Concanavalin A-induced impairment of fibroblast spreading on laminin but not on fibronectin

The effect of concanavalin A (Con A) on the adhesion of 8-day-old chick embryo fibroblasts (CEFs) to fibronectin (FN) and laminin (LM) was studied. Con A was shown to inhibit the spreading of CEF on a LM substrate. In contrast, no inhibition of CEF spreading on the FN substrate could be detected when the quantity of FN coated varied from 0.5 to 4 pmoles. The effect induced by Con A was specific, since it was abolished by 100 mM alpha-methylmannopyranoside. The inhibition of CEF spreading was only observed when the lectin was added during the 20 min following cell plating. In addition, the effect of Con A on CEF spreading on the LM substrate was shown to be dependent upon its presence at the cell surface, since under conditions which accelerate the uptake of the lectin, the effect on cell spreading is no longer detectable. Furthermore, the number of CEFs attached to LM was not modified by the lectin. The molecular weight of the isolated Con A binding sites revealed glycoproteins ranging from 30,000 to 72,000. On the other hand, these Con A binding sites did not interact with LM-Sepharose. Only a protein with a molecular weight of 68,000 which did not express affinity for Con A bound tightly to the LM-Sepharose. These data suggested that cell surface Con A binding sites do not interfere with the initial step of CEF adhesion to LM but play a key role during their spreading on this glycoprotein.     

A Mr 72K cell surface concanavalin A binding glycoprotein is specifically involved in the spreading of chick embryo fibroblasts onto laminin substrate

In the present study we have identified a 72-kDa cell surface concanavalin A binding glycoprotein (cbg 72) involved in the chick embryo fibroblast (CEF) adhesion onto laminin (LM) substrate. The cbg 72 was shown to interact specifically with immobilized laminin and to be resistant to Triton X-100 extraction when CEF were plated on laminin substrate but not on fibronectin (FN) substrate. This behavior suggested that cbg 72 could interact with cytoskeletal elements during cell spreading onto LM. This assumption is also in good agreement with the partitioning of cbg 72 in Triton X-114. Isolated cbg 72 specifically inhibited CEF spreading onto LM after their initial attachment, whereas cbg 72 did not impair the spreading of CEF onto FN. These data provide a molecular explanation to the inhibition of CEF spreading onto LM observed in the presence of the lectin concanavalin A (P. Codogno, M.-A. Doyennette-Moyne, J. Botti, and M. Aubery, 1988, J. Cell Physiol. 136, 463-470). Moreover, these results provide evidence for the role of a novel LM binding glycoprotein during the adhesion of mesenchymal derived cells. The relationship between cbg 72 and other known cell surface LM binding sites or receptors is discussed.     

Alterations of fibroblast interactions with fibronectin and laminin during chick embryo development

Eight day (8-d CEF) and 16 day old chick embryo fibroblasts (16-d CEF) obtained after a mild trypsin treatment (50 micrograms/ml in Ca2+ and Mg2+-free PBS, plus 10 mM EDTA) for 10 min at 37 degrees C present the same number of fibronectin (FN) binding sites at their surface (approximately 550,000 sites per cell) with a Kd approximately equal to 1.40 microM in both cases. Furthermore, FN interacted with high molecular weight plasma membrane proteins (150,000 and 125,000) insensitive to trypsin treatment. Both 8-d and 16-d CEF adhered and spread to the same extent on a fibronectin coated substratum (80% of the CEF adhered in 60 min). In contrast, 8-d and 16-d CEF behaved differently towards laminin (LM). 8-d CEF exhibited approximately 5500 binding sites per cell with a Kd of 1.5 nM (Codogno P., Doyennette, M.-A. and Aubery M., 1987, Experimental Cell Research, 169, 478-489.) and were highly sensitive to trypsin treatment, whereas 16-d CEF do not express cell surface binding sites for laminin. Differences were also observed in the adhesive capacities of 8-d and 16-d CEF on LM substrata: 8-d CEF adhered and spread on LM in a very specific manner (60% of the cells adhere in 60 min) and 16-d CEF did not adhere to LM even after long periods of incubation exceeding 360 min.     

