RESUMO
Polycystic ovary syndrome (PCOS) pathophysiology is poorly understood, due partly to lack of PCOS animal models fully recapitulating this complex disorder. Recently, a PCOS rat model using letrozole (LET), a nonsteroidal aromatase inhibitor, mimicked multiple PCOS phenotypes, including metabolic features absent in other models. Given the advantages of using genetic and transgenic mouse models, we investigated whether LET produces a similar PCOS phenotype in mice. Pubertal female C57BL/6N mice were treated for 5 wk with LET, which resulted in increased serum testosterone and normal diestrus levels of estradiol, similar to the hyperandrogenemia and follicular phase estrogen levels of PCOS women. As in PCOS, ovaries from LET mice were larger, polycystic, and lacked corpora lutea versus controls. Most LET females were acyclic, and all were infertile. LET females displayed elevated serum LH levels and higher Lhb mRNA in the pituitary. In contrast, serum FSH and Fshb were significantly reduced in LET females, demonstrating differential effects on gonadotropins, as in PCOS. Within the ovary, LET females had higher Cyp17, Cyp19, and Fsh receptor mRNA expression. In the hypothalamus, LET females had higher kisspeptin receptor mRNA expression but lower progesterone receptor mRNA levels. LET females also gained more weight than controls, had increased abdominal adiposity and adipocyte size, elevated adipose inflammatory mRNA levels, and impaired glucose tolerance, mirroring the metabolic phenotype in PCOS women. This is the first report of a LET paradigm in mice that recapitulates both reproductive and metabolic PCOS phenotypes and will be useful to genetically probe the PCOS condition.
Assuntos
Inibidores Enzimáticos/toxicidade , Nitrilas/toxicidade , Síndrome do Ovário Policístico/induzido quimicamente , Síndrome do Ovário Policístico/patologia , Reprodução/efeitos dos fármacos , Triazóis/toxicidade , Animais , Corpo Lúteo/metabolismo , Diestro/metabolismo , Ciclo Estral/efeitos dos fármacos , Feminino , Hiperandrogenismo/sangue , Hiperandrogenismo/induzido quimicamente , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Kisspeptinas/biossíntese , Kisspeptinas/genética , Letrozol , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Síndrome do Ovário Policístico/metabolismo , Gravidez , Testosterona/sangueRESUMO
Mutations in neurokinin B (NKB) and its receptor, NK3R, were identified in human patients with hypogonadotropic hypogonadism, a disorder characterized by lack of puberty and infertility. Further studies have suggested that NKB acts at the level of the hypothalamus to control GnRH neuron activity, either directly or indirectly. We recently reported that treatment with senktide, a NK3R agonist, induced GnRH secretion and expression of c-fos mRNA in GT1-7 cells. Here, we map the responsive region in the murine c-fos promoter to between -400 and -200 bp, identify the signal transducer and activator of transcription (STAT) (-345) and serum response element (-310) sites as required for induction, a modulatory role for the Ets site (-318), and show that induction is protein kinase C dependent. Using gel shift and Gal4 assays, we further show that phosphorylation of Elk-1 leads to binding to DNA in complex with serum response factor at serum response element and Ets sites within the c-fos promoter. Thus, we determine molecular mechanisms involved in NKB regulation of c-fos induction, which may play a role in modulation of GnRH neuron activation.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Neurocinina B/fisiologia , Neurônios/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Fator de Resposta Sérica/metabolismo , Transcrição Gênica/fisiologia , Proteínas Elk-1 do Domínio ets/fisiologia , Animais , Linhagem Celular Transformada , Camundongos , Neurocinina B/farmacologia , Neurônios/metabolismo , Ratos , Transdução de Sinais , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Reproduction is integrated by interaction of neural and hormonal signals converging on hypothalamic neurons for controlling gonadotropin-releasing hormone (GnRH). Kisspeptin, the peptide product of the kiss1 gene and the endogenous agonist for the GRP54 receptor, plays a key role in the regulation of GnRH secretion. In the present study, we investigated the interaction between kisspeptin, estrogen and BMPs in the regulation of GnRH production by using mouse hypothalamic GT1-7 cells. Treatment with kisspeptin increased GnRH mRNA expression and GnRH protein production in a concentration-dependent manner. The expression levels of kiss1 and GPR54 were not changed by kisspeptin stimulation. Kisspeptin induction of GnRH was suppressed by co-treatment with BMPs, with BMP-4 action being the most potent for suppressing the kisspeptin effect. The expression of kisspeptin receptor, GPR54, was suppressed by BMPs, and this effect was reversed in the presence of kisspeptin. It was also revealed that BMP-induced Smad1/5/8 phosphorylation and Id-1 expression were suppressed and inhibitory Smad6/7 was induced by kisspeptin. In addition, estrogen induced GPR54 expression, while kisspeptin increased the expression levels of ERα and ERß, suggesting that the actions of estrogen and kisspeptin are mutually enhanced in GT1-7 cells. Moreover, kisspeptin stimulated MAPKs and AKT signaling, and ERK signaling was functionally involved in the kisspeptin-induced GnRH expression. BMP-4 was found to suppress kisspeptin-induced GnRH expression by reducing ERK signaling activity. Collectively, the results indicate that the axis of kisspeptin-induced GnRH production is bi-directionally controlled, being augmented by an interaction between ERα/ß and GPR54 signaling and suppressed by BMP-4 action in GT1-7 neuron cells.
