Proteins with affinities for specific glycosaminoglycans (GAG'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 proteoglycan 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.Fibronectin is an extracellular matrix-associated glycoprotein found in vivo that mediates fibroblast attachment, spreading, motility, and longterm survival in vitro when adsorbed to tissue culture substrata. Fibronectin consists of dimeric subunits (54) and has the potential to interact with several cell surfaceassociated macromolecules including collagen (l 3), certain glycosaminoglycans (24, 56), and possibly gangliosides (21), and another unidentified cell surface receptor (37,43,45,56). Accumulating evidence indicates that cell surface glycosaminoglycans and proteoglycans mediate fibroblast attachment and possibly more complex adhesive responses on fibronectincontaining extracellular matrices (10, 41,47). Hyaluronic acid and heparan sulfate are two particular classes of cell surface glycosaminoglycan that have received considerable attention as potential determinants of the state of cellular adhesiveness and motility in numerous experimental systems (2, I l, 25, 34, 44). It has yet to be determined, however, if direct interactions between cell surface glycosaminoglycans and the extracellular matrix determine prop...

When normal or SV40-transformed Balb/c 3T3 cells are treated with the Ca++-specific chelator EGTA, they round up and pull away from their footpad adhesion sites to the serum-coated tissue culture substrate, as shown by scanning electron microscope studies. Elastic membranous retraction fibers break upon culture agitation, leaving adhesion sites as substrate-attached material (SAM) (Cells leave "footprints" of substrate adhesion sites during movement by a very similar process.) SAM contains 1-2% of the cell's total protein and phospholipid content and 5-10% of its glucosamine-radiolabeled polysaccharide, most of which is glycosaminoglycan (GAG). By one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there is considerable enrichment in SAM for specific GAGs; for the glycoprotein fibronectin; and for the cytoskeletal proteins actin, myosin, and the subunit protein of the 10 nm-diameter filaments. Fibrillar fibronectin of cellular origin and substratum-bound fibronectin of serum origin (cold-insoluble globulin, CIg) have been visualized by immunofluorescence microscopy. The GAG composition in SAM has been examined under different cellular growth and attachment conditions. Heparan sulfate content correlates with glycopeptide content (derived from glycoprotein). Newly attaching cells deposit SAM with principally heparan sulfate and fibronectin and little of the other GAGs. Hyaluronate and chrondroitin proteoglycans are coordinately deposited in SAM as cells begin spreading and movement over the substrate. Cells attaching to serum-coated or CIg-coated substrates deposited SAM with identical compositions. The proteoglycan nature of the GAGs in SAM has been examined, as well as the ability of proteoglycans to form two classes of reversibly dissociable "supramolecular complexes" - one class with heparan sulfate and glycopeptide-containing material and the second with hyaluronate-chondroitin complexes. Enzymatic digestion of "intact" SAM with trypsin or testicular hyaluronidase indicates that (1) only a small portion of long-term radiolabeled fibronectin and cyto-skeletal protein is bound to the substrate via hyaluronate or chondroitin classes of GAG; (2) most of the fibronectin, cytoskeletal protein and heparan sulfate coordinately resist solubilization; and (3) newly synthesized fibronectin, which is metabolically labile in SAM, is linked to SAM by hyaluronate- and/or chondroitin-dependent binding. All of our studies indicate that heparan sulfate is a direct mediator of adhesion of cells to the substrate, possibly by binding to both cell-surface fibronectin and substrate-bound CIg in the serum coating; hyaluronate-chondroitin complexes in SAM appear to be most important in motility of cells by binding and labilizing fibronectin at the periphery of footpad adhesions, with subsequent cytoskeletal disorganization.

