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PubMed1997

Vitronectin is expressed in the ventral region of the neural tube and promotes the differentiation of motor neurons.

The extracellular matrix protein vitronectin and its mRNA are present in the embryonic chick notochord, floor plate and in the ventral neural tube at the time position of motor neuron generation. When added to cultures of neural tube explants of developmental stage 9, vitronectin promotes the generation of motor neurons in the absence of either notochord or exogenously added Sonic hedgehog. Conversely, the neutralisation of endogenous vitronectin with antibodies inhibits over 90% motor neuron differentiation in co-cultured neural tube/notochord explants, neural tube explants cultured in the presence of Sonic hedgehog, and in committed (stage 13) neural tube explants. Furthermore, treatment of embryos with anti-vitronectin antibodies results in a substantial and specific reduction in the number of motor neurons generated in vivo. These results demonstrate that vitronectin stimulates the differentiation of motor neurons in vitro and in vivo. Since the treatment of stage 9 neural tube explants with Sonic hedgehog resulted in induction of vitronectin mRNA expression before the expression of floor plate markers, we conclude that vitronectin may act either as a downstream effector in the signalling cascade induced by Sonic hedgehog, or as a synergistic factor that increases Shh-induced motor neuron differentiation.

Amino Acid Sequence↗
PubMed1989

A comparison of the biological activities of the cell-adhesive proteins vitronectin and fibronectin.

The effects of vitronectin and fibronectin upon the attachment and growth of bovine corneal endothelial cells (BCE) and BHK-21 cells were compared. Similar dose-response curves for cell attachment to the substratum were obtained for both molecules and both cell types, although BCE cells exhibited a slight preference for vitronectin, and BHK cells for fibronectin. When, however, cells were plated in medium containing bovine serum stripped of fibronectin, they attached and grew normally, whereas in medium containing serum stripped of vitronectin, cells either failed to attach (BHK-21) or attached but exhibited poor cell spreading and growth. This dependence of cells upon vitronectin, rather than fibronectin, in serum for cell attachment, was shown to be due to a failure of fibronectin to coat the substratum in the presence of other serum proteins. Vitronectin was able to coat the substratum efficiently in the presence of other serum proteins. Although dependent upon vitronectin for adhesion to the substratum, bovine endothelial cells were unable to synthesize endogenous vitronectin.

Animals↗
PubMed1988

Novel purification of vitronectin from human plasma by heparin affinity chromatography.

The glycoprotein vitronectin (also called S-protein, serum spreading factor, or epibolin) promotes spreading of a variety of cultured cells, inhibits the cytotoxicity of membrane attack complex C5b-9, and modulates thrombin-antithrombin III activity. We developed a strikingly simple method to purify vitronectin from human plasma by heparin affinity chromatography. Serum was obtained from plasma by adding calcium and then centrifuging. The heparin-binding activity of vitronectin in human serum was activated with 8 M urea. The activated vitronectin specifically bound to heparin-Sepharose in 8 M urea and was eluted with 0.5 M NaCl containing 8 M urea. This procedure resulted in an approximately 250-fold purification of vitronectin with a 15-30% recovery; 3-6 mg of pure vitronectin were obtained from 100 ml human plasma within 2 days. The purified vitronectin preparations promoted spreading of BHK fibroblastic cells on substrates with a half-maximal activity at only 0.1 microgram/ml. This new method is very simple, rapid, inexpensive, and flexible. It could probably be readily scaled up for commercial applications.

Chromatography, Affinity↗
PubMed1993

Two collagen-binding domains of vitronectin.

Vitronectin is a cell-adhesive glycoprotein present in animal blood and extracellular matrix. To establish the molecular basis of vitronectin interactions with extracellular matrix macromolecules, the binding site of vitronectin to collagen has been investigated. Vitronectin fragments obtained by formic acid cleavage were separated by heparin-affinity chromatography followed by gel filtration chromatography. The collagen-binding activity of the fragments was assayed in terms of inhibitory activity on the binding of 125I-vitronectin to immobilized collagen. There were two groups of collagen-binding fragments. One group consisted of 5 heparin-binding fragments with estimated molecular masses of 12 kDa, 14 kDa, 16 kDa, 18 kDa, and 19 kDa in SDS-polyacrylamide gel electrophoresis. The other group consisted of 2 heparin-nonbinding fragments migrating at 18 kDa and 40 kDa. These results indicate that there are two collagen-binding sites in the vitronectin molecule; one located close to the heparin-binding domain in the COOH-terminal half and the other located in the NH2-terminal half of vitronectin.

Amino Acid Sequence↗
PubMed1992

Vitronectin regulates the synthesis and localization of urokinase-type plasminogen activator in HT-1080 cells.

The effect of extracellular matrix composition on the location, amount, and activity of cell-associated urokinase-type plasminogen activator was tested using HT-1080 cells adherent to either fibronectin or vitronectin. Specific immunoprecipitation of newly synthesized urokinase indicated that cells adherent to fibronectin synthesized 2-3-fold more urokinase than cells adherent to vitronectin. Complexes of urokinase and plasminogen activator inhibitor type 1 (PAI-1) were detected in cell layers of vitronectin-adherent but not fibronectin-adherent cells. Inhibition of PAI-1 using a neutralizing monoclonal antibody resulted in a 3-fold increase in urokinase enzymatic activity on vitronectin adherent cells. Urokinase activity on fibronectin adherent cells was only slightly increased following PAI-1 neutralization. Examination of both HT-1080 and normal human fibroblast cells by immunofluorescent microscopy localized urokinase-type plasminogen activator to discrete, focal areas underneath cells adherent to vitronectin. Urokinase was not detectable by immunofluorescence on cells adherent to fibronectin. The addition of exogenous prourokinase to locate urokinase receptors on adherent HT-1080 cells indicated that the focal localization of cell-surface urokinase resulted from the clustering of urokinase receptors following adhesion to vitronectin but not fibronectin-coated substrates. These results suggest that vitronectin can contribute to the control of cell-surface plasmin activity by regulating the synthesis of urokinase and directing the localization of urokinase receptors.

Antibodies, Monoclonal↗
PubMed1990

[Effect of vitronectin on the healing of rabbit corneal epithelial damage].

Vitronectin is one of the major cell-adhesive glycoproteins in mammalian plasma. We investigated the effect of topically applied vitronectin on the healing of rabbit corneal epithelial damage. Vitronectin was purified from rabbit serum by heparin-Sepharose affinity chromatography and autoclaved at 121 degrees C for 20 min after adjusting the concentration to 0.2 mg/ml with saline. Rabbit corneal epithelium was abraded with ethanol and a laser blade in the central portion of the cornea which had been demarcated with a 6.5 mm trephine. Vitronectin eye drops were instilled into the right eye, and sterile saline drops into the left eye as control. The dimensions of the circular epithelial defect were measured at various times after wounding. The area of epithelial damage of vitronectin-treated eyes was smaller than that of control eyes at 2hr-12hr after abrasion (p less than 0.01). These results suggested that topically applied vitronectin might accelerate the corneal epithelial wound healing at an early stage. Vitronectin can be considered as a candidate for treatment of corneal epithelial damage.

Animals↗
PubMed1993

[Expression of fibronectin receptor and vitronectin receptor in diseased human kidneys].

Expression of fibronectin and vitronectin receptors was studied in normal human kidney tissues and renal tissue biopsies from patients with several types of glomerulonephritis. Immunofluorescent staining of the normal kidneys showed that fibronectin receptor was present along the glomerular capillary walls, and that vitronectin receptor was present in the vessel walls, but was almost absent from the glomerulus. In kidney tissues biopsied from patients with various renal diseases, expression of fibronectin and vitronectin receptors was correlated with increase in the mesangial expansion. Expression of these receptors was decreased in the sclerosed area and hyalinized glomeruli compared with normal tissues. Fibronectin and vitronectin receptors were occasionally colocalized in the glomeruli with fibronectin and vitronectin, respectively. In situ hybridization showed that fibronectin receptor mRNA and vitronectin receptor mRNA were expressed in the diseased glomeruli. These findings indicate that expression of fibronectin and vitronectin receptors was altered in human glomerulonephritis, and that integrin expression may be important in cell-matrix interaction in the diseased glomeruli.

Glomerulonephritis↗
PubMed1994

Localization of vitronectin binding domain in plasminogen activator inhibitor-1.

