Binding of plasma fibronectin to cell layers of human skin fibroblasts

Human plasma fibronectin bound to confluent cell layers of cultured human-skin fibroblasts in two distinct pools. Initial binding of fibronectin occurred in a deoxycholate-soluble pool (Pool I). Binding in Pool I was reversible and reached a steady state after 3 h. After longer periods of incubation, fibronectin became bound in a deoxycholate-insoluble pool (Pool II). Binding in Pool II was irreversible and proceeded at a linear rate for 30 h. After 30 h of incubation, a significant proportion of fibronectin bound in Pool II was present as disulfide-bonded multimers. HT1080 cells, a human sarcoma cell line, did not bind fibronectin in either pool. Also, isolated cell matrices prepared by deoxycholate extraction did not bind fibronection. Binding of fibronectin in Pool I of normal fibroblasts occurred via specific, saturable receptors. There were 128,000 binding sites per cell, and KDiss was 3.6 X 10(-8) M. Fluorescence microscopic localization of fibronectin bound in Pool I and Pool II was performed using fluorescein-conjugated fibronectin. Fluorescent staining in Pool I was present in a punctate pattern and in short, fine fibrils. Pool II fluorescence was exclusively in coarse, dense fibrils. These data indicate that plasma fibronectin may become incorporated into the tissue extracellular matrix via specific cell-surface receptors.     

Factor XIII cross-linking of fibronectin at cellular matrix assembly sites

We describe the effect of activated Factor XIII (Factor XIIIa, plasma transglutaminase) on the incorporation of plasma fibronectin into extracellular matrix by cultured human fibroblasts. In the absence of added Factor XIIIa, fibronectin binds to cultured fibroblast cell layers and is assembled into disulfide-bonded multimers of the extracellular matrix. When Factor XIIIa was included in the binding medium of skin fibroblasts, accumulation of 125I-fibronectin in the deoxycholate-insoluble matrix was increased. Fibronectin accumulating in the cell layer was cross-linked into nonreducible high molecular weight aggregates. The 70-kDa amino-terminal fragment of fibronectin inhibited the binding and cross-linking of 125I-fibronectin to cell layers, whereas fibrinogen had little effect. When 125I-fibronectin was incubated with isolated matrices or with cell layers pretreated with cytochalasin B, it did not bind and could not be cross-linked by Factor XIIIa into the matrix. HT-1080 human fibrosarcoma cells bound exogenous fibronectin following treatment with dexamethasone; Factor XIIIa cross-linked the bound fibronectin and caused its efficient transfer to the deoxycholate-insoluble matrix. These results indicate that exogenous fibronectin is susceptible to Factor XIIIa-catalyzed cross-linking at cellular sites of matrix assembly. Thus, Factor XIIIa-mediated fibronectin cross-linking complements disulfide-bonded multimer formation in the stabilization of assembling fibronectin molecules and thus enhances the formation of extracellular matrix.     

Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts.

Transforming growth factor beta (TGF-beta) enhances the cell surface binding of 125I-fibronectin by cultured human fibroblasts. The effect of TGF-beta on cell surface binding was maximal after 2 h of exposure to TFG-beta and did not require epidermal growth factor or protein synthesis. The enhancement was dose dependent and was found with the 125I-labeled 70-kilodalton amino-terminal fragment of fibronectin as well as with 125I-fibronectin. Treatment of cultures with TGF-beta for 6 h resulted in a threefold increase in the estimated number of fibronectin binding sites. The increase in number of binding sites was accompanied by an increased accumulation of labeled fibronectin in detergent-insoluble extracellular matrix. The effect of TGF-beta was biphasic; after 6 h of exposure, less labeled fibronectin bound to treated cultures than to control cultures. Exposure of cells to TGF-beta for greater than 6 h caused a two- to threefold increase in the accumulation of cellular fibronectin in culture medium as detected by a quantitative enzyme-linked immunosorbent assay. The second phase of the biphasic effect and the increase in soluble cellular fibronectin were blocked by cycloheximide. Immunofluorescence staining of fibroblast cultures with antifibronectin revealed that TGF-beta caused a striking increase in fibronectin fibrils. The 70-kilodalton amino-terminal fragment of fibronectin, which blocks incorporation of fibronectin into extracellular matrix, blocked anchorage-independent growth of NRK-49F cells in the presence of epidermal growth factor. Our results show that an increase in the binding and rate of assembly of exogenous fibronectin is an early event preceding the increase in expression of extracellular matrix proteins. Such an early increase in cell surface binding of exogenous fibronectin may be a mechanism whereby TGF-beta can modify extracellular matrix characteristics rapidly after tissue injury or during embryonic morphogenesis.     

