B. Katzenellenbogen - Academia.edu (original) (raw)
Papers by B. Katzenellenbogen
Journal of Biological Chemistry, 1993
Journal of Biological Chemistry, 1991
We have carried out experiments to determine the role of the cysteines in the hormone-binding dom... more We have carried out experiments to determine the role of the cysteines in the hormone-binding domain (HBD) of the human estrogen receptor (ER) in receptor function. In each mutant receptor, 1 of the 4 cysteines in the HBD (cysteines 381, 417, 447, and 530) was changed by in vitro mutagenesis of the ER cDNA (containing Gly4Oo) from cysteine to alanine; was also mutated to a serine. The mutant and wild-type receptor cDNAs were expressed in Chinese hamster ovary cells using an expression vector containing the Rous sarcoma virus promoter. The mutant and wildtype receptors were assayed for hormone binding and for their ability to activate estrogen-responsive reporter plasmids. All ER mutants bound estradiol (E2) with affinity similar to wild-type ER, displaying a Kd between 0.3 and 0.8 nM (wild-type ER Kd = 0.45 2 0.10 nM). All were capable of covalent labeling by the affinity ligands ketononestrol aziridine, an estrogen agonist, and tamoxifen aziridine, an antagonist. Since in previous work we identified Cys5"0 as the site of covalent attachment of these ligands (Harlow, K.
Molecular and cellular endocrinology, Jan 15, 2017
Estrogen Receptor-β (ERβ) has been implicated in many cancers. In prostate and breast cancer its ... more Estrogen Receptor-β (ERβ) has been implicated in many cancers. In prostate and breast cancer its function is controversial, but genetic studies implicate a role in cancer progression. Much of the confusion around ERβ stems from antibodies that are inadequately validated, yet have become standard tools for deciphering its role. Using an ERβ-inducible cell system we assessed commonly utilized ERβ antibodies and show that one of the most commonly used antibodies, NCL-ER-BETA, is non-specific for ERβ. Other antibodies have limited ERβ specificity or are only specific in one experimental modality. ERβ is commonly studied in MCF-7 (breast) and LNCaP (prostate) cancer cell lines, but we found no ERβ expression in either, using validated antibodies and independent mass spectrometry-based approaches. Our findings question conclusions made about ERβ using the NCL-ER-BETA antibody, or LNCaP and MCF-7 cell lines. We describe robust reagents, which detect ERβ across multiple experimental approac...
Molecular and Cellular Biology, 2000
We find that prothymosin alpha (PTα) selectively enhances transcriptional activation by the estro... more We find that prothymosin alpha (PTα) selectively enhances transcriptional activation by the estrogen receptor (ER) but not transcriptional activity of other nuclear hormone receptors. This selectivity for ER is explained by PTα interaction not with ER, but with a 37-kDa protein denoted REA, for repressor of estrogen receptor activity, a protein that we have previously shown binds to ER, blocking coactivator binding to ER. We isolated PTα, known to be a chromatin-remodeling protein associated with cell proliferation, using REA as bait in a yeast two-hybrid screen with a cDNA library from MCF-7 human breast cancer cells. PTα increases the magnitude of ERα transcriptional activity three- to fourfold. It shows lesser enhancement of ERβ transcriptional activity and has no influence on the transcriptional activity of other nuclear hormone receptors (progesterone receptor, glucocorticoid receptor, thyroid hormone receptor, or retinoic acid receptor) or on the basal activity of ERs. In cont...
Cancer research, 1984
A substantial proportion of human breast cancers contain estrogen receptors, and it is believed t... more A substantial proportion of human breast cancers contain estrogen receptors, and it is believed that the growth of some of these tumors and their synthesis of specific proteins are stimulated by estrogens. Since natural estrogens, such as estradiol, react reversibly with estrogen receptors, it was of interest to determine the biological consequences that would result from very strong, possibly irreversible interaction of an estradiol-based ligand with the estrogen receptor of breast cancer cells. For these studies, we have examined the receptor interactions and biological character of 11 beta- chloromethylestradiol (CME) and 11 beta- bromomethylestradiol (BME) as potential estradiol-based affinity labeling ligands in MCF-7 human breast cancer cells which contain high levels of estrogen receptors. The apparent relative binding affinities of CME and BME for MCF-7 estrogen receptor measured by competitive binding assay are 230 and 15%, respectively, whereas the affinity of estradiol is...
Science translational medicine, Jan 21, 2015
Estrogenic and inflammatory components play key roles in a broad range of diseases including endo... more Estrogenic and inflammatory components play key roles in a broad range of diseases including endometriosis, a common estrogen-dependent gynecological disorder in which endometrial tissue creates inflammatory lesions at extrauterine sites, causing pelvic pain and reduced fertility. Current medical therapies focus primarily on reducing systemic levels of estrogens, but these are of limited effectiveness and have considerable side effects. We developed estrogen receptor (ER) ligands, chloroindazole (CLI) and oxabicycloheptene sulfonate (OBHS), which showed strong ER-dependent anti-inflammatory activity in a preclinical model of endometriosis that recapitulates the estrogen dependence and inflammatory responses of the disease in immunocompetent mice and in primary human endometriotic stromal cells in culture. Estrogen-dependent phenomena, including cell proliferation, cyst formation, vascularization, and lesion growth, were all arrested by CLI or OBHS, which prevented lesion expansion a...
