RESUMO
BACKGROUND AND PURPOSE: Evidence indicates nociceptin/orphanin FQ (N/OFQ) may participate in the pathology of cardiac arrhythmias associated with myocardial infarction. But the role of N/OFQ in the arrhythmogenesis in acute myocardial infarction is unclear. The aim of this study was to investigate the effects of endogenous N/OFQ on infarction-associated arrhythmias. EXPERIMENTAL APPROACH: The expression of N/OFQ, PKC activity and ventricular arrhythmias in presence and absence of UFP-101, a specific antagonist of N/OFQ receptor, were examined following permanent coronary artery occlusion in anaesthetized rats. The effect of N/OFQ on action potential duration was examined in isolated rat cardiomyocytes. KEY RESULTS: It was observed that N/OFQ was increased by 41% in the myocardium after coronary artery occlusion (P < 0.01 vs. control). Pretreatment with UFP-101 (10(-7) mol·kg(-1) , i.v.) reduced the incidence of ventricular ectopic beats by 70% and ventricular tachycardia by 51% respectively (all P < 0.05 vs. control). Meanwhile, PKC activity was elevated in the rats treated with UFP-101 (by 35%, P < 0.05 vs. control). A selective PKC inhibitor, calphostin C, completely abolished the anti-arrhythmic effects of UFP-101 (P < 0.01). N/OFQ (at 10(-11) , 10(-9) and 1 × 10(-7) mol·L(-1) ) shortened the action potential duration by 3% (P > 0.05), 10% (P < 0.05) and 22% (P < 0.01), respectively, via N/OFQ receptor. CONCLUSIONS AND IMPLICATIONS: Antagonism of endogenous N/OFQ produces anti-arrhythmic effects on ventricular arrhythmias in acute myocardial infarction, possibly via modulating PKC activity and action potential of myocytes.
Assuntos
Antiarrítmicos/farmacologia , Infarto do Miocárdio/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Antagonistas de Entorpecentes , Peptídeos Opioides/farmacologia , Proteína Quinase C/metabolismo , Taquicardia Ventricular/prevenção & controle , Complexos Ventriculares Prematuros/prevenção & controle , Potenciais de Ação , Animais , Modelos Animais de Doenças , Masculino , Infarto do Miocárdio/complicações , Infarto do Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Opioides/metabolismo , Transdução de Sinais/efeitos dos fármacos , Taquicardia Ventricular/enzimologia , Taquicardia Ventricular/etiologia , Fatores de Tempo , Complexos Ventriculares Prematuros/enzimologia , Complexos Ventriculares Prematuros/etiologia , Receptor de NociceptinaRESUMO
Perturbations in secretory function activate stress response pathways critical for yeast survival. Here we report the identification of the Hos2p/Set3p deacetylase complex (SET3C) as an essential component of the secretory stress response. Strains lacking core components of the Hos2p/Set3p complex exhibit hypersensitivity to secretory stress. Although not required for the unfolded protein response (UPR) and ribosomal gene repression, the Hos2p complex is required for proper activation of the Mpk1p/Slt2p cell integrity kinase cascade. Disruption of the Hos2p complex results in abrogated Mpk1p phosphorylation, whereas constitutive activation of the Mpk1p pathway rescues the hos2Delta mutant growth defect in response to secretory stress. Furthermore, Hos2p activity is required for the Mpk1p-mediated activation of stress-responsive transcription factor Rlm1p, but not for the stress-induced degradation of the C-type cyclin Ssn8p. Our results identify the Hos2p complex as a critical component of the secretory stress response and support the existence a coordinated stress response consisting of the UPR, ribosomal gene repression, and mitogen-activated protein kinase signaling in response to defects in secretory function.
