Detachment of cells from culture substrate by soluble fibronectin peptides

The synthetic cell attachment-promoting peptides from fibronectin (Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.)., 309:30-33) were found to detach cultured cells from the substratum when added to the culture in a soluble form. Peptides ranging in length from tetrapeptide to heptapeptide and containing the active L-arginyl-glycyl-L-aspartic acid (Arg-Gly-Asp) sequence had the detaching activity, whereas a series of different peptides with chemically similar structures had no detectable effect on any of the test cells. The Arg-Gly-Asp-containing peptides caused detachment of various cell lines of different species and histogenetic origin. Studies with defined substrates showed that the active peptides could inhibit the attachment of cells to vitronectin in addition to fibronectin, indicating that vitronectin is recognized by cells through a similar mechanism as fibronectin. The peptides did not inhibit the attachment of cells to collagen. However, cells cultured on collagen-coated plastic for 24-36 h, as well as cells with demonstrable type I or type VI collagen in their matrix, were susceptible to the detaching effect of the peptides. These results indicate that the recognition mechanism(s) by which cells bind to fibronectinand vitronectin plays a major role in the substratum attachment of cells and that collagens may not be directly involved in cell-substratum adhesion. Since vitronectin is abundant in serum, it is probably an important component in mediating the attachment of cultured cells. The independence of the effects of the peptide on the presence of serum and the susceptibility of many different cell types to detachment by the peptide show that the peptides perturb an attachment mechanism that is intrinsic to the cells and fundamentally significant to their adhesion.     

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

The interaction of cells with extracellular matrix components such as fibronectin, vitronectin, and type I collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) amino acid sequence within each protein. Synthetic peptides containing the RGD sequence can inhibit these receptor-ligand interactions. Here, we use novel RGD-containing synthetic peptides with different inhibition properties to investigate the role of the various RGD receptors in tumor cell invasion. The RGD-containing peptides used include peptides that inhibit the attachment of cells to fibronectin and vitronectin, a peptide that inhibits attachment to fibronectin but not to vitronectin, a cyclic peptide with the opposite specificity, and a peptide, GRGDTP, that inhibits attachment to type I collagen in addition to inhibiting attachment to fibronectin and vitronectin. The penetration of two human melanoma cell lines and a glioblastoma cell line through the human amniotic basement membrane and its underlying stroma was inhibited by all of the RGD-containing peptides except for the one that inhibits only the vitronectin attachment. Various control peptides lacking RGD showed essentially no inhibition. This inhibitory effect on cell invasion was dose-dependent and nontoxic. A hexapeptide, GRGDTP, that inhibits the attachment of cells to type I collagen in addition to inhibiting fibronectin- and vitronectin-mediated attachment was more inhibitory than those RGD peptides that inhibit only fibronectin and vitronectin attachment. Analysis of the location of these cells that were prevented from invading indicated that they attached to the amniotic basement membrane but did not proceed further into the tissue. These results suggest that interactions between RGD-containing extracellular matrix adhesion proteins and cells are necessary for cell invasion through tissues and that fibronectin and type I collagen are important for this process.     

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

Extracellular matrix fibronectin fibrils serve as passive structural supports for the organization of cells into tissues, yet can also actively stimulate a variety of cell and tissue functions, including cell proliferation. Factors that control and coordinate the functional activities of fibronectin fibrils are not known. Here, we compared effects of cell adhesion to vitronectin versus type I collagen on the assembly of and response to, extracellular matrix fibronectin fibrils. The amount of insoluble fibronectin matrix fibrils assembled by fibronectin-null mouse embryonic fibroblasts adherent to collagen- or vitronectin-coated substrates was not significantly different 20 h after fibronectin addition. However, the fibronectin matrix produced by vitronectin-adherent cells was ~ 10-fold less effective at enhancing cell proliferation than that of collagen-adherent cells. Increasing insoluble fibronectin levels with the fibronectin fragment, anastellin did not increase cell proliferation. Rather, native fibronectin fibrils polymerized by collagen- and vitronectin-adherent cells exhibited conformational differences in the growth-promoting, III-1 region of fibronectin, with collagen-adherent cells producing fibronectin fibrils in a more extended conformation. Fibronectin matrix assembly on either substrate was mediated by α5β1 integrins. However, on vitronectin-adherent cells, α5β1 integrins functioned in a lower activation state, characterized by reduced 9EG7 binding and decreased talin association. The inhibitory effect of vitronectin on fibronectin-mediated cell proliferation was localized to the cell-binding domain, but was not a general property of αvβ3 integrin-binding substrates. These data suggest that adhesion to vitronectin allows for the uncoupling of fibronectin fibril formation from downstream signaling events by reducing α5β1 integrin activation and fibronectin fibril extension.     