Assuntos
Proteína Morfogenética Óssea 4/fisiologia , Estrogênios/fisiologia , Kisspeptinas/fisiologia , Receptores LHRH/metabolismo , Animais , Linhagem Celular , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Expressão Gênica , Hormônio Liberador de Gonadotropina/metabolismo , Hipocampo/citologia , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Kisspeptina-1 , Receptores LHRH/genéticaRESUMO
Genetic studies in human patients with idiopathic hypogonadotropic hypogonadism (IHH) identified mutations in the genes that encode neurokinin B (NKB) and the neurokinin 3 receptor (NK3R). However, determining the mechanism whereby NKB regulates gonadotropin secretion has been difficult because of conflicting results from in vivo studies investigating the luteinizing hormone (LH) response to senktide, a NK3R agonist. NK3R is expressed in a subset of GnRH neurons and in kisspeptin neurons that are known to regulate GnRH secretion. Thus, one potential source of inconsistency is that NKB could produce opposing direct and indirect effects on GnRH secretion. Here, we employ the GT1-7 cell model to elucidate the direct effects of NKB on GnRH neuron function. We find that GT1-7 cells express NK3R and respond to acute senktide treatment with c-Fos induction and increased GnRH secretion. In contrast, long-term senktide treatment decreased GnRH secretion. Next, we focus on the examination of the mechanism underlying the long-term decrease in secretion and determine that senktide treatment represses transcription of GnRH. We further show that this repression of GnRH transcription may involve enhanced c-Fos protein binding at novel activator protein-1 (AP-1) half-sites identified in enhancer 1 and the promoter, as well as chromatin remodeling at the promoter of the GnRH gene. These data indicate that NKB could directly regulate secretion from NK3R-expressing GnRH neurons. Furthermore, whether the response is inhibitory or stimulatory toward GnRH secretion could depend on the history or length of exposure to NKB because of a repressive effect on GnRH transcription.
Assuntos
Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/metabolismo , Neurocinina B/metabolismo , Neurônios/metabolismo , Transcrição Gênica , Animais , Pareamento de Bases/genética , Sítios de Ligação , Linhagem Celular , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/genética , Elementos Facilitadores Genéticos/genética , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Receptores da Neurocinina-3/metabolismo , Deleção de Sequência/genética , Substância P/análogos & derivados , Substância P/farmacologia , Fatores de Tempo , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
In addition to its role in reproduction, estradiol-17ß is critical to the regulation of energy balance and body weight. Estrogen receptor α-null (Erα-/-) mutant mice develop an obese state characterized by decreased energy expenditure, decreased locomotion, increased adiposity, altered glucose homeostasis, and hyperleptinemia. Such features are reminiscent of the propensity of postmenopausal women to develop obesity and type 2 diabetes. The mechanisms by which ERα signaling maintains normal energy balance, however, have remained unclear. Here we used knockin mice that express mutant ERα that can only signal through the noncanonical pathway to assess the role of nonclassical ERα signaling in energy homeostasis. In these mice, we found that nonclassical ERα signaling restored metabolic parameters dysregulated in Erα-/- mutant mice to normal or near-normal values. The rescue of body weight and metabolic function by nonclassical ERα signaling was mediated by normalization of energy expenditure, including voluntary locomotor activity. These findings indicate that nonclassical ERα signaling mediates major effects of estradiol-17ß on energy balance, raising the possibility that selective ERα agonists may be developed to reduce the risks of obesity and metabolic disturbances in postmenopausal women.
