Stimulation of urokinase-type plasminogen activator receptor expression by PMA requires JNK1-dependent and -independent signaling modules R Gum 4 , J Juarez 1 , H Allgayer 1 , A Mazar 2 , Y Wang 3 and D Boyd 1 1 Department of Tumor Biology/Head & Neck Surgery, M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas, USA; 2 Angstrom Pharmaceuticals, 11772 Sorrento Valley Road, #101, San Diego, California, USA; 3 Department of Renal Medicine, Westmead Hospital, The University of Sydney, Westmead NSW 2145, Sydney, Australia; 4 Department of Molecular Immunology, SmithKline Beecham, King of Prussia, Pennsylvania 19406, USA 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 eect 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 eect 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-de®cient 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 (7184) 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 expres- sion 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 eectively 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 signi®cance 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. Keywords: urokinase receptor; proteolysis; JNK; MAPK Introduction The urokinase-type plasminogen activator is a serine protease that converts the inert zymogen plasminogen into plasmin a protease with broad substrate speci®city (Nielsen et al., 1982; Robbins et al., 1967). Urokinase can bind speci®cally and with high anity (K D '0.5 nM) to a 45 ± 60 kDa heavily glycosylated cell surface receptor (u-PAR) (Stoppelli et al., 1986; Vassalli et al., 1985). The receptor is comprised of three similar repeats approximately 90 residues each (Behrendt et al., 1991; Riittinen et al., 1996). The aminoterminal domain binds the plasminogen activator with the carboxyterminus domain serving to anchor the binding protein to the cell surface via a glycosyl- phosphatidylinositol chain (Behrendt et al., 1991; Riittinen et al., 1996). The u-PAR gene is 7 exons long and is located on chromosome 19q13 (Vagnarelli et al., 1992; Casey et al., 1994). Transcription of the u- PAR gene gives rise to a 1.4 kb mRNA or an alternatively spliced variant lacking the membrane attachment peptide sequence (Roldan et al., 1990; Pyke et al., 1993). The amounts of u-PAR are controlled mainly at the transcriptional level through 398 base pairs of upstream sequence but altered message stability and receptor recycling may represent other means of controlling the amount of this gene product at the cell surface (Lengyel et al., 1996; Shetty et al., 1997; Lund et al., 1995; Nykjaer et al., 1997). The u-PAR has been implicated in a number of physiological and pathological processes involving tissue remodeling. For example, subsequent to injury, regenerating epithelial outgrowths express u-PAR mRNA in keratinocytes at the leading edge (Romer et al., 1994). Similarly, wounded endothelial cells in monolayer rapidly up-regulate their u-PAR expression (Pepper et al., 1993). In cancer, it appears that the u- PAR plays a prominent role in tumor cell invasion and metastasis and this may very well represent an Correspondence: D Boyd, Department of Tumor Biology, Box 108, M.D. Anderson Cancer Center, 1515 Holcombe Blvd. Houston, Texas, USA Received 29 September 1997; revised 20 February 1998; accepted 20 February 1998 Oncogene (1998) 17, 213 ± 225  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc