Model of integrin-mediated cell adhesion strengthening

Cell adhesion to extracellular matrix components involves integrin binding, receptor clustering, and recruitment of cytoskeletal elements, leading to the formation of discrete adhesive structures (focal adhesions). A force balance, macroscopic-to-microscopic model of these adhesive events is presented in the context of experimentally measured parameters. Integrin bond force, bond numbers, and distribution along the contact area strongly modulated the resulting adhesive force. Furthermore, focal adhesion assembly enhanced adhesion strength by 30% over integrin clustering alone. Predicted values are in excellent agreement with experimental results. This model provides a simple framework to systematically analyze the contributions of different adhesive parameters to overall adhesion strength.     

P-selectin activates integrin-mediated colon carcinoma cell adhesion to fibronectin

During hematogenous cancer metastasis, tumor cells separate from a primary mass, enter the bloodstream, disperse throughout the body, migrate across vessel walls, and generate distant colonies. The later steps of metastasis superficially resemble leukocyte extravasation, a process initiated by selectin-mediated cell tethering to the blood vessel wall followed by integrin-mediated arrest and transendothelial migration. Some cancer cells express P-selectin ligands and attach to immobilized P-selectin, suggesting that these cells can arrest in blood vessels using sequential selectin- and integrin-mediated adhesion, as do leukocytes. We hypothesize that selectin binding may regulate subsequent integrin-mediated steps in metastasis. Using a model system of cultured Colo 320 human colon adenocarcinoma cells incubated with soluble P-selectin-IgG chimeric protein, we have found that P-selectin can stimulate activation of the alpha(5)beta(1) integrin resulting in a specific increase of adhesion and spreading of these cells on fibronectin substrates. P-selectin binding also induced activation of p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (PI3-K). PI3-K inhibitors blocked P-selectin-mediated integrin activation, cell attachment, and cell spreading. Inhibition of p38 MAPK activation blocked cell spreading, but not cell attachment. P-selectin binding also resulted in formation of a signaling complex containing PI3-K and p38 MAPK. These results suggest that P-selectin binding to tumor cells can activate alpha(5)beta(1) integrin via PI3-K and p38 MAPK signaling pathways leading to increased cell adhesion. We propose that P-selectin ligands are important tumor cell signaling molecules that modulate integrin-mediated cell adhesion in the metastatic process.     

Irisin supports integrin-mediated cell adhesion of lymphocytes

Irisin, a myokine released from skeletal muscle, has recently been found to act as a ligand for the integrins αVβ5, αVβ1, and α5β1 expressed on mesenchymal cells, thereby playing an important role in the metabolic remodeling of the bone, skeletal muscle and adipose tissues. Although the immune-modulatory effects of irisin in chronic inflammation have been documented, its interactions with lymphocytic integrins have yet to be elucidated. Here, we show that irisin supports the cell adhesion of human and mouse lymphocytes. Cell adhesion assays using a panel of inhibitory antibodies to integrins have shown that irisin-mediated lymphocyte adhesion involves multiple integrins including not only α4β1 and α5β1, but also leukocyte-specific αLβ2 and α4β7. Importantly, mouse lymphocytic TK-1 cells that lack the expression of β1 integrins have exhibited αLβ2- and α4β7-mediated cell adhesion to irisin. Irisin has also been demonstrated to bind to purified recombinant integrin αLβ2 and α4β7 proteins. Thus, irisin represents a novel ligand for integrin αLβ2 and α4β7, capable of supporting lymphocyte cell adhesion independently of β1 integrins. These results suggest that irisin may play an important role in regulating lymphocyte adhesion and migration in the inflamed vasculature.     

Transforming JB6 cells exhibit enhanced integrin-mediated adhesion to osteopontin

