RESUMO
The exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2) are expressed in a cell specific manner in the liver, but their biological functions in this tissue are poorly understood. The current study was undertaken to begin to determine the potential roles of Epac1 and Epac2 in liver physiology and disease. Male C57BL/6J mice in which expression of Epac1 and/or Epac2 are deleted, were subjected to partial hepatectomy and the regenerating liver was analyzed with regard to lipid accumulation, cell replication and protein expression. In response to partial hepatectomy, deletion of Epac1 and/or Epac2 led to increased hepatocyte proliferation 36 h post surgery, and the transient steatosis observed in wild type mice was virtually absent in mice lacking both Epac1 and Epac2. The expression of the protein cytochrome P4504a14, which is implicated in hepatic steatosis and fibrosis, was substantially reduced upon deletion of Epac1/2, while a number of factors involved in lipid metabolism were significantly decreased. Moreover, the number of Küpffer cells was affected, and Epac2 expression was increased in the liver of wild type mice in response to partial hepatectomy, further supporting a role for these proteins in liver function. This study establishes hepatic phenotypic abnormalities in mice deleted for Epac1/2 for the first time, and introduces Epac1/2 as regulators of hepatocyte proliferation and lipid accumulation in the regenerative process.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Regeneração Hepática/fisiologia , Animais , Proliferação de Células/fisiologia , Fígado Gorduroso/metabolismo , Fibrose/metabolismo , Hepatectomia/métodos , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- (Rapgef4-/-) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.
Assuntos
AMP Cíclico/metabolismo , Exocitose , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Insulina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Dinamina I/metabolismo , Humanos , Camundongos KnockoutRESUMO
Epac1 (Exchange protein directly activated by cAMP 1) limits fluid loss from the circulation by tightening the endothelial barrier. We show here that Epac1-/- mice, but not Epac2-/- mice, have prolonged bleeding time, suggesting that Epac1 may limit fluid loss also by restraining bleeding. The Epac1-/- mice had deficient in vitro secondary hemostasis. Quantitative comprehensive proteomics analysis revealed that Epac1-/- mouse platelets (thrombocytes) had unbalanced expression of key components of the glycoprotein Ib-IX-V (GPIb-IX-V) complex, with decrease of GP1bß and no change of GP1bα. This complex is critical for platelet adhesion under arterial shear conditions. Furthermore, Epac1-/- mice have reduced levels of plasma coagulation factors and fibrinogen, increased size of circulating platelets, increased megakaryocytes (the GP1bß level was decreased also in Epac1-/- bone marrow) and higher abundance of reticulated platelets. Viscoelastic measurement of clotting function revealed Epac1-/- mice with a dysfunction in the clotting process, which corresponds to reduced plasma levels of coagulation factors like factor XIII and fibrinogen. We propose that the observed platelet phenotype is due to deficient Epac1 activity during megakaryopoiesis and thrombopoiesis, and that the defects in blood clotting for Epac1-/- is connected to secondary hemostasis.
Assuntos
Plaquetas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/deficiência , Hemorragia/sangue , Hemorragia/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Fatores de Coagulação Sanguínea/metabolismo , Plaquetas/ultraestrutura , Tamanho Celular , Colágeno/farmacologia , Exocitose , Feto/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fígado/embriologia , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Camundongos Endogâmicos C57BL , Selectina-P/metabolismo , Fenótipo , Trombina/farmacologiaRESUMO
Resistance towards antiparasitic agents in the salmon louse (Lepeophtheirus salmonis) is a widespread problem along the Norwegian coast, reducing treatments efficacies and slowing down the envisioned expansion of Norwegian salmon production. The present study was conducted in order to assess the efficacies of two of the most widely used anti-parasitic substances-azamethiphos and deltamethrin-as well as assessing the benefit of having a resistant genotype compared to being fully sensitive when exposed to one of these substances. Atlantic salmon were exposed to a mix of salmon lice copepodids from a fully sensitive, a double resistant and a multi-resistant strain. Once the lice reached pre-adult stages, one group was exposed to 100 µg/L azamethiphos for 60 minutes, the other to 2 µg/L deltamethrin for 30 minutes, and the last was kept in a seawater control. Detached lice were collected at a series of time points following exposure, and all lice (immobilized and surviving) were analysed for both pyrethroid (sensitive "S" and resistant "R") and azamethiphos (fully sensitive "SS", heterozygous resistant "RS" and fully resistant "RR") resistance markers. We found that the efficacies of deltamethrin on parasites with genotype S and R were 70.3 and 13.2%, respectively. The overall efficacy of the deltamethrin treatment was 32.3%. The efficacies of azamethiphos on parasites with genotype SS, RS and RR were 100, 80 and 19.1%, respectively. The overall efficacy of the azamethiphos treatment was 80.4%. Survival analyses revealed that the median survival time in deltamethrin-sensitive and-resistant parasites were 16.8 and >172 hours, respectively. The differences were even more pronounced in the azamethiphos-treated group, where SS, RS and RR parasites survived for 0.26, 6.6 and >172 hours, respectively. The substantial differences in survival between sensitive and resistant lice following treatment demonstrate the ability of medicinal treatments to drive genetic selection towards a much more resistant salmon lice population within a very short time span if there is no influx of sensitive genotypes.
