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PubMed1995

Expression of fibronectin, fibronectin isoforms and integrin receptors in melanocytic lesions.

In vitro studies have demonstrated that fibronectin (FN) can deliver a mitogenic signal to quiescent human melanoma cells and that the alpha 5/beta 1-integrin receptor mediates this stimulus. In view of this finding we have analysed the in vivo expression of FN, and of ED-A and ED-B FN isoforms, in benign and malignant lesions of melanocyte origin. In the same specimens the expression of fibronectin integrin receptors was evaluated. The results demonstrate that, while detection of FN does not correlate with transformation and tumour progression, the expression of the two isoforms is associated with transformation and that only the ED-A variant is found in metastases. Integrin phenotyping disclosed that alpha 3/beta 1 expression is associated with tumour progression, alpha v/beta 3 is a marker of transformation, alpha 4 is rarely expressed and alpha 5 is expressed by about 50% and 30% of the primary and metastatic lesions respectively. Taken together, the results of this study demonstrate that transformation and tumour progression of the melanocyte lineage are associated with modulation of expression of FN isoforms and FN integrin receptors. Furthermore, the expression of alpha 5-integrin in a considerable percentage of primary and metastatic lesions indicates that FN may deliver a proliferative stimulus to melanoma cells in vivo.

Animals↗
PubMed2004

Interaction of Staphylococcus aureus fibronectin-binding protein with fibronectin: affinity, stoichiometry, and modular requirements.

The repetitive D1, D2, and D3 elements of Staphylococcus aureus fibronectin-binding protein FnBPA each bind the N-terminal 29-kDa fragment (N29) of fibronectin with low micromolar dissociation constants (Kd), but in tandem they compose a high affinity domain, D1-3. An additional seven Fn-binding segments have been predicted in FnBPA in a region N-terminal of the D-repeats (Schwarz-Linek, U., Werner, J. M., Pickford, A. R., Gurusiddappa, S., Kim, J. H., Pilka, E. S., Briggs, J. A., Gough, T. S., Hook, M., Campbell, I. D., and Potts, J. R. (2003) Nature 423, 177-181). We have evaluated the requirements for high affinity binding of N29 to the D-repeat domain and determined the affinity and stoichiometry of N29 binding to segments that are N-terminal of the D-repeats in the related FnBPB adhesin. We confirmed that D1-3 has two equivalent high affinity sites (Kd, approximately 1 nm) and provided evidence for one or more lower affinity sites (Kd, approximately 0.5 microm). Bimodular D1-2 and D2-3 exhibit intermediate affinity sites with respective Kd values of 0.25 and 0.044 microm, as well as a low affinity site with a Kd value of 2.2-2.5 microm. We also identified two binding domains that are N-terminal of the D-repeats, designated DuB and DuA. Segments internal to these domains individually bound N29 with similar Kd values of approximately 2 microm, whereas the DuBA polypeptide possessing both segments and other intervening sites bound four molecules of N29 with much higher affinity (Kd, approximately 10 nm). DuBAD, a larger polypeptide harboring all of the known or predicted binding motifs in FnBPB, bound seven to eight molecules of N29, with a Kd of approximately 7 nm. Because most of the isolated binding segments display low affinity for N29 and lack motifs for binding of one or both of the 1F1 and 5F1 modules in the N-terminal domain of Fn, we propose that high affinity is achieved in part as a consequence of self-interaction between bound molecules of N29.

Adhesins, Bacterial↗
PubMed1999

Bacterial fibronectin-binding proteins and endothelial cell surface fibronectin mediate adherence of Staphylococcus aureus to resting human endothelial cells.

