RESUMO
The DNA-binding domain of nuclear hormone receptors functions as an interaction interface for other transcription factors. Using the DNA-binding domain of TRbeta1 as bait in the yeast two-hybrid system, we cloned the Tat binding protein-1 that was originally isolated as a protein binding to the human immunodeficiency virus type 1 Tat transactivator. Tat binding protein-1 has subsequently been identified as a member of the ATPase family and a component of the 26S proteasome. Tat binding protein-1 interacted with the DNA-binding domain but not with the ligand binding domain of TR in vivo and in vitro. TR bound to the amino-terminal portion of Tat binding protein-1 that contains a leucine zipper-like structure. In mammalian cells, Tat binding protein-1 potentiated the ligand-dependent transactivation by TRbeta1 and TRalpha1 via thyroid hormone response elements. Both the intact DNA-binding domain and activation function-2 of the TR were required for the transcriptional enhancement in the presence of Tat binding protein-1. Tat binding protein-1 did not augment the transactivation function of the RAR, RXR, PPARgamma, or ER. The intrinsic activation domain in Tat binding protein-1 resided within the carboxyl-terminal conserved ATPase domain, and a mutation of a putative ATP binding motif but not a helicase motif in the carboxyl-terminal conserved ATPase domain abolished the activation function. Tat binding protein-1 synergistically activated the TR-mediated transcription with the steroid receptor coactivator 1, p120, and cAMP response element-binding protein, although Tat binding protein-1 did not directly interact with these coactivators in vitro. In contrast, the N-terminal portion of Tat binding protein-1 directly interacted in vitro and in vivo with the TR-interacting protein 1 possessing an ATPase activity that interacts with the activation function-2 of liganded TR. Collectively, Tat binding protein-1 might function as a novel DNA-binding domain-binding transcriptional coactivator specific for the TR probably in cooperation with other activation function-2-interacting cofactors such as TR-interacting protein 1.
Assuntos
Proteínas de Ligação a DNA/metabolismo , HIV-1/química , Complexo de Endopeptidases do Proteassoma , Receptores dos Hormônios Tireóideos/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Clonagem Molecular , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/farmacologia , Dimerização , Sinergismo Farmacológico , Proteínas Fúngicas/genética , Expressão Gênica , Glutationa Transferase/genética , Proteína Vmw65 do Vírus do Herpes Simples/genética , Humanos , Luciferases/genética , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/fisiologia , Proteínas Recombinantes de Fusão , Elementos de Resposta , Hormônios Tireóideos/farmacologia , Transativadores/genética , Transativadores/farmacologia , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional , TransfecçãoRESUMO
We recently reported that TRH-deficient mice showed characteristic tertiary hypothyroidism. In the present study, we investigated how this tertiary hypothyroidism occurred particularly in pre- and postnatal stages. Immunohistochemical analysis revealed a number of TSH-immunopositive cells in the TRH-/- pituitary on embryonic day 17.5 and at birth. The mutant pituitary at birth in pups born from TRH-deficient dams also showed no apparent morphological changes, indicating no requirement of either maternal or embryonic TRH for the development of pituitary thyrotrophs. In contrast, apparent decreases in number and level of staining of TSH-immunopositive cells were observed after postnatal day 10 in mutant pituitary. Similar decreases were observed in the 8-week-old mutant pituitary, while no apparent changes were observed in other pituitary hormone-producing cells, and prolonged TRH administration completely reversed this effect. Consistent with these morphological results, TRH-/- mice showed normal thyroid hormone levels at birth, but the subsequent postnatal increase was depressed, resulting in hypothyroidism. As expected, TSH content in the TRH-/- pituitary showed a marked reduction to only 40% of that in the wild type. Despite hypothyroidism in the mutant mice, both the pituitary TSHbeta and alpha mRNA levels were lower than those of the wild-type pituitary. These phenotypic changes were specific to the pituitary thyrotrophs. These findings indicated that 1) TRH is essential only for the postnatal maintenance of the normal function of pituitary thyrotrophs, including the normal feedback regulation of the TSH gene by thyroid hormone; 2) neither maternal nor embryonic TRH is required for normal development of the fetal pituitary thyrotroph; and 3) TRH-deficient mice do not exhibit hypothyroidism at birth. Moreover, reflecting its name, TRH has more critical effects on the pituitary thyrotrophs than on other pituitary hormone-producing cells.