Assuntos
Copépodes/efeitos dos fármacos , Organofosfatos/farmacologia , Piretrinas/farmacologia , Salmo salar/parasitologia , Animais , Copépodes/genética , Copépodes/crescimento & desenvolvimento , Resistência a Medicamentos , Genótipo , Nitrilas/farmacologia , Organotiofosfatos/farmacologiaRESUMO
BACKGROUND: Hypoxia- and Myc-dependent transcriptional regulatory pathways are frequently deregulated in cancer cells. These pathways converge in many cellular responses, but the underlying molecular mechanisms are unclear. METHODS: The ability of Miz-1 and Arnt to interact was identified in a yeast two-hybrid screen. The mode of interaction and the functional consequences of complex formation were analyzed by diverse molecular biology methods, in vitro. Statistical analyses were performed by Student's t-test and ANOVA. RESULTS: In the present study we demonstrate that the aryl hydrocarbon receptor nuclear translocator (Arnt), which is central in hypoxia-induced signaling, forms a complex with Miz-1, an important transcriptional regulator in Myc-mediated transcriptional repression. Overexpression of Arnt induced reporter gene activity driven by the proximal promoter of the cyclin-dependent kinase inhibitor 2B gene (CDKN2B), which is an established target for the Myc/Miz-1 complex. In contrast, mutated forms of Arnt, that were unable to interact with Miz-1, had reduced capability to activate transcription. Moreover, repression of Arnt reduced endogenous CDKN2B expression, and chromatin immunoprecipitation demonstrated that Arnt interacts with the CDKN2B promoter. The transcriptional activity of Arnt was counteracted by Myc, but not by a mutated variant of Myc that is unable to interact with Miz-1, suggesting mutually exclusive interaction of Arnt and Myc with Miz-1. Our results also establish CDKN2B as a hypoxia regulated gene, as endogenous CDKN2B mRNA and protein levels were reduced by hypoxic treatment of U2OS cells. CONCLUSIONS: Our data reveal a novel mode of regulation by protein-protein interaction that directly ties together, at the transcriptional level, the Myc- and hypoxia-dependent signaling pathways and expands our understanding of the roles of hypoxia and cell cycle alterations during tumorigenesis.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Carcinogênese/genética , Inibidor de Quinase Dependente de Ciclina p15/biossíntese , Genes myc/genética , Fator 1 Induzível por Hipóxia/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Inibidor de Quinase Dependente de Ciclina p15/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Imunoprecipitação , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
The nuclear receptor steroidogenic factor 1/adrenal 4 binding protein (SF-1/Ad4BP) is an essential regulator of endocrine development and function, and the expression of the corresponding gene (sf-1/ad4bp) is precisely regulated in a time- and tissue-dependent manner. We previously demonstrated that the basal promoter of sf-1/ad4bp is controlled by DNA methylation and that its methylation status reflects the expression pattern of SF-1/Ad4BP. Recently, three intronic enhancers were identified in the sf-1/ad4bp gene that target SF-1/Ad4BP expression to the fetal adrenal (FAdE; fetal adrenal-specific enhancer), to pituitary gonadotropes (PGE; pituitary gonadotrope-specific enhancer), and to the ventromedial hypothalamic nucleus (VMHE; ventromedial hypothalamic nucleus-specific enhancer). Here, we demonstrate that the activity of these enhancers is correlated with their DNA methylation status. We show that they are hypomethylated in tissues where they are active and generally hypermethylated in tissues where they are not active. Furthermore, we demonstrate in transient transfection experiments that forced DNA methylation represses reporter gene activity driven by these enhancers. These data directly demonstrate a functional significance for the enhancers' methylation status. Intriguingly, further analyses of the basal promoter in gonadotropes revealed that it is methylated in these cells, in contrast to other SF-1/Ad4BP-expressing tissues. Consistent with this, sf-1/ad4bp is transcribed from an alternative promoter in gonadotropes. Taken together, our experiments show that the tissue-specific expression of SF-1/Ad4BP is epigenetically regulated and identify tissue-specific differentially methylated regions within the sf-1/ad4bp locus that are essential for its transcriptional control.
Assuntos
Metilação de DNA , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Fator Esteroidogênico 1/genética , Glândulas Suprarrenais/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Ilhas de CpG/genética , Feminino , Imunofluorescência , Gonadotrofos/metabolismo , Íntrons/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas/genéticaRESUMO
In the adrenal cortex, the biosynthesis of steroid hormones is controlled by the pituitary-derived hormone ACTH. The functions of ACTH are principally relayed by activating cAMP-dependent signaling pathways leading to the induction of genes encoding enzymes involved in the conversion of cholesterol to steroid hormones. Previously, protein kinase A (PKA) was thought to be the only direct effector of cAMP. However, the discovery of the cAMP sensors, exchange proteins directly activated by cAMP (EPAC1 and 2), has led to a reevaluation of this assumption. In the present study, we demonstrate the occurrence of the EPAC2 splicing variant EPAC2B in adrenocortical cancer cells. Immunocytochemistry demonstrated that EPAC2B is localized predominantly in the nucleus. EPAC2B is functional because it activates Rap1 in these cells. Using the cAMP analogs 8-p-chlorophenylthio-2'-O-methyl-cAMP and N6-benzoyl-cAMP, which specifically activate EPAC1/2 and PKA, respectively, we evaluated the contribution of these factors in steroid hormone production, cell morphology, actin reorganization, and migration. We demonstrate that the expression of cAMP-inducible factors involved in steroidogenesis (steroidogenic acute regulatory protein, cytochrome P450 11A1 and 17, and nerve growth factor-induced clone B) and the cAMP-induced biosynthesis of steroid hormones (cortisol and aldosterone) are mediated by PKA and not by EPAC2B. In contrast, both PKA- and EPAC-specific cAMP analogs induced cell rounding, loss of stress fibers, and blocked migration. Taken together, the presented data confirm PKA as the central cAMP mediator in steroid hormone production and reveal the involvement of EPAC2B in cAMP-induced effects on cytoskeleton integrity and cell migration.
Assuntos
Núcleo Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/patologia , Aldosterona/biossíntese , Processamento Alternativo , Animais , Western Blotting , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Chlorocebus aethiops , AMP Cíclico/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Hidrocortisona/biossíntese , Imuno-Histoquímica , Microscopia Confocal , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
Steroidogenic factor 1 (SF1) is expressed in a time- and cell-specific manner in the endocrine system. In this study we present evidence to support that methylation of CpG sites located in the proximal promoter of the gene encoding SF1 contributes to the restricted expression pattern of this nuclear receptor. DNA methylation analyses revealed a nearly perfect correlation between the methylation status of the proximal promoter and protein expression, such that it was hypomethylated in cells that express SF1 but hypermethylated in nonexpressing cells. Moreover, in vitro methylation of this region completely repressed reporter gene activity in transfected steroidogenic cells. Bisulfite sequencing of DNA from embryonic tissue demonstrated that the proximal promoter was unmethylated in the developing testis and ovary, whereas it was hypermethylated in tissues that do not express SF1. Together these results indicate that the DNA methylation pattern is established early in the embryo and stably inherited thereafter throughout development to confine SF1 expression to the appropriate tissues. Chromatin immunoprecipitation analyses revealed that the transcriptional activator upstream stimulatory factor 2 and RNA polymerase II were specifically recruited to this DNA region in cells in which the proximal promoter is hypomethylated, providing functional support for the fact that lack of methylation corresponds to a transcriptionally active gene. In conclusion, we identified a region within the SF1/Sf1 gene that epigenetically directs cell-specific expression of SF1.