Fibronectin biosynthesis: influence on fibroblast adhesion during chick embryo development

Eight d (8d) and 16d (16d) chick embryo fibroblasts (CEF) exhibited marked differences in their adhesive capacity on plastic support, but not on fibronectin substratum. This suggests differences in fibronectin (FN) expression and/or FN receptor expression. Both 8d and 16d CEF expressed an identical number of membrane receptors for FN with similar affinity. In contrast, the newly synthesized FN appeared de novo in 30 min in 8d CEF versus 60 min in 16d CEF. This difference is not due to a modification of the polypeptide chain biosynthetic rate. The FN synthesized in 8d CEF became insensitive to endo beta-N-acetyl-glucosaminidase H (endo H) treatment after 20 min, whereas it remained sensitive to endo H until 60 min in 16d CEF. Post-translational modifications of N-linked mannose-rich chains to complex type chain may account for the difference in the expression of cell surface FN and thus for the difference in cell adhesion capacity to plastic.     

Cell surface heparan sulfate mediates some adhesive responses to glycosaminoglycan-binding matrices, including fibronectin

Proteins with affinities for specific glycosaminoglycans (GAC's) were used as probes for testing the potential of cell surface GAG's to mediate cell adhesive responses to extracellular matrices (ECM). Plasma fibronectin (FN) and proteins that bind hyaluronate (cartilage proteo-glycan core and link proteins) or heparan sulfate (platelet factor 4 [PF4]) were adsorbed to inert substrata to evaluate attachment and spreading of several 3T3 cell lines. Cells failed to attach to hyaluronate-binding substrata. The rates of attachment on PF4 were identical to those on FN; however, PF4 stimulated formation of broad convex lamellae but not tapered cell processes fibers during the spreading response. PF4-mediated responses were blocked by treating the PF4-adsorbed substratum with heparin (but not chondroitin sulfate), or alternatively the cells with Flavobacter heparinum heparinase (but not chondroitinase ABC). Heparinase treatment did not inhibit cell attachment to FN but did inhibit spreading. Cells spread on PF4 or FN contained similar Ca2+-independent cell-substratum adhesions, as revealed by EGTA-mediated retraction of their substratum-bound processes. Microtubular networks reorganized in cells on PF4 but failed to extend into the broadly spread lamellae, where fine microfilament bundles had developed. Stress fibers, common on FN, failed to develop on PF4. These experiments indicate that (a) heparan sulfate proteoglycans are critical mediators of cell adhesion and heparan sulfate-dependent adhesion via PF4 is comparable in some, but not all, ways to FN-mediated adhesion, (b) the uncharacterized and heparan sulfate-independent "cell surface" receptor for FN permits some but not all aspects of adhesion, and (c) physiologically compatible and complete adhesion of fibroblasts requires binding of extracellular matrix FN to both the unidentified "cell surface" receptor and heparan sulfate proteoglycans.     

