Estrogen Receptor Ligand Binding Research Articles

Repression of AP-1 function: a mechanism for the regulation of Blimp-1 expression and B lymphocyte differentiation by the B cell lymphoma-6 protooncogene.

The B cell lymphoma-6 (BCL-6) transcriptional repressor protein is an important regulator of B cell differentiation and is strongly implicated in the development of B cell lymphoma. Expression of the Blimp-1 transcription factor, which is critical for promoting B cell differentiation into plasma cells, is repressed by BCL-6. We have investigated the mechanism for how BCL-6 represses Blimp-1 transcription, and have found that BCL-6 regulates the Blimp-1 promoter through a novel mechanism involving AP-1 elements. Specifically, BCL-6 is a potent repressor of transcriptional activity mediated by AP-1 factors. We found that the zinc-finger region of BCL-6 interacts with c-Jun, JunB, and JunD proteins but does not bind c-Fos or Fra-2 proteins. An estrogen receptor ligand binding domain fusion with the BCL-6 zinc finger domain can act as a estrogen-inducible dominant negative protein and increase AP-1 activity in BCL-6(+) cells but not in BCL-6(-) cells, indicating that endogenous BCL-6 represses AP-1 activity. Additionally, we have confirmed a specific interaction between c-Jun and the zinc finger domain of BCL-6 in vivo using a mammalian two-hybrid assay. Repression of AP-1 function by BCL-6 may be a key mechanism for how BCL-6 regulates gene expression to control inflammation, lymphocyte differentiation, and lymphomagenesis.

Protection against Anoikis and Down-regulation of Cadherin Expression by a Regulatable β-Catenin Protein

beta-Catenin signaling plays a key role in a variety of cellular contexts during embryonic development and tissue differentiation. Aberrant beta-catenin signaling has also been implicated in promoting human colorectal carcinomas as well as a variety of other cancers. To study the molecular and cellular biological functions of beta-catenin in a controlled fashion, we created a regulatable form of activated beta-catenin by fusion to a modified estrogen receptor (ER) ligand binding domain (G525R). Transfection of tissue culture cells with expression vectors encoding this hybrid protein allows the signal transduction function of beta-catenin to be induced by the synthetic estrogen, 4-hydroxytamoxifen, leading to regulated activation of a beta-catenin-lymphocyte enhancer-binding factor-dependent reporter gene as well as induction of endogenous cyclin D1 expression. The activation of ER-beta-catenin signaling rescues RK3E cells from anoikis and correlates with an increased phosphorylation of mitogen-activated protein kinase. The inhibition of anoikis by ER-beta-catenin can be abolished by a mitogen-activated protein kinase pathway inhibitor, PD98059. Evidence is also provided to show that ER-beta-catenin down-regulates cadherin protein levels. These findings support a key role for activated beta-catenin signaling in processes that contribute to tumor formation and progression.

Estrogen regulation of gene expression in the brain: a possible mechanism altering the response to psychostimulants in female rats

Acute behavioral responses to cocaine are more pronounced in female than in male rats. We have shown that 3 weeks of treatment with 17β-estradiol (E 2) implants significantly enhanced the hyperactivity induced by cocaine in ovariectomized (OVX) rats. The ligand-bound estrogen receptor (ER) functions as a transcription factor to regulate the expression of E-responsive genes. Thus, we hypothesized that estrogen (E) modulates the behavioral response to cocaine via regulation of expression of components of dopamine (DA) and serotonin (5-HT) systems in mesolimbic nuclei important in the response to cocaine as well as the hypothalamus, a brain area known to be E-responsive. Adult female Sprague–Dawley rats were OVX; half of them then received E 2 implant (OVX+E) ( n=8/group, two groups). Twenty-seven days later, brain tissue was collected to study E 2 effects on mRNA expression for DA D 1-like (D 1) and D 2-like (D 2S, D 2L, D 3) receptors, DA transporter (DAT), 5-HT 1A, 5-HT 1B, 5-HT 2A, 5-HT 2C receptors, and 5-HT transporter (SERT) as well as ERα and ERβ in amygdala, hypothalamus, nucleus accumbens, midbrain, and ventral tegmental area (VTA). We found that E 2 implants in OVX rats increased mRNA levels for D 1 receptor in hypothalamus, D 2L receptor in midbrain, and D 3 receptor in VTA, and decreased D 3 receptor mRNA levels in midbrain relative to OVX controls. E 2 also increased 5-HT 2C receptor mRNA levels in midbrain and hypothalamus. In addition, E 2 decreased mRNA levels for ERα in amygdala and hypothalamus and ERβ in amygdala. The present study demonstrates that E can regulate mRNA expression for specific DA and 5-HT receptors in a region-specific manner and suggests that such modifications may contribute to the behavioral response to cocaine.