Assuntos
Metabolismo Energético/fisiologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Transdução de Sinais/fisiologia , Animais , Peso Corporal/genética , Ingestão de Alimentos , Feminino , Homeostase , Humanos , Leptina/metabolismo , Camundongos , Camundongos Knockout , Camundongos Obesos , Atividade Motora , Obesidade/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
Aromatase (CYP19A1) catalyzes the conversion of C(19) steroids to estrogens. Aromatase and its product estradiol (E(2)) are crucial for the sexually dimorphic development of the fetal brain and the regulation of gonadotropin secretion and sexual interest in adults. The regulation of aromatase expression in the brain is not well understood. The aromatase (Cyp19a1) gene is selectively expressed in distinct neurons of the hypothalamus through a distal brain-specific promoter I.f located approximately 36 kb upstream of the coding region. Here, we investigated a short feedback effect of E(2) on aromatase mRNA expression and enzyme activity using estrogen receptor alpha (ESR1; also known as ER alpha)-positive or ESR1-negative mouse embryonic hypothalamic neuronal cell lines that express aromatase via promoter I.f. Estradiol regulated aromatase mRNA expression and enzyme activity in a time- and dose-dependent manner, whereas an E(2) antagonist reversed these effects. The nucleotide -200/-1 region of promoter I.f conferred E(2) responsiveness. Two activator protein 1 (AP-1) elements in this region were essential for induction of promoter activity by E(2). ESR1 and JUN (c-Jun) bound to these AP-1 motifs in intact cells and under cell-free conditions. The addition of an ESR1 mutant that interacts with JUN but not directly with DNA enhanced E(2)-dependent promoter I.f activity. Independently, we demonstrated an interaction between ESR1 and JUN in hypothalamic cells. Knockdown of ESR1 abolished E(2)-induced aromatase mRNA and enzyme activity. Taken together, E(2) regulates Cyp19a1 expression via promoter I.f by enhanced binding of an ESR1/JUN complex to distinct AP-1 motifs in hypothalamic cells. We speculate that this mechanism may, in part, regulate gonadotropin secretion and sexual activity.
Assuntos
Aromatase/metabolismo , Receptor alfa de Estrogênio/metabolismo , Hipotálamo/metabolismo , Neurônios/metabolismo , Regiões Promotoras Genéticas/fisiologia , Análise de Variância , Animais , Aromatase/genética , Western Blotting , Linhagem Celular , Células Cultivadas , Imunoprecipitação da Cromatina , Relação Dose-Resposta a Droga , Ensaio de Desvio de Mobilidade Eletroforética , Ensaios Enzimáticos , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Estrogênios/farmacologia , Fulvestranto , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , TransfecçãoRESUMO
Kisspeptin is a product of the Kiss1 gene and is expressed in the forebrain. Neurons that express Kiss1 play a crucial role in the regulation of pituitary luteinizing hormone secretion and reproduction. These neurons are the direct targets for the action of estradiol-17beta (E(2)), which acts via the estrogen receptor alpha isoform (ER alpha) to regulate Kiss1 expression. In the arcuate nucleus (Arc), where the dynorphin gene (Dyn) is expressed in Kiss1 neurons, E(2) inhibits the expression of Kiss1 mRNA. However, E(2) induces the expression of Kiss1 in the anteroventral periventricular nucleus (AVPV). The mechanism for differential regulation of Kiss1 in the Arc and AVPV by E(2) is unknown. ER alpha signals through multiple pathways, which can be categorized as either classical, involving the estrogen response element (ERE), or nonclassical, involving ERE-independent mechanisms. To elucidate the molecular basis for the action of E(2) on Kiss1 and Dyn expression, we studied the effects of E(2) on Kiss1 and Dyn mRNAs in the brains of mice bearing targeted alterations in the ER alpha signaling pathways. We found that stimulation of Kiss1 expression by E(2) in the AVPV and inhibition of Dyn in the Arc required an ERE-dependent pathway, whereas the inhibition of Kiss1 expression by E(2) in the Arc involved ERE-independent mechanisms. Thus, distinct ER alpha signaling pathways can differentially regulate the expression of identical genes across different brain regions, and E(2) can act within the same neuron through divergent ER alpha signaling pathways to regulate different neurotransmitter genes.
