LFA-1–mediated Adhesion Is Regulated by Cytoskeletal Restraint and by a Ca2+-dependent Protease, Calpain

The activity of integrins on leukocytes is kept under tight control to avoid inappropriate adhesion while these cells are circulating in blood or migrating through tissues. Using lymphocyte function-associated antigen-1 (LFA-1) on T cells as a model, we have investigated adhesion to ligand intercellular adhesion molecule-1 induced by the Ca²⁺ mobilizers, ionomycin, 2,5-di-t-butylhydroquinone, and thapsigargin, and the well studied stimulators such as phorbol ester and cross-linking of the antigen-specific T cell receptor (TCR)– CD3 complex. We report here that after exposure of T cells to these agonists, integrin is released from cytoskeletal control by the Ca²⁺-induced activation of a calpain-like enzyme, and adhesive contact between cells is strengthened by means of the clustering of mobilized LFA-1 on the membrane. We propose that methods of leukocyte stimulation that cause Ca²⁺ fluxes induce LFA-1 adhesion by regulation of calpain activity. These findings suggest a mechanism whereby engagement of the TCR could promote adhesion strengthening at an early stage of interaction with an antigen-presenting cell.     

Angiopoietin-2 Stimulation of Endothelial Cells Induces αvβ3 Integrin Internalization and Degradation

The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence. Ang-2 interferes negatively with Ang-1/Tie2 signaling, thereby antagonizing the Ang-1/Tie2 axis. Here, we show that both Ang-1 and Ang-2 recruit β3 integrins to Tie2. This co-localization is most prominent in cell-cell junctions. However, only Ang-2 stimulation resulted in complex formation among Tie2, αvβ3 integrin, and focal adhesion kinase as evidenced by co-immunoprecipitation experiments. Focal adhesion kinase was phosphorylated in the FAT domain at Ser⁹¹⁰ upon Ang-2 stimulation and the adaptor proteins p130Cas and talin dissociated from αvβ3 integrin. The αvβ3 integrin was internalized, ubiquitinylated, and gated toward lysosomes. Taken together, the experiments define Tie2/αvβ3 integrin association-induced integrin internalization and degradation as mechanistic consequences of endothelial Ang-2 stimulation.     

The regulation of integrin function by divalent cations

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca²⁺-binding motifs that have essential roles in integrin biogenesis. The function of another Ca²⁺-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions.     

Novel function for beta 1 integrins in keratinocyte cell-cell interactions.

We have examined the expression, localization, and function of beta 1 integrins on cultured human epidermal keratinocytes using polyclonal and monoclonal antibodies against the beta 1, alpha 2, alpha 3, and alpha 5 integrin subunits. The beta 1 polypeptide, common to all class 1 integrins, was localized primarily in areas of cell-cell contacts of cultured keratinocytes, as were alpha 2 and alpha 3 polypeptides, suggesting a possible role in cell-cell adhesion for these integrin polypeptides. In contrast, the fibronectin receptor alpha 5 subunit showed no such accumulations in regions of cell-cell contact but was more diffusely distributed in the keratinocyte plasma membrane, consistent with the absence of fibronectin at cell-cell contact sites. Colonies of cultured keratinocytes could be dissociated by treatment with monoclonal antibody specific to the beta 1 polypeptide. Such dissociation of cell-cell contacts also occurred under conditions where the monoclonal antibody had no effect on cell-substrate adhesion. Therefore, beta 1 integrin-dependent cell-cell adhesion can be inhibited without affecting other cell-adhesive interactions. Antibody treatment of keratinocytes maintained in either low (0.15 mM) or high (1.2 mM) CaCl2 also resulted in the loss of organization of intracellular F-actin filaments and beta 1 integrins, even when the anti-beta 1 monoclonal antibody had no dissociating effect on keratinocyte colonies at the higher calcium concentration. Our results indicate that beta 1 integrins play roles in the maintenance of cell-cell contacts between keratinocytes and in the organization of intracellular microfilaments. They suggest that in epithelial cells integrins can function in cell-cell interactions as well as in cell-substrate adhesion.     

