Different Mechanisms Contribute to Simultaneous Inhibition of Urokinase and Tissue-Type Plasminogen Activators by Glucocorticoids in Human Ovarian Carcinoma Cells

Previous studies from our laboratory have demonstrated that OVCA 433 human ovarian carcinoma cells are glucocorticoid responsive by several criteria and contain high affinity, saturable, steroidspecific glucocorticoid receptors. These cells secrete both mammalian plasminogen activators (PAs), urokinase (uPA) and tissue-type PA (tPA). Treatment of OVCA 433 cells with 1 × 10−7m dexamethasone (Dex) for 4 days led to 77% and 83% reductions in the extracellular activities of uPA and tPA, respectively, released into serum-free conditioned medium during a 1-h period. Dex treatment led to a 71% decrease in the rate of extracellular uPA antigen accumulation, as determined by enzyme-linked immunosorbent assay, as well as a 73% reduction in steady state uPA mRNA levels. In contrast, Dex treatment led to only a 42% decrease in the rate of extracellular tPA antigen accumulation and a 48% decrease in tPA mRNA levels; such decreases were insufficient to account for the 83% reduction in tPA activity. Thus, while Dex-induced decreases in uPA antigen and mRNA levels accounted for all but 6% of the decrease in uPA activity, a large discrepancy existed between the magnitudes of decreased tPA activity and decreased tPA antigen and mRNA levels. OVCA 433 cells produce both PAI-1 and PAI-2, two specific PA inhibitors. Treatment of cells with 1 × 10−7 M Dex for 4 days led to a 3.3-fold increase in the rate of extracellular PAI-1 accumulation, with little or no effect on PAI-2 accumulation. In addition, Dex treatment led to marked amplification of a 120K PA activity band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography, similar to the sodium dodecyl sulfate-stable tPA:PAI-1 complexes reported by others. The mol wt of this band was consistent with complexes between 65K tPA and 54K PAI-1. Thus, despite equivalent relative reductions of extracellular uPA and tPA activities after glucocorticoid treatment of OVCA 433 cells, different combinations of mechanisms account for inhibition of these two tPAs. For uPA, extracellular activity inhibition results almost entirely from decreased extracellular enzyme accumulation secondary to decreased mRNA levels. In contrast, inhibition of extracellular tPA activity involves only modest decreases in extracellular enzyme accumulation and mRNA levels; our data indicate that stimulated production of PAI-1 with subsequent formation of tPA:PAI-1 complexes may be the additional inhibitory component required to account for the full inhibition of tPA activity observed.