Assuntos
Hipotireoidismo/metabolismo , Hipófise/embriologia , Hipófise/metabolismo , Hormônio Liberador de Tireotropina/fisiologia , Tireotropina/metabolismo , Animais , Northern Blotting , Hipotireoidismo Congênito , Genótipo , Hipotireoidismo/genética , Imuno-Histoquímica , Camundongos , Hipófise/patologia , Hormônios Hipofisários/metabolismo , RNA Mensageiro/metabolismo , Recombinação Genética , Hormônios Tireóideos/metabolismo , Tireotropina/genética , Hormônio Liberador de Tireotropina/biossíntese , Hormônio Liberador de Tireotropina/genética , Fatores de TempoRESUMO
Histone acetylation status influences transcriptional activity, and the mechanism of negative gene regulation by thyroid hormone remains unclear, although its impairment by a mutant thyroid hormone receptor (TR) is critical for resistance to thyroid hormone (RTH). We found a novel RTH mutant, F455S, that exhibited impaired repression of the TRH gene and had a strong dominant-negative effect on the gene. F455S strongly interacted with nuclear receptor corepressor (NCoR) and was hard to dissociate from it. To analyze the dynamics of histone acetylation status in vivo, we established cell lines stably expressing the TRH promoter and wild-type or F455S TR. Treatment with a histone deacetylase (HDAC) inhibitor completely abolished the repression of the gene by T3. The histones H3 and H4 at the TRH promoter were acetylated, and addition of T3 caused recruitment of HDACs 2 and 3 within 15 min, resulting in a transient deacetylation of the histone tails. TR and NCoR were located on the promoter, and T3 caused NCoR dissociation and steroid receptor coactivator-1 recruitment. In the presence of F455S, the histones were hyperacetylated, and HDAC recruitment and histone deacetylation were significantly impaired. This is the first report demonstrating the direct involvement of aberrant dynamics of chromatin modification in RTH.
Assuntos
Histonas/metabolismo , Síndrome da Resistência aos Hormônios Tireóideos/genética , Hormônio Liberador de Tireotropina/genética , Acetilação , Animais , Linhagem Celular , Criança , DNA/metabolismo , Dimerização , Feminino , Histona Acetiltransferases , Inibidores de Histona Desacetilases , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Luciferases/genética , Luciferases/metabolismo , Proteínas Nucleares/metabolismo , Correpressor 1 de Receptor Nuclear , Coativador 1 de Receptor Nuclear , Mutação Puntual , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Síndrome da Resistência aos Hormônios Tireóideos/tratamento farmacológico , Síndrome da Resistência aos Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/uso terapêutico , Hormônio Liberador de Tireotropina/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Tri-Iodotironina/metabolismoRESUMO
Retinoic acid (RA) has been reported to inhibit the secretion and synthesis of the pituitary TSH in vivo and in vitro. However, little is known about the influence of RA on the expression of the prepro-TRH gene. We therefore investigated whether the promoter activity of the mouse TRH gene is directly regulated by RA using a transient transfection assay into CV-1 cells. In the absence of cotransfected RA receptor (RAR), all-trans-RA did not affect the promoter activity. In contrast, the cotransfected RARalpha significantly stimulated promoter activity in the absence of ligand, and all-trans-RA reversed basal promoter activation. The cotransfected thyroid hormone receptor-beta (TRbeta), but not 9-cis-RA receptor (RXR), had an additive effect on the RAR-dependent stimulation. TR and RAR can similarly interact with the corepressor proteins, and the cotransfected nuclear receptor corepressor (N-CoR) has been demonstrated to augment the transcriptional stimulation of the TRH gene by unliganded TR. As observed with TR, the coexpression of a N-CoR variant significantly enhanced the ligand-independent stimulation by RAR. A mutant RAR (RAR403) lacking the C-terminal activation function-2 (AF-2) activation domain that was essential for ligand-induced corepressor release constitutively stimulated the promoter activity. The constitutive stimulation by RAR403 was augmented by the cotransfected N-CoR variant. A deletion analysis of the 5'-flanking region of the TRH gene revealed that the minimal promoter region for the regulation by RAR was -83 to +53, with a consensus half-site motif for the thyroid hormone response element at -57. In contrast to the strong binding of TR to the thyroid hormone response element half-site in gel retardation assays, no binding of RAR homodimer, RAR/ RXR heterodimer, or RAR/TR heterodimer was observed to the minimal promoter region. These results collectively suggest that RAR without heterodimerization with RXR and TR regulates transcription of the mouse TRH gene in cooperation with the corepressor, and that the DNA binding of RAR appeared to be unnecessary for regulation of the TRH gene promoter.
