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

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 sur...

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Published inProceedings 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
LanguageEnglish
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
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Summary: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.
ISSN:0027-8424
1091-6490