Identification of a novel nuclear factor-kappaB sequence involved in expression of urokinase-type plasminogen activator receptor

We have previously defined the promoter of human urokinase-type plasminogen activator receptor (uPAR) gene in a 188-bp fragment between bases -141 and +47 relative to the translation start site. Here, we report that a novel n uclear factor-kappaB (NF-kappa B)-like sequence (5'-GGGAGGAGTC-3') at -45 i s located in the uPAR promoter and one of the two DNase I-protected regions , region I between bases -51 and -30. This NF-kappa B-like motif differs at positions 7-9 from the decameric consensus sequences of NF-kappa B (5'-GGG RNNYYCC-3' where R indicates A or G, Y indicates C or T, and N indicates an y nucleotide) and at positions 1 and 7-9 from the kappa B-like motifs (5'-H GGARNYYCC-3' where H indicates A, C, or T, R indicates A or G, Y indicates C or T, and N indicates any nucleotide). Nuclear extracts from HCT116 cells contain proteins that specifically bind to the NF-kappa B-like site at pos ition -45. Mutation of the NF-kappa B-like motif decreased the binding of t ranscription factor NF-kappa B and reduced the uPAR promoter activity in co mparison with the wild-type sequences. Co-transfection with a dominant nega tive I-kappa B kinase-2 expression vector reduced uPAR promoter activity by 65-75%. These results demonstrate that a previously uncharacterized NF-kap pa B motif is required for uPAR promoter activity.