Eosinophil Adhesion and Maturation is Modulated by Laminin

Eosinophils (Eo) participate in the inflammatory response to parasites, allergins, toxins, and epitopes recognized by autoimmune antibodies. Nonetheless, little attention has heretofore been paid to the interactions of Eo with extracellular matrix (ECM) proteins during their migration through the subendothelial basement membrane and into the surrounding tissue. Therefore, we have studied the adhesion of Eo to specific ECM proteins and the effect of this adhesion on Eo viability and maturation. Control Eo (from normal donors) adhere no better to substrates coated with laminin (LM), fibronectin (FN), cytotactin (CT), or collagen types I or IV (Col IV) than they do to human serum albumin coated substrates. In contrast, Eo activated in vitro with IL-5 or in vivo in patients with eosinophilia bind well to LM, FN and Col IV. LM is by far the most avid ligand among these molecules. For example, 43% of input cells bind to a substrate bearing 200 fmol/cm² of LM; a similar level of adhesion to FN requires 30 times as much adsorbed protein. Antibody inhibition experiments suggest that the αβ1 integrin heterodimer is the predominant LM receptor on these cells. Flow cytometry showed similar levels of these subunits on control and activated Eo, suggesting that Eo adhesion to LM is not regulated simply by cell surface integrin concentration. The effects of ECM proteins on Eo behavior were also examined. A LM-coated substrate (with no added cytokine) was found to be almost as effective as IL-5 in maintaining Eo viability while an equally adhesive FN-coated substrate had much less effect. Normally, even in the presence of 10% serum, no Eo survive a 5-day incubation in vitro unless IL-3, IL-5, or GM-CSF is added to the medium. Conditions that inhibit adhesion to LM (anti-integrin antibodies in the medium or CT on the substrate) and certain anti-cy-tokine antibodies inhibited the promotion of Eo viability by LM. During incubation on LM, Eo become hypodense, as they do in the presence of IL-5, indicating that they have become activated. These observations suggest that the interactions of Eo and ECM proteins may be important both for their potential to direct Eo migration and for their ability to regulate Eo viability, cytokine production, and maturation.     

Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.     

IFN-gamma and transforming growth factor-beta 1 differently regulate fibronectin and laminin receptors of human differentiating monocytic cells.

The regulation of extracellular matrix (ECM) protein receptor expression was followed in the human promonocytic cell line U937 before and after stimulation either with PMA or various cytokines implicated in monocytopoiesis. On undifferentiated U937 cells, alpha-chains of very late Ag (VLA)-4, VLA-5, and VLA-6 were constitutively expressed whereas alpha-chains of VLA-2 (alpha 2) and vitronectin receptor (alpha V) were not. Maturation of U937 cells with PMA resulted in a marked decrease in alpha 4 expression (25% of control by day 5), and a small but significant increase in the expression of alpha 2 and alpha v over 4 days of stimulation. Unstimulated U937 cells attached to fibronectin (FN) but not to laminin (LM), collagens I/IV-coated surfaces. After PMA stimulation, U937 cells exhibited enhanced adherence on FN and expressed the ability to adhere to LM. PMA stimulation also promoted U937 spreading both on FN and LM. Adhesion on FN all along the maturation pathway was specifically and totally inhibited by anti-alpha 5 mAb but not by anti-alpha 4 mAb. Anti-beta 1, anti-alpha 6, anti-alpha 2, and anti-alpha v mAb, as well as Tyr-Ile-Gly-Ser-Arg and Arg-Gly-Asp synthetic peptides from LM, had no effect on adhesion of PMA-stimulated cells on LM, implying that U937 cell adherence to LM is mediated through hitherto distinct receptors. In the presence of rIFN-gamma, differentiating U937 cells did not adhere to LM and lost the capacity to bind to FN. Loss of adhesion to FN was correlated with the concomitant decrease in the expression of alpha 4 and alpha 5 integrin subunits. In contrast, TGF-beta 1 mimicked most of the effects of PMA by enhancing the attachment of maturating U937 cells on FN through alpha 5 receptors and by promoting adherence to LM. TGF-beta 1 stimulation also promoted U937 cell spreading on both FN- and LM-coated surfaces. The data suggest that inflammatory cytokines such as IFN-gamma and TGF-beta 1 may be critically important in the homing of monocytic cells at sites of inflammation by modulating cell-surface expression of ECM receptors.     

A comparison of fibronectin, laminin, and galactosyltransferase adhesion mechanisms during embryonic cardiac mesenchymal cell migration in vitro

Embryonic hearts contain a homogeneous population of mesenchymal cells which migrate through an extensive extracellular matrix (ECM) to become the earliest progenitors of the cardiac valves. Since these cells normally migrate through an ECM containing several adhesion substrates, this study was undertaken to examine and compare three ECM binding mechanisms for mesenchymal cell migration in an in vitro model. Receptor mechanisms for the ECM glycoproteins fibronectin (FN) and laminin (LM) and the cell surface receptor galactosyltransferase (GalTase), which binds an uncharacterized ECM substrate, were compared. Primary cardiac explants from stage 17 chick embryos were cultured on three-dimensional collagen gels. Mesenchymal cell outgrowth was recorded every 24 hr and is reported as a percentage of control. Migration was perturbed using specific inhibitors for each of the three receptor mechanisms. These included the hexapeptide GRGDSP (300-1000 micrograms/ml), which mimics a cell binding domain of FN, the pentapeptide YIGSR (300-1000 micrograms/ml), which mimics a binding domain of LM, and alpha-lactalbumin (1-10 mg/ml), a protein modifier of GalTase activity. The functional role of these adhesion mechanisms was further tested using antibodies to avian integrin (JG22) and avian GalTase. While the FN-related peptide had no significant effect on cell migration it did produce a rounded cellular morphology. The LN-related peptide inhibited mesenchymal migration 70% and alpha-lactalbumin inhibited cell migration 50%. Antibodies against integrin and GalTase inhibited mesenchymal cell migration by 80 and 50%, respectively. The substrate for GalTase was demonstrated to be a single high molecular weight substrate which was not LM or FN. Control peptides, proteins and antibodies demonstrated the specificity of these effects. These data demonstrate that multiple adhesion mechanisms, including cell surface GalTase, are potentially functional during cardiac mesenchymal cell migration. The sensitivity of cell migration to the various inhibitors suggests that occupancy of specific ECM receptors can modulate the activity of other, unrelated, ECM adhesion mechanisms utilized by these cells.     

Intracellular events are responsible for the differential expression of fibronectin on the fibroblast surface during chick embryo development

We previously showed that differences in the adhesive behaviour of fibroblasts obtained from 8-day-old (8-day CEF) and 16-day-old chick embryos (16-day CEF) were not due to alterations of cell surface fibronectin receptors. Herein we show that fibronectin (FN) was expressed more rapidly on the 8-day CEF surface (30 min) than on the 16-day CEF surface (60 min). In order to elucidate the mechanism responsible for these differences in the expression of cell surface FN we investigated the biosynthesis and the post-translational modifications of FN in 8- and 16-day CEF. Pulse-chase experiments revealed that FN was processed more slowly to an endo-beta-N-acetylglucosaminidase H (endo H)-resistant form in 16-day CEF than in 8-day CEF, whereas the kinetic of FN biosynthesis was similar in both cell populations. This difference was not related to a differential retention of FN in endoplasmic reticulum (ER) as determined after saponin-permeabilization. These results suggested that the rate-limiting step in the transport of FN to the cell surface in 16-day cells occurred between the ER and the medial part of the Golgi apparatus. It seems that the delay in the processing of endo H-resistant N-glycans was sufficient to account for differences between 8- and 16-day CEF in the rate of surface expression of FN and CEF adhesion to a plastic substratum.     

