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Muller C Cluzeaud F Pinon GM Rafestin-Oblin ME Morfin R 《The Journal of steroid biochemistry and molecular biology》2004,92(5):469-476
The human brain is a target tissue for glucocorticoids (GC). Dehydroepiandrosterone (DHEA) is a neurosteroid produced in the brain where it is transformed into 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA. The antiglucocorticoid effects of both 7-hydroxylated metabolites have been investigated with evidence in mice that neither form of DHEA interfered with the binding of GC to its glucocorticoid receptor (GR), but contributed to a decreased nuclear uptake of the activated GR. Our objective was to use COS-7 cell culture to research DHEA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA interferences with GR trafficking. These cells did not carry out the 7alpha-hydroxylation of DHEA and the oxidation of cortisol into cortisone. The cDNA of the human GR was inserted into pcDNA3 for a transient transfection of COS-7 cells. Human GR transactivation activity was measured from a luciferase-MMTV reporter gene. The transfected COS-7 cells were cultured using 10(-12) to 10(-5) M dexamethasone (DEX) or cortisol, which triggered the reporter expression. Treatment with 10(-12) to 10(-5) M DHEA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA caused no change in the GC-induced GR transactivation. A reconstruction of the process associated EGFP to the human GR cDNA. Confocal microscopic examination of COS-7 cells transiently expressing the fusion protein EGFP-GR showed nuclear fluorescence 60 min after incubation with 10(-8) M DEX or cortisol. The addition of 10(-5) M DHEA, 7alpha-hydroxy-DHEA or 7beta-hydroxy-DHEA did not change its kinesis and intensity. These results contribute to the knowledge of DHEA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA, in relation to antiglucocorticoid activity. We conclude that direct interference with GR trafficking can be discounted in the case of these hormones, therefore proposing new possibilities of investigation. 相似文献
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Elizabeth S. Browne Johnny R. Porter Gonzalo Correa Jude Abadie Frank Svec 《The Journal of steroid biochemistry and molecular biology》1993,45(6):517-524
Dehydroepiandrosterone (DHEA) decreases the activity of hepatic tyrosine aminotransferase (TAT), a glucocorticoid-inducible enzyme, in the obese, hypercorticosteronemic Zucker rat. To investigate the mechanism of this antiglucocorticoid action, the effect of exogenous DHEA on hepatic glucocorticoid receptor (GC) number and affinity was quantitated. Food supplementation with DHEA (0.6% w/w) for 1 or 7 days had no effect on either receptor number or affinity in obese Zucker rats. After 28 days, however, DHEA treatment resulted in a nearly 40% decrease in cytosolic hepatic receptor content (Bmax; fmol/mg cytosolic protein) without any change in affinity (Kd) in both lean and obese rats. DHEA treatment for 28 days also resulted in an increased liver size and cytosolic protein content. When the hepatic GC receptor content was normalized based on the change in liver size and protein content, the apparent number of GC binding sites per liver was not affected by DHEA treatment. This observation suggests that DHEA's effect on GC receptor content may not be a specific action and that downregulation of the GC receptor is not the mechanism of DHEA action on GC induced TAT activity. This is supported by the effect of DHEA on obese rat TAT activity in the same experiment where the greatest inhibition occurred after only 1 day of treatment. From these experiments it is concluded that although long-term DHEA treatment may decrease the relative concentration of GC receptors in rat liver, this change is not the mechanism through which DHEA mediates its acute antiglucocorticoid action. 相似文献
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Cardounel A Regelson W Kalimi M 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1999,222(2):145-149
