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.     

Role of α5β1 and αvβ3 integrins in relation to adhesion and spreading dynamics of prostate cancer cells interacting with fibronectin under in vitro conditions

Interaction of cell integrins with the ECM (extracellular matrix) proteins is commonly assumed to be associated with cell dissemination and tumour metastases. Since these processes depend on the mechanism of cell-protein interaction, we have attempted to show the contribution of α5β1 and αvβ3 integrins of the prostate cancer PC-3 cells in in vitro interaction with FN (fibronectin) adsorbed on defined polystyrene surfaces. Cell adhesion, spreading and cytoskeleton organization were studied using antibodies against integrins or a GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) peptide. The results show that blocking the α5β1 integrin causes: (i) a decrease in the number of the adherent cells in the early phase of adhesion and (ii) a decrease in the dynamics of cell spreading and cell shape changes, and weaker reorganization of cytoskeletal proteins than in the control cells. Conversely, the blocking of the αvβ3 integrin: (i) causes no observable effect on the number of the adhered cells; however, (ii) causes an increase in the dynamics of cell spreading and cell shape changes, and stronger reorganization of cytoskeletal proteins than in the control cells. Interestingly, the blocking of integrins with a GRGDSP peptide strongly decreases the number of the adhered cells, and a complete inhibition of cell spreading. Our results strongly suggest that the α5β1 integrin plays the main role in the adhesion and spreading of PC-3 cells interacting with FN, whereas the αvβ3 integrin seems to regulate other receptors in the spreading process. Moreover, integrin-FN interaction through the RGD sequence evidently curbed the cell adhesion and spreading.     

Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.     

Single cell tracking assay reveals an opposite effect of selective small non-peptidic α5β1 or αvβ3/β5 integrin antagonists in U87MG glioma cells

BackgroundIntegrins are extracellular matrix receptors involved in several pathologies. Despite homologies between the RGD-binding α5β1 and αvβ3 integrins, selective small antagonists for each heterodimer have been proposed. Herein, we evaluated the effects of such small antagonists in a cellular context, the U87MG cell line, which express both integrins. The aim of the study was to determine if fibronectin-binding integrin antagonists are able to impact on cell adhesion and migration in relationships with their defined affinity and selectivity for α5β1 and αvβ3/β5 purified integrins.MethodsSmall antagonists were either selective for α5β1 integrin, for αvβ3/β5 integrin or non-selective. U87MG cell adhesion was evaluated on fibronectin or vitronectin. Migration assays included wound healing recovery and single cell tracking experiments. U87MG cells stably manipulated for the expression of α5 integrin subunit were used to explore the impact of α5β1 integrin in the biological assays.ResultsU87MG cell adhesion on fibronectin or vitronectin was respectively dependent on α5β1 or αvβ3/β5 integrin. Wound healing migration was dependent on both integrins. However U87MG single cell migration was highly dependent on α5β1 integrin and was inhibited selectively by α5β1 integrin antagonists but increased by αvβ3/β5 integrin antagonists.ConclusionsWe provide a rationale for testing new integrin ligands in a cell-based assay to characterize more directly their potential inhibitory effects on integrin cellular functions.General significanceOur data highlight a single cell tracking assay as a powerful cell-based test which may help to characterize true functional integrin antagonists that block α5β1 integrin-dependent cell migration.     

αv integrin processing interferes with the cross-talk between αvβ5/β6 and α2β1 integrins

Background information. Previous studies have reported that cross‐talk between integrins may be an important regulator of integrin—ligand binding and subsequent signalling events that control a variety of cell functions in many tissues. We previously demonstrated that αvβ5/β6 integrin represses α2β1‐dependent cell migration. The αv subunits undergo an endoproteolytic cleavage by protein convertases, whose role in tumoral invasion has remained controversial. Results. Inhibition of convertases by the convertase inhibitor α1‐PDX (α1‐antitrypsin Portland variant), leading to the cell‐surface expression of an uncleaved form of the αv integrin, stimulated cell migration toward type I collagen. Under convertase inhibition, α2β1 engagement led to enhanced phosphorylation of both FAK (focal adhesion kinase) and MAPK (mitogen‐activated protein kinase). This outside‐in signalling stimulation was associated with increased levels of activated β1 integrin located in larger than usual focal‐adhesion structures and a cell migration that was independent of the PI3K (phosphoinositide 3‐kinase)/Akt (also called protein kinase B) pathway. Conclusions. The increase in cell migration observed upon convertases inhibition appears to be due to the up‐regulation of β1 integrins and to their location in larger focal‐adhesion structures. The endoproteolytic cleavage of αv subunits is necessary for αvβ5/β6 integrin to control α2β1 function and could thus play an essential role in colon cancer cell migration.     