The TRAP/Mediator coactivator complex interacts directly with estrogen receptors alpha and beta through the TRAP220 subunit and directly enhances estrogen receptor function in vitro.

Target gene activation by nuclear hormone receptors, including estrogen receptors (ERs), is thought to be mediated by a variety of interacting cofactors. Here we identify a number of nuclear extract-derived proteins that interact with immobilized ER ligand binding domains in a 17beta-estradiol-dependent manner. The most prominent of these are components of the thyroid hormone receptor-associated protein (TRAP)/Mediator coactivator complex, which interacts with ERalpha and ERbeta in both unfractionated nuclear extracts and purified form. Studies with extracts from TRAP220(-/-) fibroblasts reveal that these interactions depend on TRAP220, a TRAP/Mediator subunit previously shown to interact with ER and other nuclear receptors in a ligand-dependent manner. The physiological relevance of the in vitro interaction is documented further by the isolation of an ERalpha-TRAP/Mediator complex from cultured cells expressing an epitope-tagged ERalpha. Finally, the complete TRAP/Mediator complex is shown to enhance ER function directly in a highly purified cell-free transcription system. These studies firmly establish a direct role for TRAP/Mediator, through TRAP220, in ER function.

Estrogen Receptor Binding to DNA Is Not Required for Its Activity through the Nonclassical AP1 Pathway

In the classical signaling pathway, the estrogen receptor (ER) binds directly to estrogen response elements (EREs) to regulate gene transcription. To test the hypothesis that the nonclassical pathway involves ER interactions with other proteins rather than direct binding to DNA, mutations were introduced into the DNA binding domain (DBD) of the mouse ERalpha. The effects of these DBD mutations were examined in DNA binding assays using reporter constructs containing either EREs (classical) or AP1 (nonclassical) response elements. Using the AP1 reporter, there was a reversal of ER action relative to that seen with the ERE reporter. Estradiol induced suppression, and the antiestrogen ICI 182,780 stimulated transcription of the AP1 reporter. DBD mutations in the proximal (P-box) of the first zinc finger of the ER (E207A/G208A and E207G/G208S) eliminated ERE binding. These mutants were inactive using the ERE reporter but retained partial or full activity with the AP1 reporter. The DBD mutant ERs interacted with Jun when tested in mammalian cell two-hybrid assays. Two mutations (K366D and I362R) in the ER ligand binding domain known to alter coactivator interactions impaired transcriptional responses using either the ERE or AP1 reporters. We concluded that ER action through the AP1 response element involves interactions with other promoter-bound proteins instead of, or in addition to, direct binding to DNA. Interactions with coactivators were required for both pathways. These data supported a model in which ER-mediated transcriptional activation or repression is dependent on the ligand and the nature of the response element in the target gene.

A versatile framework for the design of ligand-dependent, transgene-specific transcription factors.

The ability to regulate transgene expression will be essential for the safety and efficacy of many gene therapies. Various ligand-dependent transcription factors, including steroid hormone receptors, have been modified to enable transgene-specific regulation. To minimize effects on cellular gene expression, chimeric steroid receptors have been constructed by replacing their native DNA binding domain (DBD) with a heterologous DBD, like that from the yeast transcription factor GAL4. This approach has limitations for human gene therapy, including the potential immunogenicity of the GAL4 domain and the inability to discriminate between different GAL4-linked transgenes in the same cell. To address this, we have constructed chimeric regulators containing the human estrogen receptor (ER) ligand binding domain (LBD) and a Cys(2)-His(2)-type zinc finger DBD. Cys(2)-His(2) zinc finger domains are common among human DNA binding proteins and can be engineered to selectively bind different DNA sequences. We demonstrate over 500-fold drug-dependent transgene induction with these chimeric regulators in vitro and the ability to regulate an adenovirus-delivered transgene in mice. Two chimeras containing different Cys(2)-His(2) domains displayed highly sequence-specific binding and regulation. Incorporating a point mutation in the ER LBD that ablates estrogen binding enables selective in vivo regulation with the clinically useful anti-estrogen tamoxifen. These Cys(2)-His(2)-ER LBD chimeras represent a versatile framework for creating transgene-specific regulators potentially useful for human gene therapy applications.

