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PubMed1988

Evaluation of fibronectin gene expression by in situ hybridization. Differential expression of the fibronectin gene among populations of human alveolar macrophages.

Populations of alveolar macrophages recovered from the lower respiratory tract secrete fibronectin, a multi-functional glycoprotein capable of influencing cell migration, attachment, differentiation, and proliferation. Using in situ hybridization of 35S-labeled antisense and sense RNA fibronectin probes, the present study demonstrates that most, but not all, normal alveolar macrophages contain fibronectin mRNA transcripts, and that among those macrophages expressing this gene, the relative amount of fibronectin mRNA transcripts varies from cell to cell. Interestingly, while 66 +/- 3% of normal alveolar macrophages contain fibronectin mRNA transcripts, this is increased to 82 +/- 2% (P less than 0.01) of alveolar macrophages recovered from the lungs of individuals with idiopathic pulmonary fibrosis (IPF), a chronic inflammatory disorder associated with exaggerated amounts of fibronectin in the lower respiratory tract. Furthermore, of the macrophages expressing the fibronectin gene, those from IPF patients contain more fibronectin mRNA transcripts than those from normals. Consistent with this observation, evaluation of tissue samples from IPF patients demonstrated that of all cells present, alveolar macrophages showed the greatest numbers of fibronectin mRNA transcripts per cell. These observations demonstrate that there can be marked cell-to-cell variation in the expression of the gene for a macrophage product such as fibronectin, suggesting that there are processes that modulate similar cells in the same anatomic compartment to vary their expression of the same gene.

Adult↗
PubMed1981

Gelatin-binding domain-specific anti-human plasma fibronectin Fab' inhibits fibronectin-mediated gelatin binding but not cell spreading.

Antisera against a Mr = 60,000 peptide containing the gelatin-binding domain of human plasma fibronectin (McDonald, J. A., and Kelley, D. G. (1980) J. Biol. Chem. 255, 8848-8858) bound the Mr = 60,000 peptide and intact fibronectin but not three other fragments released by leukocyte elastase proteinolysis (the Mr = 25,000 amino-terminal sequence, Mr = 140,000 sequence containing cell adhesive activity, and a Mr = 31,000 fragment). Affinity-purified Fab' blocked Mr = 60,000 peptide binding to gelatin and inhibited plasma and cellular fibronectin gelatin binding without affecting fibronectin-mediated cell spreading. In contrast, antifibronectin Fab' absorbed with the gelatin-binding fragment completely blocked fibronectin-mediated cell spreading. These data indicate that the gelatin-binding domain of fibronectin is immunogenic, and antisera against this domain recognize cellular fibronectin gelatin-binding sites. Inhibition of gelatin binding but not cell spreading by anti-gelatin binding domain Fab' confirms the hypothesis that fibronectin has separate sites mediating these activities. Selective inhibition of fibronectin-collagen binding by domain-specific antisera may help elucidate the role of fibronectin in organization of the extracellular matrix.

Animals↗
PubMed1986

Variants of BALB/c 3T3 cells lacking complex gangliosides retain a fibronectin matrix and spread normally on fibronectin-coated substrates.

Evidence has accumulated that di- and trisialogangliosides are involved in the interaction of cells with fibronectin. We have therefore tested the ability of variants of BALB/c 3T3 deficient in such gangliosides to organize a fibronectin matrix and to spread on fibronectin-coated substrates. Whereas BALB/c 3T3 cells contained gangliosides GM3, GM1, and GD1a, direct chemical analysis showed that five out of six variants isolated contained no detectable GD1a. By the overlaying of thin layer chromatograms of cellular gangliosides with 125I-cholera toxin, these variants were also found to lack ganglioside GM1. In contrast, the sialogalactoprotein profile of these cells, analyzed using an 125I-ricin/SDS polyacrylamide gel overlay technique, was similar to that of the parent cell line. All variants organized an extensive fibronectin matrix comparable to that of BALB/c 3T3, as shown using either immunofluorescence or lactoperoxidase-catalyzed iodination. The variants could also spread on fibronectin-coated substrates and adopt a morphology similar to that of BALB/c 3T3 cells, with little or no difference in the concentration of fibronectin required for 50% cell spreading. Cell spreading of the variants was accompanied by the formation of focal contacts and microfilament bundles, in a manner closely resembling that seen with BALB/c 3T3 cells. Treatment of BALB/c 3T3 cells with neuraminidase, which converts much of the cellular GD1a to GM1, did not affect cell spreading on fibronectin. The results clearly demonstrate that complex gangliosides are not essential for retention of a fibronectin matrix or for spreading on fibronectin-coated substrates.

