Human parechovirus 1 utilizes integrins alphavbeta3 and alphavbeta1 as receptors

Human parechovirus 1 (HPEV1) displays an arginine-glycine-aspartic acid (RGD) motif in the VP1 capsid protein, suggesting integrins as candidate receptors for HPEV1. A panel of monoclonal antibodies (MAbs) specific for integrins alphavbeta3, alphavbeta1, and alphavbeta5, which have the ability to recognize the RGD motif, and also a MAb specific for integrin alpha2beta1, an integrin that does not recognize the RGD motif, were tested on A549 cells. Our results showed that integrin alphav-specific MAb reduced infectivity by 85%. To specify which alphav integrins the virus utilizes, we tested MAbs specific to integrins alphavbeta3 and alphavbeta1 which reduced infectivity significantly, while a MAb specific for integrin alphavbeta5, as well as the MAb specific for alpha2beta1, showed no reduction. When a combination of MAbs specific for integrins alphavbeta3 and alphavbeta1 were used, virus infectivity was almost completely inhibited; this shows that integrins alphavbeta3 and alphavbeta1 are utilized by the virus. We therefore proceeded to test whether alphav integrins' natural ligands fibronectin and vitronectin had an effect on HPEV1 infectivity. We found that vitronectin reduced significantly HPEV1 infectivity, whereas a combination of vitronectin and fibronectin abolished infection. To verify the use of integrins alphavbeta3 and alphavbeta1 as HPEV1 receptors, CHO cells transfected and expressing either integrin alphavbeta3 or integrin alphavbeta1 were used. It was shown that the virus could successfully infect these cells. However, in immunoprecipitation experiments using HPEV1 virions and allowing the virus to bind to solubilized A549 cell extract, we isolated and confirmed by Western blotting the alphavbeta3 heterodimer. In conclusion, we found that HPEV1 utilises both integrin alphavbeta3 and alphavbeta1 as receptors; however, in cells that express both integrins, HPEV1 may preferentially bind integrin alphavbeta3.     

The extracellular matrix and cytokines regulate microglial integrin expression and activation

Microglia are the primary immune effector cells resident within the CNS, whose activation into migratory, phagocytic cells is associated with increased expression of cell adhesion molecules of the integrin family. To determine which specific factors are important regulators of microglial activation and integrin expression, we have examined the influence of individual cytokines and extracellular matrix (ECM) substrates by quantifying cell surface expression of MHC and individual integrins by flow cytometry. We found that the proinflammatory cytokines TNF and IFN-alpha promoted microglial activation, as assessed by amoeboid morphology and increased expression of MHC class I, and also increased expression of the alpha(4)beta(1) and Mac-1 integrins. In contrast, TGF-beta1 had the opposite effect and was dominant over the other cytokines. Furthermore, the ECM substrates fibronectin and vitronectin, but not laminin, also promoted microglial activation and increased expression of the alpha(4)beta(1), alpha(5)beta(1) and Mac-1 integrins, but significantly, the influence of fibronectin and vitronectin was not diminished by TGF-beta1. Taken together, this work suggests that, in addition to cytokines, the ECM represents an important regulatory influence on microglial activity. Specifically, it implies that increases in the local availability of fibronectin or vitronectin, as a result of blood-brain barrier breakdown or increased expression in different pathological states of the CNS, could induce microglial activation and increased expression of integrins.     

Role of hypoxia and extracellular matrix-integrin binding in the modulation of angiogenic growth factors secretion by retinal pigmented epithelial cells.