Dehydroepiandrosterone (DHEA), an adrenal cortex hormone secreted in large quantities in humans, protects cells of the clonal mouse hippocampal cell line HT-22 against the excitatory amino acid glutamate (5 mM), and amyloid beta-protein (2 microM) toxicity in a dose-dependent manner with optimum protection obtained at 5 microM concentration of DHEA. The protective effects of DHEA appear to be specific in that other related steroids and metabolites of DHEA, such as 5-androstene-3beta,17beta-diol, etiocholan-3alpha-ol-17-one, etiocholan-3beta-ol-17-one, testosterone, and 5alpha-androstane-3, 17-dione, offered no protection even at 50 microM concentrations. In addition, using immunocytochemical techniques, we observed that 20 hr of treatment with 5 mM glutamate remarkably increased glucocorticoid receptor (GR) nuclear localization in neuronal cells. Interestingly, 5 microM DHEA treatment for 24 hr, followed by 5 mM glutamate treatment for 20 hr almost completely reversed the copious nuclear localization of GR observed by glutamate treatment alone. Results obtained suggest that DHEA protects hippocampal neurons, at least in part, by its antiglucocorticoid action via decreasing hippocampal cells nuclear GR levels. 相似文献
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Riml S Schmidt S Ausserlechner MJ Geley S Kofler R 《Cell death and differentiation》2004,11(Z1):S65-S72
Glucocorticoids (GC) induce apoptosis in malignant lymphoblasts, but the mechanism of this process as well as that of the clinically important GC resistance is unknown. We investigated GC resistance in Jurkat T-ALL cells in which ectopic GC receptor (GR) restores GC sensitivity, suggesting deficient GR expression. Jurkat cells expressed one wild-type and one mutated (R477H) GR allele. GR(R477H) ligand-binding-dependent nuclear import, as revealed by live-cell microscopy of YFP-tagged GR, was unaffected. Transactivation and transrepression were markedly impaired; however, GR(R477H) did not act in a dominant-negative manner, that is, did not prevent cell death, when introduced into a GC-sensitive cell line by retroviral gene transfer. Contrary to another GR heterozygous, but GC-sensitive, T-ALL model (CCRF-CEM), Jurkats expressed lower basal GR levels and did not auto-induce their GR, as revealed by 'real-time' RT-PCR and immunoblotting. Absent GR auto-induction could not be restored by transgenic GR and, hence, was not caused by reduced basal GR levels. Thus, inactivation of one GR gene results in haploinsufficiency if associated with lack of GR auto-induction. 相似文献
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The microtubule-associated protein doublecortin-like regulates the transport of the glucocorticoid receptor in neuronal progenitor cells 总被引:1,自引:0,他引:1
Fitzsimons CP Ahmed S Wittevrongel CF Schouten TG Dijkmans TF Scheenen WJ Schaaf MJ de Kloet ER Vreugdenhil E 《Molecular endocrinology (Baltimore, Md.)》2008,22(2):248-262
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Hager GL Fletcher TM Xiao N Baumann CT Müller WG McNally JG 《Biochemical Society transactions》2000,28(4):405-410
Activation of the murine-mammary-tumour virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). We have reconstituted this nucleoprotein transition with chromatin assembled on MMTV LTR DNA with Drosophila embryo extracts, purified GR, and HeLa nuclear extract. Chromatin remodelling in vitro is ATP-dependent and maps to a region identical with that found in vivo. We demonstrate specific, glucocorticoid response element dependent, binding of purified GR to a large, multi-nucleosome MMTV chromatin array and show that GR-dependent chromatin remodelling is a multistep process. In the absence of ATP, GR binds to multiple sites on the chromatin array and inhibits nuclease access to GR recognition sites. On the addition of ATP, GR induces remodelling resulting in a large increase in access of enzymes to their sites within the transition region. These findings are complemented by studies in living cells; using a tandem array of MMTV-Ras reporter elements and a form of GR labelled with the green fluorescent protein, we have observed direct targeting of the receptor to response elements in live mouse cells. Whereas the ligand-activated receptor is associated with the MMTV promoter for observable periods, photobleaching experiments provide direct evidence that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment. The results both in vitro and in vivo are consistent with a dynamic model ('hit and run') in which GR first binds to chromatin after ligand activation, recruits a remodelling activity and is then lost from the template. 相似文献