Cell adhesion and communication (Cell Adhes Comm )

Description

Discontinued. Continued by Cell Communication & Adhesion (1541-9061).

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  • Other titles
    Cell communication & adhesion (Online), Cell communication & adhesion, Cell communication and adhesion
  • ISSN
    1061-5385
  • OCLC
    50409812
  • Material type
    Document, Periodical, Internet resource
  • Document type
    Internet Resource, Computer File, Journal / Magazine / Newspaper

Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: Chemotaxis, the directed migration of leukocytes towards a chemoattractant gradient, is a key phenomenon in the immune response. During lymphocyte-endothelial and – extracellular matrix interactions, chemokines induce the polarization of T lymphocytes. with generation of specialized cell compartments. The chemokine receptors involved in detection of the chemoattractant gradients concentrate at the leading edge (advancing front or anterior pole) of the cell. The adhesion molecules ICAM- 1, -3, CD44 and CD43 redistribute to the uropod, an appendage at the posterior pole of migrating T lymphocyte that protrudes from the contact area with endothelial or extracellular matrix substrates. Whereas chemokine receptors sense the direction of migration, the uropod is involved in the recruitment of bystander leukocytes through LFA-1/ICAM-dependent cell cell interactions. While β-actin concentrates preferentially at the cell's leading edge, the motor protein myosin II and a microtubule organizing center (MTOC) are packed in the uropod. The actin-binding protein moesin, which belongs to the ERM family of ezrin, radixin and moesin, redistributes to the distal portion of uropods and physically interacts with ICAM-3, CD44 and CD43, thus acting as a physical link between the membrane molecules and the actin cytoskeleton. Moreover, the moesin-ICAM-3 association correlates with the degree of cell polarity. The redistribution of the chemokine receptors and adhesion molecules to opposite poles of the cell in response to a chemoattractant gradient may guide cell migration and cell-cell interactions during lymphoid cell trafficking in immune and inflammatory responses.
    Cell adhesion and communication 07/2009; 6(2-3):125-133.
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    ABSTRACT: We have identified a novel gene, EMSl, that is consistently amplified and overexpressed in human carcinomas with an amplification of the chromosome 11q13 region. Comparisons of the EMSl sequences with those present in the GenBank databases revealed a high identity with chicken cortactin. Southern and western blot analyses confirm the high sequence conservation during evolution. An antiserum specific for human cortactin, showed in gene transfer experiments that both human p80 and p85 isoforms are encoded by the EMSl cDNA. Further comparisons demonstrated an high sequence and structural homology with HSl that is implicated in signal transduction in lymphoid cells only. Expression of EMSl/cortactin mRNA was restricted to tumor cell lines derived from non-lymphoid origin. Cortactin contains (i) a filamentous actin binding tandem repeat domain, (ii) a proline-rich SH3-binding and (iii) a SH3 domain that is common in proteins involved in signal transduction. Our data suggest that human EMSl/cortactin has a function in signal transmission between cell-matrix contact sites and the cytoskeleton and, as such, its overexpression due to 11q13 amplification might effect adhesive properties of human carcinomas.
    Cell adhesion and communication 07/2009; 6(2-3):185-209.
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    ABSTRACT: The integrin alpha4beta1(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human alpha4 and beta1 subunits were fused to the genomic DNA encoding the human gamma1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote alpha4beta1 heterodimer formation. The soluble alpha4beta1-Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent Kd for VCAM-1 binding were similar for both the soluble and native forms of alpha4beta1. In addition, the integrin-Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble alpha4beta1 should be generally applicable to a range of integrins.
    Cell adhesion and communication 06/2000; 7(5):377-90.
