Integrin receptor specificity for human red cell ICAM-4 ligand. Critical residues for alphaIIbeta3 binding.

The red cell intercellular adhesion molecule-4 (ICAM-4) binds to different members of the integrin receptor families. To better define the ICAM-4 integrin receptor specificity, cell transfectants individually expressing various integrins were used to demonstrate that alphaLbeta2, alphaMbeta2, and alphaIIbbeta3 (activated) bind specifically and dose dependently to the recombinant ICAM-4-Fc protein. We also show that cell surface ICAM-4 interacts with the cell surface alphaVbeta3 integrin. In addition, using a alpha4beta1 cell transfectant and beta2 integrin-deficient LAD cells, we show here that ICAM-4 failed to interact with alpha4beta1 even after alpha4beta1 activation by phorbol ester or with the monoclonal antibody TS2/16 (+ Mn2+). ICAM-4 amino acids that are critical for alphaIIbbeta3 and alphaVbeta3 interaction were identified by domain deletion analysis, site-directed mutagenesis and synthetic peptide inhibition. Our results provide evidence that the beta3 integrin binding sites encompass the first and second Ig-like domains of ICAM-4. However, while the alphaIIbbeta3 contact site comprises the ABED face of domain D1 with an extension in the C'-E loop of domain D2, the alphaVbeta3 contact site comprises residues on both faces of D1 and in the C'-E loop of D2. These data, together with our previous results, demonstrate that different integrins bind to different but partly overlapping sites on ICAM-4, and that ICAM-4 may accommodate multiple integrin receptors present on leukocytes, platelets and endothelial cells.     

Cartilage oligomeric matrix protein/thrombospondin 5 supports chondrocyte attachment through interaction with integrins

Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP5) is a major component of the extracellular matrix of the musculoskeletal system. Although COMP/TSP5 abnormalities are associated with several pathological conditions, its normal function remains unclear. This study was undertaken to delineate the function(s) of COMP/TSP5 in cartilage, especially regarding its interaction with chondrocytes. We show that COMP/TSP5 can support chondrocyte attachment and that the RGD sequence in COMP/TSP5 and the integrin receptors alpha5beta1 and alphaVbeta3 on the chondrocytes are involved in mediating this attachment. The interactions of COMP/TSP5 with the integrins are dependent on COMP/TSP5 conformation. Chondrocyte attachment to COMP/TSP5 in the calcium-replete conformation was inhibited by function-blocking integrin alpha5 and beta1 antibodies, suggesting the involvement of the alpha5beta1 integrin. Under this condition, a function-blocking antibody against alphaVbeta3 did not have any effect on cell attachment. On the other hand, chondrocyte attachment to reduced COMP/TSP5 was instead sensitive to alphaVbeta3 function-blocking antibodies, suggesting that COMP/TSP5 mediates attachment through chondrocyte alphaVbeta3 integrin under this condition. Cell attachment to reduced COMP/TSP5 was not inhibited by beta1 antibodies. These data indicate that COMP/TSP5 in different conformations can utilize different integrin receptors. These results are the first to demonstrate that COMP/TSP5 can mediate chondrocyte attachment through interactions with integrins. Through these interactions, COMP/TSP5 may be able to regulate cellular activities and respond to environment in the surrounding cartilage matrix.     

The selective regulation of alpha Vbeta 1 integrin expression is based on the hierarchical formation of alpha V-containing heterodimers