Assuntos
Regulação da Expressão Gênica , Precursores de Proteínas/genética , Receptores do Ácido Retinoico/fisiologia , Hormônio Liberador de Tireotropina/genética , Animais , Sítios de Ligação , DNA/metabolismo , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Humanos , Camundongos , Mutagênese , Regiões Promotoras Genéticas , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/genética , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/fisiologia , Receptores X de Retinoides , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transfecção , Tretinoína/farmacologiaRESUMO
Cyclo(His-Pro) or CHP was initially discovered as a metabolite of thyrotropin-releasing hormone (TRH) resulting from the action of the enzyme Pyroglutamyl aminopeptidase. Physiologic and pharmacologic studies that followed this initial discovery provided indirect evidence that all CHP may not be derived from TRH. However, the recent availability of a TRH-deficient mouse has made it possible to reinvestigate whether CHP is derived from TRH. In the present study, we examined distribution of CHP and TRH in TRH-deficient mice. Northern blot analysis confirmed the absence of preproTRH mRNA in both the hypothalamus and the cortex of TRH-deficient mice. Brains from the wild-type and TRH-deficient mice were dissected into 7 regions, and TRH and CHP concentrations were determined by specific radioimmunoassay (RIA) in each region. Whereas TRH was identified in all regions of the wild-type brain, with the highest concentration in the hypothalamus, no detectable TRH was observed in any region in the TRH-deficient mice. While CHP-like immunoreactivity (CHP-LI) was present in all regions in the wild-type brain, its concentration was reduced by approximately 50% in the hypothalamus and cerebral cortex of TRH-deficient mice, with no change in other brain regions. Furthermore, the CHP-LI present in the brain of TRH-deficient mice was immunologically and chromatographically identical to synthetic CHP. These findings strongly suggest that a portion of the CHP in the brain is derived from sources other than TRH.
Assuntos
Antioxidantes/metabolismo , Encéfalo/metabolismo , Inibidores da Captação de Neurotransmissores/metabolismo , Peptídeos Cíclicos/metabolismo , Piperazinas/metabolismo , Animais , Camundongos , Camundongos Mutantes , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , Radioimunoensaio , Hormônio Liberador de Tireotropina/deficiência , Hormônio Liberador de Tireotropina/genéticaRESUMO
We measured the amounts of TRH receptor (TRHR) messenger ribonucleic acid (mRNA) in human normal pituitary and pituitary tumors and found a novel transcript of the TRHR gene. Competitive PCR revealed expression of the TRHR mRNA in all pituitary adenomas examined, and its level was variable and similar to that in the normal pituitary. When the C-terminal region was amplified by PCR, an additional short product was observed. Cloning and sequence analysis of this short fragment revealed that the deleted sequence corresponded exactly to the 5'-sequence of exon 3, indicating alternative splicing of the TRHR mRNA. This alternative splicing resulted in a frame shift, yielding a C-terminal truncated protein (HTRHR2) on translation. Expression analysis of HTRHR2 in Chinese hamster ovary cells showed no significant binding to [3HIMeTRH or response of intracellular calcium to TRH administration. However, the mRNA ratio of HTRHR2 vs. the wild type (HTRHR1) was significantly different among pituitary tumors. The highest ratio was observed in prolactinomas (30%), and almost no detectable expression was found in GH-producing tumors. These findings indicate that this novel transcript of the human TRH receptor gene is produced in a tumor-specific manner and may be a useful parameter for evaluation of individual pituitary tumors.
Assuntos
Adenoma/metabolismo , Hipófise/metabolismo , Neoplasias Hipofisárias/metabolismo , Receptores do Hormônio Liberador da Tireotropina/genética , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Transcrição Gênica/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO/metabolismo , Cricetinae , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismoRESUMO
We cloned and characterized the mouse uncoupling protein 2 (UCP2) gene and its promoter region. The gene spans approximately 6.3 kb and contains eight exons and seven introns. Two short exons are located in the 5' untranslated region, and each of the remaining exons encodes one of the transmembrane domains. 3'-RACE analysis showed that a polyadenylation signal 257 bp downstream from the stop codon was functional. Primer extension analysis indicated a single transcriptional start site 369 bp upstream from the translational start site. The promoter region lacks both TATA and CAAT boxes but is GC-rich. A construct containing 1250 bp of the promoter region showed significant activity in all 6 cell lines examined, and the region between -160 and -678 bp exhibited strong positive regulatory activity. These features of the UCP2 gene are different from those of the UCP1 gene and may contribute to its ubiquitous expression.
