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B S Katzenellenbogen

Publications and source records attributed to B S Katzenellenbogen.

At least 55 records · Page 3Linked to original sources
PubMed1999

The estrogen receptor enhances AP-1 activity by two distinct mechanisms with different requirements for receptor transactivation functions.

Estrogen receptors (ERs alpha and beta) enhance transcription in response to estrogens by binding to estrogen response elements (EREs) within target genes and utilizing transactivation functions (AF-1 and AF-2) to recruit p160 coactivator proteins. The ERs also enhance transcription in response to estrogens and antiestrogens by modulating the activity of the AP-1 protein complex. Here, we examine the role of AF-1 and AF-2 in ER action at AP-1 sites. Estrogen responses at AP-1 sites require the integrity of the ERalpha AF-1 and AF-2 activation surfaces and the complementary surfaces on the p160 coactivator GRIP1 (glucocorticoid receptor interacting protein 1), the NID/AF-1 region, and NR boxes. Thus, estrogen-liganded ERalpha utilizes the same protein-protein contacts to transactivate at EREs and AP-1 sites. In contrast, antiestrogen responses are strongly inhibited by ERalpha AF-1 and weakly inhibited by AF-2. Indeed, ERalpha truncations that lack AF-1 enhance AP-1 activity in the presence of antiestrogens, but not estrogens. This phenotype resembles ERbeta, which naturally lacks constitutive AF-1 activity. We conclude that the ERs enhance AP-1 responsive transcription by distinct mechanisms with different requirements for ER transactivation functions. We suggest that estrogen-liganded ER enhances AP-1 activity via interactions with p160s and speculate that antiestrogen-liganded ER enhances AP-1 activity via interactions with corepressors.

Binding Sites↗
PubMed1999

Coactivator peptides have a differential stabilizing effect on the binding of estrogens and antiestrogens with the estrogen receptor.

The effectiveness of estrogens in stimulating gene transcription mediated by the estrogen receptor (ER) appears to depend on ER interactions with coactivator proteins. These coactivators bind to ER when it is liganded with an estrogen agonist, but not when it is liganded with an estrogen antagonist. Because estrogen agonists are known to induce a conformation in ER that stabilizes coactivator binding, we asked whether coactivator binding to ER causes a reciprocal stabilization of agonist ligand binding. We used a fluorescent ligand for ER, tetrahydrochrysene-ketone, to monitor the rates of ligand dissociation from ERalpha and ERbeta, and to see how this process is affected by the p160-class coactivator, steroid receptor coactivator-1 (SRC-1). We used a 15-amino acid peptide corresponding to the second nuclear receptor box LXXLL motif in SRC-1 (NR-2 peptide), which is known to interact with the ER ligand-binding domain, a mutant peptide with an LXXAL sequence (NR-2A peptide), and a 203-amino acid fragment of SRC-1, termed the nuclear receptor domain (SRC1-NRD), embodying all three of the internal NR boxes of this protein. Both the NR-2 peptide and the SRC1-NRD fragment markedly slow the rate of dissociation of the agonist ligands tetrahydrochrysene-ketone, estradiol, and diethylstilbestrol, increasing the half-life of the ER-agonist complex by up to 50- to 60-fold. The SRC1-NRD has much higher potency in retarding ligand dissociation than does the NR-2 peptide; it is maximally effective at 30 nM, and it appears to bind with the stoichiometry of one SRC1-NRD per ER dimer. The peptides had little effect on the dissociation rate of antagonist ligands. Consistent with these results, we find that increasing the concentration of SRC-1 in cells by transfection of an expression plasmid encoding SRC-1 causes a 17-fold increase in the potency of estradiol in an estrogen-responsive reporter gene transcription assay. Thus, there is multifactorial control over receptor-coactivator interaction, its strength being determined by the agonist vs. antagonist nature of the ligand and the particular structure of the agonist ligand, and by the receptor subtype and the NR box sequence. The stabilizing effect of coactivator on ER-agonist ligand complexes may be important in determining the potency of estrogen agonists in a cell and may also underlie the tissue-selective pharmacology of certain synthetic estrogens.