INTEGRIN SYNTHESIS AND UTILIZATION IN CULTURED HUMAN OSTEOBLASTS

This study describes the adhesion of human osteoblasts, culturedin vitro, to proteins of the extracellular matrix, the biosynthesis of integrins, their topography and organization in focal contacts. The adhesion of osteoblasts to laminin, type I collagen, vitronectin and fibronectin was 77–100%, in 2h and at 55nm substrata concentration, and it was accompained by spreading of the cells. Adhesion to fibronectin (FN), laminin (LN) and type I collagen (COL) was inhibited by antibodies to the β1 integrin and antibodies to the α5 chain affected adhesion only to fibronectin. Using a panel of polyclonal antibodies against α2, α3, α5, αv, β1 andβ3 integrins we detected synthesis of α3β1, α5β1, αvβ3, and an αvβ1-like dimer by immunoprecipitation of metabolically labelled cell lysates. Studies of immunolocalization demonstrated the presence of the same integrins identified in lysates, plus α4, α1 and β5 subunits. In cells adhering in the presence of serum we showed organization of β3 and αv integrins in focal contacts. In cells adhering to fibronectin α5 and β1 integrins were localized in focal contacts. In cells spread on laminin or type I collagen none of the integrins investigated was localized in focal contacts.     

Selective binding and lateral clustering of α5β1 and αvβ3 integrins: Unraveling the spatial requirements for cell spreading and focal adhesion assembly

ABSTRACT

Coordination of the specific functions of α5β1 and αvβ3 integrins is crucial for the precise regulation of cell adhesion, spreading and migration, yet the contribution of differential integrin-specific crosstalk to these processes remains unclear. To determine the specific functions of αvβ3 and α5β1 integrins, we used nanoarrays of gold particles presenting immobilized, integrin-selective peptidomimetic ligands. Integrin binding to the peptidomimetics is highly selective, and cells can spread on both ligands. However, spreading is faster and the projected cell area is greater on α5β1 ligand; both depend on ligand spacing. Quantitative analysis of adhesion plaques shows that focal adhesion size is increased in cells adhering to αvβ3 ligand at 30 and 60 nm spacings. Analysis of αvβ3 and α5β1 integrin clusters indicates that fibrillar adhesions are more prominent in cells adhering to α5β1 ligand, while clusters are mostly localized at the cell margins in cells adhering to αvβ3 ligand. αvβ3 integrin clusters are more pronounced on αvβ3 ligand, though they can also be detected in cells adhering to α5β1 ligand. Furthermore, α5β1 integrin clusters are present in cells adhering to α5β1 ligand, and often colocalize with αvβ3 clusters. Taken together, these findings indicate that the activation of αvβ3 integrin by ligand binding is dispensable for initial adhesion and spreading, but essential to formation of stable focal adhesions.     

Recombinant disintegrin targets α(v) β(3) integrin and leads to mediator production

Integrin αvβ3 is most likely the foremost modulator of angiogenesis among all known integrins. Recombinant disintegrin DisBa-01, originally obtained from snake venom glands, binds to αvβ3, thereby significantly inhibiting adhesion and generating in vivo anti-metastatic ability. However, its function in mediator production is not clear. Here, we observed that the mediators VEGF-A, IL-8, and TGF-β are not produced by human umbilical vein endothelial cells (HUVEC cell line) or monocyte/macrophage cells (SC cell line) when cells adhered to vitronectin. However, when exposed to DisBa-01, HUVECs produced higher levels of TGF-β, and SC cells produced higher levels of VEGF-A. Nonetheless, HUVECs also showed an enhancement of apoptosis after losing adherence when exposed to disintegrin, which is a characteristic of anoikis. We propose that disintegrin DisBa-01 could be used to modulate integrin αvβ3 functions.     

