Asunto(s)
Endocrinología/tendencias , Biología Molecular/tendencias , Publicaciones Periódicas como Asunto/historia , Publicaciones Periódicas como Asunto/tendencias , Congresos como Asunto/historia , Endocrinología/historia , Historia del Siglo XX , Historia del Siglo XXI , Biología Molecular/historia , Sociedades Científicas/historiaRESUMEN
Gonadotropin-releasing hormone (GnRH) and activin regulate synthesis of FSH and ultimately fertility. Recent in vivo studies cast SMAD4 and FOXL2 as master transcriptional mediators of activin signaling that act together and independently of GnRH to regulate Fshb gene expression and female fertility. Ovarian hormones regulate GnRH and its receptor (GNRHR) through negative and positive feedback loops. In contrast, the role of ovarian hormones in regulating activin, activin receptors, and components of the activin signaling pathway, including SMAD4 and FOXL2, remains understudied. The widespread distribution of activin and many of its signaling intermediates complicates analysis of the effects of ovarian hormones on their synthesis in gonadotropes, one of five pituitary cell types. We circumvented this complication by using a transgenic model that allows isolation of polyribosomes selectively from gonadotropes of intact females and ovariectomized females treated with or without a GnRH antagonist. This paradigm allows assessment of ovarian hormonal feedback and distinguishes responses that are either independent or dependent on GnRH. Surprisingly, our results indicate that Foxl2 levels in gonadotropes decline significantly in the absence of ovarian input and independently of GnRH. Expression of the genes encoding other members of the activin signaling pathway are unaffected by loss of ovarian hormonal feedback, highlighting their selective effect on Foxl2. Expression of Gnrhr, a known target of FOXL2, also declines upon ovariectomy consistent with reduced expression of Foxl2 and loss of ovarian hormones. In contrast, Fshb mRNA increases dramatically post-ovariectomy due to increased compensatory input from GnRH. Together these data suggest that ovarian hormones regulate expression of Foxl2 thereby expanding the number of genes controlled by the hypothalamic-pituitary-gonadal axis that ultimately dictate reproductive fitness.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Hormonas Gonadales/metabolismo , Gonadotrofos/metabolismo , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Animales , Retroalimentación Fisiológica , Femenino , Proteína Forkhead Box L2 , Factores de Transcripción Forkhead/genética , Ratones , Ovariectomía , Ovario/metabolismoRESUMEN
Ovarian follicles lacking FSH or FSH receptors fail to progress to a preovulatory stage, resulting in infertility. One hallmark of the preovulatory follicle is the presence of luteinizing hormone/choriogonadotropin receptors (LHCGR) on granulosa cells (GCs). However, the mechanisms by which FSH induces Lhcgr gene expression are poorly understood. Our results show that protein kinase A (PKA) and phosphoinositide 3-kinase (PI3K)/AKT pathways are required for FSH to activate both the murine Lhcgr-luciferase reporter and expression of Lhcgr mRNA in rat GCs. Based on results showing that an adenovirus (Ad) expressing a steroidogenic factor 1 (SF1) mutant that cannot bind ß-catenin abolished FSH-induced Lhcgr mRNA, we evaluated the role of ß-catenin in the regulation of Lhcgr gene expression. FSH promoted the PKA-dependent, PI3K-independent phosphorylation of ß-catenin on Ser552 and Ser665. FSH activated the ß-catenin/T-cell factor (TCF) artificial promoter-reporter TOPFlash via a PKA-dependent, PI3K-independent pathway, and dominant-negative (DN) TCF abolished FSH-activated Lhcgr-luciferase reporter and induction of Lhcgr mRNA. Microarray analysis of GCs treated with Ad-DN-TCF and FSH identified the Lhcgr as the most down-regulated gene. Chromatin immunoprecipitation results placed ß-catenin phosphorylated on Ser552 and Ser675 and SF1 on the Lhcgr promoter in FSH-treated GCs; TCF3 was constitutively associated with the Lhcgr promoter. Transduction with an Ad-phospho-ß-catenin mutant (Ser552/665/Asp) enhanced Lhcgr mRNA expression in FSH-treated cells greater than 3-fold. Finally, we identified a recognized PI3K/AKT target, forkhead box O1, as a negative regulator of Lhcgr mRNA expression. These results provide new understanding of the complex regulation of Lhcgr gene expression in GCs.