Transformation of preneoplastic epidermal JB6 cells with tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is an in vitro model of late-stage tumor promotion. Osteopontin (OPN) is a secreted, adhesive protein that is highly expressed in JB6 cells with TPA treatment, and its expression persists for at least 4 days, which is the time required for subsequent expression of transformed phenotype. These observations suggest that OPN may play a role in promoting JB6 cell transformation. To function in transformation of JB6 cells, OPN must bind to the surface of the JB6 cell and subsequently signal within the cell. Therefore, we investigated whether JB6 cells adhere to OPN and, if so, to which surface receptors. TPA-treated JB6 cells had significantly (P < 0.05) increased adherence to OPN compared with dimethylsulfoxide-treated control cells. Enhanced attachment of JB6 cells to OPN was also observed after treatment with another tumor promoter phorbol dibutyrate but not with nontumor promoters (phorbol and 1alpha,25-dihydroxyvitamin D(3)), suggesting that tumor promoters specifically modulate attachment to OPN. The argininylglycylaspartic acid (RGD) cell-binding region of OPN mediates attachment of TPA-treated JB6 cells because RGD, but not argininylglycylglutamic acid (RGE), peptides inhibited adherence of these cells to OPN in a dose-dependent manner. Flow cytometric analyses, blocking adhesion assay using anti-alpha(v) antibody, and co-immunoprecipitation assay all indicated that TPA-treated cells had similar levels of alpha(v) and beta(5) but decreased levels of beta(1) compared with untreated cells and that cell adhesion to OPN is most likely mediated through the alpha(v)beta(5). Furthermore, calphostin C, a specific protein kinase C (PKC) inhibitor, decreased TPA-treated JB6 cell adhesion to OPN by 50%, suggesting that TPA increased integrin affinity or avidity for OPN through a PKC-mediated pathway. Collectively, these results indicate that transforming JB6 cells adhere to OPN through its RGD sequence. The most likely OPN receptor is the alpha(v)beta(5) integrin, which increases the affinity or avidity for OPN through a PKC-dependent pathway rather than increasing the number of receptors.     

Rac1 links integrin-mediated adhesion to the control of lactational differentiation in mammary epithelia

The expression of tissue-specific genes during mammary gland differentiation relies on the coincidence of two distinct signaling events: the continued engagement of beta1 integrins with the extracellular matrix (ECM) and a hormonal stimulus from prolactin (Prl). How the integrin and Prl receptor (PrlR) systems integrate to regulate milk protein gene synthesis is unknown. In this study, we identify Rac1 as a key link. Dominant-negative Rac1 prevents Prl-induced synthesis of the milk protein beta-casein in primary mammary epithelial cells cultured as three-dimensional acini on basement membrane. Conversely, activated Rac1 rescues the defective beta-casein synthesis that occurs under conditions not normally permissive for mammary differentiation, either in beta1 integrin-null cells or in wild-type cells cultured on collagen. Rac1 is required downstream of integrins for activation of the PrlR/Stat5 signaling cascade. Cdc42 is also necessary for milk protein synthesis but functions via a distinct mechanism to Rac1. This study identifies the integration of signals provided by ECM and hormones as a novel role for Rho family guanosine triphosphatases.     

H-Ras signals to cytoskeletal machinery in induction of integrin-mediated adhesion of T cells

The adhesive function of integrins is regulated through cytoplasmic signaling. The present study was performed to investigate the relevance of cytoplasmic signaling and cytoskeletal assembly to integrin-mediated adhesion induced by chemokines. Adhesion of T cells induced by chemokines macrophage inflammatory protein (MIP)-1alpha and MIP-1beta was inhibited by pertussis toxin, wortmannin, and cytochalasin B, suggesting that both G protein-sensitive phosphatidylinositol (PI) 3-kinase activation and cytoskeletal assemblies are involved. The chemokine-induced T cell adhesion could be mimicked by expression of small G proteins, fully activated H-RasV12, or H-RasV12Y40C mutant, which selectively binds to PI 3-kinase, in T cells, inducing activated form of LFA-1alpha and LFA-1-dependent adhesion to ICAM-1. H-Ras expression also induced F-actin polymerization which colocalized with profilin in T cells. Adult T cell leukemia (ATL) cells spontaneously adhered to ICAM-1, which depended on endogenous MIP-1alpha and MIP-1beta through activation of G protein-sensitive PI 3-kinase. H-Ras signal pathway, leading to PI 3-kinase activation, also induced active configuration of LFA-1 and LFA-1-mediated adhesion of ATL cells, whereas expression of a dominant-negative H-Ras mutant failed to do. Profilin-dependent spontaneous polymerization of F-actin in ATL cells was reduced by PI 3-kinase inhibitors. In this paper we propose that H-Ras-mediated activation of PI 3-kinase can be involved in induction of LFA-1-dependent adhesion of T cells, which is relevant to chemokine-mediated signaling, and that profilin may form an important link between chemokine- and/or H-Ras-mediated signals and F-actin polymerization, which results in triggering of LFA-1 on T cells or leukemic T cells.     