Regulation of cell surface tissue transglutaminase: effects on matrix storage of latent transforming growth factor-beta binding protein-1.

Using a cytochemical approach, we examined the role of tissue transglutaminase (tTgase, Type II) in the incorporation of latent TGF-beta binding protein-1 (LTBP-1) in the extracellular matrix of Swiss 3T3 fibroblasts in which tTgase expression can be modulated through a tetracycline-controlled promoter. Increased tTgase expression led to an increased rate of LTBP-1 deposition in the matrix, which was accompanied by an increased pool of deoxycholate-insoluble fibronectin. Matrix deposition of LTBP-1 could also be reduced by the competitive amine substrate putrescine. Immunolocalization at the fluorescence and electron microscopic level showed that extracellular tTgase is located at the basal and apical surfaces of cells and at cell-cell contacts, and that LTBP-1 is co-distributed with cell surface tTgase, suggesting an early contribution of tTgase to the binding of LTBP-1 to matrix proteins. LTPB-1 was also found to co-localize with both intracellular and extracellular fibronectin, and increased immunoreactivity for LTBP-1 and fibronectin was found in large molecular weight polymers in the deoxycholate-insoluble matrix of fibroblasts overexpressing tTgase. We conclude that regulation of tTgase expression is important for controlling matrix storage of latent TGF-beta1 complexes and that fibronectin may be one extracellular component to which LTBP-1 is crosslinked when LTBP-1 and tTgase interact at the cell surface. (J Histochem Cytochem 47:1417-1432, 1999)     

Functional changes in cellular fibronectin from late passage fibroblasts in vitro

Analysis of fibronectin synthesized by human fibroblasts, at different times during serial subcultivation, reveals functional differences. Fibronectin isolated from late passage cells is defective in promoting cell adhesion, cell spreading, and the formation of focal contacts. These changes are not the result of an inability of late passage cells to interact with fibronectin, since late passage cells become adhesive and form focal contacts in the presence of fibronectin isolated from early passage cells. Therefore, we conclude that late passage cellular fibronectin derived from late passage cells cannot support the cell substrate interactions.     

Enhanced synthesis of a Mr = 55,000 dalton peptide by senescent human fibroblasts

The secretory protein profiles of early and late passage cultures of human fibroblasts were compared using polyacrylamide gel electrophoresis. In comparison with early passage cell cultures (40-50% lifespan completed), late passage (greater than 80% of lifespan completed) cell cultures exhibited enhanced production of several peptides in the Mr range 55-60,000. One of those peptides had an apparent molecular weight of Mr = 55,000 and was constitutively present in the late passage cell conditioned medium. Late passage cell cultures synthesized the Mr = 55,000 peptide in the presence or absence of fetal bovine serum. Serum did not enhance its production by early passage cells. Further, production of the peptide was not induced in early passage cell cultures whose proliferation was arrested either by serum starvation or by contact inhibition. Pulse chase studies demonstrated that the peptide appears in the culture medium within 60 min of labeling. There was no evidence that it is derived via degradation of other proteins present either in early passage or late passage cell conditioned media. Further, the production of the 55,000 dalton peptide did not appear to be regulated by factors present in conditioned media. The peptide was detected in the conditioned media produced by late passage cultures of several different cell strains.     

Effect of an anti-insulin-like growth factor I receptor antibody on insulin-like growth factor II stimulation of DNA synthesis in human fibroblasts

To investigate the role of insulin-like growth factor II in the control of DNA synthesis in human fibroblasts, dose-response curves for insulin-like growth factor I and II stimulation of [3H]thymidine incorporation were compared in the absence and presence of alpha IR-3, a highly specific monoclonal antibody directed against the type I insulin-like growth factor receptor. Specific binding of [125I]insulin-like growth factor I to human fibroblast monolayer cultures was inhibited 60-70% in the presence of alpha IR-3. alpha IR-3 had no effect on [125I]insulin-like growth factor II binding to human fibroblasts. However, alpha IR-3 inhibited both insulin-like growth factor I and II stimulated [3H]thymidine incorporation. These data indicate that the type II insulin-like growth factor receptor does not function as a transducer of insulin-like growth factor II's mitogenic effect in human fibroblasts.     

