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Adsorption of fibronectin and vitronectin onto Primaria and tissue culture polystyrene and relationship to the mechanism of initial attachment of human vein endothelial cells and BHK-21 fibroblasts.

The two cell culture substrata, tissue culture polystyrene (TCPS) and Primaria, were compared in order to determine whether a nitrogen-containing surface such as Primaria attracts cells by a different mechanism to an oxygen-containing surface (TCPS). The amounts of vitronectin (Vn) and fibronectin (Fn) which adsorb from the fetal bovine serum (FBS) component of the culture medium onto Primaria and TCPS were determined. Primaria adsorbed two- to threefold more Fn than TCPS, but adsorbed similar amounts of Vn from medium containing FBS. The Fn and Vn binding sites on Primaria were distinct, as adsorption was non-competitive between these two proteins. The amounts of Fn and Vn that adsorbed onto the two surfaces were compared to the concentration dependence of the cell attachment activities of Fn and Vn. Whereas the amounts of Fn which adsorbed onto TCPS were suboptimal for cell attachment, Primaria adsorbed an Fn surface density that was supraoptimal for attachment of human vein endothelial cells and BHK-21 fibroblasts. We conclude that Primaria differs from TCPS in that both Fn and Vn mediate initial cell attachment to Primaria when the culture medium contains FBS, whereas cell attachment to TCPS is dependent upon Vn.

Adsorption↗

Vitronectin interacts with Candida albicans and augments organism attachment to the NR8383 macrophage cell line.

Candida albicans is an increasingly important cause of mucocutaneous and bloodstream infections. The potential role of circulating adhesive glycoproteins such as vitronectin (Vn) in host defense against C. albicans is currently unknown. Accordingly, we investigated the binding of plasma-derived Vn with C. albicans strain 36082. Vn specifically bound to C. albicans in a concentration-dependent fashion. Higher affinity binding sites numbered 9.8 x 10(4) sites per organism, with a dissociation constant, Kd of 3.5 x 10(-7) M. Vn binding with C. albicans was significantly inhibited by heparin, suggesting interaction of the organism with Vn's glycosaminoglycan-binding region. To further determine which molecule(s) on the fungus interacted with Vn, C. albicans components were extracted, separated by SDS and blotted with radiolabeled Vn. These studies revealed that Vn binds to a 30 kDa molecule on C. albicans. Finally, we investigated the role of Vn in promoting interaction of C. albicans with phagocytic cells. Incubation of C. albicans in the presence of Vn significantly increased binding of the organism to cultured NR8383 macrophages compared to incubations performed in the absence of Vn. These data demonstrate that C. albicans interacts with the heparin-binding domain Vn and further suggest that Vn augments organism uptake by phagocytic cells.

Amino Acid Sequence↗

New sites of cellular vitronectin receptor immunoreactivity detected with osteoclast-reacting monoclonal antibodies 13C2 and 23C6.

The immunohistochemical profile of osteoclast-reacting monoclonal antibodies, 13C2 and 23C6, known to detect the alpha-chain of the vitronectin receptor, is described. Both antibodies reacted with several cell types apart from osteoclasts including megakaryocytes, smooth muscle cells, endothelial cells, thyroid follicular epithelium, renal glomeruli and tubular epithelium, myoepithelial and epithelial cells in the breast and prostate, and both cytotrophoblast and syncytiotrophoblast. In addition, macrophage polykaryons, synovial lining cells, a small number of mononuclear cells in buffy coats, and a few macrophage-like cells in the stroma of various tissues were also stained. The epitopes recognized by these antibodies are thus not osteoclast-specific and are present on other cells of the mononuclear phagocyte system. The implications of these results for osteoclast ontogeny, the nature of the antigens described and the question of osteoclast-specific antibodies are discussed.

Antibodies, Monoclonal↗

Organization of the gene encoding mouse vitronectin.

The gene (Vn) encoding the mouse vitronectin was isolated and its nucleotide sequence determined. The gene covers approximately 3 kb of genomic DNA. Alignment of the genomic sequence with that of the cDNA revealed that Vn consists of eight exons, interrupted by seven introns ranging in size from 78 to 723 bp.

Amino Acid Sequence↗

Interactions between type 1 plasminogen activator inhibitor, extracellular matrix and vitronectin.