Assuntos
Regulação Fúngica da Expressão Gênica , Histona Desacetilases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais , Antimetabólitos/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Histona Desacetilases/genética , Proteínas de Domínio MADS , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Fenótipo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tunicamicina/farmacologiaRESUMO
The painful sensation during acute myocardial ischemia or infarction is a common symptom and results from neural activity in humans. Little is known about the role of neuropeptides in this effect of myocardial ischemia. The aim of the current study was to investigate the role of substance P in mediating the noxious neural signals in spinal cord in acute myocardial ischemia by exploring the change in substance P and its mRNA in thoracic dorsal root ganglia and spinal dorsal horn (T1-T5) after coronary artery occlusion. The experiment was performed with immunohistochemistry, enzyme immunoassay and real time reverse transcription-polymerase chain reaction techniques on rats' hearts. In acute myocardial ischemia (<6 h), substance P and preprotachykinin mRNA were up-regulated in the neurons of the dorsal root ganglia and spinal dorsal horn. The increase in the density of immunoreactive material was mainly observed in small-diameter neurons of the dorsal root ganglia and the superficial laminae (I and II) of the spinal cord. The increase in the expressions was statistically significant compared with the control and the sham surgery groups (P<0.05). The results suggest that substance P is involved in the mediation of the noxious neural signals of acute myocardial ischemia in spinal cord. The pathophysiological role and significance need to be investigated.
Assuntos
Vias Aferentes/metabolismo , Angina Pectoris/metabolismo , Isquemia Miocárdica/metabolismo , Nociceptores/metabolismo , Células do Corno Posterior/metabolismo , Substância P/metabolismo , Angina Pectoris/genética , Angina Pectoris/fisiopatologia , Animais , Estenose Coronária/genética , Estenose Coronária/metabolismo , Estenose Coronária/fisiopatologia , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica/fisiologia , Coração/inervação , Masculino , Isquemia Miocárdica/genética , Isquemia Miocárdica/fisiopatologia , Neurônios Aferentes/metabolismo , Precursores de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Substância P/genética , Taquicininas/metabolismoRESUMO
The Nf2 tumor suppressor codes for merlin, a protein whose function is largely unknown. We have previously demonstrated a novel interaction between merlin and TRBP, which inhibits the oncogenic activity of TRBP. In spite of the significance of their functional interaction, its molecular mechanism still remains to be elucidated. In this report, we investigated how merlin inhibits the oncogenic activity of TRBP in association with cell growth conditions. In the human embryonic kidney 293 cell line, the level of endogenous merlin increased, whereas that of endogenous TRBP significantly decreased along with the increase in cell confluence. We demonstrated that the carboxyl-terminal region of TRBP was responsible for this phenomenon using stable cell lines expressing deletion mutants of TRBP. The overexpression of merlin decreased the protein level of TRBP, and the ubiquitin-like subdomain of merlin's FERM domain was important for this activity. We also demonstrated that TRBP is ubiquitinylated and the ubiquitinylated forms of TRBP are accumulated by ectopically expressed merlin or cell confluence in the presence of MG132, a proteasome inhibitor. Furthermore, we showed that the regulation of TRBP in response to cell confluence was abolished upon knockdown of merlin expression by specific small interfering RNA. Finally, we showed that ectopically expressed merlin restored cell-cell contact inhibition in cells stably expressing TRBP but not in TRBPDeltac. These results suggest that merlin is involved in the regulation of TRBP protein level by facilitating its ubiquitination in response to such cues as cell-cell contacts.
Assuntos
Neurofibromina 2/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ubiquitina/metabolismo , Animais , Western Blotting , Adesão Celular , Humanos , Imunoprecipitação , Rim/citologia , Rim/metabolismo , Leupeptinas/farmacologia , Camundongos , Células NIH 3T3 , Neurofibromina 2/antagonistas & inibidores , Neurofibromina 2/genética , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA/genética , Deleção de Sequência , Ativação Transcricional , TransfecçãoRESUMO
In C2C12 myoblasts, endogenous histone deacetylase HDAC4 shuttles between cytoplasmic and nuclear compartments, supporting the hypothesis that its subcellular localization is dynamically regulated. However, upon differentiation, this dynamic equilibrium is disturbed and we find that HDAC4 accumulates in the nuclei of myotubes, suggesting a positive role of nuclear HDAC4 in muscle differentiation. Consistent with the notion of regulation of HDAC4 intracellular trafficking, we reveal that HDAC4 contains a modular structure consisting of a C-terminal autonomous nuclear export domain, which, in conjunction with an internal regulatory domain responsive to calcium/calmodulin-dependent protein kinase IV (CaMKIV), determines its subcellular localization. CaMKIV phosphorylates HDAC4 in vitro and promotes its nuclear-cytoplasmic shuttling in vivo. However, although 14-3-3 binding of HDAC4 has been proposed to be important for its cytoplasmic retention, we find this interaction to be independent of CaMKIV. Rather, the HDAC4.14-3-3 complex exists in the nucleus and is required to confer CaMKIV responsiveness. Our results suggest that the subcellular localization of HDAC4 is regulated by sequential phosphorylation events. The first event is catalyzed by a yet to be identified protein kinase that promotes 14-3-3 binding, and the second event, involving protein kinases such as CaMKIV, leads to efficient nuclear export of the HDAC4.14-3-3 complex.
