RESUMEN
Brain glucose-sensing neurons detect glucose fluctuations and prevent severe hypoglycemia, but mechanisms mediating functions of these glucose-sensing neurons are unclear. Here we report that estrogen receptor-α (ERα)-expressing neurons in the ventrolateral subdivision of the ventromedial hypothalamic nucleus (vlVMH) can sense glucose fluctuations, being glucose-inhibited neurons (GI-ERαvlVMH) or glucose-excited neurons (GE-ERαvlVMH). Hypoglycemia activates GI-ERαvlVMH neurons via the anoctamin 4 channel, and inhibits GE-ERαvlVMH neurons through opening the ATP-sensitive potassium channel. Further, we show that GI-ERαvlVMH neurons preferentially project to the medioposterior arcuate nucleus of the hypothalamus (mpARH) and GE-ERαvlVMH neurons preferentially project to the dorsal Raphe nuclei (DRN). Activation of ERαvlVMH to mpARH circuit and inhibition of ERαvlVMH to DRN circuit both increase blood glucose. Thus, our results indicate that ERαvlVMH neurons detect glucose fluctuations and prevent severe hypoglycemia in mice.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Glucosa/metabolismo , Neuronas/metabolismo , Animales , Electrofisiología , Endocrinología , Femenino , Ratones , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Psychological stress contributes to the development of hypertension in humans. The ovarian hormone, estrogen, has been shown to prevent stress-induced pressor responses in females by unknown mechanisms. Here, we showed that the antihypertensive effects of estrogen during stress were blunted in female mice lacking estrogen receptor-α in the brain medial amygdala. Deletion of estrogen receptor-α in medial amygdala neurons also resulted in increased excitability of these neurons, associated with elevated ionotropic glutamate receptor expression. We further demonstrated that selective activation of medial amygdala neurons mimicked effects of stress to increase blood pressure in mice. Together, our results support a model where estrogen acts on estrogen receptor-α expressed by medial amygdala neurons to prevent stress-induced activation of these neurons, and therefore prevents pressor responses to stress.
Asunto(s)
Amígdala del Cerebelo/metabolismo , Estradiol/farmacología , Hipertensión/fisiopatología , Receptores de Estrógenos/metabolismo , Estrés Fisiológico , Amígdala del Cerebelo/efectos de los fármacos , Animales , Determinación de la Presión Sanguínea , Western Blotting , Corticosterona/sangre , Modelos Animales de Enfermedad , Femenino , Hipertensión/metabolismo , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Valores de ReferenciaRESUMEN
To further define the utility of the Ishikawa cells as a reliable in vitro model to determine the potential estrogenic activity of chemicals of interest, transcriptional changes induced by genistein (GES) in Ishikawa cells at various doses (10 pM, 1 nM, 100 nM, and 10 µM) and time points (8, 24, and 48 h) were identified using a comprehensive microarray approach. Trend analysis indicated that the expression of 5342 unique genes was modified by GES in a dose- and time-dependent manner (P ≤ 0.0001). However, the majority of gene expression changes induced in Ishikawa cells were elicited by the highest dose of GES evaluated (10 µM). The GES' estrogenic activity was identified by comparing the Ishikawa cells' response to GES versus 17 α-ethynyl estradiol (EE, at equipotent doses, ie, 10 µM vs 1 µM, respectively) and was defined by changes in the expression of 284 unique genes elicited by GES and EE in the same direction, although the magnitude of the change for some genes was different. Further, comparing the response of the Ishikawa cells exposed to high doses of GES and EE versus the response of the juvenile rat uterus exposed to EE, we identified 66 unique genes which were up- or down regulated in a similar manner in vivo as well as in vitro Genistein elicits changes in multiple molecular pathways affecting various biological processes particularly associated with cell organization and biogenesis, regulation of translation, cell proliferation, and intracellular transport; processes also affected by estrogen exposure in the uterus of the rat. These results indicate that Ishikawa cells are capable of generating a biologically relevant estrogenic response and offer an in vitro model to assess this mode of action.