Asunto(s)
Células de la Granulosa/metabolismo , Folículo Ovárico/metabolismo , Receptores de Gonadotropina/metabolismo , Receptores de HL/metabolismo , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación hacia Abajo , Femenino , Hormona Folículo Estimulante/metabolismo , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores de Gonadotropina/biosíntesis , Receptores de Gonadotropina/genética , Receptores de HL/biosíntesis , Receptores de HL/genética , Transducción de Señal , Factor Esteroidogénico 1/genética , Factor Esteroidogénico 1/metabolismo , Proteína 1 Similar al Factor de Transcripción 7/metabolismo , Transfección , beta Catenina/metabolismoRESUMEN
GnRH binds to its receptor on gonadotropes and activates multiple members of the MAPK signaling family that in turn regulates the expression of several immediate early genes (IEGs) including Jun, Fos, Atf3, and Egr1. These IEGs confer hormonal responsiveness to gonadotrope-specific genes including Gnrhr, Cga, Fshb, and Lhb. In this study we tested the hypothesis that GnRH specifically regulates the accumulation of Jun and Atf3 mRNA through a pathway that includes intracellular Ca²âº, calcineurin, and nuclear factor of activated T cells (NFAT). Our results indicate that pretreatment of murine LßT2 cells with 1, 2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)-ester, a Ca²âº chelator, reduced the expression of all the IEGs to varying degrees, whereas treatment with thapsigargin, an intracellular Ca²âº protein pump inhibitor, increased the expression of the IEG. Furthermore, cyclosporin A, a calcineurin-specific inhibitor, reduced the ability of GnRH to regulate accumulation of Jun and Atf3 mRNA and to a lesser extent Fos. In contrast, Egr1 mRNA was unaffected. NFATs are transcription factors regulated by calcineurin and were detected in LßT2 cells. GnRH increased luciferase activity of an NFAT-dependent promoter reporter that was dependent on intracellular Ca²âº and calcineurin activity. Additionally, although small interfering RNA specific for Nfat4 only marginally reduced GnRH regulation of Jun, Fos, and Atf3 mRNA accumulation, activity of an activator protein-1-responsive reporter construct was reduced by 48%. Together these data suggest that calcineurin and NFAT are new members of the gonadotrope transcriptional network that confer hormonal responsiveness to several key genes required for gonadotropin synthesis and secretion.
Asunto(s)
Factor de Transcripción Activador 3/metabolismo , Calcineurina/metabolismo , Señalización del Calcio , Gonadotrofos/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Factores de Transcripción NFATC/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción Activador 3/antagonistas & inhibidores , Factor de Transcripción Activador 3/genética , Animales , Inhibidores de la Calcineurina , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Gonadotrofos/efectos de los fármacos , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Ratones , Factores de Transcripción NFATC/antagonistas & inhibidores , Factores de Transcripción NFATC/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Gonadotropin-releasing hormone (GnRH), a hypothalamic neurohormone, regulates transcription of Lhb in gonadotrophs indirectly through transient induction and accumulation of EGR1, a zinc finger transcription factor. AlphaT3 and LbetaT2 cell lines model gonadotrophs at two distinct stages of development, prenatal and postnatal expression of Lhb. Although GnRH induces EGR1 in both cell lines, the levels of the DNA-binding protein are lower and disappear more quickly in alphaT3 than in LbetaT2 cells. Herein we show that overexpression of Egr1 in alphaT3 cells rescues activity of a transfected LHB promoter-reporter, suggesting that its transcription is dependent on EGR1 crossing a critical concentration threshold. We also show that Csda, a gene that encodes an RNA-binding protein and is a member of the cold-shock-domain (CSD) family, is expressed at higher levels in LbetaT2 compared to alphaT3 cells. Transient expression studies indicate that at least one Csd element, residing in the 3' untranslated region of Egr1 mRNA, increases activity of a chimeric pGL3 luciferase reporter vector in LbetaT2 cells. Additional experiments indicate that CSDA physically interacts with Egr1 mRNA. Furthermore, siRNA-mediated reduction of endogenous Csda mRNA attenuates GnRH regulation of a transiently transfected LHB reporter vector. Taken together, these studies suggest that CSDA contributes posttranscriptionally to GnRH-regulated expression of Egr1, thereby enabling the transcription factor to cross a critical concentration threshold necessary for maximal accumulation of Lhb mRNA in response to the neurohormone.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Proteínas de Choque Térmico/metabolismo , Hormona Luteinizante de Subunidad beta/metabolismo , Adenohipófisis/metabolismo , Animales , Línea Celular , Humanos , Ratones , Adenohipófisis/citología , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Factores de Transcripción , Transcripción GenéticaRESUMEN
Follicle-stimulating hormone (FSH) regulation of aromatase gene expression in vitro requires the transcriptional coactivator beta-catenin. To ascertain the physiological significance of beta-catenin in granulosa cells during folliculogenesis, mice homozygous for floxed alleles of beta-catenin were intercrossed with Amhr2cre mice. Conditional deletion of beta-catenin in 8-wk-old females occurred in derivatives of the Müllerian duct, granulosa cells and, surprisingly, in brain, pituitary, heart, liver, and tail. Female mice deficient for beta-catenin were infertile, despite reaching puberty and ovulating at the expected age, indications of apparently normal ovarian function. In contrast, their oviducts were grossly distended, with fewer but healthy oocytes. In addition, their uteri lacked implantation sites. Together, these two phenotypes could explain the complete loss of fertility. Nevertheless, although the ovary appeared normal, with serum estradiol concentrations in the normal range, there was marked animal-to-animal variation of mRNAs encoding beta-catenin and aromatase. Similarly, inhibin-alpha and luteinizing hormone receptor mRNAs varied considerably in whole ovaries, whereas pituitary Fshb mRNA was significantly reduced. Collectively, these features suggested cyclization recombination (CRE)-mediated recombination of beta-catenin may be unstable in proliferating granulosa cells, and therefore may mask the suspected steroidogenic requirement for beta-catenin. We tested this possibility by transducing primary cultures of granulosa cells from mice homozygous for floxed alleles of beta-catenin with a CRE-expressing adenovirus. Reduction of beta-catenin significantly compromised FSH stimulation of aromatase mRNA and subsequent production of estradiol. Collectively, these data suggest that FSH regulation of steroidogenesis requires beta-catenin, a role that remains hidden when tested through Amhr2cre-mediated recombination in vivo.
Asunto(s)
Trompas Uterinas/crecimiento & desarrollo , Infertilidad Femenina/metabolismo , Receptores de Péptidos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Útero/crecimiento & desarrollo , beta Catenina/metabolismo , Animales , Aromatasa/metabolismo , Proliferación Celular , Células Cultivadas , AMP Cíclico/metabolismo , Femenino , Hormona Folículo Estimulante/metabolismo , Hormonas Esteroides Gonadales/biosíntesis , Integrasas , Ratones , Ratones Transgénicos , Conductos Paramesonéfricos/crecimiento & desarrollo , Ovario/fisiología , Receptores de Péptidos/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Recombinación Genética , Elementos de Respuesta , beta Catenina/genéticaRESUMEN
GnRH regulates gonadotrope function through a complex transcriptional network that includes three members of the immediate early gene family: Egr1, Jun, and Atf3. These DNA-binding proteins act alone or in pairs to confer hormonal responsiveness to Cga, Lhb, Fshb, and Gnrhr. Herein we suggest that the transcriptional response of Jun requires a functional interaction between the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of DNA-binding proteins and beta-catenin (officially CTNNB1), a coactivator of TCF/LEF. Supporting data include demonstration that GnRH increases activity of TOPflash, a TCF/LEF-dependent luciferase reporter, in LbetaT2 cells, a gonadotrope-derived cell line. Additional cotransfection experiments indicate that a dominant-negative form of TCF7L2 (TCFDN) that binds DNA, but not beta-catenin, blocks GnRH induction of TOPflash. Overexpression of AXIN, an inhibitor of beta-catenin, also reduces GnRH stimulation of TOPflash. Transduction of LbetaT2 cells with TCFDN adenoviruses diminishes GnRH stimulation of Jun mRNA without altering expression of Egr1 and Atf3, two other immediate early genes that confer GnRH responsiveness. Reduction of beta-catenin in LbetaT2 cells, through stable expression of short hairpin RNA, also selectively compromises GnRH regulation of Jun expression and levels of JUN protein. Finally, overexpression of TCFDN attenuates GnRH regulation of Cga promoter activity, a known downstream target of JUN. Together, these results indicate that GnRH regulation of Jun transcription requires a functional interaction between TCF/LEF and beta-catenin and that alteration of either impacts expression of JUN downstream targets such as Cga.
Asunto(s)
Redes Reguladoras de Genes/efectos de los fármacos , Genes jun/efectos de los fármacos , Gonadotrofos/efectos de los fármacos , Hormona Liberadora de Gonadotropina/farmacología , Factores de Transcripción TCF/metabolismo , beta Catenina/metabolismo , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Genes Dominantes , Genes jun/fisiología , Hormonas Glicoproteicas de Subunidad alfa/genética , Gonadotrofos/metabolismo , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Factores de Transcripción TCF/genética , Factores de Transcripción TCF/fisiología , Transfección , beta Catenina/fisiologíaRESUMEN
GnRH binds its G-coupled protein receptor, GnRHR, on pituitary gonadotropes and stimulates transcription of Cga, Lhb, and Fshb. These three genes encode two heterodimeric glycoprotein hormones, LH and FSH, that act as gonadotropins by regulating gametogenesis and steroidogenesis in both the testes and ovary. GnRH also regulates transcription of Gnrhr. Thus, regulated expression of Cga, Lhb, Fshb, and Gnrhr provides a genomic signature unique to functional gonadotropes. Steadily increasing evidence now indicates that GnRH regulates transcription of its four signature genes indirectly through a hierarchical transcriptional network that includes distinct subclasses of DNA-binding proteins that comprise the immediate early gene (IEG) family. These IEGs, in turn, confer hormonal responsiveness to the four signature genes. Although the IEGs confer responsiveness to GnRH, they cannot act alone. Instead, additional DNA-binding proteins, including the orphan nuclear receptor steroidogenic factor 1, act permissively to allow the four signature genes to respond to GnRH-induced changes in IEG levels. Emerging new findings now indicate that beta-catenin, a transcriptional coactivator and member of the canonical WNT signaling pathway, also plays an essential role in transducing the GnRH signal by interacting with multiple DNA-binding proteins in gonadotropes. Herein we propose that these interactions with beta-catenin define a multicomponent transcriptional network required for regulated expression of the four signature genes of the gonadotrope, Cga, Lhb, Fshb, and Gnrhr.