EMBO molecular medicine, Jan 11, 2014
Estetrol (E4) is a natural estrogen with a long half-life produced only by the human fetal liver ... more Estetrol (E4) is a natural estrogen with a long half-life produced only by the human fetal liver during pregnancy. The crystal structures of the estrogen receptor α (ERα) ligand-binding domain bound to 17β-estradiol (E2) and E4 are very similar, as well as their capacity to activate the two activation functions AF-1 and AF-2 and to recruit the coactivator SRC3. In vivo administration of high doses of E4 stimulated uterine gene expression, epithelial proliferation, and prevented atheroma, three recognized nuclear ERα actions. However, E4 failed to promote endothelial NO synthase activation and acceleration of endothelial healing, two processes clearly dependent on membrane-initiated steroid signaling (MISS). Furthermore, E4 antagonized E2 MISS-dependent effects in endothelium but also in MCF-7 breast cancer cell line. This profile of ERα activation by E4, uncoupling nuclear and membrane activation, characterizes E4 as a selective ER modulator which could have medical applications tha...
The Journal of Steroid Biochemistry and Molecular Biology, 2000
Estrogens exert profound effects on the physiology of diverse target cells and these effects appe... more Estrogens exert profound effects on the physiology of diverse target cells and these effects appear to be mediated by two estrogen receptor (ER) subtypes, ERa and ERb. We have investigated how ER ligands, ranging from pure agonists to antagonists, interact with ERa and ERb, 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 ERa and others that are full agonists through ERa while being full antagonists through ERb. Antiestrogens such as hydroxytamoxifen, which are mixed agonist/antagonists through ERa, are pure antagonists through ERb at estrogen response element-containing gene sites. Studies with ERa/b chimeric proteins reveal that tamoxifen agonism requires the activation function 1 region of ERa. 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 (ERa or ERb) 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 ERa or ERb should allow optimized tissue selectivity of these agents for menopausal hormone replacement therapy and the treatment and prevention of breast cancer.
Proceedings of the National Academy of Sciences, 2010
Estrogen receptors (ERs) protect pancreatic islet survival in mice through rapid extranuclear act... more Estrogen receptors (ERs) protect pancreatic islet survival in mice through rapid extranuclear actions. ERα also enhances insulin synthesis in cultured islets. Whether ERα stimulates insulin synthesis in vivo and, if so, through which mechanism(s) remain largely unknown. To address these issues, we generated a pancreas-specific ERα knockout mouse (PERαKO −/− ) using the Cre-loxP strategy and used a combination of genetic and pharmacologic tools in cultured islets and β cells. Whereas 17β-estradiol (E2) treatment up-regulates pancreatic insulin gene and protein content in control ERαlox/lox mice, these E2 effects are abolished in PERαKO −/− mice. We find that E2-activated ERα increases insulin synthesis by enhancing glucose stimulation of the insulin promoter activity. Using a knock-in mouse with a mutated ERα eliminating binding to the estrogen response elements (EREs), we show that E2 stimulation of insulin synthesis is independent of the ERE. We find that the extranuclear ERα inter...
Proceedings of the National Academy of Sciences, 2005
Thyroid hormones (T) and estrogens (E) are nuclear receptor ligands with at least two molecular m... more Thyroid hormones (T) and estrogens (E) are nuclear receptor ligands with at least two molecular mechanisms of action: ( i ) relatively slow genomic effects, such as the regulation of transcription by cognate T receptors (TR) and E receptors (ER); and ( ii ) relatively rapid nongenomic effects, such as kinase activation and calcium release initiated at the membrane by putative membrane receptors. Genomic and nongenomic effects were thought to be disparate and independent. However, in a previous study using a two-pulse paradigm in neuroblastoma cells, we showed that E acting at the membrane could potentiate transcription from an E-driven reporter gene in the nucleus. Because both T and E can have important effects on mood and cognition, it is possible that the two hormones can act synergistically. In this study, we demonstrate that early actions of T via TRα1 and TRβ1 can potentiate E-mediated transcription (genomic effects) from a consensus E response element (ERE)-driven reporter ge...
Proceedings of the National Academy of Sciences, 2006
Estrogen receptor (ER)-mediated gene expression plays an essential role in mammary gland morphoge... more Estrogen receptor (ER)-mediated gene expression plays an essential role in mammary gland morphogenesis, function, and carcinogenesis. The repressor of ER activity (REA) is an ER-interactive protein that counterbalances estrogen-induced ER transcriptional activity. Our previous study showed that genetic deletion of both REA alleles resulted in embryonic lethality. This study demonstrates that REA and ERα are coexpressed in mammary epithelial cells. REA heterozygous (REA +/− ) mutant mice exhibit faster mammary ductal elongation in virgin animals, increased lobuloalveolar development during pregnancy, and delayed mammary gland involution after weaning. These morphological phenotypes of REA +/− mice are associated with significantly increased cell proliferation and ER transcriptional activities, as indicated by the estrogen response element (ERE)-luciferase reporter in the WT/ERE-Luc and REA +/− /ERE-Luc bigenic mice and by the higher expression levels of estrogen-responsive genes such...