Redox regulation of beta-actin during integrin-mediated cell adhesion

Redox sensitivity of actin toward an exogenous oxidative stress has recently been reported. We report here the first evidence of in vivo actin redox regulation by a physiological source of reactive oxygen species, specifically those species generated by integrin receptors during cell adhesion. Actin oxidation takes place via the formation of a mixed disulfide between cysteine 374 and glutathione; this modification is essential for spreading and for cytoskeleton organization. Impairment of actin glutathionylation, either through GSH depletion or expression of the C374A redox-insensitive mutant, greatly affects cell spreading and the formation of stress fibers, leading to inhibition of the disassembly of the actinomyosin complex. These data suggest that actin glutathionylation is essential for cell spreading and cytoskeleton organization and that it plays a key role in disassembly of actinomyosin complex during cell adhesion.     

A screen to identify Drosophila genes required for integrin-mediated adhesion.

Drosophila integrins have essential adhesive roles during development, including adhesion between the two wing surfaces. Most position-specific integrin mutations cause lethality, and clones of homozygous mutant cells in the wing do not adhere to the apposing surface, causing blisters. We have used FLP-FRT induced mitotic recombination to generate clones of randomly induced mutations in the F1 generation and screened for mutations that cause wing blisters. This phenotype is highly selective, since only 14 lethal complementation groups were identified in screens of the five major chromosome arms. Of the loci identified, 3 are PS integrin genes, 2 are blistered and bloated, and the remaining 9 appear to be newly characterized loci. All 11 nonintegrin loci are required on both sides of the wing, in contrast to integrin alpha subunit genes. Mutations in 8 loci only disrupt adhesion in the wing, similar to integrin mutations, while mutations in the 3 other loci cause additional wing defects. Mutations in 4 loci, like the strongest integrin mutations, cause a "tail-up" embryonic lethal phenotype, and mutant alleles of 1 of these loci strongly enhance an integrin mutation. Thus several of these loci are good candidates for genes encoding cytoplasmic proteins required for integrin function.     

Cell adhesion molecules and the transition from short- to long-term memory

Training chicks on a one-trial passive avoidance task results in a cascade of molecular and cellular processes in two forebrain regions, culminating within 60–90 min in post-translational glycosylation of synaptic membrane proteins and expression of immediate early genes c-fos and c-jun. We have now found a second window of vulnerability of memory to the protein synthesis inhibitor anisomycin, 4 h downstream of training. By 5.5 h post-training this window closes, to be replaced by a window of sensitivity to blockade of glycoprotein synthesis, presumably representing post-translational modification of the newly synthesised proteins. Amongst the pre- and post-synaptic membrane glycoproteins involved at both first and second time windows are the cell adhesion molecules, L1 (at both times) and NCAM (at the later). Molecular dissection of the external membrane domains of L1 distinguishes between a requirement for the IgG domain at the early time, the fibronectin-like domain at the later. The second time window only occurs if the animal is trained on a stimulus strong enough to be remembered for a long period. Weak memories do not persist beyond 6–8 h and the second wave of glycoprotein synthesis does not occur. Thus the second wave may represent the molecular processes required for the alterations in synaptic configuration, by way of the adhesion molecules amongst others, required for the morphological changes in neuronal connectivity hypothesised to encode memory.     

Inhibition of integrin-mediated adhesion and signaling disrupts retinal development

Integrins are the major family of cell adhesion receptors that mediate cell adhesion to the extracellular matrix (ECM). Integrin-mediated adhesion and signaling play essential roles in neural development. In this study, we have used echistatin, an RGD-containing short monomeric disintegrin, to investigate the role of integrin-mediated adhesion and signaling during retinal development in Xenopus. Application of echistatin to Xenopus retinal-derived XR1 glial cells inhibited the three stages of integrin-mediated adhesion: cell attachment, cell spreading, and formation of focal adhesions and stress fibers. XR1 cell attachment and spreading increased tyrosine phosphorylation of paxillin, a focal adhesion associated protein, while echistatin significantly decreased phosphorylation levels of paxillin. Application of echistatin or beta(1) integrin function blocking antibody to the embryonic Xenopus retina disrupted retinal lamination and produced rosette structures with ectopic photoreceptors in the outer retina. These results indicate that integrin-mediated cell-ECM interactions play a critical role in cell adhesion, migration, and morphogenesis during vertebrate retinal development.     

