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 determinants of nuclear receptor-corepressor interaction.

Retinoic acid and thyroid hormone receptors can act alternatively as ligand-independent repressors or ligand-dependent activators, based on an exchange of N-CoR or SMRT-containing corepressor complexes for coactivator complexes in response to ligands. We provide evidence that the molecular basis of N-CoR recruitment is similar to that of coactivator recruitment, involving cooperative binding of two helical interaction motifs within the N-CoR carboxyl terminus to both subunits of a RAR-RXR heterodimer. The N-CoR and SMRT nuclear receptor interaction motifs exhibit a consensus sequence of LXX I/H I XXX I/L, representing an extended helix compared to the coactivator LXXLL helix, which is able to interact with specific residues in the same receptor pocket required for coactivator binding. We propose a model in which discrimination of the different lengths of the coactivator and corepressor interaction helices by the nuclear receptor AF2 motif provides the molecular basis for the exchange of coactivators for corepressors, with ligand-dependent formation of the charge clamp that stabilizes LXXLL binding sterically inhibiting interaction of the extended corepressor helix.

Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation.

Ligand-dependent activation of gene transcription by nuclear receptors is dependent on the recruitment of coactivators, including a family of related NCoA/SRC factors, via a region containing three helical domains sharing an LXXLL core consensus sequence, referred to as LXDs. In this manuscript, we report receptor-specific differential utilization of LXXLL-containing motifs of the NCoA-1/SRC-1 coactivator. Whereas a single LXD is sufficient for activation by the estrogen receptor, different combinations of two, appropriately spaced, LXDs are required for actions of the thyroid hormone, retinoic acid, peroxisome proliferator-activated, or progesterone receptors. The specificity of LXD usage in the cell appears to be dictated, at least in part, by specific amino acids carboxy-terminal to the core LXXLL motif that may make differential contacts with helices 1 and 3 (or 3') in receptor ligand-binding domains. Intriguingly, distinct carboxy-terminal amino acids are required for PPARgamma activation in response to different ligands. Related LXXLL-containing motifs in NCoA-1/SRC-1 are also required for a functional interaction with CBP, potentially interacting with a hydrophobic binding pocket. Together, these data suggest that the LXXLL-containing motifs have evolved to serve overlapping roles that are likely to permit both receptor-specific and ligand-specific assembly of a coactivator complex, and that these recognition motifs underlie the recruitment of coactivator complexes required for nuclear receptor function.

Transcription activation by the human estrogen receptor subtype beta (ER beta) studied with ER beta and ER alpha receptor chimeras.

We have studied the two estrogen receptor (ER) subtypes, ER alpha and ER beta, and chimeric constructs with ER alpha and ER beta to examine the bioactivities of these receptors and their responses to estrogen and antiestrogen ligands. Transcriptional activity of ER beta is highly dependent on cell/promoter context and on the nature of the ligand. ER beta activated significant levels of transcription in response to estrogens in certain cell types, but showed only moderate activity compared with ER alpha in others. Antiestrogens such as tamoxifen and 2-phenylbenzofuran, which show some agonistic activity with ER alpha, exhibit no agonistic activity with ER beta. Alteration of the amino-terminal A/B receptor domain can result in a dramatic change in cell type- and ligand-specific transcriptional activity of ER beta. Upon replacing the A/B domain of ER beta with the A/B domain of ER alpha, this receptor chimera not only exhibits an improved transcriptional response to estrogens, but also is now able to activate transcription upon treatment with these antiestrogens. As antiestrogen agonism was lacking in ER beta and the ER beta/alpha chimera containing the amino-terminal A/B domain of ER beta fused to domains C through F of ER alpha, but was restored in an ER alpha/beta chimera containing the A/B domain of ER alpha, antiestrogen agonism was shown to depend on the A/B domain (activation function-1-containing region) of ER alpha. Together, these results indicate that the differences in the amino-terminal regions of ER alpha and ER beta contribute to the cell- and promoter-specific differences in transcriptional activity of these receptors, and their ability to respond to different ligands, thus providing a mechanism for differentially regulated transcription by these two ERs.