Matrix assembly sites for exogenous fibronectin are decreased on human fibroblasts after treatment with agents which increase intracellular cAMP

Studies with cultured fibroblasts have shown that plasma as well as cellular fibronectin can be organized into fibrillar structures and that this organization is mediated by sites at the cell surface. Treatment of human skin fibroblasts with cholera toxin resulted in a prompt decrease in the number of binding sites for 125I-labeled plasma fibronectin and a 125I-labeled 70-kDa amino-terminal fragment of fibronectin. This decrease was accompanied by less incorporation of labeled fibronectin into deoxycholate-insoluble extracellular matrix. Binding of 125I-fibronectin was also decreased in cultures treated with epinephrine, isoproterenol, or forskolin. These results, therefore, indicate that G proteins and the adenylate cyclase system are involved in regulation of fibronectin matrix assembly sites may be one mechanism whereby hormones or growth factors can modify extracellular matrix characteristics.     

Glycosaminoglycan production in cultures of early and late passage human endothelial cells: the influence of an anionic endothelial cell growth factor and the extracellular matrix

An endothelial cell (EC) growth factor isolated from bovine brain stimulates in vitro growth of human umbilical vein endothelial cells, and permits long term serial propagation. In the presence of increasing concentrations of EC growth factor, confluent cultures of early (CPDL less than or equal to 20) and late (CPDL greater than 20) passage human endothelial cells exhibit an increased incorporation of 3H-glucosamine and Na235SO4 into the glycosaminoglycans (GAG), hyaluronic acid, chondroitin, chondroitin-4-sulfate, dermatan-4-sulfate, and chondroitin-6-sulfate. An increase in both labelled sulfated and nonsulfated GAG was observed in the cytosol, membrane, secreted and extracellular matrix fractions. In contrast, endothelial cells grown in the presence of EC growth factor contained decreased amounts of labelled heparan sulfate than cells grown without EC growth factor. Confluent cultures of early passage cells had significantly more labelled GAG but significantly less heparan sulfate than cultures of late passage cells on a per cell basis. Extracellular matrix from early passage cells contained about two- to seven-fold more labelled GAG than extracellular matrix from late passage cells, but only about half as much labelled heparan sulfate. Cell adhesion was enhanced when cells were grown in the presence of EC growth factor as compared to adhesion of cells grown without EC growth factor. Conversely, trypsin-mediated detachment of cells grown in the presence of growth factor was inhibited as compared to detachment of cells grown in medium without EC growth factor. The composition of the extracellular matrix influenced incorporation of labelled GAG into extracellular matrix. Early passage cells grown to confluence on a matrix from late passage cells incorporated significantly less labelled GAG into extracellular matrix than when grown to confluence on matrix from early passage cells. Incorporation of labelled GAG into extracellular matrix was significantly higher when late passage cells were grown on a matrix from early passage endothelial cells than when grown on matrix from late passage cells. We conclude that EC growth factor selectively stimulates incorporation of isotopic precursors into GAG in cultures of early and late passage endothelial cells but inhibits incorporation of radiolabel into heparan sulfate; early passage cells contain more GAG but less heparan sulfate than late passage cells, extracellular matrix controls the amount of GAG and heparan sulfate incorporated into matrix.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modifications of proteoglycans extracted from monolayer cultures of young and senescent human skin fibroblasts

Proteoglycans (PGs) were extracted from culture monolayers of human skin fibroblasts (HFs) at early and late passages. Total PGs from senescent cells had markedly reduced abilities to bind type I collagen and hyaluronic acid, but retained normal binding properties with fibronectin and laminin. The constituent polysaccharides of PGs were comparatively characterised. PGs recovered from young and senescent HF cultures had equivalent total polyanionic charges and similar size distributions of the glycosaminoglycan chains. This applied to both types of polysaccharide chains found in PGs, namely the galactosaminoglycuronans (GalN-GAGs) and the glucosaminoglycuronans (GlcN-GAGs). However, senescent HFs produced a greater proportion of PGs containing GlcN-GAG chains and increased the sulphation of the remainding PG fraction with GalN-GAG moieties, yielding a major gain of C6-sulphate groups in the galactosamine residues.     

Role of the I-9 and III-1 modules of fibronectin in formation of an extracellular fibronectin matrix.