Fibronectin associated with the glial component of embryonic brain cell cultures

In a basic approach to investigations of neuronal–glial interactions during both normal brain development and its pathogenesis, embryonic brain cell populations were fractionated into purified neuronal and glial components. Using separation procedures based on differential adhesion and cytotoxicity, the isolated neuronal and glial phenotypes could be identified by distinct morphological and biochemical characteristics, including the visualization of glial fibrillary acid protein (GFA) within glial cells in immunohistochemical assays with monospecific anti-GFA serum. When unfractionated cerebrum cells dissociated from 10-day chick or 14-day mouse embryos were plated as monolayers and cultured for 1-14 days, monospecific antiserum against fibronectin (LETS glycoprotein) was found to react with many, but not all, of the cells as revealed by indirect immunofluorescence microscopy. The isolated neuronal and glial components of these populations were used to determine whether the appearance of membrane-associated fibronectin was characteristic of one cell type or the other, or both, and if neuronal–glial cell interaction was required for its expression. It was found that the surfaces of glial cells, completely isolated from neurons, showed an intense fluorescent reaction to the anti-fibronectin serum. In contrast, the purified neuronal cultures showed no fluorescence with either the anti-GFA or anti-fibronectin sera. These results demonstrate fibronectin as a cell surface protein associated primarily with glial cells and independent of neuronal–glial cell interaction for its expression. Furthermore, the results indicate that the fibronectin observed on glial cell surfaces in these cultures is produced endogenously and is not due to the preferential binding of fibronectin present in the culture medium. The role of fibronectin as an adhesive molecule in neuronal–glial interactions is discussed.     

IL-3-dependent mast cells attach to plate-bound vitronectin. Demonstration of augmented proliferation in response to signals transduced via cell surface vitronectin receptors.

The adhesive interactions of activated mast cells with the extracellular matrix play an important role in anchorage and cellular motility. In this report we demonstrate that IL-3-dependent bone marrow-derived mast cells adhere to plate-bound vitronectin with high affinity in a saturable and dose-dependent manner. This adhesion interaction is unique in that it does not require prior mast cell activation through Fc epsilon RI or after treatment with PMA. It is inhibited by divalent cation chelation and by competitive inhibition with a synthetic Arginine-Glycine-Aspartate-Serine tetrapeptide. Polyclonal antisera for alpha v beta 3, an integrin known to bind vitronectin, inhibits attachment to plate-bound vitronectin in a dose-dependent manner. Comparison of the adhesion interactions for vitronectin, fibronectin, and laminin indicate that adhesion to vitronectin is greater than that seen with either fibronectin or laminin, either in the presence or absence of PMA. FACS analysis using a monoclonal hamster anti-murine vitronectin receptor (alpha v) antibody followed by a fluorescein-conjugated rabbit anti-hamster IgG revealed no change in surface vitronectin receptor expression after Fc epsilon RI-mediated cell activation. Proliferation assays with correction for cell viability revealed a 25% increase in cell number above the maximal IL-3 response over a 24-h period of adhesion to a vitronectin-coated surface and a 41% increase over 96 h of adhesion to vitronectin. Binding to plate-bound vitronectin was not able to sustain cell viability in the absence of IL-3. Thus, IL-3-dependent bone marrow-derived mast cells adhere to vitronectin, an extracellular matrix protein present throughout connective tissues. This interaction generates a signal that results in the augmentation of the maximal IL-3-dependent mast cell proliferative response, thus demonstrating at least one way in which the interaction between mast cells and extracellular matrix alter the biologic responsiveness of the mast cell.     

