD47 and integrin alpha(V)beta(3)-reactive antibodies reduced chemotaxis as well as the rise in [Ca(2+)](i) in response to ALC-COL IV or the alpha3(IV)185-203 peptide but not to Engelbreth-Holm-Swarm-COL IV or fibronectin. The alpha3(IV)185-203 synthetic peptide stimulated an increase in calcium from intracellular stores exclusively, whereas ALC-COL IV, Engelbreth-Holm-Swarm-COL IV, and fibronectin stimulated Ca(2+) flux from both internal and external stores. Furthermore, treatment of the cells with Ca(2+) chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraaceticacid- acetomethoxy ester inhibited chemotaxis toward both ALC-COL IV and the alpha3(IV)185-203 peptide. These data indicate that CD47 and integrin alpha(V)beta(3) regulate tumor cell chemotaxis in response to COL IV and the alpha3(IV)185-203 peptide through a Ca(2+)-dependent mechanism.     

Vascular endothelial growth factor-induced migration of multiple myeloma cells is associated with beta 1 integrin- and phosphatidylinositol 3-kinase-dependent PKC alpha activation.

In multiple myeloma (MM), migration is necessary for the homing of tumor cells to bone marrow (BM), for expansion within the BM microenvironment, and for egress into the peripheral blood. In the present study we characterize the role of vascular endothelial growth factor (VEGF) and beta(1) integrin (CD29) in MM cell migration. We show that protein kinase C (PKC) alpha is translocated to the plasma membrane and activated by adhesion of MM cells to fibronectin and VEGF. We identify beta(1) integrin modulating VEGF-triggered MM cell migration on fibronectin. We show that transient enhancement of MM cell adhesion to fibronectin triggered by VEGF is dependent on the activity of both PKC and beta(1) integrin. Moreover, we demonstrate that PKC alpha is constitutively associated with beta(1) integrin. These data are consistent with PKC alpha-dependent exocytosis of activated beta(1) integrin to the plasma membrane, where its increased surface expression mediates binding to fibronectin; conversely, catalytically active PKC alpha-driven internalization of beta(1) integrin results in MM cell de-adhesion. We show that the regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85) is constitutively associated with FMS-like tyrosine kinase-1 (Flt-1). VEGF stimulates activation of PI 3-kinase, and both MM cell adhesion and migration are PI 3-kinase-dependent. Moreover, both VEGF-induced PI 3-kinase activation and beta(1) integrin-mediated binding to fibronectin are required for the recruitment and activation of PKC alpha. Time-lapse phase contrast video microscopy (TLVM) studies confirm the importance of these signaling components in VEGF-triggered MM cell migration on fibronectin.     

Cooperation between thrombospondin-1 type 1 repeat peptides and alpha(v)beta(3) integrin ligands to promote melanoma cell spreading and focal adhesion kinase phosphorylation.

CD47-binding sequences from the carboxyl-terminal domain of thrombospondin-1 (TSP1) are known to regulate activity of the alpha(v)beta(3) integrin (Gao, G., Lindberg, F. P., Dimitry, J. M., Brown, E. J., and Frazier, W. A. (1996) J. Cell Biol. 135, 533-544). Here we show that peptides from the type 1 repeats of TSP1 also stimulate alpha(v)beta(3) integrin function in melanoma cells. Addition of soluble peptide 246 (KRFKQDGGWSHWSPWSS) enhances spreading of A2058 melanoma cells on several alpha(v)beta(3) integrin ligands, including vitronectin, recombinant TSP1 fragments containing the Arg-Gly-Asp sequence, and native TSP1. This activity requires the Trp residues and is independent of CD36-binding sequences in the type 1 repeats. Recombinant type 1 repeats expressed as a glutathione S-transferase fusion protein also enhance spreading on vitronectin and TSP1. Activation of alpha(v)beta(3) integrin by the soluble peptide 246 stimulates organization of F-actin and increases tyrosine phosphorylation of focal adhesion kinase. In contrast, direct adhesion of melanoma cells on immobilized peptide 246 inhibits tyrosine phosphorylation of focal adhesion kinase. Stimulation of alpha(v)beta(3) integrin function by the type 1 repeat peptide differs from that induced by CD47-binding TSP1 peptides in that heparan sulfate proteoglycans are required and pertussis toxin does not inhibit the former activity. Thus, the type 1 repeats contain a second sequence of TSP1 that can enhance alpha(v)beta(3) integrin signaling, and these two sequences stimulate recognition of both vitronectin and TSP1 by the alpha(v)beta(3) integrin.     