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    ABSTRACT: To evaluate the role of MacMARCKS, a major substrate of protein kinase C, in cell adhesion, we selected a macrophage cell line, Wehi 274.1.7. Although surface expression of beta2-integrins can be detected on these cells, they lack the phorbol ester- or chemokine-induced adhesion to ICAM-1-coated surface, an event mediated by beta2-integrins. Concomitantly, these cells lack expression of both MacMARCKS and its homologue, MARCKS. When wild type MacMARCKS was expressed in these cells, the phorbol ester-induced adhesion to ICAM-1-coated surface increased approximately 5-fold compared to vector transfected control cells. To further investigate the potential physiological role of MacMARCKS in this adhesion event, we also tested the effect of monocyte chemotactic protein-1, and a 3-fold increase in the adhesion to ICAM-1-coated surface was observed with MacMARCKS-transfected cells. Therefore, these data suggest that MacMARCKS is an essential component in regulating cell adhesion.
    Cell adhesion and communication 06/2000; 7(5):359-66.
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    ABSTRACT: Gap junction intercellular communication (GJIC) consists of intercellular exchange of low molecular weight molecules. Chemically induced alterations of this communication have been suggested to result in abnormal cell growth and tumour promotion. Several in vitro assays have been developed to determine the effect of chemicals on gap junction communication in cultured cells. The scrape loading dye transfer technique is based on studying the transfer of the fluorescent dye Lucifer Yellow in cells where the dye is loaded through a cut in the cell monolayer. This technique is rapid and relatively uncomplicated, but has only been used to qualitatively demonstrate communication, due to lack of an appropriate method for quantification of the dye spreading. We show here that analysis of digital fluorescence images of cells scrape loaded with Lucifer Yellow can be used for quantitative determination of GJIC. We have analysed the images both by means of distance of diffusion of the dye in the cell monolayer, as well as by area of dye-coupled cells. The results are consistent with that obtained using microinjection of Lucifer Yellow and the method offers a simple way for quantitative determination of GJIC.
    Cell adhesion and communication 06/2000; 7(5):367-75.
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    ABSTRACT: Expression of the Polyoma Middle T (PyMT) antigen in endothelial cells results in single-step transformation to hemangioma producing malignant cells. To study the mechanism of PyMT transformation, we used the PyMT induced mouse brain endothelial cell line, bEND.3, expressing constitutively active and dominant negative mutants of the small GTPase Rac. The bEND.3 cell phenotype of tumorigenesis, loss of normal growth control and formation of cysts rather than capillary tubes in fibrin gels was reversed by expression of dominant negative Rac. The mechanism of N17 Rac action in blocking the endothelial cell transformant, PyMT, did not involve effects of Rac on the actin cytoskeleton since this component of the bEND.3 cell phenotype was not affected. Furthermore, the PyMT induced activation of the plasminogen activator (PA)/plasmin system was not affected by Rac inhibition. Inhibition of the downstream effectors of Rac, phosphatidylinositol 3-kinase (PI3-K) and p70S6k, which are known to be constitutively activated by PyMT transformation, inhibited bEND.3 cell proliferation and cyst formation in fibrin gels even in cells expressing V12 constitutively active Rac, but they did not restore capillary tube formation. These results demonstrate that middle T antigen induced endothelial cell transformation requires signal transduction by Rac. The downstream Rac effectors, P13-K and p70S6k, mediate PyMT/Rac effects on cell proliferation and cyst formation, but other unknown effectors of PyMT are required for the cytoskeletal changes and activation of the PA/plasmin system.
    Cell adhesion and communication 06/2000; 7(5):409-22.
