Integrin alpha 8 beta 1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin.

The integrin alpha 8 subunit, isolated by low stringency hybridization, is a novel integrin subunit that associates with beta 1. To identify ligands, we have prepared a function-blocking antiserum to the extracellular domain of alpha 8, and we have established by transfection K562 cell lines that stably express alpha 8 beta 1 heterodimers on the cell surface. We demonstrate here by cell adhesion and neurite outgrowth assays that alpha 8 beta 1 is a fibronectin receptor. Studies on fibronectin fragments using RGD peptides as inhibitors show that alpha 8 beta 1 binds to the RGD site of fibronectin. In contrast to the endogenous alpha 5 beta 1 fibronectin receptor in K562 cells, alpha 8 beta 1 not only promotes cell attachment but also extensive cell spreading, suggesting functional differences between the two receptors. In chick embryo fibroblasts, alpha 8 beta 1 is localized to focal adhesions. We conclude that alpha 8 beta 1 is a receptor for fibronectin and can promote attachment, cell spreading, and neurite outgrowth on fibronectin.     

Human microvascular endothelial cells use beta 1 and beta 3 integrin receptor complexes to attach to laminin

Microvascular endothelial cells (MEC) use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured human MEC interact with laminin-rich basement membranes. By using a panel of monoclonal antibodies, we found that MEC cells express a number of integrin-related receptor complexes, including alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha V beta 3. Attachment to laminin, a major adhesive protein in basement membranes, was studied in detail. Blocking monoclonal antibodies specific to different integrin receptor complexes showed that the alpha 6 beta 1 complex was important for MEC adhesion to laminin. In addition, blocking antibody also implicated the vitronectin receptor (alpha V beta 3) in laminin adhesion. We used ligand affinity chromatography of detergent-solubilized receptor complexes to further define receptor specificity. On laminin-Sepharose columns, we identified several integrin receptor complexes whose affinity for the ligand was dependent on the type of divalent cation present. Several beta 1 complexes, including alpha 1 beta 1, alpha 2 beta 1, and alpha 6 beta 1 bound strongly to laminin. In agreement with the antibody blocking experiments, alpha V beta 3 was found to bind well to laminin. However, unlike binding to its other ligands (e.g., vitronectin, fibrinogen, von Willebrand factor), alpha V beta 3 interaction with laminin did not appear to be Arg-Gly-Asp (RGD) sensitive. Finally, immunofluorescent staining demonstrated both beta 1 and beta 3 complexes in vinculin-positive focal adhesion plaques on the basal surface of MEC adhering to laminin-coated substrates. The results indicate that both these subfamilies of integrin heterodimers are involved in promoting MEC adhesion to laminin and the vascular basement membrane.     

Covalent surface immobilization of Arg-Gly-Asp- and Tyr-Ile-Gly-Ser-Arg-containing peptides to obtain well-defined cell-adhesive substrates

The synthetic peptides Gly-Arg-Gly-Asp-Tyr and Gly-Tyr-Ile-Gly-Ser-Arg-Tyr, which contain Arg-Gly-Asp (RGD) and Tyr-Ile-Gly-Ser-Arg (YIGSR), the ligands for two important classes of cell adhesion receptors, were covalently coupled to a nonadhesive modified glass surface by the N-terminal Gly. The N-terminal Gly served as a spacer, and the C-terminal Y served as a site for radioiodination. These modified substrates supported the adhesion and spreading of cultured human foreskin fibroblasts (HFFs) independently of adsorbed proteins and, it was demonstrated that a covalently immobilized YIGSR-containing peptide has biological activity. The surface concentration of grafted peptide on the glass was measured by 125I radio-labeling and was 12.1 pmol/cm2. HFFs spread on both immobilized peptide substrates, but at much slower rates on grafted YIGSR glass surfaces than on the RGD-containing substrates. Cells formed focal contacts on the RGD-derivatized substrates in the presence or absence of serum. Focal contacts formed on the YIGSR-grafted surfaces only when serum was present in the medium and had morphologies different from those observed on the RGD-containing substrates. Serum influenced the organization of microfilaments and the extent of spreading of adherent cells, although adsorption of adhesion proteins was minimal on all substrates. This derivatization method produced chemically stable substrates which may be useful in studying receptor-mediated cell adhesion, as the quantity of peptide available at the surface may be precisely measured and controlled.     