Assuntos
Histona Desacetilases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas 14-3-3 , Animais , Transporte Biológico , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Diferenciação Celular , Núcleo Celular/enzimologia , Citoplasma/metabolismo , Histona Desacetilases/química , Humanos , Fosforilação , Proteínas Repressoras/química , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
How cytokines control differentiation of helper T (TH) cells is controversial. We show that T-bet, without apparent assistance from interleukin 12 (IL-12)/STAT4, specifies TH1 effector fate by targeting chromatin remodeling to individual interferon-gamma (IFN-gamma) alleles and by inducing IL-12 receptor beta2 expression. Subsequently, it appears that IL-12/STAT4 serves two essential functions in the development of TH1 cells: as growth signal, inducing survival and cell division; and as trans-activator, prolonging IFN-gamma synthesis through a genetic interaction with the coactivator, CREB-binding protein. These results suggest that a cytokine does not simply induce TH fate choice but instead may act as an essential secondary stimulus that mediates selective survival of a lineage.
Assuntos
Interferon gama/biossíntese , Interleucina-12/metabolismo , Células Th1/imunologia , Fatores de Transcrição/metabolismo , Alelos , Animais , Proteína de Ligação a CREB , Diferenciação Celular , Divisão Celular , Linhagem da Célula , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Interferon gama/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12 , Fator de Transcrição STAT4 , Transdução de Sinais , Proteínas com Domínio T , Células Th1/citologia , Células Th1/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
The tumor suppressor p53 is activated in response to many types of cellular and environmental insults via mechanisms involving post-translational modification. Here we demonstrate that, unlike phosphorylation, p53 invariably undergoes acetylation in cells exposed to a variety of stress-inducing agents including hypoxia, anti-metabolites, nuclear export inhibitor and actinomycin D treatment. In vivo, p53 acetylation is mediated by the p300 and CBP acetyltransferases. Overexpression of either p300 or CBP, but not an acetyltransferase-deficient mutant, efficiently induces specific p53 acetylation. In contrast, MDM2, a negative regulator of p53, actively suppresses p300/CBP-mediated p53 acetylation in vivo and in vitro. This inhibitory activity of MDM2 on p53 acetylation is in turn abrogated by tumor suppressor p19(ARF), indicating that regulation of acetylation is a central target of the p53-MDM2-p19(ARF) feedback loop. Functionally, inhibition of deacetylation promotes p53 stability, suggesting that acetylation plays a positive role in the accumulation of p53 protein in stress response. Our results provide evidence that p300/CBP-mediated acetylation may be a universal and critical modification for p53 function.
