Glucocorticoid Receptor mRNA Levels Research Articles

Chronic Intermittent Hypoxia Induces 11β-Hydroxysteroid Dehydrogenase in Rat Heart

Corticosteroids are known to not only regulate electrolyte homeostasis but also play a role in the cardiovascular system, including myocardial remodeling. Because transgenic mice that overexpress 11beta-hydroxysteroid dehydrogenase (11HSD) type 2 in cardiomyocytes have been shown to spontaneously develop cardiac hypertrophy and fibrosis, we investigated whether changes in the cardiac metabolism of glucocorticoids accompany remodeling of the heart under physiological conditions. In the present study, glucocorticoid metabolism and 11HSD2 were explored in the hearts of rats exposed to chronic intermittent hypobaric hypoxia (CIH), which induces hypertrophy and fibrosis of the right and less of the left ventricle. We first demonstrated that adaptation to CIH led to a significant increase in 11HSD2 transcript levels and activity in the myocardium. In contrast, neither 11HSD1 activity and mRNA level nor the abundance of mineralocorticoid and glucocorticoid receptor mRNA were up-regulated. The adaptation to CIH also led to an increase of 11HSD2 mRNA in isolated cardiomyocytes, whereas 11HSD1, glucocorticoid receptor, and mineralocorticoid receptor mRNA levels were not changed in comparison with the cardiomyocytes of control normoxic rats. The changes in cardiac metabolism of glucocorticoids were accompanied by inflammatory responses. The expression levels of the proinflammatory markers cyclooxygenase-2 and osteopontin were significantly increased in both the myocardium and the cardiomyocytes isolated from rats exposed to CIH. These findings suggest that myocardial remodeling induced by CIH is associated with the up-regulation of cardiac 11HSD2. Consequently, local metabolism of glucocorticoids could indeed play a role in cardiac hypertrophy and fibrosis.

Glucocorticoid receptor mRNA levels are selectively decreased in neutrophils of children with sepsis

ObjectiveCorticosteroids are used in sepsis treatment to benefit outcome. However, discussion remains on which patients will benefit from treatment. Inter-individual variations in cortisol sensitivity, mediated through the glucocorticoid receptor, might play a role in the observed differences. Our aim was to study changes in mRNA levels of three glucocorticoid receptor splice variants in neutrophils of children with sepsis.Patients and designTwenty-three children admitted to the pediatric intensive care unit with sepsis or septic shock were included. Neutrophils were isolated at days 0, 3 and 7, and after recovery (>3 months). mRNA levels of the glucocorticoid receptor splice variants GR-α (determining most of the cortisol effect), GR-P (increasing GR-α effect) and GR-β (inhibitor of GR-α) were measured quantitatively.Main resultsNeutrophils from sepsis patients showed decreased levels of glucocorticoid receptor mRNA of the GR-α and GR-P splice variants on day 0 compared to after recovery. GR-α and GR-P mRNA levels showed a gradual recovery on days 3 and 7 and normalized after recovery. GR-β mRNA levels did not change significantly during sepsis. GR expression was negatively correlated to interleukin-6 (a measure of disease severity, r = −0.60, P = 0.009).ConclusionsChildren with sepsis or septic shock showed a transient depression of glucocorticoid receptor mRNA in their neutrophils. This feature may represent a tissue-specific adaptation during sepsis leading to increased cortisol resistance of neutrophils. Our study adds to understanding the mechanism of cortisol sensitivity in immune cells. Future treatment strategies, aiming at timing and tissue specific regulation of glucocorticoids, might benefit patients with sepsis or septic shock.

Maternal postpartum learned helplessness (LH) affects maternal care by dams and responses to the LH test in adolescent offspring

It is known that the early environment affects the mental development of rodent and human offspring. However, it is not known specifically whether a postpartum depressive state influences the depressive state in offspring. Using learned helplessness (LH) in rats as an animal model of depression, we examined the influence of maternal postpartum LH on responses to the LH test of offspring. Dam rats were judged as LH or non-helpless (nLH) on postnatal days (PN) 2–3, and maternal behavior was recorded during PN2–14. On PN 45–46, offspring were subjected to the LH test. Plasma corticosterone (CORT) levels, hippocampal levels of glucocorticoid receptor (GR) and brain-derived neurotrophic factor (BDNF) mRNA were measured before and after the LH test in offspring. Active nursing in LH dams was significantly lower than that in nLH dams. Susceptibility to LH in the offspring of LH dams was significantly higher than in those of nLH dams, and was negatively correlated with active nursing by LH dams. The GR mRNA levels before and after the LH test were lower in the offspring of LH dams than in those of nLH dams, and the reduced basal GR mRNA and protein might have resulted in the higher CORT response after the LH test. There was no significant difference in BDNF mRNA in the offspring of LH and nLH dams. These findings suggest that early postpartum LH decreased active nursing and increased depression-like behavior in the adolescent offspring via dysfunction of the hypothalamic–pituitary–adrenal axis.

Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse

Maternal care influences hypothalamic-pituitary-adrenal (HPA) function in the rat through epigenetic programming of glucocorticoid receptor expression. In humans, childhood abuse alters HPA stress responses and increases the risk of suicide. We examined epigenetic differences in a neuron-specific glucocorticoid receptor (NR3C1) promoter between postmortem hippocampus obtained from suicide victims with a history of childhood abuse and those from either suicide victims with no childhood abuse or controls. We found decreased levels of glucocorticoid receptor mRNA, as well as mRNA transcripts bearing the glucocorticoid receptor 1F splice variant and increased cytosine methylation of an NR3C1 promoter. Patch-methylated NR3C1 promoter constructs that mimicked the methylation state in samples from abused suicide victims showed decreased NGFI-A transcription factor binding and NGFI-A-inducible gene transcription. These findings translate previous results from rat to humans and suggest a common effect of parental care on the epigenetic regulation of hippocampal glucocorticoid receptor expression.

Role of MicroRNAs in Resistance to Dexamethasone in Multiple Myeloma Cell Lines

Multiple Myeloma is a B-cells malignant disease of unknown etiology. It accounts for 1% of all cancers and 14% of hematologic malignancies. Despite the advances in treatment it remains an incurable disease. One of the commonly used drugs in the treatment of multiple myeloma is Dexamethasone. Most patients respond to this drug, but eventually most then become refractory to the treatment with steroids. MicroRNAs (miRNA) are a newly identified class of small RNAs that play important regulatory roles at the post transcriptional level. They usually act by binding to the 3′untranslated region (3′UTR) of messenger RNA (mRNA) and either block their translation or lead to degradation. MM1 cell line is a well known myeloma cell line. By continued exposure to Dexamethasone two variants of MM1 cell line i.e.; MM1.S and MM1.R were developed. MM1.S remains sensitive while MM1.R is resistant to the action of Dexamethasone. It has been reported that MM1.R cell line expresses very minimal levels of glucocorticoid receptor (GR) mRNA and protein, hence rendering it resistant to the action of steroids. We hypothesized that altered regulation of microRNAs between the two cell lines leads to the down regulation of glucocorticoid receptor hence contributing to resistance to the action of steroids. QRT-PCR and Western blot were conducted to demonstrate decreased expression of GR mRNA and protein in MM1.R cell line. We identified certain microRNAs based on bioinformatics with putative docking sites in 3′UTR of GR mRNA. DNA sequencing was used to rule out any mutation in binding sites of these microRNAs. No such mutation was found in MM1.S or MM1.R cell line. qRT-PCR was than used to analyze the differential expression of predicted microRNAs. Several microRNAs were found to be differentially expressed between the two cell lines, including microRNA-183 (p < 0.05). We then transfected inhibitors and mimics of pertinent microRNAs and were able to up or down regulate the GR mRNA. We were also able to show some evidence of apoptosis in MM1.R cell line with Dexamethasone exposure. These studies suggest that microRNAs regulate expression of GR and play an important role in conferring resistance to the action of steroids. These findings shed further light on the underlying mechanisms of steroid resistance and may have important therapeutic implications in the treatment of multiple myeloma.