Asunto(s)
Adenocarcinoma/genética , Neoplasias Endometriales/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genisteína/farmacología , Fitoestrógenos/farmacología , Transcripción Genética/efectos de los fármacos , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Estradiol/farmacología , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Útero/efectos de los fármacos , Útero/metabolismoRESUMEN
Estrogens act upon estrogen receptor (ER)α to inhibit feeding and improve glucose homeostasis in female animals. However, the intracellular signals that mediate these estrogenic actions remain unknown. Here, we report that anorexigenic effects of estrogens are blunted in female mice that lack ERα specifically in proopiomelanocortin (POMC) progenitor neurons. These mutant mice also develop insulin resistance and are insensitive to the glucose-regulatory effects of estrogens. Moreover, we showed that propyl pyrazole triol (an ERα agonist) stimulates the phosphatidyl inositol 3-kinase (PI3K) pathway specifically in POMC progenitor neurons, and that blockade of PI3K attenuates propyl pyrazole triol-induced activation of POMC neurons. Finally, we show that effects of estrogens to inhibit food intake and to improve insulin sensitivity are significantly attenuated in female mice with PI3K genetically inhibited in POMC progenitor neurons. Together, our results indicate that an ERα-PI3K cascade in POMC progenitor neurons mediates estrogenic actions to suppress food intake and improve insulin sensitivity.
Asunto(s)
Glucemia/metabolismo , Ingestión de Alimentos/genética , Receptor alfa de Estrógeno/genética , Estrógenos/metabolismo , Resistencia a la Insulina , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasa/genética , Proopiomelanocortina/metabolismo , Animales , Ingestión de Alimentos/efectos de los fármacos , Receptor alfa de Estrógeno/agonistas , Receptor alfa de Estrógeno/metabolismo , Conducta Alimentaria/fisiología , Femenino , Glucosa/metabolismo , Homeostasis , Ratones , Ratones Noqueados , Células-Madre Neurales/efectos de los fármacos , Neuronas/efectos de los fármacos , Fenoles/farmacología , Fosfatidilinositol 3-Quinasa/efectos de los fármacos , Pirazoles/farmacología , Transducción de SeñalRESUMEN
Estrogen receptor-α (ERα) activity in the brain prevents obesity in both males and females. However, the ERα-expressing neural populations that regulate body weight remain to be fully elucidated. Here we showed that single-minded-1 (SIM1) neurons in the medial amygdala (MeA) express abundant levels of ERα. Specific deletion of the gene encoding ERα (Esr1) from SIM1 neurons, which are mostly within the MeA, caused hypoactivity and obesity in both male and female mice fed with regular chow, increased susceptibility to diet-induced obesity (DIO) in males but not in females, and blunted the body weight-lowering effects of a glucagon-like peptide-1-estrogen (GLP-1-estrogen) conjugate. Furthermore, selective adeno-associated virus-mediated deletion of Esr1 in the MeA of adult male mice produced a rapid body weight gain that was associated with remarkable reductions in physical activity but did not alter food intake. Conversely, overexpression of ERα in the MeA markedly reduced the severity of DIO in male mice. Finally, an ERα agonist depolarized MeA SIM1 neurons and increased their firing rate, and designer receptors exclusively activated by designer drug-mediated (DREADD-mediated) activation of these neurons increased physical activity in mice. Collectively, our results support a model where ERα signals activate MeA neurons to stimulate physical activity, which in turn prevents body weight gain.