Regulation of plasminogen activation is a key process in controlling proteolytic events in the extracellular matrix (ECM) and this regulation is achieved through the action of specific plasminogen activator (PA) inhibitors (PAIs). Type I PAI (PAI-1) is the physiological inhibitor both of urinary-type PA (u-PA) and tissue-type PA (t-PA) (Loskutoff et al., 1989) and is a major component of the ECM of cultured cells. This inhibitor may protect ECM constituents against cellular proteases and thus influence the cell migration and tissue destruction that occurs during development, inflammation and tumor metastasis. In this review, we discuss the properties of PAI-1 and the evidence that the binding of PAI-1 to ECM is mediated by serum-derived vitronectin (Vn).

Animals↗

The effect of the surface modification of titanium using a recombinant fragment of fibronectin and vitronectin on cell behavior.

The surface of titanium implants is in direct contact with host tissue and plays a critical role in determining biocompatibility. Fibronectin (FN) and vitronectin (VN) are major cell adhesive proteins found in the extracellular matrix (ECM) of various tissues, and in circulating blood. The aim of this study was to evaluate the engineered biomimetic surface of titanium by using recombinant fragment of FN(8-10) and VN(NTD) that contains the binding site for integrins. MC3T3-E1 cells seeded upon the FN(8-10)-coated titanium showed a marked increase in cell adhesion, proliferation, and differentiation over VN(NTD)-coated titanium. In addition, we confirmed that the surface properties of titanium prefer for FN(8-10) over VN(NTD) (p<0.05) in protein adhesion. These results suggest that the FN(8-10)-modified titanium surface can be used to improve the osseointegration of titanium implants by enhancing bone formation.

3T3 Cells↗

Vitronectin supports migratory responses of corneal epithelial cells to substrate bound IGF-I and HGF, and facilitates serum-free cultivation.

Vitronectin (VN) is a multi-functional glycoprotein best known for its effects on cell attachment and spreading, but has more recently been shown to mediate cellular responses to growth factors. The presence of VN within the tear film and expression of required receptors (alpha v integrins) on corneal epithelial cells suggests the potential for a similar role within the ocular surface. Thus we have studied the ability of VN to alter the metabolic (MTT assay) and migratory (trans-membrane migration) responses of corneal epithelial cells to growth factors associated with the ocular surface including epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and insulin-like growth factor-I (IGF-I). Our hypothesis was that culture surfaces coated with VN might selectively facilitate responses to growth factors which are known to bind VN including EGF, IGF-I (via IGF binding protein) and HGF. Metabolic responses were observed towards each growth factor when applied to the culture medium, but not towards culture plastic pre-treated with VN and, or growth factors. Optimal metabolic responses were observed towards IGF-I applied in conjunction with EGF. Migration through porous polycarbonate membrane was significantly increased when the substrate had been pre-coated with VN and IGF-I (applied in conjunction with IGFBP-3) or VN and HGF. This finding is consistent with the ability of IGF-I (via an IGFBP) and HGF to form complexes with VN and suggests that integrin/growth factor receptor co-activation is required for corneal epithelial cell migration. In further studies, VN applied in conjunction with IGF-I, IGFBP-3 and EGF (both to the culture plastic and in the culture medium) was found to support the establishment and serial propagation of limbal-corneal epithelial cell cultures in the absence of serum, but irradiated 3T3 cells (i3T3) were still necessary for culture expansion. Immunocytochemistry of resulting cultures for keratin 3 and p63 revealed a similar phenotype to those established under current best-practice conditions (i3T3, foetal bovine serum, EGF and insulin). In conclusion, our novel findings suggest a role for VN-growth factor complexes in stimulating corneal epithelial migration within the provisional wound bed and demonstrate that VN-growth factors interactions can be exploited to enable manufacture of bioengineered ocular surface tissue under serum-free conditions.

Cell Culture Techniques↗

TLR3 activation inhibits human mast cell attachment to fibronectin and vitronectin.

Mast cells are involved in both the genesis of allergic inflammation and in host defense; and reside in tissues where their location and responsiveness is regulated in part by adhesion to extracellular matrix proteins (ECM). We have reported that human mast cells (huMC) express TLR1-7, and 9 and respond to toll-like receptors (TLR) ligands by releasing cytokines and leukotriene C4. To determine if TLR ligation could similarly affect mast cells via an influence on adhesion, we employed huMC; and as substrates, fibronectin (FN) and vitronectin (VN). huMC were thus treated with double-stranded RNA (dsRNA) and adhesion to ECM was quantified. FcvarepsilonRI dependent mast cell degranulation was assessed. Adhesion molecule expression and activation was measured by flow cytometry. Activation of huMC through TLR3 with increasing amounts of polyI:C inhibited mast cell adhesion in a dose-dependent manner. This decrease in adhesion was accompanied by a similar decrease in IgE-mediated mast cell degranulation. Activation of TLR3 on huMC resulted in a change in the conformation of CD29, the receptor for FN, to an inactive form. Thus, TLR3 activation decreases mast cell attachment to VN and FN through an active process and one, which would abrogate mast cell attachment dependent potentiation of IgE-mediated responses.