Oncogene, 2005
The proliferative action of ERa largely accounts for the carcinogenic activity of estrogens. By c... more The proliferative action of ERa largely accounts for the carcinogenic activity of estrogens. By contrast, recent data show that ERb displays tumor-suppressor properties, thus supporting the interest to identify compounds that could increase its activity. Here, we show that histone deacetylase inhibitors (HDI) upregulated ERb protein levels, whereas it decreased ERa expression. Part of this regulation took place at the mRNA level through a mechanism independent of de novo protein synthesis. In addition, we found that, in various cancer cells, the treatment with different HDI enhanced the liganddependent activity of ERb more strongly than that of ERa. On the other hand, in MDA-MB231 and HeLa cells, the expression of ERs modified the transcriptional response to HDI. The use of deletion mutants of both receptors demonstrated that AF1 domain of the receptors was required. Finally, we show that ERb expression led to a dramatic increased in the antiproliferative activity of HDI, which correlated with a modification of the transcription of genes involved in cell cycle control by HDI. Altogether, these data demonstrate that the interference of ERb and HDAC on the control of transcription and cell proliferation constitute a promising approach for cancer therapy.
Nature Chemical Biology, 2008
Understanding how steroid hormones regulate physiological functions has been significantly advanc... more Understanding how steroid hormones regulate physiological functions has been significantly advanced by structural biology approaches. However, progress has been hampered by significant misfolding of the ligand binding domains in heterologous expression systems and by conformational flexibility that interferes with crystallization. Here, we show that protein folding problems common to steroid hormone receptors are circumvented by a mutations that stabilize well-characterized conformations of the receptor. We use this approach to present the first structure of an apo steroid receptor, revealing a ligand-accessible channel, allowing soaking of preformed crystals. Furthermore, crystallization of different pharmacological classes of compounds allowed us to define the structural basis of NFκB selective signaling through ER, revealing a unique conformation of the receptor that allows selective suppression of inflammatory gene expression. The ability to crystallize many receptor-ligand complexes with distinct pharmacophores allows one to define the structural features of signaling specificity that would not be apparent in a single structure.
Molecular Endocrinology, 1994
Progesterone receptors (PRs) mediate the actions of progestin hormones and play important roles d... more Progesterone receptors (PRs) mediate the actions of progestin hormones and play important roles during the reproductive cycle and pregnancy. Since PR expression is known to be regulated by estrogen, we have undertaken studies to examine the mechanisms underlying this regulation. We have identified multiple distinct regions of the rat PR gene, widely spaced and spread throughout the V-flanking region, the S-untranslated region, and the first exon (between-2264 and +2241), that can form a strong estrogen-responsive enhancer when linked together. Estrogen-responsive activities for two of the regions in isolation (+461/+636 and +2176/ +2241) were demonstrated in one or more homologous or heterologous promoter contexts. The contributions of the other regions (-2264/-1970,-11671-957 and +2068/+2110) to the overall activity of the assembled enhancer were cryptic in that they were only observed in the context of the other PR gene fragments, not in isolation. We identified four weak, but functional, imperfect estrogen response elements (EREs) in these regions of the PR gene, each differing from the consensus by 2 base pairs. In addition, we identified four ERE half-sites in the PR gene, three of which are paired (i.e. cl50 base pairs away) with the EREs in the estrogenresponsive regions. Competitive gel shift assays demonstrated weak, but detectable, binding of estrogen receptor to the EREs. Of note, the estrogenresponsive enhancer assembled from the five regions of the PR gene exhibited promoter specificity; it conferred estrogen responsiveness on the distal PR gene promoter, but it failed to enhance the endogenous estrogen responsiveness of the proximal PR gene promoter. The positioning of response elements in the rat PR gene, which we show to be unique among steroid hormone-regulated genes, may have functional consequences for the regulation of the magnitude and timing of PR gene expression by estrogen.