Plasminogen activator inhibitor type 1 (PAI-1) is the rapid physiologic inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). In plasma and the extracellular matrix, PAI-1 is associated with the adhesive glycoprotein vitronectin. In order to characterize the PAI-1 structural domain responsible for binding to vitronectin, the segment of the PAI-1 cDNA encoding amino acids 13-147 (nucleotides 248-650) was randomly mutagenized and subcloned into a bacterial expression vector containing the mature PAI-1 coding sequence. Recombinant PAI-1 mutants were expressed in Escherichia coli and bacterial lysates assayed in duplicate for uPA inhibitory activity and vitronectin binding. Of 190 clones screened, six consistently demonstrated decreased vitronectin binding relative to uPA inhibitory activity. DNA sequence analysis of four of these clones identified 10 unique missense mutations, all located between base pairs 298 and 641, with each clone containing between one and four substitutions. Each substitution was expressed independently by site-directed mutagenesis and again analyzed for uPA inhibitory activity and vitronectin binding. Five point mutations that selectively disrupt vitronectin binding were identified. All 5 residues are located on the exterior of the PAI-1 structure. These findings appear to define a complex binding surface that bridges alpha-helices C and E to beta-strand 1A and includes amino acids 55, 109, 110, 116, and 123. These results suggest that vitronectin binding may stabilize the active conformation of PAI-1 by restricting the movement of beta-sheet A and thereby preventing insertion of the reactive center loop.

Amino Acid Sequence↗
PubMed1994

[Vitronectin in plasma and colonic mucosa of patients with ulcerative colitis].

Plasma levels and localization of vitronectin in patients with ulcerative colitis (UC) were investigated. Plasma vitronectin levels in patients with UC were significantly lower than those of healthy persons, in proportion to clinical activity and severity. In addition, plasma vitronectin levels at the remission phase were high when compared with those at the active phase. Vitronectins, in addition, were found in mucosa of the active phase where a lot of inflammatory cells were found by immunohistological staining. Based on the above results, vitronectins were considered to leak from blood vessel and to be consumed for mucosa recovery in the active phase of UC, and plasma vitronectin levels were lowering. Therefore, determination of plasma vitronectin levels were considered to be useful for an index of clinical activity and severity of patients with UC.

Adult↗
PubMed1997

Plasma collagen-binding vitronectin activated by heparin and dextran sulfate in chronic liver disease.

Plasma collagen-binding vitronectin was assayed in 62 patients with chronic liver disease and 14 healthy control subjects. It was measured by an enzyme immunoassay using type I collagen and monoclonal antibody to vitronectin before and after treatment with heparin or dextran sulfate in vitro. The pretreatment level of plasma collagen-binding vitronectin (mean +/- S.E.M.) was 5.5 +/- 0.5 micrograms/ml in the controls, 8.2 +/- 0.3 micrograms/ml in chronic persistent hepatitis, 8.3 +/- 0.7 micrograms/ml in chronic active hepatitis, 7.9 +/- 0.7 micrograms/ml in liver cirrhosis, and 8.2 +/- 0.5 micrograms/ml in hepatocellular carcinoma with cirrhosis. After treatment with heparin, the percent collagen-binding vitronectin to total vitronectin was 20.6 +/- 2.0% in the controls, 24.7 +/- 4.1% in chronic persistent hepatitis, 28.6 +/- 2.5% in chronic active hepatitis, 42.6 +/- 4.5% in liver cirrhosis, and 31.8 +/- 2.3% in hepatocellular carcinoma. All percents were significantly increased compared to the pretreatment percent. The same pattern was also found after dextran sulfate treatment. Compared to that in the pretreatment state, the collagen-binding vitronectin after these treatments was more closely correlated with the serum levels of 7S collagen and hyaluronic acid. These results suggest that the collagen-binding activity of vitronectin may play an important role in the progression of liver disease and/or fibrosis through its activation with some glycosaminoglycans.

Adult↗
PubMed1995

Bovine ovarian follicular fluid vitronectin content is influenced by the follicle size.

Vitronectin was quantified in the follicular fluid aspirated from bovine follicles with diameters of 3 to 15 mm (as determined by ultrasonography) using a specific enzyme-linked immunosorbent assay (ELISA) validated for bovine vitronectin. The primary antibody was raised in rabbit against vitronectin purified from bovine plasma. Vitronectin quantified in serial dilutions of bovine plasma and ovarian follicular fluid was highly correlated with the volume of each sample assayed. In addition, known amounts of purified bovine vitronectin added to samples of plasma or follicular fluid were accurately recovered. Follicular fluid concentrations of vitronectin were positively correlated with the follicle diameter (r = 0.8; P < 0.01). These data indicate that bovine follicular fluid concentration of vitronectin is influenced by the stage of follicular development.

Journal Article↗
PubMed1995

Isolation and characterisation of a vitronectin-binding surface protein from Staphylococcus aureus.