The vitronectin receptor alpha v beta 3 binds fibronectin and acts in concert with alpha 5 beta 1 in promoting cellular attachment and spreading on fibronectin

The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily of adhesive protein receptors that mediate a wide spectrum of adhesive cellular interactions, including attachment to vitronectin, von Willebrand factor, fibrinogen, and thrombospondin. We have studied the binding of fibronectin to the purified vitronectin receptor, and the role of this receptor in the attachment of cells to fibronectin. A solid-phase microtiter assay was developed to investigate the binding properties of the vitronectin receptor. Purified alpha v beta 3 bound fibronectin with high affinity in a saturable, divalent cation- dependent manner. Binding was inhibited by soluble vitronectin, by RGD- containing peptides, and by LM609, a monoclonal antibody against the vitronectin receptor known to inhibit the binding of adhesive proteins to alpha v beta 3. Immunoinhibition experiments showed that M21 human melanoma cells, which express the fibronectin receptor, alpha 5 beta 1, as well as alpha v beta 3, used both of these integrins to attach and spread on fibronectin. In support of this finding, M21-L cells, a variant cell line that specifically lacks alpha v beta 3 but expresses alpha v beta 1, attached and spread poorly on fibronectin. In addition, alpha v beta 3 from surface-labeled M21 cells was retained, and selectively eluted by RGDS from a fibronectin affinity column. These results indicate that alpha v beta 3 acts in concert with alpha 5 beta 1 in promoting fibronectin recognition by these cells. We conclude that fibronectin binds to the alpha v beta 3 vitronectin receptor specifically and with high affinity, and that this interaction is biologically relevant in supporting cell adhesion to matrix proteins.     

Human endothelial cells grow poorly on vitronectin: role of PAI-1

The cell adhesive protein vitronectin is a common component of interstitial extracellular matrix and circulates in plasma. It competes effectively with other plasma proteins to adsorb to certain biomaterial surfaces, and is likely to represent an important cell adhesion mediator on the luminal surface of vascular grafts. It is also found associated with certain vascular pathologies. We have shown previously that human endothelial cells grow poorly on a vitronectin surface compared with other extracellular matrix molecules. In this paper we show that endothelial cells seeded on vitronectin and fibronectin produced substantially different profiles of extracellular matrix molecules. The most outstanding difference was in the amount of matrix-localised plasminogen activator-inhibitor-1 which was high on vitronectin and negligible on fibronectin. This was correlated with a small but significant inhibition of cell adhesion to vitronectin compared with fibronectin, and very significant interference with dissociation of cell: extracellular matrix contacts, resulting either from direct inhibition of the proteolytic activity of urokinase, or from interference with urokinase-receptor signaling and consequent focal adhesion turnover. Such interference would inhibit cell proliferation by disabling the cells from loosening their matrix contacts in order to proceed through mitosis. This would seriously compromise endothelial recovery in cases of damage to the vascular wall and placement of stents or grafts, where the presence of surface-adsorbed vitronectin is likely to modulate the tissue response.     

Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells

The biomimetic approach of tissue engineering exploits the favorable properties of the extracellular matrix (ECM), to achieve better scaffold performance and tissue regeneration. ECM proteins regulate cell adhesion and differentiation through integrin mediated signal transduction. In the present study, we have examined the role of ECM proteins such as collagen type I, fibronectin, laminin and vitronectin in regulating the proliferation and osteogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs were grown on selected ECM protein treated tissue culture plates. The growth kinetics was assessed by calculating the doubling time of the cells on different ECM treated plates. The cells were directed to osteoblast lineage by growing them in osteogenic induction media for 21 day. Differentiation was evaluated at different time points by osteoblast differentiation associated gene expression, alkaline phosphatase (ALP) activity, histochemical staining for mineralized matrix and calcium quantification. The doubling time of hMSCs cultured on collagen type I was significantly low, which was followed by laminin and fibronectin treated plates. However, doubling time of hMSCs cultured on vitronectin treated plate was not significantly different than that of the untreated control. High ALP gene (ALPL) expression and associated enhancement of mineralization were observed on collagen type I, fibronectin and vitronectin treated plates. Collagen type I showed early onset of mineralization with high ALP activity and up-regulation of osteopontin, ALPL, bone sialoprotein and osteocalcin genes. Vitronectin also up-regulated these genes and showed the highest amount of calcium in the secreted mineral matrix. Therefore, we conclude that, ECM proteins indeed modified the growth patterns and induced the osteoblast differentiation of hMSCs. Our findings have significant implication for bone tissue engineering applications.     

c-Src-dependent cross-talk between CEACAM6 and alphavbeta3 integrin enhances pancreatic adenocarcinoma cell adhesion to extracellular matrix components