Amino Acid Motifs↗
PubMed1999

Adenovirus-mediated delivery of a dominant negative estrogen receptor gene abrogates estrogen-stimulated gene expression and breast cancer cell proliferation.

Dominant negative estrogen receptors are transcriptionally inactive, altered forms of the estrogen receptor (ER) that can dimerize with the ER and have the potential to inactivate the biological functions of this receptor. Here, we provide the first report that adenoviral delivery of a dominant negative ER to ER-positive breast cancer cells is able to effectively suppress estrogen-stimulated cell proliferation and the hormonal induction of endogenous genes. We constructed recombinant adenoviral vectors expressing a dominant negative ER (S554 fs, Ad-fs) or, for comparison, antisense ER (Ad-AS), or the sense wild-type ER (Ad-WT). Expression of the dominant negative ER or antisense ER, but not wild-type ER, blocked estradiol stimulation of the estrogen-responsive genes pS2 and c-myc. The dominant negative ER also fully abolished the estradiol-induced increase in proliferation of MCF-7 breast cancer cells, as did the antisense ER. The antiproliferative effects of the dominant negative and antisense ERs are explained by an increase in the number of cells in the G0/G1 stage of the cell cycle and decrease in the number of cells in G2/M as determined by flow cytometry, and also by a significant increase in the percentage of cells undergoing apoptosis. Our data strongly support the idea that targeting ER action using recombinant viral delivery of dominant negative ERs is an effective way to suppress ER-positive breast cancer cell proliferation and suggests the potential attractiveness of dominant negative gene therapy approaches targeted to the ER for the treatment of hormone-responsive breast cancer.

Adenoviridae↗
PubMed1998

Transcriptional regulation of the human quinone reductase gene by antiestrogen-liganded estrogen receptor-alpha and estrogen receptor-beta.

We have previously reported that antiestrogens stimulate quinone reductase (NAD(P)H:(quinone-acceptor) oxidoreductase (QR or NQO1); EC 1.6.99.2) enzymatic activity, an action that may provide protective effects against the toxicity and mutagenicity caused by quinones. We have now investigated the transcriptional regulation of the QR gene by antiestrogens. In transfection experiments employing the 5'-flanking (863-base pair) region of the human QR gene promoter with its electrophile/antioxidant response element (EpRE/ARE) or deleted or mutated constructs, we observe that antiestrogens induced an increase in QR gene promoter reporter activity in estrogen receptor (ER) negative breast cancer and endometrial cancer cells transfected with ER, and this induction by antiestrogens was repressed by estradiol. The stimulation of QR transcriptional activity required the 31-base pair electrophile-responsive region from the human QR gene promoter and a functional ER. Intriguingly, antiestrogens were stronger activators of the QR EpRE via the ER subtype ERbeta than ERalpha. Oligonucleotide gel mobility and antibody shift assays reveal that the ER binds to the EpRE but is only a minor component of the proteins bound to the EpRE in ER-containing MCF-7 breast cancer cells. While binding of ERbeta to the estrogen response element was weaker when compared with ERalpha, ERbeta and ERalpha showed similar binding to the EpRE. Together these findings provide evidence that QR gene regulation by the antiestrogen-occupied ER is mediated by the EpRE-containing region of the human QR gene and indicate that the ER is one of the complex of proteins that binds to the EpRE. In addition, that ERbeta is a more potent activator at EpRE elements than is ERalpha suggests that the different levels of these two receptors in various estrogen target cells could impact importantly on the antioxidant potency of antiestrogens in different target cells. These findings have broad implications regarding the potential beneficial effects of antiestrogens since EpREs mediate the transcriptional induction of numerous genes, including QR, which encode chemoprotective detoxification enzymes.

