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PubMed2001

Cell-surface transglutaminase promotes fibronectin assembly via interaction with the gelatin-binding domain of fibronectin: a role in TGFbeta-dependent matrix deposition.

Assembly of fibronectin into a fibrillar matrix is critical for regulation of cell growth and migration, embryogenesis and wound healing. We have previously shown that cell-surface tissue transglutaminase serves as an integrin-binding adhesion coreceptor for fibronectin. Here we report that transglutaminase strongly promotes fibronectin assembly mediated by alpha5beta1 integrin. This effect is independent from transglutaminase-mediated enzymatic crosslinking of fibronectin and separate from the ability of transglutaminase to stimulate cell spreading. Surface transglutaminase increases the binding of fibronectin to cells via interaction with its gelatin-binding domain that contains modules I6II1,2I7-9 and lacks integrin-binding motifs. The gelatin-binding fragment of fibronectin binds to surface transglutaminase on cells in suspension but does not interact with cell monolayers where surface transglutaminase is occupied by fibronectin. Surface transglutaminase colocalizes with growing fibronectin fibrils at early timepoints of matrix formation and remains codistributed with fibronectin matrices thereafter. The observed stimulation of matrix assembly by transglutaminase is blocked by the gelatin-binding fragment of fibronectin, but is not strongly perturbed by its N-terminal fragment consisting of modules I1-5. These results implicate an interaction between transglutaminase and the gelatin-binding domain of fibronectin in matrix assembly and suggest its role in initiation of fibrillogenesis. However, blocking antibodies against alpha5beta1 integrin or the cell-binding fragment of fibronectin that contains modules III2-11 most strongly suppress matrix formation and abolish the effects of transglutaminase. Hence, transglutaminase cooperates with but can not substitute for alpha5beta1 integrin in fibronectin assembly. Treatment of fibroblasts with transforming growth factor beta (TGFbeta) significantly increases surface expression of transglutaminase and its association with beta1 integrins, but not with alphaVbeta3 integrin. TGFbeta enhances the binding of fibronectin to the cell surface and elevates matrix formation, whereas antibody against transglutaminase or the gelatin-binding fragment of fibronectin suppresses these effects, indicating an involvement of transglutaminase in TGFbeta-dependent fibronectin assembly. Therefore, TGFbeta-induced fibronectin matrix deposition during normal wound healing or fibrotic disorders may depend on upregulation of integrin-associated surface transglutaminase.

Animals↗
PubMed2005

Distinct mechanisms of integrin binding by Yersinia pseudotuberculosis adhesins determine the phagocytic response of host macrophages.

The enteropathogenic yersiniae express two outer membrane adhesins, invasin and YadA, that contribute to pathogenesis. While invasin binds directly to beta1 integrin receptors with high affinity, YadA binds indirectly through extracellular matrix (ECM) components. In this study, Yersinia pseudotuberculosis inv and yadA mutants were used to investigate how these distinct binding mechanisms compare and potentially compete in activating signalling pathways and promoting bacterial uptake by host macrophages. The efficiency of adhesin-mediated phagocytic responses was found to be dependent on the relative expression of invasin and YadA on the bacterial surface as well as the expression of ECM proteins in the extracellular milieu. Under conditions of low concentrations of ECM, invasin was found to be the dominant adhesin, promoting high levels of phagocytosis coincident with robust and sustained activation of the protein tyrosine kinases Fak and Pyk2, phosphorylation of the adaptor molecule Cas and activation of the small GTPase Rac1. In the presence of higher concentrations of ECM, YadA became the dominant functional adhesin through its ability to engage integrin receptors via an ECM bridge. We propose a model whereby invasin promotes robust and prolonged activation of phagocytic signalling cascades by inducing a 'high-affinity' integrin conformation as well as integrin clustering. We postulate that YadA-ECM promotes phagocytosis through a more transient activation of signalling cascades that arises from integrin clustering in the context of a cross-linked fibrillar ECM network.

Adhesins, Bacterial↗
PubMed2005

Integrin alpha1beta1 mediates collagen induction of MMP-13 expression in MC615 chondrocytes.