Embryonic and regenerating Xenopus retinal fibers are intrinsically different

Growth and guidance behavior of Xenopus embryonic (ER) (optic vesicle stage 25/26) and regenerating retinal fibers (stage 47/50 newly regenerating NR, and actively regenerating RR, respectively) have been studied in vitro on a variety of substrates in serum-free media. RR retinas receive a prior conditioning lesion 12-14 days before explantation while NR retinas are explanted immediately after axotomy. The substrates include plastic (UN), polylysine (PL), polyornithine (PO), laminin (LM), fibronectin (FN), and collagen type I (CO). Two kinds of experimental situations were tested, one in which substrates were derivatized to plastic as a planar surface, while the second involved the addition of a substrate as a soluble supplement to dishes derivatized with PL. A neurite growth index (NGI), based on density of neurite outgrowth and axon lengths, is determined for each fiber type on all substrates. Embryonic and regenerating fibers are phenotypically different fiber types; each displays a specific "substrate preference profile" (SPP), reflecting differential growth on each substrate. ER neurites grow equally well on all planar substrates, including plastic, but do not grow on CO (SPP, LM = FN = PL = PO = UN greater than CO). Both NR and RR neurites show distinct substrate preferences, but RR neurites grow more vigorously (SPP, LM greater than CO greater than PL = PO greater than FN). In media supplemented with LM, FN or CO, the SPPs showed little change but the neurite bundle patterns were qualitatively different. Only regenerating neurites display clockwise growth in laminin (LM) and fibronectin (FN)-supplemented media. Under no conditions do embryonic fibers exhibit this pattern which suggests that embryonic and regenerating retinal fibers also differ in cytoskeletal organization. Evidence of intrinsic growth differences in vitro suggest that embryonic and regenerating retinal fibers may not respond to identical guidance cues during in vivo development and regeneration of retinotectal connections.     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

The Effect of Extracellular Matrix Molecules on the in Vitro Behavior of Bovine Endothelial Cells

Extracellular matrix (ECM) is an important mediator of endothelial functions such as adhesion, spreading, migration, proliferation, and maintenance of differentiated functions. Attachment of cultured cells to tissue culture polystyrene (TCPS) is dependent on vitronectin which adsorbs onto the surface from the serum in the culture medium. Vitronectin (VN) will adsorb efficiently to TCPS even if the latter has been coated with another matrix molecule and blocked with albumin. This means that studies of the interactions of cells with individual coated ECM molecules will be confounded by the presence of adsorbed VN if serum is present in the culture medium. In this study, the adhesion, spreading, growth, and output of endogenous matrix molecules by bovine corneal endothelial (BCE) cells were measured on five different matrix substrates using medium which had been depleted of vitronectin to avoid such confounding effects. The same cell adhesion and spreading maxima were achieved on vitronectin, fibronectin (FN), laminin (LM), and types I and IV collagen (col I, col IV). The coating concentrations required to achieve these maxima, however, differed among the substrates, LM needing considerably higher concentrations than the other substrates for both maximal adhesion and spreading and FN needing higher concentrations for cell spreading. When cells were continuously passaged on each of the five substrates coated at concentrations optimal for cell spreading, no differences in cell proliferation rates or cell morphology were observed. Significant differences, however, were observed in the subcellular output of endogenous matrix molecules (FN, LM, col IV, and thrombospondin) between the different substrates. Col I was a poor substrate for the production of all ECM molecules tested over the 10 passages of the experiment, whereas col IV was a consistently good substrate. LM and FN substrates displayed differential effects on the output of different ECM molecules. VN was unique in that BCE cells at early passage on this substrate produced high levels of endogenous matrix molecules, whereas with continued passage on this substrate, a progressive decline in ECM secretion was observed. These results show that incorporation of individual molecules into the ECM by BCE cells in culture is significantly affected by the nature of the substratum. They further suggest that passage of endothelial cells in media containing serum (which results in coating of VN onto the substrate) may result in a progressive reduction of ECM output.     

Human neutrophil adherence to thrombospondin occurs through a CD11/CD18-independent mechanism.

