Prourokinas 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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 87; no. 6; pp. 2230 - 2234
• Main Authors: Pollanen, Jari, Vaheri, Antti, Tapiovaara, Hannele, Riley, Elizabeth, Bertram, Ken, Woodrow, Graeme, Stephens, Ross W.
• Format: Journal Article
• Language: English
• Published: National Academy of Sciences of the United States of America 01.03.1990
• Subjects: Antibodies; Cell culture techniques; Cells; Cultured cells; Focal adhesions; Monoclonal antibodies; Plasminogen activators; Plasminogen inactivators; Rhabdomyosarcoma; Tumors
• ISSN: 0027-8424

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.