During endochondral ossification, type I collagen is synthesized by osteoblasts together with some hypertrophic chondrocytes. Type I collagen has also been reported to be progressively synthesized in degenerative joints. Because Matrix Metalloproteinase-13 (MMP-13) plays an active role in remodeling cartilage in fetal development and osteoarthritic cartilage, we investigated whether type I collagen could activate MMP-13 expression in chondrocytes. We used a well-established chondrocytic cell line (MC615) and we found that MMP-13 expression was induced in MC615 cells cultured in type I collagen gel. We also found that alpha1beta1 integrin, a major collagen receptor, was expressed by MC615 cells and we further assessed the role of alpha1beta1 integrin in conducting MMP-13 expression. Induction of MMP-13 expression by collagen was potently and synergistically inhibited by blocking antibodies against alpha1 and beta1 integrin subunits, indicating that alpha1beta1 integrin mediates the MMP-13-inducing cellular signal generated by three-dimensional type I collagen. We also determined that activities of tyrosine kinase and ERK and JNK MAP kinases were required for this collagen-induced MMP-13 expression. Interestingly, bone morphogenetic protein (BMP)-2 opposed this induction, an effect that may be related to a role of BMP-2 in the maintenance of cartilage matrix.

Animals↗
PubMed2004

Evaluation of sarcoglycans, vinculin-talin-integrin system and filamin2 in alpha- and gamma-sarcoglycanopathy: an immunohistochemical study.

The sarcoglycan subcomplex (SGC) is a well-known system of interaction between extracellular matrix and sarcolemma-associated cytoskeleton in skeletal and cardiac muscle. The SGC is included in the DGC made up of sarcoplasmic subcomplex and a dystroglycan subcomplex. Recent developments in molecular genetics have demonstrated that the mutation of each single sarcoglycan gene, causes a series of recessive autosomal muscular dystrophies, dystrophin-positive, called sarcoglycanopathies or limb girdle muscular dystrophies. Our recent studies have demonstrated that costameres are a proteic machinery made up of DGC and vinculin-talin-integrin system, also revealing the colocalization of sarcoglycans and integrins in adult human skeletal muscle. These results may support the hypothesis of the existence of a bidirectional signalling between sarcoglycans and integrins in cultured L6 myocytes. The hypothesis of bidirectional signalling between sarcoglycans and integrins could be supported by the identification of a skeletal and cardiac muscle filamin2 as a gamma-sarcoglycan, delta-sarcoglycan and, hypothetically, beta1 integrin interacting protein. Our results, acquired with an immunofluorescence study on adult human skeletal muscle affected by LGMD type 2D and 2C, showed that in LGMD2D: a) alpha-sarcoglycan staining is severely reduced; b) the beta-gamma-delta-sarcoglycan subunit and all tested integrins staining are clearly detectable; c) filamin2 is normal and shows a costameric distribution. In LGMD2C: a) alpha-sarcoglycan staining is preserved; b) the beta-gamma-delta-sarcoglycan subunit staining is severely reduced; c) the alpha7B-integrin is slightly reduced and beta1D-integrin is severely reduced; d) filamin2 is severely reduced. Other tested proteins of the two systems show a normal staining pattern in both sarcoglycanopathies. Our study seems to confirm, for the first time on adult human skeletal muscle of subjects affected by LGMDs, the hypo-theses of: a) the existence, in mouse myotubes in culture, of two distinct subunits in sarcoglycans subcomplex; b) the presence of a bidirectional signalling between sarcoglycans and integrins, previously demonstrated on rat cultured L6 myocytes; c) the interaction of FLN2 with both sarcoglycans and integrins. These results may stimulate the search of yet unidentified common interactors of both fiber-extracellular matrix interaction systems.

Biopsy↗
PubMed1997

Fibronectin suppresses apoptosis in normal human melanocytes through an integrin-dependent mechanism.

Recent reports show that components of the extracellular matrix function as cell survival factors through the suppression of apoptosis (programmed cell death). In this report we show that attachment to fibronectin suppresses apoptosis of normal human fetal and neonatal melanocytes in vitro and that prevention of attachment to underlying matrix or attachment to poly-L-lysine is a potent inducer of apoptosis in melanocytes. A role for the beta1-integrin family in mediating cell survival signals was shown by the ability of beta1-blocking antibodies to enhance apoptosis in melanocytes attached to fibronectin, and by the ability of anti-beta1 antibodies immobilized on solid supports to suppress apoptosis in melanocytes. Cytochalasin D reversed the effect of fibronectin on the suppression of apoptosis in melanocytes, suggesting that an intact cytoskeleton is required for transduction of survival signals. A human metastatic melanoma cell line, SKMEL28, was resistant to apoptosis when grown in suspension or on poly-L-lysine, even after 4 d in culture in the absence of exogenous growth factors. These results suggest that fibronectin suppresses apoptosis in normal human melanocytes through an integrin-dependent pathway and that significant differences in the control of anchorage-dependent regulation of apoptosis exist in melanocytes and melanoma cells.