Asunto(s)
Peso Corporal/fisiología , Complejo Nuclear Corticomedial/metabolismo , Receptor alfa de Estrógeno/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Complejo Nuclear Corticomedial/citología , Complejo Nuclear Corticomedial/efectos de los fármacos , Metabolismo Energético , Receptor alfa de Estrógeno/deficiencia , Receptor alfa de Estrógeno/genética , Estrógenos/administración & dosificación , Femenino , Péptido 1 Similar al Glucagón/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Actividad Motora/fisiología , Neuronas/metabolismo , Obesidad/etiología , Obesidad/metabolismo , Obesidad/patología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Caracteres Sexuales , Transducción de Señal , Aumento de Peso/fisiologíaRESUMEN
Reduced bioavailability of estrogen increases skeletal fracture risk in postmenopausal women, but the mechanisms by which estrogen regulates bone mass are incompletely understood. Because estrogen signaling in bone acts, in part, through estrogen receptor alpha (ERα), mice with global deletion of ERα (ERαKO) have been used to determine the role of estrogen signaling in bone biology. These animals, however, have confounding systemic effects arising from other organs, such as increased estrogen and decreased insulin-like growth factor 1 (IGF-1) serum levels, which may independently affect bone. Mice with tissue-specific ERα deletion in chondrocytes, osteoblasts, osteocytes, or osteoclasts lack the systemic effects seen in the global knockout, but show that presence of the receptor is important for the function of each cell type. Although bone mass is reduced when ERα is deleted from osteoblasts, no study has determined if this approach reduces whole bone strength. To address this issue, we generated female osteoblast-specific ERαKO mice (pOC-ERαKO) by crossing mice expressing a floxed ERα gene (ERα(fl/fl)) with mice transgenic for the osteocalcin-Cre promoter (OC-Cre). Having confirmed that serum levels of estrogen and IGF-1 were unaltered, we focused on relating bone mechanics to skeletal phenotype using whole bone mechanical testing, microcomputed tomography, histology, and dynamic histomorphometry. At 12 and 18 weeks of age, pOC-ERαKO mice had decreased cancellous bone mass in the proximal tibia, vertebra, and distal femur, and decreased cortical bone mass in the tibial midshaft, distal femoral cortex, and L5 vertebral cortex. Osteoblast activity was reduced in cancellous bone of the proximal tibia, but osteoclast number was unaffected. Both femora and vertebrae had decreased whole bone strength in mechanical tests to failure, indicating that ERα in osteoblasts is required for appropriate bone mass and strength accrual in female mice. This pOC-ERαKO mouse is an important animal model that could enhance our understanding of estrogen signaling in bone cells in vivo.
Asunto(s)
Huesos/metabolismo , Receptor alfa de Estrógeno/metabolismo , Fracturas Óseas/metabolismo , Osteoblastos/metabolismo , Osteoporosis Posmenopáusica/metabolismo , Animales , Huesos/patología , Modelos Animales de Enfermedad , Receptor alfa de Estrógeno/genética , Femenino , Fracturas Óseas/genética , Fracturas Óseas/patología , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Noqueados , Osteoblastos/patología , Osteocalcina/genética , Osteocalcina/metabolismo , Osteoporosis Posmenopáusica/genética , Osteoporosis Posmenopáusica/patologíaRESUMEN
BACKGROUND: The receptor tyrosine kinase family includes many transmembrane proteins with diverse physiological and pathophysiological functions. The involvement of tyrosine kinase signaling in promoting a more aggressive tumor phenotype within the context of chemotherapeutic evasion is gaining recognition. The Ron receptor is a tyrosine kinase receptor that has been implicated in the progression of breast cancer and evasion of tamoxifen therapy. RESULTS: Here, we report that Ron expression is correlated with in situ, estrogen receptor alpha (ERα)-positive tumors, and is higher in breast tumors following neoadjuvant tamoxifen therapy. We also demonstrate that the majority of mammary tumors isolated from transgenic mice with mammary specific-Ron overexpression (MMTV-Ron mice), exhibit appreciable ER expression. Moreover, genetic-ablation of ERα, in the context of Ron overexpression, leads to delayed mammary tumor initiation and growth, but also results in an increased metastasis. CONCLUSIONS: Ron receptor overexpression is associated with ERα-positive human and murine breast tumors. In addition, loss of ERα on a Ron overexpressing background in mice leads to the development of breast tumors which grow slower but which exhibit more metastasis and suggests that targeting of ERα, as in the case of tamoxifen therapy, may reduce the growth of Ron overexpressing breast cancers but may cause these tumors to be more metastatic.
Asunto(s)
Receptor alfa de Estrógeno/genética , Eliminación de Gen , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Proteínas Tirosina Quinasas Receptoras/genética , Animales , Proliferación Celular , Receptor alfa de Estrógeno/metabolismo , Femenino , Expresión Génica , Neoplasias Mamarias Animales/mortalidad , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Metástasis de la Neoplasia , Estadificación de Neoplasias , Fenotipo , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
Estrogens regulate body weight and reproduction primarily through actions on estrogen receptor-α (ERα). However, ERα-expressing cells mediating these effects are not identified. We demonstrate that brain-specific deletion of ERα in female mice causes abdominal obesity stemming from both hyperphagia and hypometabolism. Hypometabolism and abdominal obesity, but not hyperphagia, are recapitulated in female mice lacking ERα in hypothalamic steroidogenic factor-1 (SF1) neurons. In contrast, deletion of ERα in hypothalamic pro-opiomelanocortin (POMC) neurons leads to hyperphagia, without directly influencing energy expenditure or fat distribution. Further, simultaneous deletion of ERα from both SF1 and POMC neurons causes hypometabolism, hyperphagia, and increased visceral adiposity. Additionally, female mice lacking ERα in SF1 neurons develop anovulation and infertility, while POMC-specific deletion of ERα inhibits negative feedback regulation of estrogens and impairs fertility in females. These results indicate that estrogens act on distinct hypothalamic ERα neurons to regulate different aspects of energy homeostasis and reproduction.