Expression of genes related to corticotropin production and glucocorticoid feedback in corticotroph adenomas of dogs with Cushing’s disease

Cushing’s disease caused by pituitary corticotroph adenoma is a common endocrine disease in dogs. A characteristic biochemical feature of corticotroph adenomas is their relative resistance to negative feedback by glucocorticoids. In this study, we examined gene expression related to adrenocorticotropic hormone (ACTH) production and secretion, and the negative feedback by glucocorticoids in canine corticotroph adenoma. We used resected corticotroph adenomas from 10 dogs with Cushing’s disease. In order to investigate the alteration of gene expression between corticotroph adenoma and normal corticotrophic cells, ACTH-positive cells in the anterior lobe were microdissected using a laser-capture microdissection system, and mRNA levels of proopiomelanocortin (POMC), corticotropin releasing hormone receptor 1 (CRHR1), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11 beta hydroxysteroid dehydrogenase (11HSD) type 1 and type 2 were determined using real-time RT-PCR. POMC, CRHR1, and 11HSD2 mRNA levels in corticotroph adenoma were greater than those in normal corticotrophic cells (POMC, 5.5-fold; CRHR1, 4.9-fold; 11HSD2, 4.2-fold, P < 0.01, respectively). MR and 11HSD1 mRNA levels in corticotroph adenoma were lower than those in normal corticotrophic cells (MR, 2.2-fold; 11HSD1, 2.9-fold, P < 0.01, respectively). GR mRNA levels did not differ between corticotroph adenoma and normal corticotrophic cells. Our results may help to understand the increased ACTH production and the resistance to negative feedback suppression by glucocorticoids in canine corticotroph adenomas. These changes in gene expression may have a role in the growth of canine corticotroph adenoma, and help elucidate the pathophysiology of dogs with Cushing’s disease.

Electroconvulsive stimulations normalizes stress-induced changes in the glucocorticoid receptor and behaviour

Animal models of chronic stress, such as 21 days of 6h/daily restraint stress cause changes in neuronal morphology in the hippocampus and alter behaviour. These changes are partly mediated by the glucocorticoids.The objective of this study was threefold: (1) to study how this particular chronic stress paradigm influences expression of hippocampal glucocorticoid receptor mRNA, (2) to study the effect of previous repeated restraint stress on the behaviours executed in the forced swim test (FST) (e.g. a novel inescapable stress situation) and (3) to investigate the modulating effect of electroconvulsive stimulations (ECS) on the neural and behavioural effects of the stress paradigm.The study shows that restraint stress lowered glucocorticoid receptor mRNA levels in all hippocampal regions, including the CA3 region which is the site of the characteristic dendritic reorganization seen in this model. Furthermore, stressed rats displayed higher increases in immobility and decreased latency to immobility subjected to the novel stressor of the FST than non-stressed rats. ECS abolished both the neural and behavioural effects of the restraint stress and thus protected against the deleterious effects of the stress paradigm. The clinical relevance of these findings is discussed.

The effect of long-term insulin treatment with and without antecedent hypoglycemia on neuropeptide and corticosteroid receptor expression in the brains of diabetic rats

We previously demonstrated that while diabetic animals receiving long-term insulin treatment exhibited some impairment in their corticosterone response to hypoglycemia, the stress response to hypoglycemia was completely absent when these animals were subjected to recurrent hypoglycemia. In the current study, we examined potential mechanisms that may contribute to defects in the adrenocortical response to hypoglycemia in long-term insulin-treated diabetic animals exposed to antecedent hypoglycemia. Whereas insulin-treated diabetic animals exhibited a significant rise in corticotrophin-releasing hormone (CRH) mRNA levels during hypoglycemia, exposure to antecedent hypoglycemia completely abolished this response. Moreover, expression of hippocampal mineralocorticoid receptors (MR) mRNA, which normally act to suppress hypothalamo–pituitary–adrenal activity, decreased in the normal control and insulin-treated diabetic groups in response to hypoglycemia, whereas MR mRNA levels remained at baseline in animals subjected to antecedent hypoglycemia. Interestingly, hippocampal glucocorticoid receptor (GR) mRNA levels decreased in all three treatment groups following the hypoglycemic clamp. While GR mRNA levels in the paraventricular nucleus were lower in normal controls following hypoglycemia, this trend just failed to reach statistical significance in the two diabetic groups. These data suggest that (1) recurrent hypoglycemia, much like uncontrolled diabetes, has a pronounced effect on hippocampal mineralocorticoid receptor mRNA expression that may prevent it, and presumably also the stress axis, from responding properly to a subsequent bout of hypoglycemia, and (2) while long-term insulin treatment was sufficient to restore some of these responses in diabetic animals, tighter glycemic control may be necessary to see full restoration of the stress response.

