Estrogen receptors: selective ligands, partners, and distinctive pharmacology.

The action of nuclear hormone receptors is tripartite, involving the receptor, its ligands, and its co-regulator proteins. The estrogen receptor (ER), a member of this superfamily, is a hormone-regulated transcription factor that mediates the effects of estrogens and anti-estrogens (e.g., tamoxifen) in breast cancer and other estrogen target cells. This chapter presents our recent work on several aspects of estrogen action and the function of the ER: 1) elucidation of ER structure-function relationships and development of ligands that are selective for one of the two ER subtypes, ERalpha or ERbeta; 2) identification of ER-selective co-regulators that potentiate the inhibitory effectiveness of anti-estrogens and dominant-negative ERs and modulate the activity of estrogens; 3) characterization of genes that are regulated by the anti-estrogen-ER versus the estrogen-ER complex; and 4) elucidation of the intriguing pharmacology of these ER complexes at different gene regulatory sites. These findings indicate that different residues of the ER hormone-binding domain are involved in the recognition of structurally distinct estrogens and anti-estrogens and highlight the exquisite precision of the regulation of ER activities by ligands, with small changes in ligand structure resulting in major changes in receptor character. Studies also explore the biology and distinct pharmacology mediated by ERalpha and ERbeta complexed with different ligands through different target genes. The upregulation of the anti-oxidant detoxifying phase II enzyme, quinone reductase, by the anti-estrogen-occupied ER, mediated via the electrophile response element in the QR gene, may contribute to the beneficial antioxidant effects of anti-estrogens in breast cancer and illustrates the activation of some genes by ER via non-estrogen response element sequences. The intriguing biology of estrogen in its diverse target cells is thus determined by the structure of the ligand, the ER subtype involved, the nature of the hormone-responsive gene promoter, and the character and balance of co-activators and co-repressors that modulate the cellular response to the ER-ligand complex. The continuing development of novel ligands and the study of how they function as selective agonists or antagonists through ERalpha or ERbeta should allow optimized tissue selectivity of these agents for hormone replacement therapy and treatment and prevention of breast cancer.

Molecular mechanisms of estrogen action: selective ligands and receptor pharmacology.

Estrogens exert profound effects on the physiology of diverse target cells and these effects appear to be mediated by two estrogen receptor (ER) subtypes, ERalpha and ERbeta. We have investigated how ER ligands, ranging from pure agonists to antagonists, interact with ERalpha and ERbeta, and regulate their transcriptional activity on different genes. Mutational mapping-structure activity studies indicate that different residues of the ER ligand binding domain are involved in the recognition of structurally distinct estrogens and antiestrogens. We have identified from ligands of diverse structure, several particularly interesting ones that are high potency selective agonists via ERalpha and others that are full agonists through ERalpha while being full antagonists through ERbeta. Antiestrogens such as hydroxytamoxifen, which are mixed agonist/antagonists through ERalpha, are pure antagonists through ERbeta at estrogen response element-containing gene sites. Studies with ERalpha/beta chimeric proteins reveal that tamoxifen agonism requires the activation function 1 region of ERalpha. Through two-hybrid assays, we have isolated an ER-specific coregulator that potentiates antiestrogen antagonist effectiveness and represses ER transcriptional activity. We have also focused on understanding the distinct pharmacologies of antiestrogen- and estrogen-regulated genes. Although antiestrogens are thought to largely act by antagonizing the actions of estrogens, we have found among several new ER-regulated genes, quinone reductase (QR), a detoxifying phase II antioxidant enzyme, that has its activity up-regulated by antiestrogens in an ER-dependent manner in breast cancer cells. This response is antagonized by estrogens, thus showing 'reversed pharmacology'. Increased QR activity by antiestrogens requires a functional ER (ERalpha or ERbeta) and is, interestingly, mediated via the electrophile response element in the QR gene 5' regulatory region. The up-regulation of QR may contribute to the beneficial effects of tamoxifen, raloxifene, and other antiestrogens in breast cancer prevention and treatment. Estrogens rapidly up-regulate expression of several genes associated with cell cytoarchitectural changes including NHE-RF, the sodium hydrogen exchanger regulatory factor, also known as EBP50. NHE-RF/EBP50 is enriched in microvilli, and may serve as a scaffold adaptor protein in regulating early changes in cell architecture and signal transduction events induced by estrogen. Analyses of the regulatory regions of these primary response genes, and the antioxidant and other signaling pathways involved, are providing considerable insight into the mechanisms by which ligands, that function as selective estrogen receptor modulators or SERMs, exert their marked effects on the activities and properties of target cells. The intriguing biology of estrogens in its diverse target cells is thus determined by the structure of the ligand, the ER subtype involved, the nature of the hormone-responsive gene promoter, and the character and balance of coactivators and corepressors that modulate the cellular response to the ER-ligand complex. The continuing development of ligands that function as selective estrogens or antiestrogens for ERalpha or ERbeta should allow optimized tissue selectivity of these agents for menopausal hormone replacement therapy and the treatment and prevention of breast cancer.