Asunto(s)
Metabolismo Energético/fisiología , Receptor alfa de Estrógeno/metabolismo , Hipotálamo/metabolismo , Neuronas/metabolismo , Animales , Estradiol/sangre , Receptor alfa de Estrógeno/deficiencia , Receptor alfa de Estrógeno/genética , Femenino , Hiperfagia/etiología , Infertilidad Femenina/etiología , Masculino , Ratones , Ratones Noqueados , Obesidad/etiología , Proopiomelanocortina/metabolismo , Factor Esteroidogénico 1/metabolismoRESUMEN
Estrogen receptor alpha (ERalpha) plays an important role in the onset and progression of breast cancer, whereas p53 functions as a major tumor suppressor. We previously reported that ERalpha binds to p53, resulting in inhibition of transcriptional regulation by p53. Here, we report on the molecular mechanisms by which ERalpha suppresses p53's transactivation function. Sequential ChIP assays demonstrated that ERalpha represses p53-mediated transcriptional activation in human breast cancer cells by recruiting nuclear receptor corepressors (NCoR and SMRT) and histone deacetylase 1 (HDAC1). RNAi-mediated down-regulation of NCoR resulted in increased endogenous expression of the cyclin-dependent kinase (CDK)-inhibitor p21(Waf1/Cip1) (CDKN1A) gene, a prototypic transcriptional target of p53. While 17beta-estradiol (E2) enhanced ERalpha binding to p53 and inhibited p21 transcription, antiestrogens decreased ERalpha recruitment and induced transcription. The effects of estrogen and antiestrogens on p21 transcription were diametrically opposite to their known effects on the conventional ERE-containing ERalpha target gene, pS2/TFF1. These results suggest that ERalpha uses dual strategies to promote abnormal cellular proliferation: enhancing the transcription of ERE-containing proproliferative genes and repressing the transcription of p53-responsive antiproliferative genes. Importantly, ERalpha binds to p53 and inhibits transcriptional activation by p53 in stem/progenitor cell-containing murine mammospheres, suggesting a potential role for the ER-p53 interaction in mammary tissue homeostasis and cancer formation. Furthermore, retrospective studies analyzing response to tamoxifen therapy in a subset of patients with ER-positive breast cancer expressing either wild-type or mutant p53 suggest that the presence of wild-type p53 is an important determinant of positive therapeutic response.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Animales , Secuencia de Bases , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Cartilla de ADN/genética , Estradiol/farmacología , Moduladores de los Receptores de Estrógeno/farmacología , Femenino , Genes p53 , Histona Desacetilasa 1/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Células Madre Neoplásicas/metabolismo , Regiones Promotoras Genéticas , Tamoxifeno/farmacología , Activación Transcripcional , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
A reliable in vitro model to determine the potential estrogenic activity of chemicals of interest is still unavailable. To further investigate the usefulness of a human-derived cell line, we determined the transcriptional changes induced by bisphenol A (BPA) in Ishikawa cells at various doses (1 nM, 100 nM, 10 microM, and 100 microM) and time points (8, 24 and 48 h) by comparing the response of approximately 38,500 human genes and ESTs between treatment groups and controls (vehicle-treated). By trend analysis, we determined that the expression of 2794 genes was modified by BPA in a dose- and time-dependent manner (p< or =0.0001). However, the majority of gene expression changes induced in Ishikawa cells were elicited by the highest doses of BPA evaluated (10-100 microM), while the genomic response of the cells exposed to low doses of BPA was essentially negligible. By comparing the Ishikawa cells' response to BPA vs.17 alpha-ethynyl estradiol we determined that the change in the expression of 307 genes was identical in the direction of the change, although the magnitude of the change for some genes was different. Further, the response of Ishikawa cells to high doses of BPA shared similarities to the estrogenic response of the rat uterus, specifically, 362 genes were regulated in a similar manner in vivo as well as in vitro. Gene ontology analysis indicated that BPA results in changes to multiple molecular pathways affecting various biological processes particularly associated with cell organization and biogenesis, regulation of translation, cell proliferation, and intracellular transport; processes also affected by estrogen exposure in the uterus of the rat. These results indicate that Ishikawa cells are capable of generating a biologically relevant estrogenic response after exposure to chemicals with varied estrogenic activity, and offer an in vitro model to assess this mode of action.
