Dexamethasone 21-mesylate: an affinity label of glucocorticoid receptors from rat hepatoma tissue culture cells (original) (raw)
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De Gruyter eBooks, 1985
The role of the glucucorticoid receptor in the expression ofa~fig~ucocortjcoid action has been investigated with a chemically-reactive derivative of three glucocorticoid steroids with differing biological potencies, i.e. the C-21 mesylates of cortisol. dexamethasone and deacylcortivazol. Dexamethasone 21-mesylate (Dex-Mes) was the most useful derivative due to its favorable balance of high receptor affinity and predominantly irreversible antiglucocorticoid activity. A number of criteria have been used to conclude that [3H]Dex-Mes covalently labels glucocorticoid receptors In the steroid-binding cavity. The available data indicate that covalent Dex-Mes-labeled receptors (mol. wt % 98,000) are responsible for the irreversible antiglucocort~~o~d activity while the partial agonist activity of Dex-Mes is due to non-covalent Dex-~~-bound receptors. Further support fcrr this hypothesis comes from the observations that dea~y~~ortiva~ol21mesyfate was a full glucocortitoid and did not afhnity label receptors (and marginahy labeled cytosol proteins) although it was capable of cova~ently-labeling bovine serum albumin. Severaf mechanisms for the expression of irreversible antiglucocorticoid activity by covalent Dex-Mes-labeled receptors were examined and can be eliminated. Covalent receptor-Dex-Mes complexes formed in whole HTC cells were found to have a decreased capacity for nuclear binding. This decreased nuclear-binding capacity could be responsible for the whole-cell irreversible antiglucocarticoid activity of Dex-Mes.
Analysis of the glucocorticoid antagonist action of dexamethasone 21-mesylate in HeLa S3 cells
Journal of Steroid Biochemistry, 1987
Several properties of human glucocorticoid receptors complexed to the synthetic glucocorticoid agonists dexamethasone (DEX) and triamcinolone acetonide (TA) and the antagonist dexamethasone 21-mesylate (DM) are compared in an attempt to define the mode of action of DM. Both DEX and TA induce an increase in alkaline phosphatase activity in HeLa S, cells. Not only is DM without effect on alkaline phosphatase activity at concentrations as great as lo-' M, it blocks the action of DEX and TA on enzyme induction, thus acting as a pure antagonist in this system. DM-receptor complexes, like agonist-receptor complexes, are recovered in the cytosol when cells are incubated with ligand at 0°C but are recovered from the nucleus when incubation is shifted to 37°C suggesting that activation of the antagonist-r~ptor complex occurs in oivo. The molecular species that undergoes this temperaturedependent shift from the cytosolic compartment to the nuclear compartment exhibits saturable binding to the antagonist. Both the cytosolic and nuclear species exhibit a relative molecular mass of-94,000 Daltons when anaIysed by SDS-polyacrylamide gel electrophoresis. Receptors labeled in intact cells with [3H]DM at 0°C sediment at N 8s in sucrose gradients, shifting to 4s when the gradients contain 0.4 M KCl. DEX-and TA-labeled receptors show the same sedimentation behavior, which has been accepted as one criterion of receptor subunit dissociation, or activation.
Molecular Endocrinology, 1987
The actions of dexamethasone 21-mesylate (DM) have been studied in two recently developed cultured murine cell lines containing approximately 200 copies of episomal minichromosome. This minichromosome contains the glucocorticoid regulatory element in the mouse mammary tumor virus long terminal repeat fused upstream of v-ras H sequences in a totally defined primary sequence environment. The levels of v-ras H mRNA were measured as an index of glucocorticoid regulated expression of this chimeric gene. In addition, expression of the endogenous single copy mouse metallothionein I (MT-I) gene was monitored simultaneously. DM was found to be an essentially pure antagonist of dexamethasone (dex)-stimulated expression of both the episomal chimeric gene and the endogenous MT-I gene. The covalent labeling efficiency by DM of glucocorticoid receptors in intact cells approached 100%, surpassing previously observed whole cell DM labeling efficiencies. These results strengthen the hypothesis that covalent complex formation is responsible for antiglucocorticoid action. The efficiency of whole cell nuclear binding of covalent receptor-DM complexes was found to be approximately 50% of that seen with receptor-dex complexes. Analyses of long terminal repeat initiated v-ras H mRNA and MT-I mRNA inductions by dex in cells previously exposed to a subsaturating concentration of DM indicated that receptor-DM complexes do not inhibit by a competitive mechanism the transcriptional activation of these glucocorticoid responsive genes by receptor-dex complexes.
The native anti-glucocorticoid paradigm
The Journal of Steroid Biochemistry and Molecular Biology, 2006
Circulating 3beta-hydroxysteroids including dehydroepiandrosterone (DHEA) are 7alpha-hydroxylated by the cytochrome P450-7B1 in the liver, skin and brain, which are the target organs of glucocorticoids. Anti-glucocorticoid effects with 7alpha-hydroxy-DHEA were observed in vivo without an interference with glucocorticoid binding to its receptor. In the organs mentioned above, the circulating inactive cortisone was reduced into active cortisol by the 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). We demonstrated that 7alpha-hydroxy-DHEA was also a substrate for this enzyme. Studies of the 11beta-HSD1 action on 7alpha-hydroxy-DHEA showed the reversible production of 7beta-hydroxy-DHEA through an intermediary 7-oxo-DHEA, and the kinetic parameters favored this production over that of active glucocorticoids. Both the production of 7alpha-hydroxysteroids and their interference with the activation of cortisone into cortisol are basic to the concept of native anti-glucocorticoids efficient at their production site. This opens a promising new area for research.