Adhesion of Staphylococcus aureus to human endothelial cells is implicated in the pathogenesis of invasive staphylococcal disease. The adhesion to endothelial cells of isogenic mutants defective in defined surface structures was studied. Three strains of S. aureus defective in fibronectin-binding proteins FnBPA and FnBPB showed reduced adhesion. This was fully restored by complementation of a FnBPA- FnBPB- mutant derived from strain 8325-4 with a multicopy plasmid encoding FnBPA or FnBPB. Adhesion of mutants defective in other surface structures was unaffected. Anti-fibronectin antibodies blocked adhesion of 8325-4 to endothelial cells, while adhesion of strains 8325-4, P1 and five clinical isolates was inhibited by the recombinant form of the binding domain of FnBPB (rFNBD) from Streptococcus dysgalactiae. Adherence of bacterial aggregates resulting from the presence of purified fibrinogen was also inhibited by rFNBD protein. Three strains of S. aureus defective in FnBPA and FnBPB were not internalized by endothelial cells. S. aureus FnBPs mediate adhesion to human endothelial cells and are required for subsequent internalization, interactions of potential relevance to pathogenesis and treatment.

Adhesins, Bacterial↗
PubMed1997

Identification of D motif epitopes in Staphylococcus aureus fibronectin-binding protein for the production of antibody inhibitors of fibronectin binding.

A fibronectin-binding protein (FnBP) adhesin of Staphylococcus aureus possesses three 37- or 38-amino-acid motifs (D1, D2, and D3) that can each bind fibronectin (Fn) with low affinity and that in tandem comprise D1-3, a high-affinity Fn-binding domain. To identify epitopes for the generation of adhesion-blocking antibodies, rabbits were immunized with recombinant D1-3 or with a glutathione S-transferase fusion protein, GSTD1-3. Affinity-purified antibodies from the D1-3 immunization were poor inhibitors of Fn binding to S. aureus and recognized several different epitopes, with a preference for clusters of acidic amino acids that do not contribute to Fn binding. Antibodies generated with GSTD1-3 as an immunogen were more effective inhibitors, but concentrations in excess of 20 microg x ml-1 did not promote more than 50% inhibition. These antibodies were highly specific for amino acids 21 to 34 of D1 (D1(21-34)), which contain a sequence that is essential for Fn binding and are identical to D2 at 12 of 14 residues. Neither antibody preparation recognized D3(20-33) of the D3 motif, where the only homology to D1(21-34) and D2(21-34) comprises a sequence motif, GG(X3,4)(I/V)DF, that is critical to Fn binding. However, antibodies specific for both D1(21-34) and D3(20-33) could be obtained by using synthetic peptides corresponding to these sequences as immunogens. F(ab')2 fragments derived from these antibodies each caused 40 to 50% inhibition of Fn binding to S. aureus, and their ability to bind to purified FnBP was eliminated by competing Fn. However, mixtures of the two F(ab')2 preparations did not provide additive or synergistic inhibition of Fn binding. Therefore, inhibition of Fn binding to S. aureus requires antibodies specific for D1(21-34) and D3(20-33), but a mixture of antibodies specific for both sequences did not provide complete inhibition.

Adhesins, Bacterial↗
PubMed1991

Immobilization of soluble fibrin on factor XIIIa-coated polystyrene beads mediated by N-terminal fibronectin fragments. II. Demonstration of covalent adducts of fibrin peptide chains and fibronectin fragments.

Previous experiments had shown that the free N-terminal fibronectin 30-kDa-domain mediates binding of soluble 125I-fibrin to transamidase-coated polystyrene beads (Hörmann et al., Biol. Chem. Hoppe-Seyler 368, 669-674, 1987). Now, the formation of covalent adducts of the N-terminal fragment with fibrin peptide chains is demonstrated. Binding of soluble 125I-fibrin was performed in presence of N-terminal fibronectin 30-kDa or 70-kDa fragments. The material adsorbed was removed from the beads under reducing conditions and analysed by dodecylsulfate gel electrophoresis followed by autoradiography. The 30-kDa fragment gave rise to bands of 80 kDa and 180-200 kDa which were lacking in the products of the 70-kDa compound. Instead, they showed bands at 120 kDa and ca. 280 kDa. Evidently, those bands represented covalent adducts of fibrin peptide chains or their dimers with the 30-kDa or the 70-kDa fragment, respectively. In addition, dimeric gamma-chains and alpha-chain polymers of fibrin were present indicating partial polymerization of bead-attached fibrin.