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

Collaborative role of various fibronectin-binding integrins (α5β1, αvβ1 and αvβ6) 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 αvβ6 integrin was strongly and specifically upregulated by transforming growth factor-β1 (TGFβ1) 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 TGFβ1. Based on antibody blocking experiments, both untreated and TGFβ1-treated HaCaT cells used αvβ6 integrin as their main fibronectin receptor for cell spreading. In contrast to TGFβ1-treated cells, the untreated cells also needed α5β1 integrin for maximal cell spreading on fibronectin. Combinations of antibodies blocking both of these receptors totally prevented spreading of both untreated and TGFβ1-treated cells. Haptotactic motility of individual HaCaT cells through fibronectin-coated membranes was again mainly dependent on αvβ6 integrin, while αvβ1 and α5β1 integrins played a lesser role both in untreated and TGFβ1-treated HaCaT cells. However, unlike haptotaxis, lateral migration of HaCaT cell sheet was mainly mediated by β1 integrins, and αvβ6 integrin showed a minor role. The migration process appeared to involve a number of β1 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.     

Aggregatibacter actinomycetemcomitans outer membrane protein 29 (Omp29) induces TGF‐β‐regulated apoptosis signal in human gingival epithelial cells via fibronectin/integrinβ1/FAK cascade

Gingival junctional epithelial cell apoptosis caused by periodontopathic bacteria exacerbates periodontitis. This pathological apoptosis is involved in the activation of transforming growth factor β (TGF‐β). However, the molecular mechanisms by which microbes induce the activation of TGF‐β remain unclear. We previously reported that Aggregatibacter actinomycetemcomitans (Aa) activated TGF‐β receptor (TGF‐βR)/smad2 signalling to induce epithelial cell apoptosis, even though Aa cannot bind to TGF‐βR. Additionally, outer membrane protein 29 kDa (Omp29), a member of the Aa Omps family, can induce actin rearrangements via focal adhesion kinase (FAK) signalling, which also plays a role in the activation of TGF‐β by cooperating with integrin. Accordingly, we hypothesized that Omp29‐induced actin rearrangements via FAK activity would enhance the activation of TGF‐β, leading to gingival epithelial cell apoptosis in vitro. By using human gingival epithelial cell line OBA9, we found that Omp29 activated TGF‐βR/smad2 signalling and decreased active TGF‐β protein levels in the extracellular matrix (ECM) of cell culture, suggesting the transactivation of TGF‐βR. Inhibition of actin rearrangements by cytochalasin D or blebbistatin and knockdown of FAK or integrinβ1 expression by siRNA transfection attenuated TGF‐βR/smad2 signalling activity and reduction of TGF‐β levels in the ECM caused by Omp29. Furthermore, Omp29 bound to fibronectin (Fn) to induce its aggregation on integrinβ1, which is associated with TGF‐β signalling activity. All the chemical inhibitors and siRNAs tested blocked Omp29‐induced OBA9 cells apoptosis. These results suggest that Omp29 binds to Fn in order to facilitate Fn/integrinβ1/FAK signalling‐dependent TGF‐β release from the ECM, thereby inducing gingival epithelial cell apoptosis via TGF‐βR/smad2 pathway.     

