RESUMO
Cell growth and differentiation are opposite events in the myogenic lineage. Growth factors block the muscle differentiation program by inducing the expression of transcription factors that negatively regulate the expression of muscle regulatory genes like MyoD. In contrast, extracellular clues that induce cell cycle arrest promote MyoD expression and muscle differentiation. Thus, the regulation of MyoD expression is critical for muscle differentiation. Here we show that estrogen induces MyoD expression in mouse skeletal muscle in vivo and in dividing myoblasts in vitro by relieving the MyoD promoter from AP-1 negative regulation through a mechanism involving estrogen receptor/AP-1 protein-protein interactions but independent of the estrogen receptor DNA binding activity.
Assuntos
Regulação da Expressão Gênica , Desenvolvimento Muscular/genética , Proteína MyoD/genética , Receptores de Estrogênio/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Estrogênios/metabolismo , Estrogênios/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Mioblastos Esqueléticos/efeitos dos fármacos , Mioblastos Esqueléticos/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Little is known about the temporal relationship and the sequential steps for peptide biosynthesis during the terminal differentiation of the peptide phenotype in central nervous system. Analysis of the TRH phenotype in primary cultures of rat fetal day 17 hypothalamic cells has shown that TRH levels start increasing only after a week in culture, in contrast with in vivo data showing a steady increase during late fetal life. The purpose of this study was to compare the developmental patterns of TRH and pro-TRH mRNA levels in vitro to determine whether the initial low and steady levels of TRH are due to deficient transcription. Pro-TRH mRNA levels were detected by semi-quantitative RT-PCR through the development of primary cultures of serum-supplemented hypothalamic fetal cells from 17 day old embryos. Pro-TRH mRNA levels per dish increased steadily since the beginning of the culture. In contrast, TRH levels per dish were low and stable during the first week increasing afterwards, but remaining low compared to equivalent in vivo values. Pro-TRH mRNA levels per hypothalamus increased between fetal day 17 and postnatal 14, suggesting that the in vitro pattern of pro-TRH mRNA development mimics that occurring in vivo. These data show that pro-TRH gene expression does not limit TRH accumulation in vitro suggesting that the transcriptional and post-transcriptional programs leading to peptide accumulation are established independently.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hipotálamo/citologia , Neurônios/fisiologia , Precursores de Proteínas/genética , Hormônio Liberador de Tireotropina/genética , Animais , Células Cultivadas , Feminino , Feto/citologia , Hipotálamo/embriologia , Técnicas In Vitro , Neurônios/citologia , Gravidez , Ácido Pirrolidonocarboxílico/análogos & derivados , RNA Mensageiro/análise , Ratos , Ratos WistarRESUMO
Thyrotropin releasing hormone (TRH) present in several brain areas has been proposed as a neuromodulator. Its administration produces opposite effects to those observed with acute ethanol consumption. Opioid peptides, in contrast, have been proposed to mediate some of the effects of alcohol intoxication. We measured TRH content and the levels of its mRNA in hypothalamic and limbic zones 1-24 h after acute ethanol injection. We report here fast and transient changes in the content of TRH and its mRNA in these areas. The levels of proenkephalin mRNA varied differently from those of proTRH mRNA, depending on the time and region studied. Wistar rats were administered one dose of ethanol (intraperitoneal, 3 g/kg body weight) and brains dissected in hypothalamus, hippocampus, amygdala, n. accumbens and frontal cortex, for TRH quantification by radioimmunoassay or for proTRH mRNA measurement by RT-PCR. After 1 h injection, TRH levels were increased in hippocampus and decreased in n. accumbens; after 4 h, it decreased in the hypothalamus, frontal cortex and amygdala, recovering to control values in all regions at 24 h. ProTRH mRNA levels increased at 1 h post-injection in total hypothalamus and hippocampus, while they decreased in the frontal cortex. The effect of ethanol was also studied in primary culture of hypothalamic cells; a fast and transient increase in proTRH mRNA was observed at 1 h of incubation (0.001% final ethanol concentration). Changes in the mRNA levels of proTRH and proenkephalin were quantified by in situ hybridization in rats administered ethanol intragastrically (2.5 g/kg). Opposite alterations were observed for these two mRNAs in hippocampus and frontal cortex, while in n. accumbens and the paraventricular nucleus of the hypothalamus, both mRNA levels were increased but with different kinetics. These results give support for TRH and enkephalin neurons as targets of ethanol and, as possible mediators of some of its observed behavioral effects.