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    ABSTRACT: Because of the known property of spontaneous regression in stage IVS of neuroblastoma all attempts are made to elucidate whether differentiation inducers possibly could be applied for neuroblastoma therapy. Here we examined the influence of retinoic acid (RA) in vitro on differentiation, proliferation and adhesion of 10 permanent and 4 primary cell lines as well as of several SCID-mouse tumour transplants. In general, after RA treatment morphologically different cell types which are characteristic for neuroblastoma cells have changed. N (neuronal)-type cells prolonged their neuronal processes, whereas S (epithelial, substrate-adherent, Schwann cell-like)-type cells lost their adherence to substratum and became apoptotic. Additionally, the reactions of all neuroblastoma cell lines with monoclonal antibodies against beta-tubulin (for neuronal cells) and glial fibrillary acidic protein (for epithelial cells) were determined. The anti-proliferative effect of all-trans-RA as well as 13-cis-RA was more profound in S-type cells (up to 40% in primary cell lines). To elucidate the role of adhesion molecules during neuronal cell differentiation, we have analysed the adhesion of neuroblastoma cells on poly-D-lysin-precoated plates under RA influence. While N-type cells displayed an increased adhesion, all S-type cell lines as well as all primary cell lines exhibited a reduced adhesion (IMR-5 and IMR-32: p < 0.001; JW, SR and PM: p < 0.05). RA treatment increased predominantly the tested antigens (HCAM, ICAM-1, NCAM, PECAM-1, VCAM-1, cadherin, FGF-R, IGF-R, NGF-R, TGF-beta/1, NF200, NF160, NF68, NSE, HLA-ABC) in all cell lines independently of their phenotypes (TGF-beta/1: p < 0.001; NF68: p < 0.01; PECAM-1 and NGF-R: p < 0.05). In recultured SCID-mouse-passaged tumour cells antigens were down-regulated (FGF-R: p < 0.01), but increased again after RA influence (TGF-beta/1: p < 0.05). In summary, the RA differentiation model demonstrates the possibility to interfere in cell adhesion and to diminish growth potential both in N-type as well as S-type neuroblastoma cells.
    Cell adhesion and communication 06/2000; 7(5):423-40.
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    ABSTRACT: E-cadherin participates in homophilic cell-to-cell adhesion and is localized to intercellular junctions of the adherens type. In the present study, we investigated the localization of adherens junction components in cells expressing mutant E-cadherin derivatives which had been previously cloned from diffuse-type gastric carcinoma. The mutations are in frame deletions of exons 8 or 9 and a point mutation in exon 8 and affect the extracellular domain of E-cadherin. Our findings indicate that E-cadherin mutated in exon 8 causes beta-catenin staining at lateral cell-to-cell contact sites and, in addition, abnormally located beta-catenin in the perinuclear region. Moreover, the various mutant E-cadherin derivatives increased the steady-state levels of alpha- and beta-catenin and were found in association with these catenins even after induction of tyrosine phosphorylation by pervanadate. Sustained pervanadate treatment led, however, to rounding-up of cells and induction of filopodia, changes which were first detectable in cells expressing E-cadherin mutated in exon 8. The deterioration of the cell contact was not accompanied with disassembly of the E-cadherin-catenin complex. Based on these observations, we propose a model whereby in the presence of mutant E-cadherin tyrosine phoshorylation of components of the cell adhesion complex triggers loss of cell-to-cell contact and actin cytoskeletal changes which are not caused by the disruption of the E-cadherin-catenin complex per se, but instead might be due to phosphorylation of other signaling molecules or activation of proteins involved in the regulation of the actin cytoskeleton.
    Cell adhesion and communication 06/2000; 7(5):391-408.
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    ABSTRACT: Loss of adherence to substrate can, by itself, induce apoptosis (anoikis) in epithelial cells, but does not do so in fibroblasts. To test the idea that adherence transmits signals that inhibit apoptosis even in fibroblasts, we took advantage of the greatly increased adherence to the substratum observed in NIH3T3 cell lines that overexpress thymosin beta four. We treated overexpressing (OE) and vector control lines with either ultraviolet light (UV) or tumor necrosis factor alpha (TNF alpha). When the cells were on a substratum, the more adherent OE cells were 2-fold more resistant to apoptosis induced by either treatment than vector controls. In contrast, when the cells were treated with either agent while in suspension, the difference in resistance between OE cells and vector controls was lost. Thus the increased resistance to apoptosis was dependent on adherence. There was no difference in the content of bcl-2 in the OE cells vs the controls. A connection between pp125FAK and resistance to apoptosis has been previously shown in primary cultures of fibroblasts. The Tbeta4 overexpressing cells have approximately 1.4x more pp125FAK than the controls, and the kinase is approximately 2-fold more phosphorylated in adherent OE cells than in the vector controls. The phosphorylation of pp125FAK decreased strikingly when the cells were put into suspension. In addition, twice as much paxillin associated with pp125FAK in OE adherent cells as in vector controls, but this difference was also lost in suspended cells. Our results support the concept of an adherence dependent pp125FAK-paxillin signalling pathway in fibroblasts that inhibits damage-induced apoptosis.