Electrophoresis, Polyacrylamide Gel↗
PubMed2001

Fluorescence polarization assay and SDS-PAGE confirms matrilysin degrades fibronectin and collagen IV whereas gelatinase A degrades collagen IV but not fibronectin.

Matrilysin and gelatinase A are hypothesized to have significant roles in uterine and ovarian function. However, proteolytic activity assays for these enzymes are limited. We describe the development of simple and rapid assays for the proteolysis of fluorescein-labeled full-length substrates, collagen IV (Col-IV) and fibronectin (FN), and demonstrate the selectivity of matrilysin (MMP-7) compared to gelatinase A (MMP-2) for fibronectin. Changes in fluorescence intensity (FIU) and fluorescence polarization (mP) resulting from the protease activity of matrilysin and gelatinase A were measured. These studies show that the fluorescently labeled substrates, Col-IV and FN, are as reliable and amenable to rapid in vitro assay as peptide substrates. In addition, they are easier to use than previously described, non-fluorescent methods. The results demonstrate that assays using full-length, biological matrix proteins are more sensitive indicators of MMP-specific substrate activity than peptide based assays.

Animals↗
PubMed1994

[Circulating complexes of plasma fibronectin (fibronectin-fibrin) in human diseases].

The study of circulating complexes fibronectin-fibrin in 21 patients with immunocomplex affections revealed their high levels which occurred in inverse relationships with fibronectin concentrations in the blood, but in direct correlation with indicators of inflammation (fibrinogen, cialic acid). It is suggested that the complexes are involved in the development of fibrosis at the sites of chronic inflammation in immune disorders.

Adult↗
PubMed1999

The tetraspanin CD9 influences the adhesion, spreading, and pericellular fibronectin matrix assembly of Chinese hamster ovary cells on human plasma fibronectin.

The role of CD9 in cell adhesion and spreading on adhesive proteins was investigated using a transfected Chinese hamster ovary (CHO) cell system. CD9 cell surface expression resulted in reduced adhesion and increased spreading on fibronectin (Fn). Whereas mock-transfected (mock CHO) and naïve CHO cells assumed a typical fibroblast spindle shape morphology, CD9-transfected (CD9-CHO) cells were polygonal with many filipodial projections and exhibited a twofold greater surface area. The spread morphology of CD9-CHO cells, but not mock CHO cells, was inhibited by PB1 mAb blockade of alpha(5)beta(1), suggesting that the coexpression of alpha(5)beta(1) and CD9 influenced cell activity on Fn. The second extracellular loop of CD9 was implicated in regulation of adhesion since reduced CD9-CHO cell adhesion on Fn was reversed by either anti-CD9 antibody ligation to the second extracellular loop or with cells expressing a CD9 mutant lacking the second extracellular loop domain. Using cell adhesion assays and ELISA, we demonstrated CD9 binding to the HEP2/IIICS region of Fn. Finally, CD9 expression resulted in a twofold reduction in Fn-rich pericellular matrix assembly. Our observations show that CD9 dramatically influences CHO cell interactions with Fn and suggest that CD9 has an important role in modulating cell-extracellular matrix interactions.

Animals↗
PubMed1995

Fibronectin binding by Streptococcus milleri group strains and partial characterisation of the fibronectin receptor of Streptococcus anginosus F4.

The Streptococcus milleri group were shown to bind fibronectin (Fn) to their cell-surface and this binding increased the adhesion of cells to hydroxyapatite. The binding of Fn to Streptococcus anginosus F4 was studied in more detail. Fn binding to bacterial cells increased the association of the bacteria with the polymorphonuclear leukocytes obtained from the peritoneal cavity of rats but did not increase killing of the bacteria. The cell-surface receptor was a protein of M(r) 14,000 which was released from cells after mutanolysin digestion. The binding was specific, with cells having a maximum number of binding sites per cell of 770. Electron microscopy, using gold-labelled Fn, localised the receptor to areas between daughter cells.

