STIMULATION OF UROKINASE-TYPE PLASMINOGEN-ACTIVATOR RECEPTOR EXPRESSION BY PMA REQUIRES JNK1-DEPENDENT AND JNK1-INDEPENDENT SIGNALING MODULES

The urokinase-type plasminogen activator receptor (u-PAR) has been imp licated in tumor progression, and previous studies have shown that the expression of this gene is strongly up-regulated by PMA, Although the signaling mechanism by which PMA modulates u-PAR expression is not kn own, the effect of this phorbol ester on the expression of other genes has been ascribed to activation of the c-Raf-1-ERK signaling pathway. However, in the current study we examined an alternate possibility th at the inductive effect of PMA on u-PAR expression also required a JNK 1-dependent signaling cascade usually associated with stress-inducing stimuli. PMA treatment of the u-PAR-deficient OVCAR-3 ovarian cancer c ells, which contain low JNK activities, resulted in a rapid (5 min) in crease in JNK activity, Maximal JNK activity (12-fold induction) occur red after 30 min; this preceding the earliest detected rise in u-PAR p rotein (2 h), Dose-response studies with PMA also indicated that the i ncreased JNK activity was tightly correlated with elevated u-PAR prote in levels. The stimulation of u-PAR promoter activity by PMA required an intact upstream AP-1 motif (- 184) and in PMA-treated cells this mo tif was bound with c-Jun as indicated from mobility shift assays, PMA up-regulated the c-Jun trans acting activity as indicated by the highe r activity of a GAL4-regulated luciferase reporter in phorbol-ester-tr eated cells co-transfected with an expression vector encoding the c-Ju n transactivation domain fused to the GAL4 DNA-binding domain, The abi lity of PMA to stimulate u-PAR promoter activity was effectively titra ted out by the co-expression of either a kinase-defective JNK1 or a do minant negative MEKK1 the latter being an upstream activator of JNK1, Conversely, u-PAR promoter activity was stimulated by the co-expressio n of a constitutively active MEKK1 and this induction was antagonized by the inclusion of the kinase-defective JNK1 plasmid, We also determi ned the biological significance of the JNK1-dependent signaling cascad e in regulating u-PAR promoter activity by c-Ha-ras since this oncogen e is activated and/or overexpressed in a variety of tumors including o varian cancer. Transfection of an activated c-Ha-ras into OVCAR-3 cell s stimulated u-PAR promoter activity over 20-fold and this could be co untered by the individual expression of dominant negative expression c onstructs to Rac-1, MEKK1 or JNK1, Taken together, these data suggest that the PMA- or c-Ha-Ras-dependent stimulation of u-PAR gene expressi on requires a JNK1-dependent signaling module and that, at least for P MA, the concurrent stimulation of a JNK1-independent signaling module is also required. Thus, caution should be exercised in invoking linear signaling modules to account for the regulation of inducible gene exp ression.