Molecular Endocrinology, 1993
We have shown previously that exposure of rat uterine cells in primary culture to estradiol (E2),... more We have shown previously that exposure of rat uterine cells in primary culture to estradiol (E2), insulin-like growth factor-l (IGF-I), or agents which alter intracellular CAMP levels, such as cholera toxin plus isobutylmethylxanthine (CT + IBMX) and 8-Br-CAMP, results in the up-regulation of cellular levels of the progesterone receptor, an effect believed to be mediated through the activation of estrogen receptor (ER) and phosphorylation pathways. We have therefore undertaken studies using transient transfection of these uterine cell cultures with a simple estrogen-responsive reporter gene in order to determine the ability of these agents to stimulate ERmediated gene transcription directly. We also compared the ability of these same agents to alter the phosphorylation state of the endogenous uterine ER protein. Plasmid DNA containing two tandem estrogen responsive elements and a TATA box linked to the chloramphenicol acetyl transferase (CAT) gene was introduced into immature rat uterine cells grown in primary culture. Treatment of transfected cells with lo-' M EP, CT (1 Fg/ml) + IBMX (lo-" M), 8-Br-CAMP (10m4 M), or IGF-I (20 rig/ml) resulted in an 8-to lofold induction of CAT activity. CAT activity stimulated by all agents was nearly completely suppressed by coincubation with the antiestrogen ICI 184,384 (ICI) or the protein kinase (PK) inhibitor H8. CAT activity induced by 8-Br-CAMP was more readily suppressed by ICI than that induced by EP, indicating that ER in cells exposed to 8-Br-CAMP is either unoccupied or minimally occupied by ligand. The level of ER phosphorylation in uterine cells was increased 3-to 5-fold upon exposure to EP, CT
Journal of Biological Chemistry, 1996
The human estrogen receptor (ER) is a ligand-inducible transcription factor that contains two tra... more The human estrogen receptor (ER) is a ligand-inducible transcription factor that contains two transcriptional activation functions, one located in the NH 2-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 NH 2-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 E 2 , 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, hormonedependent 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.
Endocrinology, 2013
Successful implantation and maintenance of pregnancy require the transformation of uterine endome... more Successful implantation and maintenance of pregnancy require the transformation of uterine endometrial stromal cells into distinct decidualized cells. Although estrogen and progesterone (P4) receptors are known to be essential for decidualization, the roles of steroid receptor coregulators in this process remain largely unknown. In this study, we have established a key role for the coregulator, repressor of estrogen receptor activity (REA), in the decidualization of human endometrial stromal cells (hESCs) in vitro and of the mouse uterus in vivo. Our studies revealed that the level of REA normally decreases to half as hESC decidualization proceeds and that uterine reduction of REA in transgenic heterozygous knockout mice or small interfering RNA knockdown of REA in hESC temporally accelerated and strongly enhanced the differentiation process, as indicated by changes in cell morphology and increased expression of biomarkers of decidualization, including P4 receptor. Findings in hESC ...
Endocrinology, 1981
Studies were undertaken to compare the effects of the estrogen ethinyl estradiol [llα-ethinylestr... more Studies were undertaken to compare the effects of the estrogen ethinyl estradiol [llα-ethinylestradiol-17β (EE2)] and the antiestrogen U11.100A [l-(2-[p-(3,4-dihydro-6-methoxy- 2 - phenyl -1 - naphthyl) phenoxy] ethyl) pyrrolidine hydrochloride (UA)] on rat liver. These compounds were assessed for their abilities to bind to estrogen-specific binding sites in liver cytosol, to translocate these sites to the nucleus, and to induce elevations of hepatic production of plasma renin substrate. Both compounds were shown to cause significant 2- to 4-fold increases in plasma renin substrate levels after sc injections at dosages of 25, 100, and 250 μg daily for 2 days to mature female rats, and two related triarylethylene antiestrogens, tamoxifen [trans-l-(4β-dimethylaminoethoxyphenyl) l,2-diphenylbut-l-ene] and CI628 [α-(p-(2-(l-pyrrolidino)ethoxy)phenyl)4-methoxy-α′-nitrostilbene], were equally potent stimulators of elevated plasma renin substrate leve s. Similar approximately 3-fold increases in plasma renin sub...
Endocrinology, 1999
We report on the identification of novel, nonsteroidal ligands that show pronounced subtype-selec... more We report on the identification of novel, nonsteroidal ligands that show pronounced subtype-selective differences in ligand binding and transcriptional potency or efficacy for the two estrogen receptor (ER) subtypes, ER␣ and ER. An aryl-substituted pyrazole is an ER␣ potency-selective agonist, showing higher binding affinity for ER␣ and 120-fold higher potency in stimulation of ER␣ vs. ER in transactivation assays in cells. A tetrahydrochrysene (THC) has a 4-fold preferential binding affinity for ER; it is an agonist on ER␣, but a complete antagonist on ER. Intriguingly, the antagonist activity of
Endocrinology, 1999
To better understand the actions of estrogens and antiestrogens in estrogen target cells, we have... more To better understand the actions of estrogens and antiestrogens in estrogen target cells, we have searched for estrogen-regulated genes in human breast cancer cells, in which the number of genes known to be directly activated by estrogen is quite small. Using differential display RNA methods, we have identified the human homolog of the Na ϩ-H ϩ exchanger regulatory factor (NHE-RF), an approximately 50-kDa protein that is also an ezrin-radixin-moesin-binding phosphoprotein, as being under rapid and direct regulation by estrogen in estrogen receptor (ER)-containing breast cancer cells. Stimulation by estrogen of NHE-RF RNA is rapid, being near maximal (ϳ6-fold) by 1 h, and is not blocked by cycloheximide, indicating that it is a primary response. Stimulation is selective for estrogen ligands, with no stimulation by other classes of steroid hormones, and stimulation by estrogen is suppressed by the antiestrogens tamoxifen and ICI 182,780. Induction is shown to require an active ER through several Materials and Methods Chemicals and materials Cell culture media were purchased from Life Technologies, Inc. (Gaithersburg, MD). Calf serum was obtained from HyClone Laboratories, Inc.