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an immunoglobulin superfamily member with a diversity of extracellular ligands that is implicated in the initiation and progression of a variety of malignancies. We sought to characterize the effects of CEACAM6 crosslinking on pancreatic adenocarcinoma cellular interaction with the extracellular matrix (ECM) components fibronectin and vitronectin. Antibody-mediated CEACAM6 crosslinking was performed and the ability of BxPC3 cells, which inherently overexpress CEACAM6, to adhere to fibronectin and vitronectin was quantified. The roles of the archetypal fibronectin (alpha5beta1 integrin) and vitronectin (alphavbeta3 integrin) receptors were determined. The effects of c-Src inhibition were investigated using the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and c-Src specific RNA interference. CEACAM6 crosslinking initiates c-Src-dependent cross-talk between CEACAM6 and alphavbeta3 integrin, leading to increased ECM component adhesion. CEACAM6-mediated signaling events may contribute to the invasive and metastatic potential of pancreatic adenocarcinoma cells by promoting their interaction with ECM components.     

Type I collagen preparations inhibit DNA synthesis in glial cells of the peripheral nervous system

The mechanisms underlying cessation of glial proliferation in the developing peripheral nervous system are obscure. One possibility, as yet little explored, is that mitotic inhibitory signals play a part in regulating glial cell numbers. In this study we demonstrate that type I collagen preparations from several different sources can inhibit the rate of DNA synthesis in purified populations of enteric glia and both short-term and long-term secondary Schwann cells in dissociated cell cultures. When these cells are grown on gelled or dried type I collagen substrata, they proliferate at substantially lower rates than on polylysine substrata. In contrast, type III or V collagen preparations do not inhibit glial DNA synthesis and laminin, fibronectin, type IV collagen, and secreted matrix from bovine corneal endothelial cells all stimulate thymidine incorporation. The inhibitory effect is not observed with heat denatured type I collagen preparations, but is seen equally in serum-containing medium, in medium containing fibronectin-free serum, or in serum-free medium, suggesting that the interaction of collagen with the cells requires structurally intact collagen molecules and does not occur via intermediary linkage to fibronectin. The inhibition on collagen is accompanied by a shape change from a more flattened morphology to a narrow spindle form. The labeling index of a rat Schwannoma cell line, 33B, is not inhibited on type I collagen substrata. These results demonstrate that type I collagen preparations inhibit the DNA synthesis levels of early postnatal peripheral glial cells in vitro. It remains to be determined whether this effect occurs via direct collagen-cell membrane interactions or whether it depends on accessory molecules, perhaps present in the collagen preparations themselves, since these are not purified to absolute homogeneity.     

Role of hypoxia and extracellular matrix-integrin binding in the modulation of angiogenic growth factors secretion by retinal pigmented epithelial cells.

The retinal pigmented epithelium (RPE) is a monolayer of polarized cells located between retinal photoreceptors and blood vessels of the choroid. The basal surface of RPE cells rests on Bruch's membrane, a complex extracellular matrix structure which becomes abnormal in several disease processes, including age-related macular degeneration (AMD). Ruptures or abnormalities in Bruch's membrane are frequently accompanied by choroidal neovascularization. Disturbed interaction of RPE cells with their extracellular matrix (ECM) could play a role in this process. The present study was undertaken to examine the complex interactions between hypoxia, integrin, and ECM in the regulation of RPE functions. Antibody blocking experiments demonstrated that RPE cell adhesion to vitronectin is mediated primarily through alphavbeta5 and adhesion to fibronectin occurs through alpha5beta1. RPE adhesion to immobilized laminin demonstrated highest level of non-RGD-mediated adhesion as compared to that with collagen IV or the RGD matrices such as vitronectin (alphavalpha5) , fibronectin (alpha5beta1), or thrombospondin (alpha5beta1 + alphavbeta5). Addition of soluble vitronectin, or fibrinogen to RPE cell cultures resulted in a small to moderate increase in VEGF and FGF2 in the media, while each of these growth factors was dramatically increased after addition of thrombospondin 1 (TSP1). In contrast, soluble fibronectin resulted in differential upregulation of VEGF but not FGF2. Similarly, immobilized TSP1 resulted in differential greater upregulation in VEGF but not FGF2 release from RPE as compared to other ECMs under either normoxic or hypoxic conditions. Additionally, hypoxia resulted in a time-dependent increase in VEGF, but not FGF2 release in the media. RPE cells grown on TSP1-coated plates showed increased VEGF and FGF2 in their media compared to cells grown on plates coated with type IV collagen, laminin, vitronectin, or fibronectin. The TSP1-induced increase in secretion of growth factors was partially blocked by anti-alpha5beta1, anti-alphavbeta3, and anti-alphavbeta5 antibodies indicating that it may be mediated in part by TSP1 binding to those integrins. These data suggest that alterations in oxygen levels (hypoxia/ischemia) and ECM of RPE cells, a prominent feature of AMD, can cause increased secretion of angiogenic growth factors that might contribute to the development of choroidal neovascularization. These data also suggest the potential modulatory role of VEGF release from RPE by ECM and alphavbeta5 and alpha5beta1 integrins.     

