Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of glucocorticoids on the regulation of stably transfected human glucocorticoid receptors has been examined. Exposure of a Chinese hamster ovary-derived cell line containing stably transfected human glucocorticoid receptor genes and glucocorticoid-responsive dihydrofolate reductase genes to 5 nM dexamethasone resulted in a rapid, time-dependent reduction in the level of glucocorticoid receptor protein to 50% of control levels within 5 h of steroid treatment. This decrease in receptor protein was persistent, with a maximal 70% reduction observed even after 4 weeks of dexamethasone treatment. Immunocytochemical analysis of the influence of dexamethasone on stably transfected glucocorticoid receptors revealed efficient translocation of receptors to the nucleus within 1 h of hormone treatment. However, upon longer exposure to dexamethasone (5 h), immunoreactive glucocorticoid receptors were localized primarily to the cytoplasm. By 24 h of treatment, glucocorticoid receptors were absent from the cytoplasm and the nucleus, suggesting that the ligand-induced loss of glucocorticoid receptors may be a cytoplasmic event. The decrease in transfected glucocorticoid receptor protein was largely reflected by similar changes in steady state levels of human glucocorticoid receptor mRNA; however, the effects of hormone on receptor protein levels were more profound than on receptor mRNA. There was an initial rapid reduction in transfected glucocorticoid receptor mRNA to 50% of control levels within 2 h of dexamethasone treatment. This reduction was followed by a transient rise in mRNA expression after 12 h of hormone treatment. With prolonged exposure to dexamethasone (> 12 h) a second, more gradual decline in human glucocorticoid receptor mRNA was observed. This biphasic pattern of glucocorticoid receptor gene expression was not reflected at the level of receptor protein, suggesting that both transcriptional and translational control mechanisms may be involved in ligand-dependent receptor regulation. When cells were removed from dexamethasone after up to 48 h of treatment, glucocorticoid receptor mRNA levels fully recovered within 12 h. Receptor protein recovered only partially during this same time period. Down-regulation of glucocorticoid receptor protein and mRNA levels by dexamethasone in stably transfected cells led to corresponding reductions in the hormone sensitivity to two glucocorticoid-regulated genes: a transiently transfected chloramphenicol acetyltransferase receptor gene and a stably integrated dihydrofolate reductase gene. These results demonstrate that stably transfected human glucocorticoid receptors are subject to ligand-induced down-regulation in a heterologous cell line. Moreover, glucocorticoid receptor autoregulation appears to be a highly conserved mechanism for attenuating cellular responsiveness to hormone.
Mol Endocrinol 1992 Dec
PMID:Ligand-dependent down-regulation of stably transfected human glucocorticoid receptors is associated with the loss of functional glucocorticoid responsiveness. 149 90

Mammary epithelial cells terminally differentiate in response to lactogenic hormones. We present evidence that oncoprotein overexpression is incompatible with this hormone-inducible differentiation and results in striking cellular morphological changes. In mammary epithelial cells in culture, lactogenic hormones (glucocorticoid and prolactin) activated a transfected beta-casein promoter and endogenous beta-casein gene expression. This response to lactogenic hormone treatment was paralleled by a decrease in cellular AP-1 DNA-binding activity. Expression of the mos, ras, or src (but not myc) oncogene blocked the activation of the beta-casein promoter induced by the lactogenic hormones and was associated with the maintenance of high levels of AP-1. Mos expression also increased c-fos and c-jun mRNA levels. Overexpression of Fos and Jun from transiently transfected constructs resulted in a functional inhibition of the glucocorticoid receptor in these mouse mammary epithelial cells. This finding clearly suggests that glucocorticoid receptor inhibition arising from oncogene expression will contribute to the block in hormonally induced mammary epithelial cell differentiation. Expression of Src resulted in the loss of the normal organization and morphological phenotype of mammary epithelial cells in the epithelial/fibroblastic line IM-2. Activation of a conditional c-fos/estrogen receptor gene encoding an estrogen-dependent Fos/estrogen receptor fusion protein also morphologically transformed mammary epithelial cells and inhibited initiation of mammary epithelial differentiation-associated expression of the beta-casein and WDNM 1 genes. In response to estrogen treatment, the cells displayed a high level of AP-1 DNA-binding activity. Our results demonstrate that high cellular AP-1 levels contribute to blocking the ability of mammary epithelial cells in culture to respond to lactogenic hormones. This and other studies indicate that the oncogene products Mos, Ras, and Src exert their effects, at least in part, by stimulating cellular Fos and probably cellular Jun activity.
Mol Cell Biol 1992 Sep
PMID:Overexpression of Mos, Ras, Src, and Fos inhibits mouse mammary epithelial cell differentiation. 150 91