The integrin beta1 subunit can form a heterodimer with 12 different alpha subunits. According to the present model, the expression level of any alphabeta complex is regulated by the availability of the specific alpha subunit, whereas beta1 subunit is constantly present in a large excess. The expression of several heterodimers containing the alphaV subunit seems to be regulated by an identical mechanism. The fact that many cells express alphaVbeta1 heterodimer, and that this fibronectin/vitronectin receptor may be selectively regulated, compromises the present model of the regulation of beta1 and alphaV integrins. We have tried to solve this problem by assuming that distinct alphabeta heterodimers are formed with different tendency. To test the hypothesis, we analyzed WM-266-4 melanoma cells transfected with a cDNA construct coding for an intracellular single-chain anti-alphaV integrin antibody. We could see 70-80% reduction in the cell surface expression of alphaV subunit. However, the only one of the alphaV integrins reduced on the cell surface was alphaVbeta1. This suggests that the cell surface expression level of alphaVbeta1 is dependent on the number of alphaV subunits available after the formation of other alphaV-containing heterodimers. Thus, there seems to be a hierarchy in the complex formation between alphaV and its different beta-partners. These observations explain how alphaVbeta1 can be specifically regulated without concomitant changes in the expression of other alphaV or beta1 integrins.     

Interactions of foot-and-mouth disease virus with soluble bovine alphaVbeta3 and alphaVbeta6 integrins

At least four members of the integrin family of receptors, alphaVbeta1, alphaVbeta3, alphaVbeta6, and alphaVbeta8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. Our investigators have recently shown that the efficiency of receptor usage appears to be related to the viral serotype and may be influenced by structural differences on the viral surface (H. Duque and B. Baxt, J. Virol. 77:2500-2511, 2003). To further examine these differences, we generated soluble alphaVbeta3 and alphaVbeta6 integrins. cDNA plasmids encoding the individual complete integrin alphaV, beta3, and beta6 subunits were used to amplify sequences encoding the subunits' signal peptide and ectodomain, resulting in subunits lacking transmembrane and cytoplasmic domains. COS-1 cells were transfected with plasmids encoding the soluble alphaV subunit and either the soluble beta3 or beta6 subunit and labeled with [35S]methionine-cysteine. Complete subunit heterodimeric integrins were secreted into the medium, as determined by radioimmunoprecipitation with specific monoclonal and polyclonal antibodies. For the examination of the integrins' biological activities, stable cell lines producing the soluble integrins were generated in HEK 293A cells. In the presence of divalent cations, soluble alphaVbeta6 bound to representatives of type A or O viruses, immobilized on plastic dishes, and significantly inhibited viral replication, as determined by plaque reduction assays. In contrast, soluble alphaVbeta3 was unable to bind to immobilized virus of either serotype; however, virus bound to the immobilized integrin, suggesting that FMDV binding to alphaVbeta3 is a low-affinity interaction. In addition, soluble alphaVbeta3 did not neutralize virus infectivity. Incubation of soluble alphaVbeta6 with labeled type A12 or O1 resulted in a significant inhibition of virus adsorption to BHK cells, while soluble alphaVbeta3 caused a low (20 to 30%), but consistent, inhibition of virus adsorption. Virus incubated with soluble alphaVbeta6 had a lower sedimentation rate than native virus on sucrose density gradients, but the particles retained all of their structural proteins and still contained bound integrin and, therefore, were not exhibiting characteristics of a picornavirus A particle.     

Alpha5beta1, alphaVbeta3 and the platelet-associated integrin alphaIIbbeta3 coordinately regulate adhesion and migration of differentiating mouse trophoblast cells

During blastocyst implantation, interaction between integrins on the apical surface of the trophoblast and extracellular matrix (ECM) in the endometrium anchors the embryo to the uterine wall. Strong adhesion of the blastocyst to fibronectin (FN) requires integrin signaling initiated by exogenous fibronectin. However, it is not known how integrin signaling enhances blastocyst adhesion. We present new evidence that the integrin, alphaIIbbeta3, plays a key role in trophoblast adhesion to fibronectin during mouse peri-implantation development. Trafficking of alphaIIb to the apical surface of the trophoblast increased dramatically after blastocysts were exposed to fibronectin, whereas other fibronectin-binding integrins, alpha5beta1 and alphaVbeta3, were resident at the apical surface before ligand exposure. Functional comparisons among the three integrins revealed that ligation of alpha5beta1 most efficiently strengthened blastocyst fibronectin-binding activity, while subsequent trophoblast cell migration was dependent primarily on the beta3-class integrins. In vivo, alphaIIb was highly expressed by invasive trophoblast cells in the ectoplacental cone and trophoblast giant cells of the parietal yolk sac. These data demonstrate that trafficking of alphaIIb regulates adhesion between trophoblast cells and fibronectin as invasion of the endometrium commences.     

