REQUIREMENT OF AN UPSTREAM AP-1 MOTIF FOR THE CONSTITUTIVE AND PHORBOL ESTER-INDUCIBLE EXPRESSION OF THE UROKINASE-TYPE PLASMINOGEN-ACTIVATOR RECEPTOR GENE

The urokinase-type plasminogen activator receptor (u-PAR) facilitates extracellular matrix proteolysis by accelerating plasmin formation at the cell surface, The present study was undertaken to identify element s in the u-PAR promoter required for the elevated expression of this b inding site, Toward this end, we used two cultured colon cancer cell l ines; one (RKO) has a transcriptionally activated u-PAR gene, and the other (GEO) overexpresses the receptor only after phorbol ester treatm ent. A chloramphenicol acetyltransferase (CAT) reporter driven by 398 nucleotides of 5' regulatory sequence of the u-PAR gene was strongly a ctivated in the RKO cells, which displays approximately 3 x 10(5) rece ptors/cell. A region of this promoter between -197 and -8 was required for optimal expression, as indicated using a CAT reporter driven by 5 ' deleted fragments. DNase I footprinting revealed three protected reg ions (I, -190 to -171; II, -148 to -124; and III, -99 to -70) in this part of the promoter, Mutation of an AP-1 binding site at -184 within region I reduced activation of the promoter by 85%. Deletion of either region II or III also reduced promoter activity by over 60%. An oligo nucleotide spanning the AP-1 motif at -184 bound, specifically, nuclea r factors from RKO cells, and antibodies specific for Jun-D, c-Jun, or Fra-1 proteins supershifted the complex indicating the presence of th ese proteins. The amount of these factors was reduced in GEO cells in which the u-PAR gene is only weakly transcriptionally activated, Expre ssion of a vector encoding a wild-type Jun-D cDNA increased u-PAR prom oter activity in GEO cells, Conversely, transfection of RKO cells with a transactivation domain-lacking Jun-D expression construct resulted in a dose-dependent decrease in u-PAR promoter activity, Treatment of GEO cells with phorbol ester increased u-PAR mRNA and the activity of a CAT reporter driven by the wild-type but not the AP-1 (-184)mutated u-PAR promoter, and this was associated with a strong induction in the amount of Jun-D, c-Jun, and c-Fos. Methylation interference studies u sing a fragment of the u-PAR promoter (spanning -201 to -150) bound wi th nuclear extracted proteins from RKO cells, and phorbol 12-myristate 13 acetate-treated and -untreated GEO cells showed that the contact p oints corresponded 60 the AP-1 binding site at -184, Thus, the elevate d expression of u-PAR in RKO cells, which constitutively produces this binding site, as well as in phorbol 12-myristate 13-acetate stimulate d GEO cells requires an AP-1 motif located 184 bp upstream of the tran scriptional start site.