Base Sequence↗
PubMed1998

Determinants of ligand specificity of estrogen receptor-alpha: estrogen versus androgen discrimination.

We have been interested in understanding how the estrogen receptor (ER) binds estrogens and discriminates between different classes of steroids with closely related structures. Using insights from our prior studies on ER and from sequence comparisons of steroid receptors, we identified three residues in the hormone-binding domain of the human ER, Leu345, Thr347, and Glu353, that we considered were likely to be involved in steroid A-ring recognition and therefore estrogen versus androgen discrimination. We then tested the effect on ER activity of mutating these ER residues to the corresponding androgen receptor residues. Specifically, we examined the ability of the mutant receptors to bind and be activated by 17beta-estradiol and three different androgens. No change in receptor activity was observed with the T347N mutation, while the L345S mutation greatly reduced ER activity in response to all ligands. Interestingly, the E353Q substitution behaved as expected, causing a 9-fold reduction in the transactivation potency of estradiol and a concomitant 10-140-fold increase in the transactivation potency of different androgens. These reciprocal changes in the transcriptional effectiveness of estrogens and androgens correlated with a decreased affinity of the E353Q ER for estradiol binding and an increased affinity for androgen binding. Therefore, amino acid Glu353 appears to be playing a significant role in binding the A-ring phenolic group of estradiol and in receptor discrimination between estrogens and the most closely structurally related steroids, androgens. Based on this data and our earlier observations, we propose a model for the orientation of ligand within the binding pocket of ER in which the A-ring 3-phenol of estradiol is hydrogen bonded to Glu353 in helix-3 and the 17beta-hydroxyl of estradiol is hydrogen bonded to His524 in helix-11. Our findings with estrogen and androgen suggest that this orientation of the steroid in the ligand-binding pocket, with the steroid A-ring in contact with helix-3 and the D-ring in contact with helix-11 residues, is likely to be general for all the steroid hormone receptors.

Amino Acid Sequence↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1998

Estrogen receptor activation function 1 works by binding p160 coactivator proteins.

Estrogen receptor-alpha contains two transactivation functions, a weak constitutive activation function (AF-1) and a hormone-dependent activation function (AF-2). AF-2 works by recruiting a large coactivator complex, composed of one or more p160s, CREB-binding protein (CBP)/p300, and P/CAF (p300 and CBP-associated factor), via direct contacts with the p160s. We report here that independent AF-1 activity also requires p160 contacts. Unlike AF-2, which binds signature NR boxes in the center of the p160 molecule, AF-1 binds to sequences near the p160 C terminus. We propose that the ability of AF-1 and AF-2 to interact with separate surfaces of the same coactivator is important for the ability of these transactivation functions to synergize.

Acetyltransferases↗
PubMed1998

Hormone induction of progesterone receptor (PR) messenger ribonucleic acid and activation of PR promoter regions in ovarian granulosa cells: evidence for a role of cyclic adenosine 3',5'-monophosphate but not estradiol.