Assuntos
Acetiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Genes Supressores de Tumor , Humanos , Proteínas Proto-Oncogênicas c-mdm2 , Supressão Genética , Proteína Supressora de Tumor p14ARFRESUMO
The cAMP response element binding protein (CREB)-binding protein (CBP)/p300 family of coactivator proteins regulates gene transcription through the integration of multiple signal transduction pathways. Here, we show that induction of tumor necrosis factor alpha (TNF-alpha) gene expression in T cells stimulated by engagement of the T cell receptor (TCR) or by virus infection requires CBP/p300. Strikingly, in mice lacking one copy of the CBP gene, TNF-alpha gene induction by TCR activation is inhibited, whereas virus induction of the TNF-alpha gene is not affected. Consistent with these findings, the transcriptional activity of CBP is strongly potentiated by TCR activation but not by virus infection of T cells. Thus, CBP gene dosage and transcriptional activity are critical in TCR-dependent TNF-alpha gene expression, demonstrating a stimulus-specific requirement for CBP in the regulation of a specific gene.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas Nucleares/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Transativadores/fisiologia , Fator de Necrose Tumoral alfa/genética , Animais , Proteína de Ligação a CREB , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Células HeLa , Humanos , Camundongos , Fatores de Transcrição NFATC , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Mice with monoallelic inactivation of the CBP gene develop highly penetrant, multilineage defects in hematopoietic differentiation and, with advancing age, an increased incidence of hematologic malignancies. The latter are characterized, at least in some cases, by loss of heterozygosity (LOH) at the CBP locus. No such pathology was observed in wild-type or p300 heterozygous null mice of the same age and genetic background. Thus, a full complement of CBP, but not p300, is required for normal hematopoietic differentiation. These results also provide the first experimental evidence for the hypothesis that CBP has tumor-suppressing activity.
Assuntos
Genes Supressores de Tumor/genética , Neoplasias Hematológicas/genética , Hematopoese/genética , Proteínas Nucleares/genética , Transativadores/genética , Animais , Southern Blotting , Western Blotting , Transplante de Medula Óssea , Proteína de Ligação a CREB , Transplante de Células , Proteína p300 Associada a E1A , Neoplasias Hematológicas/patologia , Heterozigoto , Perda de Heterozigosidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/metabolismo , Fenótipo , Baço/citologia , Transativadores/metabolismoRESUMO
The Drosophila ecdysone receptor (EcR)/ultraspiracle (USP) heterodimer is a key regulator in molting and metamorphoric processes, activating and repressing transcription in a sequence-specific manner. Here, we report the isolation of an EcR-interacting protein, SMRTER, which is structurally divergent but functionally similar to the vertebrate nuclear corepressors SMRT and N-CoR. SMRTER mediates repression by interacting with Sin3A, a repressor known to form a complex with the histone deacetylase Rpd3/HDAC. Importantly, we identify an EcR mutant allele that fails to bind SMRTER and is characterized by developmental defects and lethality. Together, these results reveal a novel nuclear receptor cofactor that exhibits evolutionary conservation in the mechanism to achieve repression and demonstrate the essential role of repression in hormone signaling.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila , Drosophila melanogaster/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/fisiologia , Sequência de Aminoácidos , Animais , Evolução Biológica , Linhagem Celular , Mapeamento Cromossômico , Cromossomos/genética , Cromossomos/ultraestrutura , Proteínas Correpressoras , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Variação Genética , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/química , Correpressor 1 de Receptor Nuclear , Correpressor 2 de Receptor Nuclear , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Repressoras/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , VertebradosRESUMO
During melanocyte development, the cytokine Steel factor activates its receptor c-Kit, initiating a signal transduction cascade, which is vital for lineage determination via unknown downstream nuclear targets. c-Kit has recently been found to trigger mitogen-activated protein kinase-mediated phosphorylation of Microphthalmia (Mi), a lineage-restricted transcription factor, which, like Steel factor and c-Kit, is essential for melanocyte development. This cascade results in increased Mi-dependent transcriptional reporter activity. Here we examine the mechanism by which Mi is activated by this pathway. Phosphorylation does not significantly alter Mi's nuclear localization, DNA binding, or dimerization. However, the transcriptional coactivator p300/CBP selectively associates with mitogen-activated protein kinase-phosphorylated Mi, even under conditions in which non-MAPK phospho-Mi is more abundant. Moreover, p300/CBP coactivates Mi transcriptional activity in a manner dependent upon this phosphorylation. Mi thus joins CREB as a transcription factor whose signal-responsive phosphorylation regulates coactivator recruitment, in this case modulating lineage development in melanocytes.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Melanócitos/fisiologia , Proteínas Nucleares/fisiologia , Transdução de Sinais , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Animais , Proteína de Ligação a CREB , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cricetinae , Proteínas de Ligação a DNA/genética , Dimerização , Proteína p300 Associada a E1A , Ativação Enzimática , Humanos , Camundongos , Fator de Transcrição Associado à Microftalmia , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-kit/fisiologia , Coelhos , Fator de Células-Tronco/fisiologia , Ativação Transcricional , Células Tumorais CultivadasRESUMO