Estrogen receptor-mediated cross-talk with growth factor signaling pathways.

Estrogen (E2) palys critical roles in the development of tumors in female reproductive organs. Development of most breast cancers is dependent on E&sub2; in most cases. Most E&sub2; actions are considered to be exerted through two subtypes of Estrogen receptors (ERs), ERalpha and ERbeta. ERs belong to the nuclear receptor superfamily, and act as ligand-inducible transcription factors to activate transcription of a particular set of the target genes. Ligand-bound ER recruits at least two distinct classes of coactivator complexes. In estrogen-dependent breast cancer, growth factors are shown to often act synergisticaly with E&sub2;, and the breast cancer often become resistant to treatment of estogen antagonists. However, the molecular basis of this coupled regulation of growth factor and ER-mediated signaling and hormone-resistance are largely unknown. We have previously shown that MAP (mitogen-activated protein) kinase (MAPK) activated by growth factors phosphorylates and potentiates the N-terminal transactivation function (AF-1), indicating a possible molecular mechanism of a novel cross-talk between two signalings (Kato et al, 1995). Furthermore, we have identified a coactivator that specifically interacts with ER alpha AF-1 (Endoh et al, 1999). In this review, this cross-talk is discussed in terms of the transactivation function of ERs and their coactivators.

Triarylpyrazoles with basic side chains: development of pyrazole-based estrogen receptor antagonists

Recently, we developed a novel triaryl-substituted pyrazole ligand system that has high affinity for the estrogen receptor (ER) (Fink, B. E.; Mortenson, D. S.; Stauffer, S. R.; Aron, Z. D.; Katzenellenbogen, J. A. Chem. Biol. 1999, 6, 205). Subsequent work has shown that some analogues in this series are very selective for the ERα subtype in terms of binding affinity and agonist potency (Stauffer, S. R.; Coletta, C. J.; Tedesco, R.; Sun, J.; Katzenellenbogen, J. A. J. Med. Chem. 2000, submitted). We now investigate how this pyrazole ER agonist system might be converted into an antagonist or a selective estrogen receptor modifier (SERM) by incorporating a basic or polar side chain like those typically found in antiestrogens and known to be essential determinants of their mixed agonist/antagonist character. We selected an N-piperidinyl-ethyl chain as a first attempt, and introduced it at the four possible sites of substitution on the pyrazole core structure to determine the orientation that the pyrazole might adopt in the ER ligand binding pocket. Of these four, the C(5) piperidinyl-ethoxy-substituted pyrazole 5 had by far the highest affinity. Also, it bound to the ER subtype alpha (ERα) with 20-fold higher affinity than to ERβ. In cell-based transcription assays, pyrazole 5 was an antagonist on both ERα and ERβ, and it was also more potent on ERα. Based on structure-binding affinity relationships and on molecular modeling studies of these pyrazoles in a crystal structure of the ERα–raloxifene complex, we propose that pyrazoles having a basic substituent on the C(5) phenyl group adopt a binding mode that is different from that of the pyrazole agonists that lack this group. The most favorable orientation appears to be one which places the N(1) phenol in the A-ring binding pocket so that the basic side chain can adopt an orientation similar to that of the basic side chain of raloxifene.

A computationally based identification algorithm for estrogen receptor ligands: part 2. Evaluation of a hERalpha binding affinity model.