Expression of progesterone receptor (PR) mRNA in granulosa cells of ovarian preovulatory follicles is induced by LH (1, 2) and is essential for ovulation (3). Although 17beta-estradiol (E) can induce PR mRNA and activate PR promoter-reporter constructs in other cell types, the effects of E in granulosa cells appear to be indirect. We show herein that E alone does not induce the expression of PR mRNA in preovulatory granulosa cells. Rather, induction of PR mRNA depends on the differentiation of granulosa cells in response to E and a physiological amount of FSH followed by exposure to agonists (elevated levels of LH, FSH, and forskolin) that markedly increase cAMP. Induction of PR mRNA by forskolin is blocked by the A-kinase inhibitor H89 and cycloheximide but not by the E antagonist, ICI 164,384. These results indicate that phosphorylation and synthesis of some regulatory factor(s) other than or in addition to the estrogen receptor (ER) are essential for transactivation of the PR gene. When distal and proximal PR promoter-reporter constructs that are responsive to E in other cell types were transiently transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Likewise, when a vector containing the consensus vitellogenin B1 gene estrogen response element (ERE) was transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Using electrophoretic mobility shift assays, the consensus ERE was shown to bind ERbeta, the predominant subtype present in rat granulosa cells, and ERalpha, the predominant subtype present in luteal cells, whereas the putative ERE-like region (ERE3) of the proximal PR promoter did not bind either ER subtype. Although the identity of the specific factors binding to the ERE3 site remain to be determined, mutation of this region abolished forskolin-induced activity of ERE3-PR-CAT constructs. The GC-rich region of the distal PR promoter bound Sp1 and Sp3 but not C/EBPalpha/beta, indicating that factors binding to ERE3 interact synergistically with Sp1/Sp3 to confer increased responsiveness of the distal promoter to forskolin. Taken together, these results indicate that activation of the A-kinase pathway leads to the phosphorylation of some transcription factor(s) other than or in addition to ER that is (are) critical for the transactivation of the PR gene and that this mechanism is selectively activated in differentiated granulosa cells possessing a preovulatory phenotype.

Adenosine Monophosphate↗
PubMed1997

Altered ligand binding properties and enhanced stability of a constitutively active estrogen receptor: evidence that an open pocket conformation is required for ligand interaction.

To elucidate the ligand binding properties of the estrogen receptor (ER) and how ligand access to and release from the ligand binding pocket is affected by the conformational state of the receptor, we have measured the rates of estradiol association and dissociation, the equilibrium binding, and the stability of estradiol binding to denaturants, comparing wild-type human ER and a point mutant (Y537S ER) that shows full constitutive activity, i.e., the same full transcriptional activity in the absence or presence of estrogen. Ligand binding kinetics and affinity were measured with the full-length (1-595) ERs and with truncated forms of both receptors containing domains C through F (including the DNA binding, hinge, and ligand binding domains, amino acids 175-595) or domains E and F (the ligand binding domain; amino acids 304-595). With all ERs, the rates of ligand association and dissociation were considerably slower with the Y537S mutant ER than with wild-type ER (6-fold and 3-4-fold, respectively). These marked differences in ligand on and off rates for the wild-type and Y537S receptors result in a predicted (k-1/k+1) and measured Kd that is 2-fold lower for Y537S ER compared to wild-type ER. The binding of estradiol by wild-type ER was disrupted by high concentrations of urea (above 2 M), whereas the Y537S ER was distinctly more resistant to this disruption. These results are consistent with a model in which wild-type ER in the absence of ligand adopts a transcriptionally inactive collapsed pocket conformation, stabilized by specific interactions of Y537 with nearby regions of ER. When estradiol is bound, the wild-type ER adopts a transcriptionally active, closed pocket (ligand occupied) conformation. By contrast, the Y537S mutant ER favors the transcriptionally active closed pocket conformation, whether occupied by ligand or not, the latter state (closed pocket but unoccupied) accounting for its constitutive activity. Our findings suggest that the entry or exit of ligand from the binding pocket requires that ER adopt an open pocket conformation. The reduced rates of ligand association and dissociation in the constitutively active form of the ER, as well as its greater resistance to disruption of ligand binding by urea, support the supposition that the rate at which this open pocket conformation can be accessed from the unoccupied or ligand-occupied Y537S ER is slower than from the unoccupied or occupied forms of wild-type ER. Thus, the binding and release of ligand by ER require that the receptor access an open pocket state, and the ease with which this state can be accessed is affected by mutations that alter receptor conformation.

Binding Sites↗
PubMed1997

17 alpha (haloacetamidoalkyl) estradiols alkylate the human estrogen receptor at cysteine residues 417 and 530.