alphaVbeta6 is a novel receptor for human fibrillin-1. Comparative studies of molecular determinants underlying integrin-rgd affinity and specificity

Human fibrillin-1, the major structural protein of connective tissue 10-12 nm microfibrils, contains multiple calcium binding epidermal growth factor-like domains interspersed with transforming growth factor beta-binding protein-like (TB) domains. TB4 contains a flexible RGD loop that mediates cell adhesion via alphaVbeta3 and alpha5beta1 integrins. This study identifies integrin alphaVbeta6 as a novel cellular receptor for fibrillin-1 with a K(d) of approximately 0.45 mum. Analyses of this interaction by surface plasmon resonance and immunocytochemistry reveal different module requirements for alphaVbeta6 activation compared with those of alphaVbeta3, suggesting that a covalent linkage of an N-terminal calcium binding epidermal growth factor-like domain to TB4 can modulate alphaV integrin binding specificity. Furthermore, our data suggest alpha5beta1 is a low affinity fibrillin-1 receptor (K(d) > 1 mum), thus providing a molecular explanation for the different alpha5beta1 distribution patterns seen when human keratinocytes and fibroblasts are plated on recombinant fibrillin fragments versus those derived from the physiological ligand fibronectin. Non-focal contact distribution of alpha5beta1 suggests that its engagement by fibrillin-1 may elicit a lesser degree and/or different type of intracellular signaling compared with that seen with a high affinity ligand.     

Integrin alpha(v)beta1 is an adenovirus coreceptor

The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor alpha(v)beta3 or alpha(v)beta5 integrins, yet these cells are efficiently infected by Ad vectors. Here we demonstrate that Ad binds to HEK293 cells via the fiber receptor CAR and is subsequently internalized via interaction with integrin alpha(v)beta1. Function-blocking antibodies directed against alpha(v) or beta1, but not beta3, beta5, or alpha5, integrin subunits block Ad infection and viral endocytosis. Therefore, alpha(v)beta1 serves as a coreceptor for Ad infection, and the lack of beta3 and/or beta5 but the relatively high expression of alpha(v)beta1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.     

Regulation of expression of fibronectin and its receptor, alpha 5 beta 1, during development and regeneration of peripheral nerve.

The extracellular matrix glycoprotein, fibronectin, is a potent promoter of peripheral neurite outgrowth. Interactions of peripheral neurons with fibronectin have been shown to be primarily mediated by the beta 1 class of integrin heterodimers. In the present study, we have examined the expression and regulation of fibronectin and its integrin receptor, alpha 5 beta 1, in developing and regenerating chick peripheral nerve. We show that fibronectin and alpha 5 beta 1 are expressed at comparatively high levels in developing nerve with alpha 5 beta 1 expression on axons and non-neuronal cells. With nerve maturation, both proteins are less prominently expressed and the cellular pattern of alpha 5 beta 1 expression becomes more restricted. Following lesion of mature nerve, both fibronectin and alpha 5 beta 1 are strongly induced with prominent expression of alpha 5 beta 1 on regenerating neurites and Schwann cells. The elevation in fibronectin levels in the regenerating nerve is highest in the vicinity of the lesion, an area undergoing extensive cellular remodeling including Schwann cell migration and growth cone extension. Our results suggest that fibronectin and its receptor, alpha 5 beta 1, may mediate functionally important interactions in the development and regeneration of peripheral nerve.     

