Transcriptional activation of transforming growth factor α by estradiol:: requirement for both a GC-rich site and an estrogen response element half-site

17 beta-Estradiol (E2) induces transforming growth factor alpha (TGF alpha) gene expression in MCF-7 cells and previous studies have identified a 53 bp (- 252 to -200) sequence containing two imperfect estrogen responsive elements (EREs) that contribute to E2 responsiveness. Deletion analysis of the TGF alpha gene promoter in this study identified a second upstream region of the promoter (- 623 to - 549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and transacting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor alpha (ER alpha)/Sp1 complex interacting with an Sp1(N)(30) ERE half-site (1/2) motif in which both ER alpha and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ER alpha or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)ERE1/2 motif identified in the TGF alpha gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.