In a previous study we demonstrated that cells of Staphylococcus aureus strain V8 bind 125I-labelled vitronectin in a receptor-ligand type of interaction, and a protein having a molecular mass of 60 kDa was identified as a putative high-affinity staphylococcal vitronectin-binding protein (Liang, O.D. et al. (1993) Biochim. Biophys. Acta 1225, 57-63). In the present communication we report on the isolation and preliminary characterisation of the 60 kDa vitronectin-binding protein. The bacterial cell surface proteins were released by stirring bacteria with 1 M LiCl at 37 degrees C for 2 h and separated on an FPLC Mono-Q column with a gradient of 0-0.5 M NaCl in 20 mM Tris buffer at pH 9.0. Fractions containing vitronectin-binding activity, assayed on microtiter plates with immobilised human vitronectin, were collected and SDS-PAGE analysis showed the content to be a single protein band at the 60 kDa position. In Western blot experiments the protein transblotted onto nitrocellulose membranes could bind soluble vitronectin. Its amino-terminal amino acid sequences showed a striking similarity with those of a 60 kDa heparan sulfate-binding protein from the same staphylococcal strain (Liang, O.D. et al. (1992) Infect. Immun. 60, 899-906), suggesting that they are identical molecules. This was supported by ligand blotting experiments where both vitronectin and heparan sulfate were shown to bind to the same protein band in parallel strips.

Amino Acid Sequence↗
PubMed1999

Migration properties of the human ovarian adenocarcinoma cell line IGROV1: importance of alpha(v)beta3 integrins and vitronectin.

Cell migration of ovarian tumoral cells is essential for cell dissemination and for invasion of the submesothelial extracellular matrix (ECM). We have conducted a study of the migratory properties of an ovarian adenocarcinoma cell line (IGROV1) by using 2 distinct methods for the evaluation of cell migration. We found that in a short-term transfilter migration assay, IGROV1 cells migrated toward vitronectin, fibronectin, type IV collagen and laminin in an integrin-dependent manner. When migration was evaluated in a wound healing assay, the restitution of the wounded area was stimulated solely by added, exogenous vitronectin and was almost totally dependent on alpha(v)beta3 integrin function. Moreover, we demonstrated that alpha(v)beta3 was localized in focal contacts restricted to the leading edge of migrating cells, whereas vitronectin notably localized with actin stress fibers and cortical actin. On the other hand, several kinase inhibitors were found to impede migration of IGROV1 induced by vitronectin. It thus appears that alpha(v)beta3-vitronectin interactions lead to the activation of multiple signaling pathway including activation of protein kinase C, phosphatidyl-inositol-3-phosphate kinase and protein tyrosine kinase. The "alpha(v)beta3-vitronectin system" is therefore essential to the migration of human ovarian carcinoma cells.

Adenocarcinoma↗
PubMed1995

Plasmin abrogates alpha v beta 5-mediated adhesion of a human keratinocyte cell line (HaCaT) to vitronectin.

At cellular surfaces, urokinase-type plasminogen activator (uPA) is bound to a specific receptor (uPA-R). When bound to this receptor, uPA activates plasminogen, which is derived from plasma or the interstitial fluids. Thus, plasmin is provided for proteolysis of pericellular proteinaceous substrates. Here we demonstrate by immunocytology and laser scan microscopy that in the human keratinocyte cell line HaCaT uPA-R and uPA are localized together with the integrin alpha v beta 5 in focal contacts. Via the integrin alpha v beta 5, HaCaT cells adhere to vitronectin in a RGD-dependent manner. Plasmin interfered with the alpha v beta 5-mediated keratinocyte adhesion to vitronectin, most likely via cleavage of vitronectin and destruction of its cell binding function. Our findings demonstrate that plasmin, when generated by the uPA-dependent cell surface-associated pathway of plasminogen activation, can abrogate the cell-binding function of vitronectin and can thus disturb the adhesive interaction with this matrix molecule. In focal contacts molecules are assembled that are crucial for adhesion to vitronectin (i.e., the integrin alpha v beta 5), as well as for the generation of plasmin (i.e., uPA-R and uPA), which can negatively influence the binding interaction. We suggest that the plasmin-mediated abrogation of the interaction between the integrin alpha v beta 5 and vitronectin is a pathway of negative regulation; the codistribution of uPA-R/uPA and alpha v beta 5 in focal contacts may restrict this process to areas of cell/matrix contact.

Amino Acid Sequence↗
PubMed2000

Attachment of primary neonatal rat astrocytes to vitronectin is mediated by integrins alphavbeta5 and alpha8beta1: modulation by the type 1 plasminogen activator inhibitor.