Integrin-using rotaviruses bind alpha2beta1 integrin alpha2 I domain via VP4 DGE sequence and recognize alphaXbeta2 and alphaVbeta3 by using VP7 during cell entry

Integrins alpha2beta1, alphaXbeta2, and alphaVbeta3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the alpha2beta1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the alphaXbeta2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of alpha2beta1, alphaXbeta2, and alphaVbeta3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound alpha2beta1, and VP7 interacted with alphaXbeta2 and alphaVbeta3 at a postbinding stage. DGEA inhibited rotavirus binding to alpha2beta1 and infectivity, whereas GPRP binding to alphaXbeta2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed alpha2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the alpha2 I domain. In a novel process, integrin-using viruses bind the alpha2 I domain of alpha2beta1 via DGE in VP4 and interact with alphaXbeta2 (via GPR) and alphaVbeta3 by using VP7 to facilitate cell entry and infection.     

Axonal regulation of Schwann cell integrin expression suggests a role for alpha 6 beta 4 in myelination

Ensheathment and myelination of axons by Schwann cells in the peripheral nervous system requires contact with a basal lamina. The molecular mechanism(s) by which the basal lamina promotes myelination is not known but is likely to reflect the activity of integrins expressed by Schwann cells. To initiate studies on the role of integrins during myelination, we characterized the expression of two integrin subunits, beta 1 and beta 4, in an in vitro myelination system and compared their expression to that of the glial adhesion molecule, the myelin-associated glycoprotein (MAG). In the absence of neurons, Schwann cells express significant levels of beta 1 but virtually no beta 4 or MAG. When Schwann cells are cocultured with dorsal root ganglia neurons under conditions promoting myelination, expression of beta 4 and MAG increased dramatically in myelinating cells, whereas beta 1 levels remained essentially unchanged. (In general agreement with these findings, during peripheral nerve development in vivo, beta 4 levels also increase during the period of myelination in sharp contrast to beta 1 levels which show a striking decrease.) In cocultures of neurons and Schwann cells, beta 4 and MAG appear to colocalize in nascent myelin sheaths but have distinct distributions in mature sheaths, with beta 4 concentrated in the outer plasma membrane of the Schwann cell and MAG localized to the inner (periaxonal) membrane. Surprisingly, beta 4 is also present at high levels with MAG in Schmidt-Lanterman incisures. Immunoprecipitation studies demonstrated that primary Schwann cells express beta 1 in association with the alpha 1 and alpha 6 subunits, while myelinating Schwann cells express alpha 6 beta 4 and possibly alpha 1 beta 1. beta 4 is also downregulated during Wallerian degeneration in vitro, indicating that its expression requires continuous Schwann cell contact with the axon. These results indicate that axonal contact induces the expression of beta 4 during Schwann cell myelination and suggest that alpha 6 beta 4 is an important mediator of the interactions of myelinating Schwann cells with the basal lamina.     

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.     

Integrins alpha5beta1, alphavbeta1, and alphavbeta6 collaborate in squamous carcinoma cell spreading and migration on fibronectin

The expression of alphavbeta6 fibronectin/tenascin receptor integrin is induced in malignant transformation of oral epithelium. In this study, we demonstrate the contribution of alphavbeta6 as well as other fibronectin receptor integrins in squamous cell carcinoma (SCC) cell adhesion and migration. Of 11 SCC cell lines isolated from the head and neck area, 8 (73%) expressed alphavbeta6 integrin on the cell surface. Three cell lines were chosen for further functional experiments: 1 with relatively high, 1 with moderate, and 1 with minimal surface expression of alphavbeta6 integrin. In addition to alphavbeta6, all 3 cell lines expressed alpha5beta1 and alphavbeta1 fibronectin receptor integrins. Function-blocking experiments with inhibitory anti-integrin antibodies showed that all these three integrins were functional in SCC cell spreading on fibronectin. Integrin alphavbeta6, however, was not used as a primary but as an alternative fibronectin receptor by SCC cells, as the inhibitory anti-beta6 integrin antibody alone had no effect on spreading. In migration, however, alphavbeta6, alpha5beta1, and alphavbeta1 integrins were all used in cooperation. The presence of alphavbeta1 integrin in SCC cells is a novel finding as is its contribution to SCC cell migration. When one or two of these three receptors were blocked, the cells demonstrated an adaptive ability to remain migratory using integrins that were not targeted by antibodies. Utilization of a combination of receptors of different affinities may be beneficial for SCC cell migration versatility.     