A Role for the αvβ3 Integrin in the Transmigration of Monocytes

The β2 integrins and intercellular adhesion molecule-1 (ICAM-1) are important for monocyte migration through inflammatory endothelium. Here we demonstrate that the integrin αvβ3 is also a key player in this process. In an in vitro transendothelial migration assay, monocytes lacking β3 integrins revealed weak migratory ability, whereas monocytes expressing β3 integrins engaged in stronger migration. This migration could be partially blocked by antibodies against the integrin chains αL, β2, αv, or IAP, a protein functionally associated with αvβ3 integrin. Transfection of β3 integrin chain cDNA into monocytes lacking β3 integrins resulted in expression of the αvβ3 integrin and conferred on these cells an enhanced ability to transmigrate through cell monolayers expressing ICAM-1. These monocytes also engaged in αLβ2-dependent locomotion on recombinant ICAM-1 which was enhanced by αvβ3 integrin occupancy. Antibodies against IAP were able to revert this αvβ3 integrin-dependent cell locomotion to control levels. Finally, adhesion assays revealed that occupancy of αvβ3 integrin could decrease monocyte binding to ICAM-1.In conclusion, we show that αvβ3 integrin modulates αLβ2 integrin-dependent monocyte adhesion to and migration on ICAM-1. This could represent a novel mechanism to promote monocyte motility on vascular ICAM-1 and initiate subsequent transendothelial migration.     

α-Catenin and Vinculin Cooperate to Promote High E-cadherin-based Adhesion Strength

Maintaining cell cohesiveness within tissues requires that intercellular adhesions develop sufficient strength to support traction forces applied by myosin motors and by neighboring cells. Cadherins are transmembrane receptors that mediate intercellular adhesion. The cadherin cytoplasmic domain recruits several partners, including catenins and vinculin, at sites of cell-cell adhesion. Our study used force measurements to address the role of αE-catenin and vinculin in the regulation of the strength of E-cadherin-based adhesion. αE-catenin-deficient cells display only weak aggregation and fail to strengthen intercellular adhesion over time, a process rescued by the expression of αE-catenin or chimeric E-cadherin·αE-catenins, including a chimera lacking the αE-catenin dimerization domain. Interestingly, an αE-catenin mutant lacking the modulation and actin-binding domains restores cadherin-dependent cell-cell contacts but cannot strengthen intercellular adhesion. The expression of αE-catenin mutated in its vinculin-binding site is defective in its ability to rescue cadherin-based adhesion strength in cells lacking αE-catenin. Vinculin depletion or the overexpression of the αE-catenin modulation domain strongly decreases E-cadherin-mediated adhesion strength. This supports the notion that both molecules are required for intercellular contact maturation. Furthermore, stretching of cell doublets increases vinculin recruitment and α18 anti-αE-catenin conformational epitope immunostaining at cell-cell contacts. Taken together, our results indicate that αE-catenin and vinculin cooperatively support intercellular adhesion strengthening, probably via a mechanoresponsive link between the E-cadherin·β-catenin complexes and the underlying actin cytoskeleton.     

Expression and In Vitro Regulation of Integrins by Normal Human Urothelial Cells

Integrins are thought to be essential adhesion receptors for the maintenance of tissue hisr tioarchitecture. The purpose of this study was to determine integrin expression patterns in the human stratified transitional epithelium of the urinary tract (urothelium). In situ expression patterns were compared with in vitro expression, using a normal cell culture model system in which the effects of cell stratification can be studied independently of differentiation. By immunohistological criteria, the urothelia of bladder, ureter and renal pelvis expressed α2β1 and α3β1 integrins in all layers at intercellular junctions, and cytoplasmically in the lower strata. By contrast, α6β4 and occasionally αvβ4 were expressed only by basal cells and localised to the basal lamina. These expression patterns were unaltered in specimens where an inflammatory cell infiltrate was present. In long-term cultures of normal urothelial cells maintained in a low-Ca⁺⁺serum-free medium, the monolayer cultures expressed α2β1, α3β1 and α5β1 integrins at intercellular junctions and in cytoplasmic inclusions, whereas α6β4 was distributed in a random pattern over the substratum. Increasing exogenous Ca⁺⁺concentrations induced cell stratification and desmosome formation, but not cytodifferentiation. Under these conditions, α6β4 became cell-, rather than substratum-associated, localising particularly to filopodia and lamellipodia. Quantitation of integrin expression by flow cytometry confirmed increased surface expression of α6β4 in high Ca⁺⁺media, and also of α3 and α5, but not α2, subunits. These results suggest that α2β1 and α3β1 integrins, although differentially regulated, are mainly involved in homotypic cell-cell interactions and the maintenance of a stratified morphology, whereas α6β4 is the principal integrin involved in substratum adhesion.     