The retinal pigmented epithelium (RPE) is a monolayer of polarized cells located between retinal photoreceptors and blood vessels of the choroid. The basal surface of RPE cells rests on Bruch's membrane, a complex extracellular matrix structure which becomes abnormal in several disease processes, including age-related macular degeneration (AMD). Ruptures or abnormalities in Bruch's membrane are frequently accompanied by choroidal neovascularization. Disturbed interaction of RPE cells with their extracellular matrix (ECM) could play a role in this process. The present study was undertaken to examine the complex interactions between hypoxia, integrin, and ECM in the regulation of RPE functions. Antibody blocking experiments demonstrated that RPE cell adhesion to vitronectin is mediated primarily through alphavbeta5 and adhesion to fibronectin occurs through alpha5beta1. RPE adhesion to immobilized laminin demonstrated highest level of non-RGD-mediated adhesion as compared to that with collagen IV or the RGD matrices such as vitronectin (alphavalpha5) , fibronectin (alpha5beta1), or thrombospondin (alpha5beta1 + alphavbeta5). Addition of soluble vitronectin, or fibrinogen to RPE cell cultures resulted in a small to moderate increase in VEGF and FGF2 in the media, while each of these growth factors was dramatically increased after addition of thrombospondin 1 (TSP1). In contrast, soluble fibronectin resulted in differential upregulation of VEGF but not FGF2. Similarly, immobilized TSP1 resulted in differential greater upregulation in VEGF but not FGF2 release from RPE as compared to other ECMs under either normoxic or hypoxic conditions. Additionally, hypoxia resulted in a time-dependent increase in VEGF, but not FGF2 release in the media. RPE cells grown on TSP1-coated plates showed increased VEGF and FGF2 in their media compared to cells grown on plates coated with type IV collagen, laminin, vitronectin, or fibronectin. The TSP1-induced increase in secretion of growth factors was partially blocked by anti-alpha5beta1, anti-alphavbeta3, and anti-alphavbeta5 antibodies indicating that it may be mediated in part by TSP1 binding to those integrins. These data suggest that alterations in oxygen levels (hypoxia/ischemia) and ECM of RPE cells, a prominent feature of AMD, can cause increased secretion of angiogenic growth factors that might contribute to the development of choroidal neovascularization. These data also suggest the potential modulatory role of VEGF release from RPE by ECM and alphavbeta5 and alpha5beta1 integrins.     

Integrin alphavbeta3 binding to human alpha5-laminins facilitates FGF-2- and VEGF-induced proliferation of human ECV304 carcinoma cells

Human ECV304 cells respond reproducibly by tube formation to complex basement membrane matrices. Laminins are major glycoproteins of basement membranes. We therefore studied the ability of ECV304 cells to attach to defined laminin isoforms and to fibronectin, and identified the involved laminin receptors. The cells bound poorly to fibronectin, to some extent to laminin-1, whereas laminin-2/4 and -10/11 were strong adhesive substrates. Antibody perturbation assays showed that adhesion to laminin-1 was mediated by integrin alpha6beta1, and adhesion to laminin-2/4 by cooperative activity of integrins alpha3beta1 and alpha6beta1. Adhesion of ECV 304 cells to laminin-10/11 was mainly mediated by integrins alpha3beta1, with minor involvement of alpha6beta1/4 and alphavbeta3. Solid-phase binding assays confirmed that integrin alphavbeta3 binds human laminin-10/11 and -10, in an RGD-dependent fashion. Although integrin alphavbeta3 played a very minor role in cell adhesion to laminin-10/11, this interaction facilitated growth factor-induced proliferation of ECV304 cells. In response to FGF-2 or VEGF, the cells proliferated better when attached on laminin-10/11 than on laminin-1, -2/4, or gelatin. The proliferation induced by the joint application of laminin-10/11 and either one of the growth factors could be blocked by antibodies against integrin alphavbeta3. Fragments of several other basement membrane components are known to interact with alphavbeta3. The current data show that that integrin alphavbeta3 can bind intact alpha5-containing laminin trimers. Since the laminin alpha5 chain is broadly expressed in adult basement membranes, this interaction could be physiologically important. Our data suggest that this interaction is involved in the regulation of cellular responses to growth factors known to be involved in epithelial and endothelial development.     

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.     

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.     