Syndecans promote integrin-mediated adhesion of mesenchymal cells in two distinct pathways

Syndecans are transmembrane proteoglycans that support integrin-mediated adhesion. Well documented is the contribution of syndecan-4 that interacts through its heparan sulphate chains to promote focal adhesion formation in response to fibronectin domains. This process has requirements for integrin and signaling through the cytoplasmic domain of syndecan-4. Here an alternate pathway mediated by the extracellular domains of syndecans-2 and -4 is characterized that is independent of both heparan sulphate and syndecan signaling. This pathway is restricted to mesenchymal cells and was not seen in any epithelial cell line tested, apart from vascular endothelia. The syndecan ectodomains coated as substrates promoted integrin-dependent attachment, spreading and focal adhesion formation. Syndecan-4 null cells were competent, as were fibroblasts compromised in heparan sulphate synthesis that were unable to form focal adhesions in response to fibronectin. Consistent with actin cytoskeleton organization, the process required Rho-GTP and Rho kinase. While syndecan-2 and -4 ectodomains could both promote integrin-mediated adhesion, their pathways were distinct, as shown by competition assays. Evidence for an indirect interaction of beta1 integrin with both syndecan ectodomains was obtained, all of which suggests a distinct mechanism of integrin-mediated adhesion.     

Changes in the integrin-mediated adhesion of human neutrophils in the cold and after rewarming

Changes in the mechanical and adhesive properties of neutrophils may modify perfusion of the microcirculation in cooled tissue. We tested how integrin-mediated adhesion of isolated human neutrophils was altered by cooling, or cooling and rewarming. First, adhesion was tested in a static assay. In the presence or absence of integrin-activating agents (formyl peptide, fMLP or Mn(++)), there were significant reductions in adhesion to immobilised albumin at 10 degrees C or 0 degrees C compared to 37 degrees C, although a slight increase in adhesion was induced by fMLP or Mn(++) at 10 degrees C or 0 degrees C. If cells were cooled for 5 or 20 min at 10 degrees C and rewarmed (in the absence of activators) there was >100% increase in adhesion compared to cells held at 37 degrees C. In a flow assay, neutrophils perfused over P-selectin at 37 degrees C formed rolling attachments, but if neutrophils were cooled to 10 degrees C and rewarmed for 1 or 5 min, there was transformation to stationary adhesion, which was reversed by antibody against CD18. After 20 minutes of rewarming, rolling was restored. Cooling and rewarming did not cause de novo expression of CD11b/CD18, and so appears to transiently activate constitutively-expressed integrin. Thus, integrin-mediated adhesion may be impaired in cold tissue but on return to normal temperature, neutrophils may transiently adhere locally or in remote vessels.     

Effect of hypoxia on integrin-mediated adhesion of endothelial progenitor cells

Homing of endothelial progenitor cells (EPCs) is crucial for neoangiogenesis, which might be negatively affected by hypoxia. We investigated the influence of hypoxia on fibronectin binding integrins for migration and cell‐matrix‐adhesion. AMP‐activated kinase (AMPK) and integrin‐linked kinase (ILK) were examined as possible effectors of hypoxia.Human EPCs were expanded on fibronectin (FN) and integrin expression was profiled by flow cytometry. Cell‐matrix‐adhesion‐ and migration‐assays on FN were performed to examine the influence of hypoxia and AMPK‐activation. Regulation of AMPK and ILK was shown by Western blot analysis. We demonstrate the presence of integrin β₁, β₂ and α₅ on EPCs. Adhesion to FN is reduced by blocking β₁ and α₅ (49% and 2% of control, P < 0.05) whereas α₄‐blockade has no effect. Corresponding effects were shown for migration. Hypoxia and AMPK‐activation decrease adhesion on FN. Although total AMPK‐expression remains unchanged, phospho‐AMPK increases eightfold.The EPCs require α₅ for adhesion on FN. Hypoxia and AMPK‐activation decrease adhesion. As α₅ is the major adhesive factor for EPCs on FN, this suggests a link between AMPK and α₅‐integrins. We found novel evidence for a connection between hypoxia, AMPK‐activity and integrin activity. This might affect the fate of EPCs in ischaemic tissue.     