Thrombospondin (TSP), a 450-kDa trimeric glycoprotein secreted by platelets and endothelial cells at sites of tissue injury or inflammation, may play an important role in polymorphonuclear leukocyte (PMN) adherence to blood vessel walls before diapedesis. We have examined the adherence of PMN to TSP and compared it to adherence to other extracellular matrix proteins. PMN adherence to TSP-coated plastic was complete by 60 min with spreading completed by 2 h. The kinetics of adhesion and spreading on TSP were similar to that of vitronectin (VN), laminin (LN), and fibronectin (FN). Activation of PMN with the calcium ionophore A23187 or the chemotactic peptide FMLP increased PMN adherence to LN and FN, but not to TSP or VN, suggesting that PMN activation may differentially regulate expression of TSP and VN receptors as compared to LN and FN receptors. The specificity of PMN adherence to TSP was confirmed by competition with saturating amounts of TSP and inhibition with anti-TSP antibodies. mAb A6.1, which binds to the protease-resistant core of TSP, was the most effective in blocking PMN adherence to TSP. Using TSP proteolytic fragments, we demonstrated that the primary interaction of PMN with TSP was mediated through the 140-kDa COOH-terminal domain. Inasmuch as the 140-kDa fragment of TSP contains an Arg-Gly-Asp sequence similar to the cell recognition site of FN and VN, we determined whether RGDS peptides would inhibit PMN adhesion. RGDS did not significantly inhibit PMN adhesion to TSP, VN, or LN, but reduced PMN adhesion to FN by 50%. To determine if PMN adhesion to TSP was mediated by a beta 2 integrin receptor such as LFA-1, MO-1, or p150,95, we performed adhesion assays using PMN isolated from patients with leukocyte adhesion deficiency that lack beta 2 receptors. Leukocyte adhesion deficiency PMN exhibited normal adherence to TSP. In contrast, adherence to VN, LN, and FN was reduced by 95%. Therefore, adherence to TSP is probably not mediated by a beta 2 integrin receptor. These data contribute to the accumulating evidence that PMN can interact with extracellular matrix proteins through a CD11/CD18-independent process.     

The effects of pertussis toxin and cholera toxin on mitogen-induced interleukin-2 production: evidence for G protein involvement in signal transduction

GTP-binding proteins, known as G proteins, play important roles in transducing signals generated by the binding of specific ligands to cell surface receptors. We examined the possibility that a G protein is involved in transducing the concanavalin A (Con A) signal for IL-2 production using a T-cell hybridoma, FS6-14.13, and the bacterial toxins, pertussis toxin (PTX) and cholera toxin (CTX). These toxins are known to interact with and modify the functions of G proteins. High concentrations of PTX (25-50 micrograms/ml) stimulated IL-2 production in the FS-6 cells in the absence of Con A, presumably due to the ability of its B subunit to crosslink membrane proteins. However, in the presence of Con A, PTX inhibited IL-2 production at concentrations ranging from 0.05 to 50 micrograms/ml. It is unlikely that this inhibition was due to a competitive interaction between Con A and PTX for binding sites at the cell surface, since high concentrations of PTX only minimally reduced Con A-FITC binding, evaluated by FACS analysis. In addition, concentrations of PTX which were not able to stimulate IL-2 production in the absence of Con A, retained their ability to inhibit IL-2 production in the presence of Con A. These data suggest the involvement of the PTX A subunit in this activity. In support of this possibility, PTX catalyzed ADP-ribosylation of a Mr = 41,000-Da protein in FS-6 membranes. This strongly suggests that a PTX substrate is involved in transducing the Con A signal for IL-2 production in FS-6 cells. CTX also inhibited Con A-induced IL-2 production, an effect mimicked by the addition of dibutyryl-cAMP. This suggests that a CTX substrate linked to the adenylyl cyclase-cAMP pathway is probably not involved in transducing the stimulatory Con A signal, but may play a role in downregulating T-cell activation.     

Concanavalin A binding to human erythrocytes leads to alterations in properties of the membrane skeleton.

