Control of AKR fibroblast phenotype by fibronectin: Regulation of cell-surface fibronectin binding receptor by fibronectin

Results of previous studies show that the expression of fibronectin and its cell-surface fibronectin binding receptor is coregulated in 3-methylchloranthrene transformation of normal AKR-2B cells to form AKR-MCA cells and in N, N,-dimethylformamide (DMF) induction of differentiation of transformed AKR-MCA cells (1990, J. Cell. Physiol., 143:445). In this study, we tested the corgulation hypothesis by transfection experiments using an antisense fibronectin expression vector. We determined the effect of antisense fibronectin RNA expression on untransformed AKR-2B cells, and on the responses of transformed AKR-MCA cells to DMF treatment. Expression of antisense fibronectin RNA in AKR-2B cells down-modulated fibronectin production, reduced adhesion to extracellular fibronectin, and altered cellular morphology Saturation binding and Scatchard analyses using radiolabelled fibronectin revealed a concurrent down-modulation of cell-surface fibronectin binding sites, but the binding affinity of the receptor for the ligand was not affected. Immunoblotting and immunostaining revealed down-modulation of the expression of α5β1 integrins. Expression of antisense fibronectin RNA in AKR-MCA cells down-modulated the ability of DMF to restore normal fibronectin production, cell-surface fibronectin binding receptor, adhesion to extracellular fibronectin, and cellular morphology. These studies show that both fibronectin and its cell-surface fibronectin binding receptor were tightly regulated during transformation and induction of differentiation in these cells, that the ligand and its cell-surface fibronectin binding receptor worked together to bring about phenotypic changes, and that fibronectin production regulated the expression of its cell-surface fibronectin binding receptor. © 1994 Wiley-Liss, Inc.     

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.     

Inhibition of binding of fibronectin to matrix assembly sites by anti- integrin (alpha 5 beta 1) antibodies

Fibroblasts have cell surface sites that mediate assembly of plasma and cellular fibronectin into the extracellular matrix. Cell adhesion to fibronectin can be mediated by the interaction of an integrin (alpha 5 beta 1) with the Arg-Gly-Asp-Ser (RGDS)-containing cell adhesion region of fibronectin. We have attempted to elucidate the role of the alpha 5 beta 1 fibronectin receptor in assembly of fibronectin in matrices. Rat monoclonal antibody mAb 13, which recognizes the integrin beta 1 subunit, completely blocked binding and matrix assembly of 125I-fibronectin as well as binding of the 125I-70-kD amino-terminal fragment of fibronectin (70 kD) to fibroblast cell layers. Fab fragments of the anti-beta 1 antibody were also inhibitory. Antibody mAb 16, which recognizes the integrin alpha 5 subunit, partially blocked binding of 125I-fibronectin and 125I-70-kD. When cell layers were coincubated with fluoresceinated fibronectin and either anti-beta 1 or anti-alpha 5, anti-beta 1 was a more effective inhibitor than anti-alpha 5 of binding of labeled fibronectin to the cell layer. Inhibition of 125I-fibronectin binding by anti-beta 1 IgG occurred within 20 min. Inhibition of 125I-fibronectin binding by anti-beta 1 Fab fragments or IgG could not be overcome with increasing concentrations of fibronectin, suggesting that anti-beta 1 and exogenous fibronectin may not compete for the same binding site. No beta 1-containing integrin bound to immobilized 70 kD. These data indicate that the beta 1 subunit plays an important role in binding and assembly of exogenous fibronectin, perhaps by participation in the organization, regeneration, or cycling of the assembly site rather than by a direct interaction with fibronectin.     

Transforming growth factors from human colonic carcinoma cells induced molecular alterations in untransformed mouse AKR embryo fibroblasts

Over 700 polypeptide spots could be detected by two-dimensional electrophoretic analyses of membranes prepared from the murine AKR fibroblastic cells. Out of this abundance of polypeptides, only 9 polypeptide spots were found to be differentially expressed between the untransformed AKR-2B cells and their methylcholanthrene-transformed counterparts, the AKR-MCA cells. Treatment of the untransformed AKR-2B cells with transforming growth factors, prepared from the serum-free conditioned medium of HCT 116 MOSER human colonic carcinoma cells, induced the altered expression of 6 of these polypeptides which paralleled the electrophoretic profile of their permanently transformed counterparts, the AKR-MCA cells.     