佛波酯对SMMC-7721人肝癌细胞粘附及整合蛋白基因表达的调控作用

用促癌剂佛波酯（ＰＭＡ）作用于ＳＭＭＣ－７７２１人肝癌细胞,研究细胞表面的主要粘附分子α５β１整合蛋白基因表达及相应细胞粘附行为的改变．用１００ｎｍｏｌ／ＬＰＭＡ作用ＳＭＭＣ－７７２１人肝癌细胞,发现其作用因时间的长短而异,作用３０、６０、１２０ｍｉｎ分别增加细胞与纤连蛋白（Ｆｎ）粘附１８．８％、３８．７％和５６．６％,作用６、１２ｈ分别降低４４．０％、３７．４％,而不影响与多聚赖氨酸的粘附．使用足量的抗α５和／或抗β１单抗预先封闭细胞与Ｆｎ的结合点,再将细胞与Ｆｎ粘附,发现α５单抗单独使用可将ＳＭＭＣ－７７２１细胞与Ｆｎ的粘附抑制２０％左右,β１单抗则抑制１４％,两者联合使用时可封闭４０％左右的粘附,提示该细胞表面存在除α５β１外的其它整合蛋白在介导着细胞与Ｆｎ的粘附．进一步应用Ｎｏｒｔｈｅｒｎｂｌｏｔ方法,分析整合蛋白基因表达,发现１００ｎｍｏｌ／ＬＰＭＡ抑制α５亚基转录,以３０ｍｉｎ最明显,抑制达８３．１％,作用６、１２ｈ抑制率仍为４６．６％、４３．６％．还就ＰＭＡ影响细胞粘附和整合蛋白基因表达的可能机理作了讨论．     

Structural insights into the extra cellular segment of integrinβ5 and molecular interaction studies

Abstract

Primary tumor cells often spread to other organs by metastasis. Despite of it, primary tumor cells break their surrounding extra cellular matrix (ECM) proteins and reach the destination organ by the process of intravasation and extravasation. Metastasized tumor cells induce the process of angiogenesis, this highly regulated process involves several ECM proteins. However, integrins are primarily involved in the blood vessel growth and repair. Therefore, integrins are promising angiogenesis targets. Integrins are receptors on cell surface, involved in signal transduction and attachments in extra cellular matrix (ECM). IntegrinαVβ3 and αVβ5 are implicated in tumor angiogenesis, metastasis, inflammation and bone resorption. The crystal structure of integrinαvβ5 is not available in protein structural databases, therefore; molecular model of integrinβ5 structure was prepared and stereo chemical model quality was checked. Integrin β5 active sites were identified based on insilico analysis tools. Further, molecular level interactions between integrinβ5 and ECM proteins were predicted. In the present study ECM proteins such as focal adhesion kinase 1 (FAK1), annexin A5 and P21 activated kinase 4 (PAK4) were considered for protein-protein docking, to understand inter molecular interactions. The predicted model is conceived to be stereo chemically good and can be used for molecular interaction studies of angiogenic inhibitors.     

Fibronectin-mediated upregulation of α5β1 integrin and cell adhesion during differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells have a broad potential application in regenerative medicine and can be differentiated into cells of all three germ layers. Adhesion of ES cells to extracellular matrix (ECM) proteins is essential for the differentiation pathway; Cell-ECM adhesion is mediated by integrins that have the ability to activate many intracellular signaling pathways. Therefore, we hypothesize that the expression and function of integrin receptors is a critical step in ES differentiation. Using functional cell adhesion assays, our study demonstrates that α5β1 is a major functional integrin receptor expressed on the cell surface of undifferentiated mouse ES-D3 cells, which showed significantly higher binding to fibronectin as compared to collagens. This adhesion was specific mediated by integrin α5β1 as evident from the inhibition with a disintegrin selective for this particular integrin. Differentiation of ES-D3 cells on fibronectin or on a collagen type1/fibronectin matrix, caused further selective up-regulation of the α5β1 integrin. Differentiation of the cells, as evaluated by immunofluorescence, FACS analysis and quantitative RT-PCR, was accompanied by the upregulation of mesenchymal (Flk1, isolectin B4, α-SMA, vimentin) and endodermal markers (FoxA2, SOX 17, cytokeratin) in parallel to increased expression of α5β1 integrin. Taken together, the data indicate that fibronectin-mediated, upregulation of α5β1 integrin and adhesion of ES-D3 cells to specific ECM molecules are linked to early stages of mouse embryonic stem cells commitment to meso-endodermal differentiation.     