Antibodies↗
PubMed2002

Involvement of integrin alphavbeta3 in the pathogenesis of human immunodeficiency virus type 1 infection in monocytes.

Attachment of HIV to macrophages is a critical early event in the establishment of infection. In the present study, we demonstrate the involvement of integrin alphavbeta3 (vitronectin receptor) in HIV infection of peripheral blood monocyte-derived macrophages. Culturing monocytes in the presence of M-CSF for 3 days upregulated expression of the alphav-containing integrins, alphavbeta3 and alphavbeta5. The increase in alphavbeta3 expression was accompanied by increased HIV-1 replication by monocytes. Immunoblot analysis showed that purified HIV-gp120 protein interacted with CD4 and alphavbeta3 in immunoprecipitation experiments. Neutralizing antibodies against the alphavbeta3 integrin interfered with the coprecipitation of alphavbeta3 with an anti-gp120 antibody and substantially inhibited HIV infection of monocytes. Neutralizing antibodies against alphavbeta5 or beta1 integrins did not significantly affect HIV infection. These results indicate that HIV infection of primary monocytes requires differentiation of these cells and may involve alphavbeta3 interaction with the HIV-1 envelope protein gp120 for productive infection.

Antibodies↗
PubMed2006

Effect of homocysteine on platelet activation induced by collagen.

OBJECTIVE: The present study investigated the effects of homocysteine on platelet activation induced by collagen and the downstream signaling pathways potentially involved in these effects. METHODS: Washed human platelets were incubated with homocysteine and collagen type I. The effects of homocysteine on platelet aggregation and adhesion and the tyrosine phosphorylation of total platelet proteins, Src kinase, and phospholipase-Cgamma2 (PLCgamma2) were studied. RESULTS: Homocysteine (10 to 100 microM) increased collagen-induced aggregation of washed platelets. Upon homocysteine (50 to 100 microM) treatment, platelet deposition to collagen-coated surface was significantly augmented under the low shear rate model (100/s) but not under the high shear rate model (1600/s). Collagen-stimulated total protein tyrosine phosphorylation in platelets was further enhanced by incubation with homocysteine. This effect was almost abrogated by genistein. Homocysteine potentiated collagen-stimulated tyrosine phosphorylation of the Src kinase and PLCgamma2, which was partly decreased by integrin beta1 blocking antibody. CONCLUSION: Homocysteine (at 10 to 100 microM) potentiates collagen type I induced-platelet activation through signaling components of glycoprotein VI and integrin alpha2beta1 pathway. Our results suggested that upregulation of tyrosine phosphorylation of proteins such as Src and PLCgamma2 is involved in the downstream signaling events of homocysteine stimulation in human platelets.

Blotting, Western↗
PubMed1998

Effect of ligand conformation on melanoma cell alpha3beta1 integrin-mediated signal transduction events: implications for a collagen structural modulation mechanism of tumor cell invasion.