Comparison of the effects of transforming growth factor beta, N,N-dimethylformamide, and retinoic acid on transformed and nontransformed fibroblasts

In order to compare the effects of transforming growth factor (TGF beta) with those of the differentiation promoters N,N-dimethylformamide (DMF) and retinoic acid (RA), the antiproliferative and fibronectin-inducing activities of the three agents were examined. AKR-2B mouse embryo fibroblasts and their chemically transformed counterpart AKR-MCA cells were used as the model system. Growth in monolayer culture of both cell lines was inhibited by TGF beta (EC50 approximately 1 ng/ml), DMF (EC50 approximately 0.5%), and RA (EC50 approximately 1 microM) in a concentration-dependent manner. Time-dependent elevation in fibronectin expression was also observed with all three agents. The EC50 for growth inhibition of both cell lines by TGF beta agreed well with that obtained for stimulation of fibronectin synthesis. A 3-h exposure to TGF beta is sufficient to obtain the maximal fibronectin level observed at 48 h in AKR-2 B cells but not in AKR-MCA cells. Our results indicate that in this system the effects of TGF beta are similar to those of the chemical differentiation inducers DMF and RA. Furthermore, our data also suggest that the TGF beta signal may be processed differently by nontransformed and transformed fibroblasts.     

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.     

Regulation of growth inhibitory activity in transformed mouse embryo fibroblasts

Treatment of the transformed mouse embryo fibroblast cell line AKR-MCA with 1% N,N-dimethylformamide (DMF) resulted in the restoration of a nontransformed phenotype in these cells. In order to determine if an increase in growth inhibitory peptides might be responsible for these changes in growth properties of the DMF-treated AKR-MCA cells we examined the serum-free conditioned medium for its ability to inhibit the anchorage-independent growth of a human colon carcinoma cell line. The extracellular levels of inhibitory activity were two-fold higher in conditioned medium derived from AKR-MCA cells than in AKR-MCA cells grown in 1% DMF (AKR-MCA/DMF). Fractionation of the crude conditioned medium indicated the presence of an Mr 20,000 inhibitory fraction in AKR-MCA/DMF conditioned medium which was reduced in AKR-MCA cells. This Mr 20,000 inhibitory activity was acid and heat stable and sensitive to dithiothreitol and trypsin. In addition to inhibiting the growth of a human colon carcinoma cell line this protein induced colony formation in AKR-2B cells and competed for binding to the transforming growth factor beta (TGF-beta) receptor. Therefore, this Mr 20,000 inhibitory polypeptide induced by DMF is probably TGF-beta. TGF-beta was also shown to inhibit the growth of AKR-MCA cells in monolayer culture.     

Identification of Porphyromonas gingivalis components that mediate its interactions with fibronectin.

Porphyromonas (Bacteroides) gingivalis W12 binds and degrades human plasma fibronectin. In the presence of the protease inhibitor N-alpha-p-tosyl-L-lysyl chloromethyl ketone, P. gingivalis cells accumulated substantial amounts of 125I-fibronectin as a function of incubation time. Fibronectin binding was specific, reversible, and saturable. The Kd for the reaction was estimated to be on the order of 100 nM, and there was an average of 3.5 x 10(3) fibronectin binding sites per cell. Unlabeled fibronectin inhibited the binding of 125I-fibronectin to bacteria; however, fibrinogen was an even more efficient inhibitor of 125I-fibronectin binding. Unrelated proteins were without effect on fibronectin binding. A fibronectin-binding component (Mr, 150,000) was identified in sodium dodecyl sulfate-solubilized P. gingivalis. Fibronectin was degraded into discrete peptides by P. gingivalis W12. The degradation of fibronectin was inhibited by N-alpha-p-tosyl-L-lysyl chloromethyl ketone. Two P. gingivalis components (Mrs, 120,000 and 150,000) degraded fibronectin in substrate-containing gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a previous study (M. S. Lantz, R. D. Allen, T. A. Vail, L. M. Switalski, and M. Hook, J. Bacteriol. 173:495-504, 1991), we found that the same strain of P. gingivalis bound and subsequently degraded human fibrinogen via apparently distinct cell surface components of molecular sizes similar to those of components now implicated in the binding and degradation of fibronectin. These results raise the possibility that the two ligands are recognized and modified by the same components on P. gingivalis W12. In support of this hypothesis, unlabeled fibrinogen effectively inhibited the binding of 125I-fibronectin to bacteria and blocked 125I-fibronectin binding to a P. gingivalis ligand-binding component (Mr, 150,000 immobilized on a nitrocellulose membrane.     

Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells

Suramin, a polyanionic compound, has previously been shown to dissociate platelet-derived growth factor (PDGF) from its receptor. In the present study suramin was found to inhibit the growth of sparse cultures of AKR-2B cells in fetal bovine serum (FBS)-supplemented medium in a dose-dependent, reversible fashion. Suramin also inhibited the ability of FBS, transforming growth factor beta (TGF beta), heparin-binding growth factor type-2 (HBGF-2), and epidermal growth factor (EGF) to stimulate DNA synthesis in density-arrested cultures of AKR-2B cells. The inhibition of growth factor-stimulated mitogenicity was directly correlated to the dose of suramin required to inhibit the binding of 125I-labeled TGF beta, HBGF-2, and EGF to their cell surface receptors. Suramin affected TGF beta and HBGF-2-related events at a 10-15-fold lower dose than that required for EGF-related events. It was also noted that suramin inhibited TGF beta-stimulated soft agar colony formation of AKR-2B (clone 84A) cells as well as the spontaneous colony formation of AKR-MCA cells, a chemically transformed derivative of AKR-2B cells. This demonstrates that suramin's spectrum of action for growth factors and their receptors should be extended to include TGF beta, HBGF-2, and EGF as well as PDGF. The data further suggest that the spontaneous growth of AKR-MCA cells in soft agar is dependent on growth factor binding to cell surface receptors.     

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.     

Binding of extracellular matrix proteins to the surface of Bacteroides spp

The binding of fibronectin an vitronectin to 207 Bacteroides strains and the binding of collagen and sialoproteins to 55 Bacteroides strains were investigated by means of latex agglutination tests. The binding of fibronectin, collagen and lactoferrin to the same 55 strains was also tested by using 125I-labelled proteins. The 207 strains, belonging to ten Bacteroides species, were isolated from different infections (51%) and from faeces of healthy subjects (49%). Most of the strains displaying fibronectin binding belonged in the species B. fragilis or B. vulgatus. The binding could be inhibited by preincubation of the cells with an excess amount of fibronectin. No inhibition of the binding was observed with carbohydrates. The vitronectin binding of the strains was less common, but was always observed to accompany fibronectin binding. None of the examined 55 strains exhibited any binding to fetuin or asialofetuin. The radiolabelling method indicated a low binding to 125I-fibronectin. The binding of 125I-collagen-I and 125I-lactoferrin in the Bacteroides strains tested was higher than that of 125I-fibronectin.     

The heparin III-binding domain of fibronectin (III4–5 repeats) binds to fibronectin and inhibits fibronectin matrix assembly

Fibronectin matrix assembly involves interactions among various regions of the molecule, which contribute to elongation and stabilization of the fibrils. In this study, we examined the possible role of the heparin III domain of fibronectin (repeats III4-5) in fibronectin fibrillogenesis. We show that a recombinant fragment comprising these repeats (FNIII4-5 fragment) blocked fibronectin fibril formation and the incorporation of 125I-fibronectin into cell layers. Binding assays using a biosensor revealed that FNIII4-5 bound fibronectin and the amino-terminal 70 kDa and 29 kDa fragments. It also bound to itself, indicating a previously unidentified self-association site in repeats III4-5. These interactions were specific since FNIII4-5 did not bind to the FNIII7-10 fragment, representing a central region in fibronectin. The fibronectin-binding property of the III4-5 domain, but not its matrix assembly inhibitory function, was apparently cryptic in larger fragments. By mutating the arginine residues in the WTPPRAQITGYRLTVGLTRR proteoglycan-binding sequence (HBP/III5 site) of FNIII4-5 [Moyano, J.V., Carnemolla, B., Albar, J.P., Leprini, A., Gaggero, B., Zardi, L., Garcia-Pardo, A., 1999. Cooperative role for activated alpha4beta1 integrin and chondroitin sulfate proteoglycans in cell adhesion to the heparin III domain of fibronectin. Identification of a novel heparin and cell binding sequence in repeat III5. J. Biol. Chem. 274, 135-142.], we found that the first two arginine residues in HBP/III5 were involved in the fibronectin-binding property of FNIII4-5, while the last two arginine residues in HBP/III5 were required for inhibition of matrix assembly and the binding of 125I-fibronectin to cell layers. Both properties appear to function independently from each other, depending on the conformation of the fibronectin dimer.     