Asunto(s)
Contaminantes Ocupacionales del Aire/toxicidad , Endometrio/patología , Estrógenos no Esteroides/toxicidad , Fenoles/toxicidad , Animales , Compuestos de Bencidrilo , Línea Celular , Dermatoglifia del ADN , Interpretación Estadística de Datos , Relación Dosis-Respuesta a Droga , Endometrio/citología , Endometrio/efectos de los fármacos , Femenino , Humanos , Embarazo , ARN/biosíntesis , ARN/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Útero/efectos de los fármacos , Útero/metabolismoRESUMEN
We have determined the gene expression profile induced by 17 alpha-ethynyl estradiol (EE) in Ishikawa cells, a human uterine-derived estrogen-sensitive cell line, at various doses (1 pM, 100 pM, 10 nM, and 1 microM) and time points (8, 24, and 48 h). The transcript profiles were compared between treatment groups and controls (vehicle-treated) using high-density oligonucleotide arrays to determine the expression level of approximately 38,500 human genes. By trend analysis, we determined that the expression of 2560 genes was modified by exposure to EE in a dose- and time-dependent manner (p = 0.0001). The annotation available for the genes affected indicates that EE exposure results in changes in multiple molecular pathways affecting various biological processes, particularly associated with development, morphogenesis, organogenesis, cell proliferation, cell organization, and biogenesis. All of these processes are also affected by estrogen exposure in the uterus of the rat. Comparison of the response to EE in both the rat uterus and the Ishikawa cells showed that 71 genes are regulated in a similar manner in vivo as well as in vitro. Further, some of the genes that show a robust response to estrogen exposure in Ishikawa cells are well known to be estrogen responsive, in various in vivo studies, such as PGR, MMP7, IGFBP3, IGFBP5, SOX4, MYC, EGR1, FOS, CKB, and CCND2, among others. These results indicate that transcript profiling can serve as a viable tool to select reliable in vitro systems to evaluate potential estrogenic activities of target chemicals and to identify genes that are relevant for the estrogen response.
Asunto(s)
Etinilestradiol/farmacología , Expresión Génica/efectos de los fármacos , Útero/metabolismo , Animales , Línea Celular , Bases de Datos Genéticas , Relación Dosis-Respuesta a Droga , Femenino , Expresión Génica/genética , Perfilación de la Expresión Génica , Genes/genética , Genes/fisiología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/análisis , Ratas , Factores de Tiempo , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo , Neoplasias Uterinas/metabolismoRESUMEN
The role of the carboxyl terminal F-domain of estrogen receptor (ERalpha) is uncertain, but evidence suggests that this region may impart internal restraint on ER dimerization in the presence of 17beta-estradiol (E2). To identify the C-terminal residues affecting human ERalpha activation, we created a series of deletions and examined E2 induced receptor dimerization and transactivation. Deletion of the final 24 C-terminal amino acids of the F-domain (Delta7b) yielded a fivefold increase in dimerization, when compared to wild type (wt) ERalpha in the presence of 2nM E2, utilizing a yeast two-hybrid assay. This increase in dimerization is similar to that observed when the entire F-domain was deleted. Measurement of mutant:mutant homodimer formation yielded similar increases compared to mutant:wt interactions. Interestingly, a point mutation at the C-terminus (mut 3) showed increases in dimerization comparable to that of Delta7b in the presence of nanomolar amounts of E2. However, at sub-nanomolar levels of E2, mut 3 behaved similarly to wt ERalpha, whereas Delta7b maintained striking increases in dimerization. Determination of E2 binding affinity (Kd) constants revealed only marginal differences for wt and F-domain mutants, suggesting that the F-domain affects dimerization directly. We also observed enhanced interaction of F domain mutants with p160 family coactivator SRC1. Finally, transcriptional regulation of estrogen responsive reporters, 2XERE-LacZ and 3XERE-Luc in yeast and mammalian cells, respectively, reflected the increased propensity for dimerization by F domain mutants. Together, these data indicate that the C-terminal amino acids of ERalpha are critical for attenuation of E2 induced receptor dimerization and transcriptional activity.