Cell Adhesion↗

Expression, production, and characterization of full-length vitronectin in Escherichia coli.

Vitronectin (VN) is one of the primary adhesive proteins in serum and serves to promote the attachment and spreading of a wide variety of cell types to tissue culture plastic. In this study, the pGEX2t expression vector was used to express full-length human VN as a GST-tagged fusion protein in Escherichia coli. GST/VN production was induced with IPTG and the protein was found to localize to inclusion bodies. The inclusion bodies were isolated from cell lysates, washed once with 2 M urea and Triton X-100, and then solubilized with 8 M urea in the presence of a reducing compound. Solubilized GST/VN was purified by heparin affinity chromatography and refolded by dialysis against phosphate buffered saline. Approximately 40 mg of GST/VN was recovered from 1L of bacterial culture. Purified GST/VN migrated at the predicted molecular mass on SDS-PAGE and was recognized by both anti-GST and anti-VN antibodies. GST/VN bound to heparin and promoted cell adhesion, spreading, and growth to a similar extent as that observed with plasma-derived VN. As such, the production of recombinant VN in bacteria represents a rapid and convenient method to produce large quantities of VN for cellular studies.

Animals↗

Responses of keratinocytes to substrate-bound vitronectin: growth factor complexes.

Insulin-like growth factor-1 (IGF-I) can associate with the extracellular matrix protein vitronectin (VN) via select IGF-binding proteins, and the resulting complex stimulates responses in a variety of cell types. As VN can also associate with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), we hypothesized that the multimeric nature of VN could be exploited to deliver multiple growth factors to the cell surface. We report here that VN enhances bFGF but not EGF stimulated [(3)H]-leucine incorporation in the HaCAT keratinocyte cell line, with VN synergistically enhancing cell migration in response to both EGF and bFGF when presented as a VN-bound complex. Furthermore, the addition of EGF and/or bFGF to IGF-I:IGFBP-5:VN complexes significantly enhances both [(3)H]-leucine incorporation and migration of HaCAT cells above that induced by IGF:IGFBP-5:VN complexes alone. Indeed, similar responses are observed in primary cultures of human skin keratinocytes, highlighting the potential use of these novel complexes for a wide range of tissue repair applications.

Cell Adhesion↗

Vitronectin is a substrate for transglutaminases.

Vitronectin (VN) was found to be a substrate for both plasma transglutaminase (Factor XIIIa) and guinea pig liver transglutaminase (TG). Incorporation of [3H]-putrescine indicated the presence of reactive glutaminyl residues in VN. When VN was incubated with TG or Factor XIIIa, in the absence of putrescine, multimeric covalent complexes were identified, indicating that VN can also contribute lysyl residues to the bond catalyzed by transglutaminases. Cross-linking of VN by TG and Factor XIIIa may modulate the effects of VN on the complement and coagulation systems in hemostatic plugs and extracellular matrix.

Cross-Linking Reagents↗

Vitronectin is phosphorylated by a cAMP-dependent protein kinase released by activation of human platelets with thrombin.

Activation of freshly isolated human platelets with a physiological stimulant (thrombin) causes them to release a cAMP-dependent protein kinase which specifically phosphorylates one plasma protein (Mr 75000). This protein is immunochemically and biochemically identified as vitronectin (also know as S protein), which was previously implicated in blood clotting, complement function and cell adhesion.

Adenosine Triphosphate↗

Evidence showing that the two-chain form of vitronectin is produced in the liver by a selective furin cleavage.

The adhesive protein vitronectin (75 kDa) occurs in human blood fluid in a one-chain (Vn75) or a two-chain form (Vn65-10), and is produced by a specific cleavage (at Arg379-Ala380), by a proteinase not identified hitherto. These two forms were shown to be functionally different and therefore, this cleavage may have a regulatory significance in vivo. Here, we report the use of a tailored one-chain recombinant Vn, a specific protein kinase A phosphorylation at Ser378, and sequence analysis to show: (1) that none of the proteinases originating from blood, previously thought to be the endogenous proteinase (plasmin, thrombin, tPA, and uPA), is indeed the in vivo convertase; and (2) that furin, a serine endoproteinase residing in the secretory pathway of hepatocytes, where Vn is synthesized, specifically cleaves Vn at the endogenous cleavage site. Consequently, we propose that the Vn75 to Vn65-10 conversion takes place in the liver (not in blood) and is carried out by furin.

