Glucocorticoid Receptor Protein Research Articles

Mechanisms Underlying the Increased Plasma ACTH Levels in Chronic Psychosocially Stressed Male Mice

Mice exposed to chronic subordinate colony housing (CSC, 19 days), an established paradigm for chronic psychosocial stress, show unaffected basal morning plasma corticosterone (CORT) concentrations, despite enlarged adrenal glands and an increased CORT response to an acute heterotypic stressor. In the present study we investigate the mechanisms underlying these phenomena at the level of the pituitary. We show that both basal and acute stressor-induced (forced swim (FS), 6 min) plasma adrenocorticotropic hormone (ACTH) concentrations, the number of total and corticotroph pituitary cells, and relative protein expression of pituitary mineralocorticoid receptor and FK506-binding protein 51 was increased in CSC compared with single-housed control (SHC) mice, while relative corticotropin releasing hormone (CRH) receptor 1 (CRH-R1) and glucocorticoid receptor protein expression was down-regulated. Relative pituitary pro-opiomelanocortin and arginine vasopressin (AVP) receptor 1b (AVPR-1b) protein expression, FS (6 min)-induced ACTH secretion in dexamethasone-blocked mice, and the number of AVP positive magnocellular and parvocellular neurons in the paraventricular hypothalamic nucleus (PVN) was unaffected following CSC. Taken together, the data of the present study indicate that 19 days of CSC result in pituitary hyperactivity, under both basal and acute heterotypic stress conditions. Although further studies have to assess this in detail, an increased number of pituitary corticotrophs together with unaffected relative pituitary AVPR-1b and decreased CRH-R1 protein expression following CSC suggests that pituitary hyperdrive is mediated by newly formed corticotrophs that are more sensitive to AVP than CRH. Moreover, our data indicate that changes in PVN AVP and negative feedback inhibition seem not to play a major role in pituitary hyperactivity following CSC.

Long-Term Effects of Subacute Ruminal Acidosis (SARA) on Milk Quality and Hepatic Gene Expression in Lactating Goats Fed a High-Concentrate Diet

PurposeThe mechanism underlying the decline in milk quality during periods of feeding high-concentrate diets to dairy ruminants is not well documented. The aim of this study was to investigate the metabolic changes in the liver that contribute to the input of substrate precursors to the mammary gland after feeding a high-concentrate diet to lactating goats for a long period.Experimental DesignEight mid-lactating goats with rumen cannulas were randomly assigned to two groups. For 9 weeks, the treatment group was fed a high-concentrate diet (60% concentrate of dry matter, HC) and the control group was fed a low-concentrate diet (40% concentrate of dry matter, LC). Ruminal fluid, plasma, and liver tissues were sampled, microarray techniques and real-time polymerase chain reaction were used to evaluate metabolic parameters and gene expression in liver.ResultsFeeding a 60%-concentrate diet for 9 weeks resulted in a significant decrease in rumen pH. Changes in fat and protein content also occurred, which negatively affected milk quality. Plasma levels of leptin (p = 0.058), non-esterified fatty acid (p = 0.071), and glucose (p = 0.014) increased markedly in HC group. Plasma cortisol concentration was significantly elevated in the treatment group (p<0.05). Expression of the glucocorticoid receptor protein gene was significantly down-regulated (p<0.05) in the liver. The expression of genes for interleukin 1β, serum amyloid A, C-reactive protein, and haptoglobin mRNA was significantly increased (p<0.05) in the HC group. GeneRelNet analysis showed that gene expression involved in inflammatory responses and the metabolism of lipids, protein, and carbohydrate were significantly altered by feeding a high-concentrate diet for 9 weeks.ConclusionsActivation of the acute phase response and the inflammatory response may contribute to nutrient partitioning and re-distribution of energy in the liver, and ultimately lead to a decline in milk quality.

Lose dose genistein inhibits glucocorticoid receptor and ischemic brain injury in female rats

Although acute bolus of genistein treatment has been shown to protect against neuronal damage in experimental brain injury animal models, chronic continuous low dose treatment of genistein on ischemic brain injury has not been well elucidated. In the present study, female rats were received either pure genistein (0.1mg/kg/day via osmotic minipumps) or placebo at the time of ovariectomy, and transient forebrain ischemia was induced 7days later. Results demonstrated that genistein treatment for 14days significantly improved ischemic neuronal survival in hippocampal CA1 region of ovariectomized rats. Glucocorticoid receptor (GR) is a member of the adrenal steroid hormone receptor, which is highly expressed in the rat hippocampus. Activation of the GR plays a critical role in the neuronal stress responses, including ischemic brain damage. This study therefore examined the potential mechanisms by which genistein regulates GR signaling, including the protein distribution and receptor activation in hippocampus following ischemic reperfusion (I/R). Results showed that GR expression in the ovariectomized rats was excessively increased both in neurons (I/R 6h) and activated microglial cells (I/R 7d) in hippocampal CA1 region. Genistein treatment significantly attenuated GR induction and the enhanced GR nuclear translocation and DNA-binding capacity. The effects of genistein on the GR levels was accompanied with decreased blood plasma levels of corticosterone (primary glucocorticoid in rodents) and coupled to an E3 ubiquitin ligase Mdm2 targeted proteasomal degradation of GR, because genistein treatment could enhance the GR–Mdm2 interaction and the ubiquitination level of GR protein. In addition, our results indicated that genistein markedly prevented the excessive activation of microglia in CA1 sector. These results demonstrate the neuroprotective action of chronic low dose genistein replacement against ischemic brain damage, and a potential mechanism associated with the inhibition of both neuronal and microglial GR signaling following ischemic stress.

