RESUMEN
P-glycoprotein (pgp), an efflux transporter localized in a variety of tissues including the intestinal mucosa, renal tubules and bile canaliculi, is known to participate in the disposition of a variety of chemicals, including steroid hormones. This study examined the relationship of pgp to the movement into the bile of the hormone estradiol (E2), and the potential for transport interactions between the environmental pollutant nonylphenol ethoxylate (NPE) and E2. Biliary-cannulated in situ-prepared isolated perfused livers were used to assess pgp transport function. E2, in competitive transport preparations with Rhodamine 123 (Rho123), a pgp substrate, demonstrated significant decreases in Rho123 transport into bile, as did the prototypic inhibitor and substrate verapamil. [3H]E2 (0.28 nM) transport into bile was significantly reduced with either 20 M NPE or verapamil. These results suggest that E2 is a substrate and/or modulator for the catfish biliary pgp transporter, and that NPE potentially influences biliary transport and excretion of E2.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Bilis/metabolismo , Bagres/metabolismo , Estradiol/metabolismo , Glicoles de Etileno/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Transporte Biológico Activo/efectos de los fármacos , Antagonistas de Estrógenos/farmacología , Glicoles de Etileno/metabolismo , Hígado/metabolismo , Rodamina 123/metabolismo , Verapamilo/metabolismo , Verapamilo/farmacología , Contaminantes Químicos del Agua/metabolismoRESUMEN
The POU-domain transcription factor Oct4 is essential for the maintenance of the mammalian germline. In this study, we show that the germ cell nuclear factor (GCNF), an orphan nuclear receptor, represses Oct4 gene activity by specifically binding within the proximal promoter. GCNF expression inversely correlates with Oct4 expression in differentiating embryonal cells. GCNF overexpression in embryonal cells represses Oct4 gene and transgene activities, and we establish a link to transcriptional corepressors mediating repression by GCNF. In GCNF-deficient mouse embryos, Oct4 expression is no longer restricted to the germ cell lineage after gastrulation. Our studies suggest that GCNF is critical in repressing Oct4 gene activity as pluripotent stem cells differentiate and in confining Oct4 expression to the germline.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/fisiología , Células Germinativas/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Antineoplásicos/farmacología , Diferenciación Celular/fisiología , Línea Celular , Linaje de la Célula , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Factores de Transcripción Fushi Tarazu , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes Reporteros , Proteínas de Homeodominio , Hibridación in Situ , Sustancias Macromoleculares , Ratones , Ratones Noqueados , Proteínas Nucleares/metabolismo , Co-Represor 1 de Receptor Nuclear , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares , Factor 3 de Transcripción de Unión a Octámeros , Regiones Promotoras Genéticas/genética , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , Factor Esteroidogénico 1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transgenes/genética , Tretinoina/farmacología , Técnicas del Sistema de Dos HíbridosRESUMEN
GCNF is an orphan member of the nuclear receptor superfamily. The nuclear receptor superfamily is a large superfamily of transcription factors, the majority of which are designated as orphan receptors because their ligands and functions are currently unknown. GCNF (Germ Cell Nuclear Factor) is so named because of its restricted expression pattern in the adult. In the testis, GCNF is expressed only in the post meiotic round spermatids. Likewise in the ovary, GCNF's expression is restricted to the growing oocyte. To date nothing is known of GCNF's putative ligand; however, much is known about its physiological function through the use of gene targeting. Inactivation of the GCNF gene showed that it was essential for normal embryonic development. In addition to being expressed in the germ cells of the adult, it is expressed widely throughout the embryo after gastrulation. Significant strides have also been made in understanding GCNF's mechanism of action using molecular biology. The DNA binding properties of GCNF have been investigated and its response element identified. GCNF binds as a homodimer to a direct repeat element with zero nucleotides between the reiterated sequence AGGTCA. GCNF target genes have been identified that contain this DR0 element in their promoters. Such genes as Protamines 1 and 2 and Oct4 are regulated by GCNF through this element. GCNF has been shown to be a repressor of the protamine and Oct4 genes. GCNF's repression function has been shown to be mediated by interaction with the co-repressors N-CoR and SMRT in the absence of ligand. Our current efforts are to explore GCNF function in the adult germ cells using tissue specific gene targeting to specifically knock out the GCNF gene in oocytes and spermatogenic cells. In addition, efforts are being made to identify the endogenous ligand that regulates GCNF's transcriptional properties.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Desarrollo Embrionario y Fetal/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Proteínas Represoras/fisiología , Animales , Desarrollo Embrionario y Fetal/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares , Transcripción GenéticaRESUMEN
Proper expression of the protamine genes is an important event in the terminal differentiation of the male gametes in mammals. Here we present evidence that the novel orphan member of the nuclear receptor gene superfamily, Germ Cell Nuclear Factor (GCNF), may play a role in the regulation of these genes. Previously, we have shown that high-level expression of GCNF mRNA is restricted to spermatids (stages 1-8) in the adult male mouse, which makes it temporally and spatially available to regulate the mouse protamine genes. Furthermore, we have previously identified a sequence to which GCNF can bind, which consists of a direct repeat of the core halfsite AGGTCA with zero base pairs spacing the repeats (DRO). We have identified several genes that contain DRO sequences in their 5' promoter regions, including the protamines. The mouse protamine 1 and 2 (Prm1 and Prm2) genes therefore are potential target genes for GCNF regulation. We show that GCNF binds to one of the two DRO sequences in the Prm1 promoter, and to the DRO sequence in the Prm2 promoter in a specific manner. Furthermore, by using antibodies directed against GCNF, we detect endogenous GCNF in testis nuclear extracts and elutriated round spermatid nuclear extracts in Western blots. Additionally, by using these antibodies in gel-shift assays, we show that this endogenous GCNF can bind to both the Prm1 and Prm2 promoters. This evidence supports the hypothesis that GCNF mediates a novel signaling pathway, two targets of which may be the Prm1 and Prm2 genes in spermatids.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Protaminas/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Proteínas de Unión al ADN/genética , Femenino , Masculino , Ratones , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares , Conejos , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
We have shown that the orphan receptor Germ Cell Nuclear Factor (GCNF) binds to a direct repeat of the sequence AGGTCA with zero base pair spacing (DR0). Here, we further characterize the binding characteristics of GCNF. We demonstrate that GCNF binds specifically to DR0s as a homodimer, and does not bind with high affinity to DR1-DR6 sequences. GCNF is the first nuclear receptor shown to bind specifically to DR0s. The wild type GCNF is unable to transactivate the reporter plasmid DR0(2)tkCAT. Lacking a ligand to activate GCNF, we fused the activation domain from the viral protein VP16 to GCNF, and observed activation of DR0(2)tkCAT. This activation is specifc to DR0s, and is not observed when that sequence is replaced by DR1-DR6 sequences. In addition GCNF does not transactivate through an SF-1 response element. At increasing concentrations, wild type GCNF is able to repress basal transcription. Repression is again specific to DR0s. The preference of GCNF for the DR0 sequence both in vitro and in transfections suggests that GCNF defines a novel nuclear receptor signaling pathway.