Galectin binding to Mgat5-modified N-glycans regulates fibronectin matrix remodeling in tumor cells

Oncogenic signaling stimulates the dynamic remodeling of actin microfilaments and substrate adhesions, essential for cell spreading and motility. Transformation is associated with increased expression of beta1,6GlcNAc-branched N-glycans, products of Golgi beta1,6-acetylglucosaminyltransferase V (Mgat5) and the favored ligand for galectins. Herein we report that fibronectin fibrillogenesis and fibronectin-dependent cell spreading are deficient in Mgat5(-/-) mammary epithelial tumor cells and inhibited in Mgat5(+/+) cells by blocking Golgi N-glycan processing with swainsonine or by competitive inhibition of galectin binding. At an optimum dosage, exogenous galectin-3 added to Mgat5(+/+) cells activates focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K), recruits conformationally active alpha5beta1-integrin to fibrillar adhesions, and increases F-actin turnover. RGD peptide inhibits PI3K-dependent fibronectin matrix remodeling and fibronectin-dependent cell motility, while galectin-3 stimulates and overrides the inhibitory effects of RGD. Antibodies to the galectin-3 N-terminal oligomerization domain stimulate alpha5beta1 activation and recruitment to fibrillar adhesions in Mgat5(+/+) cells, an effect that is blocked by disrupting galectin-glycan binding. Our results demonstrate that fibronectin polymerization and tumor cell motility are regulated by galectin-3 binding to branched N-glycan ligands that stimulate focal adhesion remodeling, FAK and PI3K activation, local F-actin instability, and alpha5beta1 translocation to fibrillar adhesions.     

Human neural cell adhesion molecule L1 and rat homologue NILE are ligands for integrin alpha v beta 3

Integrin alpha v beta 3 is distinct in its capacity to recognize the sequence Arg-Gly-Asp (RGD) in many extra-cellular matrix (ECM) components. Here, we demonstrate that in addition to the recognition of ECM components, alpha v beta 3 can interact with the neural cell adhesion molecule L1-CAM; a member of the immunoglobulin superfamily (IgSF). M21 melanoma cells displayed significant Ca(++)-dependent adhesion and spreading on immunopurified rat L1 (NILE). This adhesion was found to be dependent on the expression of the alpha v-integrin subunit and could be significantly inhibited by an antibody to the alpha v beta 3 heterodimer. M21 cells also displayed some alpha v beta 3-dependent adhesion and spreading on immunopurified human L1. Ligation between this ligand and alpha v beta 3 was also observed to promote significant haptotactic cell migration. To map the site of alpha v beta 3 ligation we used recombinant L1 fragments comprising the entire extracellular domain of human L1. Significant alpha v beta 3-dependent adhesion and spreading was evident on a L1 fragment containing Ig-like domains 4, 5, and 6. Importantly, mutation of an RGD sequence present in the sixth Ig-like domain of L1 abrogated M21 cell adhesion. We conclude that alpha v beta 3-dependent recognition of human L1 is dependent on ligation of this RGD site. Despite high levels of L1 expression the M21 melanoma cells did not display significant adhesion via a homophilic L1-L1 interaction. These data suggest that M21 melanoma cells recognize and adhere to L1 through a mechanism that is primarily heterophilic and integrin dependent. Finally, we present evidence that melanoma cells can shed and deposit L1 in occluding ECM. In this regard, alpha v beta 3 may recognize L1 in a cell-cell or cell- substrate interaction.     

Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement

Cell adhesion-dependent signaling implicates cytoplasmic proteins interacting with the intracellular tails of integrins. Among those, the integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha) has been shown to interact specifically with the beta(1) integrin cytoplasmic domain. Although it is likely that this protein plays an important role in controlling cell adhesion and migration, little is known about its actual function. To search for potential ICAP-1alpha-binding proteins, we used a yeast two-hybrid screen and identified the human metastatic suppressor protein nm23-H2 as a new partner of ICAP-1alpha. This direct interaction was confirmed in vitro, using purified recombinant ICAP-1alpha and nm23-H2, and by co-immunoprecipitation from CHO cell lysates over-expressing ICAP-1alpha. The physiological relevance of this interaction is provided by confocal fluorescence microscopy, which shows that ICAP-1alpha and nm23-H2 are co-localized in lamellipodia during the early stages of cell spreading. These adhesion sites are enriched in occupied beta(1) integrins and precede the formation of focal adhesions devoid of ICAP-1alpha and nm23-H2, indicating the dynamic segregation of components of matrix adhesions. This peripheral staining of ICAP-1alpha and nm23-H2 is only observed in cells spreading on fibronectin and collagen and is absent in cells spreading on poly-l-lysine, vitronectin, or laminin. This is consistent with the fact that targeting of both ICAP-1alpha and nm23-H2 to the cell periphery is dependent on beta(1) integrin engagement rather than being a consequence of cell adhesion. This finding represents the first evidence that the tumor suppressor nm23-H2 could act on beta(1) integrin-mediated cell adhesion by interacting with one of the integrin partners, ICAP-1alpha.     

Deposition of laminin 5 by keratinocytes regulates integrin adhesion and signaling

Deposition of laminin 5 over exposed dermal collagen in epidermal wounds is an early event in repair of the basement membrane. We report that deposition of laminin 5 onto collagen switches adhesion and signaling from collagen-dependent to laminin 5-dependent. Ligation of laminin 5 by integrin alpha(6)beta(4) activates phosphoinositide 3-OH-kinase (PI3K) signaling. This activation allows for adhesion and spreading via integrin alpha(3)beta(1) on laminin 5 independent of RhoGTPase, a regulator of actin stress fibers. In contrast, adhesion and spreading on collagen via alpha(2)beta(1) is Rho-dependent and is inhibited by toxin B, a Rho inhibitor. Deposition of laminin 5 and ligation of alpha(6)beta(4) increases PI3K-dependent production of phosphoinositide di- and triphosphates, PI3K activity, and phosphorylation of downstream target protein c-Jun NH(2)-terminal kinase. Conversely, blocking laminin 5-deposition with brefeldin A, an inhibitor of vesicle transport, or with anti-laminin 5 monoclonal antibodies abolishes the PI3K-dependent spreading mediated by alpha(3)beta(1) and phosphorylation of c-Jun NH(2)-terminal kinase. Studies with keratinocytes lacking alpha(6)beta(4) or laminin 5 confirm that deposition of laminin 5 and ligation by alpha(6)beta(4) are required for PI3K-dependent spreading via alpha(3)beta(1). We suggest that deposition of laminin 5 onto the collagen substratum, as in wound repair, enables human foreskin keratinocytes to interact via alpha(6)beta(4) and to switch from a RhoGTPase-dependent adhesion on collagen to a PI3K-dependent adhesion and spreading mediated by integrin alpha(3)beta(1) on laminin 5.     

Distinct functions of integrin alpha and beta subunit cytoplasmic domains in cell spreading and formation of focal adhesions

Integrin-mediated cell adhesion often results in cell spreading and the formation of focal adhesions. We exploited the capacity of recombinant human alpha IIb beta 3 integrin to endow heterologous cells with the ability to adhere and spread on fibrinogen to study the role of integrin cytoplasmic domains in initiation of cell spreading and focal adhesions. The same constructs were also used to analyze the role of the cytoplasmic domains in maintenance of the fidelity of the integrin repertoire at focal adhesions. Truncation mutants of the cytoplasmic domain of alpha IIb did not interfere with the ability of alpha IIb beta 3 to initiate cell spreading and form focal adhesions. Nevertheless, deletion of the alpha IIb cytoplasmic domain allowed indiscriminate recruitment of alpha IIb beta 3 to focal adhesions formed by other integrins. Truncation of the beta 3 subunit cytoplasmic domain abolished cell spreading mediated by alpha IIb beta 3 and also abrogated recruitment of alpha IIb beta 3 to focal adhesions. This truncation also dramatically impaired the ability of alpha IIb beta 3 to mediate the contraction of fibrin gels. In contrast, the beta 3 subunit cytoplasmic truncation did not reduce the fibrinogen binding affinity of alpha IIb beta 3. Thus, the integrin beta 3 subunit cytoplasmic domain is necessary and sufficient for initiation of cell spreading and focal adhesion formation. Further, the beta 3 cytoplasmic domain is required for the transmission of intracellular contractile forces to fibrin gels. The alpha subunit cytoplasmic domain maintains the fidelity of recruitment of the integrins to focal adhesions and thus regulates their repertoire of integrins.     

