Prourokinase activation on the surface of human rhabdomyosarcoma cells: localization and inactivation of newly formed urokinase-type plasminogen activator by recombinant class 2 plasminogen activator inhibitor.
Recombinant class 2 plasminogen activator inhibitor (PAI-2) was used in an approach to probe the formation and location of enzymatically active urokinase-type plasminogen activator (u-PA) sites on the surface of cultured human rhabdomyosarcoma cells (RD cells). Activation of pro-u-PA on the cell surface and consequent binding of PAI-2 was dependent on the addition of native plasminogen to serum cultures of the cells. Inhibition of the enzyme activity of surface-bound u-PA by the added PAI-2 resulted in a 79% reduction in the capacity of the RD cells to generate cell surface-associated plasmin activity from bound plasminogen. Under these conditions, the PAI-2 probe was localized at focal adhesions of RD cells, where it colocalized with both extracellular u-PA and intracellular vinculin antigens in double immunofluorescence labeling. Specificity of the probe's interaction with cell surface-bound u-PA was confirmed by blocking with a monoclonal antibody to human u-PA, which could also inhibit the formation of bound plasmin activity. These results showed the assembly of the plasmin-generating system at focal adhesions and the accessibility of bound u-PA on which it depends to added PAI-2. Therefore, PAI-2 has the potential both to localize at sites of tumor expression of functionally active u-PA and simultaneously to inhibit cell surface plasminogen activation.
Pöllänen
J
J
Department of Virology, University of Helsinki, Finland.
Vaheri
A
A
Tapiovaara
H
H
Riley
E
E
Bertram
K
K
Woodrow
G
G
Stephens
R W
RW
D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007700:Kinetics; D010960:Plasminogen Activators / Q000378:metabolism*; D015849:Plasminogen Inactivators / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000201:enzymology; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D004792:Enzyme Precursors; D015849:Plasminogen Inactivators; D011994:Recombinant Proteins; D010960:Plasminogen Activators; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator