No abstract

Cell surface plasminogen activators have been proposed to participate in cell migration and invasion by activating both intracellular signaling pathways and extracellular proteolysis. Urokinase-type plasminogen activator (uPA) is secreted from many cell types and localizes to focal contact areas when cells are seeded onto the plasma protein vitronectin. Induction of vitronectin synthesis during migration of neural crest cells and growth of certain tumors suggests that the de novo synthesis and deposition of vitronectin into the tissue matrix may remodel the matrix to provide an environment suitable for cell migration and (or) tumor invasion. To investigate the effects of vitronectin secretion and matrix deposition on the localization and activity of cell-associated uPA, HT-1080 fibrosarcoma cells were transfected with the Rc/CMV expression vector containing a vitronectin cDNA insert and stable cell lines expressing vitronectin were selected. Vitronectin-secreting cells were allowed to attach and spread on collagen- and fibronectin-coated substrates. Within 6 h, vitronectin was detected on the substrate; vitronectin synthesis was accompanied by the clustering of both the alpha v beta 5 vitronectin receptor and uPA into vinculin-containing focal adhesions. Although mock transfected cells formed small focal adhesions on both collagen and fibronectin, no co-localization of uPA or alpha v beta 5 to focal adhesions was evident in these cells. Vitronectin-secreting cells also exhibited decreased levels of plasminogen activation and increased levels of cell adhesion as compared with the mock transfected cells. These data demonstrate that the synthesis of vitronectin and its matrix association by transfected HT-1080 fibrosarcoma cells results in localization of uPA to alpha v beta 5 containing focal adhesions, decreased cell surface uPA activity, and an increase in cell adhesion.

During the process of tissue remodeling, vitronectin (Vn) is deposited in the extracellular matrix where it plays a key role in the regulation of pericellular proteolysis and cell motility. In previous studies we have shown that extracellular levels of vitronectin are controlled by receptormediated endocytosis and that this process is dependent upon vitronectin binding to sulfated proteoglycans. We have now identified vitronectin's 12 amino acid "basic domain" which is contained within the larger 40 amino acid heparin binding domain, as a syndecan binding site. Recombinant vitronectins representing wild type vitronectin (rVn) and vitronectin with the basic domain deleted (rVn 347-358) were prepared in a baculoviral expression system. The rVn as well as a glutathione S-transferase (GST) fusion protein, consisting of vitronectin's 40 amino acid heparin binding domain (GST-VnHBD), exhibited dose dependent binding to HT-1080 cell surfaces, which was attenuated following deletion of the basic domain. In addition, GST-VnHBD supported both HT-1080 and dermal fibroblast cell adhesion, which was also dependent upon the basic domain. Similarly, ARH-77 cells transfected with syndecans -1, -2, or -4, but not Glypican-1, adhered to GST-VnHBD coated wells, while adhesion of these same cells was lost following deletion of the basic domain. HT-1080 cells were unable to degrade rVn 347-358. Degradation of rVn 347-358 was completely recovered in the presence of GST-VnHBD but not in the presence of GST-VnHBD 347-358. These results indicate that turnover of soluble vitronectin requires ligation of vitronectin's basic domain and that this binding event can work in trans to regulate vitronectin degradation.