The importance of three-dimensional interactions between receptors with their respective ligands has been extensively explored during the binding process, but considerably less so for postbinding events such as induction of signaling pathways. Tumor cell receptor association with basement membrane proteins is believed to facilitate the metastatic process. Melanoma and ovarian carcinoma cells have been shown to utilize the alpha3beta1 integrin to bind to models of the alpha1(IV)531-543 sequence from basement membrane (type IV) collagen [Miles, A. J., et al. (1994) J. Biol. Chem. 269, 30939-30945; Miles, A. J., et al. (1995) J. Biol. Chem. 270, 29047-29050]. In the present study, the effects of ligand three-dimensional structure on possible signal transduction pathways induced by alpha3beta1 integrin binding have been evaluated. Human melanoma cell binding to type IV collagen resulted in Tyr phosphorylation of p125(FAK), consistent with prior studies correlating beta1 integrin subunit binding to collagen and p125(FAK) Tyr phosphorylation. Cross-linking of an anti-alpha3 integrin subunit monoclonal antibody also induced p125(FAK) Tyr phosphorylation. Incubation of melanoma cells with single-stranded or triple-helical peptide models of alpha1(IV)531-543 induced Tyr phosphorylation of intracellular proteins. Immunoprecipitation analysis identified one of these proteins as pp125(FAK). Induction of p125(FAK) Tyr phosphorylation was enhanced and the time of induction was shortened when the ligand was used in triple-helical conformation. Subsequent clustering of either the single-stranded or the triple-helical ligand also increased the level of p125(FAK) phosphorylation compared to unclustered ligand. The clustered triple-helical peptide ligand induced more rapid paxillin Tyr phosphorylation than the single-stranded ligand. In addition, the induction of activated proteases was found to be more rapid due to ligand triple helicity. Overall, these studies have shown that (i) a model of an isolated sequence from type IV collagen, alpha1(IV)531-543, can induce alpha3beta1 integrin-mediated signal transduction in melanoma cells and (ii) ligand conformation (secondary, tertiary, and/or quaternary structure) can directly influence several alpha3beta1 integrin-mediated signal transduction events. The effects of ligand conformation suggest that a "collagen structural modulation" mechanism may exist for tumor cell invasion, whereby triple-helical collagen promotes cell binding and induction of signal transduction, subsequently leading to collagen dissolution by proteases, decreased signal transduction, and enhanced tumor cell motility.

Cell Adhesion↗
PubMed1999

Integrin alpha4beta1/VCAM-1 pathway mediates primary adhesion of RAW117 lymphoma cells to hepatic sinusoidal endothelial cells under flow.

Adhesion and stabilization of circulating tumor cells to endothelial cells in target blood vessels play an important role in the complex process of metastasis. We examined the cell surface receptors involved in the liver-metastatic adhesive interactions of murine RAW117 large-cell lymphoma cells to unstimulated hepatic sinusoidal endothelial cells (HSE) under physiological flow conditions. Flow cytometric analysis indicated that VCAM-1, ICAM-1 and PECAM-1 are constitutively expressed on the surfaces of both HSE and RAW117 cells. However, monoclonal antibody (mAb) blockade studies showed that ICAM-1 and PECAM-1 affected neither the attachment nor the stabilization step of the adhesion of RAW117 cells to HSE cell monolayers under flow. In contrast, RAW117 cells required a significantly lower shear stress to establish adhesion to HSE cells when VCAM-1 receptors on HSE cells were blocked with mAb. Furthermore, the presence of the anti-VCAM-1 mAb significantly decreased the extent of adhesion compared to that of the control, without affecting adherent cell stabilization times. Blocking the alpha4 integrin subunits present mainly on RAW117 cells produced similar results to those previously observed with anti-VCAM-1 mAb. Although constitutively present mainly on the surfaces of RAW117 cells, MAdCAM-1 and beta7 integrin subunit do not appear to play a role in either the arrest or stabilization of RAW117 cells on HSE cell monolayers. However, blocking the beta1 integrin subunit on the RAW117-H10 cells reduced adhesion to the same extent as anti-alpha4 and anti-VCAM-1 treatments. These observations suggest that an interaction of integrin alpha4/beta1 on RAW117 cells with liver endothelial VCAM-1 occurs during the early stages of the adhesion process and may be important in liver metastasis.

Animals↗
PubMed1998

P-selectin and MAC-1 mediate monocyte rolling and adhesion to ECM-bound platelets under flow conditions.

Accumulation of monocyte-derived foam cells in focal areas of the atherosclerotic (A.S.-) lesion is one of the key events in early atherogenesis. Using a flow model for the damaged vessel wall, we examined the ability of ECM-bound platelets to induce monocyte tethering and adhesion. Whereas ECM-proteins alone induced monocyte adhesion only at low shear stresses (< 100 mPa), ECM-bound platelets induced monocyte rolling and adhesion at shear stresses up to 240 mPa. Studies with specific antibodies showed that monocyte adhesion to platelets was mainly mediated by P-selectin and monocyte PSGL-1 (maximum inhibition 90%). beta2-Integrin blocking CD18 and CD11b antibodies partly inhibited the arrest of rolling cells. Antibodies against other adhesion molecules such as LFA-1, PECAM-1, and beta1-integrins had no effect. Even sparsely adhered platelets (approximately 10% coverage of the surface) already strongly supported monocyte tethering. In conclusion, activated platelets present on ECM are a powerful adhesive substrate for monocyte recruitment under flow conditions.