Actin Cytoskeleton↗
PubMed1992

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.

Binding Sites↗
PubMed1981

Fibronectin from human germ-cell tumors resembles amniotic fluid fibronectin.

Three human germ-cell tumors established as xenografts in immunosuppressed mice were shown to produce fibronectin. Immunoperoxidase tissue staining with a specific antiserum revealed human fibronectin in some tumor cells. The cyst fluid which accumulated in the tumors contained human fibronectin as shown by a species-specific radioimmunoassay. Human fibronectin was isolated from two of the tumor fluids and compared to previously characterized fibronectins. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and gel electrofocusing showed that the germ-cell fibronectins resembled amniotic fluid fibronectin with regard to subunit molecular weight and isoelectric point. The distinct features of the amniotic fluid and germ-cell tumor fibronectin suggest that such fibronectins may provide new oncodevelopmental markers.

Amniotic Fluid↗
PubMed1994

Binding of hyaluronan to plasma fibronectin increases the attachment of corneal epithelial cells to a fibronectin matrix.

We wished to determine whether hyaluronan would affect the attachment of epithelial cells to extracellular matrix proteins. Multiwell tissue culture plates were coated with human plasma fibronectin, laminin, or collagen type IV (0.01-10.0 micrograms/ml). Single-cell suspensions of rabbit corneal epithelial cells were placed in the wells, and after 45 minutes incubation the cells adhering to the matrix proteins were stained and counted. Cells attached to all three types of proteins. Preincubation of the matrix proteins with hyaluronan (0.1-1.0 mg/ml) significantly increased the number of cells attached to the fibronectin matrix, but it did not increase the numbers of cells attached to laminin or collagen type IV. Hyaluronidase inhibited this stimulatory effect. Glycosaminoglcyans other than hyaluronan (chondroitin sulfate, keratan sulfate, or heparan sulfate) failed to increase the numbers of attached cells. Treatment of the fibronectin matrix with monoclonal antibodies against the cell-binding domain of fibronectin (FN12-8 or FN30-8, 0.03-0.3 mg/ml, for 1 hour), before or after hyaluronan treatment, significantly decreased the numbers of attached cells. Monoclonal antibody against the fibrin- and heparin-binding domain at the N-terminal (FN9-1), however, significantly decreased the number of attached cells only when this antibody treatment preceded the hyaluronan treatment. Preincubation of the cells with hyaluronan had no effect; preincubation with GRGDSP (1 mg/ml), a synthetic peptide that blocks the cell surface receptor for fibronectin, significantly decreased cell attachment whether the fibronectin matrix was treated with hyaluronan or not. Further studies demonstrated that monoclonal antibody against the fibrin- and heparin-binding domain at the N-terminal of plasma fibronectin prevented radiolabeled hyaluronan from binding to fibronectin; likewise, the isolated N-terminal fragment, coupled with Sepharose 4B, bound to hyaluronan in columns. We conclude that hyaluronan binds to a fibrin- and heparin-binding domain at the N-terminal of plasma fibronectin and facilitates the attachment of epithelial cells.

Animals↗
PubMed1987

Developmental study of human fetal placental fibronectin: alterations in carbohydrates of tissue fibronectin during gestation.

