Isolation and characterization of rat liver nuclear glucocorticoid receptor

The rat liver nuclear glucocorticoid receptor has a molecular weight of 90 000. Using antibody bound to the stationary matrix, the cytosol and nuclear glucocorticoid receptors from rat liver were purified. The translocation of glucocorticoid receptor from rat liver cytosol into the nucleus was studied using immunoaffinity chromatography. Immediately after the intraperitoneal injection of rats with the hormone, the receptor translocation started and was complete within 10 min. The 90 000 dalton nuclear receptor component is identical to the 90 000 dalton cytosol component. They have identical molecular weights in the same gel electrophoresis system and produce identical peptide fragments after digestion with Staphyolococcal aureus V8 protease. The receptor component enriched by immunoaffinity chromatography from cytosol of adrenalectomised rats contained mainly a 45 000 dalton component.     

The effects of serotonin on glucocorticoid receptor binding in rat raphe nuclei and hippocampal cells in culture

The raphe-hippocampal serotonin (5-HT) system is involved in the regulation of the hypothalamus-pituitary-adrenal axis. The purpose of this study was to determine and compare the roles of 5-HT in the regulation of glucocorticoid receptor (GR) binding in the raphe nuclei and in the hippocampus. The effects of 5-HT, 5-HT agonists, and the 5-HT reuptake inhibitor citalopram on GR binding sites were studied in primary cultures of the fetal raphe nuclei and the hippocampus. Exposure of hippocampal cells to 5-HT, (+/-)-2,5-dimethoxy-4-iodoamphetamine (DOI; a 5-HT2 agonist), or citalopram resulted in an increase in number of GR binding sites. The effect of DOI was blocked by ketanserin (a 5-HT2 antagonist). Specific and saturable GR binding was found in raphe cells. Exposure of raphe cells to 5-HT, (+/-)-8 hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; a 5-HT1A agonist), or citalopram induced a significant decrease in number of GR binding sites. The effect of 8-OH-DPAT was reversed by WAY 100135 [N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropiona mide; a 5-HT1A antagonist]. These results show that the regulation of GRs during fetal life is structure-dependent and involves different 5-HT receptor subtypes. Moreover, the regulation of hippocampal GRs by citalopram suggests an action of antidepressants independent of their effects on monoamines.     

Photo-CIDNP study of the interaction between the glucocorticoid receptor DNA-binding domain and glucocorticoid response elements

Summary Photo-CIDNP studies were performed on two protein fragments that both contain the double zinc-finger DNA-binding domain of the glucocorticoid receptor. In the absence of DNA, Tyr⁴⁵² and Tyr⁴⁷⁴ are polarised in both fragments while Tyr⁴⁹⁷ is not. Addition of a palindromic glucocorticoid response element (GRE) results in the suppression of Tyr⁴⁷⁴ polarization while the polarization of Tyr⁴⁵² is unaffected. The same result is observed upon adding a half GRE to the protein fragment indicating that the suppression of Tyr⁴⁷⁴ polarization is not due to protein-protein contacts but to interaction with DNA.     

Peroxisome proliferator-activated receptor alpha agonist-induced down-regulation of hepatic glucocorticoid receptor expression in SD rats

It was reported that glucocorticoid production was inhibited by fenofibrate through suppression of type-1 11β-hydroxysteroid dehydrogenase gene expression in liver. The inhibition might be a negative-feedback regulation of glucocorticoid receptor (GR) activity by peroxisome proliferator-activated receptor alpha (PPARα), which is quickly induced by glucocorticoid in the liver. However, it is not clear if GR expression is changed by fenofibrate-induced PPARα activation. In this study, we tested this possibility in the liver of Sprague-Dawley rats. GR expression was reduced by fenofibrate in a time- and does-dependent manner. The inhibition was observed in liver, but not in fat and muscle. The corticosterone level in the blood was increased significantly by fenofibrate. These effects of fenofibrate were abolished by PPARα inhibitor MK886, suggesting that fenofibrate activated through PPARα. In conclusion, inhibition of GR expression may represent a new molecular mechanism for the negative feedback regulation of GR activity by PPARα.     

Interaction of glucocorticoid · receptor complexes with rat liver nuclei

Some previous reports on acellular binding of glucocorticoid · receptor complexes to rat liver nuclei have pointed to the conclusion that there exists a small number of high affinity nuclear “receptor” sites. Various investigations lead us to the opposite conclusion and suggest that these results were actually due to the presence, in the cytosol, of one or several macromolecules which inhibited the binding to nuclei of steroid · receptor complexes. The mechanism of this inhibition was examined. It appeared to be due not to a competition between both molecules for the same nuclear acceptor site but to an interaction in the cytosol between teh inhibitor and the steroid · receptor complex which prevented the binding of the latter to the nuclei. The search for high affinity specific acceptor sites was also negative for physiological saline conditions and for the non-salt-extractable fraction of the nuclear receptor. When 940-fold purified receptor · steroid complexes were used, very high concentrations of complexes could be achieved and saturation of nuclei was then observed, but only under physiological ionic strength conditions. However, the interaction was of relatively low affinity (K_A = 3.8 · 10⁷ M^?1) and to a great number of acceptor sites (N = 26.2 pmol/mg DNA), largely exceeding the cellular concentration of receptor (5.8 pmol/mg DNA).These results suggested that saturation of nuclei by steroid · receptor complexes should not occur in the intact liver cell. They were confirmed by studies on the distribution of steroid · receptor complexes in liver slices incubated with various concentrations of [³H]dexamethasone. For all hormone concentrations a constant proportion (90%) of the complexes was found in the nuclei, thus showing no saturation of the nuclear acceptor sites.     

Immunocytochemical localization of angiotensinogen in rat liver and kidney

Cell and Tissue Research - The renin substrate, angiotensinogen, was localized by immunocytochemistry in liver and kidney of normal rats by the use of an antiserum directed against pure rat...     

Immunocytochemical localization of glucocorticoid receptor in rat skin

The aim of the present study was to map immunohistochemically the distribution of the glucocorticoid receptor (GR) in rat skin. Nuclear GR-like immunoreactivity (LI) was found in both epidermis and dermis. In the epidermis, the basal cell layer showed an intense immunoreaction; the lower part of the spinous layer was also labelled. In the dermis, the fibroblasts as well as the sweat glands, sebocytes and adipocytes were GR-immunoreactive (IR). In the root sheath of the hair follicle the staining was most intensive in the outer layer. The endothelial cells comprising the smooth muscle cells of the blood vessels, as well as the arrector pili muscle, showed GR-LI. In the peripheral nerves, the immunoreaction was localized to the nuclei of the Schwann cells and in the perineurial fibroblasts. Mast cells did not show nuclear GR-LI. Based on our immunocytochemical findings that several cell types of the skin are GR-IR, the variable physiological and pharmacological effects of glucocorticoids are easier to understand.     

Discovery and optimisation of a selective non-steroidal glucocorticoid receptor antagonist

High-throughput screening of 3.87 million compounds delivered a novel series of non-steroidal GR antagonists. Subsequent rounds of optimisation allowed progression from a non-selective ligand with a poor ADMET profile to an orally bioavailable, selective, stable, glucocorticoid receptor antagonist.     