Reduction of hepatic glucocorticoid receptor and hexose-6-phosphate dehydrogenase expression ameliorates diet-induced obesity and insulin resistance in mice

Intracellular glucocorticoid (GC) receptor (GR) function determines tissue sensitivity to GCs and strongly affects the development of type 2 diabetes and obesity. 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) mediates intracellular steroid exposure to mouse liver GR by prereceptor reactivation of GCs and is crucially dependent on hexose-6-phosphate dehydrogenase (H6PDH)-generating NADPH system. Pharmacological inhibition of 11beta-HSD1 improves insulin intolerance and obesity. Here, we evaluated the potential beneficial effects of 11beta-HSD1 inhibitor carbenoxolone (CBX) in diet-induced obese (DIO) and insulin-resistant mice by examining the possible influence of CBX on the expression of GR, 11beta-HSD1, and H6PDH in vivo and in vitro in hepatocytes. Treatment of DIO mice with CBX markedly reduced hepatic GR mRNA levels and reduced weight gain, hyperglycemia, and insulin resistance. The reduction of hepatic GR gene expression was accompanied by CBX-induced inhibition of both 11beta-HSD1 and H6PDH activity and mRNA in the liver. Moreover, CBX treatment also suppressed the expression of both phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase enzyme (G6Pase) mRNA and improved hepatic [1, 2-(3)H] deoxy-d-glucose uptake in DIO mice. In addition, the treatment of primary cultures of hepatocytes with increasing concentrations of CBX led to a dose-dependent downregulation of GR mRNA levels, which correlated with the suppression of both 11beta-HSD1 and H6PDH activity and their gene expression. Addition of CBX to primary hepatocytes also resulted in suppression of both PEPCK and G6Pase mRNA levels. These findings suggest that CBX exerts some of its beneficial effects, at least in part, by inhibiting hepatic GR and H6PDH expression.

Dexamethasone allows sugar‐induced trafficking of the glucocorticoid receptor in rat small intestine

Fructose (F) consumption has increased dramatically and is linked to childhood obesity but mechanisms regulating neonatal F absorption are unknown. The intestinal F transporter GLUT5 cannot be enhanced by luminal F in rats &lt; 14 d old unless the gut is primed with dexamethasone (D). The effect of D requires transcription and is mediated by the glucocorticoid receptor (GR). We tested the hypothesis that in 10 d old pups, D‐ and F‐induced GLUT5 upregulation involves trafficking of the GR from cytoplasm to nucleus. Intestines of 10 d pups (+ D), were perfused in vivo with F or glucose. GR mRNA levels were similar in all 4 groups but GR levels in the nucleus increased markedly only in D‐primed, F‐perfused pups. Regulation of KPNA2, a nuclear importin whose mRNA levels are age‐sensitive and increase with F perfusion, seems linked with that of GLUT5. KPNA2 is also transcriptionaly upregulated and levels are high in nuclei of F‐perfused, D‐primed 10d pups, indicating that KPNA2 could be the importin carrying GR to the GRE in the GLUT5 promoter. Ser‐211 is phosphorylated when GR is active. Using a specific Ser211‐GRp antibody, we failed to observe GRp in the nucleus, indicating that phosphorylation is not necessary in D‐allowed, F‐induced GR trafficking into the nucleus. In 10 d pups, regulation of GLUT5 involves KPNA2‐mediated trafficking of the GR to the nucleus. Support: NIH RDK075617A, NSF IOS‐0722365

Melatonin prevents glucocorticoid inhibition of cell proliferation and toxicity in hippocampal cells by reducing glucocorticoid receptor nuclear translocation

Glucocorticoids are the main product of the adrenal cortex and participate in multiple cell functions as immunosupressors and modulators of neural function. Within the brain, glucocorticoid activity is mediated by high-affinity mineralocorticoid and low-affinity glucocorticoid receptors. Among brain cells, hippocampal cells are rich in glucocorticoid receptors where they regulate excitability and morphology. Also, elevated glucocorticoid levels suppress hippocampal neurogenesis in adults. The pineal neuroindole, melatonin, reduces the affinity of glucocorticoid receptors in rat brain and prevents glucocorticoid-induced apoptosis. Here, the ability of melatonin to prevent glucocorticoid-induced cell death in hippocampal HT22 cells was investigated in the presence of neurotoxins. Results showed that glucocorticoids reduce cellular growth and also enhance sensitivity to neurotoxins. We found a G 1 cell cycle arrest mediated by an increase of cyclin/cyclin-dependent kinase inhibitor p21 WAF1/CIP1 protein after dexamethasone treatment and incremental change in amyloid β protein and glutamate toxicity. Melatonin prevents glucocorticoids inhibition of cell proliferation and reduces the toxicity caused by glucocorticoids when cells were treated with dexamethasone in combination with neurotoxins. Although, melatonin does not reduce glucocorticoid receptor mRNA or protein levels, it decreases receptor translocation to nuclei in these cells.