Asunto(s)
Redes Reguladoras de Genes , Gonadotrofos/metabolismo , Hormona Liberadora de Gonadotropina/genética , beta Catenina/genética , Animales , Regulación de la Expresión Génica , Hormona Liberadora de Gonadotropina/metabolismo , Humanos , Modelos Biológicos , Transducción de Señal , Factor Esteroidogénico 1/fisiología , Factores de Transcripción TCF/fisiología , beta Catenina/metabolismo , beta Catenina/fisiologíaRESUMEN
GnRH regulates expression of LHB via transcriptional regulation of early growth response 1 (EGR1), an immediate early gene that encodes a zinc-finger DNA-binding protein. EGR1 interacts functionally with the orphan nuclear receptor steroidogenic factor 1 (SF1) and pituitary homeobox 1, a member of the paired-like homeodomain family. The functional synergism of this tripartite interaction defines the maximal level of LHB transcription that can occur in response to GnRH. Results presented herein provide new evidence that the interaction between SF1 and EGR1 also requires beta-catenin, a transcriptional coactivator and member of the canonical Wnt signaling pathway. For instance, targeted reduction of beta-catenin attenuates activity of a GnRH-primed LHB promoter. Additional gene reporter assays indicate that overexpression of beta-catenin, or its targeted reduction by small interfering RNA, modulates activity of both SF1 and EGR1 as well as their functional interaction. beta-Catenin coimmunoprecipitates with SF1. Moreover, an SF1 mutant that lacks a beta-catenin binding domain has compromised transcriptional activity and fails to interact synergistically with EGR1. Finally, GnRH promotes beta-catenin colocalization with SF1 and EGR1 on the endogenous mouse Lhb promoter-regulatory region. Taken together, these data suggest that beta-catenin binds to SF1 and that this interaction is required for subsequent functional interaction with EGR1. Thus, these data identify beta-catenin as a new and required member of the basal transcriptional complex that allows the LHB promoter to achieve maximal activity in response to GnRH.
Asunto(s)
Regulación de la Expresión Génica , Hormona Liberadora de Gonadotropina/fisiología , Proteínas de Homeodominio/metabolismo , Hormona Luteinizante de Subunidad beta/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , beta Catenina/metabolismo , Animales , Células Cultivadas , Proteína 1 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Genes Reporteros , Hormona Liberadora de Gonadotropina/farmacología , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Inmunoprecipitación , Ratones , Mutación , Factores de Transcripción Paired Box/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Estructura Terciaria de Proteína/genética , ARN Interferente Pequeño/farmacología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Factor Esteroidogénico 1 , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genéticaRESUMEN
Estrogens profoundly influence the physiology and pathology of reproductive and other tissues. Consequently, emphasis has been placed on delineating the mechanisms underlying regulation of estrogen levels. Circulating levels of estradiol in women are controlled by follicle-stimulating hormone (FSH), which regulates transcription of the aromatase gene (CYP19A1) in ovarian granulosa cells. Previous studies have focused on two downstream effectors of the FSH signal, cAMP and the orphan nuclear receptor steroidogenic factor-1 (NR5A1). In this report, we present evidence for beta-catenin (CTNNB1) as an essential transcriptional regulator of CYP19A1. FSH induction of select steroidogenic enzyme mRNAs, including Cyp19a1, is enhanced by beta-catenin. Additionally, beta-catenin is present in transcription complexes assembled on the endogenous gonad-specific CYP19A1 promoter, as evidenced by chromatin immunoprecipitation assays. Transient expression and RNAi studies demonstrate that FSH- and cAMP-dependent regulation of this promoter is sensitive to alterations in the level of beta-catenin. The stimulatory effect of beta-catenin is mediated through functional interactions with steroidogenic factor-1 that involve four acidic residues within its ligand-binding domain, mutation of which attenuates FSH/cAMP-induced Cyp19a1 mRNA accumulation. Together, these data demonstrate that beta-catenin is essential for FSH/cAMP-regulated gene expression in the ovary, identifying a central and previously unappreciated role for beta-catenin in estrogen biosynthesis, and a potential broader role in other aspects of follicular maturation.