Animals↗
PubMed1987

Adhesion of lymphoid cell lines to fibronectin-coated substratum: biochemical and physiological characterization and the identification of a 140-kDa fibronectin receptor.

Little information is available on the interaction between lymphocytes and fibronectin (fn). To gain a better understanding on this issue we examined the adhesion of 12 lymphoid cell lines, each exhibiting different phenotypic characteristics, to fn-coated substratum. Of the cell lines tested, five that adhered to fn possessed B-cell characteristics, while neither the T-cell lines nor the pre-B-cell line adhered. The physiology and biochemistry of adhesion of a B-cell line, MOPC 315, were examined in detail. Our results indicated that (1) the adhesion was a specific and time-dependent process, (2) the adhesion was temperature-dependent and inhibited by metabolic inhibitors, such as KCN and 2-deoxyglucose, (3) the presence of cycloheximide and pretreatment of cells with trypsin inhibited adhesion, (4) a 140-kDa surface protein was immunoprecipitated by anti-fn receptor antibodies, (5) the presence of divalent cations was essential for adhesion, (6) the presence of colchicine had no effect on adhesion, while cytochalasin B partially inhibited adhesion, and (7) the treatment of cells by both phorbol 12-myristate 13-acetate and calcium ionophore A23187 enhanced adhesion. In this study, we have established the interaction between lymphoid cell lines and fn. Such an interaction might play an important role in the behavior of lymphocytes in tissues.

Animals↗
PubMed1991

Phorbol ester-induced differentiation of U937 cells enhances attachment to fibronectin and distinctly modulates the alpha 5 beta 1 and alpha 4 beta 1 fibronectin receptors.

Monocyte interaction with fibronectin (Fn) involves specific cell surface receptors and results in cell attachment and differentiation. We have studied the regulation of these receptors using the promonocytic cell line U937 and its PMA-induced differentiation as a model. We recently reported that U937 cells interact with two sites in Fn, RGD and CS-1, via two independent receptors (O. C. Ferreira, A. Garcia-Pardo, and C. Bianco (1990) J. Exp. Med. 171, 351). In this study we have determined the effects of PMA on the interaction of U937 cells with both sites in Fn. PMA-U937 cells showed an enhanced attachment to Fn and to an RGD-containing 80-kDa Fn fragment. This enhancement paralleled a two- to threefold increase in the surface expression of the RGD-dependent receptor alpha 5 beta 1. An anti-alpha 5 beta 1 mAb completely inhibited cell adhesion to Fn and to the 80-kDa fragment. alpha 5 beta 1 receptors from untreated and PMA-treated U937 cells were isolated on 80-kDa-Sepharose columns and shown to contain a similar complex of 152/125-kDa proteins, although proteins from PMA-treated cells had slightly faster mobility on SDS-gels. In contrast, the total number of PMA-U937 cells adhering to a 38-kDa Fn fragment (containing the CS-1 site) was lower when compared to that of untreated cells. This decrease was accompanied by a 50% loss of cell surface alpha 4 beta 1, the specific receptor for CS-1. Our results indicate that differentiation of U937 cells enhances adhesion to Fn primarily by up-regulating the alpha 5 beta 1 Fn receptor. PMA also induces a down-regulation of alpha 4 beta 1, suggesting that these two integrins play different roles during monocyte differentiation.

Amino Acid Sequence↗
PubMed2005

Fibronectin fragmentation promotes alpha4beta1 integrin-mediated contraction of a fibrin-fibronectin provisional matrix.