    Cell adhesion and communication 02/2000; 7(4):311-20.
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    ABSTRACT: The alphav integrins present on the membrane of numerous cells, mediate attachment to matrix proteins, cell proliferation, migration and survival. We studied the expression of alphav integrinis and CD47 (a beta3 chain integrin associated protein) in various forms of glomerulonephritis (GN) characterized by mesangial proliferation and/or increased mesangial matrix. In normal glomeruli, epithelial cells expressed alphavbeta3, alphavbeta5 and CD47; endothelial cells expressed alpha5beta1 and CD47; mesangial cells expressed alphavbeta5, CD47, and to a less extent alphavbeta3. In acute post infectious GN (APIGN), membrano-proliferative GN (MPGN) and diabetic nephropathy(DN), we observed that the beta3 chain, normally expressed by mesangial cells, was not detectable in the mesangium while its expression by epithelial cells was not modified. Parallel to the disappearance of alphavbeta3, the CD47 expression was decreased on the mesangial cells in MPGN, APIGN and DN. The expression of alphavbeta5 was clearly increased on podocytes and on proliferating mesangial cells in APIGN. By contrast, the mesangial expression of alphavbeta was normal or decreased in DN. The alpha5 chain of integrin, absent on normal mesangial cell, was expressed on proliferating mesangial cells in MPGN and APIGN. Thus, we observed modifications of alphavbeta3 and alphavbeta5 expression during human GN. The modulations of alphavbeta3 and alphavbeta5 expression differed according to the different glomerular cell types and were not parallel in glomerular cells: alphavbeta3 was decreased (and alphavbeta5 unchanged) on proliferating mesangial cells and alphavbeta5 was increased (and alphavbeta3 unchanged) in podocytes. This may reflect the existence of two distinct regulatory pathways.
    Cell adhesion and communication 02/2000; 7(6):441-51.
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    ABSTRACT: The Rac1 small GTP-binding protein is known to be involved in reorganization of the actin cytoskeleton and in regulation of intracellular signal transduction. The assembly and maintenance of cadherin-based cell cell junctions in epidermal keratinocytes is thought to be dependent on activity of Rac1. In this study we have generated green fluorescent protein (GFP)-tagged wild type, dominant negative and constitutively active Rac1 expression vectors and analyzed distribution of Rac1 following microinjection of human SCC12F epidermal keratinocytes. Wild type, dominant negative and constitutively active GFP Rac1 proteins distribute to sites of cell cell adhesion and co-localize with E-cadherin and the catenins. Disruption of cadherin-based junctions by reduction in extracellular calcium concentrations, or by use of antibodies to E-cadherin, results in redistribution of Rac1 away from sites of cell cell interaction but the co-localization with E-cadherin is maintained. In addition, expression of constitutively active GFP Rac1 results in formation of membrane ruffles on the apical surface of cells and intracellular vesicles. Interestingly, co-localization of Rac1 with E-cadherin is maintained in these structures. In contrast to previously published work we find that expression of dominant negative Rac1 neither disrupts cell cell adhesion nor prevents assembly of new cadherin-based adhesion structures.
    Cell adhesion and communication 02/2000; 7(6):465-76.