Asunto(s)
Aromatasa/metabolismo , AMP Cíclico/metabolismo , Hormona Folículo Estimulante/metabolismo , Regulación Enzimológica de la Expresión Génica , beta Catenina/metabolismo , Animales , Aromatasa/genética , Células Cultivadas , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Estradiol/sangre , Femenino , Células de la Granulosa/citología , Células de la Granulosa/fisiología , Humanos , Ovario/citología , Ovario/fisiología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Factor Esteroidogénico 1RESUMEN
Normal reproductive function in mammals requires precise control of LH synthesis and secretion by gonadotropes of the anterior pituitary. Synthesis of LH requires expression of two genes [alpha-glycoprotein subunit (alphaGSU) and LHbeta] located on different chromosomes. Hormones from the hypothalamus and gonads modulate transcription of both genes as well as secretion of the biologically active LH heterodimer. In males and females, the transcriptional tone of the genes encoding alphaGSU and LHbeta reflects dynamic integration of a positive signal provided by GnRH from hypothalamic neurons and negative signals emanating from gonadal steroids. Although alphaGSU and LHbeta genes respond transcriptionally in the same manner to changes in hormonal input, different combinations of regulatory elements orchestrate their response. These hormone-responsive regulatory elements are also integral members of much larger combinatorial codes responsible for targeting expression of alphaGSU and LHbeta genes to gonadotropes. In this review, we will profile the genomic landscape of the promoter-regulatory region of both genes, depicting elements and factors that contribute to gonadotrope-specific expression and hormonal regulation. Within this context, we will highlight the different combinatorial codes that control transcriptional responses, particularly those that mediate the opposing effects of GnRH and one of the sex steroids, androgens. We will use this framework to suggest that GnRH and androgens attain the same transcriptional endpoint through combinatorial codes unique to alphaGSU and LHbeta. This parallelism permits the dynamic and coordinate regulation of two genes that encode a single hormone.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Código Genético , Hormona Luteinizante/genética , Regiones Promotoras Genéticas/fisiología , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Animales , Femenino , Humanos , Hormona Luteinizante/metabolismo , MasculinoRESUMEN
Stringent regulation of LH secretion from the pituitary is vital to ovarian function in mammals. Two rodent models of LH hypersecretion are the transgenic LHbeta-C-terminal peptide (LHbetaCTP) and estrogen receptor-alpha (ERalpha)-null (alphaERKO) mice. Both exhibit ovarian phenotypes of chronic anovulation, cystic and hemorrhagic follicles, lack of corpora lutea, interstitial/stromal hyperplasia, and elevated plasma estradiol and testosterone. Because ERbeta is highly expressed in granulosa cells of the ovary, we hypothesized the intraovarian actions of ERbeta may be necessary for full manifestation of phenotypes associated with LH hyperstimulation. To address this question, we generated female mice that possess elevated LH, but lack ERbeta, by breeding the LHbetaCTP and ERbeta-null (betaERKO) mice. A comparison of LHbetaCTP, alphaERKO, and betaERKO(LHCTP) females has allowed us to elucidate the contribution of each ER form to the pathologies and endocrinopathies that occur during chronic LH stimulation of the ovary. alphaERKO ovaries respond to elevated LH by exhibiting an amplified steroidogenic pathway characteristic of the follicular stage of the ovarian cycle, whereas wild-type(LHCTP) and betaERKO(LHCTP) females exhibit a steroidogenic profile more characteristic of the luteal stage. In addition, the hemorrhagic and cystic follicles of the LHbetaCTP and alphaERKO ovaries require the intraovarian actions of ERbeta for manifestation, because they were lacking in the betaERKO(LHCTP) ovary. In turn, ectopic expression of the Leydig cell-specific enzyme, Hsd17b3, and male-like testosterone synthesis in the alphaERKO ovary are unique to this genotype and are therefore the culmination of elevated LH and the loss of functional ERalpha within the ovary.
Asunto(s)
Hormona Luteinizante/metabolismo , Quistes Ováricos/etiología , Quistes Ováricos/metabolismo , Folículo Ovárico , Receptores de Estrógenos/metabolismo , Animales , Receptor beta de Estrógeno , Femenino , Expresión Génica , Hormonas/sangre , Hormona Luteinizante/sangre , Ratones , Ratones Noqueados , Ratones Transgénicos , Quistes Ováricos/sangre , Quistes Ováricos/patología , Folículo Ovárico/metabolismo , Folículo Ovárico/patología , Ovario/patología , Receptores de Estrógenos/deficiencia , Receptores de Estrógenos/genéticaRESUMEN
Targeted overexpression of LH in transgenic mice causes hyperproliferation of Pit-1-positive pituitary cells and development of functional adenomas. To characterize gene expression changes associated with pituitary tumorigenesis, we performed microarray studies using Affymetrix GeneChips comparing expression profiles from pituitary tumors in LH-overexpressing mice to wild-type control pituitaries. We identified a number of candidate genes with altered expression in pituitary tumors. One of these, p8 (candidate of metastasis-1), encodes a native high-mobility group-like transcription factor previously shown to be necessary for ras-mediated transformation of mouse embryonic fibroblasts and also implicated in breast cancer progression. Herein, we show that expression of p8, normally quiescent in adult pituitary, localizes to tumor foci containing lactotropes, suggesting a linkage with their transformation. To further establish the functional significance of p8 in pituitary tumorigenesis, we constructed several clonal cell lines with reduced expression of p8 from a parent GH3 somatolactotrope cell line. These clonal derivates, along with the parent cell line, were tested for tumorigenicity by injection into athymic mice. When compared with wild-type GH3 with higher levels of p8, GH3 cells with reduced expression of p8 displayed attenuated tumor development or failed to develop tumors at all. Similar results were obtained with gonadotrope-derived cell lines that displayed reduced expression of p8. Together, these data suggest that maintenance of the transformed phenotype of pituitary GH3 cells requires expression of p8 and that it may play a similar role when reexpressed in a subset of lactotropes that form prolactinomas in vivo.