In injured tissues, the fibrin-fibronectin (FN) provisional matrix provides a framework for cell adhesion, migration, and repair. Effective repair and remodeling require a proper balance between extracellular matrix (ECM) deposition, contraction, and turnover. We utilized a three-dimensional (3D) fibrin-FN provisional matrix model to determine the contributions of the FN-binding integrin receptors alpha5beta1 and alpha4beta1 to matrix contraction. CHOalpha5 cells expressing alpha5beta1, a receptor for FN's RGD cell-binding domain, were highly contractile, and cells were well spread on a 3D fibrin-FN matrix. In contrast, CHOalpha4 cells expressing the alpha4beta1 receptor for FN's alternatively spliced V region attached less efficiently to FN and were deficient in fibrin-FN matrix contraction. Surprisingly, cell adhesion and matrix contraction by CHOalpha4 cells were dramatically enhanced, to levels equivalent to CHOalpha5 cells, when proteolyzed FN was used in place of intact FN in the fibrin-FN matrix. Similar enhancement was observed when ligand binding by alpha4beta1 integrins was activated by treatment with Mn(++), but not by stimulation of actin organization with LPA. Therefore, alpha4beta1-dependent cell responses to the provisional matrix are modulated by cleavage of matrix components.

Animals↗
PubMed2001

Identification of a novel heparin-binding site in the alternatively spliced IIICS region of fibronectin: roles of integrins and proteoglycans in cell adhesion to fibronectin splice variants.

The extracellular matrix molecule fibronectin (FN) is a glycoprotein whose major functional property is to support cell adhesion. FN contains at least two distinct cell-binding domains: the central cell-binding domain and the HepII/IIICS region. The HepII region comprises type III repeats 12-14 and contains proteoglycan-binding sites, while the alternatively spliced IIICS segment possesses the major alpha4beta1 integrin-binding sites. Both cell surface proteoglycans and integrins are important for mediating the adhesion of cells to this region of FN. By comparing heparin binding to different recombinant splice variants of the HepII/IIICS region, evidence was obtained for the existence of a novel heparin-binding site in the centre of the IIICS. Site-directed mutagenesis of basic amino acid sequences in this region reduced heparin binding to recombinant HepII/IIICS proteins and, in conjunction with mutations in the HepII region, caused a synergistic loss of activity. Using the H/120 variant of FN, which contains type III repeats 12-15 and the full-length IIICS region, and the H/95 variant of FN, which contains type III repeats 12-15 but lacks the high affinity integrin-binding LDV sequence, the relative roles played by cell-surface proteoglycans and integrins in mediating cell adhesion have been investigated. This was achieved by studying the effects of anti-integrin antibodies and exogenous heparin on A375 melanoma cell attachment to the wild-type and three different mutants of H/120 and H/95 in which the potential proteoglycan-binding sites were partially or completely removed. A375 cell adhesion to H/120 and its mutants was found to involve the co-operative action of both integrin and cell-surface proteoglycan binding, although integrin made a dominant contribution. Anti-integrin antibodies and exogenous heparin were capable of inhibiting melanoma cell adhesion to H/95 and in this case adhesion was due primarily to cell-surface proteoglycan and not integrin binding.

Alternative Splicing↗
PubMed2000

Levels of the bcl-2 protein, fibronectin and alpha(5)beta(1) fibronectin receptor in HIV-1-infected patients with Kaposi's sarcoma.

Kaposi's sarcoma (KS) is an angioproliferative disease characterized by proliferation of neoplastic cells (spindle cells) mixed with endothelial and inflammatory cells. In this study we evaluated the role of the adhesive glycoprotein, fibronectin (FN) and its receptor alpha(5)beta(1) (FNR), and the proto-oncogene bcl-2, an anti-apoptotic protein. Significantly decreased serum levels of FN were noted in HIV-1-infected patients with KS, whereas serum levels of FNR were significantly increased in the same patients. Furthermore, increased FNR expression was observed on CD4 cells from KS patients. Serum levels of bcl-2 protein were significantly decreased in asymptomatic seropositive patients, whereas HIV-1-infected patients with KS showed increased serum levels of bcl-2. These results provide further information about interaction between integrins and the extracellular matrix and bcl-2 protein that can support cell survival either of neoplastic cells or endothelial and inflammatory cells.

AIDS-Related Opportunistic Infections↗
PubMed1985

Fibronectin-mediated keratinocyte migration and initiation of fibronectin receptor function in vitro.

