INCREASE IN CYTOSOLIC CALCIUM UP-REGULATES THE SYNTHESIS OF TYPE-1 PLASMINOGEN-ACTIVATOR INHIBITOR IN THE HUMAN HISTIOCYTIC CELL-LINE U937

In the U937 histiocytic cell line, we investigated the effect of calci um-mobilizing agents with or without tumor necrosis factor-alpha (TNF) on the regulation of the synthesis of plasminogen activator inhibitor -type 1 (PAI-1). cultured U937 cells were stimulated with ionophore A2 3187 and thapsigargin with or without TNF. The response was analyzed i n terms of cytosolic calcium mobilization, PAI-1 accumulation in the m edium, and PAI-1 mRMa expression. The study was extended to urokinase (uPA) secretion and surface expression of its receptor (uPAR). Using F luo-3 as a calcium-indicator dye to measure cytosolic calcium mobiliza tion, we showed by flow cytometry that both agents mobilized calcium i n a dose-dependent manner. TNF provoked a slight calcium mobilization that was also observed by digital imaging microscopy. Association of T NF with the calcium-mobilizing agents potentiated the calcium mobiliza tion. Both calcium-mobilizing agents induced at 18 hours a dose-depend ent accumulation of PAI-1 in culture medium, whereas uPA was not affec ted. TNF alone induced a more marked accumulation of PAI-1 than of uPA . association of TNF with the agents induced a PAI-1 response that was more than additive of the two, whereas the secretion of uPA was not e nhanced. Membrane expression of uPAR, measured by flow cytometry, tend ed to be slightly augmented by the calcium-mobilizing agents only. All the treatments resulted in a significant increase in PAI-1 mRNA level at 3 hours after the stimulation, which was very marked when calcium mobilizing agents were present. Incubation of U937 cells in a calcium- free medium totally prevented both the mRNA expression and accumulatio n of PAI-1 induced by calcium-mobilizing agents and, to lesser extent, that induced by INF. The increase in PAI-1 mRNA expression did not re quire de novo protein synthesis, as cycloheximide did not suppress the increase in PAI-1 mRMA induced by calcium-mobilizing agents. It is co ncluded that, in U937 cells, calcium triggers a pathway that upregulat es PAI-1 synthesis and positively interacts with the TNF-induced pathw ay that stimulates PAI-1 synthesis. As uPA and uPAR were differently a ffected, it is suggested that an increase in cytosolic calcium leads t o a reduced pericellular proteolysis. (C) 1996 by The American Society of Hematology.