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.     

The membrane-spanning domain of CD98 heavy chain promotes alpha(v)beta3 integrin signals in human extravillous trophoblasts

CD98 heavy chain (CD98hc) is expressed highly in developing human placental trophoblast. CD98hc is an amino acid transporter and is thought to function in cell fusion, adhesion, and invasion by interacting with integrins. In invasive extravillous trophoblast, alpha(v)beta(3) integrin is expressed in a temporally and spatially specific manner, which prompted us to investigate the potential role of CD98hc in signal transduction of alpha(v)beta(3) integrin. Immunocytochemistry of extravillous trophoblast derived from human placenta revealed that CD98hc colocalized with alpha(v)beta(3) integrin and with alpha(v)beta(3)-associated cytoplasmic proteins including paxillin, vinculin, and focal adhesion kinase. Coimmunoprecipitation of CD98hc and its mutants revealed that the transmembrane domain of CD98hc is necessary for the association of CD98hc with alpha(v)beta(3) integrin. When CD98hc negative liver cells (FLC4) were stably transfected with CD98hc and the extracellular domain of CD98hc was cross-linked by anti-CD98 antibody, FLC4 cells binding affinity to fibronectin and cell motility increased. The anti-CD98 antibody cross-linking promoted actin stress fiber formation and activation of signal transduction downstream of RhoA GTPase, and elevated the phosphorylation of focal adhesion kinase, paxillin, and protein kinase B. Pretreatment of transfected FLC4 cells with specific inhibitors for alpha(v)beta(3)integrin, phosphatidylinositol 3-kinase, and RhoA diminished these effects caused by anti-CD98 antibody cross-linking. These results suggest that notoriously invasive activity of extravillous trophoblast is mediated by CD98hc, which promotes alpha(v)beta(3) integrin-dependent signals.     

Alpha 4 integrin signaling activates phosphatidylinositol 3-kinase and stimulates T cell adhesion to intercellular adhesion molecule-1 to a similar extent as CD3, but induces a distinct rearrangement of the actin cytoskeleton.

Dynamic regulation of beta(2) integrin-dependent adhesion is critical for a wide array of T cell functions. We previously showed that binding of high-affinity alpha(4)beta(1) integrins to VCAM-1 strengthens alpha(L)beta(2) integrin-mediated adhesion to ICAM-1. In this study, we compared beta(2) integrin-mediated adhesion of T cells to ICAM-1 under two different functional contexts: alpha(4) integrin signaling during emigration from blood into tissues and CD3 signaling during adhesion to APCs and target cells. Cross-linking either alpha(4) integrin or CD3 on Jurkat T cells induced adhesion to ICAM-1 of comparable strength. Adhesion was dependent on phosphatidylinositol (PI) 3-kinase but not p44/42 mitogen-activated protein kinase (extracellular regulated kinase 1/2), because it was inhibited by wortmannin and LY294002 but not U0126. These data suggest that PI 3-kinase is a ubiquitous regulator of beta(2) integrin-mediated adhesion. A distinct morphological change consisting of Jurkat cell spreading and extension of filopodia was induced by alpha(4) integrin signaling. In contrast, CD3 induced radial rings of cortical actin polymerization. Inhibitors of PI 3-kinase and extracellular regulated kinase 1/2 did not affect alpha(4) integrin-induced rearrangement of the actin cytoskeleton, but treatment with ionomycin, a Ca(2+) ionophore, modulated cell morphology by reducing filopodia and promoting lamellipodia formation. Qualitatively similar morphological and adhesive changes to those observed with Jurkat cells were observed following alpha(4) integrin or CD3 stimulation of human peripheral blood T cells.     

Differential modulation of human melanoma cell metalloproteinase expression by alpha2beta1 integrin and CD44 triple-helical ligands derived from type IV collagen

Tumor cell binding to components of the basement membrane is well known to trigger intracellular signaling pathways. Signaling ultimately results in the modulation of gene expression, facilitating metastasis. Type IV collagen is the major structural component of the basement membrane and is known to be a polyvalent ligand, possessing sequences bound by the alpha1beta1, alpha2beta1, and alpha3beta1 integrins, as well as cell surface proteoglycan receptors, such as CD44/chondroitin sulfate proteoglycan (CSPG). The role of alpha2beta1 integrin and CD44/CSPG receptor binding on human melanoma cell activation has been evaluated herein using triple-helical peptide ligands incorporating the alpha1(IV)382-393 and alpha1(IV)1263-1277 sequences, respectively. Gene expression and protein production of matrix metalloproteinases-1 (MMP-1), -2, -3, -13, and -14 were modulated with the alpha2beta1-specific sequence, whereas the CD44-specific sequence yielded significant stimulation of MMP-8 and lower levels of modulation of MMP-1, -2, -13, and -14. Analysis of enzyme activity confirmed different melanoma cell proteolytic potentials based on engagement of either the alpha2beta1 integrin or CD44/CSPG. These results are indicative of specific activation events that tumor cells undergo upon binding to select regions of basement membrane collagen. Based on the present study, triple-helical peptide ligands provide a general approach for monitoring the regulation of proteolysis in cellular systems.     

Alpha(v)beta3 and alpha(v)beta5 integrins bind both the proximal RGD site and non-RGD motifs within noncollagenous (NC1) domain of the alpha3 chain of type IV collagen: implication for the mechanism of endothelia cell adhesion

The NC1 domains of human type IV collagen, in particular alpha3NC1, are inhibitors of angiogenesis and tumor growth (Petitclerc, E., Boutaud, A., Prestayko, A., Xu, J., Sado, Y., Ninomiya, Y., Sarras, M. P., Jr., Hudson, B. G., and Brooks, P. C. (2000) J. Biol. Chem. 275, 8051-8061). The recombinant alpha3NC1 domain contained a RGD site as part of a short collagenous sequence at the N terminus, designated herein as RGD-alpha3NC1. Others, using synthetic peptides, have concluded that this RGD site is nonfunctional in cell adhesion, and therefore, the anti-angiogenic activity is attributed exclusively to alpha(v)beta(3) integrin interactions with non-RGD motifs of the RGD-alpha3NC1 domain (Maeshima, Y., Colorado, P. C., and Kalluri, R. (2000) J. Biol. Chem. 275, 23745-23750). This nonfunctionality is surprising given that RGD is a binding site for alpha(v)beta(3) integrin in several proteins. In the present study, we used the alpha3NC1 domain with or without the RGD site, expressed in HEK 293 cells for native conformation, as an alternative approach to synthetic peptides to assess the functionality of the RGD site and non-RGD motifs. Our results demonstrate a predominant role of the RGD site for endothelial adhesion and for binding of alpha(v)beta(3) and alpha(v)beta(5) integrins. Moreover, we demonstrate that the two non-RGD peptides, previously identified as the alpha(v)beta(3) integrin-binding sites of the alpha3NC1 domain, are 10-fold less potent in competing for integrin binding than the native protein, indicating the importance of additional structural and/or conformational features of the alpha3NC1 domain for integrin binding. Therefore, the RGD site, in addition to non-RGD motifs, may contribute to the mechanisms of endothelial cell adhesion in the human vasculature and the anti-angiogenic activity of the RGD-alpha3NC1 domain.     

