Transcriptional activation of transforming growth factor alpha 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 b p (- 252 to -200) sequence containing two imperfect estrogen responsive ele ments (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 specif ic cis-elements and transacting factors were determined by promoter analysi s in transient transfection experiments, gel mobility shift assays and in v itro 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-DN A complex is formed using nuclear extracts from MCF-7 cells but not with re combinant human ER alpha or Sp1 proteins, suggesting that other nuclear fac tor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)E RE1/2 motif identified in the TGF alpha gene promoter has also been charact erized 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.