Alanine↗

Construction of a plasminogen activator inhibitor-1 variant without measurable affinity to vitronectin but otherwise normal.

Vitronectin (VN) and plasminogen activator inhibitor-1 (PAI-1) have important functional interactions: VN stabilises the protease inhibitory activity of PAI-1 and PAI-1 inhibits binding of adhesion receptors to VN. Having previously mapped the PAI-1 binding area for VN, we have now constructed a PAI-1 variant, R103A-M112A-Q125A, without measurable affinity to VN, but with full protease inhibitory activity and endocytosis receptor binding. As a tool for evaluating the physiological and pathophysiological functions of the PAI-1-VN interaction, our new variant is far superior to the previously widely used PAI-1 variant Q125K, which we have found possesses an only about 10-fold reduced affinity to VN.

Amino Acid Substitution↗

Vitronectin-dependent invasion of epithelial cells by Neisseria gonorrhoeae involves alpha(v) integrin receptors.

Binding of vitronectin (VN) to Neisseria gonorrhoeae expressing the heparan sulfate proteoglycan (HSPG) specific Opa50 protein was recently shown to trigger bacterial internalization into distinct epithelial cell lines. We have investigated the role of VN-binding integrin receptors and protein kinase C (PKC) in VN-triggered bacterial uptake. Blocking integrin function by RGDS peptides or by antibodies specific to alpha(v)beta5 or alpha(v)beta3 resulted in an abrogation of VN-triggered bacterial internalization. Moreover, inhibitors of PKC were found to block VN-triggered uptake. The essential role of alpha(v) integrins and the presumable involvement of PKC in VN-triggered gonococcal uptake are discussed.

Antigens, Bacterial↗

Activation of the cyclic AMP pathway in cells adhering to biomaterials: regulation by vitronectin- and fibronectin-integrin binding.

Our previous studies have shown that cells adhering to biomaterials in serum-free conditions increase their content of cyclic AMP (cAMP) and become aggregated. In cells on an acrylonitrile membrane (AN69), these biochemical and morphological changes are prevented by adding 10% foetal calf serum (FCS) to the medium; cells on the cellulose membrane Cuprophan (CU) remain unaffected. The present study examines the roles of vitronectin (VN)- and/or fibronectin (FN)-integrin binding in this inhibition. Competitively blocking VN- and FN-receptors with echistatin increased intracellular cAMP significantly and caused cells on AN69 to aggregate, but did not modify cAMP-dependent cell aggregation on CU. VN or FN adsorbed onto CU also inhibited cAMP production by attached cells and prevented their aggregation, whereas adsorbed BSA had no effect. Therefore, the binding of VN or FN to cell-surface integrins seems to limit the activation of the cAMP pathway initiated by the substratum itself.

1-Methyl-3-isobutylxanthine↗

Caught in the matrix: how vitronectin controls neuronal differentiation.

Cerebellar granule cells are the most abundant neurons in the brain and are crucial to the circuitry that controls motor coordination. The proliferation of granule cell precursors (GCPs) is controlled by the secreted signaling molecule Sonic hedgehog (Shh), but the factors that regulate GCP differentiation remain a mystery. A recent study suggests that the extracellular matrix protein vitronectin might tell GCPs when to stop dividing and begin differentiation.

Animals↗

The role of beta-strand 5A of plasminogen activator inhibitor-1 in regulation of its latency transition and inhibitory activity by vitronectin.

Plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. It circulates in plasma in a complex with vitronectin (VN). We have studied biochemical mechanisms for PAI-1 neutralisation and its modulation by VN, using site-directed mutagenesis and limited proteolysis. We demonstrate that VN, besides delaying conversion of PAI-1 to the inactive latent form, also protects PAI-1 against cold- and detergent-induced substrate behaviour and counteracts conversion of PAI-1 to inert forms by certain amphipathic organochemical compounds. VN protection against cold- and detergent-induced substrate behaviour is associated with inhibition of the proteolytic susceptibility of beta-strand 5A. Alanine substitution of a lysine residue placed centrally in beta-strand 5A implied a VN-induced acceleration of latency transition, instead of the normal delay. This substitution not only protects PAI-1 against neutralisation, but also counteracts VN-induced protection against neutralisation. We conclude that beta-strand 5A plays a crucial role in VN-regulation of PAI-1 activity.

Amino Acid Substitution↗