Dexamethasone treatment affects nuclear glucocorticoid receptor and glucocorticoid response element binding activity in liver of rats (Rattus norvegicus) during aging

Aging is associated with marked changes in the biochemical processes of many organs. Basal and glucocorticoid induced of liver nuclear glucocorticoid receptor (GR) on the level of protein expression and DNA-binding activity were investigated at different ages (3, 6, 12, 18 and 24 months old) in two groups of rats in: untreated and dexamethasone treated. The results showed a significant decline of GR protein immunopurified from untreated rats of advanced age. In dexamethasone-treated rats, the quantity of GR protein was lower than in controls at all ages. The interactions of liver nuclear proteins with radioactively labelled synthetic oligonucleotide analogue containing consensus GRE sequence were analysed during aging. The results showed that GRE binding activity demonstrated a decrease both in untreated and in dexamethasone treated rats. However, relative to untreated rats, dexamethasone treatment resulted in a significant increase in GRE binding at all ages, except that of three months old animals. In conclusion, the observed alterations in GR protein expression and its DNA binding activity may play a role in the changes of the cell response to glucocorticoid during aging.     

Hyperthermic stress modulates the functions of rat liver glucocorticoid receptor

A mild whole body hyperthermic stress causes a rapid and reversible reduction of rat liver glucocorticoid receptor (GR) binding capacity and affects the stability of the GR-DNA complexes formed after thermal transformation of the receptor. These changes appear to be physiologically relevant, since they are accompanied by a decrease in dexamethasone induction of hepatic tyrosine aminotransferase (TAT). In spite of the decreased rate of the GR degradation in liver cytosol of hyperthermic as compared to control rats, the total amount of the GR and its proteolytic products recognized by BuGR2 monoclonal antibody was found to be lower in the former cytosol, but higher in the respective nuclei.     

Identification and characterization of glucocorticoid receptors in liver of nude mice

Glucocorticoids regulate the expression of many liver-specific genes via glucocorticoid receptors. The presence of glucocorticoid receptors in liver has been reported in many mammalian species but not in nude mice. In the present study, we demonstrate the presence of specific glucocorticoid receptors in nude mouse liver. The binding of ligands to these receptors could be completely inhibited by RU486, and partially blocked by hydrocortisone and progesterone, whereas estrogen and testosterone had no effect. Hydrocortisone down-regulated the level of glucocorticoid receptors in livers of nude mice and correspondingly enhanced the activities of tyrosine aminotransferase and -glutamyltransferase. Our results indicate that glucocorticoid receptors in nude mouse liver are specific, fully functional, and present at levels 28.5-fold higher than in the liver of normal inbred mice. We suggest that the nude mouse is a valuable model for studies of hepatic glucocorticoid action and may provide a clue to a putative hepatic-thymic interaction.     

Expression of glucocorticoid receptor isoforms in human adrenocortical adenomas

Introduction

Glucocorticoid receptor (GR) is expressed in the normal human adrenal gland, however, no study has been performed to evaluate the separate expression of α- and β-isoforms (GRα and GRβ) in normal human adrenals and in adrenocortical adenomas.

Experimental

GRα and GRβ mRNA expression was examined by quantitative real-time PCR in 31 adrenal tissues including 19 non-functioning adenomas (NFA), 6 cortisol-producing adenomas (CPA) and 6 normal adrenocortical tissues. In addition, the presence and cellular localization of GRα and GRβ proteins in adrenal tissues were studied by immunohistochemistry.

Results

Compared to normal adrenocortical tissues, both GRα and GRβ mRNAs were significantly increased in CPA but not in NFA. Using anti-GRα antibody a strong nuclear staining was observed in NFA and CPA, and a less remarkable immunoreactivity was detected in some nuclei of normal adrenocortical cells. GRβ immunostaining was absent in normal adrenal tissues and NFA, while a strong cytoplasmic and nuclear immunoreaction was found in CPA.

Conclusions

Altered expression of GRα and GRβ in CPA raises their possible role in the pathophysiology of these adrenal tumors, although further studies are needed to elucidate the potential significance of these findings.     