Estrogen receptor regulation of the Na+/H+ exchange regulatory factor.

To better understand the actions of estrogens and antiestrogens in estrogen target cells, we have searched for estrogen-regulated genes in human breast cancer cells, in which the number of genes known to be directly activated by estrogen is quite small. Using differential display RNA methods, we have identified the human homolog of the Na+ -H+ exchanger regulatory factor (NHE-RF), an approximately 50-kDa protein that is also an ezrin-radixin-moesin-binding phosphoprotein, as being under rapid and direct regulation by estrogen in estrogen receptor (ER)-containing breast cancer cells. Stimulation by estrogen of NHE-RF RNA is rapid, being near maximal (approximately 6-fold) by 1 h, and is not blocked by cycloheximide, indicating that it is a primary response. Stimulation is selective for estrogen ligands, with no stimulation by other classes of steroid hormones, and stimulation by estrogen is suppressed by the antiestrogens tamoxifen and ICI 182,780. Induction is shown to require an active ER through several approaches, including the use of ER-negative breast cancer cells containing a stably integrated ER. NHE-RF protein levels, monitored using antibodies specific for this protein, increase after estrogen and reach maximal levels at 24-48 h. Interestingly, NHE-RF is a PDZ domain-containing protein that is enriched in polarized epithelia, where it is known to be localized in microvilli. Among various human tissues we have examined, we found that NHE-RF is expressed at a fairly high level in mammary tissue. NHE-RF regulates protein kinase A inhibition of the Na+ -H+ exchanger and may serve as a scaffold adaptor protein that contributes to the specificity of signal transduction events. Our findings suggest that the early, known effects of estrogen on cell cytoarchitecture (e.g. increasing microvilli on breast cancer cells) and on some cell signaling pathways (e.g. those involving cAMP) may involve rapid estrogen-mediated changes in the production of NHE-RF.

Mechanistic aspects of estrogen receptor activation probed with constitutively active estrogen receptors: correlations with DNA and coregulator interactions and receptor conformational changes.

The estrogen receptor (ER) belongs to a large family of nuclear receptors, many of whose members function as ligand-dependent transcriptional activators. The mechanism by which the receptor is converted from an inactive into an activated state is not yet completely understood. To investigate the kind of changes in receptor conformation and interactions that are involved in this activation, we have used the wild type ER and a set of constitutively active ER point mutants that show from 20% to nearly 100% activity in the absence of estrogen. These mutants are of particular interest as they could mimic, in the absence of ligand, the activated state of the wild type receptor. We have analyzed several transcriptional steps that could be involved in the activation: the ability of these receptors 1) to interact with several coactivators (steroid receptor coactivator-1, SRC-1; transcription intermediary factor-1, TIF-1; and estrogen receptor-associated protein 140, ERAP 140) and with members of the preinitiation complex [TATA box-binding protein (TBP), transcription factor IIB (TFIIB)]; 2) to exhibit conformational changes revealed by proteolytic digest patterns similar to those observed for the wild type hormone-occupied ER; and 3) to bend estrogen response element-containing DNA, which is thought to be one of the important phenomena triggering transcriptional activation. Our results demonstrate that the interaction of these mutant receptors with coactivators is likely to be one of the features of the activated step, as the mutant receptors interacted with some coactivators in a ligand-independent manner in proportion to their extent of constitutive activity. However, the different degrees of ligand-independent interaction of the mutant ERs with the three coactivators suggest that SRC-1, TIF-1, and ERAP 140 may play different roles in receptor activity. Limited proteolytic digest experiments reveal that the activated state of the receptor corresponds to a particular conformation of the receptor, which is fully observed with the mutant ER showing the highest activity in the absence of estrogen. Finally, it appears that in inactive or active states, the receptor exhibits distinctly different DNA-bending abilities. Addition of estradiol is able to modify the bending ability of only the wild type receptor, whereas estradiol has no influence on the constitutive receptors, which exhibited the same bending ability as that observed for the ligand-occupied wild type receptor. These data document that the ER undergoes major changes in its conformation and also in its functional properties when it is turned from an inactive into an active state and that mutational changes in the ER protein that result in constitutive, hormone-independent activation mimic many of the changes in ER properties that are normally under hormone regulation.

Cloning and structural organization of the gene encoding the murine nuclear receptor transcription factor, NURR1.

NURR1 is an immediate early gene product and a member of the nuclear receptor superfamily of transcription factors. Using the NURR1 cDNA as a probe, we isolated the genomic DNA encoding NURR1 from a mouse 129SvEv genomic library. The NURR1 gene is approximately 6.2 kb long and is organized into 7 exons separated by 6 introns. Structural analysis of the NURR1 reveals that this gene shares a similar structure with that of the nuclear receptor NUR77/NGF1-B. As in NUR77, the promoter region of NURR1 lacks an identifiable TATA box, but is GC-rich. The proximal promoter region also contains an ATF/CREB consensus binding site that may participate in cAMP-mediated induction of this immediate early gene product. Isolation and structural characterization of the NURR1 gene provides information for further developmental and transcriptional regulation studies of this gene.