β1‐integrin‐mediated adhesion of melanoma cells to the propolypeptide of von Willebrand factor

Abstract

Cell-adhesion activity of the bovine propolypeptide of von Willebrand factor (pp-vWF) was assessed by means of an in vitro assay with several cell lines of both normal and tumor-cell origin. pp-vWF promoted adhesion and spreading of B16 mouse melanoma cells and G-361 human melanoma cells. However, it could not induce adhesion of any other cell lines tested including endothelial cells, normal fibroblasts, and tumor cells of sarcoma, carcinoma, neuroblastoma and leukemia origin. A monospecific polyclonal antibody against pp-vWF, but not against fibronectin, laminin, and von Willebrand factor (vWF), completely blocked the pp-vWF-mediated adhesion, indicating that the cell adhesion was due to the pp-vWF molecule and not due to possible contamination of these three well-known adhesive proteins. The cell-adhesion activity was also observed with human ppvWF and, furthermore, the adhesion to both bovine and human pp-vWF was not affected by a peptide containing the Arg-Gly-Asp sequence while the peptide abolished the cell adhesion to vWF. The adhesion was completely dependent on Mg2+ and inhibited by Ca"'. Inhibition by an anti-(pl integrin) mAb (4B4) indicates that the receptor for this protein belongs to the pl-integrin family. A monoclonal antibody (TC4) among several antibodies directed against bovine pp-vWF inhibited the B16 adhesion to immobilized pp-vWF. The epitope for this monoclonal antibody lies in a central 8-kDa portion of pp-vWF, suggesting that this region is important for the cell-adhesion activity. This idea was supported by the finding that purified 8-kDa fragment promoted adhesion of B16 cells in a concentration-dependent manner. As pp-vWF shows unique cell-type specificity in its adhesion activity, which is completely different from that of fibronectin, laminin, vWF and collagen, it may be a novel type of adhesive glycoprotein that utilizes a Dl-integrin receptor.The adhesion of cells to extracellular matrix (ECM) proteins is a key step involved in modulating the morphology, proliferation, differentiation, and migration of cells. This process is mediated by interactions between specific cell surface receptors and specific matrix proteins. Different cells adhere to different adhesive proteins due to the differential expression of surface receptors. It is well known that many cells adhere to fibronectin, laminin and collagen through more than one class of receptors including various integrins [ l , 21. Although these three proteins are abundant in ECM and thought to be primarily important in tissue organization under physiological conditions, there are a number of other proteins that have cell-adhesion activity in vitro and are thought to play a crucial role in more specialized stages of cellular events. [7]. A large amount of evidence is accumulating that these adhesive glycoproteins play dynamic roles in embryo-

Tissue Transglutaminase, Coagulation Factor XIII, and the Pro-polypeptide of von Willebrand Factor Are All Ligands for the Integrins α9β1 and α4β1

“…We also reported that G-361 cells adhered to pp-vWF (30) and that this adhesion was not inhibited at all by an anti-␣ 4 mAb, but we were unable to define the responsible integrin(s). By analogy with tTG and FXIII, we have suspected that this adhesion might be mediated by ␣ 9 ␤ 1 .…”

“…In the case of MOLT-3 cells, in addition to 0.25 mM MnCl 2 , 5 g/ml TS2/16, a ␤ 1 -integrin activating mAb, was added to the cell suspension. Cell adhesion assays were performed according to the method described previously (30). In brief, 6-mm square chips cut from bacteriologic plastic dishes (Falcon 1029) were coated for 16 h at 4°C with 50 l of solution containing adhesive proteins at the following concentrations diluted in 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl.…”

Propolypeptide of von Willebrand Factor Is a Novel Ligand for Very Late Antigen-4 Integrin

“…Furthermore, we have found that pp-vWF serves as a substrate for transglutaminase and is specifically cross-linked to laminin (7), suggesting a possibility that it acts as transient matrix protein upon secretion from platelets and endothelial cells at the site of vascular injury. In a previous paper (8), we reported that pp-vWF promoted the attachment and spreading of melanoma cells. The receptor responsible for this adhesion was the ␤1 class of integrin but the corresponding ␣ subunit could not be identified.…”

“…Integrinexpressing Leukemia Cells-In a previous study (8), we have found that only a limited number of cell lines are capable of adhering to pp-vWF, i.e. only two cell lines of melanoma origin out of more than 15 cell lines of both normal and tumor tissue origin adhered well to pp-vWF.…”

Expression of the Molecule Detectable by Anti-Propolypeptide of von Willebrand Factor Antibody in Rat Mesangial Cells in Anti-Thy 1.1 mAb 1-22-3 Induced Glomerulonephritis: A Marker of Injured Mesangial Cells

“…We have shown that pp-vWF binds to collagen with an affinity comparable to that of mature vWF and inhibits collagen-induced platelet aggregation [9, 10]and that pp-vWF is cross-linked to laminin [11]. Recently, we have also found that pp-vWF serves as a ligand for very-late antigen 4 integrin [12, 13]. These observations from in vitro experiments suggest that pp-vWF is incorporated in the extracellular matrix and affects the cell-matrix interaction and that pp-vWF functions in leukocyte recruitment to inflammatory or vascular injury sites.…”