Embryonic exposure to corticosterone modifies aggressive behavior through alterations of the hypothalamic pituitary adrenal axis and the serotonergic system in the chicken

Exposure to excess glucocorticoids (GCs) during embryonic development influences offspring phenotypes and behaviors and induces epigenetic modifications of the genes in the hypothalamic–pituitary–adrenal (HPA) axis and in the serotonergic system in mammals. Whether prenatal corticosterone (CORT) exposure causes similar effects in avian species is less clear. In this study, we injected low (0.2μg) and high (1μg) doses of CORT into developing embryos on day 11 of incubation (E11) and tested the changes in aggressive behavior and hypothalamic gene expression on posthatch chickens of different ages. In ovo administration of high dose CORT significantly suppressed the growth rate from 3weeks of age and increased the frequency of aggressive behaviors, and the dosage was associated with elevated plasma CORT concentrations and significantly downregulated hypothalamic expression of arginine vasotocin (AVT) and corticotropin-releasing hormone (CRH). The hypothalamic content of glucocorticoid receptor (GR) protein was significantly decreased in the high dose group (p<0.05), whereas no changes were observed for GR mRNA. High dose CORT exposure significantly increased platelet serotonin (5-HT) uptake, decreased whole blood 5-HT concentration (p<0.05), downregulated hypothalamic tryptophan hydroxylase 1 (TPH1) mRNA and upregulated 5-HT receptor 1A (5-HTR1A) and monoamine oxidase A (MAO-A) mRNA, but not monoamine oxidase B (MAO-B). High dose CORT also significantly increased DNA methylation of the hypothalamic GR and CRH gene promoters (p<0.05). Our findings suggest that embryonic exposure to CORT programs aggressive behavior in the chicken through alterations of the HPA axis and the serotonergic system, which may involve modifications in DNA methylation.

Glucocorticoid receptors on and in a unicellular organism, Cryptobia salmositica

This is the first report to our knowledge that demonstrates a functional steroid hormone receptor in a protozoon. The study used Cryptobia salmositica, a pathogenic haemoflagellate found in salmonid fishes. It has been previously shown that cortisol and dexamethasone (a synthetic glucocorticoid) enhanced the multiplication of C. salmositica under in vitro conditions indicating the presence of glucocorticoid receptors on/in the parasite. Also, the glucocorticoid receptor antagonist, mifepristone (RU486), inhibited the stimulatory effect of the two glucocorticoids on parasite multiplication. In the present study, we used an antibody (produced in a rabbit against glucocorticoid receptor protein) agglutination test and confocal microscopy with immunohistofluorescence staining to demonstrate cortisol-glucocorticoid receptor-like protein receptors on the plasma membrane and in the cytoplasm of the parasite. In two in vitro studies, the addition of 50ngml−1 of RU486 was more effective in inhibiting parasite replication in cultures with 7,000parasitesml−1 than in cultures with 14,000parasitesml−1. Also, 100ngml−1 of RU486/ml was more effective than 50ngml−1 in inhibiting parasite multiplication in the 14,000 parasitesml-1 cultures. These in vitro studies indicate that the number of binding sites on/in the parasite is finite. The findings may be important in future studies especially on steroid receptor signalling pathways and dissection of ligand–receptor interactions, and for evaluating the adaptations that develop in pathogens as part of the host–parasite interaction.

All-trans retinoic acid-induced hypothalamus–pituitary–adrenal hyperactivity involves glucocorticoid receptor dysregulation