Cultured fibroblasts bind soluble protomeric fibronectin and mediate its conversion to insoluble disulfide-bonded multimers. The disulfide-bonded multimers are deposited in fibrillar pericellular matrix. Antifibronectin monoclonal antibodies were analyzed to identify domains of fibronectin required for assembly into matrix. Two antibodies, L8 and 9D2, inhibited binding and insolubilization of 125I-labeled plasma fibronectin by fibroblasts but did not inhibit binding of labeled amino-terminal 70-kDa fragment of fibronectin to matrix assembly sites. Immunoblotting of fibronectin fragments showed that the epitope for 9D2 is in the first type III homology sequence (III-1) whereas the epitope for L8 requires that the last type I sequence of the gelatin binding region (I-9) be contiguous to III-1 and is sensitive to reduction of disulfides in I-9. A 56-kDa gelatin-binding thermolysin fragment of fibronectin that contains III-1 and the L8 and 9D2 epitopes inhibited binding of fibronectin to cell layers 10-fold better than a 40-kDa gelatin-binding fragment that lacks III-1 and the antigenic sites. This 56-kDa fragment, however, did not bind specifically to cell layers. These results indicate that the I-9 and III-1 modules of fibronectin form a functional unit that mediates an interaction, perhaps between protomers, important in the assembly of fibronectin.     

Fibronectin from aged fibroblasts is defective in promoting cellular adhesion

Late passage fibroblasts show decreased cell-substrate adhesion. We provide evidence that the reduced adhesion is due to a defect in the adhesive glycoprotein fibronectin. Late passage cells become more adhesive in culture media that has been conditioned by the growth of early passage cells. Analysis of fibronectins purified from early and late passage cell conditioned media indicates that there are striking differences in their abilities to promote cell adhesion. Young cell fibronectin supports the maximal adhesion of both young and old cells. However, old cells require quantitatively more fibronectin. In contrast, old cell fibronectin is less effective in supporting the adhesion of either cell type. In addition, neither cell type achieves a normal morphology in the presence of old cell fibronectin. The results support the conclusion that the fibronectin released by late passage cells is defective and does not support normal cell-substrate interactions.     

Myocilin binding to Hep II domain of fibronectin inhibits cell spreading and incorporation of paxillin into focal adhesions

Myocilin, a novel matricellular protein found in the human eye, can modify signaling events mediated by the Heparin II domain of fibronectin. Using myocilin produced in sf9 insect cells, myocilin inhibited spreading of cycloheximide-treated human skin fibroblasts plated on substrates co-coated with myocilin and either fibronectin or its Heparin II domain. Cell spreading could be rescued by adding back either substrate adsorbed or soluble Heparin II domains. Myocilin did not inhibit cell attachment to fibronectin even in the presence of a 2400 M excess of myocilin. Myocilin impaired focal adhesion formation and specifically blocked the incorporation of paxillin, but not vinculin, into focal adhesions. The Heparin II domain mediated the incorporation of paxillin into focal adhesions, since paxillin was not assembled into focal adhesions unless the Heparin II domain was present. The effect of myocilin on focal adhesions could be overcome by treating cells with either phorbol 12-myristate (PMA) or oleoyl-L-alpha-lysophosphatidic acid (LPA). Myocilin bound to the fibroblast cell surface, but its binding could not be competed with excess fibronectin, suggesting that myocilin does not compete for cell surface binding sites of fibronectin. Myocilin therefore appears to specifically block functions mediated by the Heparin II domain possibly through direct interactions with it.     

Fibronectin's amino-terminal matrix assembly site is located within the 29-kDa amino-terminal domain containing five type I repeats

Fibronectin is organized into disulfide cross-linked, insoluble pericellular matrix fibrils by fibroblasts in vitro. Two sites, the Arg-Gly-Asp-Ser-containing cell attachment domain and a site located in the first 70 kDa of fibronectin, are required for matrix assembly. The first 70 kDa of fibronectin contain two structural motifs termed type I and type II homologies, which are repeated nine and two times, respectively. Previous work has implicated the amino-terminal region and the carboxyl terminus containing three type I repeats in matrix assembly, suggesting that type I repeats possess binding activity essential for fibronectin matrix assembly. To test this hypothesis, we developed a sensitive capture immunoassay to quantify insoluble matrix fibronectin and tested a panel of fibronectin fragments, containing all of the type I repeats found in the intact protein, for their ability to inhibit matrix assembly. Only fragments containing the first five type I repeats inhibited fibronectin matrix assembly, although sequences carboxyl-terminal to this domain enhanced this activity. Additional evidence for the specific recognition of the amino-terminal type I repeats by matrix assembling cells was found when the reversible, detergent-sensitive binding of a 125I-labeled fragment containing the first five type I repeats (29 kDa) to cell monolayers was studied. Only monolayers of cell lines that incorporate fibronectin into a fibrillar matrix specifically bound 125I-labeled 29 kDa. Binding of the radiolabeled amino-terminal fragment to matrix-forming cells was inhibited by unlabeled fragments containing the first five type I repeats but not by unlabeled fragments containing the remaining seven type I repeats. Matrix assembly is therefore not a generalized property of type I repeats. Rather, a critical site is located within the first 29 kDa of fibronectin.     