Asunto(s)
Proteínas de Unión al ADN/genética , Hormona Luteinizante/metabolismo , Proteínas de Neoplasias/genética , Neoplasias Hipofisarias/genética , Prolactinoma/genética , Animales , Secuencia de Bases , Cartilla de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Plásmidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Transgenic mice with engineered disruptions in bidirectional endocrine signaling between the pituitary and gonad have shed light on the specific effects of the loss of function of gonadotropins and inhibins. These models are valuable tools for studying ovarian biology because they phenocopy specific pathological states and have variations in ovarian tissue composition that allow us to identify genes expressed in specific cell types. We have used emerging mRNA expression profiling technologies to gain a more comprehensive view of genes that are expressed in the mammalian ovary and adrenal gland in the FSHbeta and inhibin alpha knockout mouse models. Oligonucleotide array hybridization experiments using Affymetrix GeneChip technology and NIA 15K murine cDNA microarray studies identified hundreds of transcripts differentially expressed compared with wild type, over 30 of which were selected for further characterization by Northern blot analyses. Additionally, we performed in situ hybridization studies to localize 10 mRNAs, melanocyte-specific gene 1, amino acid transporter SN2, overexpressed and amplified in teratocarcinoma (Bcat1), Forkhead box protein FOXO1, 24p3, vascular cell adhesion molecule, epiregulin, Bcl2-like10, PC3B, and retinoblastoma binding protein 7. These 10 genes have expression patterns and postulated functions suggesting that they mediate important processes in the physiology and pathology of ovarian and adrenal tissue.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/etiología , Perfilación de la Expresión Génica , Gonadotropinas/metabolismo , Inhibinas/deficiencia , Proteínas Nucleares , Neoplasias Ováricas/etiología , Andrógenos/biosíntesis , Animales , Proteínas Portadoras/fisiología , Ciclo Celular , Proteínas de Ciclo Celular/fisiología , Femenino , Hormona Folículo Estimulante de Subunidad beta/deficiencia , Gonadotropinas Equinas/farmacología , Células de la Granulosa/metabolismo , Hormona Luteinizante de Subunidad beta/metabolismo , Masculino , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Oocitos/citología , Ovario/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , ARN Mensajero/metabolismo , Proteína 7 de Unión a Retinoblastoma , Células de Sertoli/metabolismo , Células Tecales/efectos de los fármacosRESUMEN
The majority of pituitary adenomas in humans are nonmetastasizing, monoclonal neoplasms that occur in approximately 20% of the general population. Their development has been linked to a combination of extrinsic factors and intrinsic defects. We now demonstrate with transgenic mice that targeted and chronic overexpression of LH causes ovarian hyperstimulation and subsequent hyperproliferation of Pit-1-positive cells that culminates in the appearance of functional pituitary adenomas ranging from focal to multifocal expansion of lactotropes, somatotropes, and thyrotropes. Tumors fail to develop in ovariectomized mice, indicating that contributions from the ovary are necessary for adenoma development. Although the link between chronic ovarian hyperstimulation and PRL-secreting adenomas was expected, the involvement of somatotropes and thyrotropes was surprising and suggests that multiple ovarian hormones may contribute to this unusual pathological consequence. In support of this idea, we have found that ovariectomy followed by estrogen replacement results in the expansion of lactotropes selectively in LH overexpressing mice, but not somatotropes and thyrotropes. Collectively, these data indicate that estrogen is sufficient for the formation of lactotrope adenomas only in animals with a hyperstimulated ovary, whereas the appearance of GH- and TSH-secreting adenomas depends on multiple ovarian hormones. Together, our data expand current models of pituitary tumorigenesis by suggesting that chronic ovarian hyperstimulation may underlie the formation of a subset of pituitary adenomas containing lactotropes, somatotropes, and thyrotropes.