Journal of Biological Chemistry, 1993
Journal of Biological Chemistry, 1991
We have carried out experiments to determine the role of the cysteines in the hormone-binding dom... more We have carried out experiments to determine the role of the cysteines in the hormone-binding domain (HBD) of the human estrogen receptor (ER) in receptor function. In each mutant receptor, 1 of the 4 cysteines in the HBD (cysteines 381, 417, 447, and 530) was changed by in vitro mutagenesis of the ER cDNA (containing Gly4Oo) from cysteine to alanine; was also mutated to a serine. The mutant and wild-type receptor cDNAs were expressed in Chinese hamster ovary cells using an expression vector containing the Rous sarcoma virus promoter. The mutant and wildtype receptors were assayed for hormone binding and for their ability to activate estrogen-responsive reporter plasmids. All ER mutants bound estradiol (E2) with affinity similar to wild-type ER, displaying a Kd between 0.3 and 0.8 nM (wild-type ER Kd = 0.45 2 0.10 nM). All were capable of covalent labeling by the affinity ligands ketononestrol aziridine, an estrogen agonist, and tamoxifen aziridine, an antagonist. Since in previous work we identified Cys5"0 as the site of covalent attachment of these ligands (Harlow, K.
Molecular and cellular endocrinology, Jan 15, 2017
Estrogen Receptor-β (ERβ) has been implicated in many cancers. In prostate and breast cancer its ... more Estrogen Receptor-β (ERβ) has been implicated in many cancers. In prostate and breast cancer its function is controversial, but genetic studies implicate a role in cancer progression. Much of the confusion around ERβ stems from antibodies that are inadequately validated, yet have become standard tools for deciphering its role. Using an ERβ-inducible cell system we assessed commonly utilized ERβ antibodies and show that one of the most commonly used antibodies, NCL-ER-BETA, is non-specific for ERβ. Other antibodies have limited ERβ specificity or are only specific in one experimental modality. ERβ is commonly studied in MCF-7 (breast) and LNCaP (prostate) cancer cell lines, but we found no ERβ expression in either, using validated antibodies and independent mass spectrometry-based approaches. Our findings question conclusions made about ERβ using the NCL-ER-BETA antibody, or LNCaP and MCF-7 cell lines. We describe robust reagents, which detect ERβ across multiple experimental approac...
Molecular and Cellular Biology, 2000
We find that prothymosin alpha (PTα) selectively enhances transcriptional activation by the estro... more We find that prothymosin alpha (PTα) selectively enhances transcriptional activation by the estrogen receptor (ER) but not transcriptional activity of other nuclear hormone receptors. This selectivity for ER is explained by PTα interaction not with ER, but with a 37-kDa protein denoted REA, for repressor of estrogen receptor activity, a protein that we have previously shown binds to ER, blocking coactivator binding to ER. We isolated PTα, known to be a chromatin-remodeling protein associated with cell proliferation, using REA as bait in a yeast two-hybrid screen with a cDNA library from MCF-7 human breast cancer cells. PTα increases the magnitude of ERα transcriptional activity three- to fourfold. It shows lesser enhancement of ERβ transcriptional activity and has no influence on the transcriptional activity of other nuclear hormone receptors (progesterone receptor, glucocorticoid receptor, thyroid hormone receptor, or retinoic acid receptor) or on the basal activity of ERs. In cont...
Cancer research, 1984
A substantial proportion of human breast cancers contain estrogen receptors, and it is believed t... more A substantial proportion of human breast cancers contain estrogen receptors, and it is believed that the growth of some of these tumors and their synthesis of specific proteins are stimulated by estrogens. Since natural estrogens, such as estradiol, react reversibly with estrogen receptors, it was of interest to determine the biological consequences that would result from very strong, possibly irreversible interaction of an estradiol-based ligand with the estrogen receptor of breast cancer cells. For these studies, we have examined the receptor interactions and biological character of 11 beta- chloromethylestradiol (CME) and 11 beta- bromomethylestradiol (BME) as potential estradiol-based affinity labeling ligands in MCF-7 human breast cancer cells which contain high levels of estrogen receptors. The apparent relative binding affinities of CME and BME for MCF-7 estrogen receptor measured by competitive binding assay are 230 and 15%, respectively, whereas the affinity of estradiol is...
Science translational medicine, Jan 21, 2015
Estrogenic and inflammatory components play key roles in a broad range of diseases including endo... more Estrogenic and inflammatory components play key roles in a broad range of diseases including endometriosis, a common estrogen-dependent gynecological disorder in which endometrial tissue creates inflammatory lesions at extrauterine sites, causing pelvic pain and reduced fertility. Current medical therapies focus primarily on reducing systemic levels of estrogens, but these are of limited effectiveness and have considerable side effects. We developed estrogen receptor (ER) ligands, chloroindazole (CLI) and oxabicycloheptene sulfonate (OBHS), which showed strong ER-dependent anti-inflammatory activity in a preclinical model of endometriosis that recapitulates the estrogen dependence and inflammatory responses of the disease in immunocompetent mice and in primary human endometriotic stromal cells in culture. Estrogen-dependent phenomena, including cell proliferation, cyst formation, vascularization, and lesion growth, were all arrested by CLI or OBHS, which prevented lesion expansion a...