The related proteins p300 and CBP (cAMP-response-element-binding protein (CREB)-binding protein)) are transcriptional co-activators that act with other factors to regulate gene expression and play roles in many cell-differentiation and signal transduction pathways. Both proteins have intrinsic histone-acetyltransferase activity and may act directly on chromatin, of which histone is a component, to facilitate transcription. They are also involved in growth control pathways, as shown by their interaction with the tumour suppressor p53 and the viral oncogenes E1A and SV40 T antigen. Here we report functional differences of p300 and CBP in vivo. We examined their roles during retinoic-acid-induced differentiation, cell-cycle exit and programmed cell death (apoptosis) of embryonal carcinoma F9 cells, using hammerhead ribozymes capable of cleaving either p300 or CBP messenger RNAs. F9 cells expressing a p300-specific ribozyme became resistant to retinoic-acid-induced differentiation, whereas cells expressing a CBP-specific ribozyme were unaffected. Similarly, retinoic-acid-induced transcriptional upregulation of the cell-cycle inhibitor p21Cip1 required normal levels of p300, but not CBP, whereas the reverse was true for p27Kip1. In contrast, both ribozymes blocked retinoic-acid-induced apoptosis, indicating that both co-activators are required for this process. Thus, despite their similarities, p300 and CBP have distinct functions during retinoic-acid-induced differentiation of F9 cells.
Assuntos
Proteínas de Ciclo Celular , Diferenciação Celular/fisiologia , Proteínas Nucleares/fisiologia , Transativadores , Fatores de Transcrição/fisiologia , Tretinoína/farmacologia , Proteínas Supressoras de Tumor , Apoptose , Proteína de Ligação a CREB , Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/biossíntese , Ciclinas/genética , Regulação da Expressão Gênica , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , RNA Catalítico/metabolismo , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
The transcriptional coactivator and integrator p300 and its closely related family member CBP mediate multiple, signal-dependent transcriptional events. We have generated mice lacking a functional p300 gene. Animals nullizygous for p300 died between days 9 and 11.5 of gestation, exhibiting defects in neurulation, cell proliferation, and heart development. Cells derived from p300-deficient embryos displayed specific transcriptional defects and proliferated poorly. Surprisingly, p300 heterozygotes also manifested considerable embryonic lethality. Moreover, double heterozygosity for p300 and cbp was invariably associated with embryonic death. Thus, mouse development is exquisitely sensitive to the overall gene dosage of p300 and cbp. Our results provide genetic evidence that a coactivator endowed with histone acetyltransferase activity is essential for mammalian cell proliferation and development.
Assuntos
Desenvolvimento Embrionário e Fetal/genética , Dosagem de Genes , Transativadores/fisiologia , Animais , Encéfalo/anormalidades , Proteínas Estimuladoras de Ligação a CCAAT , Divisão Celular , Células Cultivadas , Cruzamentos Genéticos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Coração/embriologia , Heterozigoto , Histona Acetiltransferases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Defeitos do Tubo Neural/genética , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Coativador 3 de Receptor Nuclear , Receptores do Ácido Retinoico/genética , Transativadores/genética , Transcrição GênicaRESUMO
Differentiation of skeletal muscle cells and B lymphocytes is regulated by basic helix-loop-helix (bHLH) proteins. Both differentiation programs are inhibited by the adenovirus E1A oncoprotein. Analysis of E1A mutants has implicated two of its cellular-binding proteins, p300 and CBP, in controlling certain aspects of differentiation. We find that p300 can cooperate with tissue-specific bHLH proteins in activating target genes and requires only the bHLH domain of such proteins to stimulate E box-directed transcription. Importantly, the ability of bHLH proteins to activate transcription correlates with the presence of p300/CBP in E box-dependent DNA-binding complexes, because both phenomena require at least two adjacent E-box motifs. Microinjection of p300/CBP antibodies into myoblasts blocks terminal differentiation, cell fusion, and transcriptional activity of myogenic bHLH proteins. These results suggest that the function of p300/CBP is essential for the execution of key aspects of cellular differentiation.