Steroids, 1994
HeLa $3 cells that contain endogenous glucocorticoid receptors (GR) were treated with dexamethasone (DEX) for periods of time ranging from 24 h to 2 weeks or chronically over a 2-year period. Regulation of GR protein and mRNA were examined by affinity labeling, Western blotting, and Northern blotting. Relatively short-term treatment of cells with DEX for 24 or 48 h revealed more profound down-regulation of GR protein than of GR mRNA. However, by 2 weeks of DEX treatment, the levels of both receptor protein and mRNA were both maximally down-regulated. Cells that had been chronically DEX treated (for up to 2 years) had no measurable GR protein or mRNA. The down-regulation of receptor protein and RNA that occurred after 2 weeks of DEX treatment is completely reversible upon DEX removal, whereas reversibility did not occur with cells that had been chronically treated with DEX. Furthermore, transfection of a glucocorticoid responsive reporter plasmid into these chronically DEX-treated cells demonstrated that these cells were no longer responsive to steroid treatment. However, cotransfection of a plasmid encoding the human GR into these chronically DEX-treated cells resulted in restored production of GR and responsiveness to hormone, indicating that the defect in these cells occurs only at the receptor level.
The 11-ketosteroid 11-ketodexamethasone is a glucocorticoid receptor agonist
Molecular and Cellular Endocrinology, 2004
Dexamethasone (Dex) is a potent and long-acting glucocorticoid in terms of anti-inflammatory activity without substantial sodium retaining effect. Here, we examine the ability of the 11β-hydroxyglucocorticoids Dex and cortisol and their 11-keto forms 11-ketodexamethasone (11-ketoDex) and cortisone to bind to glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) and to mediate nuclear translocation and transactivation of a reporter-gene. Unlike cortisone, the 11-ketosteroid 11-ketoDex acts as a potent GR agonist, comparable to Dex and cortisol. Transactivation of MR by Dex or 11-ketoDex was weak or undetectable, despite efficient binding and induction of nuclear translocation. 11β-HSD2 protects MR and GR from inappropriate occupation by cortisol; it is, however, unable to prevent activation of GR by 11-ketoDex. The finding that 11-ketoDex is a specific GR agonist may explain the potent glucocorticoid effect of Dex in tissues expressing 11β-HSD2 including kidney and colon and also in certain tumor cells.
Glucocorticoids and Their Actions: An Introduction
Annals of the New York Academy of Sciences, 2004
Glucocorticoids regulate a variety of growth, metabolic, developmental, and immune functions and play a pivotal role in preserving basal and stress-related homeostasis. 1-3 They also represent one of the most widely prescribed drugs worldwide. During the last 50 years, pharmacologic doses of glucocorticoids have been used in the treatment of inflammatory, autoimmune, and lymphoproliferative diseases, and in the prevention of graft rejection, while substitution doses have been employed in the management of adrenocortical insufficiency states. Glucocorticoids exert their effects through the glucocorticoid receptor (GR), which belongs to the superfamily of nuclear receptors that function as ligand-dependent transcription factors. 4,5 Alternative splicing of the human (h) GR in exon 9 generates two highly homologous receptor isoforms, hGRα and hGRβ, which are identical through amino acid 727, but differ at their carboxyl-termini. hGRα is ubiquitously expressed in almost all human tissues and cells and represents the classic GR that functions as a ligand-dependent transcription factor, whereas hGRβ does not bind glucocorticoids and inhibits the transcriptional activity of hGRα in a dose-dependent manner. In the absence of ligand, hGRα resides mostly in the cytoplasm of cells as part of a large multiprotein complex, which consists of the receptor polypeptide, two molecules of heat-shock protein (hsp)-90, and several other proteins. Upon ligand binding, hGRα dissociates from the hsps and translocates into the nucleus of cells, where it modulates the transcriptional activity of glucocorticoid-responsive genes in either of two ways: by binding to specific sequences in the promoter region of target genes, the glucocorticoid-response elements (GREs), or through protein-protein interactions with other transcription factors, such as nuclear factor (NF)-κB, activator protein-1 (AP-1), and several signal transducers and activators of transcription (STATs). 9-11 Newly characterized nuclear receptor coregulators (coactivators or corepressors) may also enhance or attenuate glucocorticoid signal transduction
The Journal of Steroid Biochemistry and Molecular Biology, 2004
The human brain is a target tissue for glucocorticoids (GC). Dehydroepiandrosterone (DHEA) is a neurosteroid produced in the brain where it is transformed into 7␣-hydroxy-DHEA and 7-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, 7␣-hydroxy-DHEA and 7-hydroxy-DHEA interferences with GR trafficking. These cells did not carry out the 7␣-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, 7␣hydroxy-DHEA and 7-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, 7␣-hydroxy-DHEA or 7-hydroxy-DHEA did not change its kinesis and intensity. These results contribute to the knowledge of DHEA, 7␣-hydroxy-DHEA and 7-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.