Regulation of integrin-mediated adhesion at focal contacts by cyclic AMP

Cyclic AMP (cAMP) elevation causes diverse types of cultured cells to round partially and develop arborized cell processes. Renal glomerular mesangial cells are smooth, muscle-like cells and in culture contain abundant actin microfilament cables that insert into substratum focal contacts. cAMP elevation causes adhesion loss, microfilament cable fragmentation, and shape change in cultured mesangial cells. We investigated the roles of the classical vitronectin (αVβ3 integrin) and fibronectin (α5β1 integrin) receptors in these changes. Mesangial cells on vitronectin-rich substrata contained microfilament cables that terminated in focal contacts that stained with antibodies to vitronectin receptor. cAMP elevation caused loss of focal contact and associated vitronectin receptor. Both fibronectin and its receptor stained in a fibrillary pattern at the cell surface under control conditions but appeared aggregated along the cell processes after cAMP elevation. This suggested that cAMP elevation caused loss of adhesion mediated by vitronectin receptor but not by fibronectin receptor. We plated cells onto fibronectin-coated slides to test the effect of ligand immobilization on the cellular response to cAMP. On fibronectin-coated slides fibronectin receptor was observed in peripheral focal contacts where actin filaments terminated, as seen with vitronectin receptor on vitronectin-coated substrata, and in abundant linear arrays distributed along microfilaments as well. Substratum contacts mediated by fibronectin receptor along the length of actin filaments have been termed fibronexus contacts. After cAMP elevation, microfilaments fragmented and fibronectin receptor disappeared from peripheral focal contacts, but the more central contacts along residual microfilament fragments appeared intact. Also, substratum adhesion was maintained after cAMP elevation on fibronectin—but not on vitronectincoated surfaces. Although other types of extracellular matrix receptors may also be involved, our observations suggest that cAMP regulates adhesion at focal contacts but not at fibronexus-type extracellular matrix contacts. © 1993 Wiley-Liss, Inc.     

Silencing of VAMP3 inhibits cell migration and integrin-mediated adhesion

Integrins are transmembrane receptors for cell adhesion to the extracellular matrix. In cell migration, integrins are endocytosed from the plasma membrane or the cell surface, transported in vesicles and exocytosed actively at the cell front. In the present study, we examined the roles of VAMP3, a SNARE protein that mediates exocytosis, in cell migration and integrin trafficking. Small interfering RNA (siRNA)-induced silencing of VAMP3 inhibited chemotactic cell migration by more than 60% without affecting cell proliferation. VAMP3 silencing reduced the levels of β1 integrin at the cell surface but had no effect on total cellular β1 integrin, indicating that VAMP3 is required for trafficking of β1 integrin to the plasma membrane. Furthermore, VAMP3 silencing diminished cell adhesion to laminin but not to fibronectin or collagen. Taken together, these data suggest that VAMP3-dependent integrin trafficking is crucial in cell migration and cell adhesion to laminin.     

A role for caveolin and the urokinase receptor in integrin-mediated adhesion and signaling

The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with beta1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with beta1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes beta1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, beta1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with beta1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal beta1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.     

Adhesive F-actin waves: a novel integrin-mediated adhesion complex coupled to ventral actin polymerization

At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in "ventral F-actin waves" that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the extracellular matrix downstream of ventral F-actin waves in several mammalian cell lines as well as in primary mouse embryonic fibroblasts. These "adhesive F-actin waves" require a cycle of integrin engagement and disengagement to the extracellular matrix for their formation and propagation, and exhibit morphometry and a hierarchical assembly and disassembly mechanism distinct from other integrin-containing structures. After Arp2/3-mediated actin polymerization, zyxin and VASP are co-recruited to adhesive F-actin waves, followed by paxillin and vinculin, and finally talin and integrin. Adhesive F-actin waves thus represent a previously uncharacterized integrin-based adhesion complex associated with Arp2/3-mediated actin polymerization.     

Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components

Episialin (MUC1) is a transmembrane molecule with a large mucin-like extracellular domain protruding high above the cell surface. The molecule is located at the apical side of most glandular epithelial cells, whereas in carcinoma cells it is often present at the entire surface and it is frequently expressed in abnormally large quantities. We have previously shown that overexpression of episialin reduces cell- cell interactions. Here we show that the integrin-mediated adhesion to extracellular matrix of transfectants of a melanoma cell line (A375), a transformed epithelial cell line (MDCK-ras-e) and a human breast epithelial cell line (HBL-100) is reduced by high levels of episialin. This reduction can be reversed by inducing high avidity of the beta 1 integrins by mAb TS2/16 (at least for beta 1-mediated adhesion). The adhesion can also be restored by redistribution of episialin on the cell surface by monoclonal antibodies into patches or caps. Similarly, capping of episialin on ZR-75-1 breast carcinoma cells, growing in suspension, caused adherence and spreading of these cells. We propose that there is a delicate balance between adhesion and anti-adhesion forces in episialin expressing cells, which can be shifted towards adhesion by strengthening the integrin-mediated adhesion, or towards anti-adhesion by increasing the level of expression of episialin.