Antibodies, Monoclonal↗
PubMed1997

Upregulation of CD9 expression during TPA treatment of K562 cells.

The CD9 antigen, a major platelet glycoprotein, is a member of the tetraspan superfamily. We show that treatment of K562 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) which induces megakaryocytic differentiation, leads to a seven-fold increase in CD9 expression, which becomes associated with the integrin beta1, suggesting that it is functionally relevant. The upregulation of CD9 expression precedes the appearance of the megakaryocytic-specific marker GPIIb (CD41) as well as integrins beta3 (GPIIIa/CD61), alpha v (CD51) and VLA-2 (CD49b). Both GPIIb/IIIa expression and CD9 upregulation are dependent on protein kinase C (PKC) activation since they are blocked by the specific inhibitor GF109203X. Steady-state levels of CD9 and GPIIb mRNA were also measured by quantitative RT-PCR. Both messengers were detected on resting cells and were shown to accumulate during TPA treatment. However, the increase of the CD9 mRNA was detected much earlier than the increase of GPIIb mRNA (1-2 h vs 24-48 h). Using different constructs of the 5'-flanking domain of the CD9 gene cloned ahead of the CAT reporter gene, we could demonstrate that a responsive element was located in a 52 bp fragment of the promoter of the CD9 gene. Altogether, these data suggest that CD9 upregulation in the megakaryocytic lineage could occur at early stages of differentiation.

Antigens, CD↗
PubMed2001

The cribriform features of adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma: cytokeratin and integrin expression.

Cribriform areas are common features of both adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma. Both are malignant salivary gland tumors that share similar histologic patterns, but with marked distinct clinical behavior. This study was undertaken to improve the accuracy of the histopathology diagnostic process, using an immunohistochemical panel to differentiate adenoid cystic carcinoma from polymorphous low-grade adenocarcinoma, with special concern to the common cribriform areas shared by these tumors. Three-microm serial sections of these tumors were submitted to the streptavidin-biotin peroxidase immunotechnique against the monoclonal antibodies anticytokeratins 7, 8, 14 and 19, and anti-integrins beta1, beta3, and beta4. In the neoplastic lobules of adenoid cystic carcinoma cribriform type, the spaces were mainly surrounded by cells negative for the cytokeratins and integrins studied. In the solid type of adenoid cystic carcinoma, the microcystic areas were caused by spaces lined by neoplastic luminal cells positive for cytokeratins and presenting integrins concentrated in the apical pole of these cells. The cribriform areas of polymorphous low-grade adenocarcinoma were composed of cords of luminal cells, positive for cytokeratins and showing integrins disposed in a bipolar pattern. We concluded that cribriform areas of adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma are histologically similar, although not identical. Indeed, their cellular composition is distinct and can be distinguishable from one another by the proteins of the cytoskeleton, by the integrins, or both.

Adenocarcinoma↗
PubMed1999

Microfibril-associated glycoprotein-2 specifically interacts with a range of bovine and human cell types via alphaVbeta3 integrin.