Interactions controlling the assembly of nuclear-receptor heterodimers and co-activators.

Retinoic-acid receptor-alpha (RAR-alpha) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) are members of the nuclear-receptor superfamily that bind to DNA as heterodimers with retinoid-X receptors (RXRs). PPAR-RXR heterodimers can be activated by PPAR or RXR ligands, whereas RAR-RXR heterodimers are selectively activated by RAR ligands only, because of allosteric inhibition of the binding of ligands to RXR by RAR. However, RXR ligands can potentiate the transcriptional effects of RAR ligands in cells. Transcriptional activation by nuclear receptors requires a carboxy-terminal helical region, termed activation function-2 (AF-2), that forms part of the ligand-binding pocket and undergoes a conformational change required for the recruitment of co-activator proteins, including NCoA-1/SRC-1. Here we show that allosteric inhibition of RXR results from a rotation of the RXR AF-2 helix that places it in contact with the RAR coactivator-binding site. Recruitment of an LXXLL motif of SRC-1 to RAR in response to ligand displaces the RXR AF-2 domain, allowing RXR ligands to bind and promote the binding of a second LXXLL motif from the same SRC-1 molecule. These results may partly explain the different responses of nuclear-receptor heterodimers to RXR-specific ligands.

Signal-specific co-activator domain requirements for Pit-1 activation.

POU-domain proteins, such as the pituitary-specific factor Pit-1, are members of the homeodomain family of proteins which are important in development and homeostasis, acting constitutively or in response to signal-transduction pathways to either repress or activate the expression of specific genes. Here we show that whereas homeodomain-containing repressors such as Rpx2 seem to recruit only a co-repressor complex, the activity of Pit-1 is determined by a regulated balance between a co-repressor complex that contains N-CoR/SMRT, mSin3A/B and histone deacetylases, and a co-activator complex that includes the CREB-binding protein (CBP) and p/CAF. Activation of Pit-1 by cyclic AMP or growth factors depends on distinct amino- and carboxy-terminal domains of CBP, respectively. Furthermore, the histone acetyltransferase functions of CBP or p/CAF are required for Pit-1 function that is stimulated by cyclic AMP or growth factors, respectively. These data show that there is a switch in specific requirements for histone acetyltransferases and CBP domains in mediating the effects of different signal-transduction pathways on specific DNA-bound transcription factors.

Transcription factor-specific requirements for coactivators and their acetyltransferase functions.

Different classes of mammalian transcription factors-nuclear receptors, cyclic adenosine 3',5'-monophosphate-regulated enhancer binding protein (CREB), and signal transducer and activator of transcription-1 (STAT-1)-functionally require distinct components of the coactivator complex, including CREB-binding protein (CBP/p300), nuclear receptor coactivators (NCoAs), and p300/CBP-associated factor (p/CAF), based on their platform or assembly properties. Retinoic acid receptor, CREB, and STAT-1 also require different histone acetyltransferase (HAT) activities to activate transcription. Thus, transcription factor-specific differences in configuration and content of the coactivator complex dictate requirements for specific acetyltransferase activities, providing an explanation, at least in part, for the presence of multiple HAT components of the complex.

William L. McGuire Memorial Lecture. Antiestrogens: mechanisms of action and resistance in breast cancer.