Results obtained in a previous study suggested that cysteine residues in the estrogen receptor were covalent attachment sites for four 17 alpha-(haloacetamidoalkyl) estradiols (halo, bromo or iodo; alkyl, methyl, ethyl, or propyl). To identify the putative concerned cysteines, we expressed wild-type and various cysteine --> alanine mutants of the human estrogen receptor in COS cells and determined their ability to be alkylated by the four electrophiles. The quadruple mutant, in which all the cysteines (residues 381, 417, 447, and 530) of the hormone-binding site were changed to alanines, showed very little electrophile labeling, whereas the four single mutants (C381A, C417A, C447A, and C530A) were alkylated as efficiently as the wild-type receptor. These results (i) demonstrate that cysteine residues were covalent attachment sites of electrophiles and (ii) indicate that more than one cysteine residue could be alkylated. Analysis of three double mutants (C381A/C530A, C417A/C530A, and C447A/C530A) provided strong evidence that only C417 and C530 were sites for electrophile covalent attachment. Since C530 was also alkylated by tamoxifen aziridine, a nonsteroidal affinity-labeling agent, we propose a selective mode of superimposition of tamoxifen-class antiestrogens with estradiol, which could account for the relative positioning of the two types of ligands in the receptor hormone-binding pocket. According to the structure of the hormone-binding pocket of nuclear receptors, as inferred from crystallographic studies and general sequence alignment of hormone-binding domains, C417 and C530 appear to be (1) located at the extreme border or in structural elements involved in delineation of the hormone-binding pocket, (2) spatially in close proximity to each other, and (3) in positions highly homologous to those of glucocorticoid receptor sites alkylated by affinity- and photoaffinity-labeling agents, respectively.

Affinity Labels↗
PubMed1997

The quinone reductase gene: a unique estrogen receptor-regulated gene that is activated by antiestrogens.

Antiestrogens are thought to exert most of their beneficial effects in breast cancer by antagonizing the actions of estrogen. We report here that antiestrogens also stimulate the expression of quinone reductase (QR) [NAD(P)H:quinone oxidoreductase, EC 1.6.99.2], which may provide protective effects against the toxicity and mutagenicity caused by quinones. QR is up-regulated by low concentrations of antiestrogens (trans-hydroxytamoxifen, tamoxifen, and ICI182,780) in estrogen receptor (ER)-containing breast cancer cells, and this increase is suppressed by estrogen via an ER-dependent mechanism. Since regulation of the QR gene, as well as other genes involved in detoxification such as the glutathione S-transferase Ya subunit (GST Ya) gene, is known to be mediated by an electrophile/antioxidant response element (EpRE/ARE), we examined the effects of antiestrogens on a 41-bp electrophile responsive region derived from the GST Ya gene. Transfection of this EpRE-containing region into ER-negative breast cancer cells in the presence or absence of an expression vector for the human ER, as well as mutagenesis studies, revealed that the EpRE-containing construct was activated by antiestrogen to the same extent as by tert-butylhydroquinone (TBHQ), a known activator of EpREs; however, only the stimulation by antiestrogen, and not TBHQ, required ER and was repressed by estradiol, although activation by both inducers mapped to the same 10-bp EpRE consensus sequence. Thus, there appear to be two pathways for QR induction, one that is activated by electrophile inducers such as TBHQ and is ER independent, and a second that is antiestrogen regulated and ER dependent; both pathways act through the EpRE. The anticancer action of antiestrogens may thus derive not only from the already well-known repression of estrogen-stimulated activities but also from the activation of detoxifying enzymes, such as QR, that may contribute to the beneficial antioxidant activity of antiestrogens.

Animals↗
PubMed1997

Different residues of the human estrogen receptor are involved in the recognition of structurally diverse estrogens and antiestrogens.

