Jadranka Dundjerski

Association of the rat liver glucocorticoid receptor with Hsp90 and Hsp70 upon whole body hyperthermic stress

The influence of whole body hyperthermic stress (41 degrees C, 15 min) on association of the glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70 was followed in rat liver cytosol during a 24 h period after the stress. Total cytosolic concentration of the GR, Hsp90 and Hsp70 and the amounts of Hsp90 and Hsp70 co-immunopurified with the GR were determined by a quantitative Western blotting using appropriate monoclonal antibodies. A significant decrease in the cytosolic GR level in response to the stress was noticed. The ratio of the amount of the GR to Hsp90 recruited by the GR was found to be unaltered by hyperthermia, in spite of the stress-induced increase in the total Hsp90 concentration in the cytosol. Hsp70 was also found in association with the GR and its 2.5-fold induction by the stress was accompanied by about 3-fold increase in its relative amount that co-immunopurified with the GR. The results suggest that heat stress influences the interaction of the GR with Hsp70 through the mechanisms controlling the untransformed rat liver GR heterocomplexes assembly process.

Glucocorticoid receptor-Hsp90 interaction in the liver cytosol of cadmium-intoxicated rats.

The influence of cadmium on the rat liver glucocorticoid receptor (GR) binding capacity, on the cytosolic level of 90 kDa heat shock protein (Hsp90), and on the association of the two proteins was investigated. The results showed that the mode of metal application led to diverse alterations in hormone binding to the GR. Reduction of the GR binding capacity observed afterin vitro treatment was proportional to the applied metal concentrations. In animals administered different doses of cadmium, GR binding capacity was not reduced, except in those that received the highest dose. A concomitant elevation of Hsp90 level was detected both in the cytosol and within the GR untransformed heterocomplexes. The results suggest that cadmium-induced reduction of the GR binding capacity seenin vitro was prevented in intact animals by the elevated level of Hsp90 within the GR heterocomplexes.

THE LEVEL AND PHOSPHORYLATION OF HSP70 IN THE RAT LIVER CYTOSOL AFTER ADRENALECTOMY AND HYPERTHERMIA

Hepatic heat shock protein Hsp70 synthesis and in vitro phosphorylation were studied in the liver cytosol of intact, adrenalectomized and dexamethasone‐administered adrenalectomized rats after 41°C whole body hyperthermic stress. Hsp70 was detected by immunoblotting with N27F3‐4 monoclonal antibody recognizing both constitutive and inducible forms of the protein. A comparison between basal and heat stress‐induced levels of the protein in the liver cytosol of the three groups of animals suggested that glucocorticoid hormones stimulate the basal synthesis of Hsp70 and inhibit its induction by stress. In both unstressed and hyperthermia‐exposed animals, hepatic Hsp70 was detected as a phosphoprotein. The extent of itsin vitro phosphorylation was found to be significantly reduced by heat stress or adrenalectomy, but dexamethasone failed to restore it to the original level.

Cadmium affects the activity of rat liver tyrosine aminotransferase and its induction by dexamethasone

Abstract  The effects of cadmium (Cd) administration to intact rats on hepatic glucocorticoid receptor (GR) steroid binding capacity and DNA-binding ability were examined and correlated with the influence of the metal on rat liver tyrosine aminotransferase (TAT) activity and its induction by dexamethasone. It was found that 24 h after i.p. administration of Cd doses ranging from 0.5 to 4 mg/kg, the GR steroid- and DNA-binding activities were significantly reduced in a dose-dependent manner. The same doses of Cd also affected the basal and dexamethasone-induced level of TAT activity, as well as the concentration of metallothionein in rat liver. The decrease in TAT activity and in its induction by dexamethasone observed in response to low Cd doses was proportional to the alterations of the GR functional properties. Higher doses of Cd, which were more effective in reducing both the GR binding of the hormone and to DNA, however, stimulated TAT activity and potentiated dexamethasone induction of the enzyme. The results led to the conclusion that Cd may alter physiological response of rat liver cells to glucocorticoids interfering with the GR-dependent transcriptional regulation of the TAT gene.

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.

Mercury inhibits rat liver and kidney glucocorticoid receptor hormone binding activity

The present study was focused on the influence of mercury on the rat liver and kidney glucocorticoid receptor (GR) binding properties. The time-course and dose-dependence of mercury effects, as well as possible involvement of thiol groups were examined afterin vivo andin vitro administration of the metal in the form of HgCl2. Mercury led to reduction of the liver and kidney GR hormone binding capacity. In both examined tissues maximal reduction was noticed 4 h after administration of the metal at 2 and 3 mg Hg/kg bw, but the effect was more prominent in kidney as compared to liver. On the other hand, binding affinity in the two tissues was similar. The complete reversal of mercury effects on GR binding capacity by 10 mmol/L DTT was achieved in liver and partially in kidney. The reversal by DTT suggested that mercury caused the decrease of GR binding activity by interacting with thiol groups. The difference in the response of the two tissues reflected the fact that kidney contained a higher mercury concentration and a lower thiol content in comparison to liver. The implicated thiols probably belong to GR, since when appliedin vitro at 0°C, mercury produced reduction of the receptor binding activity similar to that observedin vivo. GR protein level examined by quantitative Western blot was either unchanged, when determined by polyclonal antibody, or reduced, when determined by BuGR2 antibody, suggesting that Hg might affect BuGR epitope availability.