The objective of this study was to evaluate the capability of an expert system described in the previous paper (S. Bradbury et al., Toxicol. Sci. 58, 253-269) to identify the potential for chemicals to act as ligands of mammalian estrogen receptors (ERs). The basis of the expert system was a structure activity relationship (SAR) model, based on relative binding affinity (RBA) values for steroidal and nonsteroidal chemicals derived from human ERalpha (hERalpha) competitive binding assays. The expert system enables categorization of chemicals into (RBA ranges of < 0.1, 0.1 to 1, 1 to 10, 10 to 100, and >150% relative to 17ss-estradiol. In the current analysis, the algorithm was evaluated with respect to predicting RBAs of chemicals assayed with ERs from MCF7 cells, and mouse and rat uterine preparations. The best correspondence between predicted and observed RBA ranges was obtained with MCF7 cells. The agreement between predictions from the expert system and data from binding assays with mouse and rat ER(s) were less reliable, especially for chemicals with RBAs less than 10%. Prediction errors often were false positives, i.e., predictions of greater than observed RBA values. While discrepancies were likely due, in part, to species-specific variations in ER structure and ligand binding affinity, a systematic bias in structural characteristics of chemicals in the hERalpha training set, compared to the rodent evaluation data sets, also contributed to prediction errors. False-positive predictions were typically associated with ligands that had shielded electronegative sites. Ligands with these structural characteristics were not well represented in the training set used to derive the expert system. Inclusion of a shielding criterion into the original expert system significantly increased the accuracy of RBA predictions. With this additional structural requirement, 38 of 46 compounds with measured RBA values greater than 10% in hERalpha, MCF7, and rodent uterine preparations were correctly categorized. Of the remaining 129 compounds in the combined data sets, RBA values for 65 compounds were correctly predicted, with 47 of the incorrect predictions being false positives. Based upon this exploratory analysis, the modeling approach, combined with a high-quality training set of RBA values derived from a diverse set of chemical structures, could provide a credible tool for prioritizing chemicals with moderate to high ER binding affinity for subsequent in vitro or in vivo assessments.

Differential estrogen receptor binding of estrogenic substances: a species comparison

The study investigated the ability of 34 natural and synthetic chemicals to compete with [ 3H]17β-estradiol (E2) for binding to bacterially expressed glutathione- S-transferase (GST)-estrogen receptors (ER) fusion proteins from five different species. Fusion proteins consisted of the ER D, E and F domains of human alpha (GST-hERαdef), mouse alpha (GST-mERαdef), chicken (GST-cERdef), green anole (GST-aERdef) and rainbow trout ERs (GST-rtERdef). All five fusion proteins displayed high affinity for E2 with dissociation constants ( K d) ranging from 0.3 to 0.9 nM. Although, the fusion proteins exhibited similar binding preferences and binding affinities for many of the chemicals, several differences were observed. For example, α-zearalenol bound with greater affinity to GST-rtERdef than E2, which was in contrast to other GST-ERdef fusion proteins examined. Coumestrol, genistein and naringenin bound with higher affinity to the GST-aERdef, than to the other GST-ERdef fusion proteins. Many of the industrial chemicals examined preferentially bound to GST-rtERdef. Bisphenol A, 4- t-octylphenol and o,p′ DDT bound with approximately a ten-fold greater affinity to GST-rtERdef than to other GST-ERdefs. Methoxychlor, p,p′-DDT, o,p′-DDE, p,p′-DDE, α-endosulfan and dieldrin weakly bound to the ERs from the human, mouse, chicken and green anole. In contrast, these compounds completely displaced [ 3H]E2 from GST-rtERdef. These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for estrogenic compounds and that these differences may be due to the variability in the amino acid sequence within their respective ER ligand binding domains.

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, ERα and ERβ. We have investigated how ER ligands, ranging from pure agonists to antagonists, interact with ERα and ERβ, 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 ERα and others that are full agonists through ERα while being full antagonists through ERβ. Antiestrogens such as hydroxytamoxifen, which are mixed agonist/antagonists through ERα, are pure antagonists through ERβ at estrogen response element-containing gene sites. Studies with ERα/β chimeric proteins reveal that tamoxifen agonism requires the activation function 1 region of ERα. 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 (ERα or ERβ) 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 ERα or ERβ should allow optimized tissue selectivity of these agents for menopausal hormone replacement therapy and the treatment and prevention of breast cancer.