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Plasma fibronectin (pFN) adhesion mechanisms on inert substrata were evaluated for murine fibroblasts (3T3) and human neuroblastoma (Platt) cells using glass coverslips chemically derivatized with a self-assembled monolayer of aliphatic chains terminated with a specific endgroup to interact with adsorbed pFN: [CH3], [SH], [SCOCH3], [NH2], [SO3H], or underivatized glass [SiOH]. All surfaces bound similar amounts of pFN and facilitated attachment of both cell types within narrow ranges. However, spreading/differentiation responses of cells differed considerably among the surfaces. While 3T3 cells spread and developed microfilament stress fibers comparably on all surfaces, inclusion of an RGDS-containing synthetic peptide in the medium revealed variation in resistance to stress fiber formation mediated by an RGDS-recognizing integrin: [NH2] greater than [CH3] much greater than [SiOH],[SH],[SCOCH3]. Different patterns of neurite formation were observed for neuroblastoma cells: [SiOH], [SO3H] greater than [SCOCH3],[SH] much greater than [CH3] greater than [NH2]. Similarity in cell responses to both [CH3] and [NH2] surfaces argues against a pattern dependent upon the hydrophobicity of substrata. When pFN was diluted to a subsaturable concentration with albumin for adsorption, neuroblastoma responses changed significantly from those observed on pFN-saturated surfaces, for both spreading and neurite generation: [NH2],[SO3H] much greater than [SH], [SCOCH3] greater than [SiOH],[CH3]. Responses to the pFN: albumin mixture were markedly improved from responses after sequential adsorptions, demonstrating "optimization" of pFN conformation (not merely binding) by coadsorption of albumin molecules. In most cases, the [NH2] surface yielded responses distinctively different from the other surfaces. Overall, these data suggest many variations in the conformation of pFN molecules adsorbed to specific inert surfaces, as well as variations in the responses of cell surface receptors to conformationally specific pFNs. They also reveal cell-type-specific changes in differentiated cell responses on derivatized substrata, mediated by different classes of cell surface receptors for the two cell types, and provide optimism for regulating FN-dependent adhesion mechanisms in either positive or negative contexts on biomaterial surfaces derivatized with one or more of these chemical end-groups.

Abstract. Dermatan sulfate proteoglycans (DS-PGs) isolated from bovine articular cartilage have been examined for their effects on the adhesive responses of BALB/c 3T3 cells and bovine dermal fibroblasts on plasma fibronectin (pFN) and/or type I collagen matrices, and compared to the effects of the chondroitin sulfate/keratan sulfate proteoglycan monomers (CS/KSPGs) from cartilage. DS-PGs inhibited the attachment and spreading of 3T3 cells on pFN-coated tissue culture substrata much more effectively than the cartilage CS/KS-PGs reported previously; in contrast, dermal fibroblasts were much less sensitive to either proteoglycan class unless they were pretreated with cycloheximide. Both cell types failed to adhere to substrata coated only with the proteoglycans; binding of the proteoglycans to various substrata has also been quantitated. While a strong inhibitory effect was obtained with the native intact DS-PGs, little inhibitory effect was obtained with isolated DS chains (liberated by alkaline-borohydride cleavage) or with core protein preparations (liberated by chondroitinase ABC digestion). In marked contrast, DS-PGs did not inhibit attachment or spreading responses of either 3T3 or dermal fibroblasts on type I collagen-coated substrata when the collagen was adsorbed with pFN alone, DS-PGs alone, or the two in combination. These results support evidence for (a) collagen-dependent, fibronectin-independent mechanisms of adhesion of fibroblasts, and (b) different sites on the collagen fibrils where DS-PGs bind and where cell surface "receptors" for collagen bind.Experiments were developed to determine the mechanism(s) of inhibition. All evidence indicated that the mechanism using the intact pFN molecule involved the binding of the DS-PGs to the glycosaminoglycan (GAG)-binding sites of substratum-bound pFN, thereby inhibiting the interaction of the fibronectin with receptors on the cell surface. This was supported by affinity chromatography studies demonstrating that DS-PGs bind completely and effectively to pFN-Sepharose columns whereas only a subset of the cartilage CS/KS-PG binds weakly to these columns. In contrast, when a 120-kD chymotrypsin-generated cell-binding fragment of pFN (CBF which has no detectable GAG-binding activity as a soluble ligand) was tested in adhesion assays, DS-PGs inhibited 313 adherence on CBF more effectively than on intact pFN. A variety of experiments indicated that the mechanism of this inhibition also involved the binding of DS-PGs to only substratum-bound CBF due to the presence of a cryptic GAG-binding domain not observed in the soluble CBF. When a series of complementary cell-binding fragments generated from pFN by thermolysin digestion and subsequent affinity chromatography (Castellani, P., A. Siri, C. Rosellini, E. Infusini, L. Borsi, and L. , J. Cell Biol., 103:1671-1677 were tested, a graded response to inhibition by DS-PGs was observed revealing the proximity of the cryptic GAGbinding domain to the cell-binding domain of the fibronectin molecule.All of these results taken...