Heat stress affects the glucocorticoid receptor interaction with heat shock protein Hsp70 in the rat liver.

The association of glucocorticoid hormones receptor (GR) with heat shock protein Hsp70 in the liver cytosol of rats exposed to 41°C whole body hyperthermic stress was examined by quantitative immunoblotting of the two proteins within immunopurified untransformed GR multiprotein complexes. The presence of Hsp70 in the rat liver GR heterocomplexes was confirmed, and 2‐fold increase in the Hsp70 relative to the steroid binding protein content within the complexes was recorded 2 and 12 h after the stress. This increase exceeded the stress‐induced elevation in the total cytoplasmic Hsp70 level, but could not be seen 24 h after the stress, when cytoplasmic Hsp70 returned to basal level. The results suggest that hyperthermic stress alters the composition of the rat liver untransformed GR heterocomplexes increasing the Hsp70 share.

Validation of endogenous controls for gene expression studies in peripheral lymphocytes from war veterans with and without PTSD

BackgroundSelection of appropriate endogenous control is a critical step in gene expression analysis. The aim of this study was to evaluate expression stability of four frequently used endogenous controls: β-actin, glyceraldehyde-3-phosphate dehydrogenase, β2-microglobulin and RNA polymerase II polypeptide A in peripheral blood mononuclear cells from war veterans with and without posttraumatic stress disorder (PTSD). The study was designed as to identify suitable reference gene(s) for normalization of gene expression in peripheral blood mononuclear cells in response to war trauma and/or PTSD.ResultsThe variability in expression of the four endogenous controls was assessed by TaqMan Real-time RT-PCR in peripheral blood mononuclear cells from: war veterans with current PTSD, those with lifetime PTSD, trauma controls and healthy subjects. Expression stability was analyzed by GeNorm and NormFinder software packages, and by direct comparison of Ct values. Both, GeNorm and NormFinder identified β-actin and glyceraldehyde-3-phosphate dehydrogenase as a pair of genes with the lowest stability value.ConclusionsThe combination of β-actin and glyceraldehyde-3-phosphate dehydrogenase appeared to be the most suitable reference for studying alterations in gene expression in peripheral blood mononuclear cells related to vulnerability and resilience to PTSD, as well as to trauma-provoked developing of this disorder and recovery from it. Using glyceraldehyde-3-phosphate dehydrogenase, β-actin and β2-microglobulin as individual endogenous controls would provide satisfactory data, while RNA polymerase II polypeptide A could not be recommended.

HYPERTHERMIC STRESS AFFECTS GLUCOCORTICOID RECEPTOR-MEDIATED TRANSCRIPTION IN RAT LIVER

Binding capacity of the cytoplasmic and nuclear glucocorticoid receptor (GR) and the activity of tyrosine aminotransferase (TAT) were examined in the liver of intact and adrenalectomized rats exposed to 41°C whole body hyperthermic stress. In glucocorticoid‐deprived animals, stress‐induced decrease in the cytoplasmic steroid binding was followed by parallel increases in its nuclear binding and TAT activity, suggesting a stimulation of TAT gene transcription by the GR in the absence of the ligand. In intact animals, however, a diminution of the steroid binding in the cytosol, its unchanged nuclear binding and an impairment of TAT activity were observed upon the stress. The results imply that stress could elicit different structural or functional alterations of unligandedvsliganded GR.

Glucocorticoid signaling in the liver and adipose tissue of male and female fructose-fed rats

Background The rise in consumption of refined sugars high in fructose appears to be an important factor contributing to epidemic of obesity and metabolic syndrome [1]. Fructose is involved in the genesis and progression of the syndrome through deregulation of metabolic pathways in the liver and adipose tissue, as sites of insulin-modulated metabolism [2]. Enhanced regeneration of glucocorticoids within the liver and adipose tissue, mediated by the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11bHSD1), may contribute to adiposity and metabolic disease [3]. 11bHSD1 reductase activity is crucially dependent on NADPH, a cofactor generated by the enzyme hexose-6phosphate dehydrogenase (H6PDH) [4]. We hypothesized that harmful effects of high fructose consumption are mediated by alterations in prereceptor metabolism of glucocorticoids and in the level of glucocorticoid receptor (GR) expression and compartmental redistribution in the liver and adipose tissue. We also assume that high fructose intake differently affects glucocorticoid signaling in the liver and adipose tissue of male and female rats.