Stimulation of urokinase-type plasminogen activator receptor expression by PMA requires JNK1-dependent and -independent signaling modules

Oncogene. 1998 Jul 16;17(2):213-25. doi: 10.1038/sj.onc.1201917.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Calcium-Calmodulin-Dependent Protein Kinases / antagonists & inhibitors
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Dose-Response Relationship, Drug
  • Flavonoids / pharmacology
  • JNK Mitogen-Activated Protein Kinases
  • Mitogen-Activated Protein Kinases / metabolism
  • Nerve Tissue Proteins / metabolism
  • Promoter Regions, Genetic
  • Protein-Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins c-raf / metabolism
  • Proto-Oncogene Proteins p21(ras) / metabolism
  • Receptors, Cell Surface / biosynthesis*
  • Receptors, Cell Surface / genetics
  • Receptors, Urokinase Plasminogen Activator
  • Signal Transduction
  • Tetradecanoylphorbol Acetate / pharmacology*
  • Transcription Factor AP-1 / metabolism
  • Transcription, Genetic
  • Up-Regulation

Substances

  • Flavonoids
  • Nerve Tissue Proteins
  • Receptors, Cell Surface
  • Receptors, Urokinase Plasminogen Activator
  • Transcription Factor AP-1
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • Calcium-Calmodulin-Dependent Protein Kinases
  • JNK Mitogen-Activated Protein Kinases
  • Mitogen-Activated Protein Kinases
  • Proto-Oncogene Proteins p21(ras)
  • Tetradecanoylphorbol Acetate
  • 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one