The effect of progesterone on the differentiation of the 3T3-L1 preadipocytes was investigated and compared with other sex steroids (estradiol and testosterone), with cortisol, with the synthetic progestin R5020 and with the progestin/glucocorticoid antagonist RU38486. At 10(-8) M, progesterone stimulated the activity of glycerol-3-phosphate dehydrogenase and triglyceride deposition. Progesterone, R5020, cortisol, and RU38486 increased triglycerides about 2-fold at 10(-7) M. Only minimal effects were observed with testosterone and estradiol even at 10(-6) M. When the cells were cultured in presence of 10(-5) M metyrapone the effect of progesterone was unchanged, suggesting that the progesterone was not metabolized to a glucocorticoid. Progesterone, R5020 and RU38486 competed efficiently with [3H]dexamethasone for the glucocorticoid receptor in 3T3-L1 cytosol. These results indicate a significant, reproducible dose-dependent effect of progestins on differentiation of the preadipocytes, which appears to be mediated via the glucocorticoid receptor.
J Steroid Biochem Mol Biol 1992 Sep
PMID:Progestins stimulate the differentiation of 3T3-L1 preadipocytes. 152 40

We have isolated a full length cDNA that encodes a heat shock protein, hsp90, from a rat brain library and present the nucleotide sequence and deduced amino acid sequence. Comparison of the entire nucleotide sequence with mouse hsp84 and human hsp90 beta cDNAs reveal sequence similarities of 92 and 87%, respectively. The coding region of 2172 nucleotides corresponds to a polypeptide chain of 724 amino acids. Comparison with mouse hsp84 and human hsp90 beta amino acid sequences indicates a similarity of 97%, respectively. Characterization of the constitutive expression of this cDNA both by RNA blot hybridization and immunoblotting, reveals that it is expressed in all rat tissues examined. Hsp90 has been shown to form a transient complex with steroid hormone receptors. In order to further elucidate the role of hsp90 in the endocrine response of cells, we have examined the effects of dexamethasone and RU38486 on the level of hsp90 mRNA in a system in which glucocorticoids down-regulate glucocorticoid receptor mRNA levels. In this system, a subtle but reproducible approx. 2-fold decrease in hsp90 mRNA levels is observed after 48 h treatment with dexamethasone.
J Steroid Biochem Mol Biol 1992 Sep
PMID:Cloning and regulation by glucocorticoid receptor ligands of a rat hsp90. 152 42

We have used a modified cotransfection and selection strategy to create a series of mammalian cell lines that stably express high levels of intact glucocorticoid receptors. These cell lines were produced by subjecting Chinese hamster ovary (CHO) cells, which had been previously cotransfected with a glucocorticoid-responsive dihydrofolate reductase (DHFR) gene and the human glucocorticoid receptor gene, to growth in increasing concentrations of methotrexate (MTX). By linking the MTX selection process to glucocorticoid receptor function via the DHFR gene, stable cell lines resistant to a range of MTX concentrations (50 nM to 3 microM) were isolated that were strictly dependent upon glucocorticoids for growth. Quantitation of steroid binding capacity in MTX-resistant cells revealed a progressive increase in the number of glucocorticoid receptors as a function of increasing MTX concentration. This increase in receptor content was maximal at the highest level of MTX resistance examined (3 microM MTX) and represented a 25-fold elevation in glucocorticoid receptor number relative to CHO cells expressing only endogenous hamster receptor. The increases in steroid binding obtained after MTX selection were reflected by similar increases in the level of glucocorticoid receptor protein as determined by immunoblot analysis. Examination of glucocorticoid receptor structure by sucrose density gradient centrifugation revealed that oligomeric (9 S) steroid receptor complexes were formed at all levels of receptor expression. Subcellular localization of the glucocorticoid receptor protein by immunocytochemical staining revealed effective nuclear translocation of the overexpressed receptors in MTX-resistant cells. Functional transfection studies using a glucocorticoid-responsive reporter gene indicated that the additional glucocorticoid receptors in CHO cells were competent to activate transcription. To determine the molecular basis for the MTX-induced increases in functional glucocorticoid receptors, steady-state levels of glucocorticoid receptor mRNA were examined. MTX selection produced a 5- to 7-fold increase in transfected glucocorticoid receptor gene expression relative to untreated cells. MTX-resistant cells also expressed increased levels of a putative hamster glucocorticoid receptor mRNA species. Interestingly, the observed increases in receptor gene expression in these cells could not be accounted for by amplification of either the human or the hamster glucocorticoid receptor genes.
Mol Cell Endocrinol 1992 Feb
PMID:Methotrexate-induced overexpression of functional glucocorticoid receptors in Chinese hamster ovary cells. 154 9