Assuntos
Encefalinas/metabolismo , Etanol/farmacologia , Hipotálamo/metabolismo , Sistema Límbico/metabolismo , Precursores de Proteínas/metabolismo , Hormônio Liberador de Tireotropina/metabolismo , Animais , Encefalinas/genética , Injeções Intraperitoneais , Masculino , Precursores de Proteínas/genética , Ácido Pirrolidonocarboxílico/análogos & derivados , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Hormônio Liberador de Tireotropina/genética , Distribuição TecidualRESUMO
External clues for neuron development include extracellular matrix (ECM) molecules. To explore ECM influence on the early development of peptide phenotype in the CNS, we have compared pro-TRH levels in primary cultures of rat hypothalamic cells plated either on poly-lysine (PL) (control) or on PL plus one of various ECM molecules at 10 microgram/ml. Fetal day 17 cells plated at a density of 1250/mm(2) were grown in a serum free medium made of Neurobasal medium supplemented with B27 (GIBCO). Cultures, consisting mainly of neurons, were analyzed at DIV 2. ECM proteins induced morphological effects in agreement with previously published studies. The amount of pro-TRH per dish, quantified by Western blotting, was increased to 275% for laminin, 191% for fibronectin and 173% for tenascin-C (control=100%); there was no effect of vitronectin. Laminin or fibronectin did not change pro-TRH mRNA or TRH levels but enhanced levels of the pro-protein convertase PC1 suggesting that the ECM molecules did regulate the translational status of pro-TRH. In conclusion, we have shown that some ECM proteins increased pro-TRH level in vitro; this may contribute to the enhancement of pro-TRH levels observed early in vivo in the hypothalamus.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Hipotálamo/citologia , Neurônios/enzimologia , Precursores de Proteínas/metabolismo , Hormônio Liberador de Tireotropina/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Feto/citologia , Fibronectinas/análise , Regulação da Expressão Gênica no Desenvolvimento , Hipotálamo/embriologia , Técnicas In Vitro , Laminina/análise , Neurônios/química , Neurônios/citologia , Pró-Proteína Convertases , Biossíntese de Proteínas/fisiologia , Precursores de Proteínas/genética , Ácido Pirrolidonocarboxílico/análogos & derivados , RNA Mensageiro/análise , Radioimunoensaio , Ratos , Ratos Wistar , Tenascina/análise , Hormônio Liberador de Tireotropina/genética , Vitronectina/análiseRESUMO
The biosynthesis of thyrotropin-releasing hormone (TRH) in the hypothalamic paraventricular nucleus (PVN) is subject to neural and hormonal regulations. To identify some of the potential effectors of this modulation, we incubated hypothalamic dispersed cells with dexamethasone for short periods of time (1-3 h) and studied the interaction of this hormone with protein kinase C (PKC) and PKA signaling pathways. TRH mRNA relative changes were determined by the RT-PCR technique. One hour incubation with 10(-10)-10(-4) M dexamethasone produced a concentration-dependent biphasic effect: an inhibition was observed on TRH mRNA levels at 10(-10) M, an increase above control at 10(-8)-10(-6) M and a reduction at higher concentrations (10(-5)- 10(-4) M). The stimulatory effect of 10(-8) M dexamethasone on TRH mRNA was essentially independent of new protein synthesis, as evidenced by cycloheximide pretreatment. Changes in TRH mRNA levels were reflected by enhanced TRH cell content. Incubation with a cAMP analogue (8-bromo-cAMP, 8Br-cAMP) or with a PKC activator (12-O-tetradecanoylphorbol-13-acetate, TPA) increased TRH mRNA levels after 1 and 2 h, respectively. An increase in TRH mRNA expression was observed by in situ hybridization of dexamethasone or 8Br-cAMP-treated cells. The interaction of dexamethasone, PKA and PKC signaling pathways was studied by combined treatment. The stimulatory effect of 10(-7) M TPA on TRH mRNA levels was additive to that of dexamethasone; in contrast, coincubation with 10(-3) M 8-Br-cAMP and dexamethasone diminished the stimulatory effect of both drugs. An inhibition was observed when the cAMP analogue was coincubated with TPA or TPA and dexamethasone. These results demonstrate that dexamethasone can rapidly regulate TRH biosynthesis and suggest a cross talk between cAMP, glucocorticoid receptors and PKC transducing pathways.