Investigation of sex differences in behavioural, endocrine, and neural measures following repeated psychological stressor exposure

Animal models of repeated stressor exposure have generally been limited to physical stressors, despite the fact that the purpose of such models is to represent repeated stress in humans, which is usually psychological in nature. The present study was undertaken to investigate the behavioural, endocrine, and neural responses to a repeated psychological stressor exposure in male and female rats. Long-Evans rats were exposed to cat odour or a control condition for 1 h each day from Day 1 to Day 22. Every fourth day, defensive (e.g. hiding), and non-defensive (e.g. grooming) behaviour was quantified, during both the initial and the final 10 min of the hour. Defensive behaviours in cat odour-exposed animals remained vigorous during the initial 10 min of exposure across 22 exposure days. Non-defensive behaviours were suppressed during early exposures, but this suppression habituated across repeated exposures. Overall, the pattern of behavioural results indicated enhanced responses to novelty and to repeated cat odour exposure, in females, relative to males. Plasma corticosterone (CORT) levels were higher in females relative to males overall. However, males, but not females, exposed to cat odour had higher levels of CORT following exposure on Days 1 and 22, relative to controls. Finally, mRNA levels of glucocorticoid receptor, mineralocorticoid receptor, and brain-derived neurotrophic factor, all of which are modulated by CORT, were examined in hippocampus at the completion of stressor exposure, but none was affected by repeated stressor exposure. Results are discussed within the context of potential differences in effects of repeated psychological versus physical stressors.

Long-term behavioral and neuroendocrine alterations following chronic social stress in mice: Implications for stress-related disorders

The period of adolescence is characterized by a high vulnerability to stress and trauma, which might result in long-lasting consequences and an increased risk to develop psychiatric disorders. Using a recently developed mouse model for chronic social stress during adolescence, we studied persistent neuroendocrine and behavioral effects of chronic social stress obtained 12 months after cessation of the stressor. As a reference, we investigated immediate effects of chronic stress exposure obtained at the end of the chronic stress period. Immediately after the 7 week chronic stress period stressed animals show significantly increased adrenal weights, decreased thymus weight, increased basal corticosterone secretion and a flattened circadian rhythm. Furthermore, stressed animals display an increased anxiety-like behavior in the elevated plus maze and the novelty-induced suppression of feeding test. Hippocampal mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) mRNA levels were significantly decreased. To investigate persistent consequences of this early stressful experience, the same parameters were assessed in aged mice 12 months after the cessation of the stressor. Interestingly, we still found differences between formerly stressed and control mice in important stress-related parameters. MR expression levels were significantly lower in stressed animals, suggesting lasting, possibly epigenetic alterations in gene expression regulation. Furthermore, we observed long-term behavioral alterations in animals stressed during adolescence. Thus, we could demonstrate that chronic stress exposure during a crucial developmental time period results in long-term, persistent effects on physiological and behavioral parameters throughout life, which may contribute to an enhanced vulnerability to stress-induced diseases.

NALP7 and NALP11 Differential Expression Correlates with Glucocorticoid Sensitivity in Multiple Myeloma Cell Lines.