Cell-cell adhesion via the ECM: integrin genetics in fly and worm.

Integrins are essential for the development of the two genetically tractable invertebrate model organisms, the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Just two integrins are present in C. elegans: one putative RGD binding integrin alphapat-2betapat-3, corresponding to Drosophila alphaPS2betaPS and vertebrate alpha5beta1, alphaVbeta1 and alpha8beta1, and one putative laminin binding integrin alphaina-1betapat-3, corresponding to Drosophila alphaPS1betaPS and vertebrate alpha3beta1, alpha6beta1 and alpha7beta1. In this review, the function of this minimal set of integrins during the development of these two invertebrates is compared. Despite the differences in bodyplan and developmental strategy, integrin adhesion to the extracellular matrix is required for similar processes: the formation of the link that translates muscle contraction into movement of the exoskeleton, cell migration, and morphogenetic interactions between epithelia. Other integrin functions, such as regulation of gene expression, have not yet been experimentally demonstrated in both organisms. Additional proteins have been characterised in each organism that are essential for integrin function, including extracellular matrix ligands and intracellular interacting proteins, but so far different proteins have been found in the two organisms. This in part represents the fact that the characterisation of the full set of interacting proteins is not complete in either system. However, in other cases different proteins appear to be used for similar functions in the two animals. The continued use of genetic approaches to identify proteins required for integrin function in these two model organisms should lead to the identification of the minimal set of conserved components that form integrin adhesive structures.     

Beta1 integrins are distributed in adhesion structures with fibronectin and caveolin and in coated pits.

Integrins are found in adhesion structures, which link the extracellular matrix to cytoskeletal proteins. Here, we attempt to further define the distribution of beta1 integrins in the context of their association with matrix proteins and other cell surface molecules relevant to the endocytic process. We find that beta1 integrins colocalize with fibronectin in fibrillar adhesion structures. A fraction of caveolin is also organized along these adhesion structures. The extracellular matrix protein laminin is not concentrated in these structures. The alpha4beta1 integrin exhibits a distinct distribution from other beta1 integrins after cells have adhered for 1 h to extracellular matrix proteins but is localized in adhesion structures after 24 h of adhesion. There are differences between the fibronectin receptors: alpha5beta1 integrins colocalize with adaptor protein-2 in coated pits, while alpha4beta1 integrins do not. This parallels our earlier observation that of the two laminin receptors, alpha1beta1 and alpha6beta1, only alpha1beta1 integrins colocalize with adaptor protein-2 in coated pits. Calcium chelation or inhibition of mitogen-activated protein kinase kinase, protein kinase C, or src did not affect localization of alpha1beta1 and alpha5beta1 integrins in coated pits. Likewise, the integrity of coated-pit structures or adhesion structures is not required for integrin and adaptor protein-2 colocalization. This suggests a robust and possibly constitutive interaction between these integrins and coated pits.     

Membrane type-1 matrix metalloproteinase (MT1-MMP) processing of pro-alphav integrin regulates cross-talk between alphavbeta3 and alpha2beta1 integrins in breast carcinoma cells

We have recently demonstrated that in breast carcinoma MCF7 cells MT1-MMP processes the alphav, alpha3, and alpha5 integrin precursors generating the respective mature S-S-linked heavy and light alpha-chains. The precursor of alpha2 integrin subunit was found resistant to MT1-MMP proteolysis. The processing of the alphav subunit by MT1-MMP facilitated alphavbeta3-dependent adhesion, activation of FAK signaling pathway, and migration of MCF7 cells on vitronectin. To elucidate further the effects of MT1-MMP on cellular integrins, we examined the functional activity of alpha5beta1 and alpha2beta1 integrins in MCF7 cells expressing MT1-MMP. Either expression of MT1-MMP alone or its coexpression with alphavbeta3 failed to affect the functionality of alpha5beta1 integrin, and adhesion of cells to fibronectin. MT1-MMP, however, profoundly affected the cross-talk involving alphavbeta3 and alpha2beta1 integrins. In MT1-MMP-deficient cells, integrin alphavbeta3 suppressed the functional activity of the collagen-binding alpha2beta1 integrin receptor and diminished cell adhesion to type I collagen. Coexpression of MT1-MMP with integrin alphavbeta3 restored the functionality of alpha2beta1 integrin and, consequently, the ability of MCF7 cells to adhere efficiently to collagen. We conclude that the MT1-MMP-controlled cross-talk between alphavbeta3 and alpha2beta1 integrins supports binding of aggressive, MT1-MMP-, and alphavbeta3 integrin-expressing malignant cells on type I collagen, the most common substratum of the extracellular matrix.     