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    ABSTRACT: Gap junction intercellular communication (GJIC) is involved in several aspects of normal cell behaviour, and disturbances in this type of communication have been associated with many pathological conditions. Reliable and accurate methods for the determination of GJIC are therefore important in studies of cell biology. (Tomasetto, C., Neveu, M.J., Daley, J., Horan, P.K. and Sager, R. (1993) Journal of Cell Biology, 122, 157-167) reported some years ago the use of flow cytometer to determine transfer between cells of a mobile dye, calcein, as a measure of cell communication through gap junctions. In spite of this being a method with potential for quantitative and reliable determination of GJIC, it has been modestly used, possibly due to technical difficulties. In the present work we have illustrated several ways to use flow cytometric data to express cell communication through gap junctions. The recipient cells were pre-stained with the permanent lipophilic dye PKH26, and the donor cell population were loaded with the gap junction permeable dye, calcein. We show that the method may be used to measure the effect of chemicals on GJIC, and that the information is reliable, objective and reproducible due to the large number of cells studied. The data may give additional information to that obtained with other methods, since the effect observed will be on the establishment of cell communication as compared to what is observed for microinjection or scrape loading, where the effect is on already established communication. This is probably the reason for the more potent effects of DMSO on GJIC measured by the present method than on already existing GJIC measured by microinjection or quantitative scrape loading. We also show that the problem related to the mobile dye calcein not being fixable with aldehydes will not affect the results as long as the cells are kept on ice in the dark and analysed by flow cytometer within the first hours after formalin cell fixation.
    Cell adhesion and communication 02/2000; 7(6):501-12.
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    ABSTRACT: Interactions with the extracellular matrix constitute basic steps in cervix carcinoma cell invasion. In this study, we examined the adhesion and migration profiles of two human papillomavirus (HPV) DNA-transfected keratinocyte-derived cell lines, EIL8 and 18-11S3, and of the cervix adenocarcinoma SiHa cell line, towards laminin-1, and the selective effect of a 24-72 h treatment of 1000 U/ml interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), a treatment that significantly decreases cervix carcinoma cell proliferation and progression in nude mice, on these parameters. Compared to normal cervix keratinocytes (CK) and two HPV DNA-transfected keratinocyte cell lines, in basal conditions, the SiHa cell line was characterized by increased attachment (SiHa, 48.74 +/- 4.02 vs. normal keratinocytes, 4.32 +/- 0.40, EIL8, 17.80 +/- 3.03 and 18-11S3, 17.82 +/- 1.48% of attached cells after 30 min) and marked directed chemotactic migration towards laminin-1. Interestingly, treatment of the cells with the cytokines (1000 U/ml IFN-gamma and TNF-alpha) did not modulate the adhesion properties of the cells, but chemotactic migration of SiHa cells to laminin-1 was significantly decreased, while migration towards type I collagen was increased. Similar results were obtained with the Ca Ski cervix carcinoma cell line. Our results emphasize the altered pattern of interactions of cervix carcinoma cells with extracellular matrix components such as laminin-1, compared to normal and pre-neoplastic cells, and contributes to the understanding of the effects of cytokine treatment on cervix carcinoma cells.
    Cell adhesion and communication 02/2000; 7(4):321-9.
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    ABSTRACT: The migration of neutrophils from the circulation to areas of inflammation is the result of the sequential activation of multiple cellular adhesion molecules. beta1-Integrins are cell surface glycoproteins and the class of adhesion molecules responsible for binding to the extracellular matrix. The goal of this study was to determine the contribution of glycosylation, specifically the presence of sialic acid, to beta1-integrin adhesion in a neutrophil model. beta1-Integrins on differentiated HL60 cells were remodeled by treatment with the exoglycosidases, sialidase and beta-galactosidase. beta1-Integrin activity was determined by measuring adherence to the extracellular matrix protein fibronectin. The expression of beta1-integrins, beta2-integrins and activated beta1-integrins was determined by flow cytometry. Remodeling of beta1-integrins by treatment with sialidase increased adhesion by greater than 1,000%. Flow cytometric analysis of remodeled beta1-integrins demonstrated an increased expression of the activated beta1-integrin, but only minor increases in the expression of total beta1- and beta2-integrins. We postulate that glycosidase treatment increases adhesion and expression of activated beta1-integrins by exposure of the normally hidden ligand-binding site. The glycosylation of beta1-integrins on neutrophils may act to hide the ligand-binding site in unstimulated cells thereby contributing to the affinity modulation observed in neutrophil beta1-integrin function.