Fibronectin-binding properties of the purified platelet glycoprotein IIb-IIIa complex

Fibronectin binds to specific receptors on the surface of washed, thrombin-activated platelets. Evidence suggests that these receptors are closely associated with the platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa). To determine whether GP IIb-IIIa itself can form a platelet receptor for fibronectin, we used a filtration assay to examine the interaction of purified fibronectin with purified GP IIb-IIIa incorporated into phospholipid vesicles. 125I-Fibronectin binding to the phospholipid vesicles required the presence of incorporated GP IIb-IIIa and was specific, time-dependent, reversible, saturable, and divalent cation-dependent (Mg2+ greater than Ca2+). The dissociation constant for 125I-fibronectin binding to the GP IIb-IIIa-containing vesicles in the presence of 2 mM MgCl2 was 87 nM. Proteins or peptides that inhibit 125I-fibronectin binding to whole platelets also inhibited 125I-fibronectin binding to the GP IIb-IIIa vesicles. Thus, specific 125I-fibronectin binding was inhibited by excess unlabeled fibrinogen or fibronectin, the anti-GP IIb-IIIa monoclonal antibody 10E5, the decapeptide from the carboxyl terminus of the fibrinogen gamma-chain, and the tetrapeptide Arg-Gly-Asp-Ser from the cell-binding domain of fibronectin. In contrast to results obtained using whole platelets, unlabeled fibronectin inhibited 125I-fibronectin binding to the GP IIb-IIIa vesicles. These results show that 125I-fibronectin binds directly to purified GP IIb-IIIa with most of the previously reported properties of 125I-fibronectin binding to washed, thrombin-stimulated platelets. Thus, GP IIb-IIIa has the potential to function as a platelet receptor for fibronectin as well as for fibrinogen.     

Continuous maintenance of transformed fibroblasts under reduced serum conditions: utility as a model system for investigating growth factor-specific effects in nonquiescent cells

We report the continuous growth maintenance of untransformed and chemically transformed fibroblasts (AKR-2B, AKR-MCA cells) in low concentrations of serum (0.1% FBS). The cell lines established (AKR-0.1F, MCA-0.1F) proliferated at rates comparable to cells maintained under high serum conditions (10% FBS). Complete removal of serum from the cells did not induce quiescence. The MCA-0.1F cells were more similar to the untransformed AKR-2B fibroblasts in their morphology, saturation density, inability to form colonies under anchorage-independent conditions, steady-state level of c-myc expression, and kinetics of induction of c-myc in response to specific growth factors. This report demonstrates the utility of this cell line as a nonquiescent model system for investigating growth factor-specific effects in serum-free, cycling cells. Addition of transforming growth factor-beta (TGF-beta) (5 ng/ml) to proliferating MCA-0.1F cells, in the absence of any serum, induced a multilayered growth pattern at confluency, similar to that of AKR-MCA cells maintained in 10% FBS. Other growth factors tested did not elicit this effect. The induction of this growth pattern by TGF-beta was associated with a sustained induction of the c-myc proto-oncogene at confluency, but not with a restoration of anchorage-independent growth. The data suggest that TGF-beta may play a role in the up-regulation of c-myc at confluency previously described for AKR-MCA cells maintained in 10% serum.     

Basolateral distribution of fibronectin matrix assembly sites on vascular endothelial monolayers is regulated by substratum fibronectin.

Endothelial cells exhibit binding sites for the amino terminus of fibronectin that participate in subendothelial fibronectin matrix assembly. These binding sites, termed matrix assembly sites, are localized on the basolateral surface of confluent endothelial monolayers (Kowalczyk et al. Blood, 75:2335, 1990). The present study investigates the role of cell-cell and cell-substratum interactions in the localization of matrix assembly sites to the basal surface of endothelial cells. Cells were cultured in Transwell culture inserts and matrix assembly sites were detected by binding assays using an iodinated 70 Kd amino-terminal fibronectin fragment. Integrity of intercellular junctions was monitored by measuring protein flux across Transwell filters. Time course experiments demonstrated that matrix assembly site expression on the basolateral cell surface preceded intercellular junction formation. Transfer of confluent monolayers to calcium-free medium resulted in the loss of junctions and in an increase in 125I-70 kD binding from the apical medium. The increased 125I-70 kD binding resulted from increased access of 125I-70 kD to basolateral matrix assembly sites and not from the relocation of binding sites to the apical membrane. To determine the effect of matrix composition on matrix assembly site expression and localization, cells were seeded onto vitronectin- or fibronectin-coated substrates. Fibronectin increased the expression of matrix assembly sites on the apical surface within 24 hours. By 48 hours, matrix assembly sites were located only on the basolateral surface. Vitronectin had no effect on the expression or localization of matrix assembly sites. These results indicate that the expression and localization of matrix assembly sites on the surface of vascular endothelial cells can be regulated by substratum fibronectin.     