Assuntos
Linfócitos B/citologia , Proteínas de Ligação a DNA/metabolismo , Sequências Hélice-Alça-Hélice , Músculo Esquelético/citologia , Proteínas Nucleares/metabolismo , Transativadores , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Sítios de Ligação , Proteína de Ligação a CREB , Diferenciação Celular , Extratos Celulares , Fusão Celular/fisiologia , DNA/metabolismo , DNA Recombinante , Proteínas de Ligação a DNA/análise , Proteína p300 Associada a E1A , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Fatores de Transcrição MEF2 , Camundongos , Desenvolvimento Muscular , Fibras Musculares Esqueléticas , Músculo Esquelético/crescimento & desenvolvimento , Proteína MyoD/análise , Proteína MyoD/metabolismo , Fatores de Regulação Miogênica , Miogenina/análise , Miogenina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Fatores de Transcrição TCF , Proteína 1 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Ativação Transcricional/fisiologiaRESUMO
p300 and its family member, CREB-binding protein (CBP), function as key transcriptional coactivators by virtue of their interaction with the activated forms of certain transcription factors. In a search for additional cellular targets of p300/CBP, a protein-protein cloning strategy, surprisingly identified SRC-1, a coactivator involved in nuclear hormone receptor transcriptional activity, as a p300/CBP interactive protein. p300 and SRC-1 interact, specifically, in vitro and they also form complexes in vivo. Moreover, we show that SRC-1 encodes a new member of the basic helix-loop-helix-PAS domain family and that it physically interacts with the retinoic acid receptor in response to hormone binding. Together, these results implicate p300 as a component of the retinoic acid signaling pathway, operating, in part, through specific interaction with a nuclear hormone receptor coactivator, SRC-1.
Assuntos
Proteínas Nucleares/metabolismo , Transativadores , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Compartimento Celular , Clonagem Molecular , Sequência Consenso , DNA Complementar/genética , Proteína p300 Associada a E1A , Técnica Indireta de Fluorescência para Anticorpo , Histona Acetiltransferases , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Coativador 1 de Receptor Nuclear , Ligação Proteica , Receptores do Ácido Retinoico/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
During metamorphosis of Drosophila melanogaster, a cascade of morphological changes is triggered by the steroid hormone 20-OH ecdysone via the ecdysone receptor, a member of the nuclear receptor superfamily. In this report, we have transferred insect hormone responsiveness to mammalian cells by the stable expression of a modified ecdysone receptor that regulates an optimized ecdysone responsive promoter. Inductions reaching 4 orders of magnitude have been achieved upon treatment with hormone. Transgenic mice expressing the modified ecdysone receptor can activate an integrated ecdysone responsive promoter upon administration of hormone. A comparison of tetracycline-based and ecdysone-based inducible systems reveals the ecdysone regulatory system exhibits lower basal activity and higher inducibility. Since ecdysone administration has no apparent effect on mammals, its use for regulating genes should be excellent for transient inducible expression of any gene in transgenic mice and for gene therapy.