Platelet-derived growth factor receptor beta and vascular endothelial growth factor receptor 2 bind to the beta 3 integrin through its extracellular domain

Integrin-mediated cell attachment and growth factor stimulation often act synergistically on cell proliferation, differentiation, migration, and survival. Some of these synergistic effects depend on the physical interaction of integrins with growth factor receptors. Here we examine the nature of the physical interaction between the alpha(v)beta(3) integrin and two receptor tyrosine kinases (RTKs), the platelet-derived growth factor receptor beta (PDGF-Rbeta) and the vascular endothelial growth factor receptor 2 (VEGF-R2, also known as KDR and flk-1). Both of these RTKs associate with the alpha(v)beta(3) integrin but do not associate with beta(1) integrins. Furthermore, growth factor stimulation of these RTKs promotes increased cell proliferation and migration when cells are attached to the alpha(v)beta(3) ligand, vitronectin. We show that alpha(v)beta(3) in which the beta(3) cytoplasmic domain is deleted or replaced with the beta(1) cytoplasmic domain coimmunoprecipitates with PDGF-Rbeta and VEGF-R2. The beta(3) extracellular domain alone was sufficient for the PDGF-Rbeta association whereas the VEGF-R2 association required the presence of the alpha(v) subunit. Activation of the RTKs by their ligands was not required for them to associate with the integrin. Cell migration to PDGF was enhanced in the cells transfected with the chimeric subunit containing the beta(3) extracellular domain but not when that domain came from the beta(1) subunit. These results show that the interactions that lead to the association of the alpha(v)beta(3) integrin with PDGF-Rbeta and VEGF-R2 and enhancement of RTK activity take place outside the cell.     

Beta1 integrin antisense oligodeoxynucleotides: utility in controlling osteoclast function.

The involvement of beta1 integrins in osteoclast function has been investigated by utilising an antisense oligodeoxynucleotide (ODN) approach. 18-mer antisense and control phosphorothioate ODNs were made to a conserved internal region of beta1 integrin sequence (nucleotide positions 1634-1651 of the human beta1 fibronectin receptor). These were tested on rabbit osteoclasts for anti-adhesive and resorptive effects mediated by alphaVbeta3 and alpha2beta1, the major integrins of osteoclasts. Antisense, but not control, beta1 ODNs inhibited osteoclast adhesion to collagen-coated glass (by up to 70%), but not to glass coated with vitronectin, fibronectin or fibrinogen. Adhesion to dentine and subsequent resorption were also inhibited (up to 60%) in a sequence-specific manner. The mechanism of action was verified using both a melanoma cell line, DX3, which expresses multiple integrins at high level including alphaVbeta3 and alpha2beta1, and in a rabbit osteoclast marrow culture (BMC) system. Exposure of DX3 cells to antisense ODN for up to 48 hours reduced adhesion to FCS- and collagen-coated glass, and concomitantly inhibited beta1 protein expression assessed by FACS and Western blot analysis; expression of other integrin subunits, alphaV and beta3, was unaffected. Similarly, the beta1 protein levels in the BMC were reduced by > 75% without any effect on actin expression. These data reveal the utility of antisense ODNs in exploring osteoclast biology and further define the functional role of osteoclastic beta1 integrin(s).