Assuntos
Encéfalo/efeitos dos fármacos , Dinorfinas/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Proteínas/metabolismo , Animais , Núcleos Anteriores do Tálamo/efeitos dos fármacos , Núcleos Anteriores do Tálamo/metabolismo , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Núcleo Arqueado do Hipotálamo/metabolismo , Encéfalo/metabolismo , Dinorfinas/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Introdução de Genes , Kisspeptinas , Hormônio Luteinizante/sangue , Camundongos , Camundongos Transgênicos , Núcleos da Linha Média do Tálamo/efeitos dos fármacos , Núcleos da Linha Média do Tálamo/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas/genética , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
Nonclassical estrogen receptor alpha (ERalpha) signaling can mediate E(2) negative feedback actions in the reproductive axis; however, downstream pathways conveying these effects remain unclear. These studies tested the hypothesis that p21-activated kinase 1 (PAK1), a serine/threonine kinase rapidly activated by E(2) in nonneural cells, functions as a downstream node for E(2) signaling pathways in cells of the preoptic area, and it may thereby mediate E(2) negative feedback effects. Treatment of ovariectomized (OVX) rats with estradiol benzoate (EB) caused rapid and transient induction of phosphorylated PAK1 immunoreactivity in the medial preoptic nucleus (MPN) but not the arcuate nucleus. To determine whether rapid induction of PAK phosphorylation by E(2) is mediated by nonclassical [estrogen response element (ERE)-independent] ERalpha signaling, we used female ERalpha null (ERalpha(-/-)) mice possessing an ER knock-in mutation (E207A/G208A; AA), in which the mutant ERalpha is incapable of binding DNA and can signal only through membrane-initiated or ERE-independent genotropic pathways (ERalpha(-/AA) mice). After 1-h EB treatment, the number of pPAK1-immunoreactive cells in the MPN was increased in both wild-type (ERalpha(+/+)) and ERalpha(-/AA) mice but was unchanged in ERalpha(-/-) mice. Serum luteinizing hormone (LH) was likewise suppressed within 1 h after EB treatment in ERalpha(+/+) and ERalpha(-/AA) but not ERalpha(-/ -) mice. In OVX rats, 5-min intracerebroventricular infusion of a PAK inhibitor peptide but not control peptide blocked rapid EB suppression of LH secretion. Taken together, our findings implicate PAK1 activation subsequent to nonclassical ERalpha signaling as an important component of the negative feedback actions of E(2) in the brain.
Assuntos
Estradiol/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Receptor alfa de Estrogênio/deficiência , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Hormônio Luteinizante/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Ratos , Transdução de Sinais , Fatores de Tempo , Quinases Ativadas por p21/genéticaRESUMO
The estrogen receptor-alpha (ERalpha) acts through multiple pathways, including estrogen response element (ERE)-dependent (classical) and ERE-independent (nonclassical) mechanisms. We previously created a mouse model harboring a two-amino-acid mutation of the DNA-binding domain (E207A, G208A) that precludes direct binding of ERalpha to an ERE. After crossing heterozygous mutant mice with an ERalpha knockout (ERKO) line, it was possible to assess the degree of physiological rescue by the isolated ERalpha nonclassical allele (-/AA; AA) when compared with ERKO mice (-/-) and to wild type (+/+; WT). In male ERKO mice up to 8 months of age, testosterone levels were high, although LH levels were similar to WT. Testosterone was normal in the AA mice, indicating that the AA allele rescues the enhanced testosterone biosynthesis in ERKO mice. Male ERKO mice exhibited distention of the seminiferous tubules as early as 2-3 months of age as a consequence of decreased water resorption in the efferent ducts. By 3-4 months of age, ERKO mice had impaired spermatogenesis in approximately 40% of their tubules, and sperm counts and motility declined in association with the histological changes. In the AA mice, histological defects were greatly reduced or absent, and sperm counts and motility were rescued. Levels of aquaporins 1 and 9, which contribute to water uptake in the efferent ducts, were reduced in ERKO mice and partially or fully rescued in AA mice, whereas another water transporter, sodium-hydrogen exchanger-3, was decreased in both ERKO and AA mice. We conclude that non-ERE-dependent estrogen pathways are sufficient to rescue the defective spermatogenesis observed in ERKO mice and play a prominent role in ERalpha action in the testis, including pathways that regulate water resorption and androgen biosynthesis.