Multiple myeloma is the second most common hematological malignancy and remains incurable. Glucocorticoids (GC) are among the most commonly used therapy for myeloma by inducing growth arrest and apoptosis in myeloma cells. While GC treatments are usually effective at first, all patients will eventually develop resistance. Understanding the mechanism of GC resistance is an important step towards developing new therapy to overcome this significant clinical problem. In order to study the mechanism underlying the GC resistance phenotype in myeloma, we compared the gene expression profiles of the clinically relevant myeloma clones derived from the same patient (MM.1) which are either sensitive or resistant to GC-induced apoptosis and growth inhibition. By cDNA microarray analysis, we identified NALP7 and NALP11 among the few genes that showed most significant differential levels of expression between the sensitive and resistant myeloma lines. Both genes belong to the NALP (NATCH-, LRR- and pyrin domain-containing proteins) family of proteins which have been implicated in the innate immune response. However, their function and regulation in myeloma has never been explored. Using a two-step quantitative real-time RT-PCR analysis, we have demonstrated that the NALP7 expression was significantly reduced whereas the NALP11 expression was markedly increased in GC-resistant MM.1 cells compared to the GC-sensitive MM.1 cells. We then investigated the biological significance of NALP gene differential expression in myeloma GC-resistance. To test whether suppressing NALP7 expression would lead to GC-resistance, we transfected the sensitive MM.1 cells with NALP-7 siRNA which effectively knocked down NALP7 mRNA level by over 80%. The knock-down of NALP-7, however, did not appear to affect the GC sensitivity of the transfected myeloma cells as dexamethasone (dex) treatment effectively caused growth arrest and apoptosis in transfected cells as in the control cells. We have also tested whether suppressing NALP11 expression would overcome the resistance to GC treatment by transfecting the GC-resistant MM.1 cells with NALP11 siRNA. While NALP11 expression was effectively knocked down to less than 20%, the transfected cells remain resistant to GC treatment as in the control cells, suggesting that NALP11 expression is not required to maintain the GC-resistant phenotype. We further investigated the upstream signaling cascades influencing NALP7 and NALP11 expression. Interestingly, dex treatment suppressed the expression of both NALP7 and NALP11 genes in the GC-sensitive MM.1 cells in a time- and dose-dependent manner. Co-treatment with glucocorticoid receptor (GR) antagonist RU486 blocked the down-regulation of NALP7 and NALP11 by dex, suggesting that a functional GR is required to mediate this action. In GC-resistant MM.1 cells which have much reduced levels of wild type GR, NALP7 and NALP11 mRNA levels were not affected by dex treatment, suggesting further that the suppression of NALP7 by GC requires sufficient levels of GR. Our studies demonstrated that the differential expression of two NALP family genes, NALP7 and NALP11, correlates with GC sensitivity in myeloma, and that both genes are regulated by glucocorticoid signaling. The biological significance of their differential regulation in myeloma remains to be determined.

Cell Density and Serum Exposure Modify the Function of the Glucocorticoid Receptor C/EBP Complex

The glucocorticoid receptor (GR) is a major control factor for proliferation, differentiation, and inflammation. Our knowledge about the GR is focused on its function as a transcription regulator. However, cells do not always respond to steroids in the same way or develop resistance. The mechanism underlying such a modified steroid response is not well understood, and may depend on the microenvironment of the cells or on the stage of their differentiation. Therefore, we studied the effect of cell density and inflammatory conditions on the expression, compartmentalization, activation, and the anti-proliferative function of the GR in primary human lung fibroblast cultures. In subconfluent cells the GR was located perinuclear, while in confluent cells it was ubiquitously expressed. Serum stimulation up-regulated the level of GR mRNA and protein under all conditions. In subconfluent cells dexamethasone activated the nuclear accumulation and DNA binding of the GR persistently, while in confluent cells its activity declined after 6 hours. In subconfluent cells, but not in confluent cells, the GR interacted with a 42-kD, but not the 30-kD C/EBP-alpha isoprotein, which resulted in an up-regulation of p21((Waf1/Cip1)) expression and suppression of proliferation. In confluent cells, glucocorticoids induced p27((Kip1)) expression via p38 mitogen-activated protein kinase and a 52-kD C/EBP-beta isoprotein. However, p27((Kip1)) did not mediate the antiproliferative effect of glucocorticoids, but simultaneous inhibition of p21((Waf1/Cip1)) and p27((Kip1)) unlocked contact inhibition in confluent cells. Our results indicate that cell density and serum exposure alter the localization and function of the GR.

Retracted: A reduction state potentiates the glucocorticoid response through receptor protein stabilization

The intracellular redox state regulates all biological processes including gene expression. The glucocorticoid receptor (GR), a hormone-dependent transcription factor, is affected by the redox state. GR translocation from the cytoplasm to the nucleus is regulated by oxidative stress. The molecular mechanism of how the redox state affects GR transcriptional regulation, however, has not been clarified. We identified a deoxidizing agent, cobalt chloride (CoCl(2)), that potentiates the GR transcriptional effects by stabilizing endogenously expressed GR protein as well as exogenously over-expressed one without affecting GR mRNA level. Consequent GR protein stabilization enhanced co-factor recruitments on the target gene promoters. These results support the existence of a novel redox-dependent mechanism of GR transcriptional regulation mediated by receptor protein stabilization.