Human term placental tissue fibronectin contains about twice the carbohydrate content of human adult plasma fibronectin or fetal plasma fibronectin. The chief difference is the presence of substantial amounts of large N-linked polylactosamine chains on the placental fibronectin. The large carbohydrate on placental tissue fibronectin weakens the binding of fibronectin to denatured collagen. To examine whether a developmental change takes place in the placental fibronectin during gestation, fibronectin was isolated from placentas of different developmental stages beginning with the first trimester and ending with term. Polylactosamine carbohydrate, as well as total quantity of carbohydrate, increased steadily during gestation, reaching a maximum at term of more than 9% carbohydrate. Weakened binding of fibronectin to collagen occurred near the end of gestation concomitant with an increase in the quantity of larger polylactosamine glycopeptides. Relationships among these developmental changes, the impending birth, and the end of the function of the placenta remain to be investigated.

Amniotic Fluid↗
PubMed1983

Concurrent modulation of cell surface fibronectin and adhesion to fibronectin in hepatoma cells.

Rat hepatoma cells grown in vitro were poorly adhesive to plastic surfaces coated with fibronectin and lacked cell surface fibronectin matrix. They synthesized soluble fibronectin into the medium. The cell surface fibronectin matrix and the ability to attach to fibronectin-coated surface were restored in the 7777 cells upon passage as a tumor in rats and by coculturing these cells with normal liver-derived cells in vitro. Fibronectin matrix and the ability of cells to attach to fibronectin were thus modulated in a coordinated fashion, suggesting that the formation of a cell surface fibronectin matrix is dependent on the cell surface property that enables cells to interact with fibronectin.

Animals↗
PubMed1988

Fibronectin fibril formation involves cell interactions with two fibronectin domains.

Fibronectin fragments and domain-specific antibodies have been used to study the mechanism by which cells reorganize exogenous fibronectin substrata into fibrils. Fibroblasts prevented from protein synthesis, and hence not secreting endogenous fibronectin or other matrix components, reorganized exogenous fibronectin substrata into arrays resembling the matrix of normally cultured cells. Cells also formed fibrils from substrata containing mixtures of cell- and either of two different heparin-binding fibronectin fragments but not from either fragment alone. The gelatin-binding fragment alone or in conjunction with the cell-binding fragment did not promote fibril formation. Antibodies recognizing cell- and either heparin- or the gelatin-binding domains labeled fibrils formed by cells under normal culture conditions or when a substratum of intact fibronectin was used as the sole exogenous source. However, only antibodies recognizing the cell- or either heparin-binding fragment reduced fibrillogenesis from intact fibronectin substrates when added during cell spreading. These data suggest that formation of fibronectin fibrils can occur at the cell surface and that membrane components recognizing the cell- and the heparin-binding domains in fibronectin may cooperate in the assembly process.

Animals↗
PubMed1990

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

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

Antibodies, Monoclonal↗
PubMed1993

Inhibition of fibronectin binding and fibronectin-mediated cell adhesion to collagen by a peptide from the second type I repeat of thrombospondin.

The platelet and extracellular matrix glycoprotein thrombospondin interacts with various types of cells as both a positive and negative modulator of cell adhesion, motility, and proliferation. These effects may be mediated by binding of thrombospondin to cell surface receptors or indirectly by binding to other extracellular matrix components. The role of peptide sequences from the type I repeats of thrombospondin in its interaction with fibronectin were investigated. Fibronectin bound specifically to the peptide Gly-Gly-Trp-Ser-His-Trp from the second type I repeat of thrombospondin but not to the corresponding peptides from the first or third repeats or flanking sequences from the second repeat. The two Trp residues and the His residue were essential for binding, and the two Gly residues enhanced the affinity of binding. Binding of the peptide and intact thrombospondin to fibronectin were inhibited by the gelatin-binding domain of fibronectin. The peptide specifically inhibited binding of fibronectin to gelatin or type I collagen and inhibited fibronectin-mediated adhesion of breast carcinoma and melanoma cells to gelatin or type I collagen substrates but not direct adhesion of the cells to fibronectin, which was inhibited by the peptide Gly-Arg-Gly-Asp-Ser. Thus, the fibronectin-binding thrombospondin peptide Gly-Gly-Trp-Ser-His-Trp is a selective inhibitor of fibronectin-mediated interactions of cells with collagen in the extracellular matrix.