Microfibril-associated glycoprotein (MAGP)-1 and MAGP-2 are small structurally related glycoproteins that are specifically associated with fibrillin-containing microfibrils. MAGP-2, unlike MAGP-1, contains an RGD motif with potential for integrin binding. To determine if the RGD sequence is active, a series of cell binding assays was performed. MAGP-2 was shown to promote the attachment and spreading of bovine nuchal ligament fibroblasts when coated onto plastic wells in molar quantities similar to those of fibronectin. In contrast, approximately 10-fold more MAGP-1 was required to support comparable levels of cell adhesion. The fibroblast binding to MAGP-2 was completely inhibited if the peptide GRGDSP or the MAGP-2-specific peptide GVSGQRGDDVTTVTSET was added to the reaction medium at a 10 microM final concentration. The control peptide GRGESP had no effect on the interaction. These findings indicate that the cell interaction with MAGP-2 is an RGD-mediated event. A monoclonal antibody to human alphaVbeta3 integrin (LM609) almost completely blocked cell attachment to MAGP-2 when added to the medium at 0.5 microgram/ml, whereas two monoclonal antibodies specific for the human beta1 integrin subunit, 4B4 (blocking) and QE2.E5 (activating), had no effect even at 10 microgram/ml. Fetal bovine aortic smooth muscle cells, ear cartilage chondrocytes, and arterial endothelial cells and human skin fibroblasts and osteoblasts were also observed to adhere strongly to MAGP-2. In addition, each cell type was able to spread on MAGP-2 substrate, with the exception of the endothelial cells, which remained spherical after 2 h of incubation. The binding of each cell type was blocked when the anti-alphaVbeta3 integrin antibody was included in the assay, indicating that alphaVbeta3 integrin is the major receptor for MAGP-2 on several cell types. Thus, MAGP-2 may mediate interactions between fibrillin-containing microfibrils and cell surfaces during the development of a variety of tissues.

Adult↗
PubMed1997

Alphavbeta3 integrin associates with activated insulin and PDGFbeta receptors and potentiates the biological activity of PDGF.

Integrin-mediated cell attachment modulates growth responses and growth factors regulate cell attachment. Moreover, both cell attachment to extracellular matrix and mitogenic signaling by growth factors are necessary for the proliferation of most types of normal cells, suggesting that integrin and growth factor receptor signaling pathways meet at some downstream point. We report here that a small, highly tyrosine-phosphorylated fraction of PDGFbeta and insulin receptors co-immunoprecipitates with the alphavbeta3 integrin from cells. The integrin association requires growth factor stimulation of the receptors. Several signaling molecules that are known to be associated with activated growth factor receptors were present in the alphavbeta3 integrin complexes. Mitogenicity and chemotaxis induced by PDGF-BB were enhanced in cells plated on the alphavbeta3 ligand vitronectin compared with cells plated on the beta1 integrin ligand collagen. Thus, the engagement of the alphavbeta3 integrin in cell-matrix interactions appears to coordinate an intense response to growth factors, helping to explain the importance of this integrin for tissue regeneration, angiogenesis and tumor metastasis.

3T3 Cells↗
PubMed1998

Streptococcus pyogenes serotype M1 encodes multiple pathways for entry into human epithelial cells.

The ability of a serotype M1 strain of Streptococcus pyogenes to efficiently invade A549 human lung epithelial cells was previously shown to be dependent on bacterial exposure to human or bovine serum proteins or synthetic peptides containing the sequence RGD. In this study, stimulation by invasion agonists was determined to be dependent on expression of the streptococcal cell surface protein, M1. Fetal bovine serum (FBS), fibronectin (Fn), the extracellular matrix protein laminin (Lm), and RGD-containing peptides were tested for their abilities to promote epithelial cell invasion and adherence by isogenic M1(+) and M1(-) strains of S. pyogenes. In the absence of an agonist, invasion and adherence were comparable for the two bacterial strains. FBS, Fn, and Lm stimulated invasion of the M1(+) strain as much as 70-fold but failed to significantly affect invasion by the M1(-) mutant. Adherence of the wild-type strain was stimulated by these same agonists. Epithelial cell adherence by the M1(-) strain, however, was unaffected by the presence of Fn or Lm. Several RGD-containing peptides were found to promote invasion independently of M1 expression. Binding of 125I-Fn was reduced 88% by the M1(-) mutation and Fn was found to bind purified M1 protein, suggesting that Fn mediates invasion by direct binding to M1. To determine if host integrins might be involved in internalization of streptococci, several anti-integrin monoclonal antibodies (MAbs) were tested for their abilities to inhibit invasion. Antibody directed against integrin beta1 inhibited FBS-, Fn-, and Lm-mediated invasion but did not abrogate RGD-peptide-stimulated invasion. MAb directed against the epithelial cell Fn receptor, integrin alpha5beta1, inhibited Fn and FBS-mediated invasion but did not specifically inhibit Lm-mediated invasion. These results indicate that S. pyogenes has evolved multiple mechanisms for invasion of eukaryotic cells, at least two of which involve interactions between M1 protein, host integrins, and integrin ligands.