Assuntos
Metilação de DNA , Fator Esteroidogênico 1/genética , Animais , Sequência de Bases , Padronização Corporal/genética , Ilhas de CpG , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Dados de Sequência Molecular , Células NIH 3T3 , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , Fator Esteroidogênico 1/metabolismo , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
Nuclear receptors and their coactivators are key regulators of numerous physiological functions. GRIP1 (glucocorticoid receptor-interacting protein) is a member of the steroid receptor coactivator family. Here, we show that GRIP1 is regulated by cAMP-dependent protein kinase (PKA) that induces its degradation through the ubiquitin-proteasome pathway. GRIP1 was down-regulated in transiently transfected COS-1 cells after treatment with 8-para-chlorophenylthio-cAMP or forskolin and 3-isobutyl-1-methylxanthine and in adrenocortical Y1 cells after incubation with adrenocorticotropic hormone. Pulse-chase experiments with transiently transfected COS-1 cells demonstrated that the half-life of GRIP1 was markedly reduced in cells overexpressing the PKA catalytic subunit, suggesting that activation of PKA increases the turnover of GRIP1 protein. The proteasome inhibitors MG132 and lactacystin abolished the PKA-mediated degradation of GRIP1. Using ts20 cells, a temperature-sensitive cell line that contains a thermolabile ubiquitin-activating E1 enzyme, it was confirmed that PKA-mediated degradation of GRIP1 is dependent upon the ubiquitin-proteasome pathway. Coimmunoprecipitation studies of COS-1 cells transfected with expression vectors encoding GRIP1 and ubiquitin using anti-GRIP1 and anti-ubiquitin antibodies showed that the ubiquitination of GRIP1 was increased by overexpression of PKA. Finally, we show that PKA regulates the intracellular distribution pattern of green fluorescent protein-GRIP1 and stimulates recruitment of GRIP1 to subnuclear foci that are colocalized with the proteasome. Taken together, these data demonstrate that GRIP1 is ubiquitinated and degraded through activation of the PKA pathway. This may represent a novel regulatory mechanism whereby hormones down-regulate a nuclear receptor coactivator.
Assuntos
Acetilcisteína/análogos & derivados , Núcleo Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/análogos & derivados , Coativador 2 de Receptor Nuclear/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Acetilcisteína/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Animais , Western Blotting , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Colforsina/farmacologia , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Regulação para Baixo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Vetores Genéticos , Imunoprecipitação , Leupeptinas/farmacologia , Luciferases/metabolismo , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Coativador 2 de Receptor Nuclear/química , Inibidores de Proteases/farmacologia , Temperatura , Tionucleotídeos/farmacologia , Fatores de Tempo , Ativação Transcricional , TransfecçãoAssuntos
AMP Cíclico/fisiologia , Proteínas de Ligação a DNA/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Córtex Suprarrenal/metabolismo , Animais , Fatores de Transcrição COUP , Bovinos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Regiões Promotoras Genéticas/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Elementos de Resposta/fisiologia , Esteroide 17-alfa-Hidroxilase/genética , Fator Esteroidogênico 1 , Fatores de Transcrição/metabolismoRESUMO
Knockout mice lacking steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a complex phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To explore further how SF-1 regulates endocrine function, we used bacterial artificial chromosome transgenesis to develop a lineage marker for SF-1-expressing cells. A genomic fragment containing 50 kb of the mouse Nr5a1 gene was used to target enhanced green fluorescent protein (eGFP) in transgenic mice. These sequences directed eGFP to multiple cell lineages that express SF-1, including steroidogenic cells of the adrenal cortex, testes, and ovaries, neurons of the ventromedial hypothalamic nucleus, and reticuloendothelial cells of the spleen. Despite the proven role of SF-1 in gonadotrope function, eGFP was not expressed in the anterior pituitary. These experiments show that 50 kb of the mouse Nr5a1 gene can target transgenic expression to multiple cell lineages that normally express SF-1. The SF-1/eGFP transgenic mice will facilitate approaches such as fluorescence-activated cell sorting of eGFP-positive cells and DNA microarray analyses to expand our understanding of the multiple actions of SF-1 in endocrine development and function.