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

LRP16 gene expression is induced by 17-βestradiol (E2) via estrogen receptor alpha (ERα) 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 ERαand Sp1 were required for hormone-induced transactivation, which involved both ERαand Sp1 directly binding to DNA. Taken together, these findings suggest that ERαand Sp1 play a role in activation of the human LRP16 gene promoter.