Focal Adhesion Kinase pp125and the β1 Integrin Subunit Are Constitutively Complexed in HaCaT Cells

Binding of integrins to the extracellular matrix (ECM) activates various signal transduction pathways and regulates gene expression in many cell types. Integrin-dependent cytoplasmic protein/protein interactions are necessary for activation of those signal transduction cascades. In our studies we investigated a possible association of pp125^FAK, an adhesion involved tyrosine kinase, with the integrin β1 subunit. Further we wanted to know to which extent protein tyrosine phosphorylation affects cell adhesion to the ECM and the possible β1 integrin/pp125^FAKcomplex. We were able to show that in HaCaT cells (a human keratinocyte derived cell line) the integrin β1 subunit is associated with tyrosine kinase pp125^FAK. This association was observed in ECM-adherent cells and nonadherent cells and is independent of tyrosine phosphorylation. However, cell adhesion of HaCaT cells to specific substrates requires tyrosine phosphorylation since genistein treatment that blocks phosphorylation of many cellular proteins as pp125^FAKled to a reduced substrate adhesion.     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.     

Soluble Integrin Ligands and Growth Factors Independently Rescue Neuroblastoma Cells from Apoptosis under Nonadherent Conditions

We have investigated the role of extracellular matrix (ECM) and growth factors in the survival of nonadherent human neuroblastoma cells (line SK-N-BE). Cells cultured in serum-free medium under nonadherent conditions died with apoptotic-like features (cromatin condensation and nuclear fragmentation). SK-N-BE cells underwent neuronal differentiation in response to retinoic acid (RA). While RA itself did not induce apoptosis, differentiation increased the susceptibility of SK-N-BE cells to detachment-induced apoptosis. The appearance of the apoptotic-like phenotype required the maintenance in suspension of SK-N-BE cells for at least 16 h (12.43 ± 1.40% of cells undergoing apoptosis) and the percentage increased up to 46.84 ± 3.15% after 24 h. Suspension-induced apoptosis did not depend on increased intracellular Ca²⁺levels nor onde novoprotein synthesis and was not associated with extensive DNA degradation. Stimulation by soluble collagen I rescued suspended cells from apoptosis, even in the absence of cell adhesion and spreading. The survival promoting effect of ECM was mediated by the integrin receptors, since ([1]) the protective effect of soluble collagen I was blocked by anti-integrin antibodies to β₁and α₁subunits and ([2]) the antibody-induced clustering of α₁, α₃, α_v, β₁, and β₃integrins rescued SK-N-BE cells cultured in suspension from apoptosis. As expected, adhesion on immobilized ECM proteins, collagen I, or laminin (0.1 to 10 μg/ml) also rescued SK-N-BE cells from apoptosis in a dose-dependent manner. Thede novoprotein synthesis was required to promote the survival effect of ECM, since cycloheximide completely abolished the protective effect of collagen I and protection from apoptosis by ECM or by anti-β₁antibody was associated with the increased expression ofbcl-2.In addition to integrin stimulation, serum, insulin, and nerve growth factor inhibited suspension-induced apoptosis of SK-N-BE cells. The survival effect of serum and growth factors did not require the synthesis of new proteins, unlike the ECM effect. These data show that matrix proteins can promote cell survival in neuronal cells via integrin receptors. This effect does not require cell adhesion and the subsequent changes in cell shape as it can be mediated by soluble integrin ligands in suspended cells and involves a signaling pathway different from that triggered by growth factors.     

Regulation of urokinase plasminogen activator localization in keratinocytes by calcium ion and E-cadherin.

In keratinocyte culture, the cellular distribution of many adhesion markers and the organization of intercellular junctions are controlled by the calcium ion concentration of the medium. We show in the present study that urokinase plasminogen activator (uPA) localization in the human keratinocyte is similarly dependent upon calcium concentration. At 30 microM calcium, uPA is present throughout the cell, often with a perinuclear concentration. Upon calcium elevation to 1.0 mM, uPA is concentrated along the cell-cell borders, where it colocalizes (at the light microscope level) with E-cadherin. Blocking antibody to E-cadherin delays the calcium-induced redistribution of uPA, in a manner very similar to the previously observed delay in redistribution of several adhesion-related markers, including vinculin, desmoplakin, and beta 1 integrin. These data suggest a link between the redistribution of uPA to the cell-cell borders and the calcium-induced organization of intercellular junctions in the human keratinocyte. The presence of uPA along the intercellular borders suggests that this enzyme may be involved in regulation of epidermal adhesion through proteolysis.     