Antiestrogens have proven to be highly effective in the treatment of hormone-responsive breast cancer. However, resistance to antiestrogen therapy often develops. In addition, although tamoxifen-like antiestrogens are largely inhibitory and function as estrogen antagonists in breast cancer cells, they also have some estrogen-like activity in other cells of the body. Thus, recent efforts are being directed toward the development of even more tissue-selective antiestrogens, i.e. compounds that are antiestrogenic on breast and uterus while maintaining the beneficial estrogen-like actions on bone and the cardiovascular system. Efforts are also being directed toward understanding ligand structure-estrogen receptor (ER) activity relationships and characterizing the molecular changes that underlie alterations in parallel signal transduction pathways that impact on the ER. Recent findings show that antiestrogens, which are known to exert most of their effects through the ER of breast cancer cells, contact a different set of amino acids in the hormone binding domain of the ER than those contacted by estrogen, and evoke a different receptor conformation that results in reduced or no transcriptional activity on most genes. Resistance to antiestrogen therapy may develop due to changes at the level of the ER itself, and at pre- and post-receptor points in the estrogen receptor-response pathway. Resistance could arise in at least four ways: (1) ER loss or mutation; (2) Post-receptor alterations including changes in cAMP and phosphorylation pathways, or changes in coregulator and transcription factor interactions that affect the transcriptional activity of the ER; (3) Changes in growth factor production/sensitivity or paracrine cell-cell interactions; or (4) Pharmacological changes in the antiestrogen itself, including altered uptake and retention or metabolism of the antiestrogen. Model cell systems have been developed to study changes that accompany and define the antiestrogen resistant versus sensitive breast cancer phenotype. This information should lead to the development of antiestrogens with optimized tissue selectivity and agents to which resistance may develop more slowly. In addition, antiestrogens which work through somewhat different mechanisms of interaction with the ER should prove useful in treatment of some breast cancers that become resistant to a different category of antiestrogens.

A transcriptionally active estrogen receptor mutant is a novel type of dominant negative inhibitor of estrogen action.

We have characterized a human estrogen receptor (ER) mutant, V364E, which has a single amino acid substitution in its hormone-binding domain. This ER mutant is fully active or even superactive at saturating levels of estradiol (10(-8) M E2) yet has the capacity to act as a strong dominant negative inhibitor of the wild type ER. In transient transfection assays using ER-negative Chinese hamster ovary (CHO) cells and two different estrogen response element (ERE)-containing promoter reporter genes, V364E treated with 10(-8) M E2 exhibited approximately 250% and 100% of the activity of the wild type ER with these two promoter contexts, respectively. Despite the high activity of V364E when present alone in cells, coexpression of both V364E and wild type ER causes a significant decrease in overall ER-mediated transcriptional activity. On the TATA promoter, where V364E was more inhibitory, estrogen-stimulated activity was reduced by approximately 50% at a 1:1 ratio of mutant to wild type ER expression vector, and at a 10:1 ratio, 75% of ER activity was inhibited. V364E was expressed at lower levels than wild type ER and has a approximately 40-fold lower affinity for E2 compared with wild type ER. In promoter interference assays, V364E exhibited a strict dependence upon E2 for binding to an ERE. Surprisingly, even when V364E was unable to bind to ERE DNA (i.e. either at low E2 concentration or by mutation of its DNA-binding domain), this mutant retained full dominant negative activity. This highly active ER mutant is, thus, able to repress ER-mediated transcription when the mutant and wild type ER are present together in cells, even without DNA binding. Since competition for ERE binding and the formation of inactive heterodimers cannot fully account for the dominant negative activity of V364E, it is probable that altered interactions with proteins important in ER-mediated transcription play a key role in the repression of transcription by V364E. The properties and probable mechanism of action of V364E distinguish it from other previously described dominant negative inhibitors, in which competition for cis-acting DNA elements by transcriptionally inactive receptors played a large role in the resultant dominant negative phenotype.

Different regions in activation function-1 of the human estrogen receptor required for antiestrogen- and estradiol-dependent transcription activation.

The human estrogen receptor (ER) is a ligand-inducible transcription factor that contains two transcriptional activation functions, one located in the NH2-terminal region of the protein (AF-1) and the second in the COOH-terminal region (AF-2). Antiestrogens, such as trans-hydroxytamoxifen (TOT), have partial agonistic activity in certain cell types, and studies have implied that this agonism is AF-1-dependent. We have made progressive NH2-terminal and other segment deletions and ligations in the A/B domain, and studied the transcriptional activity of these mutant ERs in ER-negative MDA-MB-231 human breast cancer and HEC-1 human endometrial cancer cells. Using several estrogens and several partial agonist/antagonist antiestrogens, we find that estrogens and antiestrogens require different regions of AF-1 for transcriptional activation. Deletion of the first 40 amino acids has no effect on receptor activity. Antiestrogen agonism is lost upon deletion to amino acid 87, while estrogen agonism is not lost until deletions progress to amino acid 109. Antiestrogen agonism has been further defined to require amino acids 41-64, as deletion of only these amino acids results in an ER that exhibits 100% activity with E2, but no longer shows an agonist response to TOT. With A/B-modified receptors in which antiestrogens lose their agonistic activity, the antiestrogens then function as pure estrogen antagonists. Our studies show that in these cellular contexts, hormone-dependent transcription utilizes a range of the amino acid sequence within the A/B domain. Furthermore, the agonist/antagonist balance and activity of antiestrogens such as TOT are determined by specific sequences within the A/B domain and thus may be influenced by differences in levels of specific factors that interact with these regions of the ER.