Amino Acid Sequence↗
PubMed1984

Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function.

Fibronectin and certain polypeptide regions of this adhesive glycoprotein mediate cell attachment and spreading on various substrates. We explored the theoretical prediction that this adhesive protein could become a competitive inhibitor of fibronectin-mediated processes if present in solution at appropriately high concentrations. Fibronectin function was inhibited by purified plasma fibronectin at 5-10 mg/ml, by a 75,000-dalton cell-interaction fragment of the protein at 0.5-1 mg/ml, and even by two synthetic peptides containing a conserved, hydrophilic amino acid sequence at 0.1-0.5 mg/ml. Inhibition of fibronectin-dependent cell spreading was dose dependent, noncytotoxic, and reversible. It was competitive in nature, since increased quantities of substrate-adsorbed fibronectin or longer incubation periods decreased the inhibition. A peptide inhibitory for fibronectin-mediated cell spreading also inhibited fibronectin-mediated attachment of cells to type I collagen, but it did not affect concanavalin A-mediated spreading. These results demonstrate the potential of a cell adhesion molecule and its biologically active peptide fragments to act as competitive inhibitors, and they suggest that fibronectin may act by binding to a saturable cell surface receptor.

Amino Acid Sequence↗
PubMed1987

Effect of fibronectin therapy and fibronectin deficiency on wound healing: a study in rats.

Rats with standardized 3rd-degree burns of 1% body surface were treated intraperitoneally with different doses of human fibronectin or swine gelatin immediately after injury and on the following days. Controls received bovine albumin or no further treatment. Wound healing was assessed by planimetry (days 0, 2, and 7); additionally, plasma fibronectin was determined (days 3 and 7). On day 3 fibronectin levels were significantly elevated after albumin and the highest dosage of fibronectin (3 X 16 mg) and decreased after gelatin and the lowest dosage of fibronectin (3 X 4 mg). Wound healing was neither affected by albumin nor by human fibronectin, but it was significantly impaired by gelatin-induced lack of fibronectin. The data support the opinion that fibronectin is an essential factor for the post-traumatic clearance of gelatin-like tissue debris via the reticuloendothelial system (RES). Overload by gelatin results in fibronectin deficiency and RES dysfunction with risks of infection and poor wound healing.

Animals↗
PubMed1988

Progressive loss of fibronectin-mediated opsonic activity in plasma cryoprecipitate with storage. Role of fibronectin fragmentation.

Septic injured patients often manifest a deficiency of plasma fibronectin. Several studies have shown improvements in organ function in such patients following infusion of fibronectin-rich plasma cryoprecipitate, while other studies found no improvement. One explanation for these differences may be the use of plasma cryoprecipitate which has been stored for various time intervals prior to its use as a source of fibronectin. This investigation tested the hypothesis that the opsonic activity of fibronectin in cryoprecipitate may decline with increased storage duration. Using a bioassay of opsonic activity, we evaluated human plasma cryoprecipitate that was stored at either -20 or -80 degrees C for various intervals (2 weeks to 12 months) after its preparation from fresh donor plasma. Our findings demonstrated that the opsonic activity of fibronectin in cryoprecipitate declined with increasing time of storage. Significant loss (p less than 0.05) of opsonic activity was first evident after 2 months of storage. Storage at -80 degrees C did not prevent this decline in opsonic activity as compared to storage at -20 degrees C. Immunoblot analysis revealed extensive fragmentation of the dimeric fibronectin (440 kdaltons) and the presence of lower molecular weight fragments in 4- to 12-month-old plasma cryoprecipitate. Therefore, plasma cryoprecipitate of varying ages (storage time) when used as a source of fibronectin for replacement therapy to support phagocytic function in septic injured patients may result in different fibronectin-mediated responses. The decline in activity may be due, in part, to fragmentation of the fibronectin molecule.

Biological Assay↗
PubMed1985

Attachment of staphylococci and streptococci on fibronectin, fibronectin fragments, and fibrinogen bound to a solid phase.