Three properties related to the erythrocyte membrane skeleton are found to be altered after the binding of concanavalin A (Con A) to erythrocytes or their isolated membranes. Con A binding to normal erythrocytes imparts resistance to heat (49 degrees C)-induced fragmentation of the cells. The fragmentation, due to denaturation of spectrin at 49 degrees C, is prevented by Con A in a dose-dependent manner, but levels off at concentrations of Con A in excess of 100 micrograms/ml. The binding of Con A to ghosts isolated from normal, trypsin- or Pronase-treated cells prevents (completely or substantially) the elution of the skeletal protein complex when the membranes are extracted under low-ionic-strength conditions in the cold. The Con A-agglutinated membranes of trypsin- and Pronase-treated, but not normal, cells show cross-linking of skeletal proteins and band 3 with dimethyl adipimidate, a 0.86 nm (8.6 A)-span bifunctional reagent. The extent of cross-linking is greater in the Pronase-treated membrane than in the less-agglutinable trypsin-treated membranes. The results show that, after Con A has bound, rearrangements occur in the membrane that alter properties of the skeletal proteins. Additionally, redistribution of the skeletal proteins and the Con A receptor occurs in the lectin-agglutinated membranes.     

Matrix assembly induction and cell migration and invasion inhibition by a 13-amino acid fibronectin peptide

Fibronectin (FN) is an extracellular matrix (ECM) protein involved in tumor growth and metastasis. Five human FN cDNA segments encoding for FN fragments, all starting with the II1 repeat and ending with different C-terminal extensions, have been stably expressed in chick embryo fibroblasts (CEF). These FN cDNAs induce the formation of an organized ECM in CEF as long as they retain a sequence coding for a 13-amino acid stretch (FN13), with collagen binding activity, localized between type II2 and I7 repeats. An FN13 synthetic peptide induces in control CEF the assembly of an FN-ECM comparable with that observed in CEF-expressing FN fragments. The activity of FN13 is specific for its amino acid sequence, although the cysteine present in the 6th position can be substituted with a polar serine without affecting the induction of a fibrillar FN-ECM. A less fibrillar matrix is induced by FN13-modified peptides in which the cysteine is methylated or substituted by a non-polar alanine. FN13 induces the assembly of an FN-ECM also in Rous sarcoma virus-transformed CEF lacking the ECM and in hepatoma (SK-Hep1) and fibrosarcoma (HT-1080) human cell lines. FN13 also promotes the adhesion of CEF and Rous sarcoma virus-CEF at levels comparable with those obtained with purified intact FN. Finally, FN13 inhibits the migratory and invasive properties of tumorigenic cells, whereas intact FN favors their migration. All FN13-modified peptides show similar effects, although with reduced efficiency. None of these activities is supported by a scrambled peptide. These data suggest a possible role of FN13 in tumor growth and metastasis inhibition and its possible use as anti-tumorigenic agent.     

A fibronectin fragment induces tumor necrosis factor production of rat basophilic leukemia cells

Proteolytic digest of fibronectin (FN), but not intact FN, induced TNF-alpha secretion of rat basophilic leukemia (RBL-2H3) cells. As a result of the identification of FN fragment responsible for TNF-alpha secretion, a 30-kDa fragment derived from the carboxyl-terminal heparin-binding (Hep 2) domain of FN was isolated from the FN digest. The TNF-alpha secretion was abrogated by treatment of RBL-2H3 cells with cycloheximide, indicating the de novo synthesis of TNF-alpha, but not with polymyxin B, excluding the possible TNF-alpha induction by some contaminated lipopolysaccharides. A 22-mer synthetic peptide originated from the Hep 2 domain, termed FNIII14, which has been found to negatively modulate the beta1 integrin activation, had the ability to induce TNF-alpha production, whereas this activity of FNIII14 disappeared by shuffling a YTIYVIAL sequence essential for the integrin-inactivating activity. FNIII14 suppressed the spreading of RBL-2H3 cells on FN substrate, wherein RBL-2H3 cell proliferation was inhibited with FNIII14 in a dose-dependent manner. Thus, it appears that FN fragments containing the YTIYVIAL anti-adhesive site affect the activation status of RBL-2H3 mast cells, characterized by the stimulation of TNF-alpha production and growth suppression, probably due to negative regulation of beta1 integrin activity.     