Asunto(s)
Estradiol/metabolismo , Receptor alfa de Estrógeno/metabolismo , Sitios de Unión , Línea Celular Tumoral , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/genética , Genes Reporteros , Histona Acetiltransferasas/metabolismo , Humanos , Ligandos , Mutación , Coactivador 1 de Receptor Nuclear , Conformación Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Elementos de Respuesta , Factores de Transcripción/metabolismo , Transcripción Genética , TransfecciónRESUMEN
A number of studies have reported on the unusual pharmacological behavior of type I antiestrogens, such as tamoxifen. These agents display mixed agonist/antagonist activity in a dose-, cell-, and tissue-specific manner. Consequently, many efforts have been made to develop so-called 'pure' antiestrogens that lack mixed agonist/antagonist activity. The recent report of the structure of estrogen receptor (ER) beta with a second molecule of 4-hydroxytamoxifen (HT) bound in the coactivator-binding surface of the ligand-binding domain (LBD) represents the first direct example of a second ER ligand-binding site and provides insight into the possible origin of mixed agonist/antagonist activity of type I antiestrogens. In this review, we summarize the biological reports leading up to the structural conformation of a second ER ligand-binding site, compare the ERbeta LBD structure bound with two HT molecules to other ER structures, and discuss the potential for small molecular inhibitors designed to directly inhibit ER-coactivator and, more generally, nuclear receptor (NR)-coactivator interactions. The studies support a departure from the traditional paradigm of drug targeting to the ligand-binding site, to that of a rational approach targeting a functionally important surface, namely the NR coactivator-binding (activation function-2) surface. Furthermore, we provide evidence supporting a reevaluation of the strict interpretation of the agonist/antagonist state with respect to the position of helix 12 in the NR LBD.
Asunto(s)
Antineoplásicos Hormonales/metabolismo , Receptores de Estrógenos/metabolismo , Tamoxifeno/metabolismo , Transactivadores/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Histona Acetiltransferasas/metabolismo , Humanos , Datos de Secuencia Molecular , Coactivador 1 de Receptor Nuclear , Unión Proteica , Receptores de Estrógenos/química , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismoRESUMEN
The estrogen receptor-alpha (ERalpha) is a critical transcription factor that regulates epithelial cell proliferation and ductal morphogenesis during postnatal mammary gland development. Tissue recombination and transplantation studies using the first generation of ERalpha knockout (ERKO) mice suggested that this steroid hormone receptor is required in the mammary stroma that subsequently exerts its effect on the epithelium through additional paracrine signaling events. A more detailed analysis revealed that ERKO mice produce a truncated ERalpha protein with detectable transactivation activity, and it is likely that this functional ERalpha variant has masked the biological significance of this steroid receptor in the mammary epithelium. In this article, we describe the generation a Cre-lox-based conditional knockout of the ERalpha gene to study the biological function of this steroid receptor in the epithelial compartment at defined stages of mammary gland development. The mouse mammary tumor virus (MMTV)-Cre-mediated, epithelial-specific ablation of exon 3 of the ERalpha gene in virgin mice severely impaired ductal elongation and side branching. The conditional knockout resulted in ablation of the ERalpha protein, and the progesterone receptor (PR), whose expression is under the control of ERalpha, was largely absent. The whey acidic protein (WAP)-Cre-mediated deletion of ERalpha during successive gestation cycles resulted in a loss of ductal side-branching and lobuloalveolar structures, ductal dilation, and decreased proliferation of alveolar progenitors. These abnormalities compromised milk production and led to malnourishment of the offspring by the second lactation. These observations suggest that ERalpha expression in the mammary epithelium is essential for normal ductal morphogenesis during puberty and alveologenesis during pregnancy and lactation.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Morfogénesis , Animales , Epitelio/química , Epitelio/metabolismo , Receptor alfa de Estrógeno/análisis , Receptor alfa de Estrógeno/genética , Femenino , Expresión Génica , Inmunohistoquímica , Integrasas/metabolismo , Lactancia/genética , Lactancia/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Virus del Tumor Mamario del Ratón/fisiología , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas de la Leche/metabolismo , Morfogénesis/genéticaRESUMEN