The attachment of Staphylococcus aureus (Cowan I) and two strains of group A and G streptococci on glass cover slips coated with fibronectin, fibronectin fragments, or fibrinogen was studied. The attachment was quantitated by counting the attached bacteria on glass surfaces coated with a similar molarity of the proteins. Fibronectin was a more effective attachment factor than fibrinogen for staphylococci, while group G streptococci attached better on fibrinogen- than on fibronectin-coated cover slips. In this system, group A streptococci bound almost exclusively to substrate-bound fibrinogen. Attachment experiments involving the use of staphylococci pretreated with soluble fibronectin or fibrinogen revealed that bacterium-bound fibronectin and fibrinogen were able to enhance the adherence on cover slips coated with fibronectin. The 30-kilodalton NH2-terminal and the 120- to 140-kilodalton COOH-terminal fragments of fibronectin, both of which contain bacterial binding sites, mediated the staphylococcal attachment, suggesting that both parts of the molecule are involved in the attachment mediated by fibronectin.

Adhesiveness↗
PubMed2001

Lactobacillus casei acquires the binding activity to fibronectin by the expression of the fibronectin binding domain of Streptococcus pyogenes on the cell surface.

Fibronectin binding domain was expressed on the cell surface of Lactobacillus casei strain Shirota which hardly adheres to fibronectin. DNA for the fibronectin binding domain of the sfbl gene, which encodes a fibronectin binding protein of Streptococcus pyogenes ATCC 21059, was amplified with polymerase chain reaction, cloned into a surface display vector pSAK332, and introduced into L. casei. The fibronectin binding domain was expressed as a fusion protein consisting of staphylokinase of Staphylococcus aureus and the anchor sequence of cell wall-associated 763 proteinase of Lactococcus lactis NCDO 763. The fibronectin binding ability of the resulting L. casei was confirmed with Western blot analysis, immunoelectron microscopic analysis, and adherence to fibroblast cells. These results indicate that L. casei has acquired a new phenotype to bind fibronectin upon the expression of the fibronectin binding domain on the cell surface. This L. casei also shows binding affinity to fibrinogen, indicating that fibronectin binding domain is involved in the binding to fibrinogen as well.

Adhesins, Bacterial↗
PubMed1992

Cancer-associated changes in glycosylation of fibronectin. Immunohistological localization of oncofetal fibronectin defined by monoclonal antibodies.

The extracellular matrix adhesion molecule fibronectin exhibits different isoforms derived by alternative splicing as well as recently demonstrated variation in O-glycosylation. Although fibronectin is widely distributed in normal tissues, the individual isoforms have been found to show restricted tissue distribution and association with malignancies. The monoclonal antibody FDC-6 defines a cancer-associated de novo glycosylation of a specific threonine residue in the C-terminal region of the fibronectin molecule termed oncofetal fibronectin. Here we report an immunohistological study of oral squamous cell carcinomas (n = 33), premalignant lesions (n = 15), and normal oral mucosa (n = 10) using the FDC-6 antibody. A selective expression of the oncofetal fibronectin epitope was demonstrated in close relation to the invading carcinoma, whereas no staining was observed in premalignant lesions without epithelial dysplasia, or in normal epithelium. Furthermore, we attempted to identify additional carbohydrate-related epitopes distinguishing fibronectin of human hepatoma cell line HUH-7 from plasma fibronectin. No novel epitopes were identified, as all generated monoclonal antibodies lacking reactivity with plasma fibronectin showed the same specificity as FDC-6. Previous studies have indicated that the de novo glycosylation is induced by a novel transferase activity only found in fetal and carcinoma cell lines, placenta and hepatoma tissues. Here we provide further evidence that a purified UDP-GalNAc:peptide N-acetylgalactosaminyltransferase from normal bovine thymus and human placentae is incapable of utilizing the hexapeptide VTHPGY as a substrate. The results demonstrate that oncofetal fibronectin is highly associated with malignancy, and appears to be induced by expression of a unique glycosyltransferase or modification of the specificity of the normally expressed transferase.

Aged↗
PubMed1991

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.

Antibodies, Monoclonal↗