Different integrins mediate cell spreading, haptotaxis and lateral migration of HaCaT keratinocytes on fibronectin

Collaborative role of various fibronectin-binding integrins (alpha5beta1, alphavbeta1 and alphavbeta6) as mediators of cell adhesion and migration on fibronectin was studied using cultured HaCaT keratinocytes. This cell line spontaneously expressed all three fibronectin-binding integrins. In addition, the expression of alphavbeta6 integrin was strongly and specifically upregulated by transforming growth factor-beta1 (TGFbeta1) whereas the amount of other integrins remained practically unchanged on the cell surface. Adhesion, spreading and motility of HaCaT keratinocytes on fibronectin were promoted by TGFbeta1. Based on antibody blocking experiments, both untreated and TGFbeta1-treated HaCaT cells used alphavbeta6 integrin as their main fibronectin receptor for cell spreading. In contrast to TGFbeta1-treated cells, the untreated cells also needed alpha5beta1 integrin for maximal cell spreading on fibronectin. Combinations of antibodies blocking both of these receptors totally prevented spreading of both untreated and TGFbeta1-treated cells. Haptotactic motility of individual HaCaT cells through fibronectin-coated membranes was again mainly dependent on alphavbeta6 integrin, while alphavbeta1 and alpha5beta1 integrins played a lesser role both in untreated and TGFbeta1-treated HaCaT cells. However, unlike haptotaxis, lateral migration of HaCaT cell sheet was mainly mediated by beta1 integrins, and alphavbeta6 integrin showed a minor role. The migration process appeared to involve a number of beta1 integrins that could adaptively replace each other when blocking antibodies were present. Thus, keratinocytes appear to use different fibronectin receptors for different functions, such as cell spreading, haptotaxis and lateral migration. The cells can also adapt to a situation where one receptor is unfunctional by switching to another receptor of the same ligand.     

Basic fibroblast growth factor modulates integrin expression in microvascular endothelial cells.

During angiogenesis capillary endothelial cells undergo a coordinated set of modifications in their interactions with extracellular matrix components. In this study we have investigated the effect of the prototypical angiogenic factor basic fibroblast growth factor (bFGF) on the expression and function of several integrins in microvascular endothelial cells. Immunoprecipitation experiments with antibodies to individual subunits indicated that microvascular cells express at their surface several integrins. These include the alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1 laminin/collagen receptors; the alpha 6 beta 1 laminin receptor; the alpha 5 beta 1 and alpha v beta 1 fibronectin receptors; the alpha 6 beta 4 basement membrane receptor; and the alpha v beta 3 and alpha v beta 5 vitronectin receptors. Treatment with bFGF caused a significant increase in the surface expression of the alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha 6 beta 4, and alpha v beta 5 integrins. In contrast, the level of expression of the alpha 1 beta 1 and alpha v beta 3 integrins was decreased in bFGF-treated cells. Immunoprecipitation of metabolically labeled cells indicated that bFGF increases the biosynthesis of the alpha 3, alpha 5, alpha 6, beta 4, and beta 5 subunits and decreases the production of the alpha v and beta 3 subunits. These results suggest that bFGF modulates integrin expression by altering the biosynthesis of individual alpha or beta subunits. In accordance with the upregulation of several integrins observed in bFGF-treated cells, these cells adhered better to fibronectin, laminin, vitronectin, and type I collagen than did untreated cells. The largest differences in beta 1 integrin expression occurred approximately 72 h after exposure to bFGF, at a time when the expression of the endothelial cell-to-cell adhesion molecule endoCAM was also significantly upregulated. In contrast, a shorter exposure to bFGF (24-48 h) was required for the maximal induction of plasminogen activator production in the same cells. Taken together, these results show that bFGF causes significant changes in the level of expression and function of several integrins in microvascular endothelial cells.     