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Luminescentes/genética , Fatores de Transcrição/genética , Glândulas Suprarrenais/embriologia , Glândulas Suprarrenais/metabolismo , Animais , Biomarcadores , Linhagem da Célula , Cromossomos Artificiais Bacterianos , Proteínas de Ligação a DNA/metabolismo , Sistema Endócrino/fisiologia , Feminino , Citometria de Fluxo/métodos , Fatores de Transcrição Fushi Tarazu , Regulação da Expressão Gênica no Desenvolvimento , Engenharia Genética/métodos , Proteínas de Fluorescência Verde , Proteínas de Homeodomínio , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Ovário/embriologia , Ovário/metabolismo , Adeno-Hipófise/embriologia , Adeno-Hipófise/metabolismo , Receptores Citoplasmáticos e Nucleares , Fator Esteroidogênico 1 , Testículo/embriologia , Testículo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Steroidogenic factor-1 (SF-1) is a member of the nuclear receptor superfamily that plays essential roles in the development of endocrine organs. Steroid receptor coactivator 1 and transcription intermediary factor 2 (TIF2) belong to the p160 coactivator family that mediates transcriptional activation by several nuclear receptors, including SF-1. Here, it is reported that another of the p160 coactivators, p/CIP, interacts with SF-1 through the activation function-2 domain. Both p300/CBP/cointegrator-associated protein (p/CIP) and TIF2 potentiated SF-1-mediated transcription from two reporter gene constructs in transfected nonsteroidogenic COS-1 cells and in adrenocortical Y1 cells. PKA was shown to stimulate SF-1 transcriptional activity, and coexpression of p/CIP together with the PKA catalytic subunit stimulated SF-1-mediated transactivation even further. In contrast, PKA catalytic subunit overexpression impaired the ability of TIF2 to potentiate SF-1-dependent transcription. Activation of PKA also inhibited the TIF2-mediated coactivation of other nuclear receptors such as PPAR alpha/-gamma and liver X receptor-alpha. The TIF2 mRNA levels were not affected by PKA, but instead we found that PKA activation led to a decrease in the levels of TIF2 protein. Moreover, the C-terminal activation domain 2 of TIF2 was required for the inhibitory effect of PKA, suggesting that this region is the target for the PKA-mediated down-regulation. Thus, in contrast to the regulation of p/CIP and steroid receptor coactivator 1, we suggest that activation of PKA leads to selective down-regulation of TIF2 and subsequently repression of TIF2 coactivator function.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Animais , Baculoviridae , Células COS , AMP Cíclico/metabolismo , Fatores de Transcrição Fushi Tarazu , Histona Acetiltransferases , Proteínas de Homeodomínio , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Coativador 2 de Receptor Nuclear , Coativador 3 de Receptor Nuclear , Receptores Citoplasmáticos e Nucleares , Transdução de Sinais , Fator Esteroidogênico 1 , Células Tumorais Cultivadas , LevedurasRESUMO
The orphan nuclear receptor steroidogenic factor-1 (SF-1) plays pivotal roles in the development and function of steroidogenic organs. Here we describe the differential effect of protein kinase A (PKA) on coregulation of SF-1 dependent transcription by two p160 family members, p300/CBP co-integrator-associated protein (p/CIP) and transcription intermediary factor-2 (TIF2). Thus, whereas p/CIP-stimulated SF-1 dependent transcription is further potentiated by PKA, we show that activation of PKA leads to selective downregulation of TIF2 protein and a subsequent repression of TIF2 coactivator function. Using a yeast two-hybrid screen we also identified a novel zinc finger containing protein, which interacts with SF-1 via the AF-2 domain.