A comparison of fibronectin, laminin, and galactosyltransferase adhesion mechanisms during embryonic cardiac mesenchymal cell migration in vitro

Embryonic hearts contain a homogeneous population of mesenchymal cells which migrate through an extensive extracellular matrix (ECM) to become the earliest progenitors of the cardiac valves. Since these cells normally migrate through an ECM containing several adhesion substrates, this study was undertaken to examine and compare three ECM binding mechanisms for mesenchymal cell migration in an in vitro model. Receptor mechanisms for the ECM glycoproteins fibronectin (FN) and laminin (LM) and the cell surface receptor galactosyltransferase (GalTase), which binds an uncharacterized ECM substrate, were compared. Primary cardiac explants from stage 17 chick embryos were cultured on three-dimensional collagen gels. Mesenchymal cell outgrowth was recorded every 24 hr and is reported as a percentage of control. Migration was perturbed using specific inhibitors for each of the three receptor mechanisms. These included the hexapeptide GRGDSP (300–1000 μg/ml), which mimics a cell binding domain of FN, the pentapeptide YIGSR (300–1000 μg/ml), which mimics a binding domain of LM, and α-lactalbumin (1–10 mg/ml), a protein modifier of GalTase activity. The functional role of these adhesion mechanisms was further tested using antibodies to avian integrin (JG22) and avian GalTase. While the FN-related peptide had no significant effect on cell migration it did produce a rounded cellular morphology. The LN-related peptide inhibited mesenchymal migration 70% and α-lactalbumin inhibited cell migration 50%. Antibodies agasinst integrin and GalTase inhibited mesenchymal cell migration by 80 and 50%, respectively. The substrate for GalTase was demonstrated to be a single high molecular weight substrate which was not LM or FN. Control peptides, proteins and antibodies demonstrated the specificity of these effects. These data demonstrate that multiple adhesion mechanisms, including cell surface GalTase, are potentially functional during cardiac mesenchymal cell migration. The sensitivity of cell migration to the various inhibitors suggests that occupancy of specific ECM receptors can modulate the activity of other, unrelated, ECM adhesion mechanisms utilized by these cells.     

Cellular fibronectin binds to lysyl oxidase with high affinity and is critical for its proteolytic activation

Lysyl oxidase (LOX) is a copper-containing amine oxidase known to catalyze the covalent cross-linking of fibrillar collagens and elastin at peptidyl lysine residues. In addition, its involvement in cancer, wound healing, cell motility, chemotaxis, and differentiation reflect a remarkable functional diversity of LOX. To investigate novel mechanisms of LOX regulation and function, we performed a yeast two-hybrid screen to identify LOX-interacting proteins. Three overlapping positive clones were identified as C-terminal fragments of fibronectin (FN). Glutathione S-transferase pull-downs and solid phase binding assays confirmed this interaction. LOX binds to the cellular form of FN (cFN) with a dissociation constant (K(d)) of 2.5 nm. This was comparable with our measured K(d) of LOX binding to tropoelastin (1.9 nm) and type I collagen (5.2 nm), but LOX demonstrated a much lower binding affinity for the plasma form of FN (pFN). Immunofluorescent microscopy revealed co-localization of FN and LOX in normal human tissues, where these proteins may interact in vivo. LOX enzymatic activity assays showed that cFN does not seem to be a substrate of LOX. However, cFN can act as a scaffold for enzymatically active 30-kDa LOX. Furthermore, in FN-null mouse embryonic fibroblasts, we observed dramatically decreased proteolytic processing of the 45-kDa LOX proenzyme to the 30-kDa active form, with a corresponding decrease in LOX enzyme activity. Our results suggest that the FN matrix may provide specific microenvironments to regulate LOX catalytic activity.