Assuntos
Ecdisona/farmacologia , Expressão Gênica/efeitos dos fármacos , Animais , Sequência de Bases , Disponibilidade Biológica , Linhagem Celular , Drosophila melanogaster , Ecdisterona/análogos & derivados , Ecdisterona/farmacocinética , Ecdisterona/farmacologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores de Esteroides/efeitos dos fármacos , Receptores de Esteroides/genética , Tetraciclina/farmacologia , TransfecçãoRESUMO
Seven-up (Svp), the Drosophila homolog of the chicken ovalbumin upstream transcription factor (COUP-TF); Ultraspiracle (Usp), the Drosophila homolog of the retinoid X receptor; and the ecdysone receptor are all members of the nuclear/steroid receptor superfamily. COUP-TF negatively regulates hormonal signaling involving retinoid X receptor in tissue culture systems. Here we demonstrate that Svp, like COUP-TF, can modulate Ultraspiracle-based hormonal signaling both in vitro and in vivo. Transfection assays in CV-1 cells demonstrate that Seven-up can inhibit ecdysone-dependent transactivation by the ecdysone receptor complex, a heterodimeric complex of Usp and ecdysone receptor. This repression depends on the dose of Svp and occurs with two different Drosophila ecdysone response elements. Ectopic expression of Svp in vivo induces lethality during early metamorphosis, the time of maximal ecdysone responsiveness. Concomitant overexpression of Usp rescues the larvae from the lethal effects of Svp. DNA binding studies show that Svp can bind to various direct repeats of the sequence AGGTCA but cannot bind to one of the ecdysone response elements used in the transient transfection assays. Our results suggest that Svp-mediated repression can occur by both DNA binding competition and protein-protein interactions.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/fisiologia , Receptores de Esteroides/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Galinhas , Chlorocebus aethiops , Clonagem Molecular , Proteínas de Ligação a DNA/biossíntese , Proteínas de Drosophila , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Receptores de Esteroides/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Ativação Transcricional , TransfecçãoRESUMO
In a search for retinoid X receptor-like molecules in Drosophila, we have identified an additional member of the nuclear receptor superfamily, XR78E/F. In the DNA-binding domain, XR78E/F is closely related to the mammalian receptor TR2, as well as to the nuclear receptors Coup-TF and Seven-up. We demonstrate that XR78E/F binds as a homodimer to direct repeats of the sequence AGGTCA. In transient transfection assays, XR78E/F represses ecdysone signaling in a DNA-binding-dependent fashion. XR78E/F has its highest expression in third-instar larvae and prepupae. These experiments suggest that XR78E/F may play a regulatory role in the transcriptional cascade triggered by the hormone ecdysone in Drosophila.
Assuntos
Drosophila/metabolismo , Ecdisona/metabolismo , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Southern Blotting , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Ecdisterona/análogos & derivados , Ecdisterona/farmacologia , Genes de Insetos , Biblioteca Genômica , Hibridização In Situ , Dados de Sequência Molecular , Ligação Proteica , Receptores do Ácido Retinoico/genética , Proteínas Recombinantes/metabolismo , Receptores X de Retinoides , Análise de Sequência de DNA , Fatores de Tempo , Distribuição Tecidual , Fatores de Transcrição/genéticaRESUMO
Although the biological activity of the insect moulting hormone ecdysone, is manifested through a hormonally regulated transcriptional cascade associated with chromosomal puffing, a direct association of the receptor with the puff has yet to be established. The cloned ecdysone receptor (EcR) is by itself incapable of high-affinity DNA binding or transcriptional activation. Rather, these activities are dependent on heterodimer formation with Ultraspiracle (USP) the insect homologue of vertebrate retinoid X receptor. Here we report that native EcR and USP are co-localized on ecdysone-responsive loci of polytene chromosomes. Moreover, we show that natural ecdysones selectively promote physical association between EcR and USP, and conversely, that high-affinity hormone binding requires both EcR and USP. Replacement of USP with retinoid X receptor produces heterodimers with distinct pharmacological and functional properties. These results redefine the ecdysone receptor as a dynamic complex whose activity may be altered by combinatorial interactions among subunits and ligand.
Assuntos
Cromossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Ecdisona/metabolismo , Genes de Insetos , Receptores do Ácido Retinoico , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Ecdisterona/análogos & derivados , Ecdisterona/metabolismo , Ecdisterona/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/genética , Receptores X de Retinoides , Fatores de Transcrição/genéticaRESUMO
The vertebrate retinoid X receptor (RXR) has been implicated in the regulation of multiple hormonal signaling pathways through the formation of heteromeric receptor complexes that bind DNA with high affinity. We now demonstrate that ultraspiracle (usp), a Drosophila RXR homolog, can substitute for RXR in stimulating the DNA binding of receptors for retinoic acid, T3, vitamin D, and peroxisome proliferator activators. These observations led to the search and ultimate identification of the ecdysone receptor (EcR) as a Drosophila partner of usp. Together, usp and EcR bind DNA in a highly cooperative fashion. Cotransfection of both EcR and usp expression vectors is required to render cultured mammalian cells ecdysone responsive. These results implicate usp as an integral component of the functional EcR. By demonstrating that receptor heterodimer formation precedes the divergence of vertebrate and invertebrate lineages, these data underscore a central role for RXR and its homolog usp in the evolution and control of the nuclear receptor-based endocrine system.