Multiple binding sites in fibronectin and the staphylococcal fibronectin receptor.

The binding of fibronectin to Staphylococci exhibits the properties of a ligand-receptor interaction and has been proposed to mediate bacterial adherence to host tissues. To localize staphylococcal-binding sites in fibronectin, the protein was subjected to limited proteolysis and, of the generated fragments, Staphylococci appeared to preferentially bind to the N-terminal fragment. Different fibronectin fragments were isolated and tested for their ability to inhibit 125I-fibronectin binding to Staphylococci. The results indicate that only the N-terminal region effectively competed for fibronectin binding. However, when isolated fragments were adsorbed to microtiter wells, we found that two distinct domains, corresponding to the N-terminal fragment and to the heparin-binding peptide mapping close to the C-terminal end of fibronectin, promoted the attachment of both Staphylococcus aureus Newman and coagulase-negative strain of Staphylococcus capitis 651. These same domains were recognized by purified 125I-labeled staphylococcal receptor, either when immobilized on microtiter wells or probed after adsorption onto nitrocellulose membrane. The heparin-binding domain is comprised of type-III-homology repeats 14, 15 and 16. To determine which repeats participate in this interaction, we isolated and tested repeats type III14 and type III16. We found that the major staphylococcal binding site is located in repeat type III14. The staphylococcal receptor bound the N-terminal domain of fibronectin with a KD of 1.8 nM, whereas the dissociation constant of the receptor molecule for the internal heparin-binding domain was 10 nM. Since the fusion protein ZZ-FR, which contains the active sequences of fibronectin receptor (D1-D3) bound only to the N-terminus, it is reasonable to assume that the bacterial receptor may have additional binding sites outside the D domains, capable of interacting with the internal heparin-binding domain of fibronectin.     

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.     

Fibronectin binding to complement subcomponent C1q. Localization of their respective binding sites.

The interaction of purified human plasma fibronectin with the C1q subcomponent of complement was investigated by using a solid-phase radiobinding assay. 125I-fibronectin binding to native C1q, purified collagen domain (C1q-c) or globular domain (C1q-g) was compared. When the purified domains were insolubilized by binding to plastic, the C1q-c exhibited 59% of the binding demonstrated with intact C1q, whereas the C1q-g exhibited 35% of the binding. N-Terminal sequencing of the globular domain showed that a sequence of seven collagen-like amino acids was retained on each chain of the C1q-g fragment. 125I-fibronectin binding to C1q could be inhibited equally well by fluid-phase C1q and C1q-c, but not by fluid-phase C1q-g, implying that the collagen-like region retained on the C1q-g is masked in the fluid phase. In addition, studies were performed to determine which subunit(s) of C1q bind(s) fibronectin. The percentages of fibronectin bound by the A, B, and C chain of C1q were found to be 38, 21 and 41% respectively. Inhibition studies with purified 200-180 kDa, 50 kDa or 29 kDa fragments of fibronectin show that the binding site on fibronectin for C1q is the 50 kDa gelatin-binding domain.     

Anti-(Arg-Gly-Asp-Ser) antibody and its interaction with fibronectin, fibrinogen and platelets

An antibody population recognizing the sequence Arg-Gly-Asp-Ser (RGDS) in fibronectin, anti-(RGDS)N, was isolated by immunoadsorption. Between 2.5% and 4.9% of antibodies were obtained from two different anti-fibronectin sera indicating that this region represents an antigenic epitope in native fibronectin. Complete inhibition of binding of 125I-fibronectin to anti-(RGDS)N was produced only by nonreduced and reduced fibronectin. Fibrinogen and synthetic RGDS tetrapeptide, each at concentration of 10 microM, showed only a slight inhibition of 22% and 17%, respectively. Measurements of the conformational constant, the equilibrium constant for the interconversion of the non-native and native conformations of this epitope, showed that less than 0.0001% of the RGDS molecules adopt the native conformation in aqueous solutions. It indicates that long-range interactions in fibronectin and fibrinogen result in different conformations of the RGDS sequence in both proteins. Anti-(RGDS)N antibodies purified from anti-fibronectin serum had a strong inhibitory effect on thrombin-stimulated platelet aggregation. They also inhibited binding of fibronectin and fibrinogen to thrombin-stimulated platelets, supporting the primary role of the RGDS sequence in the direct interaction of these proteins with platelet membrane receptors.