BINDING OF UROKINASE TO PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 MEDIATES CELL-ADHESION AND SPREADING

Urokinase plasminogen activator and its receptor are both found at the surface of the cell membrane in many cell types. The plasminogen acti vator inhibitor type-1 (PAI-1) is often associated with the extracellu lar matrix. The spatial localization of these three molecules could ac count for their involvement in cell adhesion and/or migration. We have shown previously that the urokinase receptor mediates mechanical forc e transmission across the cell surface to the cytoskeleton. Here we in vestigated whether immobilized plasminogen activator inhibitor type 1 (PAI-1) could regulate cell spreading and cytoskeleton reorganization. Serum deprived human myogenic cells were plated in serum free medium onto bacteriologic dishes precoated with different extracellular matri x ligands (fibronectin, vitronectin, or type 1 collagen) or PAI-I at i ncreasing concentrations. The number of adherent cells and their proje cted area were quantitated after 3 hours of plating. PAI-1 promoted ce ll adhesion and spreading in a dose dependent manner. Addition of anti bodies to PAI-1 inhibited the adhesion on PAI-1 coated dishes in a dos e dependent way. The PAI-1 mediated cell adhesion required the presenc e of urokinase at the cell surface. Removal of the glycosylphosphatidy linositol (GPI)-linked proteins abolished cell adhesion on PAI-I dish, suggesting its dependence on the presence of the urokinase receptor, a GPI-linked receptor. Furthermore, addition of antibodies against alp ha v beta 3 integrin completely inhibited cell adhesion on PAI-I, sugg esting that alpha v beta 3 might be the transmembrane molecule that ph ysically connects the complex of PAI-1, urokinase, and urokinase recep tor to the cytoskeleton. Visualization of spread cells stained for fil amentous actin with confocal microscopy showed a dose-dependent increa se of filopodia on PAI-1 coated dishes and cytoskeletal reorganization , suggesting a migratory profile. These data indicate that PAI-I plays a direct role in dynamic cell adhesion particularly at the leading ed ge, where increased levels of urokinase plasminogen activator (uPA) an d its receptor (uPAR) are localized in migrating cells. Immobilized PA I-1 could therefore serve to bridge the cell surface with the extracel lular matrix via the formation of a multimolecular complex that includ es alpha v beta 3 integrins in myogenic cells.