EMBO molecular medicine, Jan 11, 2014
Estetrol (E4) is a natural estrogen with a long half-life produced only by the human fetal liver ... more Estetrol (E4) is a natural estrogen with a long half-life produced only by the human fetal liver during pregnancy. The crystal structures of the estrogen receptor α (ERα) ligand-binding domain bound to 17β-estradiol (E2) and E4 are very similar, as well as their capacity to activate the two activation functions AF-1 and AF-2 and to recruit the coactivator SRC3. In vivo administration of high doses of E4 stimulated uterine gene expression, epithelial proliferation, and prevented atheroma, three recognized nuclear ERα actions. However, E4 failed to promote endothelial NO synthase activation and acceleration of endothelial healing, two processes clearly dependent on membrane-initiated steroid signaling (MISS). Furthermore, E4 antagonized E2 MISS-dependent effects in endothelium but also in MCF-7 breast cancer cell line. This profile of ERα activation by E4, uncoupling nuclear and membrane activation, characterizes E4 as a selective ER modulator which could have medical applications tha...
The Journal of Steroid Biochemistry and Molecular Biology, 2000
Estrogens exert profound effects on the physiology of diverse target cells and these effects appe... more Estrogens exert profound effects on the physiology of diverse target cells and these effects appear to be mediated by two estrogen receptor (ER) subtypes, ERa and ERb. We have investigated how ER ligands, ranging from pure agonists to antagonists, interact with ERa and ERb, 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 ERa and others that are full agonists through ERa while being full antagonists through ERb. Antiestrogens such as hydroxytamoxifen, which are mixed agonist/antagonists through ERa, are pure antagonists through ERb at estrogen response element-containing gene sites. Studies with ERa/b chimeric proteins reveal that tamoxifen agonism requires the activation function 1 region of ERa. 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 (ERa or ERb) 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 ERa or ERb should allow optimized tissue selectivity of these agents for menopausal hormone replacement therapy and the treatment and prevention of breast cancer.
Proceedings of the National Academy of Sciences, 2010
Estrogen receptors (ERs) protect pancreatic islet survival in mice through rapid extranuclear act... more Estrogen receptors (ERs) protect pancreatic islet survival in mice through rapid extranuclear actions. ERα also enhances insulin synthesis in cultured islets. Whether ERα stimulates insulin synthesis in vivo and, if so, through which mechanism(s) remain largely unknown. To address these issues, we generated a pancreas-specific ERα knockout mouse (PERαKO −/− ) using the Cre-loxP strategy and used a combination of genetic and pharmacologic tools in cultured islets and β cells. Whereas 17β-estradiol (E2) treatment up-regulates pancreatic insulin gene and protein content in control ERαlox/lox mice, these E2 effects are abolished in PERαKO −/− mice. We find that E2-activated ERα increases insulin synthesis by enhancing glucose stimulation of the insulin promoter activity. Using a knock-in mouse with a mutated ERα eliminating binding to the estrogen response elements (EREs), we show that E2 stimulation of insulin synthesis is independent of the ERE. We find that the extranuclear ERα inter...
Proceedings of the National Academy of Sciences, 2005
Thyroid hormones (T) and estrogens (E) are nuclear receptor ligands with at least two molecular m... more Thyroid hormones (T) and estrogens (E) are nuclear receptor ligands with at least two molecular mechanisms of action: ( i ) relatively slow genomic effects, such as the regulation of transcription by cognate T receptors (TR) and E receptors (ER); and ( ii ) relatively rapid nongenomic effects, such as kinase activation and calcium release initiated at the membrane by putative membrane receptors. Genomic and nongenomic effects were thought to be disparate and independent. However, in a previous study using a two-pulse paradigm in neuroblastoma cells, we showed that E acting at the membrane could potentiate transcription from an E-driven reporter gene in the nucleus. Because both T and E can have important effects on mood and cognition, it is possible that the two hormones can act synergistically. In this study, we demonstrate that early actions of T via TRα1 and TRβ1 can potentiate E-mediated transcription (genomic effects) from a consensus E response element (ERE)-driven reporter ge...
Proceedings of the National Academy of Sciences, 2006
Estrogen receptor (ER)-mediated gene expression plays an essential role in mammary gland morphoge... more Estrogen receptor (ER)-mediated gene expression plays an essential role in mammary gland morphogenesis, function, and carcinogenesis. The repressor of ER activity (REA) is an ER-interactive protein that counterbalances estrogen-induced ER transcriptional activity. Our previous study showed that genetic deletion of both REA alleles resulted in embryonic lethality. This study demonstrates that REA and ERα are coexpressed in mammary epithelial cells. REA heterozygous (REA +/− ) mutant mice exhibit faster mammary ductal elongation in virgin animals, increased lobuloalveolar development during pregnancy, and delayed mammary gland involution after weaning. These morphological phenotypes of REA +/− mice are associated with significantly increased cell proliferation and ER transcriptional activities, as indicated by the estrogen response element (ERE)-luciferase reporter in the WT/ERE-Luc and REA +/− /ERE-Luc bigenic mice and by the higher expression levels of estrogen-responsive genes such...