Prostaglandin E2 promotes integrin alpha Vbeta 3-dependent endothelial cell adhesion,rac-activation,and spreading through cAMP/PKA-dependent signaling

We have recently reported that the inhibition of endothelial cell COX-2 by non-steroidal anti-inflammatory drugs suppresses alpha(V)beta(3)- (but not alpha(5)beta(1)-) dependent Rac activation, endothelial cell spreading, migration, and angiogenesis (Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041-1047). Here we investigated the role of the COX-2 metabolites PGE(2) and TXA2 in regulating human umbilical vein endothelial cell (HUVEC) adhesion and spreading. We report that PGE(2) accelerated alpha(V)beta(3)-mediated HUVEC adhesion and promoted Rac activation and cell spreading, whereas the TXA2 agonist retarded adhesion and inhibited spreading. We show that the cAMP level and the cAMP-regulated protein kinase A (PKA) activity are critical mediators of these PGE(2) effects. alpha(V)beta(3)-mediated adhesion induced a transient COX-2-dependent rise in cAMP levels, whereas the cell-permeable cAMP analogue 8-brcAMP accelerated adhesion, promoted Rac activation, and cell spreading in the presence of the COX-2 inhibitor NS-398. Pharmacological inhibition of PKA completely blocked alpha(V)beta(3)-mediated adhesion. A constitutively active Rac mutant (L61Rac) rescued alpha(V)beta(3)-dependent spreading in the presence of NS398 or, but did not accelerate adhesion, whereas a dominant negative Rac mutant (N17Rac) suppressed spreading without affecting adhesion. alpha(5)beta(1)-mediated HUVEC adhesion, Rac activation, and spreading were not affected by PGE(2), 8-brcAMP, or the inhibition of PKA. In conclusion, these results demonstrate that PGE(2) accelerates alpha(V)beta(3)-mediated endothelial cell adhesion through cAMP-dependent PKA activation and induces alpha(V)beta(3)-dependent spreading via cAMP- and PKA-dependent Rac activation and may contribute to the further understanding of the regulation of vascular integrins alpha(V)beta(3) by COX-2/PGE(2) during tumor angiogenesis and inflammation.     

The synergy peptide PHSRN and the adhesion peptide RGD mediate cell adhesion through a common mechanism

This work reports on the role of the synergy peptide PHSRN in mediating the adhesion of cells. The attachment of baby hamster kidney cells and 3T3 Swiss fibroblasts to model substrates presenting either GRGDS or PHSRN was evaluated using self-assembled monolayers of alkanethiolates on gold presenting the peptide ligands mixed with tri(ethylene glycol) groups. These substrates permit rigorous control over the structures and densities of peptide ligands and at the same time prevent nonspecific interactions with adherent cells. Both cell types attached efficiently to monolayers presenting either the RGD or the PHSRN peptide but not to monolayers presenting scrambled peptide GRDGS or HRPSN. Cell attachment was comparable on substrates presenting either peptide ligand but less efficient than on substrates presenting the protein fibronectin. The degree of cell spreading, however, was substantially higher on substrates presenting RGD relative to PHSRN. Staining of 3T3 fibroblasts with anti-vinculin and phalloidin revealed clear cytoskeletal filaments and focal adhesions for cells attached by way of either RGD or PHSRN. Inhibition experiments showed that the attachment of 3T3 fibroblasts to monolayers presenting RGD could be inhibited completely by a soluble RGD peptide and partially by a soluble PHSRN peptide. IMR 90 fibroblast attachment to monolayers presenting PHSRN could be inhibited with anti-integrin alpha(5) or anti-integrin beta(1) antibody. This work demonstrates unambiguously that PHSRN alone can support the attachment of cells and that the RGD and PHSRN bind competitively to the integrin receptors.     