Chemically Regulated Zinc Finger Transcription Factors

Ligand-dependent transcriptional regulators were generated by fusion of designed Cys(2)-His(2) zinc finger proteins and steroid hormone receptor ligand binding domains. To produce novel DNA binding domains, three-finger proteins binding specific 9-base pair sequences were constructed from modular building blocks. Fusion of these zinc finger proteins to a transcriptional activation domain and to modified ligand binding domains derived from either the estrogen or progesterone receptors yielded potent ligand-dependent transcriptional regulators. Together with optimized minimal promoters, these regulators provide 4-hydroxytamoxifen- or RU486-inducible expression systems with induction ratios of up to 3 orders of magnitude. These inducible expression systems are functionally independent, and each can be selectively switched on within the same cell. The potential use of zinc finger-steroid receptor fusion proteins for the regulation of natural promoters was also explored. A gene-specific six-finger protein binding an 18-base pair target sequence was converted into a ligand-dependent regulator by fusion with either two estrogen receptor ligand binding domains or one ecdysone receptor and one retinoid X receptor ligand binding domain. These single-chain receptor proteins undergo an intramolecular rearrangement, rather than intermolecular dimerization and are functional as monomers. Thus, the ability to engineer DNA binding specificities of zinc finger proteins enables the construction of ligand-dependent transcriptional regulators with potential for the regulation of virtually any desired artificial or natural promoter. It is anticipated that the novel chemically regulated gene switches described herein will find many applications in applied and basic research, where the specific modulation of gene expression can be exploited.

The role of CBP in estrogen receptor cross-talk with nuclear factor-kappaB in HepG2 cells.

Functional interactions or cross-talk between ligand-activated nuclear receptors and the proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) may play a major role in ligand-mediated modification of diseases processes. In particular, the cardioprotective effects of estrogen replacement therapy are thought to be due in part to the ability of ligand-bound estrogen receptor (ER) to inhibit NF-kappaB function. In the current study 17beta-estradiol-bound ERalpha interfered with cytokine-induced activation of a NF-kappaB reporter in HepG2 cells. The estrogen metabolite, 17alpha-ethinyl estradiol, and the phytoestrogen, genistein, were also effective inhibitors of NF-kappaB activation, whereas tamoxifen, 4-hydroxytamoxifen, and raloxifene were inactive. This inhibition was reciprocal, as NF-kappaB interfered with the trans-activation properties of ERalpha. Ligand-bound ERalpha did not inhibit NF-kappaB binding to DNA, but it did decrease the histone acetyltransferase activity required for NF-kappaB transcriptional activity. Coexpression of the transcription coactivator CREB binding protein (CBP), but not steroid receptor coactivator 1a, reversed the ERalpha-mediated inhibition of NF-kappaB activity. Mammalian two-hybrid experiments also revealed that ligand-bound ERalpha can interact functionally with CBP-NF-kappaB complexes. We suggest that CBP targeting by ERalpha results in the inhibition of NF-kappaB and may occur through formation of transcriptionally inert multimeric complexes that are dependent upon the nature of the ERalpha ligand.

Mutations in the Estrogen Receptor Ligand Binding Domain Discriminate between Hormone-dependent Transactivation and Transrepression

The estrogen receptor (ER) suppresses transcriptional activity of the RelA subunit of nuclear factor-kappaB in a hormone-dependent manner by a mechanism involving both the receptor DNA binding domain and ligand binding domain (LBD). In this study we examine the role of the ER LBD in mediating ligand-dependent RelA transrepression. Both ERalpha and ERbeta inhibit RelA in response to 17beta-estradiol but not in the presence of antihormones. We have identified residues within the ERalpha LBD that are responsible for receptor dimerization and show that dimerization is necessary for transactivation and transrepression. Moreover we have generated mutant receptors that have lost their ability to inhibit RelA but retain their capacity to stimulate transcription and conversely mutants that are transcriptionally defective but capable of antagonizing RelA. Overexpression of p160 and cAMP-response element-binding protein-binding protein/p300 co-activators failed to relieve repression of RelA, which is consistent with the demonstration that RelA inhibition can occur independently of these co-activators. These findings suggest it is unlikely that sequestration of these cofactors required for ER transcriptional activation can account for hormone-dependent antagonism of RelA. The identification of ER mutants that discriminate between transactivation and transrepression implies that distinct surfaces within the LBD are involved in mediating these two receptor functions.