Oncogene, 2005
The proliferative action of ERa largely accounts for the carcinogenic activity of estrogens. By c... more The proliferative action of ERa largely accounts for the carcinogenic activity of estrogens. By contrast, recent data show that ERb displays tumor-suppressor properties, thus supporting the interest to identify compounds that could increase its activity. Here, we show that histone deacetylase inhibitors (HDI) upregulated ERb protein levels, whereas it decreased ERa expression. Part of this regulation took place at the mRNA level through a mechanism independent of de novo protein synthesis. In addition, we found that, in various cancer cells, the treatment with different HDI enhanced the liganddependent activity of ERb more strongly than that of ERa. On the other hand, in MDA-MB231 and HeLa cells, the expression of ERs modified the transcriptional response to HDI. The use of deletion mutants of both receptors demonstrated that AF1 domain of the receptors was required. Finally, we show that ERb expression led to a dramatic increased in the antiproliferative activity of HDI, which correlated with a modification of the transcription of genes involved in cell cycle control by HDI. Altogether, these data demonstrate that the interference of ERb and HDAC on the control of transcription and cell proliferation constitute a promising approach for cancer therapy.
Nature Chemical Biology, 2008
Understanding how steroid hormones regulate physiological functions has been significantly advanc... more Understanding how steroid hormones regulate physiological functions has been significantly advanced by structural biology approaches. However, progress has been hampered by significant misfolding of the ligand binding domains in heterologous expression systems and by conformational flexibility that interferes with crystallization. Here, we show that protein folding problems common to steroid hormone receptors are circumvented by a mutations that stabilize well-characterized conformations of the receptor. We use this approach to present the first structure of an apo steroid receptor, revealing a ligand-accessible channel, allowing soaking of preformed crystals. Furthermore, crystallization of different pharmacological classes of compounds allowed us to define the structural basis of NFκB selective signaling through ER, revealing a unique conformation of the receptor that allows selective suppression of inflammatory gene expression. The ability to crystallize many receptor-ligand complexes with distinct pharmacophores allows one to define the structural features of signaling specificity that would not be apparent in a single structure.
Molecular Endocrinology, 1994
Progesterone receptors (PRs) mediate the actions of progestin hormones and play important roles d... more Progesterone receptors (PRs) mediate the actions of progestin hormones and play important roles during the reproductive cycle and pregnancy. Since PR expression is known to be regulated by estrogen, we have undertaken studies to examine the mechanisms underlying this regulation. We have identified multiple distinct regions of the rat PR gene, widely spaced and spread throughout the V-flanking region, the S-untranslated region, and the first exon (between-2264 and +2241), that can form a strong estrogen-responsive enhancer when linked together. Estrogen-responsive activities for two of the regions in isolation (+461/+636 and +2176/ +2241) were demonstrated in one or more homologous or heterologous promoter contexts. The contributions of the other regions (-2264/-1970,-11671-957 and +2068/+2110) to the overall activity of the assembled enhancer were cryptic in that they were only observed in the context of the other PR gene fragments, not in isolation. We identified four weak, but functional, imperfect estrogen response elements (EREs) in these regions of the PR gene, each differing from the consensus by 2 base pairs. In addition, we identified four ERE half-sites in the PR gene, three of which are paired (i.e. cl50 base pairs away) with the EREs in the estrogenresponsive regions. Competitive gel shift assays demonstrated weak, but detectable, binding of estrogen receptor to the EREs. Of note, the estrogenresponsive enhancer assembled from the five regions of the PR gene exhibited promoter specificity; it conferred estrogen responsiveness on the distal PR gene promoter, but it failed to enhance the endogenous estrogen responsiveness of the proximal PR gene promoter. The positioning of response elements in the rat PR gene, which we show to be unique among steroid hormone-regulated genes, may have functional consequences for the regulation of the magnitude and timing of PR gene expression by estrogen.
Molecular Endocrinology, 1993
We have shown previously that exposure of rat uterine cells in primary culture to estradiol (E2),... more We have shown previously that exposure of rat uterine cells in primary culture to estradiol (E2), insulin-like growth factor-l (IGF-I), or agents which alter intracellular CAMP levels, such as cholera toxin plus isobutylmethylxanthine (CT + IBMX) and 8-Br-CAMP, results in the up-regulation of cellular levels of the progesterone receptor, an effect believed to be mediated through the activation of estrogen receptor (ER) and phosphorylation pathways. We have therefore undertaken studies using transient transfection of these uterine cell cultures with a simple estrogen-responsive reporter gene in order to determine the ability of these agents to stimulate ERmediated gene transcription directly. We also compared the ability of these same agents to alter the phosphorylation state of the endogenous uterine ER protein. Plasmid DNA containing two tandem estrogen responsive elements and a TATA box linked to the chloramphenicol acetyl transferase (CAT) gene was introduced into immature rat uterine cells grown in primary culture. Treatment of transfected cells with lo-' M EP, CT (1 Fg/ml) + IBMX (lo-" M), 8-Br-CAMP (10m4 M), or IGF-I (20 rig/ml) resulted in an 8-to lofold induction of CAT activity. CAT activity stimulated by all agents was nearly completely suppressed by coincubation with the antiestrogen ICI 184,384 (ICI) or the protein kinase (PK) inhibitor H8. CAT activity induced by 8-Br-CAMP was more readily suppressed by ICI than that induced by EP, indicating that ER in cells exposed to 8-Br-CAMP is either unoccupied or minimally occupied by ligand. The level of ER phosphorylation in uterine cells was increased 3-to 5-fold upon exposure to EP, CT
Journal of Biological Chemistry, 1996
The human estrogen receptor (ER) is a ligand-inducible transcription factor that contains two tra... more The human estrogen receptor (ER) is a ligand-inducible transcription factor that contains two transcriptional activation functions, one located in the NH 2-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 NH 2-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 E 2 , 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, hormonedependent 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.