Mercury stimulates rat liver glucocorticoid receptor association with Hsp90 and Hsp70

The subject of the present study is the influence of mercury on association of rat liver glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70. The glucocorticoid receptor heterocomplexes with Hsp90 and Hsp70 were immunopurified from the liver cytosol of rats administered with different doses of mercury. The amounts of co-immunopurified apo-receptor, Hsp90 and Hsp70 were then determined by quantitative Western blotting. The ratio between the amount of heat shock protein Hsp90 or Hsp70 and the amount of apo-receptor within immunopurified heterocomplexes was found to increase in response to mercury administration. On the other hand, the levels of Hsp90 and Hsp70 in hepatic cytosol remained unaltered. The finding that mercury stimulates association of the two heat shock proteins with the glucocorticoid receptor, rendering the cytosolic heat shock protein levels unchanged, suggests that mercury affects the mechanisms controlling the assembly of the receptor heterocomplexes.     

Characterization of a novel non-steroidal glucocorticoid receptor antagonist

Selective antagonists of the glucocorticoid receptor (GR) are desirable for the treatment of hypercortisolemia associated with Cushing’s syndrome, psychic depression, obesity, diabetes, neurodegenerative diseases, and glaucoma. NC3327, a non-steroidal small molecule with potent binding affinity to GR (K_i = 13.2 nM), was identified in a high-throughput screening effort. As a full GR antagonist, NC3327 greatly inhibits the dexamethasone (Dex) induction of marker genes involved in hepatic gluconeogenesis, but has a minimal effect on matrix metalloproteinase 9 (MMP-9), a GR responsive pro-inflammatory gene. Interestingly, the compound recruits neither coactivators nor corepressors to the GR complex but competes with glucocorticoids for the interaction between GR and a coactivator peptide. Moreover, NC3327 does not trigger GR nuclear translocation, but significantly blocks Dex-induced GR transportation to the nucleus, and thus appears to be a ‘competitive’ GR antagonist. Therefore, the non-steroidal compound, NC3327, may represent a new class of GR antagonists as potential therapeutics for a variety of cortisol-related endocrine disorders.     

Dipetalonema viteae: Effects of hypo- and hyperthermic stress on microfilaremia in the Mongolian jird, Meriones unguiculatus

The effects of thermal stress on the numbers of circulating microfilariae of the filariid parasite, Dipetalonema viteae, in its host, the Mongolian jird, Meriones unguiculatus, was determined. The microfilaremia of each animal was allowed to stabilize, and the effects of stress were measured from this level. The ambient temperature was raised or lowered in 5C increments and maintained at each increment for a 24-hr period. Deep core body temperature of the jirds was recorded by the use of telemetry transmitters placed in the abdominal cavity. The transmitter signals were recorded on a Dynograph recorder. Blood samples were drawn at 0, 12, and 24 hr exposure to each ambient temperature, and microfilariae counted. Microfilaremial levels were then compared to numbers circulating prior to thermal stress. Both hypothermia and hyperthermia in the jird resulted in increased numbers of microfilariae in the peripheral blood. It is speculated that these fluctuations are changes in the percentage of the total microfilarial pool, and represent fluctuations of larvae being driven out of tissue and deep circulatory channels.     

Functional interaction between the glucocorticoid receptor and GANP/MCM3AP

Glucocorticoids are widely used to treat inflammatory diseases but have a number of side effects that partly are connected to inhibition of cell proliferation. Glucocorticoids mediated their action by binding to the glucocorticoid receptor. In the present study, we have identified by two-hybrid screens the germinal center-associated protein (GANP) and MCM3-associated protein (MCM3AP), a splicing variant of GANP, as glucocorticoid receptor interacting proteins. GANP and MCM3AP can bind to the MCM3 protein involved in initiation of DNA replication. Glutathione-S-transferase-pull-down and co-immunoprecipitation assays showed that the C-terminal domain of GANP, encompassing MCM3AP, interacts with the ligand-binding domain of the glucocorticoid receptor. Characterization of the intracellular localization of GANP revealed that GANP is shuttling between the nucleus and the cytoplasm. Furthermore, we show that glucocorticoids are unable to inhibit DNA replication in HeLa cells overexpressing MCM3AP suggesting a role for both glucocorticoid receptor and GANP/MCM3AP in regulating cell proliferation.