Identification of an Estrogen-Mediated Deoxyribonucleic Acid-Binding Independent Transactivation Pathway on the Epidermal Growth Factor Receptor Gene Promoter

To investigate the estrogenic effects on the transcriptional regulation of the epidermal growth factor (EGF) receptor (EGFR) gene, we assayed its promoter ability to direct transcription of the luciferase reporter gene after transfection into HeLa cells. Our studies demonstrated a dose-dependent activation of the EGFR gene transcription by ligand-bound estrogen receptor α (ERα). This action was retained by the 36-bp core promoter fragment and did not require the receptor DNA binding domain, as demonstrated by analyzing the role of ERα deletion mutants on EGFR gene promoter-derived constructs. The 36-bp promoter fragment does not contain an estrogen response element but an imperfect thyroid hormone response element half-site that overlaps the Sp1 binding site. ERα does not bind this imperfect thyroid hormone response element half-site but is able to enhance binding of Sp1 to its site, in gel mobility shift assays, suggesting that the mechanism by which the receptor stimulated the transcription involved protein-protein interactions that replaced DNA binding. To explain this action, we propose a model in which induction of the EGFR gene expression by estrogens in HeLa cells is dependent upon the formation of a transcriptionally active ERα-Sp1 complex that binds to the GC-rich (Sp1) region of the minimal promoter.

Identification of an Estrogen-Mediated Deoxyribonucleic Acid-Binding Independent Transactivation Pathway on the Epidermal Growth Factor Receptor Gene Promoter*

To investigate the estrogenic effects on the transcriptional regulation of the epidermal growth factor (EGF) receptor (EGFR) gene, we assayed its promoter ability to direct transcription of the luciferase reporter gene after transfection into HeLa cells. Our studies demonstrated a dose-dependent activation of the EGFR gene transcription by ligand-bound estrogen receptor alpha (ERalpha). This action was retained by the 36-bp core promoter fragment and did not require the receptor DNA binding domain, as demonstrated by analyzing the role of ERalpha deletion mutants on EGFR gene promoter-derived constructs. The 36-bp promoter fragment does not contain an estrogen response element but an imperfect thyroid hormone response element half-site that overlaps the Sp1 binding site. ERalpha does not bind this imperfect thyroid hormone response element half-site but is able to enhance binding of Sp1 to its site, in gel mobility shift assays, suggesting that the mechanism by which the receptor stimulated the transcription involved protein-protein interactions that replaced DNA binding. To explain this action, we propose a model in which induction of the EGFR gene expression by estrogens in HeLa cells is dependent upon the formation of a transcriptionally active ERalpha-Sp1 complex that binds to the GC-rich (Sp1) region of the minimal promoter.

Acyclic amides as estrogen receptor ligands: Synthesis, binding, activity and receptor interaction

We have prepared a series of bisphenolic amides that mimic bibenzyl and homobibenzyl motifs commonly found as substructures in ligands for the estrogen receptor (ER). Representative members were prepared from three classes: N-phenyl benzamides, N-phenyl acetamides, and N-benzyl benzamides; in some cases the corresponding thiocarboxamides and sulfonamides were also prepared. Of these three classes, the N-phenyl benzamides had the highest affinity for ER, the N-phenyl acetamides had lower, and the N-benzyl benzamides were prone to fragmentation via a quinone methide intermediate. In the N-phenyl benzamide series, the highest affinity analogues had bulky N-substituents; a CF3 group, in particular, conferred high affinity. The thiocarboxamides bound better than the corresponding carboxamides and these bound better than the corresponding sulfonamides. Binding affinity comparisons suggest that the p-hydroxy group on the benzoate ring, which contributes most to the binding, is playing the role of the phenolic hydroxyl of estradiol. Computational studies and NMR and X-ray crystallographic analysis indicate that the two anilide systems studied have a strong preference for the s-cis or exo amide conformation, which places the two aromatic rings in a syn orientation. We used this structural template, together with the X-ray structure of the ER ligand binding domain, to elaborate an additional hydrogen bonding site on a benzamide system that elevated receptor binding further. When assayed on the individual ER subtypes, ERα and ERβ, these compounds show modest binding affinity preference for ERα. In a reporter gene transfection assay of transcriptional activity, the amides generally have full to nearly full agonist character on ERα, but have moderate to full antagonist character on ERβ. One high affinity carboxamide is 500-fold more potent as an agonist on ERα than on ERβ. This work illustrates that ER ligands having simple amide core structures can be readily prepared, but that high affinity binding requires an appropriate distribution of bulk, polarity, and functionality. The strong conformational preference of the core anilide function in all of these ligands defines a rather rigid geometry for further structural and functional expansion of these series.