Insulin-like growth factor (IGF) binding to human fibroblast and glioblastoma cells: the modulating effect of cell released IGF binding proteins (IGFBPs)

The cell surface of human fibroblasts contains not only type I IGF receptors but at least two forms of IGFBPs. Studies were undertaken to analyze the mechanisms by which these IGFBPs alter IGF-I-cell surface interactions. Human fetal fibroblasts (GM10) and a human glioblastoma cell line (1690) were chosen for analysis. During assays to quantify [125I]-IGF-I binding, both cell lines were shown to release IGFBPs into the binding assay buffer. Under equilibrium conditions, [125I]-IGF-I preferentially associates with IGFBPs in the assay buffer (up to 40% of the [125I]-IGF-I added) since they have a higher affinity than type I IGF receptors or IGFBPs associated with the cell surface. Likewise the addition of increasing concentrations of unlabeled IGF-I results in preferential competition for binding to assay buffer IGFBPs. This results in a repartitioning of the [125I]-IGF-I that is bound to assay buffer IGFBPs onto cell surface binding sites. The degree of repartitioning is quantitatively related to the amount of [125I]-IGF-I bound to released IGFBPs. When cultures are exposed to cycloheximide before the binding assay, both the amount of IGFBPs that are released into the assay buffer and the amount of [125I]-IGF-I that is repartitioned are decreased. In contrast when [Gln3, Ala4, Tyr15, Leu16]-IGF-I ([QAYL]-IGF-I, an IGF analog that has unaltered affinity for type I IGF receptors) is iodinated and tested, the competition curve with unlabeled IGF-I shows no repartitioning effect. This form of IGF can be used to quantify type I receptor number independent of the presence of IGFBPs. IGF-I and the [QAYL]-IGF-I compete equally with the [125I]-[QAYL]-IGF-I for binding to cell surfaces, whereas unlabeled [QAYL]-IGF-I is greater than 25-fold less potent compared to IGF-I in competing with [125I]-IGF-I for cell surface binding. Specific binding of [125I]-[QAYL]-IGF-I to GM10 and 1690 cell surfaces is less than 20% of [125I]-IGF-I binding. These findings suggest that IGFBPs that are present on human fibroblast surfaces represent a large portion of the IGF binding sites. We conclude that the amount of IGFBPs released into assay buffer is a major determinant of the repartitioning of [125I]-IGF-I to cell surface binding sites and that both cell surface and assay buffer IGFBPs modulate type I IGF receptor binding.     

Influence of tumor promoter on binding and release of fibronectin added to human lung fibroblasts

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) did not alter the binding and release kinetics of externally added 125I-labeled plasma fibronectin to human lung fibroblasts in culture. Cell layer-bound plasma fibronectin was found to be chased into the medium at the same rate in tumor-promoter-treated as in non-treated cells. Unlabeled fibronectin accumulated to a much higher degree in the medium when tumor promoter was present. We conclude that TPA does not interfere with the fibronectin receptor on substrate-attached fibroblasts, but may influence intracellular fibronectin before it is bound to the extracellular matrix.     