Inhibition of Cancer Cell Adhesion,Migration and Proliferation by a Bispecific Antibody that Targets two Distinct Epitopes on αv Integrins

Members of the αv family of integrins regulate activation of transforming growth factor beta (TGFβ) and are directly involved in pro-tumorigenic phenotypes. Thus, αv integrins may be therapeutic targets for fibrosis and cancer, yet the isolation of selective inhibitors is currently a challenge. We generated synthetic antibodies selective for αv integrins by phage display selections on cell lines that displayed integrin heterodimers. We identified antibodies that targeted two distinct epitopes on cell-surface αv integrins and partially inhibited cell adhesion mediated by interactions between integrins and the latency-associated peptide, part of the pro-form of TGFβ. Using the isolated antibody paratope sequences we engineered a bispecific antibody capable of binding to both epitopes simultaneously; this antibody potently and completely inhibited cell adhesion mediated by integrins αvβ1, αvβ3 and αvβ5. In addition, the bispecific antibody inhibited proliferation and migration of lung carcinoma lines, where the highest and lowest potencies observed correlated with integrin-αv cell surface expression levels. Taken together, our results demonstrate that phage display selections with live cells can yield high quality anti-integrin antibodies, which we used as biparatopic building blocks to construct a bispecific antibody that strongly inhibited integrin function and may be a therapeutic candidate for cancer and fibrosis.     

Localization of αvβ3-like integrin in cultivated larval cells of the mussel Mytilus trossulus during neuronal and muscle differentiation

Using immunofluorescence phenotyping, the expression of αvβ3-like integrin was examined during neuronal and muscle differentiation in cell cultures derived from trochophore larvae of the mussel Mytilus trossulus. We have demonstrated that some mussel cells grown on fibronectin in vitro express the extracellular matrix (ECM) αvβ3 integrin-like receptor. At the same time, the distribution of αvβ3-like integrin is not ubiquitous, i.e. it depends on the cell type and the time of cultivation. Using immunohistochemical staining, we have found that only in some cells this integrin is co-localized with molluscan neuronal markers, neurotransmitters serotonin (5-HT) or Phe-Met-Arg-Phe-NH(2) neuropeptide (FMRFamide), and also with filament actin but not with paramyosin. Although we have previously shown that an integrin-dependent mechanism is involved in cell adhesion and differentiation of muscle cells of Mytilus, in this study, αvβ3-like integrin has not been found to participate in fibronectin adhesion of muscle cells but may be a linking agent between the ECM and the neuron-like cells.     

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

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

In vitro model of angiogenesis using a human endothelium-derived permanent cell line: contributions of induced gene expression, G-proteins, and integrins.

The EA hy926 cell line is a continuous, clonable, human cell line that displays a number of features characteristic of vascular endothelial cells (Edgell et al., 1983). Here we report that when EA hy926 cells (EA cells) are plated on an extracellular matrix material [Matrigel], they undergo a process of morphological re-organization leading to the formation of a complex network of cord or tubelike structures. These events seem to resemble, in some respects, an in vitro process of angiogenesis. The morphological re-arrangement occurs within a 12-16 hr period and seems to require expression of new messenger RNA and protein, since it is completely blocked when actinomycin D or cycloheximide are present at the time the cells are plated on Matrigel. This is not due to overt toxicity of the drugs, since exposure of cells to actinomycin D at 2 hr or more after plating on Matrigel has little effect on the formation of the tubelike structures. The process of Matrigel-induced tube formation also apparently involves a G-protein mediated signal. Treatment of the EA cells with pertussis toxin completely blocks the process and causes the ADP-ribosylation of a 42 kD protein that is recognized by an antibody to Gi-alpha subunits. In contrast, concentrations of pertussis toxin sufficient to block tube formation have only modest effects on the adhesion or motility of EA cells on purified matrix components such as laminin or collagen IV. The process of Matrigel-induced tube formation also involves integrins since monoclonal antibodies to integrin alpha 6 or beta 1 subunits can completely block the process. The concentrations of anti-integrin antibodies needed to block tube formation are much lower than those required to block cell adhesion on purified matrix components and are sufficient to occupy less than 10% of the alpha 6 or beta 1 subunits available at the cell surface. These results suggest that integrins may be involved in this potential model of angiogenesis in processes beyond their usual role in cell adhesion. Based on these results, it seems likely that the EA hy 926 cell line will prove to be a useful model for in vitro study of angiogenic processes.     