Analysis of estrogen receptor transcriptional enhancement by a nuclear hormone receptor coactivator.

The estrogen receptor (ER), a member of a large superfamily of nuclear hormone receptors, is a ligand-inducible transcription factor that regulates the expression of estrogen-responsive genes. The ER, in common with other members of this superfamily, contains two transcription activation functions (AFs)--one located in the amino-terminal region (AF-1) and the second located in the carboxyl-terminal region (AF-2). In most cell contexts, the synergistic activity of AF-1 and AF-2 is required for full estradiol (E2)-stimulated activity. We have previously shown that a ligand-dependent interaction between the two AF-containing regions of ER was promoted by E2 and the antiestrogen trans-hydroxytamoxifen (TOT). This interaction, however, was transcriptionally productive only in the presence of E2. To explore a possible role of steroid receptor coactivators in transcriptional synergism between AF-1 and AF-2, we expressed the amino terminal (AF-1-containing) and carboxyl-terminal (AF-2-containing) regions of ER as separate polypeptides in mammalian cells, along with the steroid receptor coactivator-1 protein (SRC-1). We demonstrate that SRC-1, which has been shown to significantly increase ER transcriptional activity, enhanced the interaction, mediated by either E2 or TOT, between the AF-1-containing and AF-2-containing regions of the ER. However, this enhanced interaction resulted in increased transcriptional effectiveness only with E2 and not with TOT, consistent with the effects of SRC-1 on the full-length receptor. Our results suggest that after ligand binding, SRC-1 may act, in part, as an adapter protein that promotes the integration of amino- and carboxyl-terminal receptor functions, allowing for full receptor activation. Potentially, SRC-1 may be capable of enhancing the transcriptional activity of related nuclear receptor superfamily members by facilitating the productive association of the two AF-containing regions in these receptors.

Ligand-dependent, transcriptionally productive association of the amino- and carboxyl-terminal regions of a steroid hormone nuclear receptor.

The estrogen receptor (ER), a 66-kDa protein that mediates the actions of estrogens in estrogen-responsive tissues, is a member of a large superfamily of nuclear hormone receptors that function as ligand-activated transcription factors. ER shares a conserved structural and functional organization with other members of this superfamily, including two transcriptional activation functions (AFs), one located in its amino-terminal region (AF-1) and the second located in its carboxyl-terminal, ligand-binding region (AF-2). In most promoter contexts, synergism between AF-1 and AF-2 is required for full ER activity. In these studies, we demonstrate a functional interaction of the two AF-containing regions of ER, when expressed as separate polypeptides in mammalian cells, in response to 17 beta-estradiol (E2) and antiestrogen binding. The interaction was transcriptionally productive only in response to E2, and was eliminated by point or deletion mutations that destroy AF-1 or AF-2 activity or E2 binding. Our results suggest a definitive mechanistic role for E2 in the activity of ER--namely, to alter receptor conformation to promote an association of the amino- and carboxyl-terminal regions, leading to transcriptional synergism between AF-1 and AF-2. The productive re assembly of two portions of ER expressed in cells as separate polypeptides demonstrates the evolutionarily conserved modular structural and functional organization of the nuclear hormone receptors. The ligand-dependent interaction of the two AF-containing regions of ER allows for the assembly of a complete activation function from two distinct regions within the same protein, providing a mechanism for hormonally regulated transcription.