Vitronectin is expressed in a cell-specific manner in the developing brain and concentrated in the brain during disease processes, such as germinal matrix hemorrhage and infarction, in which there is breakdown of the blood-brain barrier. In this study, we identified the integrin receptors that mediate attachment of primary neonatal rat astrocytes to vitronectin. Using fluorescent activated cell sorter and immunoprecipitation analyses, we established that the vitronectin receptor integrins alphavbeta5 and alpha8beta1, but not alphavbeta3, are expressed on neonatal rat astrocytes. Attachment of the neonatal astrocytes to vitronectin was inhibited (85%) in an additive manner by neutralizing anti-alphavbeta5 and anti-beta1 antibodies. Attachment to vitronectin was also inhibited in a dose-dependent manner by the type I plasminogen activator inhibitor (PAI-1), a serine protease inhibitor. Our data demonstrate that unstimulated primary neonatal rat astrocytes attach to vitronectin, utilizing integrins alphavbeta5 and alpha8beta1, and that this attachment is regulated by PAI-1.

Animals↗
PubMed1985

A 125/115-kDa cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin.

Affinity chromatography was used to identify a cell surface receptor for the adhesive protein vitronectin. Detergent extracts of human osteosarcoma (MG-63) cells were chromatographed on either vitronectin-Sepharose or Sepharose linked to the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, which includes the fibronectin cell attachment sequence Arg-Gly-Asp. Two cell surface proteins with apparent molecular mass of 125 and 115 kDa bound to both columns and were specifically eluted with a solution containing the Gly-Arg-Gly-Asp-Ser-Pro peptide. These proteins could be incorporated into phosphatidylcholine liposomes and mediated the specific binding of these liposomes to vitronectin but not to fibronectin. In contrast, liposomes containing a previously identified 140-kDa fibronectin receptor, which interacts with the Arg-Gly-Asp sequence in fibronectin, did not bind to vitronectin. Thus, the fibronectin and vitronectin receptors each recognize the Gly-Arg-Gly-Asp-Ser-Pro peptide but exhibit mutually exclusive reactivities toward fibronectin and vitronectin. These receptors appear to belong to a family of proteins that mediate cell substratum adhesion via related but subtly different specificities.

Amino Acid Sequence↗
PubMed1992

Involvement of a vitronectin-like protein in attachment of Agrobacterium tumefaciens to carrot suspension culture cells.

Infections of dicotyledonous plants by Agrobacterium tumefaciens result in the formation of crown gall tumors. Attachment of the bacteria to plant host cells is required for tumor formation. Human vitronectin and antivitronectin antibodies both inhibited the binding of A. tumefaciens to carrot cells. Wild-type bacteria are able to bind radioactive vitronectin; nonattaching mutants showed a reduction in the ability to bind vitronectin. The binding of biotype 1 A. tumefaciens to carrot cells or to radioactive vitronectin was not affected by high ionic strength. Detergent extraction of carrot cells removed the receptor to which the bacteria bind. The extract was found to contain a vitronectin-like protein. These results suggest that A. tumefaciens utilizes a vitronectin-like protein on the plant cell surface as the receptor for its initial attachment to host cells.

Agrobacterium tumefaciens↗
PubMed1991

Manganese ion elicits a binding activity of placenta vitronectin receptor to fibronectin cell-binding domain.

Some members of the integrin family recognize the RGD sequence which is common to cell adhesive proteins in a divalent cation-dependent manner. In the presence of Ca2+ and Mg2+, the fibronectin receptor of placenta recognizes the RGD sequence of fibronectin, but not that of vitronectin, while the vitronectin receptor of placenta recognizes the RGD sequence of vitronectin, but not that of fibronectin, although both receptors recognize the same RGD sequence. We have found by performing an enzyme-linked immunosorbent assay (ELISA) using receptor-specific monoclonal antibodies and by electrophoretic analysis that in the presence of Mn2+ a vitronectin receptor of placenta binds to an affinity column coupled with the cell-binding domain of fibronectin. By replacing divalent cations from Mn2+ to Ca2+ and Mg2+, the vitronectin receptor was completely eluted from the column. When the synthetic peptides GRGDSP and GRGESP were applied to the column as competitors, the Mn(2+)-dependent binding was inhibited by both peptides. These results suggest that Mn2+ elicits a binding activity of the placenta vitronectin receptor to the RGD site of fibronectin. The modulation of ligand specificity by Mn2+ will provide an important clue in the elucidation of the cause of individual ligand specificity of RGD-recognizing integrins.

Antibodies, Monoclonal↗