The extracellular matrix protein, Fn, has critical functions in cell attachment, migration, differentiation, and proliferation. We have previously shown that fibronectin (Fn) is abnormally expressed and potentiates entry into the cell cycle of basal keratinocytes in uninvolved psoriatic skin, in combination with T cell lymphokines. It is not known what type of Fn is present in psoriatic skin, however, and how this Fn may regulate signaling. Embryonic forms of cellular Fn containing extra domains, designated EDA and EDB, are generated by alternative splicing and are seen in proliferating, developing tissue and in wound healing. Because the EDA segment enhances the integrin binding sequence Arg, Gly, Asp (RGD), which, when present, has been shown to be critical in integrin-extracellular matrix signaling, we were particularly interested in determining whether or not EDA-containing Fn (EDA+Fn) represented the aberrantly expressed Fn in psoriasis. Increased EDA+ Fn protein was demonstrated by immunostaining at the dermal-epidermal junction in clinically uninvolved skin from six of six patients with psoriasis, but not in skin from control subjects. Using reverse transcription polymerase chain reaction an increased ratio of EDA+ Fn versus EDA- Fn mRNA was present in epidermal samples from psoriatic but not control individuals. Interestingly, the EDA+Fn in the psoriatic epidermis had the IIICS region spliced out (EDA+, FDB-, IIICS-, III9+), which was shared with normal epidermis (EDA-, EDB-, IIICS-, III9+). These results suggest a selective predominance of the EDA+ Fn isoform at the dermal-epidermal junction of psoriatic skin. The consistent aberrant localization of EDA+ Fn at the dermal-epidermal junction in uninvolved skin of psoriatics may confer the hyperresponsiveness of psoriatic uninvolved basal keratinocytes for rapid cellular proliferation in response to T cell signals. Key words: immunohistochemistry/integrin/keratinocyte/RT-PCR.

The proteins which have been left tightly bound to the tissue culture substrate after ethylenebis (oxyethyl-enenitrilo) tetraacetic acid (EGTA)-mediated removal of normal, virus-transformed, and revertant mouse cells and which have been implicated in the substrate adhesion process have been analyzed by slab sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three size classes of hyaluronate proteoglycans were resolved in the 5% well gel; approximately half of the protein in the substrate-attached material coelectrophoresed with these polysaccharides-so-called glycosaminoglycan-associated protein(GAP). A portion of the GAP was shown to be highly heterogeneous and displaced from the polysaccharide by preincubation with calf histone before electrophoresis. The relative proportions of the proteoglycans varied in material deposited during a variety of cellular attachment and growth conditions. The remainder of the cellular protein in substrate-attached material was resolved as several major and distinct protein bands in 8 or 20% separating gels (a limited number of distinct serum proteins have also been identified as substrate bound). Protein C0 (molecular weight 220 000) was a prominent component in the material from a variety of normal and virus-transformed cells and resembled the so-called LETS or CSP glycoprotein in several respects; protein Ca was myosin-like in several respects; protein C2 was shown to be actin; and protein C1 (molecular weight 56 000) does not appear to be tubulin. Histones were also present in most preparations of substrate-attached material, particularly at high levels in transformed cell meterial, and may result from EGTA-mediated leakiness of the cell and subsequent binding to the negatively charged polysaccharide. These substrate-attached proteins were (a) prominent in substrate-attached material from many cell types in characteristic relative proportions, (b) deposited by EGTA-subcultured cells during the first hour of attachment to fresh substrate, (c) deposited by cells growing on plastic or glass substrates (three additional) components were also prominent in glass-attached material), and (d) deposited during long-term growth on or initial attachment to substrates coated wit 3T3 substrate-attached material. Pulse-chase analyses with radioactive leucine indicated that these proteins exhibit different turn-over behaviors. These results are discussed with regard to the possible involvement of these substrate-attached proteins in the substrate adhesion process, with particular interest in the interaction of cytoskeletal microfilaments with other surface membrane components and with regard to alteration of substrate adhesion by virus transformation.