Assuntos
Proteínas de Membrana Transportadoras , Proteínas Mitocondriais , Regiões Promotoras Genéticas , Proteínas/genética , Desacopladores , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Éxons , Genes , Íntrons , Canais Iônicos , Camundongos , Dados de Sequência Molecular , Poli A , Reação em Cadeia da Polimerase , Processamento Pós-Transcricional do RNA , Mapeamento por Restrição , Análise de Sequência de DNA , Transcrição Gênica , Proteína Desacopladora 2RESUMO
Thyrotropin-releasing hormone (TRH), originally isolated as a hypothalamic hormone, has been reported to be present and released from the pancreatic beta cells, affecting pancreatic functions. However, it still remains unclear whether TRH receptor is expressed in the pancreas. In the present study, we characterized TRH receptors (TRHR) in mouse pancreatic islets and HIT-T15 cells, a hamster clonal beta cell line. RT-PCR study showed significant expression of TRHR subtype 1 (TRHR1) mRNA in both mouse pancreatic islets and HIT-T15 (HIT) cells. In contrast, there was no expression of TRHR2 mRNA, a novel subtype of TRHR which is expressed predominantly in the central nervous system. Sequencing analysis demonstrated that TRHR1 of the islets was identical to that in the pituitary, and cloned hamster TRHR1 shared 93.3 % homology with that of the mouse at the nucleic acid level. Northern blot analysis of TRHR 1 mRNA in HIT-T15 cells showed a single strong hybridization signal approximately 3.7 kb in length. Furthermore, Scatchard plot analysis in HIT-T15 cells revealed that the Kd value for MeTRH was 0.63 nM. Significant elevation of intracellular calcium concentration was observed in response to as little as 10 nM TRH , and this was not affected by removal of extracellular calcium. This is the first description indicating the presence of functional TRH receptor subtype 1 in the pancreatic beta cells, and our observations suggested the regulation of pancreatic function by TRH through autocrine or paracrine mechanisms.
Assuntos
Ilhotas Pancreáticas/metabolismo , RNA Mensageiro/biossíntese , Receptores do Hormônio Liberador da Tireotropina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Cálcio/metabolismo , Células Cultivadas , Cricetinae , Expressão Gênica , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Hipófise/química , RNA/isolamento & purificação , Receptores do Hormônio Liberador da Tireotropina/biossíntese , Receptores do Hormônio Liberador da Tireotropina/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Hormônio Liberador de Tireotropina/farmacologiaRESUMO
A 66-year-old man was hospitalized for abdominal pain and diarrhea of more than 10 times a day. He had been under regular medication with prednisolone for rheumatoid arthritis. On admission, laboratory data and clinical examination indicated inflammation, dehydration, acute renal failure with a high level of serum musculogenic enzyme (creatine kinase), and ileus. Salmonella enteritidis was isolated from his fecal and blood samples. The patient died within 24 hours after admission, and autopsy showed hemorrhagic necrotic enteritis localized to the ileum. Enterocolitis due to Salmonella enteritidis, which is usually an acute self-limited gastrointestinal illness, may occasionally be a serious and lethal disease.