Urokinase receptors promote beta1 integrin function through interactions with integrin alpha3beta1

The urokinase receptor (uPAR) is linked to cellular migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate cell signaling in response to urokinase (uPA) binding. The mechanisms for these activities remain incompletely defined, although uPAR was recently identified as a cis-acting ligand for the beta2 integrin CD11b/CD18 (Mac-1). Here we show that a major beta1 integrin partner for uPAR/uPA signaling is alpha3. In uPAR-transfected 293 cells uPAR complexed (>90%) with alpha3beta1 and antibodies to alpha3 blocked uPAR-dependent vitronectin (Vn) adhesion. Soluble uPAR bound to recombinant alpha3beta1 in a uPA-dependent manner (K(d) < 20 nM) and binding was blocked by a 17-mer alpha3beta1 integrin peptide (alpha325) homologous to the CD11b uPAR-binding site. uPAR colocalized with alpha3beta1 in MDA-MB-231 cells and uPA (1 nM) enhanced spreading and focal adhesion kinase phosphorylation on fibronectin (Fn) or collagen type I (Col) in a pertussis toxin- and alpha325-sensitive manner. A critical role of alpha3beta1 in uPA signaling was verified by studies of epithelial cells from alpha3-deficient mice. Thus, uPAR preferentially complexes with alpha3beta1, promoting direct (Vn) and indirect (Fn, Col) pathways of cell adhesion, the latter a heterotrimeric G protein-dependent mechanism of signaling between alpha3beta1 and other beta1 integrins.     

Sequential binding of αVβ3 and ICAM-1 determines fibrin-mediated melanoma capture and stable adhesion to CD11b/CD18 on neutrophils

Fibrin (Fn) deposition defines several type 1 immune responses, including delayed-type hypersensitivity and autoimmunity in which polymorphonuclear leukocytes (PMNs) are involved. Fn monomer and fibrinogen are multivalent ligands for a variety of cell receptors during cell adhesion. These cell receptors provide critical linkage among thrombosis, inflammation, and cancer metastasis under venous flow conditions. However, the mechanisms of Fn-mediated interactions among immune cells and circulating tumor cells remain elusive. By using a cone-plate viscometer shear assay and dual-color flow cytometry, we demonstrated that soluble fibrinogen and Fn had different abilities to enhance heterotypic aggregation between PMNs and Lu1205 melanoma cells in a shear flow, regulated by thrombin levels. In addition, the involvement of integrin α(v)β(3), ICAM-1, and CD11b/CD18 (Mac-1) in fibrin(ogen)-mediated melanoma-PMN aggregations was explored. Kinetic studies provided evidence that ICAM-1 mediated initial capture of melanoma cells by PMNs, whereas α(v)β(3) played a role in sustained adhesion of the two cell types at a shear rate of 62.5 s(-1). Quantitative analysis of the melanoma-PMN interactions conducted by a parallel-plate flow chamber assay further revealed that at a shear rate of 20 s(-1), α(v)β(3) had enough contact time to form bonds with Mac-1 via Fn, which could not otherwise occur at a shear rate higher than 62.5 s(-1). Our studies have captured a novel finding that leukocytes could be recruited to tumor cells via thrombin-mediated Fn formation within a tumor microenvironment, and α(v)β(3) and ICAM-1 may participate in multistep fibrin(ogen)-mediated melanoma cell adhesion within the circulation.     

Divalent cations regulate the organization of integrins αvβ3 and αvβ5 on the cell surface

Extracellular divalent cations are important regulators of integrin ligand binding activity. In this study we evaluated how divalent cations affect the organization of integrins into focal adhesion sites. Integrins αvβ3 and αvβ5 were compared because they share a high degree of structural homology and because both integrins mediate cell adhesion to vitronectin. On MG-63 osteosarcoma cells, we found that both the extent and pattern of integrin organization was regulated by the type of extracellular divalent ion. Integrin αvβ3 organized in focal contacts when Mn²⁺ or Mg²⁺ was present, but not in Ca²⁺. In contrast, αvβ5 organized in focal contacts only when Ca²⁺ or Mg²⁺ was present. Integrin αvβ5 clustered in a centrally located punctate field on the ventral surface of the cell in the presence of Mn²⁺. These observations reveal a previously unappreciated role for divalent ions in regulating the organization of integrins into focal adhesion sites. © 1996 Wiley-Liss, Inc.