TRANSCRIPTIONAL REGULATION OF THE TISSUE FACTOR GENE IN HUMAN EPITHELIAL-CELLS IS MEDIATED BY SP1 AND EGR-1

Tissue factor (TF) gene expression is rapidly induced in epithelial ce lls by phorbol 12-myristate 13-acetate and serum. We have shown that t his induction is mediated by a novel serum response region (SRR) (-111 to +14 bp) within the human TF promoter. In this study, we characteri zed cis-acting genetic elements within the SRR that regulated basal an d inducible expression of the TF gene in HeLa cells, Gel mobility shif t assays using oligonucleotides spanning the entire SRR identified thr ee 12-base pair (bp) motifs within subregions 1, 2, and 3 that bound c onstitutively expressed Sp1 and inducibly expressed EGR-1. Analysis of protein binding to these 12-bp motifs by competition with Sp1 and EGR -1 sites, mutation, and antibody supershift experiments indicated that they each contained distinct EGR-1 and Sp1 sites that overlapped by 6 bp. Functional studies using HeLa cells transfected with plasmids con taining the wild-type TF promoter (-111 to +14 bp) or derivatives cont aining mutations in the three Sp1 and/or EGR-1 sites examined basal an d inducible expression, The Sp1 sites mediated basal promoter activity , and both Sp1 and EGR-1 sites were required for maximal induction of the TF promoter by phorbol 12-myristate 13 acetate or serum. These dat a indicated that TF gene expression in HeLa cells was regulated by bot h Sp1 and EGR-1.