Estrogen receptor β activates the human retinoic acid receptor α-1 promoter in response to tamoxifen and other estrogen receptor antagonists, but not in response to estrogen

Human estrogen receptor-alpha (hER alpha) or -beta (hER beta) transfected into Hep G2 or COS1 cells each responded to estrogen to increase transcription from an estrogen-responsive element (ERE)-driven reporter vector with similar fold induction through a classical mechanism involving direct receptor binding to DNA. ER antagonists inhibited this estrogen induction through both hER alpha and hER beta, although raloxifene was more potent through ER alpha than ER beta, and tamoxifen was more potent via ER beta than ER alpha. We have shown previously that estrogen stimulated the human retinoic acid receptor-alpha-1 (hRAR alpha-1) promoter through nonclassical EREs by a mechanism that was ER alpha dependent, but that did not involve direct receptor binding to DNA. We show here that in contrast to hER alpha, hER beta did not induce reporter activity driven by the hRAR alpha-1 promoter in the presence of estrogen. While hER beta did not confer estrogen responsiveness on this promoter, it did elicit transcriptional activation in the presence of 6-hydroxytamoxifen (4-OH-Tam). Additionally, this 4-OH-Tam agonist activity via ER beta was completely blocked by estrogen. Like ER alpha, transcriptional activation of this promoter by ER beta was not mediated by direct receptor binding to DNA. While hER alpha was shown to act through two estrogen-responsive sequences within the promoter, hER beta acted only at the 3'-region, through two Sp1 sites, in response to 4-OH-Tam. Other ER antagonists including raloxifene, ICI-164,384 and ICI-182,780 also acted as agonists through ER beta via the hRAR alpha-1 promoter. Through the use of mutant and chimeric receptors, it was shown that the 4-OH-Tam activity via ER beta from the hRAR alpha-1 promoter in Hep G2 cells required the amino-terminal region of ER beta, a region that was not necessary for estrogen-induced ER beta activity from an ERE in Hep G2 cells. Additionally, the progesterone receptor (PR) antagonist RU486 acted as a weak (IC50 >1 mu M) antagonist via hER alpha and as a fairly potent (IC50 similar to 200 nM) antagonist via hER beta from an ERE-driven reporter in cells that do not express PR. Although RU486 bound only weakly to ER alpha or ER beta in vitro, it did bind to ER beta in whole-cell binding assays, and therefore, it is likely metabolized to an ER beta-interacting compound in the cell. Interestingly, RU486 acted as an agonist through ER beta to stimulate the hRAR alpha-1 promoter in Rep G2 cells. These findings may have ramifications in breast cancer treatment regimens utilizing tamoxifen or other ER antagonists and may explain some of the known estrogenic or antiestrogenic biological actions of RU486.