Evidence for a differential functional regulation of the two beta(3)-integrins alpha(V)beta(3) and alpha(IIb)beta(3)

The functional regulation of integrins is a major determinant of cell adhesion, migration and tissue maintenance. The binding of cytoskeletal proteins to various sites of integrin cytoplasmic domains is a key mechanism of this functional regulation. Expression of recombinant integrin alpha(IIb)beta(3) and alpha(M)beta(2) lacking the GFFKR-region in CHO cells results in constitutively activated integrins. In contrast, CHO cells stably expressing either a GFFKR-deleted alpha(V(del))beta(3) or a FF to AA-substituted alpha(V(AA))beta(3) do not reveal a constitutively activated integrin. Adhesion to immobilized fibrinogen is strongly impaired in alpha(V(del))beta(3) or alpha(V(AA))beta(3)-expressing cells, whereas it is not impaired in alpha(IIb)beta(3) and alpha(M)beta(2), both lacking the GFFKR-region. In a parallel plate flow chamber assay, alpha(V)beta(3)-expressing cells adhere firmly to fibrinogen and spread even at shear rates of 15 to 20 dyn/cm(2), whereas alpha(V(del))beta(3) or alpha(V(AA))beta(3) cells are detached at 15 dyn/cm(2). Actin stress fiber formation and focal adhesion plaques containing alpha(V)beta(3) are observed in alpha(V)beta(3) cells but not in alpha(V(del))beta(3) or alpha(V(AA))beta(3)-expressing cells. As an additional manifestation of impaired outside-in signaling, phosphorylation of pp125(FAK) was reduced in these cells. In summary, we report that the GFFKR-region of the alpha(V)-cytoplasmic domain and in particular two phenylalanines are essential for integrin alpha(V)beta(3) function, especially for outside-in signaling. Our results suggest that the two beta(3)-integrins alpha(IIb)beta(3) and alpha(V)beta(3) are differentially regulated via their GFFKR-region.     

Interplay between PEO tether length and ligand spacing governs cell spreading on RGD-modified PMMA-g-PEO comb copolymers

The effects of tether length on cell adhesion to poly(methyl methacrylate)-graft-poly(ethylene oxide), PMMA-g-PEO, comb copolymer films functionalized with the adhesion peptide RGD were investigated. Copolymers having PEO tether lengths of 10 and 22 EO segments were synthesized and coupled with a synthetic peptide that contained both RGD and the synergy sequence PHSRN. Cell spreading assays revealed that the longer polymer tethers increased the rate of spreading and reduced the time required for fibroblasts to form focal adhesions. Fluorescence resonance energy transfer (FRET) measurements indicated a mean separation between integrin-bound peptides of 15.6 +/- 1.4 nm for combs with long (22-mer) tethers, compared with 17.5 +/- 1.3 nm for short (10-mer) tethers, on films of comparable peptide density (approximately 2500 peptides/microm2). The results suggest that the added mobility afforded by the more extensible tethers encouraged the formation of focal adhesions by allowing cells to reorganize tethered peptides on the nanometer length scale. In addition, adhesion peptides were selectively coupled to 10-mer or 22-mer PEO tethers within a bimodal brush to investigate stratification effects on cell adhesion. Peptides bound by short tethers in a bed of long unsubstituted chains resulted in surfaces that resisted, rather than promoted, cell adhesion. By contrast, when long peptide tethers were employed with short unsubstituted chains, cell attachment and spreading were comparable to that found on a monomodal brush of long chains at equivalent peptide density.     