    Cell adhesion and communication 02/2000; 7(6):491-500.
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    ABSTRACT: The alpha1beta1 and alpha2beta1 integrins, extracellular matrix receptors for collagens and/or laminins, have similarities in structure and ligand binding. Recent studies suggest that the two receptors mediate distinct post-ligand binding events and are not simply redundant receptors. To discern the mechanisms by which the two receptors differ, we focused on the roles of the cytoplasmic domains of the alpha subunits. We expressed either full-length alpha1 integrin subunit cDNA (X1C1), full-length alpha2 integrin subunit cDNA (X2C2), chimeric cDNA composed of the extracellular and transmembrane domains of alpha2 subunit and the cytoplasmic domain of alpha1 (X2C1), chimeric cDNA composed of the extracellular and transmembrane domains of alpha1 subunit and the cytoplasmic domain of alpha2 (X1C2), alpha1 cDNA truncated after the GFFKR sequence (X1C0) or alpha2 cDNA truncated after the GFFKR sequence (X2C0) in K562 cells. Although the cytoplasmic domains of the alpha1 and alpha2 subunits were not required for adhesion, the extent of adhesion at low substrate density was enhanced by the presence of either the alpha1 or alpha2 cytoplasmic tail. Spreading was also influenced by the presence of an alpha subunit cytoplasmic tail. Activation of the protein kinase C pathway with phorbol dibutyrate-stimulated motility that was dependent upon the presence of the alpha2 cytoplasmic tail. Both the phosphatidylinosotide-3-OH kinase and the mitogen-activated protein kinase pathways were required for phorbol-activated, alpha2-cytoplasmic tail-dependent migration.
    Cell adhesion and communication 02/2000; 7(4):281-97.
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    ABSTRACT: Cytokines and other paracrine or autocrine factors functionally modulate the invasion-suppressor and signal-transducing E-cadherin/catenin complex. We have used conditioned medium from human squamous carcinoma COLO 16 cells (CM COLO 16) as a source of such factors to modulate the E-cadherin/catenin complex in human breast carcinoma MCF-7 cells. CM COLO 16 induces scattering of MCF-7/AZ, but not of MCF-7/6 cells on tissue culture plastic substratum, and reduces aggregation of MCF-7/AZ cells in suspension. Insulin-like growth factor I counteracts this reduction of aggregation. Confocal laser scanning microscopy of immunocytochemical stainings shows loss of the honeycomb pattern of E-cadherin, alpha-catenin and beta-catenin, and internalization of those elements. Cell surface biotinylation shows a decrease in membrane-bound E-cadherin. Immunoprecipitation and cell fractionation show that the composition of the complex is maintained. Interleukin-1, interleukin-6, granulocyte-monocyte colony stimulating factor, stem cell factor, scatter factor/hepatocyte growth factor and transforming growth factor-beta, added separately to MCF-7/AZ cells, could not mimic the effects of CM COLO 16. Neither could we find evidence that the 80 kDa extracellular fragment of E-cadherin is implicated in scattering of MCF-7/AZ cells. This fragment is present in CM COLO 16, but it is also produced by the MCF-7/AZ cells themselves, even at higher levels. Our data point toward cytoplasmic internalization induced by paracrine factors as one of the downregulating mechanisms for the E-cadherin/catenin complex.
    Cell adhesion and communication 02/2000; 7(4):299-310.