Endocrinology, 2013
Successful implantation and maintenance of pregnancy require the transformation of uterine endome... more Successful implantation and maintenance of pregnancy require the transformation of uterine endometrial stromal cells into distinct decidualized cells. Although estrogen and progesterone (P4) receptors are known to be essential for decidualization, the roles of steroid receptor coregulators in this process remain largely unknown. In this study, we have established a key role for the coregulator, repressor of estrogen receptor activity (REA), in the decidualization of human endometrial stromal cells (hESCs) in vitro and of the mouse uterus in vivo. Our studies revealed that the level of REA normally decreases to half as hESC decidualization proceeds and that uterine reduction of REA in transgenic heterozygous knockout mice or small interfering RNA knockdown of REA in hESC temporally accelerated and strongly enhanced the differentiation process, as indicated by changes in cell morphology and increased expression of biomarkers of decidualization, including P4 receptor. Findings in hESC ...
Endocrinology, 1981
Studies were undertaken to compare the effects of the estrogen ethinyl estradiol [llα-ethinylestr... more Studies were undertaken to compare the effects of the estrogen ethinyl estradiol [llα-ethinylestradiol-17β (EE2)] and the antiestrogen U11.100A [l-(2-[p-(3,4-dihydro-6-methoxy- 2 - phenyl -1 - naphthyl) phenoxy] ethyl) pyrrolidine hydrochloride (UA)] on rat liver. These compounds were assessed for their abilities to bind to estrogen-specific binding sites in liver cytosol, to translocate these sites to the nucleus, and to induce elevations of hepatic production of plasma renin substrate. Both compounds were shown to cause significant 2- to 4-fold increases in plasma renin substrate levels after sc injections at dosages of 25, 100, and 250 μg daily for 2 days to mature female rats, and two related triarylethylene antiestrogens, tamoxifen [trans-l-(4β-dimethylaminoethoxyphenyl) l,2-diphenylbut-l-ene] and CI628 [α-(p-(2-(l-pyrrolidino)ethoxy)phenyl)4-methoxy-α′-nitrostilbene], were equally potent stimulators of elevated plasma renin substrate leve s. Similar approximately 3-fold increases in plasma renin sub...
Endocrinology, 1999
We report on the identification of novel, nonsteroidal ligands that show pronounced subtype-selec... more We report on the identification of novel, nonsteroidal ligands that show pronounced subtype-selective differences in ligand binding and transcriptional potency or efficacy for the two estrogen receptor (ER) subtypes, ER␣ and ER. An aryl-substituted pyrazole is an ER␣ potency-selective agonist, showing higher binding affinity for ER␣ and 120-fold higher potency in stimulation of ER␣ vs. ER in transactivation assays in cells. A tetrahydrochrysene (THC) has a 4-fold preferential binding affinity for ER; it is an agonist on ER␣, but a complete antagonist on ER. Intriguingly, the antagonist activity of
Endocrinology, 1999
To better understand the actions of estrogens and antiestrogens in estrogen target cells, we have... more To better understand the actions of estrogens and antiestrogens in estrogen target cells, we have searched for estrogen-regulated genes in human breast cancer cells, in which the number of genes known to be directly activated by estrogen is quite small. Using differential display RNA methods, we have identified the human homolog of the Na ϩ-H ϩ exchanger regulatory factor (NHE-RF), an approximately 50-kDa protein that is also an ezrin-radixin-moesin-binding phosphoprotein, as being under rapid and direct regulation by estrogen in estrogen receptor (ER)-containing breast cancer cells. Stimulation by estrogen of NHE-RF RNA is rapid, being near maximal (ϳ6-fold) by 1 h, and is not blocked by cycloheximide, indicating that it is a primary response. Stimulation is selective for estrogen ligands, with no stimulation by other classes of steroid hormones, and stimulation by estrogen is suppressed by the antiestrogens tamoxifen and ICI 182,780. Induction is shown to require an active ER through several Materials and Methods Chemicals and materials Cell culture media were purchased from Life Technologies, Inc. (Gaithersburg, MD). Calf serum was obtained from HyClone Laboratories, Inc.