An RGD sequence in the P2Y(2) receptor interacts with alpha(V)beta(3) integrins and is required for G(o)-mediated signal transduction

The P2Y(2) nucleotide receptor (P2Y(2)R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein-coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y(2)R to K562 erythroleukemia cells was inhibited by antibodies against alpha(V)beta(3)/beta(5) integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y(2)Rs indicated that alpha(V) integrins colocalized 10-fold better with the wild-type P2Y(2)R than with a mutant P2Y(2)R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y(2)R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal-regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca(2+). Furthermore, an anti-alpha(V) integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y(2)R. Pertussis toxin, an inhibitor of G(i/o) proteins, partially inhibited Ca(2+) mobilization mediated by the wild-type P2Y(2)R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y(2)R-mediated activation of G(o), but not G(q). Since CD47 has been shown to associate directly with G(i/o) family proteins, these results suggest that interactions between P2Y(2)Rs, integrins, and CD47 may be important for coupling the P2Y(2)R to G(o).     

An Arg-Gly-Asp sequence within thrombin promotes endothelial cell adhesion

Thrombin, in addition to its central role in hemostasis, possesses diverse cellular bioregulatory functions implicated in wound healing, inflammation, and atherosclerosis. In the present study we demonstrate that thrombin molecules modified either at the procoagulant or catalytic sites induce endothelial cell (EC) adhesion, spreading, and cytoskeletal reorganization. The most potent adhesive thrombin analogue (NO2-alpha-thrombin) was obtained by nitration of tyrosine residues. The cell adhesion promoting activity of NO2-alpha-thrombin was blocked upon the formation of thrombin-antithrombin III (ATIII) complexes and by antiprothrombin antibodies, but was unaffected by hirudin. Arg-Gly-Asp-containing peptides, fully inhibited EC adhesion to NO2-alpha-thrombin, while synthetic peptides corresponding to thrombin "Loop B" mitogenic site and the thrombin-derived chemotactic fragment "CB67-129", were uneffective. Immunofluorescence studies indicated that EC adhesion to NO2-alpha-thrombin was followed by cell spreading, actin microfilament assembly, and formation of focal contacts. By the use of specific antibodies, the vitronectin (vn) receptor (alpha v beta 3) was found to be localized in clusters upon cell adhesion to NO2-alpha-thrombin. An anti alpha v beta 3 antibody blocked EC adhesion and spreading while antifibronectin (fn) receptor (alpha 5 beta 1) antibodies were uneffective. While native thrombin exhibited a very low cell attachment activity, thrombin that was incubated at 37 degrees C before coating of plastic surfaces induced EC attachment and spreading. We propose that under certain conditions the naturally hindered RGD domain within thrombin is exposed for interaction with alpha v beta 3 on EC. This in turn promotes cell adhesion, spreading, and reorganization of cytoskeletal elements, which may altogether contribute to repair mechanisms in the disturbed vessel wall. This study defines a new biological role of thrombin and characterizes a new recognition mechanism on EC for this molecule.     

The relative importance of topography and RGD ligand density for endothelial cell adhesion

The morphology and function of endothelial cells depends on the physical and chemical characteristics of the extracellular environment. Here, we designed silicon surfaces on which topographical features and surface densities of the integrin binding peptide arginine-glycine-aspartic acid (RGD) could be independently controlled. We used these surfaces to investigate the relative importance of the surface chemistry of ligand presentation versus surface topography in endothelial cell adhesion. We compared cell adhesion, spreading and migration on surfaces with nano- to micro-scaled pyramids and average densities of 6×10(2)-6×10(11) RGD/mm(2). We found that fewer cells adhered onto rough than flat surfaces and that the optimal average RGD density for cell adhesion was 6×10(5) RGD/mm(2) on flat surfaces and substrata with nano-scaled roughness. Only on surfaces with micro-scaled pyramids did the topography hinder cell migration and a lower average RGD density was optimal for adhesion. In contrast, cell spreading was greatest on surfaces with 6×10(8) RGD/mm(2) irrespectively of presence of feature and their size. In summary, our data suggest that the size of pyramids predominately control the number of endothelial cells that adhere to the substratum but the average RGD density governs the degree of cell spreading and length of focal adhesion within adherent cells. The data points towards a two-step model of cell adhesion: the initial contact of cells with a substratum may be guided by the topography while the engagement of cell surface receptors is predominately controlled by the surface chemistry.     