The integrins

The integrins are a superfamily of cell adhesion receptors that bind to extracellular matrix ligands, cell-surface ligands, and soluble ligands. They are transmembrane αβ heterodimers and at least 18 α and eight β subunits are known in humans, generating 24 heterodimers. Members of this family have been found in mammals, chicken and zebrafish, as well as lower eukaryotes, including sponges, the nematode Caenorhabditis elegans (two α and one β subunits, generating two integrins) and the fruitfly Drosophila melanogaster (five α and one β, generating five integrins). The α and β subunits have distinct domain structures, with extracellular domains from each subunit contributing to the ligand-binding site of the heterodimer. The sequence arginine-glycine-aspartic acid (RGD) was identified as a general integrin-binding motif, but individual integrins are also specific for particular protein ligands. Immunologically important integrin ligands are the intercellular adhesion molecules (ICAMs), immunoglobulin superfamily members present on inflamed endothelium and antigen-presenting cells. On ligand binding, integrins transduce signals into the cell interior; they can also receive intracellular signals that regulate their ligand-binding affinity. Here we provide a brief overview that concentrates mostly on the organization, structure and function of mammalian integrins, which have been more extensively studied than integrins in other organisms.     

Cross Talk between Adhesion Molecules: Control of N-cadherin Activity by Intracellular Signals Elicited by β1 and β3 Integrins in Migrating Neural Crest Cells

During embryonic development, cell migration and cell differentiation are associated with dynamic modulations both in time and space of the repertoire and function of adhesion receptors, but the nature of the mechanisms responsible for their coordinated occurrence remains to be elucidated. Thus, migrating neural crest cells adhere to fibronectin in an integrin-dependent manner while maintaining reduced N-cadherin–mediated intercellular contacts. In the present study we provide evidence that, in these cells, the control of N-cadherin may rely directly on the activity of integrins involved in the process of cell motion. Prevention of neural crest cell migration using RGD peptides or antibodies to fibronectin and to β1 and β3 integrins caused rapid N-cadherin–mediated cell clustering. Restoration of stable intercellular contacts resulted essentially from the recruitment of an intracellular pool of N-cadherin molecules that accumulated into adherens junctions in tight association with the cytoskeleton and not from the redistribution of a preexisting pool of surface N-cadherin molecules. In addition, agents that cause elevation of intracellular Ca²⁺ after entry across the plasma membrane were potent inhibitors of cell aggregation and reduced the N-cadherin– mediated junctions in the cells. Finally, elevated serine/ threonine phosphorylation of catenins associated with N-cadherin accompanied the restoration of intercellular contacts. These results indicate that, in migrating neural crest cells, β1 and β3 integrins are at the origin of a cascade of signaling events that involve transmembrane Ca²⁺ fluxes, followed by activation of phosphatases and kinases, and that ultimately control the surface distribution and activity of N-cadherin. Such a direct coupling between adhesion receptors by means of intracellular signals may be significant for the coordinated interplay between cell–cell and cell–substratum adhesion that occurs during embryonic development, in wound healing, and during tumor invasion and metastasis.     

Variation in One Residue Associated with the Metal Ion-Dependent Adhesion Site Regulates αIIbβ3 Integrin Ligand Binding Affinity

The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS) divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala²⁵² and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS) of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala²⁵² to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion.     

A Cell-free System to Study Regulation of Focal Adhesions and of the Connected Actin Cytoskeleton

Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca²⁺ concentrations induce quasi-reversible diffusion of β1 integrins out of focal adhesions, whereas low Ca²⁺ concentrations induce irreversible recruitment of β1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated β1 receptors show that the cytoplasmic portion of β1 is required for low Ca²⁺-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified α-actinin colocalizes and redistributes with β1 receptors on ventral plasma membranes depleted of actin, implicating binding of α-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.     

β1 Integrin-Mediated Adhesion Signalling Is Essential for Epidermal Progenitor Cell Expansion

Background

There is a major discrepancy between the in vitro and in vivo results regarding the role of β1 integrins in the maintenance of epidermal stem/progenitor cells. Studies of mice with skin-specific ablation of β1 integrins suggested that epidermis can form and be maintained in their absence, while in vitro data have shown a fundamental role for these adhesion receptors in stem/progenitor cell expansion and differentiation.

Methodology/Principal Findings

To elucidate this discrepancy we generated hypomorphic mice expressing reduced β1 integrin levels on keratinocytes that developed similar, but less severe defects than mice with β1-deficient keratinocytes. Surprisingly we found that upon aging these abnormalities attenuated due to a rapid expansion of cells, which escaped or compensated for the down-regulation of β1 integrin expression. A similar phenomenon was observed in aged mice with a complete, skin-specific ablation of the β1 integrin gene, where cells that escaped Cre-mediated recombination repopulated the mutant skin in a very short time period. The expansion of β1 integrin expressing keratinocytes was even further accelerated in situations of increased keratinocyte proliferation such as wound healing.

Conclusions/Significance

These data demonstrate that expression of β1 integrins is critically important for the expansion of epidermal progenitor cells to maintain epidermal homeostasis.     