Exogenous cdk4 overcomes reduced cdk4 RNA and inhibition of G1 progression in hematopoietic cells expressing a dominant-negative CBF - a model for overcoming inhibition of proliferation by CBF oncoproteins.

Core Binding Factor (CBF) is required for the development of definitive hematopoiesis, and the CBF oncoproteins AML1-ETO, TEL-AML1, and CBFbeta-SMMHC are commonly expressed in subsets of acute leukemia. CBFbeta-SMMHC slows the G1 to S cell cycle transition in hematopoietic cells, but the mechanism of this effect is uncertain. We have sought to determine whether inhibition of CBF-mediated trans-activation is sufficient to slow proliferation. We demonstrate that activation of KRAB-AML1-ER, a protein containing the AML1 DNA-binding domain, the KRAB repression domain, and the Estrogen receptor ligand binding domain, also slows G1, if its DNA-binding domain is intact. Also, exogenous AML1 overcame CBFbeta-SMMHC-induced inhibition of proliferation. Representational difference analysis (RDA) identified cdk4 RNA expression as an early target of KRAB-AML1 activation. Inhibition of CBF activities by KRAB-AML1-ER or CBFbeta-SMMHC rapidly reduced endogenous cdk4 mRNA levels, even in cells proliferating at or near control rates as a result of exogenous cdk4 expression. Over-expression of cdk4, especially a variant which cannot bind p16INK4a, overcame cell cycle inhibition resulting from activation of KRAB-AML1-ER, although cdk4 did not accelerate proliferation when expressed alone. These findings indicate that mutations which alter the expression of G1 regulatory proteins can overcome inhibition of proliferation by CBF oncoproteins. Oncogene (2000).

Control of estrogen receptor ligand binding by Hsp90

The molecular chaperone Hsp90 interacts with unliganded steroid hormone receptors and regulates their activity. We have analyzed the function of yeast and mammalian Hsp90 in regulating the ability of the human estrogen receptor (ER) to bind ligands in vivo and in vitro. Using the yeast system, we show that the ER expressed in several different hsp82 mutant strains binds reduced amounts of the synthetic estrogen diethylstilbestrol compared to the wild type. This defect in hormone binding occurs without any significant change in the steady state levels of ER protein. To analyze the role of mammalian Hsp90, we synthesized the human ER in rabbit reticulocyte lysates containing geldanamycin, an Hsp90 inhibitor. At low concentrations of geldanamycin we observed reduced levels of hormone binding by the ER. At higher concentrations, we found reduced synthesis of the receptor. These data indicate that Hsp90 functions to maintain the ER in a high affinity hormone-binding conformation.

Acute effects of interferon on estrogen receptor function do not involve the extracellular signal-regulated kinases p42mapk and p44mapk.

Exposure to type I interferons (IFN) increased estrogen receptor (ER) ligand binding and induced protein kinase C (PKC) translocation within 30 min but had no effect on net incorporation of [32P] into ER in Madin Darby bovine kidney (MDBK) cells. Ligand binding was also increased within 30 min by phorbol ester and the protein phosphatase inhibitor okadaic acid. Mitogen-activated protein (MAP) kinase phosphorylation was initially inhibited between 2 and 30 min and subsequently activated between 30 and 60 min after treatment with IFN. The activatory response was blocked by the PKC inhibitor Ro 31-8220. Following transient transfection with an ERE-CAT reporter construct, IFN increased CAT expression after 6 h but decreased ER ligand binding, transcriptional activity and phosphorylation after 48 h, probably as a result of decreased ER concentrations. The results rule out rapid activation of ER ligand binding through phosphorylation at Ser118 by MAP kinase because (1) the increase in ligand binding preceded activation of MAP kinase, and (2) IFN had no short-term effect on [32P]incorporation or ER transcriptional activity. The rapid effect of IFN on ER ligand binding is postulated to reflect phosphorylation of the receptor at Tyr537 by p56lck, a member of the Src family of PKC-activated tyrosine kinases.