A novel vitronectin receptor integrin (alpha v beta x) is responsible for distinct adhesive properties of carcinoma cells

Carcinoma cells express a novel integrin involved in cell adhesion to vitronectin, but not to fibrinogen or von Willebrand factor, whereas melanoma and endothelial cells express a vitronectin receptor (alpha v beta 3) that promotes cell attachment to all of these matrix components. The integrin responsible for this adhesive phenotype of carcinoma cells is composed of an alpha subunit that is indistinguishable from the alpha v of the vitronectin receptor and a beta subunit (beta x) that is distinct from any known integrin beta subunit. Accordingly, Northern blot analysis identifies an mRNA for alpha v, but not for beta 3 in carcinoma cells. This receptor appears to mediate cell adhesion to vitronectin as well as fibronectin since an antibody directed to its alpha subunit blocked carcinoma cell adhesion to both of these matrix proteins. These results suggest that homologous integrins with identical alpha subunits and structurally distinct beta subunits can account for the functional recognition of different matrixes by two cell types.     

Adhesion of a chicken myeloblast cell line to fibrinogen and vitronectin through a beta 1-class integrin.

The adhesive interactions of circulating blood cells are tightly regulated, receptor-mediated events. To establish a model for studies on regulation of cell adhesion, we have examined the adhesive properties of the HD11 chick myeloblast cell line. Function-perturbing antibodies were used to show that integrins containing the beta 1 subunit mediate HD11 cell attachment to several distinct extracellular matrix proteins, specifically fibronectin, collagen, vitronectin, and fibrinogen. This is the first evidence that an integrin heterodimer in the beta 1 family functions as a receptor for fibrinogen. While the alpha v beta 1 heterodimer has been shown to function as a vitronectin receptor on some cells, this heterodimer could not be detected on HD11 cells. Instead, results suggest that the beta 1 subunit associates with different, unidentified alpha subunit(s) to form receptors for vitronectin and fibrinogen. Results using function-blocking antibodies also demonstrate that on these cells, additional receptors for vitronectin are formed by alpha v beta 3 and alpha v associated with an unidentified 100-kD beta subunit. The adhesive interactions of HD11 cells with these extracellular matrix ligands were shown to be regulated by lipopolysaccharide treatment, making the HD11 cell line attractive for studies of mechanisms regulating cell adhesion. In contrast to primary macrophage which rapidly exhibit enhanced adhesion to laminin and collagen upon activation, activated HD11 cells exhibited reduced adhesion to most extracellular matrix constituents.     

Galectin-8 functions as a matricellular modulator of cell adhesion

The interaction of cells with the extracellular matrix regulates cell adhesion and motility. Here we demonstrate that different cell types adhere and spread when cultured in serum-free medium on immobilized galectin-8, a mammalian beta-galactoside-binding protein. At maximal doses, galectin-8 is equipotent to fibronectin in promoting cell adhesion and spreading. Cell adhesion to immobilized galectin-8 is mediated by sugar-protein interactions with integrins, and galectin-8 triggers integrin-mediated signaling cascades including Tyr phosphorylation of focal adhesion kinase and paxillin. Cell adhesion is potentiated in the presence of Mn(2+), whereas it is interrupted in the presence of soluble galectin-8, integrin beta(1) inhibitory antibodies, EDTA, or thiodigalactoside but not by RGD peptides. Furthermore, cells readily adhere onto immobilized monoclonal galectin-8 antibodies, which are equipotent to integrin antibodies in promoting cell adhesion. Cell adhesion to immobilized galectin-8 is partially inhibited by serum proteins, suggesting that complex formation between immobilized galectin-8 and serum components generates a matrix that is less supportive of cell adhesion. Accordingly, cell motility on immobilized galectin-8 readily takes place in the presence of serum. Truncation of the C-terminal half of galectin-8, including one of its two carbohydrate recognition domains, largely abolishes its ability to modulate cell adhesion, indicating that both carbohydrate recognition domains are required to maintain a functional form of galectin-8. Collectively, our findings implicate galectin-8 as a physiological modulator of cell adhesion. When immobilized, it functions as a matrix protein equipotent to fibronectin in promoting cell adhesion by ligation and clustering of cell surface integrin receptors. In contrast, when present in excess as a soluble ligand, galectin-8 (like fibronectin) forms a complex with integrins that negatively regulates cell adhesion. Because of its dual effects on the adhesive properties of the cells and its association with fibronectin, galectin-8 might be considered a novel type of matricellular protein.     

