Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells.
We studied the immunocytochemical localization of urokinase-type plasminogen activator (u-PA) and the type 1 plasminogen activator inhibitor (PAI-1) in human fibroblasts and sarcoma cells, using both polyclonal and monoclonal antibodies. The u-PA was found to be located at discrete cell-substratum contact sites, and also at areas of cell-cell contacts, whereas PAI-1 was distributed as a homogeneous carpet excluding strialike areas on the substrate under the cells. To confirm the extracellular localization of u-PA and PAI-1, we stained the cells live at 0 degree C before fixation. A double-labeling experiment showed different distribution of u-PA and PAI-1 under the cells, and especially their peripheral parts. The staining pattern of u-PA and PAI-1 resisted treatment with 0.2% saponin followed by mechanical removal of cells, a method previously reported to isolate focal contact membranes of fibroblasts. We further demonstrated the deposition of u-PA to the contact areas of cells obtained by saponin treatment by zymography, and that of PAI-1 by metabolic labeling, reverse zymography, immunoblotting, and immunoprecipitation. Fibronectin was also present in the preparations. The deposition of both PAI-1 and fibronectin by the sarcoma cells was enhanced, after treating the cells with 10(-6) M dexamethasone. The confinement of u-PA to discrete contact sites and the more uniform distribution of PAI-1 on the cell substratum may explain how cells producing large amounts of enzyme inhibitors can produce PA-mediated focal proteolysis.
D002460:Cell Line; D004792:Enzyme Precursors / Q000032:analysis*; D005347:Fibroblasts / Q000201:enzymology / Q000648:ultrastructure; D005354:Fibrosarcoma / Q000201:enzymology* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D008854:Microscopy, Electron; D008970:Molecular Weight; D010960:Plasminogen Activators / Q000032:analysis*; D015849:Plasminogen Inactivators; D012867:Skin; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis*
D004792:Enzyme Precursors; D006023:Glycoproteins; D015849:Plasminogen Inactivators; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator