Mechanism of transcriptional regulation of LRP16 gene expression by 17-beta estradiol in MCF-7 human breast cancer cells

LRP16 gene expression is induced by 17-betaestradiol (E2) via estrogen receptor alpha (ERalpha) in MCF-7 human breast cancer cells. A previous study also demonstrated that ectopic expression of LRP16 gene promoted MCF-7 cell proliferation. To explore the mechanism of hormone-induced LRP16 gene expression, the LRP16 gene promoter region (-2600 to -24 bp upstream of the LRP16 gene translation starting site) was analyzed in the present study by using different 5'-truncated constructs, and a luciferase reporter. The 5'-flanking sequence of -676 to -24 bp (pGL3-S5) was found to be E2-responsive. After exchange of the fragment from -213 to -24 bp with the TK gene proximal promoter region in pGL3-S5, E2 still induced reporter gene activity in MCF-7 and HeLa cells. Sequence analysis showed that the pGL3-S6 (-676 to -214) sequence contains two motifs that may contribute to E2-induced transactivation; namely, an estrogen-responsive element (ERE) half-site/Sp1 at -246 to -227 bp and an E-box site at -225 to -219 bp. Further deletion and mutation analysis of these two motifs indicated that both the 1/2 ERE and Sp1 binding sites were required for E2 action, while E-box deletion did not affect the luciferase activity in MCF-7 and HeLa cells. The results of gel mobility shift and chromatin immunoprecipitation assays confirmed that both ERalphaand Sp1 were required for hormone-induced transactivation, which involved both ERalphaand Sp1 directly binding to DNA. Taken together, these findings suggest that ERalphaand Sp1 play a role in activation of the human LRP16 gene promoter.     

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

17beta-Estradiol (E2) induces transforming growth factor alpha (TGFalpha) 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 TGFalpha 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 trans-acting 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 (ERalpha)/Sp1 complex interacting with an Sp1(N)(30) ERE half-site ((1/2)) motif in which both ERalpha and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ERalpha 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 TGFalpha 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.     

A mathematical approach to predict the affinity of estrogen receptors alpha and beta binding to DNA

Estrogen receptors alpha and beta (ERalpha and ERbeta) bind to specific DNA sequences, estrogen response elements (EREs), usually located in the promoters of estrogen-regulated genes. The consensus ERE contains two inverted repeats of the 5'-AGGTCA-3' half-site (1/2 ERE) separated by three base pairs (bp). Many estrogen-responsive gene promoters contain one or more direct repeats (DR) of 1/2 ERE. Here, we examined the affinity of ERalpha and ERbeta binding and estradiol (E(2))-induced transactivation from select EREs and DRs. The affinity of ERalpha and ERbeta binding to imperfect EREs in vitro can be predicted from equations using the number of 1/2 EREs and the number of (AT)-(GC) bp substitutions within the 15-bp candidate ERE sequence as independent variables. Transactivation by ERalpha and ERbeta correlates with the affinity of ER-ERE binding with the exception of ERalpha from two low-affinity EREs. The equations developed here can be used to screen the promoters of estrogen-responsive genes for candidate ERE sequences.