Endocrine systems in juvenile anadromous and landlocked Atlantic salmon ( Salmo salar): Seasonal development and seawater acclimation

The present study compares developmental changes in plasma levels of growth hormone (GH), insulin-like growth factor I (IGF-I) and cortisol, and mRNA levels of their receptors and the prolactin receptor (PRLR) in the gill of anadromous and landlocked Atlantic salmon during the spring parr-smolt transformation (smoltification) period and following four days and one month seawater (SW) acclimation. Plasma GH and gill GH receptor (GHR) mRNA levels increased continuously during the spring smoltification period in the anadromous, but not in landlocked salmon. There were no differences in plasma IGF-I levels between strains, or any increase during smoltification. Gill IGF-I and IGF-I receptor (IGF-IR) mRNA levels increased in anadromous salmon during smoltification, with no changes observed in landlocked fish. Gill PRLR mRNA levels remained stable in both strains during spring. Plasma cortisol levels in anadromous salmon increased 5-fold in May and June, but not in landlocked salmon. Gill glucocorticoid receptor (GR) mRNA levels were elevated in both strains at the time of peak smoltification in anadromous salmon, while mineralocorticoid receptor (MR) mRNA levels remained stable. Only anadromous salmon showed an increase of gill 11β-hydroxysteroid dehydrogenase type-2 (11β-HSD2) mRNA levels in May. GH and gill GHR mRNA levels increased in both strains following four days of SW exposure in mid-May, whereas only the anadromous salmon displayed elevated plasma GH and GHR mRNA after one month in SW. Plasma IGF-I increased after four days in SW in both strains, decreasing in both strains after one month in SW. Gill IGF-I mRNA levels were only increased in landlocked salmon after 4 days in SW. Gill IGF-IR mRNA levels in SW did not differ from FW levels in either strain. Gill PRLR mRNA did not change after four days of SW exposure, and decreased in both strains after one month in SW. Plasma cortisol levels did not change following SW exposure in either strain. Gill GR, 11β-HSD2 and MR mRNA levels increased after four days in SW in both strains, whereas only the anadromous strain maintained elevated gill GR and 11β-HSD2 mRNA levels after one month in SW. The results indicate that hormones and receptors of the GH and cortisol axes are present at significantly lower levels during spring development and SW acclimation in landlocked relative to anadromous salmon. These findings suggest that attenuation of GH and cortisol axes may, at least partially, result in reduced preparatory upregulation of key gill ion-secretory proteins, possibly a result of reduced selection pressure for marine adaptations in landlocked salmon.

Peripheral mechanisms contributing to the glucocorticoid hypersensitivity in proopiomelanocortin null mice treated with corticosterone.

Proopiomelanocortin (POMC) deficiency causes severe obesity through hyperphagia of hypothalamic origin. However, low glucocorticoid levels caused by adrenal insufficiency mitigate against insulin resistance, hyperphagia and fat accretion in Pomc−/− mice. Upon exogenous glucocorticoid replacement, corticosterone-supplemented (CORT) Pomc−/− mice show exaggerated responses, including excessive fat accumulation, hyperleptinaemia and insulin resistance. To investigate the peripheral mechanisms underlying this glucocorticoid hypersensitivity, we examined the expression levels of key determinants and targets of glucocorticoid action in adipose tissue and liver. Despite lower basal expression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which generates active glucocorticoids within cells, CORT-mediated induction of 11β-HSD1 mRNA levels was more pronounced in adipose tissues of Pomc−/− mice. Similarly, CORT treatment increased lipoprotein lipase mRNA levels in all fat depots in Pomc−/− mice, consistent with exaggerated fat accumulation. Glucocorticoid receptor (GR) mRNA levels were selectively elevated in liver and retroperitoneal fat of Pomc−/− mice but were corrected by CORT in the latter depot. In liver, CORT increased phosphoenolpyruvate carboxykinase mRNA levels specifically in Pomc−/− mice, consistent with their insulin-resistant phenotype. Furthermore, CORT induced hypertension in Pomc−/− mice, independently of adipose or liver renin–angiotensin system activation. These data suggest that CORT-inducible 11β-HSD1 expression in fat contributes to the adverse cardiometabolic effects of CORT in POMC deficiency, whereas higher GR levels may be more important in liver.