Virulent Escherichia coli strains for chicks bind fibronectin and type II collagen

125I-fibronectin and 125I-collagen (type II) binding was detected in Escherichia coli strains isolated from chickens and poults. High fibronectin binding-strains also bind the 29 kD aminoterminal fragment of fibronectin. Binding properties in strain CK28 were partially characterized. The highest binding of 125I-fibronectin and 125I-collagen for strain CK28 was obtained with bacteria grown at 33 degrees C. Binding of 125I-fibronectin, its 125I-29 kD fragment, and 125I-collagen, was very rapid, reaching a maximum in 5 min. Binding of 125I-fibronectin and 125I-collagen was considerably inhibited by preincubation of bacteria with unlabelled fibronectin and unlabelled type I collagen respectively, but not inhibited with human immunoglobulin G or bovine serum albumin. Inhibition experiments showed that the reversibility of 125I-fibronectin binding was estimated at approximately 50%, while reversibility for 125I-collagen binding was higher than 90%. Receptors for fibronectin, its 29 kD fragment, and collagen were released from the bacterial surface by treatment at different temperatures, and surface material released at 100 degrees C inhibited binding. There was cross-inhibition for both fibronectin and collagen binding when unlabelled fibronectin and unlabelled collagen were used as inhibitors, suggesting that binding receptors for both proteins may be closely located.     

The interaction of human papillary and reticular fibroblasts and human keratinocytes in the contraction of three-dimensional floating collagen lattices

Fibroblasts derived from the papillary and reticular dermis of human skin and human keratinocytes show differences in their abilities to contract floating three-dimensional gels constructed from type I collagen. Reticular fibroblasts produce greater gel contraction than papillary fibroblasts. When equal numbers of papillary and reticular fibroblasts are mixed in the gels, papillary fibroblasts consistently inhibit gel contraction by reticular fibroblasts indicating interaction between these cell types in the contraction process. Surprisingly, keratinocytes alone produce greater gel contraction than that produced by either fibroblast type. Cooperativity in the gel contraction process is observed when fibroblasts are incorporated into the collagen matrix and keratinocytes are seeded onto the gel surface. Keratinocytes and dermal fibroblasts adhere to the collagen fibril to induce gel contraction by different mechanisms. Fibroblast contraction of collagen gels does not require fibronectin but is a serum-dependent reaction. In contrast, keratinocyte contraction of collagen gels occurs in a serum-free environment. Polyclonal, affinity-purified antibodies to human plasma fibronectin at high concentrations do not inhibit gel contraction by keratinocytes, making unlikely the possibility that fibronectin synthesized by the keratinocyte is a significant factor in the gel contraction process. We are currently examining the possibilities either that keratinocytes are synthesizing other adhesion proteins or that receptors on the cell surface can interact directly with the collagen fiber.     

Modulation of fibronectin synthesis and fibronectin binding during transformation and differentiation of mouse AKR fibroblasts

In previous studies it was shown that transformation of AKR fibroblasts with 3-methylcholanthrene was associated with a loss of surface fibronectin and that induction of differentiation of the transformed cells with N,N-dimethylformamide (DMF) was associated with reacquisition of surface fibronectin (Chakrabarty et al., J. Cell. Physiol. 133:415, 1987). It is shown in the present study that changes in surface fibronectin reflect altered fibronectin synthesis and altered fibronectin binding. Both the nontransformed cells (AKR-2B) and their transformed counterparts (AKR-MCA) bound 125I-fibronectin in a receptor-like fashion, but the AKR-MCA cells had only 20% of the receptors found on the AKR-2B cells. Whole cell extracts prepared from the AKR-2B cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions were examined for 125I-fibronectin binding. Under these conditions, the majority of binding occurred to moieties with molecular weights of 180 kD, 150 kD, and 97 kD. Binding to similar moieties on the AKR-MCA cells was virtually absent but occurred rapidly after treatment with DMF. The appearance of these moieties paralleled the acquisition of 125I-fibronectin binding activity by whole cells. Antibodies to the fibronectin receptor isolated from human placenta reacted with the DMF-sensitive moieties in immunoblot assays. Both the appearance of the fibronectin binding moieties and the acquisition of 125I-fibronectin binding activity by whole cells occurred within 6 hr of DMF treatment and increased over the subsequent 4 day period. The time course of these events paralleled closely the time course for induction of fibronectin biosynthesis by DMF. These changes in fibronectin binding and fibronectin production were associated with alterations in cell-substrate adhesion. The AKR-2B cells rapidly attached and spread on bovine serum albumin-coated dishes and on fibronectin-coated dishes, whereas the AKR-MCA cells were less adhesive on both substrates. Capacity to attach and spread was regained concomitantly with the induction of fibronectin binding and fibronectin production. Adhesion on both substrates was partially inhibited by antibodies to the fibronectin receptor and by RGDS. These studies suggest that fibronectin production and fibronectin binding are coregulated in AKR fibroblasts and that they function together to bring about changes in cell-substrate adhesion.