Assuntos
Injúria Renal Aguda/microbiologia , Bacteriemia/etiologia , Enterite/microbiologia , Infecções por Salmonella/etiologia , Salmonella enteritidis/isolamento & purificação , Injúria Renal Aguda/patologia , Idoso , Bacteriemia/patologia , Enterite/patologia , Evolução Fatal , Fezes/microbiologia , Humanos , Íleo/microbiologia , Íleo/patologia , Masculino , Estudos Retrospectivos , Infecções por Salmonella/patologiaRESUMO
Cone photoreceptors in the murine retina are patterned by dorsal repression and ventral activation of S opsin. TR beta 2, the nuclear thyroid hormone receptor beta isoform 2, regulates dorsal repression. To determine the molecular mechanism by which TR beta 2 acts, we compared the spatiotemporal expression of TR beta 2 and S opsin from embryonic day (E) 13 through adulthood in C57BL/6 retinae. TR beta 2 and S opsin are expressed in cone photoreceptors only. Both are transcribed by E13, and their levels increase with cone genesis. TR beta 2 is expressed uniformly, but transiently, across the retina. mRNA levels are maximal by E17 at completion of cone genesis and again minimal before P5. S opsin is also transcribed by E13, but only in ventral cones. Repression in dorsal cones is established by E17, consistent with the occurrence of patterning during cone cell genesis. The uniform expression of TR beta 2 suggests that repression of S opsin requires other dorsal-specific factors in addition to TR beta 2. The mechanism by which TR beta 2 functions was probed in transgenic animals with TR beta 2 ablated, TR beta 2 that is DNA binding defective, and TR beta 2 that is ligand binding defective. These studies show that TR beta 2 is necessary for dorsal repression, but not ventral activation of S opsin. TR beta 2 must bind DNA and the ligand T3 (thyroid hormone) to repress S opsin. Once repression is established, T3 no longer regulates dorsal S opsin repression in adult animals. The transient, embryonic action of TR beta 2 is consistent with a role (direct and/or indirect) in chromatin remodeling that leads to permanent gene silencing in terminally differentiated, dorsal cone photoreceptors.
Assuntos
Células Fotorreceptoras Retinianas Cones/embriologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Opsinas de Bastonetes/metabolismo , Receptores beta dos Hormônios Tireóideos/metabolismo , Animais , Padronização Corporal , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Opsinas de Bastonetes/genética , Receptores beta dos Hormônios Tireóideos/deficiência , Receptores beta dos Hormônios Tireóideos/genética , Tri-Iodotironina/metabolismoRESUMO
The prolactin-releasing peptide (PrRP) gene is a novel bioactive peptide expressed in very restricted regions in the brain. To explore the molecular mechanism of PrRP gene expression, we cloned and characterized the gene and its promoter region. The gene spans approximately 2.4 kb and contains three exons and two introns. 3'RACE analysis showed that a polyadenylation signal 103 bp downstream from the stop codon was functional. Primer extension analysis indicated three transcriptional start sites (TSSs) 92, 199, and 325 bp upstream from the translational start site. Interestingly, in addition to the putative binding sites for SP1-1, AP-2, and Oct-2A, three characteristic TATA boxes were identified close to these TSSs. Transient transfection study using a series of deletion mutants revealed that the middle TATA box is important for the promoter activity. Furthermore, the cloned 1.6 kb promoter region was active only in neuron- and pituitary-derived cell lines, and the promoter region -1600 approximately -800 bp worked as a negative regulatory element. We demonstrated for the first time, the genomic organization and promoter function of the PrRP gene, and this knowledge will facilitate elucidation of transcriptional control of the PrRP gene.
Assuntos
Hormônios Hipotalâmicos/genética , Neuropeptídeos/genética , Regiões Promotoras Genéticas , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Northern Blotting , Encéfalo/metabolismo , Clonagem Molecular , DNA Complementar/metabolismo , Éxons , Deleção de Genes , Biblioteca Gênica , Íntrons , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Hormônio Liberador de Prolactina , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/metabolismo , TATA Box , Transcrição Gênica , TransfecçãoRESUMO
Thyrotropin-releasing hormone (TRH) is a brain hypothalamic hormone that regulates thyrotropin (TSH) secretion from the anterior pituitary and is ubiquitously distributed throughout the brain and other tissues including pancreas. To facilitate studies into the role of endogenous TRH, we have used homologous recombination to generate mice that lack TRH. These TRH-/- mice are viable, fertile, and exhibit normal development. However, they showed obvious hypothyroidism with characteristic elevation of serum TSH level and diminished TSH biological activity. Their anterior pituitaries exhibited an apparent decrease in TSH immunopositive cells that was not due to hypothyroidism. Furthermore, this decrease could be reversed by TRH, but not thyroid hormone replacement, suggesting a direct involvement of TRH in the regulation of thyrotrophs. The TRH-/- mice also exhibited hyperglycemia, which was accompanied by impaired insulin secretion in response to glucose. These findings indicate that TRH-/- mice provide a model of exploiting tertiary hypothyroidism, and that TRH gene abnormalities cause disturbance of insulin secretion resulting in marked hyperglycemia.