Voluntary Exercise Impacts on the Rat Hypothalamic-Pituitary-Adrenocortical Axis Mainly at the Adrenal Level

Introduction: Evidence is accumulating that the regular performance of exercise is beneficial for stress coping. However, the hypothalamic-pituitary-adrenocortical (HPA) axis of voluntarily exercising rats has never been comprehensively investigated. Methods: Therefore, male Sprague-Dawley rats were given access to a running wheel in their home cage for 4 weeks in which they ran 4–7 km per night. Results: After 4 weeks, the exercising animals showed significantly less body weight gain, less abdominal fat tissue, decreased thymus weight, and increased adrenal weight (relative to body weight). Furthermore, tyrosine hydroxylase (TH) mRNA levels were selectively increased in the right adrenal me- dulla indicating an increase in sympathoadrenomedullary capacity in exercising rats. No changes were observed in paraventricular corticotropin-releasing hormone (CRH), arginine-vasopressin (AVP) and oxytocin mRNA levels. Mineralocorticoid receptor (MR) mRNA levels in hippocampus and glucocorticoid receptor (GR) mRNA levels in frontal cortex, parvocellular paraventricular nucleus and anterior pituitary were unchanged, whereas GR mRNA levels were increased in distinct hippocampal cell layers. Early morning baseline levels of plasma ACTH and corticosterone were similar in both groups. Interestingly, the response to different stressful stimuli (e.g. forced swimming, novelty) revealed that the exercising rats showed stressor-specific changes in HPA hormone responses. Forced swimming evoked a markedly enhanced response in corticosterone levels in the exercising rats. In contrast, if rats were exposed to a novel environment, exercising rats showed a much lower response in corticosterone than the control animals. However, the response in ACTH to either stressor was comparable between groups. Thus, in exercising rats physically demanding stressors evoke enhanced glucocorticoid responses whereas mild psychologically stressful stimuli such as novelty result in an attenuated glucocorticoid response. Interestingly, this attenuated hormone response corresponded with the observation that the exercising rats showed less anxious behaviour in the novelty situation. Conclusions: The differential responses in plasma corticosterone levels to different types of stress in the face of comparable responses in ACTH levels underscore the existence of critical regulatory control mechanisms at the level of the adrenal gland. We have hypothesized that changes in the sympathoadrenomedullary input may play an important role in these distinct glucocorticoid responses to stress. Our previous studies have shown similar changes in voluntarily exercising mice. Therefore, we conclude that the effects of exercise on the organism are not species-specific. Thus, our observations may have translational implications for the human situation.

Bioactive compounds from Paecilomyces tenuipes regulating the function of the hypothalamo-hypophyseal system axis in chronic unpredictable stress rats

A bioactive compound from Paecilomyces tenuipes (BCPT) has an inhibitory effect on monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) in vitro and in vivo, which indicates BCPT may be a potential antidepressant. In this study we aimed to study the antidepressant effects of BCPT in the chronic unpredictable stress (CUS) model in rats and explore underlying mechanisms in the hypothalamic-pituitary-adrenal (HPA) axis. The antidepressant effects of BCPT were studied in the chronic unpredictable stress model in rats. Animals were housed isolated, except the control group. Rats were exposed daily to different random stressors from day 1 to 21. Awarding response was detected by calculating the 24-hour consumption of sucrose water. Cortisol (CORT) and adrenocorticotropic hormone (ATCH) contents in serum and arginine vasopressin (AVP) contents in the pituitary body were detected by radio immunoassays. Total RNA of hippocampus or hypothalamus was extracted and subjected to reverse transcription-polymerase chain reaction (RT-PCR) for the measurement of corticotrophin releasing hormone (CRH) mRNA or mineralocorticoid receptor (MR) mRNA and glucocorticoid receptor (GR) mRNA levels. Statistical analyses were performed using one way analysis of variance (ANOVA) followed by Student-Newman-Keuls (SNK) test. Chronic unpredictable stress resulted in reduction of sensitivity to reward and abnormality in the HPA axis in the animal model. BCPT improved the reward reaction as measured by increasing sucrose consumption, remarkably reduced serum CORT and ACTH levels and the AVP content in the pituitary body in the CUS-treated rats, decreased the expression of CRH mRNA, enhanced the expression of hippocampus MR mRNA, GR mRNA and decreased the ratio of MR/GR. BCPT has potentially antidepressant-like activity and normalized the HPA axis hyperactivity in a CUS model of depression in rats. This may be an important mechanism of its antidepressant effect.