Asunto(s)
Adenoma/etiología , Adenoma/fisiopatología , Proteínas de Unión al ADN/metabolismo , Hormona Luteinizante/metabolismo , Ovario/fisiopatología , Neoplasias Hipofisarias/etiología , Neoplasias Hipofisarias/fisiopatología , Factores de Transcripción/metabolismo , Adenoma/metabolismo , Adenoma/patología , Animales , División Celular , Estradiol/farmacología , Femenino , Hormona del Crecimiento/metabolismo , Hiperplasia , Hipertrofia , Ratones , Ratones Transgénicos , Ovariectomía , Fenotipo , Hipófisis/metabolismo , Hipófisis/patología , Neoplasias Hipofisarias/metabolismo , Neoplasias Hipofisarias/patología , Prolactina/metabolismo , Tirotropina/metabolismo , Factor de Transcripción Pit-1RESUMEN
Chronically elevated luteinizing hormone (LH) induces significant pathology in the LHbetaCTP transgenic mouse model, which uses the bovine gonadotropin alpha (alpha)-subunit promoter to direct transgene expression specifically to gonadotropes in the anterior pituitary. Previously, it was shown that female LHbetaCTP mice are infertile due to anovulation, develop granulosa cell tumors, and undergo precocious puberty from elevated LH and steroid hormones that fail to completely repress the alpha-subunit promoter. This chapter will discuss recent studies that further elucidate the impact of chronically elevated LH on diverse physiological systems. Granulosa cell tumors induced by elevated LH are strain dependent and prevented when transgenics are treated with human chorionic gonadotropin (hCG) surges. A granulosa cell tumor-associated transcriptome is generated, revealing several possible gene candidates for ovarian granulosa cell tumorigenesis. Primordial follicles in LHbetaCTP transgenics become depleted and oocytes exhibit increased rates of meiotic segregation defects, although meiotic competency is acquired normally. Anovulation can be rescued in transgenics by superovulation, though pregnancy fails at midgestation due to maternal factors. Uterine receptivity defects prevent implantation of normal embryos following induction of pseuodpregnancy. Transgenics develop Cushing-like adrenocortical hyperfunction with increased corticosterone production following induction of adrenal LH receptor expression. Elevated LH acts as a tumor promoter in the gonads and the adrenal gland, when expressed in conjunction with the inhibin-alpha SV40 transgene. Finally, chronic elevated LH promotes mammary tumorigenesis. The understanding of multiple clinical pathologies--including ovarian cancer, perimenopausal reproductive aging, premature ovarian failure, polycystic ovarian syndrome, Cushing's syndrome, and breast cancer--may be enhanced through further study of this useful transgenic mouse model.
Asunto(s)
Modelos Animales de Enfermedad , Hormona Luteinizante/metabolismo , Enfermedades del Ovario/etiología , Envejecimiento , Animales , Gonadotropina Coriónica Humana de Subunidad beta/genética , Síndrome de Cushing/etiología , Síndrome de Cushing/fisiopatología , Femenino , Hormona Luteinizante/genética , Hormona Luteinizante de Subunidad beta/genética , Ratones , Ratones Transgénicos , Enfermedades del Ovario/fisiopatología , Neoplasias Ováricas/etiología , Neoplasias Ováricas/fisiopatología , Ovario/fisiopatología , Fragmentos de Péptidos/genética , Síndrome del Ovario Poliquístico/etiología , Síndrome del Ovario Poliquístico/fisiopatología , Insuficiencia Ovárica Primaria/etiología , Insuficiencia Ovárica Primaria/fisiopatología , Proteínas Recombinantes de FusiónRESUMEN
Inhibin and activin are members of the transforming growth factor beta (TGF-beta) family of ligands produced and secreted primarily by the gonads and adrenals. Inhibin-null (INH(-/-)) mice develop gonadal tumors and-when gonadectomized-adrenocortical carcinoma. The mechanisms leading to adrenal tumorigenesis have been proposed to involve the lack of a gonadal factor and/or a compensatory increase in gonadotropins. In order to achieve elevation of gonadotropins without the concomitant loss of a gonadal hormone, we crossed INH(-/-) mice with a transgenic mouse strain that has chronically elevated luteinizing hormone (LH) levels (LH-CTP). Compound INH(-/-)-LH-CTP mice die within 6 weeks of age from severe cancer cachexia induced by large, activin-secreting ovarian tumors. Unexpectedly, INH(-/-)-LH-CTP mice not only fail to develop adrenal tumors but have smaller adrenals, with a regressed x zone, indicating that elevated LH levels are not sufficient to induce adrenal tumor formation. However, following gonadectomy, INH(-/-)-LH-CTP mice develop large, sex steroid-producing adrenal tumors that arise from the x zone, indicating a growth-promoting effect of high levels of LH on the adrenal cortex in the absence of ovarian tumors. In addition, in vivo and in vitro data indicate that activin induces apoptosis specifically in the adrenal x zone. The restricted expression of activin receptor subunits and Smad2 in cells of the adrenal x zone, together with the elevated activin levels in INH(-/-)-LH-CTP mice, supports the conclusion that activin inhibits adrenal tumor growth by inducing x-zone regression.