Epiligrin, a new cell adhesion ligand for integrin alpha 3 beta 1 in epithelial basement membranes

Epiligrin is a new glycoprotein in most epithelial basement membranes (BMs) and is a ligand for cell adhesion via integrin alpha 3 beta 1. In the extracellular matrix of human foreskin keratinocytes (HFKs), epiligrin contains three disulfide-bonded, glycoprotein subunits, E170, E145, and E135, based on molecular size in kilodaltons. Epiligrin, immunopurified with MAb P1E1, induced cell adhesion and localization of integrin alpha 3 beta 1 in focal adhesions (FAs). Cell adhesion to epiligrin was inhibited with an anti-alpha 3 beta 1 MAb. Epiligrin also colocalized with integrin alpha 6 beta 4 in hemidesmosome-like stable anchoring contacts (SACs). alpha 3 beta 1-FAs encircled alpha 6 beta 4-SACs in a complex adhesion structure. alpha 3 beta 1 and epiligrin localized in BM junctions of epithelial cells primarily in organs of endodermal/ectodermal origin. In epidermis, epiligrin was detected in the lamina lucida of BMs. alpha 3 beta 1 localized in plasma membranes of basal cells in contact with epiligrin and also in lateral/apical membranes. Epiligrin is the ligand of an adhesion super complex composed of alpha 3 beta 1-FAs and alpha 6 beta 4-SACs (hemidesmosomes).     

Outside-in regulation of integrin clustering processes by ECM components per se and their involvement in actin cytoskeleton organization in a colon adenocarcinoma cell line

We investigated in a colon adenocarcinoma cell line, the exclusive role of extracellular matrix (ECM) components in the absence of soluble factors regarding the integrin clustering processes, and their implication in cell adhesion, spreading and organization of the actin cytoskeleton. Caco-2 cells were shown to express at the plasma membrane 11 integrins, some of which (e.g. alpha3beta1, alpha5beta1, alpha6beta1/beta4, alpha8beta1 and alpha(v)beta1/beta5/beta6) were identified for the first time in this cell line. Cell adhesion and spreading processes were governed essentially by lamellipodium, the regulation of which was shown to be induced by two types of integrin clustering processes mediated by ECM proteins alone. During these phenomena, alpha2beta1, alpha(v)beta6 and alpha6beta1 integrins, the Caco-2 cell specific receptors of type IV collagen, fibronectin and laminin, respectively, were clustered in small focal complexes (point contacts), whereas alpha(v)beta5, the vitronectin receptor in this cell line, was aggregated in focal adhesions. The two levels of integrin clustering induced only F-actin cortical web formation organized in thin radial and/or circular filaments. We conclude thus that ECM components per se through their action on integrin clustering are involved in cell adhesion, cortical actin cytoskeleton organization and cell spreading.     

The glycobiology of gametes and fertilization

In previous studies, RGD-CAP (collagen-associated protein containing the RGD sequence) isolated from a collagen fiber-rich fraction of pig cartilage was found to be orthologous to human (beta)ig-h3, which is synthesized by lung adenocarcinoma cells in response to transforming growth factor-beta. In the present study, we examined the effect of recombinant chick RGD-CAP on the spreading of chondrocytes and fibroblasts using RGD-CAP-coated dishes. When rabbit articular chondrocytes, chick embryonic sternal chondrocytes, rabbit peritoneal fibroblasts or human MRC5 fibroblasts were seeded on plastic dishes coated with RGD-CAP, cell spreading was enhanced compared with that on control dishes (bovine serum albumin- or beta-galactosidase-coated dishes). The effect of RGD-CAP on the cell spreading required divalent cations (Mg(2+) or Mn(2+)), and was reduced by EDTA. Monoclonal antibodies (mAbs) to the human integrin alpha(1) or beta(1) subunit, but not to the alpha(2), alpha(3), alpha(5) or beta(2) subunits, suppressed the RGD-CAP-induced spreading of human MRC5 fibroblasts. In a parallel experiment, the mAb to the alpha(5) subunit, but not the mAb to the alpha(1) subunit, suppressed fibronectin-induced spreading of these cells. These findings suggest that RGD-CAP is a novel ligand for integrin alpha(1)beta(1) that dose not bind to the RGD motif. Accordingly, an RGD-CAP fragment, which carries a deletion in the C-terminal region containing the RGD motif, was still capable of stimulating cell spreading.     

