Stimulation of urokinase-type plasminogen activator receptor expression by PMA requires JNK1-dependent and -independent signaling modules.
The urokinase-type plasminogen activator receptor (u-PAR) has been implicated 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 known, 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 that the inductive effect of PMA on u-PAR expression also required a JNK1-dependent signaling cascade usually associated with stress-inducing stimuli. PMA treatment of the u-PAR-deficient OVCAR-3 ovarian cancer cells, which contain low JNK activities, resulted in a rapid (5 min) increase in JNK activity. Maximal JNK activity (12-fold induction) occurred after 30 min; this preceding the earliest detected rise in u-PAR protein (2 h). Dose-response studies with PMA also indicated that the increased JNK activity was tightly correlated with elevated u-PAR protein levels. The stimulation of u-PAR promoter activity by PMA required an intact upstream AP-1 motif (-184) and in PMA-treated cells this motif was bound with c-Jun as indicated from mobility shift assays. PMA up-regulated the c-Jun trans acting activity as indicated by the higher activity of a GAL4-regulated luciferase reporter in phorbol-ester-treated cells co-transfected with an expression vector encoding the c-Jun transactivation domain fused to the GAL4 DNA-binding domain. The ability of PMA to stimulate u-PAR promoter activity was effectively titrated out by the co-expression of either a kinase-defective JNK1 or a dominant negative MEKK1 the latter being an upstream activator of JNK1. Conversely, u-PAR promoter activity was stimulated by the co-expression of a constitutively active MEKK1 and this induction was antagonized by the inclusion of the kinase-defective JNK1 plasmid. We also determined the biological significance of the JNK1-dependent signaling cascade in regulating u-PAR promoter activity by c-Ha-ras since this oncogene is activated and/or overexpressed in a variety of tumors including ovarian cancer. Transfection of an activated c-Ha-ras into OVCAR-3 cells stimulated u-PAR promoter activity over 20-fold and this could be countered by the individual expression of dominant negative expression constructs to Rac-1, MEKK1 or JNK1. Taken together, these data suggest that the PMA- or c-Ha-Ras-dependent stimulation of u-PAR gene expression requires a JNK1-dependent signaling module and that, at least for PMA, 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 expression.
Gum
R
R
Department of Molecular Immunology, SmithKline Beecham, King of Prussia, Pennsylvania 19406, USA.
Juarez
J
J
Allgayer
H
H
Mazar
A
A
Wang
Y
Y
Boyd
D
D
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005419:Flavonoids / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics; D055293:Receptors, Urokinase Plasminogen Activator; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic; D015854:Up-Regulation
D005419:Flavonoids; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D018808:Transcription Factor AP-1; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D016283:Proto-Oncogene Proteins p21(ras); D013755:Tetradecanoylphorbol Acetate; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one