Vitronectin regulates the synthesis and localization of urokinase-type plasminogen activator in HT-1080 cells.

The effect of extracellular matrix composition on the location, amount, and activity of cell-associated urokinase-type plasminogen activator was tested using HT-1080 cells adherent to either fibronectin or vitronectin. Specific immunoprecipitation of newly synthesized urokinase indicated that cells adherent to fibronectin synthesized 2-3-fold more urokinase than cells adherent to vitronectin. Complexes of urokinase and plasminogen activator inhibitor type 1 (PAI-1) were detected in cell layers of vitronectin-adherent but not fibronectin-adherent cells. Inhibition of PAI-1 using a neutralizing monoclonal antibody resulted in a 3-fold increase in urokinase enzymatic activity on vitronectin adherent cells. Urokinase activity on fibronectin adherent cells was only slightly increased following PAI-1 neutralization. Examination of both HT-1080 and normal human fibroblast cells by immunofluorescent microscopy localized urokinase-type plasminogen activator to discrete, focal areas underneath cells adherent to vitronectin. Urokinase was not detectable by immunofluorescence on cells adherent to fibronectin. The addition of exogenous prourokinase to locate urokinase receptors on adherent HT-1080 cells indicated that the focal localization of cell-surface urokinase resulted from the clustering of urokinase receptors following adhesion to vitronectin but not fibronectin-coated substrates. These results suggest that vitronectin can contribute to the control of cell-surface plasmin activity by regulating the synthesis of urokinase and directing the localization of urokinase receptors.     

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.     

Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization

We have developed two rat mAbs that recognize different subunits of the human fibroblast fibronectin receptor complex and have used them to probe the function of this cell surface heterodimer. mAb 13 recognizes the integrin class 1 beta polypeptide and mAb 16 recognizes the fibronectin receptor alpha polypeptide. We tested these mAbs for their inhibitory activities in cell adhesion, spreading, migration, and matrix assembly assays using WI38 human lung fibroblasts. mAb 13 inhibited the initial attachment as well as the spreading of WI38 cells on fibronectin and laminin substrates but not on vitronectin. Laminin-mediated adhesion was particularly sensitive to mAb 13. In contrast, mAb 16 inhibited initial cell attachment to fibronectin substrates but had no effect on attachment to either laminin or vitronectin substrates. When coated on plastic, both mAbs promoted WI38 cell spreading. However, mAb 13 (but not mAb 16) inhibited the radial outgrowth of cells from an explant on fibronectin substrates. mAb 16 also did not inhibit the motility of individual fibroblasts on fibronectin in low density culture and, in fact, substantially accelerated migration rates. In assays of the assembly of an extracellular fibronectin matrix by WI38 fibroblasts, both mAbs produced substantial inhibition in a concentration-dependent manner. The inhibition of matrix assembly resulted from impaired retention of fibronectin on the cell surface. Treatment of cells with mAb 16 also resulted in a striking redistribution of cell surface fibronectin receptors from a streak-like pattern to a relatively diffuse distribution. Concomitant morphological changes included decreases in thick microfilament bundle formation and reduced adhesive contacts of the streak-like and focal contact type. Our results indicate that the fibroblast fibronectin receptor (a) functions in initial fibroblast attachment and in certain types of adhesive contact, but not in the later steps of cell spreading; (b) is not required for fibroblast motility but instead retards migration; and (c) is critically involved in fibronectin retention and matrix assembly. These findings suggest a central role for the fibronectin receptor in regulating cell adhesion and migration.     

Interaction between collagens and glycosaminoglycans investigated using a surface plasmon resonance biosensor.

The interactions of glycosaminoglycans with collagens and other glycoproteins in extracellular matrix play important roles in cell adhesion and extracellular matrix assembly. In order to clarify the chemical bases for these interactions, glycosaminoglycan solutions were injected onto sensor surfaces on which collagens, fibronectin, laminin, and vitronectin were immobilized. Heparin bound to type V collagen, type IX collagen, fibronectin, laminin, and vitronectin; and chondroitin sulfate E bound to type II, type V, and type VII collagen. Heparin showed a higher affinity for type IX collagen than for type V collagen. On the other hand, chondroitin sulfate E showed the highest affinity for type V collagen. The binding of chondroitin sulfate E to type V collagen showed higher affinity than that of heparin to type V collagen. These data suggest that a novel characteristic sequence included in chondroitin sulfate E is involved in binding to type V collagen.     

Hepatocyte adhesion on plastic : Different mechanisms for serum- and fibronectin-mediated adhesion

Hepatocytes adhere well on plastic in the presence of serum or fibronectin and subsequent spreading is not prevented when protein synthesis was blocked by cycloheximide. Protein synthesis-independent spreading was also observed in cultures containing serum depleted of fibronectin by affinity chromatography. This indicates that serum-mediated adhesion is independent of fibronectin and suggests the existence of an adhesion factor other than fibronectin in serum. The involvement of different membrane components for fibronectin- and serum-mediated adhesion was demonstrated by experiments where the different adhesion-inhibiting activities of antisera raised against plasma membranes of rat liver and Morris hepatoma 7777 (Neumeier et al., FEBS lett 168 (1984) 241-244) were used. Whereas anti-liver antibodies inhibited both types of adhesion, anti-hepatoma antibodies were only able to prevent fibronectin-mediated adhesion. This indicates again that two different mechanisms are responsible for fibronectin- and serum-mediated adhesion. Fractionation of fetal calf serum (FCS) by size exclusion HPLC revealed that proteins of molecular weights of 60-80 kD promoted attachment and spreading of hepatocytes. Spreading was not perturbated by anti-hepatoma antibodies, indicating that an adhesion factor of 60-80 kD is responsible for serum-mediated adhesion. 'Serum-spreading factor', also called vitronectin, from human plasma has been described as having a similar molecular weight. The purified factor was found to mediate hepatocyte adhesion which was not inhibited by anti-hepatoma antibodies. This suggests that serum-mediated adhesion depends on an adhesion factor present in FCS, which is similar to or identical with vitronectin.     

Localization of urokinase type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors

Previous studies have shown that the adhesion protein, vitronectin, directs the localization of urokinase-type plasminogen activator (uPA) to areas of cell-substrate adhesion, where uPA is thought to regulate cell migration as well as pericellular proteolysis. In the present study, HT-1080 cell lines expressing either wild-type vitronectin or vitronectin containing a single amino-acid substitution in the integrin binding domain were used to assess whether ligation of the alphavbeta5 integrin was required for uPA localization to focal adhesions. The synthesis of wild-type vitronectin by HT-1080 cells adherent to either collagen or fibronectin resulted in the redistribution of both the alphavbeta5 integrin as well as uPA to focal adhesion structures. In contrast, cells synthesizing mutant vitronectin, containing the amino-acid substitution in the integrin binding domain, were unable to direct the redistribution of either alphavbeta5 or uPA to focal adhesions. Recombinant forms of wild-type and mutant vitronectin were prepared in a baculovirus system and compared for their ability to direct the redistribution of vitronectin integrin receptors as well as uPA on human skin fibroblasts. In the absence of vitronectin, fibroblast cells adherent to fibronectin assemble focal adhesions which contain the beta1 integrin but do not contain uPA. Addition of recombinant wild-type, but not mutant, vitronectin to fibroblasts adherent to fibronectin resulted in the redistribution of alphavbeta3, alphavbeta5, and uPA into focal adhesions. However, when cells were plated directly onto antibodies directed against either the alphavbeta3 or alphavbeta5 integrins, uPA was not localized on the cell surface. These data indicate that ligation of vitronectin integrin receptors is necessary but not sufficient for the localization of uPA to areas of cell matrix adhesion, and suggest that vitronectin may promote cell migration by recruiting vitronectin integrin receptors and components of the plasminogen activator system to areas of cell matrix contact.