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Activinas/metabolismo , Neoplasias de la Corteza Suprarrenal/metabolismo , Carcinoma Corticosuprarrenal/metabolismo , Apoptosis/fisiología , Inhibinas/metabolismo , Hormona Luteinizante/metabolismo , Glándulas Suprarrenales/anatomía & histología , Glándulas Suprarrenales/metabolismo , Glándulas Suprarrenales/patología , Animales , Peso Corporal , Caquexia , Proteínas de Unión al ADN/metabolismo , Estradiol/metabolismo , Femenino , Humanos , Hibridación in Situ , Inhibinas/genética , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Ovariectomía , Fenotipo , Receptores de HL/metabolismo , Proteína Smad2 , Testosterona/metabolismo , Transactivadores/metabolismoRESUMEN
Transgenic (TG) mice expressing the Simian virus 40 T-antigen under the control of the murine inhibin-alpha promoter (Inhalpha/Tag) develop granulosa and Leydig cell tumors at the age of 5-6 months, with 100% penetrance. When these mice are gonadectomized, they develop adrenocortical tumors. Suppression of gonadotropin secretion inhibits the tumorigenesis in the gonads of intact animals and in the adrenals after gonadectomy. To study further the role of luteinizing hormone (LH) in gonadal and adrenal tumorigenesis, a double TG mouse model was generated by crossing the Inhalpha/Tag mice with mice producing constitutively elevated levels of LH (bLHbeta-CTP mice). Our results show that in double TG mice (bLHbeta-CTP/Inhalpha/Tag), gonadal tumorigenesis starts earlier and progresses faster than in Inhalpha/Tag mice. Both ovarian and testicular tumors were histologically comparable with the tumors found in Inhalpha/Tag mice. In addition, adrenal tumorigenesis was found in intact double TG females, but not in Inhalpha/Tag females. Inhibin-alpha and LH receptor (LHR) were highly expressed in tumorigenic gonadal tissues, and the elevated LH levels were shown to be associated with ectopic LHR and high inhibin-alpha expression in the female adrenals. We conclude that in the Inhalpha/Tag tumor mouse model, elevated LH levels act as a tumor promoter, advancing gonadal and adrenal tumorigenesis.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/etiología , Antígenos Transformadores de Poliomavirus/genética , Inhibinas/genética , Hormona Luteinizante de Subunidad beta/genética , Neoplasias Ováricas/etiología , Neoplasias Testiculares/etiología , Neoplasias de la Corteza Suprarrenal/metabolismo , Neoplasias de la Corteza Suprarrenal/patología , Glándulas Suprarrenales/metabolismo , Animales , Fusión Artificial Génica , Gonadotropina Coriónica Humana de Subunidad beta/genética , Femenino , Tumor de Células de la Granulosa/etiología , Tumor de Células de la Granulosa/metabolismo , Tumor de Células de la Granulosa/patología , Hormonas/sangre , Inhibinas/biosíntesis , Tumor de Células de Leydig/etiología , Tumor de Células de Leydig/metabolismo , Tumor de Células de Leydig/patología , Hormona Luteinizante/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Receptores de HL/biosíntesis , Receptores de HL/genética , Virus 40 de los Simios/inmunología , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologíaRESUMEN
Transgenic (TG) female mice, expressing a chimeric bovine luteinizing hormone (LH) beta-subunit/human chorionic gonadotropin beta-subunit COOH-terminal extension (bLHbeta-CTP) gene, produce high levels of circulating LH and serve as a model for functional ovarian hyperandrogenism and follicular cysts. We report here that obesity is a typical feature of these female mice. The mean body weight of the bLHbeta-CTP females was significantly higher than in controls at, and beyond 5 wk of age, and at 5 mo, it was 32% increased. At this age, the amount of white adipose tissue in the bLHbeta-CTP females was significantly increased, as reflected by the weight difference of the retroperitoneal fat pad. In addition, the expression of leptin mRNA in white adipose tissue of the TG females was elevated about twofold. Serum leptin and insulin levels, and food intake, were also increased significantly in the TG females. Brown adipose tissue (BAT) thermogenic activity, as measured by GDP binding to BAT mitochondria, was reduced (P < 0.05). Ovariectomy at the age of 3 wk totally prevented the development of obesity. In summary, the present results show that intact female bLHbeta-CTP mice are obese, have increased food consumption, and reduced BAT thermogenic activity. The weight gain can be explained partly by elevated androgens but is probably also contributed to the increased adrenal steroidogenesis. Hence, the bLHbeta-CTP mice provide a useful model for studying obesity related to elevated LH secretion, with consequent alterations in ovarian and adrenal function.