The high mobility group A2 protein (HMGA2) has been implicated in the pathogenesis of mesenchymal tumors such as leiomyoma, lipoma and hamartoma. HMGA2 was pinpointed by mapping the breakpoints in the chromosomal translocations in 12q15, especially the t(12;14) that is commonly seen in uterine leiomyoma. It is generally assumed that altered expression of HMGA2 is an early event in the pathway to tumor formation. Here, we show evidence that three novel transcripts, A15, B6 and D12 are located within the HMGA2 gene itself and are transcribed from the opposite strand. These embedded transcripts are expressed at 6-20-fold higher levels in tumors compared to matched myometrium from the same patients. We estimate that the domain of increased expression extends 500kb on chromosome 12q15, and encompasses the majority of t(12;14) translocation breakpoints. However, a corresponding domain of consistently altered expression is not seen on chromosome 14 or outside of the chromosome 12 multiple aberration region. These data suggest that t(12;14) breakpoints contribute to the pathogenesis of uterine leiomyoma by interrupting a complex regulation of HMGA2 and other genes embedded within and around it. We also discovered a novel laminin receptor gene, transcribed from the opposite strand, within the promoter region of HMGA2. Although the roles for these embedded transcripts are still unknown, preliminary data suggest that they are members of the family of non-coding RNA and that they may play an important role in the pathology of uterine leiomyoma.
Asunto(s)
Cromosomas Humanos Par 12 , Cromosomas Humanos Par 14 , Proteína HMGA2/genética , Leiomioma/genética , Translocación Genética , Neoplasias Uterinas/genética , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Etiquetas de Secuencia Expresada , Femenino , Proteína HMGA2/metabolismo , Humanos , Leiomioma/metabolismo , Leiomioma/patología , Modelos Genéticos , Datos de Secuencia Molecular , Miometrio/metabolismo , Regiones Promotoras Genéticas , Receptores de Laminina/genética , Neoplasias Uterinas/metabolismoRESUMEN
Bisphenol-A (BPA) shows proliferative actions in uterus and mammary glands and may influence the development of male and female reproductive tracts in utero or during early postnatal life. Because of its ability to function as an estrogen receptor (ER) agonist, BPA has the potential to disrupt normal endocrine signaling through regulation of ER target genes. Some genes are regulated by both estradiol (E2) and BPA, but those exclusive to either agent have not been described. Using a yeast strain incorporating a vitellogenin A2 ERE-LacZ reporter gene into the genome, we found that BPA induced expression of the reporter in colonies transformed with the ERalpha expression plasmid, illustrating BPA-mediated regulation within a chromatin context. Additionally, a reporter gene transiently transfected into the endometrial cancer (Ishikawa) cell line also showed BPA activity, although at 100-fold less potency than E2. To compare global gene expression in response to BPA and E2, we used a variant of the MCF-7 breast cancer cell line stably expressing HA-tagged ERalpha. Cultures were treated for 3h with an ethanol vehicle, E2 (10(-8)M), or BPA (10(-6)M), followed by isolation of RNA and microarray analysis with the human U95A probe array (Affymetrix, Santa Clara, CA, USA). More than 300 genes were changed 2-fold or more by either or both agents, with roughly half being up-regulated and half down-regulated. A number of growth- and development-related genes, such as HOXC1 and C6, Wnt5A, Frizzled, TGFbeta-2, and STAT inhibitor 2, were found to be affected exclusively by BPA. We used quantitative real-time PCR to verify regulation of the HOXC6 gene, which showed decreased expression of approximately 2.5-fold by BPA. These results reveal novel effects by BPA and E2, raising interesting possibilities regarding the role of endocrine disruptors in sexual development.
Asunto(s)
Contaminantes Ambientales/toxicidad , Receptor alfa de Estrógeno/metabolismo , Estrógenos no Esteroides/toxicidad , Fenoles/toxicidad , Compuestos de Bencidrilo , Línea Celular Tumoral , Desarrollo Embrionario , Receptor alfa de Estrógeno/genética , Femenino , Perfilación de la Expresión Génica , Genes Reporteros , Genitales/efectos de los fármacos , Genitales/embriología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Exposición Materna , Análisis por Micromatrices , Embarazo , Elementos de RespuestaRESUMEN
Evidence is presented for a unified model for the reaction of antiestrogens with estrogen receptors that explains much of the unusual pharmacology of these clinically important agents. Agonist activity results from occupancy of the estradiol-binding (primary) site in the receptor and antagonism from the additional interaction with a secondary locus not recognized by hormone. In the case of type I antiestrogens, such as tamoxifen, this is weaker than primary site binding, so these substances are agonists at low concentrations and antagonists at higher levels. With type II antiestrogens, affinities for both sites are comparable, so one never has the agonist situation (only primary site occupied), and these agents are pure antagonists. Reproductive tissues of the mouse and guinea pig contain a small macromolecule that binds hydroxytamoxifen, but not estradiol, keeping the free antiestrogen concentration below that required for secondary binding. Thus, in these species, tamoxifen is a pure agonist.