Ligation of integrin alpha 3beta 1 by laminin 5 at the wound edge activates Rho-dependent adhesion of leading keratinocytes on collagen

Wounding of the epidermis signals the transition of keratinocytes from quiescent anchorage on endogenous basement membrane laminin 5 to migration on exposed dermal collagen. In this study, we attempt to characterize activation signals that transform quiescent keratinocytes into migratory leading cells at the wound edge. Previously, we reported that adhesion and spreading on collagen via integrin alpha(2)beta(1) by cultured human foreskin keratinocytes (HFKs) requires RhoGTP, a regulator of actin stress fibers. In contrast, adhesion and spreading on laminin 5 requires integrins alpha(3)beta(1) and alpha(6)beta(4) and is dependent on phosphoinositide 3-hydroxykinase (Nguyen, B. P., Gil, S. G., and Carter, W. G. (2000) J. Biol. Chem. 275, 31896-31907). Here, we report that quiescent HFKs do not adhere to collagen but adhere and spread on laminin 5. By using collagen adhesion as one criterion for conversion to a "leading wound cell," we found that activation of collagen adhesion requires elevation of RhoGTP. Adhesion of quiescent HFKs to laminin 5 via integrin alpha(3)beta(1) and alpha(6)beta(4) is sufficient to increase levels of RhoGTP required for adhesion and spreading on collagen. Consistently, adhesion of quiescent HFKs to laminin 5, but not collagen, also promotes expression of the precursor form of laminin 5, a characteristic of leading keratinocytes in the epidermal outgrowth. We suggest that wounding of quiescent epidermis initiates adhesion and spreading of keratinocytes at the wound edge on endogenous basement membrane laminin 5 via alpha(3)beta(1) and alpha(6)beta(4) in a Rho-independent mechanism. Spreading on endogenous laminin 5 via alpha(3)beta(1) is necessary but not sufficient to elevate expression of precursor laminin 5 and RhoGTP, allowing for subsequent collagen adhesion via alpha(2)beta(1), all characteristics of leading keratinocytes in the epidermal outgrowth.     

Distinct biological consequences of integrin alpha v beta 3-mediated melanoma cell adhesion to fibrinogen and its plasmic fragments.

Fibrinogen/fibrin and its proteolytic fragments serve as potential adhesive substrates during thrombosis, wound healing, and cancer. In this report we examined the biological response of human melanoma cells exposed to fibrinogen and its naturally occurring plasmic breakdown products that are known constituents of the tumor stroma. Plasmin treatment of fibrinogen first results in fragment X, which is characterized by removal of the COOH-terminal portion of the alpha chain including an RGD sequence (A alpha 572-575). Further digestion leads to fragment D comprising primarily an intact COOH-terminal stretch of the gamma chain containing the platelet adhesion sequence HHLGGAKQAGDV. In a sensitive adhesion assay M21 human melanoma cells utilized integrin alpha v beta 3 to attach to all three of these ligands. However, only intact fibrinogen promoted significant cell spreading, while fragment X produced minimal spreading and fragment D promoted only adhesion. These results indicate that fibrinogen contains at least two alpha v beta 3-dependent adhesive sites and these promote distinct biological responses of human melanoma cells. The differential functional properties of these ligands directly correlate to their relative binding affinity for purified alpha v beta 3 as measured in a solid-phase receptor binding assay. These results provide evidence that a single integrin can promote distinct biological signals depending on the molecular nature of the ligand binding event.