Antigens, Bacterial↗
PubMed1999

Identification of laminin-10/11 as a strong cell adhesive complex for a normal and a malignant human epithelial cell line.

Laminins are heterotrimeric proteins of basement membranes. More than 50 different trimers may exist. Laminin-10 (alpha5beta1gamma1 rather than laminin-1 (alpha1beta1gamma1) could be the most abundant isoform in the adult stage, and laminin-10 is made by several developing epithelial sheets. We show here that a much used commercial human preparation contains laminin-10 (alpha5beta1gamma1), some laminin-11 (alpha5beta2gamma1), but no laminin-1. Moreover, the laminin-10/11 mixture was found to be a strong adhesive for two human cell lines derived from epithelia. Antibodies against integrin beta1, alpha6 or alpha3 (at 50 microgram/ml) or dystroglycan did not inhibit cell attachment to laminin-10/11, although lower concentrations of anti-dystroglycan and integrin alpha6 antibodies inhibited cell binding to laminin-1.

Antibodies↗
PubMed2000

Localization of urokinase type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors.

Previous studies have shown that the adhesion protein, vitronectin, directs the localization of urokinase-type plasminogen activator (uPA) to areas of cell-substrate adhesion, where uPA is thought to regulate cell migration as well as pericellular proteolysis. In the present study, HT-1080 cell lines expressing either wild-type vitronectin or vitronectin containing a single amino-acid substitution in the integrin binding domain were used to assess whether ligation of the alphavbeta5 integrin was required for uPA localization to focal adhesions. The synthesis of wild-type vitronectin by HT-1080 cells adherent to either collagen or fibronectin resulted in the redistribution of both the alphavbeta5 integrin as well as uPA to focal adhesion structures. In contrast, cells synthesizing mutant vitronectin, containing the amino-acid substitution in the integrin binding domain, were unable to direct the redistribution of either alphavbeta5 or uPA to focal adhesions. Recombinant forms of wild-type and mutant vitronectin were prepared in a baculovirus system and compared for their ability to direct the redistribution of vitronectin integrin receptors as well as uPA on human skin fibroblasts. In the absence of vitronectin, fibroblast cells adherent to fibronectin assemble focal adhesions which contain the beta1 integrin but do not contain uPA. Addition of recombinant wild-type, but not mutant, vitronectin to fibroblasts adherent to fibronectin resulted in the redistribution of alphavbeta3, alphavbeta5, and uPA into focal adhesions. However, when cells were plated directly onto antibodies directed against either the alphavbeta3 or alphavbeta5 integrins, uPA was not localized on the cell surface. These data indicate that ligation of vitronectin integrin receptors is necessary but not sufficient for the localization of uPA to areas of cell matrix adhesion, and suggest that vitronectin may promote cell migration by recruiting vitronectin integrin receptors and components of the plasminogen activator system to areas of cell matrix contact.

Extracellular Matrix↗
PubMed2002

Unique ability of integrin alpha(v)beta 3 to support tumor cell arrest under dynamic flow conditions.

Shear-resistant arrest of circulating tumor cells is required for metastasis from the blood stream. Arrest during blood flow can be supported by tumor cell interaction with attached, activated platelets. This is mediated by tumor cell integrin alpha(v)beta3 and cross-linking plasma protein ligands. To analyze the mechanism of tumor cell ligand interactions under dynamic flow conditions, we used real-time video microscopy and tested human melanoma cell binding to fibrinogen, von Willebrand Factor, or fibronectin matrices in a buffer perfusion system. When perfused at venous flow, melanoma cells arrested abruptly and began to spread immediately. This was uniquely mediated by integrin alpha(v)beta3 on all tested ligands, and required alpha(v)beta3 activation and actin polymerization. Under static conditions, alpha(v)beta3 cooperated with alpha(v)beta1 and alpha5beta1 in supporting melanoma cell adhesion to fibronectin. But even when activated, beta1 integrins did not contribute to melanoma cell arrest during flow. Soluble ligand served as a cross-linker between attached and circulating tumor cells and enhanced melanoma cell arrest. Cohesion of activated melanoma cells was restricted to the matrix surface and did not occur in suspension. We conclude that the presence of alpha(v)beta3 in a functionally activated state provides a unique advantage for circulating tumor cells by promoting tumor cell arrest in the presence of flow-dependent shear forces.

Actins↗