We have previously examined, by alanine scanning mutagenesis, amino acids 515-535 of the estrogen receptor (ER) ligand binding domain to determine which of these residues are important in estradiol binding. Mutation at four sites that potentially lie along one face of an alpha-helix, Gly521, His524, Leu525, and Met528, all significantly impaired estradiol binding by the ER (Ekena, K., Weis, K. E., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. (1996) J. Biol. Chem. 271, 20053-20059). In this report, we compare the pattern of residues that are important in the recognition of several structurally diverse estrogen agonists and antagonists (the synthetic nonsteroidal agonist hexestrol, an agonist derived from the mold metabolite zearalenone, P1496, and the partial agonist-antagonist trans-hydroxytamoxifen) with those that are predicted to contact estradiol in the receptor-ligand complex. Although there are some similarities in the pattern of residue recognition among all four ligands, each ligand showed distinct differences as well. Interestingly, alanine substitution at only one residue, the leucine at position 525, was found to inhibit binding of all the ligands tested. Another residue, His524, was found to be important in the recognition of three different agonists but not trans-hydroxytamoxifen (the only ligand lacking a second hydroxyl group). The recognition of estradiol and another agonist, P1496, was impaired by the G521A mutation, whereas ligand-induced activity by the two compounds that lack B- and C-rings, hexestrol and trans-hydroxytamoxifen, was unaffected. Our findings demonstrate that these ligands fit into the ER ligand binding pocket differently and that each contacts a distinct set of amino acids. The smaller ligands (estradiol and hexestrol) have a narrower footprint of interacting residues than the larger ligands (P1496 and trans-hydroxytamoxifen). This pattern of interaction is most consistent with the amino acids within this region being in contact with the portion of these ligands that corresponds to the D-ring end of estradiol. The interplay between the shape of an ER ligand and the residues that support its binding to ER may potentially underlie the selective actions of different ER ligands in various cell and promoter contexts.

Binding Sites↗
PubMed1997

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.

Breast Neoplasms↗
PubMed1997

Identification of a novel transferable cis element in the promoter of an estrogen-responsive gene that modulates sensitivity to hormone and antihormone.

The estrogen receptor (ER) is a ligand-regulated transcription factor that acts at the promoters of estrogen-regulated genes to modulate their expression. In the present study, we examined three estrogen-regulated promoters, namely the rat progesterone receptor gene distal (PRD) and proximal (PRP) promoters and the human pS2 gene promoter, and observed marked differences in their sensitivity to stimulation by estrogen and repression of estrogen-stimulated transcription by antiestrogen (AE)-occupied ER. ER-containing MCF-7 human breast cancer cells were transfected with reporter gene constructs containing estrogen response elements upstream of the three gene promoters. In this system, PRP and PRD showed similar dose-response curves for stimulation by estradiol whereas pS2 was activated by even lower concentrations of estradiol. By contrast, PRD was much less sensitive to repression of estrogen-stimulated activity by all AEs studied, relative to the PRP and the pS2 promoters. Using deletion and mutational analysis, we have identified a transferable cis element at -131 to -94 bp in PRD that is involved in modulating the sensitivity of this promoter to both estrogens and AEs. The element reduced the magnitude of estrogen-stimulated activity, enhanced the ability of AEs to repress estrogen-stimulated activity, and elicited similiar effects when transferred to the promoter of another estrogen-responsive gene. Thus, removal of this region from PRD further accentuated the insensitivity of this promoter to AE while enhancing its sensitivity (both EC50 and fold induction) to estrogen. Gel mobility shift assays showed that proteins from nuclear extracts of MCF-7 cells interact with this element and that the binding of these proteins is inversely correlated with the transcriptional effectiveness of the ER. The findings demonstrate that a specific cis element from the promoter of an estrogen-responsive gene can alter the transcriptional activity of hormone and antihormone-occupied receptor bound at its response element near the promoter. Such ligand response modulatory elements, and changes in the levels and activity of factors that bind to such elements, may underlie the different sensitivities of steroid hormone-regulated genes to both hormones and antihormones.

Animals↗
PubMed1997

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.

Adaptor Proteins, Signal Transducing↗
PubMed1996

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.

Binding Sites↗
PubMed1996

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.

Animals↗