Cell suspensions of human keratinocytes, freshly isolated from skin specimens, did not express plasma fibronectin (pFN) receptor function in short-term assays for cell attachment and spreading on pFN-coated culture dishes and binding and phagocytosis of pFN-coated latex beads. These activities were expressed, however, by the cells harvested from primary keratinocyte cultures after 2-4 days of culture. Analysis of the cell types arising during primary culture, based on staining with antikeratin antibodies and bullous pemphigoid (BP) serum, revealed that about 90% of the originally isolated cell population consisted of keratinocytes (keratin-positive) and 30% were basal cells (BP antigen-positive). After 2 days of culture, 95% of the cells were keratinocytes and 70% were basal cells. In vitro initiation of pFN receptor function also was observed in cells harvested from epidermal explants. After 9 days in culture, the cells that migrated out of the explants also were active in short-term cell adhesion assays, while cells remaining in the central region of the explant had much less activity. In related studies, the role of pFN in epidermal cell migration was analyzed, and it was found that anti-pFN antibodies inhibited migration of keratinocytes out of epidermal explants. Addition of preimmune IgG, however, had no effect. It appears, therefore, that pFN is important in all aspects of keratinocyte adhesion, and the expression of pFN receptor function may be a critical activation step necessary for basal cell phagocytosis and migration during wound healing.

Antibodies, Anti-Idiotypic↗
PubMed1997

The 80-kD fibronectin fragment increases the production of fibronectin and tumour necrosis factor-alpha (TNF-alpha) in cultured mesangial cells.

The presence of fibronectin (FN) fragments has been demonstrated in several inflammatory processes, and they have been implicated in the recruitment of mononuclear cells. However, the interaction of these FN fragments with resident cells has hardly been studied. We have hypothesized that the 80-kD FN fragment, which includes the RGD cell binding domain of FN, could contribute to the pathogenesis of glomerular damage through the interaction with mesangial cells (MC) via alpha5beta1 integrin. Since an increase in the glomerular deposit of matrix components, particularly FN, is frequently observed in progressive glomerulonephritis, we studied whether its synthesis is modulated by the 80-kD FN fragment and the native FN molecule. While the 80-kD FN fragment stimulated FN in a dose-dependent manner, both at the mRNA and protein level, the whole FN molecule exerted a dual effect. High doses produced FN inhibition, while low doses elicited a certain increase. This stimulation was abrogated by the presence of Sam-1, a MoAb against the alpha-subunit of the alpha5beta1 integrin. Since cytokines play a fundamental role in glomerular injury, we studied the production of TNF-alpha, one of the most powerful mediators of inflammation. TNF-alpha synthesis was induced by the 80-kD FN fragment, in a dose-dependent manner, but not by native FN. When MC were incubated with the 29- and 31-kD FN fragments, which lack the RGD cell binding domain, TNF-alpha secretion was not detected. These results strongly suggest that in cultured MC, the 80-kD FN fragment induces the synthesis of matrix proteins such as FN, and cytokines such as TNF-alpha, via alpha5beta1 integrin. This mechanism could contribute to the perpetuation of renal injury.

Animals↗
PubMed1997

Establishment of a standardized assay system of fibronectin activity using fibronectin-mediated cell adhesion.

An in vitro assay of fibronectin (FN) was established based on the adhesion of baby hamster kidney (BHK) cells through the cell-binding domain of FN. Each well of a microtiter plate was coated with samples or various concentrations of standard FN. Bovine serum albumin was further coated to prevent the non-specific adhesion of the cells. Various numbers of BHK cells were plated and incubated. After washing out the non-attached cells, the number of attached cells was measured using neutral red (NR)-staining. The conditions for the assay were optimal when 1 x 10(5) cells/well were plated and incubated for 90 min. The linear relationship between the concentration of FN coated and the absorbance of NR was observed in the range of 0.1-1.0 micro/ml of FN. The inhibition of cell binding by the peptides containing an Arg-Gly-Asp (RGD) sequence demonstrated that this assay system depended on FN-mediated cell adhesion through the major cell-binding domain.

Animals↗