Asunto(s)
Antagonistas de Estrógenos/metabolismo , Modelos Biológicos , Receptores de Estradiol/metabolismo , Animales , Antagonistas de Estrógenos/farmacocinética , Femenino , Cobayas , Ratones , Receptores de Estradiol/antagonistas & inhibidoresRESUMEN
Xenoestrogens such as bisphenol-A (BPA) can mimic endogenous 17beta-estradiol (E2) in vitro and in vivo through binding the estrogen receptor (ER), and modulating target gene expression. In the present study, we compared global gene regulation by BPA and E2 in estrogen responsive (ERalpha-HA) human breast cancer cells derived from the MCF-7 cell line. The ERalpha-HA cells (stably over-expressing ERalpha) were exposed to E2 (10(-8)M) or BPA (10(-6)M), for 3h followed by analysis of global gene expression. More than 40 transcripts were significantly changed in ERalpha-HA cells, with many being unique to BPA. At least 15 genes were modulated by BPA in the ER-null C4-12 cell line, indicating ER independent activity. Utilizing quantitative reverse transcription-polymerase chain reaction (RT-PCR), we confirmed BPA and E2 mediated regulation of four selected genes. A consensus Alu-type estrogen responsive element (ERE) was found in the Wiskott-Aldrich syndrome protein (WASP) gene, which conferred responsiveness to BPA and E2 in a reporter gene assay. Significant stimulation was seen only in ERalpha expressing cells, thus indicating a functional ERE. Taken together these data illustrate novel gene regulation by BPA and E2, which has implications for in vivo actions and previous reports of additive and synergistic effects on breast cancer cell growth.
Asunto(s)
Neoplasias de la Mama/genética , Estradiol/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Fenoles/farmacología , Elementos de Respuesta/efectos de los fármacos , Elementos Alu/genética , Compuestos de Bencidrilo , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/genética , Línea Celular Tumoral , Proteínas del Citoesqueleto , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/fisiología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Familia de Multigenes/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Elementos de Respuesta/genéticaRESUMEN
OBJECTIVES: The uterus responds to an implanting blastocyst by undergoing extensive tIssue modification leading to decidualization. This modification includes differentiation and apoptosis of epithelial as well as stromal cell compartments. It is generally accepted that the decidual cell regression pattern is similar to the pattern of initial differentiation, suggesting that decidual cell death is the end point of timed differentiation. However, the molecular mechanisms controlling these events are not understood clearly. Therefore, we aimed to investigate the involvement of apoptotic factors using an in vitro cell culture system. DESIGN: In order to assess the role of apoptotic factors during decidualization, we used a decidual cell line (GG-AD) that had been transformed with a temperature-sensitive SV-40 mutant. At the non-permissive temperature (39 degrees C), these cells showed the characteristics of differentiated decidual cells. They dedifferentiated into stromal cells when the temperature was shifted back to 33 degrees C. METHODS: We performed Northern blot analysis for bax, bcl-x(L) and bcl-2 at both temperatures. The onset of apoptosis was examined by Annexin V staining. The expression of p53 protein was also determined by Western blot. RESULTS: We found an increase in the expression of bax when GG-AD cells were grown at 39 degrees C. We also showed apoptosis with Annexin V staining at 39 degrees C. The p53 protein expression was also similar to that of the animal models, suggesting that the programmed cell death of the decidual cells occurred in a p53-independent manner. CONCLUSIONS: These data indicate that a parallelism exists between the increased expression of pro-apoptotic genes and decidual cell death, similar to animal models. Therefore, an in vitro model of GG-AD cells can be used to assess directly the relationship between apoptotic regulators and decidualization and could be used to study the mechanism of decidual cell regression.