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    ABSTRACT: Dynamic interactions between cells and the extracellular matrix are essential in the regulation of a number of cellular processes including migration, adhesion, proliferation and differentiation. A variety of factors have been identified which modulate these interactions including transforming growth factor-beta, platelet-derived growth factor and others. Insulin-like growth factors have been shown to regulate collagen production by heart fibroblasts; however, the effects of this growth factor on the interactions of heart fibroblasts with the extracellular matrix have not been examined. The present studies were carried out to determine the effects of IGF-I on the ability of fibroblasts to interact with the extracellular matrix and to begin to determine the mechanisms of this response. These experiments illustrate that IGF-I treatment results in increased migration, collagen reorganization and gel contraction by heart fibroblasts. IGF-I has been shown to activate both the mitogen-activated protein kinase and phophatidylinositol-3 kinase pathways in isolated cells. Experiments with pharmacological antagonists of these pathways indicate that the mitogen-activated protein kinase pathway is essential for IGF-I stimulated collagen gel contraction by fibroblasts. These studies illustrate that IGF-I modulates the ability of fibroblasts to interact with the collagen matrix and that activation of multiple signaling pathways by IGF-I may produce distinct downstream responses in these cells.
    Cell adhesion and communication 02/2000; 7(6):513-23.
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    ABSTRACT: Collagen is a powerful platelet activating agent that promotes adhesion and aggregation of platelets. To differentiate the signals generated in these processes we have analyzed the tyrosine phosphorylation occurring in platelets after activation with collagen in suspension or under flow conditions. For the suspension studies, washed platelets were activated with different concentrations of purified type I collagen (ColI). Studies under flow conditions were performed using two different adhesive substrata: ColI and endothelial cells extracellular matrix (ECM). Coverslips coated with ColI or ECM were perfused through a parallel-plate perfusion chamber at 800 s(-1) for 5 min. After activation of platelets either in suspension or by adhesion, samples were solubilized and proteins were resolved by electrophoresis. Tyrosine-phosphorylated proteins were detected in immunoblots by specific antibodies. Activation of platelet suspensions with collagen induced tyrosine phosphorylation before aggregation could be detected. Profiles showing tyrosine-phosphorylated proteins from platelets adhered on ColI or on ECM were almost identical and lacked proteins p95, p80, p66, and p64, which were present in profiles from platelets activated in suspension. The intensity of phosphorylation was quantitatively weaker in those profiles from platelets adhered on ECM. Results from the present work indicate that activation of platelets in suspension or by adhesion induces differential tyrosine phosphorylation patterns. Phosphorylation of proteins p90 and p76 may be related to early activation events occurring during initial contact and spreading of platelets. Considering that adhesion is the first step of platelet activation, studies on signal transduction mechanisms under flow conditions may provide new insights to understand the signaling processes taking place at earliest stages of platelet activation.
    Cell adhesion and communication 02/2000; 7(4):349-58.
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    ABSTRACT: The adhesion molecule lymphocyte function-associated antigen 3 (LFA-3) (CD58) is an important regulator of immune cell function which occurs as both surface-associated and 'soluble' forms. This study has investigated the inter-relationship and the effects of cytokines on the expression of LFA-3 isoforms. The surface antigen was found to be relatively unaffected by cytokines, but the release of soluble LFA-3 (sLFA-3) was highly responsive to interleukin 1beta (IL-1beta), interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha). This modulation was cell-specific, particularly with regard to IFN-gamma, which up-regulated sLFA-3 release by A431 cells but down-regulated the release of the soluble form from HEp2 and HepG2 cells. We further demonstrated that LFA-3 is also present in a cytoplasmic 'pool' in each of the cells and, moreover, that cleavage of LFA-3 from the cell surface by phospholipase C resulted in an increase in the levels of the intracellular LFA-3 and replacement of the membrane-associated antigen. These observations suggest that the expression of the surface, soluble and intracellular forms of LFA-3 may be linked by regulatory mechanisms which are likely to exert an important influence on inflammatory interactions.
    Cell adhesion and communication 02/2000; 7(6):453-64.
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    ABSTRACT: 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.
    Cell adhesion and communication 02/2000; 7(6):477-90.

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