Assuntos
Receptor alfa de Estrogênio/fisiologia , Estrogênios/farmacologia , Elementos de Resposta/genética , Túbulos Seminíferos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting , Receptor alfa de Estrogênio/genética , Hormônio Foliculoestimulante/sangue , Imuno-Histoquímica , Hormônio Luteinizante/sangue , Masculino , Camundongos , Camundongos Knockout , Mutação , Túbulos Seminíferos/citologia , Túbulos Seminíferos/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Contagem de Espermatozoides , Motilidade dos Espermatozoides , Espermatogênese/efeitos dos fármacos , Testosterona/sangueRESUMO
During the female reproductive cycle, the neuroendocrine action of estradiol switches from negative feedback to positive feedback to initiate the preovulatory GnRH and subsequent LH surges. Estrogen receptor-alpha (ERalpha) is required for both estradiol negative and positive feedback regulation of LH. ERalpha may signal through estrogen response elements (EREs) in DNA and/or via ERE-independent pathways. Previously, a knock-in mutant allele (ERalpha-/AA) that selectively restores ERE-independent signaling onto the ERalpha-/- background was shown to confer partial negative but not positive estradiol feedback on serum LH. The current study investigated the roles of the ERE-dependent and ERE-independent ERalpha pathways for estradiol feedback at the level of GnRH neuron firing activity. The above ERalpha genetic models were crossed with GnRH-green fluorescent protein mice to enable identification of GnRH neurons in brain slices. Targeted extracellular recordings were used to monitor GnRH neuron firing activity using an ovariectomized, estradiol-treated mouse model that exhibits diurnal switches between negative and positive feedback. In wild-type mice, GnRH neuron firing decreased in response to estradiol during negative feedback and increased during positive feedback. In contrast, both positive and negative responses to estradiol were absent in GnRH neurons from ERalpha-/- and ERalpha-/AA mice. ERE-dependent signaling is thus required to increase GnRH neuron firing to generate a GnRH/LH surge. Furthermore, ERE-dependent and -independent ERalpha signaling pathways both appear necessary to mediate estradiol negative feedback on serum LH levels, suggesting central and pituitary estradiol feedback may use different combinations of ERalpha signaling pathways.
Assuntos
Receptor alfa de Estrogênio/fisiologia , Retroalimentação Fisiológica/genética , Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/fisiologia , Transmissão Sináptica/genética , Animais , Eletrofisiologia , Estradiol/sangue , Estradiol/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Retroalimentação Fisiológica/efeitos dos fármacos , Feminino , Hormônio Luteinizante/sangue , Hormônio Luteinizante/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Ovariectomia , Hipófise/metabolismo , Hipófise/fisiologia , Elementos de Resposta/efeitos dos fármacos , Elementos de Resposta/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Estrogen receptor alpha (ERalpha) mediates estrogen (E2) actions in the brain and is critical for normal reproductive function and behavior. In the classical pathway, ERalpha binds to estrogen response elements (EREs) to regulate gene transcription. ERalpha can also participate in several non-classical pathways, including ERE-independent gene transcription via protein-protein interactions with transcription factors and rapid, non-genotropic pathways. To distinguish between ERE-dependent and ERE-independent mechanisms of E2 action in vivo, we have created ERalpha null mice that possess an ER knock-in mutation (E207A/G208A; "AA"), in which the mutant ERalpha cannot bind to DNA but retains activity in ERE-independent pathways (ERalpha(-/AA) mice). Understanding the molecular mechanisms of ERalpha action will be helpful in developing pharmacological therapies that differentiate between ERE-dependent and ERE-independent processes. This review focuses on how the ERalpha(-/AA) model has contributed to our knowledge of ERalpha signaling mechanisms in estrogen regulation of the reproductive axis and sexual behavior.
