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

Abstract

LRP16 gene expression is induced by 17-estradiol (E2) via estrogen receptor alpha (ER ) 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 58-truncated constructs, and a luciferase reporter. The 58-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 ER and Sp1 were required for hormone-induced transactivation, which involved both ER and Sp1 directly binding to DNA. Taken together, these findings suggest that ER and Sp1 play a role in activation of the human LRP16 gene promoter.

Role of specificity protein transcription factors in estrogen-induced gene expression in MCF-7 breast cancer cells

“…For example, studies in this laboratory and others have demonstrated that deletion and mutational analysis of promoters derived from several E 2 -responsive genes contain GC-rich motifs that bind Sp proteins (Safe & Kim 2004). Some of the genes that fall into this category are important for cell proliferation, angiogenesis, and nucleotide metabolism and include E 2 F1, retinoic acid receptor a (RARa), carbamoylphosphate synthetase/ aspartate transcarbamylase/dihydroorotase (CAD), bcl-2, DNA polymerase a, vascular endothelial growth factor (VEGF), VEGFR receptor 2 (VEGFR2), progesterone receptor, creatine kinase B, thymidylate synthase, insulinlike growth factor binding protein 4, epidermal growth factor receptor, receptor for advanced glycation end products, low density lipoprotein receptor, vitamin D receptor, pS2, LRP16, metastasis associated protein 3, and SK3 (Porter et al 1996, Duan et al 1998, Sun et al 1998, 2005, Qin et al 1999, Xie et al 1999, 2000, Byrne et al 2000, Salvatori et al 2000, Saville et al 2000, Stoner et al 2000, Tanaka et al 2000, Castro-Rivera et al 2001, Li et al 2001, Bruning et al 2003, Jacobson et al 2003, Khan et al 2003, Ngwenya & Safe 2003, Schultz et al 2003, 2005, Fujita et al 2004, Bardin et al 2005, Zhao et al 2005, Higgins et al 2006b). In addition, RNA interference studies with a small inhibitory RNA (siRNA) for Sp1 (iSp1) show that after transfection with iSp1, there was a significant decrease in hormone-induced G 0 /G 1 to S-phase progression, suggesting that ERa/Sp1 plays an important role in this process (Abdelrahim et al 2002).…”

Differential induction of LRP16 by liganded and unliganded estrogen receptor α in SKOV3 ovarian carcinoma cells

“…A proximal region of K676 to K24 bp of the human LRP16 promoter was previously identified to be essential for estrogen induction of LRP16 expression in MCF-7 cells (Han et al 2008). Estrogen induces LRP16 gene transactivation by stimulating the interaction and recruitment of ERa and Sp1 transcription factor at a 1/2 ERE/ GC-rich site and multiple GC-rich sites present in K676 to K24 bp of the upstream regulatory region of LRP16 gene (Zhao et al 2005, Han et al 2008. By promoter analysis, we demonstrate that the fragment from K676 to K214 bp of the LRP16 upstream regulatory region mainly confers estrogen-repressed effect of LRP16 expression (Fig.…”

“…BG-1 human ovarian epithelial cancer cell line was cultured as previously described (Geisinger et al 1989). Steroid-deprived serum was prepared as previously described (Zhao et al 2005). …”

“…Mammalian expression plasmid for ERa (pS5G-hERa) was provided by Prof. Hajime Nawata at Kyushu University, and the reporter 3!ERE-TATA-Luc was provided by Prof. Donald P McDonnell at Duke University Medical Center (Norris et al 1998). The luciferase reporter constructs pGL3-S 0 , pGL3-S 2 , pGL3-S 4 , pGL3-S 5 , and pGL3-S B1 , containing the fragment of K2623 to K24 bp, K1775 to K24 bp, K1064 to K24 bp, K676 to K24 bp, K213 to K24 bp of the LRP16 upstream regulatory region respectively, have been previously described (Zhao et al 2005, Han et al 2008.…”

“…In cell culture, it has been shown that the expression level of LRP16 is strongly dependent on the estrogen actions in ERa-positive breast and endometrial cancer cell lines . A proximal region of K676 to K24 bp of the human LRP16 promoter, in which a 1/2 ERE/Sp1 site and multiple GC-rich elements that confer estrogen responsiveness have been recognized, is essential for estrogen action (Zhao et al 2005, Han et al 2008. Interestingly, estrogen-upregulated LRP16 can interact with ERa and enhance the receptor's transcriptional activity in a ligand-dependent manner, thus establishing a positive feedback regulatory loop between LRP16 and ERa signal transduction in estrogen-responsive breast cancer cells .…”