The antitumor properties of the alpha3(IV)-(185-203) peptide from the NC1 domain of type IV collagen (tumstatin) are conformation-dependent

Tumor progression may be controlled by various fragments derived from noncollagenous 1 (NC1) C-terminal domains of type IV collagen. We demonstrated previously that a peptide sequence from the NC1 domain of the alpha3(IV) collagen chain inhibits the in vitro expression of matrix metalloproteinases in human melanoma cells through RGD-independent binding to alpha(v)beta(3) integrin. In the present paper, we demonstrate that in a mouse melanoma model, the NC1 alpha3(IV)-(185-203) peptide inhibits in vivo tumor growth in a conformation-dependent manner. The decrease of tumor growth is the result of an inhibition of cell proliferation and a decrease of cell invasive properties by down-regulation of proteolytic cascades, mainly matrix metalloproteinases and the plasminogen activation system. A shorter peptide comprising the seven N-terminal residues 185-191 (CNYYSNS) shares the same inhibitory profile. The three-dimensional structures of the CNYYSNS and NC1 alpha3(IV)-(185-203) peptides show a beta-turn at the YSNS (188-191) sequence level, which is crucial for biological activity. As well, the homologous MNYYSNS heptapeptide keeps the beta-turn and the inhibitory activity. In contrast, the DNYYSNS heptapeptide, which does not form the beta-turn at the YSNS level, is devoid of inhibitory activity. Structural studies indicate a strong structure-function relationship of the peptides and point to the YSNS turn as necessary for biological activity. These peptides could act as potent and specific antitumor antagonists of alpha(v)beta(3) integrin in melanoma progression.     

Cholesterol-independent interactions with CD47 enhance alphavbeta3 avidity

Expression in OV10 cells of either wild-type CD47 or its extracellular IgV domain linked to a glycosylphosphatidylinositol anchor-(IgV-GPI) enhanced ligand-induced alpha(v)beta(3) activation as detected by the binding of LIBS1 and LIBS6 mAbs. The amplitude of LIBS binding was greater with both CD47 and IgV-GPI expression, indicating an increase in the population of "activable" integrin molecules. Expression of either CD47 species also increased alpha(v)beta(3)-mediated adhesion to vitronectin, and to surfaces coated with the anti-beta(3) antibody AP3, because of enhanced clustering of alpha(v)beta(3) as confirmed by chemical cross-linking. Cholesterol depletion with methyl-beta-cyclodextrin did not prevent the increase in anti-LIBS binding, but reduced cell adhesion to vitronectin and AP3. However, cells expressing CD47 were partially insulated against this disruption, and IgV-GPI was even more effective. Both CD47 and IgV-GPI were found in cholesterol-rich rafts prepared in the absence of detergent, but only CD47 could recruit alpha(v)beta(3) and its associated signaling molecules to these domains. Thus CD47-alpha(v)beta(3) complexes in cholesterol-rich raft domains appear to engage in G(i)-dependent signaling whereas CD47-alpha(v)beta(3) interactions that lead to integrin clustering are also detergent resistant, but are insensitive to cholesterol depletion and do not require the transmembrane region of CD47.     

A peptide of the alpha3 chain of type IV collagen protects basement membrane against damage by PMN.

We have shown that basement membrane (BM) collagen (type IV), and specifically the peptide CNYYSNSYSFWLASLNPER (a.a. 185-203), from the non-collagenous domain of the alpha3 chain inhibits PMN. We examined the role of this peptide on PMN damage to BM in a vessel wall model. The presence of the endothelial monolayer as well as treatment of PMN with the alpha3(IV) 185-203 peptide reduced damage to BM by non-activated but not by activated PMN. The damage inhibition is unique to the alpha3(IV) peptide and not exhibited by comparable alpha1(IV) and alpha2(IV) chain peptides. A shorter peptide alpha3(IV) 185-191, containing the -SNS- triplet, reduced damage, whereas the one lacking the triplet, residues 194-203, was not effective. The CD47-alphavbeta3 integrin complex is the receptor for the alpha3(IV) peptide. Incubation of PMN with CD47 reactive mAb followed by the alpha3(IV) peptide abolished its protective effect on BM damage.     