Assuntos
Estrogênios/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Estrogênios/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Camundongos , Camundongos Knockout , Reprodução/efeitos dos fármacos , Comportamento Sexual Animal/efeitos dos fármacosRESUMO
Estrogen receptor (ER)-alpha mediates estradiol (E(2)) actions in the male gonads and brain and is critical for normal male reproductive function. In the classical pathway, ERalpha binds to estrogen response elements (EREs) to regulate gene transcription. ERalpha can also regulate gene transcription independently of EREs via protein-protein interactions with transcription factors and additionally signal via rapid, nongenomic pathways originating at the cell membrane. This study assessed the degree to which ERE-independent ERalpha signaling can rescue the disrupted masculine sexual behaviors and elevated serum testosterone (T) levels that have been shown to result from ERalpha gene deletion. We utilized male ERalpha null mice that possess a ER knock-in mutation (E207A/G208A; AA), in which the mutant ERalpha is incapable of binding to DNA and can signal only through ERE-independent pathways (ERalpha(-/AA) mice). We found that sexual behavior, including mounting, is virtually absent in ERalpha(-/-) and ERalpha(-/AA) males, suggesting that ERE-independent signaling is insufficient to maintain any degree of normal sexual behavior in the absence of ERE binding. By contrast, ERE-independent signaling in the ERalpha(-/AA) mouse is sufficient to restore serum T levels to values observed in wild-type males. These data indicate that binding of ERs to EREs mediates most if not all of E(2)'s effects on male sexual behavior, whereas ERE-independent ERalpha signaling may mediate E(2)'s inhibitory effects on T production.
Assuntos
Estradiol/fisiologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/fisiologia , Elementos de Resposta/fisiologia , Comportamento Sexual Animal/fisiologia , Testosterona/metabolismo , Animais , Feminino , Hormônio Foliculoestimulante/sangue , Hormônio Luteinizante/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores Sexuais , Transdução de Sinais/fisiologia , Testosterona/sangueRESUMO
Estrogens are generated mainly by the action of aromatase, which converts testosterone to estradiol and androstenedione to estrone. However, in addition to estradiol and estrone, a variety of other steroids, whose synthesis is not dependent on aromatase, can stimulate the estrogen receptor. Here we show that testosterone is converted into such estrogenic steroids by aromatase-negative HeLa cells. This aromatase-independent generation of estrogenic steroids is seen in aromatase-positive MCF-7 cells as well. In both cell lines, the synthesis of estrogenic steroids was blocked by inhibition of testosterone conversion into dihydrotestosterone using a 5 alpha-reductase inhibitor finasteride, suggesting that they are generated downstream of dihydrotestosterone. This finding raises the possibility that the combination of a 5 alpha-reductase inhibitor and an aromatase inhibitor may reduce estrogenic steroids in vivo more completely than an aromatase inhibitor alone.
Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/farmacologia , Aromatase/metabolismo , Estrogênios/metabolismo , Esteroides/metabolismo , Testosterona/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Aromatase/farmacologia , Feminino , Genes Reporter , Células HeLa , Humanos , Modelos Biológicos , Testosterona/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
The classical pathway for estrogen receptor (ER) signaling is mediated by ER binding to an estrogen response element (ERE) in DNA. ERalpha can also act via a nonclassical pathway by altering the activities of other transcription factors (e.g., Sp1, AP-1, or NF-kappaB) at their cognate sites on DNA. We previously generated a mutant form of ERalpha (E207A/G208A) that does not bind to EREs, and therefore lacks signaling via the classical pathway but retains signaling via the nonclassical pathway. In the current study, we introduce this mutant ERalpha into MDA-MB231 ERalpha-negative breast carcinoma cells to identify nonclassical pathway genes that respond to 17beta-estradiol (E2), selective estrogen receptor modulators (SERMs) tamoxifen (TAM) or raloxifene (RAL), or the estrogen antagonist ICI 182,780 (ICI). Consistent with a role for nonclassical signaling in SERM action, microarray analyses identify 268 responsive nonclassical ERalpha pathway target genes. ICI elicits the largest number of nonclassical genes, followed by RAL, TAM, and E2. Custom microarrays containing identified nonclassical ERalpha responsive genes are used to compare gene expression in human breast tumor (n = 34) and normal mammary epithelial cell (n = 9) samples. A subset of nonclassical genes (n = 32) are differentially expressed in breast tumors. In summary, we show that nonclassical ERalpha pathway target genes exhibit a range of transcriptional responses to SERMs and identify targets of this pathway as potentially relevant to breast cancer. The identification of nonclassical ERalpha target genes offers new insight into estrogen receptor signaling and cross talk with pathways that mediate breast tumor response to SERM therapy.