LOCALIZATION OF UROKINASE TO FOCAL ADHESIONS BY HUMAN FIBROSARCOMA CELLS SYNTHESIZING RECOMBINANT VITRONECTIN

Cell surface plasminogen activators have been proposed to participate in cell migration and invasion by activating both intracellular signal ing pathways and extracellular proteolysis. Urokinase-type plasminogen activator (uPA) is secreted from many cell types and localizes to foc al contact areas when cells are seeded onto the plasma protein vitrone ctin. Induction of vitronectin synthesis during migration of neural cr est cells and growth of certain tumors suggests that the de novo synth esis 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 secretio n and matrix deposition on the localization and activity of cell-assoc iated uPA, HT-1080 fibrosarcoma cells were transfected with the Rc/CMV expression vector containing a vitronectin cDNA insert and stable cel l lines expressing vitronectin were selected. Vitronectin-secreting ce lls were allowed to attach and spread on collagen- and fibronectin-coa ted substrates. Within 6 h, vitronectin was detected on the substrate; vitronectin synthesis was accompanied by the clustering of both the a lpha(v) beta(5) vitronectin receptor and uPA into vinculin-containing focal adhesions. Although mock transfected cells formed small focal ad hesions on both collagen and fibronectin, no co-localization of uPA or alpha(v) beta(5) to focal adhesions was evident in these cells. Vitro nectin-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 v itronectin and its matrix association by transfected HT-1080 fibrosarc oma cells results in localization of uPA to alpha(v) beta(5) containin g focal adhesions, decreased cell surface uPA activity, and an increas e in cell adhesion.