Inhibitory effects of Porphyromonas gingivalis fimbriae on interactions between extracellular matrix proteins and cellular integrins

Porphyromonas gingivalis is a predominant periodontal pathogen, whose fimbriae are considered to be a major virulence factor, especially for bacterial adherence and invasion of host cells. In the present study, we investigated the influence of fimbriae on the interactions between alphavbeta3- and alpha5beta1-integrins and their ligand extracellular matrix (ECM) proteins (vitronectin and fibronectin), using human alphavbeta3- and alpha5beta1-integrin-overexpressing CHO cell lines (CHOalphavbeta3 and CHOalpha5beta1, respectively). P. gingivalis was found to have significantly greater binding to CHOalphavbeta3 and CHOalpha5beta1 than to control cells, whereas a fimbria-deficient mutant showed negligible binding to any of the tested cell lines. CHOalphavbeta3 and CHOalpha5beta1 cells attached to the polystyrene culture dishes in the presence of their ligand ECM proteins, while fimbriae markedly inhibited those attachments in a dose-dependent manner, with the highest dose of fimbriae achieving complete inhibition. In addition, the binding of vitronectin and fibronectin to CHOalphavbeta3 and CHOalpha5beta1 was inhibited by P. gingivalis cells. These results suggest that P. gingivalis fimbriae compete with ECM proteins for alphavbeta3- and alpha5beta1-integrins, and inhibit integrin/ECM protein-related cellular functions.     

The integrin beta 1 subunit associates with the vitronectin receptor alpha v subunit to form a novel vitronectin receptor in a human embryonic kidney cell line.

We describe a novel integrin heterodimer on the surface of the human embryonic kidney cell line 293. This receptor is comprised of alpha v and beta 1 subunits, each of which has been previously found in association with other integrin subunits. This alpha v.beta 1 complex was identified as the predominant vitronectin receptor (VnR) on the surface of 293 cells by immunoprecipitation with antibodies raised against the alpha v subunit. Polymerase chain reaction analysis detected mRNAs for alpha v and beta 1 subunits while no evidence was obtained for beta 2, beta 3, or alpha IIb integrin subunit mRNA. Immunoprecipitation of surface-iodinated proteins with antibodies to alpha v gave bands of 150 and 120 kDa. The 120-kDa band reacted with antibodies to beta 1 in immunoblotting experiments. 293 cells adhere to vitronectin, fibronectin, laminin, and collagen IV, while von Willebrand factor and fibrinogen, known ligands of the VnR (alpha v.beta 3), did not support adhesion. A polyclonal antibody directed against both subunits of the VnR (alpha v, beta 3) inhibits attachment of 293 cells to vitronectin but not to other adhesive proteins. A beta 1-specific monoclonal inhibited attachment to fibronectin, laminin, and collagen IV, known ligands of beta 1 integrins, as well as vitronectin. This novel (alpha v. beta 1) VnR thus appears to mediate cell adhesion exclusively to vitronectin, in contrast to previously described VnRs which have multiple ligands.     

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.     

Tumor necrosis factor alpha and interferon gamma modulate the expression of the vitronectin receptor (integrin beta 3) in human endothelial cells.

In this paper we show that tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) alter the expression of extracellular matrix receptors (integrins) in cultured human endothelial cells. Endothelial cells express at their surface integrins of the beta 1 and beta 3 groups that include receptors for fibronectin, collagen, laminin, and vitronectin. After treatment for 72 h with a combination of TNF alpha and IFN gamma, the level of the vitronectin receptor (alpha v beta 3) at the cell surface decreases by 70%, whereas the amounts of the beta 1 integrins remain unchanged. The decreased expression of the alpha v beta 3 complex at the cell surface is due to a selective effect of TNF alpha and IFN gamma on the regulation of the beta 3 subunit synthesis at the translational level. In fact, although the steady state levels of the mRNA for the beta 3 subunit are comparable in control and treated cells, the overall synthesis of the beta 3 subunit is decreased by a factor of 70%. No significant alteration of the synthesis of the companion alpha v subunit is detectable in cytokine-treated cells. As a consequence of the decreased expression of the receptor, cytokine-treated cells show decreased ability to adhere to vitronectin but adhere normally to fibronectin. These data show that two important inflammatory mediators, TNF alpha and IFN gamma, can modify the interaction of endothelial cells with the extracellular matrix by selectively altering the expression of specific cell surface integrin complexes.     

Cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta3 integrins

Hantaviruses replicate primarily in the vascular endothelium and cause two human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). In this report, we demonstrate that the cellular entry of HFRS-associated hantaviruses is facilitated by specific integrins expressed on platelets, endothelial cells, and macrophages. Infection of human umbilical vein endothelial cells and Vero E6 cells by the HFRS-causing hantaviruses Hantaan (HTN), Seoul (SEO), and Puumala (PUU) is inhibited by antibodies to alphavbeta3 integrins and by the integrin ligand vitronectin. The cellular entry of HTN, SEO, and PUU viruses, but not the nonpathogenic Prospect Hill (PH) hantavirus (i.e., a virus with no associated human disease), was also mediated by introducting recombinant alphaIIbbeta3 or alphavbeta3 integrins into beta3-integrin-deficient CHO cells. In addition, PH infectivity was not inhibited by alphavbeta3-specific sera or vitronectin but was blocked by alpha5beta1-specific sera and the integrin ligand fibronectin. RGD tripeptides, which are required for many integrin-ligand interactions, are absent from all hantavirus G1 and G2 surface glycoproteins, and GRGDSP peptides did not inhibit hantavirus infectivity. Further, a mouse-human hybrid beta3 integrin-specific Fab fragment, c7E3 (ReoPro), also inhibited the infectivity of HTN, SEO, and PUU as well as HPS-associated hantaviruses, Sin Nombre (SN) and New York-1 (NY-1). These findings indicate that pathogenic HPS- and HFRS-causing hantaviruses enter cells via beta3 integrins, which are present on the surfaces of platelets, endothelial cells, and macrophages. Since beta3 integrins regulate vascular permeability and platelet function, these findings also correlate beta3 integrin usage with common elements of hantavirus pathogenesis.     

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes

We have compared the adhesive properties and integrin expression profiles of cultured human epidermal keratinocytes and a strain of nondifferentiating keratinocytes (ndk). Both cell types adhered to fibronectin, laminin, and collagen types I and IV, but ndk adhered more rapidly and at lower coating concentrations of the proteins. Antibody blocking experiments showed that adhesion of both cell types to fibronectin was mediated by the alpha 5 beta 1 integrin and to laminin by alpha 3 beta 1 in synergy with alpha 2 beta 1. Keratinocytes adhered to collagen with alpha 2 beta 1, but an antibody to alpha 2 did not inhibit adhesion of ndk to collagen. Both cell types adhered to vitronectin by alpha v-containing integrins. Immunoprecipitation of surface-iodinated and metabolically labeled cells showed that in addition to alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1, both keratinocytes and ndk expressed alpha 6 beta 4 and alpha v beta 5. ndk expressed all these integrins at higher levels than normal keratinocytes. ndk, but not normal keratinocytes, expressed alpha v beta 1 and alpha v beta 3; they also expressed alpha 1 beta 1, an integrin that was not consistently detected on normal keratinocytes. Immunofluorescence experiments showed that in stratified cultures of normal keratinocytes integrin expression was confined to cells in the basal layer; terminally differentiating cells were unstained. In contrast, all cells in the ndk population were integrin positive. Our observations showed that the adhesive properties of ndk differ from normal keratinocytes and reflect differences in the type of integrins expressed, the level of expression and the distribution of integrins on the cell surface. ndk thus have a number of characteristics that distinguish them from normal basal keratinocytes.     