The glucocorticoid receptor belongs to a family of ligand activated nuclear receptors. This family includes, in addition to the receptors for steroid hormones, receptors for thyroid hormone, retinoic acid and 1,25-dihydroxy vitamin D3 as well as some receptors with as yet unknown ligands. The glucocorticoid receptor DNA-binding domain has been expressed in E. coli. The purified protein binds to the same DNA sequences as the native receptor and is therefore suitable for biochemical and structural studies of the DNA-binding function of the receptor protein. This protein has been shown to bind as a dimer to its DNA-binding site. Protein-protein interactions facilitate DNA-binding and a segment responsible for these interactions has been identified close to the C-terminal zinc-binding site. The family of nuclear receptors, with their related DNA-binding sites, provides an opportunity to study determinants for DNA sequence recognition. A segment close to the N-terminal zinc ion has been shown to be responsible for the target specificity of glucocorticoid and estrogen receptors. DNA-binding domains of nuclear receptors include nine conserved cysteine residues which have been shown to coordinate two zinc ions and zinc has been shown to be required for the structural integrity and DNA-binding ability of the glucocorticoid receptor DNA-binding domain. A motif for DNA recognition, based around zinc ions, was first described for transcription factor IIIA and nuclear receptors were believed to recognize DNA via a similar motif. However, the three-dimensional structure determination of the glucocorticoid receptor DNA-binding domain shows that its structure is clearly different from that of the TFIIIA type zinc-binding domains.
J Steroid Biochem Mol Biol 1992 Mar
PMID:DNA-binding by the glucocorticoid receptor: a structural and functional analysis. 156 6

In this work we have probed the mechanism responsible for two non-DNA-binding states of the mouse glucocorticoid receptor. In the first case, transformed receptors were treated with hydrogen peroxide. It is known that oxidizing agents promote the formation of disulfide bonds in the glucocorticoid receptor, but it has not been determined what domains are involved in any disulfide bond formation that leads to inactivation of DNA-binding activity. We show here that hydrogen peroxide inhibits DNA-binding by the 15-kDa tryptic fragment containing the DNA-binding fingers with the same concentration dependency as it inhibits DNA-binding by the uncleaved receptor. This suggests that all of the effect of peroxide is on sulfhydryl groups within the zinc fingers. After dissociation (transformation) of cytosolic heteromeric glucocorticoid receptor complexes, only a portion (40-60%) of the dissociated receptors can bind to DNA-cellulose. We show that the 15-kDa tryptic fragment derived from the portion of transformed receptors that do not bind to DNA is itself competent at DNA-binding.
J Steroid Biochem Mol Biol 1992 Mar
PMID:DNA-binding and non-DNA-binding forms of the transformed glucocorticoid receptor. 156 44

In a recent paper we described a system in which glucocorticoid receptors associate with particulate complexes containing tubulin [Cancer Res. 49 (1989) 2222s-2229s]. When L cell cytosol is mixed with a microtubule stabilizing buffer and heated to 37 degrees C, the receptor becomes associated with a complex that can be centrifuged out of solution at 150,000 g. In this work we show that the glucocorticoid receptor-cytoskeletal protein complex forms in a temperature and glutamate-dependent manner. Molybdate does not affect generation of the cytoskeletal protein complex but it inhibits association of the receptor with the complex. This suggests that transformation of the receptor to its DNA-binding form is required for interaction with the cytoskeletal complex. Colchicine has no effect on generation of the particulate complex or on the association of receptor with it, suggesting that formation of the complex does not represent a classic in vitro process of tubulin polymerization.
J Steroid Biochem Mol Biol 1992 Mar
PMID:Association of the transformed glucocorticoid receptor with a cytoskeletal protein complex. 156 45

Transient co-transfection of receptor cDNA and suitable reporter genes was used to study human glucocorticoid receptor (hGR) function in a neutral mammalian cell background. A variety of natural and synthetic steroids were analyzed for their ability to activate gene expression through the hGR and to bind to extracts of cells expressing the hGR cDNA. There was very good correlation between these two in vitro parameters for these compounds. Furthermore, correlation of these data with reported in vivo anti-inflammatory potencies was surprisingly close, with two exceptions. The in vitro data suggest an explanation for the discrepant compounds, consistent with published data on their metabolic fate in vivo. The co-transfection assay has utility as a quantitative predictor of in vivo glucocorticoid pharmacology.
J Steroid Biochem Mol Biol 1992 Mar
PMID:Interaction of glucocorticoid analogues with the human glucocorticoid receptor. 156 47

Recent immunohistochemical studies suggest that the unoccupied glucocorticoid receptor (GR) is cytoplasmic and that the ligand causes its translocation into the target cell nucleus. The subcellular location of GR is especially interesting in that other members of the steroid receptor superfamily appear to be nuclear. The intracellular distribution of GR was studied immunohistochemically using a new freeze-drying and vapor fixation method which eliminates the protein diffusion and redistribution possibly caused by liquid fixation techniques. We used two monoclonal antibodies against rat liver GR. Dried samples of the adrenalectomized rat brain and uterus were fixed in p-benzoquinone vapor for 3 h at 60 degrees C and embedded in paraffin. Sections were stained with a biotinylated mouse monoclonal GR antibody using the avidin-biotin-peroxidase complex. Both unoccupied and occupied GR were found in the nucleus of the target cells, fibroblasts in the uterus and nerve cells in the cortex of the brain. The staining was saturated with the cytosol of cos cells transfected with GR. No cytoplasmic staining was seen even 2 days after adrenalectomy. In conclusion we propose that GR is also located in the nucleus independently of occupation.
J Steroid Biochem Mol Biol 1992 Mar
PMID:Subcellular location of unoccupied and occupied glucocorticoid receptor by a new immunohistochemical technique. 156 49


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>