Expression of β1 Integrins by Cultured Articular Chondrocytes and in Osteoarthritic Cartilage

Expression of β1 integrins was studied in vitro as articular chondrocytes reestablished a matrix in culture and in situ in a nonhuman primate model of osteoarthritis in order to investigate a potential role for integrins in mediating cell-extracellular matrix interactions in cartilage. Chondrocytes were found to express α1β1, α3β1, and α5β1 integrins both in vitro and in situ. Cell surface expression of β1 integrins increased as chondrocytes were maintained in culture from 3 to 7 days. Increased β1 integrin expression was also observed in osteoarthritic cartilage compared with normal cartilage. The greatest relative increase in both systems was noted for the α1β1 integrin. The increase in chondrocyte β1 integrin expression in vitro was noted in both monolayer and alginate cultures and occurred prior to detectable changes in the differentiated phenotype of the chondrocyte. Disruption of the cytoskeleton with the drug dihydrocytochalasin B inhibited the cell culture induced increase in integrin expression, while treatment of cultured cells with TGF-β resulted in increased expression of the α5β1 integrin. The modulation of β1 integrin expression noted in vitro and in situ indicates that chondrocytes are capable of regulated expression of β1 integrins and suggests that β1 integrins may play an important role in mediating chondrocyte-extracellular matrix interactions in cartilage.     

Clustering of Syndecan-4 and Integrin β1 by Laminin α3 Chain–derived Peptide Promotes Keratinocyte Migration

Syndecans function as receptors for extracellular matrix (ECM) with integrins in cell spreading. However, the molecular mechanism of their specific involvement in cell migration or in wound healing has not been elucidated yet. Here, we report that a synthetic peptide, PEP75, which contains the syndecan-binding sequence of the laminin α3LG4 module, induces keratinocyte migration in in vitro and in vivo. Soluble PEP75 induced the clustering of syndecan-4 and conformation-modified integrin β1 colocalized with syndecan-4 in soluble PEP75-induced clusters. Treatment of cells in solution with PEP75 resulted in the exposure of the P4G11 antibody epitope of integrin β1 in immunostaining as well as in flow cytometry and augmented integrin β1–dependent cell adhesion to ECM. Pulldown assays demonstrated that PEP75 bound to syndecan-4, but not to integrin β1. A siRNA study revealed a role for syndecan-4 in PEP75-induced up-regulation of P4G11 antibody binding and migration of HaCaT cells. We conclude that binding of soluble PEP75 to syndecan-4 induces the coupling of integrin β1, which is associated with integrin β1-conformational changes and activation, and leads to keratinocyte migration. To activate integrin function through syndecans could be a novel therapeutic approach for chronic wound.     

Activation of beta1 integrins induces cell-cell adhesion

Integrins are highly regulated receptors that can function in both cell-substrate and cell-cell adhesion. We have found that the activating anti-beta1 mAb, 12G10, can specifically and rapidly induce both cell-substrate and cell-cell adhesion of HT-1080 human fibrosarcoma and other cell types. Binding of mAb 12G10 induced clustering of cell-surface integrins, and the preferential localization of beta1 integrins expressing the 12G10 epitope at cell-cell adhesion sites. Fab fragments of mAb 12G10 induced HT-1080 cell-cell adhesion as effectively as did intact antibodies, suggesting that integrin clustering was not due to direct antibody crosslinking. Latrunculin B, an inhibitor of F-actin polymerization, inhibited cell-cell adhesion but not the clustering of integrins. Results from a novel, two-color cell-cell adhesion assay suggested that nonactivated cells can bind to activated cells and that integrin activation-induced HT-1080 cell-cell adhesion minimally requires the interaction of activated alpha2beta1 with nonactivated alpha3beta1. These findings suggest that HT-1080 cell-cell adhesion induced by integrin activation require a signaling process involving integrin clustering and the subsequent organization of the cytoskeleton. Integrin activation could therefore play a key role in cell-cell adhesion.     

The Muscle-Specific Laminin Receptor α7β1 Integrin Negatively Regulates α5β1 Fibronectin Receptor Function

α7β1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the α7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with α7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the α7β1. α7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of α7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the α5β1 fibronectin receptor. Although cell surface expression of α5β1 was reduced by a factor of 20–25% in α7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of¹²⁵I-fibronectin for its surface receptor was decreased by 50% in α7 transfectants, indicating that the α5β1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn²⁺, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn²⁺restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in α7 transfectants. These data indicate that α7 expression leads to the functional down regulation of α5β1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of anegative cooperativitybetween α7 and α5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.