Cell adhesion and integrin expression are modulated by oxidative stress in EA.hy 926 cells

The effects of oxidative stress on integrin-mediated cell adhesion to the extracellular matrix (ECM) and related apoptosis were investigated using the EA.hy926 endothelial cells treated (or not) with two oxidants: the hypoxanthine/xanthine oxidase system (HX/XO) or the tert-butyl hydroperoxide (t-BHP) which both increased cell apoptosis. Cell adhesion onto vitronectin (Vn) and fibronectin (Fn) was increased at low concentrations of HX/XO (up to 5 mU/ml) or t-BHP (up to 125 microM) and prevented ROS-induced apoptosis. Flow cytometry analysis of integrin expression showed that the expression of integrin alphav and alpha5 subunits was, respectively, increased and decreased. Cell adhesion inhibition experiments using function-blocking monoclonal antibodies against integrin subunits indicated that alphavbeta1 and alphavbeta3 integrins were involved in adhesion of cells to Vn, and alphavbeta3 integrin played a major role in oxidant-treated cells. For adhesion to Fn, alpha5beta1 and alphavbeta1 integrins were required for oxidant-treated cells. Taken together, the results suggest that reactive oxygen species (ROS) produced either by HX/XO or t-BHP could affect expression and/or activation of specific integrins in the interaction of EA.hy926 cells with ECM.     

Matrix-specific suppression of integrin activation in shear stress signaling

Atherosclerotic plaque develops at sites of disturbed flow. We previously showed that flow activates endothelial cell integrins, which then bind to the subendothelial extracellular matrix (ECM), and, in cells on fibronectin or fibrinogen, trigger nuclear factor-kappaB activation. Additionally, fibronectin and fibrinogen are deposited into the subendothelial ECM at atherosclerosis-prone sites at early times. We now show that flow activates ECM-specific signals that establish patterns of integrin dominance. Flow induced alpha2beta1 activation in cells on collagen, but not on fibronectin or fibrinogen. Conversely, alpha5beta1 and alphavbeta3 are activated on fibronectin and fibrinogen, but not collagen. Failure of these integrins to be activated on nonpermissive ECM is because of active suppression by the integrins that are ligated. Protein kinase A is activated specifically on collagen and suppresses flow-induced alphavbeta3 activation. Alternatively, protein kinase Calpha is activated on fibronectin and mediates alpha2beta1 suppression. Thus, integrins actively cross-inhibit through specific kinase pathways. These mechanisms may determine cellular responses to complex extracellular matrices.     

The vitronectin receptor alpha v beta 3 binds fibronectin and acts in concert with alpha 5 beta 1 in promoting cellular attachment and spreading on fibronectin

The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily of adhesive protein receptors that mediate a wide spectrum of adhesive cellular interactions, including attachment to vitronectin, von Willebrand factor, fibrinogen, and thrombospondin. We have studied the binding of fibronectin to the purified vitronectin receptor, and the role of this receptor in the attachment of cells to fibronectin. A solid-phase microtiter assay was developed to investigate the binding properties of the vitronectin receptor. Purified alpha v beta 3 bound fibronectin with high affinity in a saturable, divalent cation- dependent manner. Binding was inhibited by soluble vitronectin, by RGD- containing peptides, and by LM609, a monoclonal antibody against the vitronectin receptor known to inhibit the binding of adhesive proteins to alpha v beta 3. Immunoinhibition experiments showed that M21 human melanoma cells, which express the fibronectin receptor, alpha 5 beta 1, as well as alpha v beta 3, used both of these integrins to attach and spread on fibronectin. In support of this finding, M21-L cells, a variant cell line that specifically lacks alpha v beta 3 but expresses alpha v beta 1, attached and spread poorly on fibronectin. In addition, alpha v beta 3 from surface-labeled M21 cells was retained, and selectively eluted by RGDS from a fibronectin affinity column. These results indicate that alpha v beta 3 acts in concert with alpha 5 beta 1 in promoting fibronectin recognition by these cells. We conclude that fibronectin binds to the alpha v beta 3 vitronectin receptor specifically and with high affinity, and that this interaction is biologically relevant in supporting cell adhesion to matrix proteins.     

