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

17β-Estradiol (E2) induces transforming growth factor α (TGFα) 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 TGFα 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 α (ERα)/Sp1 complex interacting with an Sp1(N)₃₀ ERE half-site ( 1/2 ) motif in which both ERα and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ERα or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)ERE 1/2 motif identified in the TGFα 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.