Phosphoinositide 3-kinase inhibition reverses platelet aggregation triggered by the combination of the neutrophil proteinases elastase and cathepsin G without impairing alpha(IIb)beta(3) integrin activation

Neutrophil elastase (NE) upregulates the fibrinogen binding activity of the platelet integrin alpha(IIb)beta(3) through proteolysis of the alpha(IIb) subunit. This cleavage allows a strong potentiation of platelet aggregation induced by low concentrations of cathepsin G (CG), another neutrophil serine proteinase. During this activation process, we observed a strong fibrinogen binding and aggregation-dependent phosphatidylinositol 3,4-bis-phosphate (PtdIns(3,4)P(2)) accumulation. PtdIns(3,4)P(2) has been suggested to play a role in the stabilization of platelet aggregation, possibly through the control of a maintained alpha(IIb)beta(3) integrin activation. Here we show that inhibition of phosphoinositide 3-kinase (PI 3-K) by very low concentrations of wortmannin or LY294002 transformed the irreversible platelet aggregation induced by a combination of NE and low concentrations of CG into a reversible aggregation. However, although inhibition of PI 3-K was very efficient in inducing platelet disaggregation, it did not modify the level of alpha(IIb)beta(3) activation as assessed by binding of an activation-dependent antibody. These results indicate that PI 3-K activity can control the irreversibility of platelet aggregation even under conditions where alpha(IIb)beta(3) integrin remains activated.     

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.     

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.     

P21-activated kinase 4 interacts with integrin alpha v beta 5 and regulates alpha v beta 5-mediated cell migration

p21-activated kinase 1 (PAK1) can affect cell migration (Price et al., 1998; del Pozo et al., 2000) and modulate myosin light chain kinase and LIM kinase, which are components of the cellular motility machinery (Edwards, D.C., L.C. Sanders, G.M. Bokoch, and G.N. Gill. 1999. Nature Cell Biol. 1:253-259; Sanders, L.C., F. Matsumura, G.M. Bokoch, and P. de Lanerolle. 1999. SCIENCE: 283:2083-2085). We here present a novel cell motility pathway by demonstrating that PAK4 directly interacts with an integrin intracellular domain and regulates carcinoma cell motility in an integrin-specific manner. Yeast two-hybrid screening identified PAK4 binding to the cytoplasmic domain of the integrin beta 5 subunit, an association that was also found in mammalian cells between endogenous PAK4 and integrin alpha v beta 5. Furthermore, we mapped the PAK4 binding to the membrane-proximal region of integrin beta 5, and identified an integrin-binding domain at aa 505-530 in the COOH terminus of PAK4. Importantly, engagement of integrin alpha v beta 5 by cell attachment to vitronectin led to a redistribution of PAK4 from the cytosol to dynamic lamellipodial structures where PAK4 colocalized with integrin alpha v beta 5. Functionally, PAK4 induced integrin alpha v beta 5-mediated, but not beta1-mediated, human breast carcinoma cell migration, while no changes in integrin cell surface expression levels were observed. In conclusion, our results demonstrate that PAK4 interacts with integrin alpha v beta 5 and selectively promotes integrin alpha v beta 5-mediated cell migration.     

Glycosylation profile of integrin alpha 3 beta 1 changes with melanoma progression

Glycosylation of integrins has been implicated in the modulation of their function. Characterisation of carbohydrate moieties of alpha(3) and beta(1) subunits from non-metastatic (WM35) and metastatic (A375) human melanoma cell lines was carried out on peptide-N-glycosidase F-released glycans using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). beta(1) integrin subunit from both cell lines displayed tri- and tetraantennary oligosaccharides complex type glycans, but only in A375 cell line was the sialylated tetraantennary complex type glycan (Hex(7)HexNAc(6)FucSia(4)) present. In contrast, only alpha(3) subunit from metastatic cells possessed beta1-6 branched structures. Our data indicate that the beta(1) and alpha(3) subunits expressed by the metastatic A375 cell line carry beta1-6 branched structures, suggesting that these cancer-associated glycan chains may modulate tumor cell adhesion by affecting the ligand binding properties of alpha(3)beta(1) integrin. In direct ligand binding assays, alpha(3)beta(1) integrin from both cell lines binds strongly to fibronectin and to much lesser degree to placental laminin. No binding to collagen IV was observed. Enzymatic removal of sialic acid residues from purified alpha(3)beta(1) integrin stimulates its adhesion to all examined ECM proteins. Our data suggest that the glycosylation profile of alpha(3)beta(1) integrin in human melanoma cells correlates with the acquisition of invasive capacity during melanoma progression.     

Identification of a unique co-operative phosphoinositide 3-kinase signaling mechanism regulating integrin alpha IIb beta 3 adhesive function in platelets

Phosphoinositide (PI) 3-kinases play an important role in regulating the adhesive function of a variety of cell types through affinity modulation of integrins. Two type I PI 3-kinase isoforms (p110 beta and p110 gamma) have been implicated in G(i)-dependent integrin alpha(IIb)beta(3) regulation in platelets, however, the mechanisms by which they coordinate their signaling function remains unknown. By employing isoform-selective PI 3-kinase inhibitors and knock-out mouse models we have identified a unique mechanism of PI 3-kinase signaling co-operativity in platelets. We demonstrate that p110 beta is primarily responsible for G(i)-dependent phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) production in ADP-stimulated platelets and is linked to the activation of Rap1b and AKT. In contrast, defective integrin alpha(IIb)beta(3) activation in p110 gamma(-/-) platelets was not associated with alterations in the levels of PI(3,4)P(2) or active Rap1b/AKT. Analysis of the effects of active site pharmacological inhibitors confirmed that p110 gamma principally regulated integrin alpha(IIb)beta(3) activation through a non-catalytic signaling mechanism. Inhibition of the kinase function of PI 3-kinases, combined with deletion of p110 gamma, led to a major reduction in integrin alpha(IIb)beta(3) activation, resulting in a profound defect in platelet aggregation, hemostatic plug formation, and arterial thrombosis. These studies demonstrate a kinase-independent signaling function for p110 gamma in platelets. Moreover, they demonstrate that the combined catalytic and non-catalytic signaling function of p110 beta and p110 gamma is critical for P2Y(12)/G(i)-dependent integrin alpha(IIb)beta(3) regulation. These findings have potentially important implications for the rationale design of novel antiplatelet therapies targeting PI 3-kinase signaling pathways.     

Integrin alpha3beta1, a novel receptor for alpha3(IV) noncollagenous domain and a trans-dominant Inhibitor for integrin alphavbeta3

Exogenous soluble human alpha3 noncollagenous (NC1) domain of collagen IV inhibits angiogenesis and tumor growth. These biological functions are attributed to the binding of alpha3NC1 to integrin alphavbeta3. However, in some tumor cells that express integrin alphavbeta3, the alpha3NC1 domain does not inhibit proliferation, suggesting that integrin alphavbeta3 expression is not sufficient to mediate the anti-tumorigenic activity of this domain. Therefore, in the present study, we searched for novel binding receptors for the soluble alpha3NC1 domain in cells lacking alphavbeta3 integrin. In these cells, soluble alpha3NC1 bound integrin alpha3beta1; however, unlike alphavbeta3, alpha3beta1 integrin did not mediate cell adhesion to immobilized alpha3NC1 domain. Interestingly, in cells lacking integrin alpha3beta1, adhesion to the alpha3NC1 domain was enhanced due to activation of integrin alphavbeta3. These findings indicate that integrin alpha3beta1 is a receptor for the alpha3NC1 domain and transdominantly inhibits integrin alphavbeta3 activation. Thus integrin alpha3beta1, in conjunction with integrin alphavbeta3, modulates cellular responses to the alpha3NC1 domain, which may be pivotal in the mechanism underpinning its anti-angiogenic and anti-tumorigenic activities.     

Purification and functional characterization of integrin alpha v beta 5. An adhesion receptor for vitronectin.

We have purified a novel member of the integrin gene family from placenta that serves as a vitronectin receptor. This integrin is composed of the alpha v subunit and a beta subunit that we designate beta 5. Purification was accomplished by immunodepleting a placental extract of integrin alpha v beta 3, allowing us to purify alpha v beta 5 from the remaining extract by monoclonal antibody affinity chromatography on LM 142-Sepharose, which binds to the alpha v subunit. Purification to homogeneity was subsequently achieved by affinity chromatography on wheat germ lectin-Sepharose. Western blot analysis with antibodies raised against alpha v beta 5 and alpha v beta 3 demonstrated that beta 3 and beta 5 were distinct but confirmed that the alpha subunit of the two integrins were immunologically identical. Similarly, antibodies that bind beta 3 proximal to the ligand-binding site failed to react with beta 5, indicating an architectural difference at the ligand-binding site of these related integrins. This structural difference apparently results in a functional distinction, since purified alpha v beta 3 bound to vitronectin, fibrinogen, von Willebrand factor, and fibronectin, whereas integrin alpha v beta 5 bound preferentially to vitronectin. Finally, we demonstrate by three criteria that beta 5 and beta x, the latter of which was identified in lung carcinoma cells (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), are identical. First, peptide maps of beta x and beta 5 are identical. Secondly, polyclonal antibodies raised against alpha v beta 5 immunoprecipitate both beta 5 and beta x, and finally, the amino-terminal amino acid sequences of beta x and beta 5 are identical.     

Vascular endothelial cell adhesion and spreading promoted by the peptide REDV of the IIICS region of plasma fibronectin is mediated by integrin alpha 4 beta 1.

We have recently reported the attachment and spreading of human umbilical vein endothelial cells (HUVECs) upon substrates containing covalently grafted Arg-Glu-Asp-Val (REDV) peptide (Hubbell, J. A., Massia, S. P., Desai, N. P., and Drumheller, P. D. (1991) Bio/Technology 9, 568-572). This peptide has been reported to be the minimal active sequence within the CS5 site of the alternatively spliced type III connecting segment (IIICS) region of fibronectin, and the integrin alpha 4 beta 1 has been identified as the receptor on melanoma cells for this site. The integrin alpha 4 beta 1 has also been identified as the receptor for the CS1 site in the IIICS region on cells of neural crest origin, melanoma cells, lymphocytes, and hematopoietic stem cells. In this study, we demonstrate that this integrin also serves as a receptor on HUVECs for the peptide REDV from the CS5 site. The alpha 4 subunit was shown to be expressed upon HUVEC membranes by whole-cell enzyme-linked immunosorbent assay. Antifunctional antibodies directed against integrin subunits alpha 4 and beta 1 inhibited cell adhesion on REDV-grafted substrates, but not on RGD-grafted substrates. The alpha 4 subunit localized into fibrillar structures within spread cells on the REDV-grafted substrates, but not within spread cells on RGD-grafted substrates. Two proteins (144 and 120 kDa) were isolated from HUVEC extracts by REDV ligand affinity chromatography and were demonstrated by immunoprecipitation and Western blot to be the integrin subunits alpha 4 (144 kDa) and beta 1 (120 kDa); furthermore, the immunoprecipitation analyses demonstrated that the subunits formed a complex. HUVEC binding to REDV-grafted substrates was inhibited by both soluble REDV and RGD, demonstrating that adhesion was biospecific and that the REDV peptide is RGD-like. In this report we demonstrate for the first time that alpha 4 is present in the endothelial cell membrane, in contrast to previous reports by others, and that integrin alpha 4 beta 1 is the receptor for REDV-mediated adhesion to the IIICS region of region of plasma fibronectin.