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.     

Alterations in integrin receptor expression on chemically transformed human cells: specific enhancement of laminin and collagen receptor complexes

The abilities of malignant tumor cells to bind and migrate through basement membranes are important steps in invasion and metastasis. Malignant tumor cells would therefore be expected to express receptors on their surfaces for basement membrane and stromal components, such as collagens, laminin, and fibronectin, although the pattern of expression of these receptors on the malignant cells may be different from that on their normal progenitors. We report here that chemically transformed tumorigenic human cells express an altered pattern of integrin receptors on their cell surfaces as compared with their untransformed nontumorigenic counterparts. Specifically, N-methyl-N'-nitro-N-nitrosoguanidine transformation of HOS cells into highly tumorigenic cells results in a significant specific increase in the expression of (in descending order of level of cell surface expression) the integrins alpha 6/beta 1, alpha 2/beta 1, and alpha 1/beta 1, which are receptors for laminin, collagens, and collagen type IV and laminin, respectively. The level of expression of two fibronectin receptor integrins, alpha 5/beta 1 and alpha 3/beta 1, are, however, unaltered, whereas the level of expression of vitronectin receptor integrin, alpha v/beta 3, is drastically reduced on the transformed cells. Consistent with the increased expression of laminin and collagen receptors and the decreased expression of vitronectin receptors on the transformed cells, these cells attached three- to fivefold more strongly to laminin and collagen but attached very poorly to vitronectin. The MNNG-HOS cells were also found to have a greater potential for invasion through reconstituted basement membrane, matrigel, the major components of which are laminin and type IV collagen. The invasion of both the HOS and MNNG-HOS cells was inhibited 45-50% by a polyclonal anti-fibronectin receptor antibody. However, although the invasion of HOS cells could be inhibited up to 75% by an anti-alpha 6 monoclonal antibody, a similar concentration of this antibody had no effect on the alpha 6-overproducing MNNG-HOS cells. A fivefold higher concentration of this antibody did result in partial inhibition of MNNG-HOS invasion. These data indicate a critical role for the alpha 6/beta 1 laminin receptor in the invasion of these cells through basement membranes and demonstrate that chemical transformation of nontumorigenic human cells to highly tumorigenic cells is associated with an altered pattern of integrin expression which may play a direct role in the increased capacity of these cells to bind and invade through basement membranes.     

Fibronectin- and vitronectin-induced microglial activation and matrix metalloproteinase-9 expression is mediated by integrins alpha5beta1 and alphavbeta5

Early in the pathogenesis of multiple sclerosis, the blood-brain barrier is compromised, which leads to deposition of the plasma proteins fibronectin and vitronectin in cerebral parenchyma. In light of our previous finding that microglial activation in vitro is strongly promoted by fibronectin and vitronectin, we set out to examine the possibility that modulation of microglial activation by fibronectin or vitronectin is an important regulatory mechanism in vivo. In an experimental autoimmune encephalomyelitis mouse model of demyelination, total brain levels of fibronectin and vitronectin were strongly increased and there was a close relationship between fibronectin and vitronectin deposition, microglial activation, and microglial expression of matrix metalloproteinase-9. In murine cell culture, flow cytometry for MHC class I and gelatin zymography revealed that microglial activation and expression of pro-matrix metalloproteinase-9 were significantly increased by fibronectin and vitronectin. Function-blocking studies showed that the influence of fibronectin and vitronectin was mediated by the alpha(5)beta(1) and alpha(v)beta(5) integrins, respectively. Taken together, this work suggests that fibronectin and vitronectin deposition during demyelinating disease is an important influence on microglial activation state. Furthermore, it provides the first evidence that the alpha(5)beta(1) and alpha(v)beta(5) integrins are important mediators of microglial activation.