Clinical reports have highlighted a role for retinoids in the etiology of mood disorders. Although we had shown that recruitment of the nuclear receptor retinoic acid receptor-α (RAR-α) to corticotropin-releasing hormone (CRH) promoter is implicated in activation of the hypothalamus–pituitary–adrenal (HPA) axis, further insight into how retinoids modulate HPA axis activity is lacking. Here we show that all-trans retinoic acid (RA)-induced HPA activation involves impairments in glucocorticoid receptor (GR) negative feedback. RA was applied to rats chronically through intracerebroventricular injection. A 19-day RA exposure induced potent HPA axis activation and typical depression-like behavior. Dexamethasone failed to suppress basal corticosterone (CORT) secretion, which is indicative of a disturbed GR negative feedback. In the hypothalamic paraventricular nucleus, increased CRH+ and c-fos+ cells were found while a negative R−2+/ER+ correlation was present between the number of RAR-α+ and GR+ cells. This was paralleled by increased RAR-α and decreased GR protein expression in the hypothalamus. Additional in vitro studies confirmed that RA abolished GR-mediated glucocorticoid-induced suppression of CRH expression, indicating a negative cross-talk between RAR-α and GR signaling pathways. Finally, the above changes could be rapidly normalized by treatment with GR antagonist mifepristone. We conclude that in addition to the ‘classic' RAR-α-mediated transcriptional control of CRH expression, disturbances in GR negative feedback constitute a novel pathway that underlies RA-induced HPA axis hyperactivity. The rapid normalization by mifepristone may be of potential clinical interest in this respect.

Differential expression of hypothalamic fear- and stress-related genes in broiler chickens showing short or long tonic immobility

The serotonin system and the hypothalamic-pituitary-adrenal axis play important roles in modulating fear and stress-coping characteristics. Tonic immobility (TI) is a fear-related phenotype, and previously we have shown that broiler chickens showing short TI (STI) duration experience better growth performance and higher adaptability to stress. Here, we sought to further elucidate the central mechanisms underlying the phenotypic differences between chickens showing STI and long TI duration, by comparing the hypothalamic expression of genes in the serotonergic system and the hypothalamic-pituitary-adrenal axis under basal and corticosterone-exposed situations. The STI broilers had significantly lower (P < 0.01) hypothalamic expression of serotonin reuptake transporter and serotonin receptor 1A. Moreover, 11β-hydroxysteroid dehydrogenase type 2 was expressed significantly lower in STI chickens at the level of both mRNA (P < 0.01) and protein (P < 0.05). Hypothalamic expression of glucocorticoid receptor (GR) mRNA tended to be higher (P < 0.059) in long TI chickens, but the protein content was approximately 2 times higher (P < 0.01) in STI chickens. The uncoupled expression of GR mRNA and protein was associated with significantly lower (P < 0.05) expression of gga-miR-181a, gga-miR-211, and gga-miR-22, which are predicted to target GR, in STI chickens. Corticosterone administration reduced the mRNA expression of postsynaptic serotonin receptors, 5-hydroxytryptamine receptor 1B (P = 0.059) and 5-hydroxytryptamine receptor 7 (P < 0.05), yet significantly increased the protein content of 11β-hydroxysteroid dehydrogenase type 2 (P < 0.05). These results suggest that broilers of different TI phenotypes have a distinct pattern of hypothalamic expression of fear- and stress-related genes.

Direct Reversal of Glucocorticoid Resistance by AKT Inhibition in Acute Lymphoblastic Leukemia

Glucocorticoid resistance is a major driver of therapeutic failure in T cell acute lymphoblastic leukemia (T-ALL). Here, we identify the AKT1 kinase as a major negative regulator of the NR3C1 glucocorticoid receptor protein activity driving glucocorticoid resistance in T-ALL. Mechanistically, AKT1 impairs glucocorticoid-induced gene expression by direct phosphorylation of NR3C1 at position S134 and blocking glucocorticoid-induced NR3C1 translocation to the nucleus. Moreover, we demonstrate that loss of PTEN and consequent AKT1 activation can effectively block glucocorticoid-induced apoptosis and induce resistance to glucocorticoid therapy. Conversely, pharmacologic inhibition of AKT with MK2206 effectively restores glucocorticoid-induced NR3C1 translocation to the nucleus, increases the response of T-ALL cells to glucocorticoid therapy, and effectively reverses glucocorticoid resistance in vitro and in vivo.

Susceptibility to stress-induced visceral sensitivity: a bad legacy for next generations

Despite high prevalence, the precise irritable bowel syndrome (IBS) pathophysiology remains poorly understood likely due to the heterogeneity of IBS populations and the multifactorial etiology of this disorder. Among risk factors, genetic predisposition and early life trauma have been reported. In this context, the debate on genetic or environmental influences in the IBS pathogenesis is still open. The study by van der Wijngaard etal., reporting for the first time that susceptibility to stress-induced visceral hypersensitivity in maternally separated rats can be transferred to the next generation without any further exposure of F2 individuals to maternal separation, supports the importance of environmental influence in the IBS phenotype. Epigenetic mechanisms such as hypermethylation in the promoter region of the glucocorticoid receptor gene mediating the long-term and transgenerational behavioral effects of maternal care on the development have been shown in some but not in all studies. Van der Wijngaard etal. incriminated maternal care in the transmitted susceptibility to stress-induced visceral hypersensitivity, but not changes in glucocorticoid receptor protein expression in the brain. This finding opens a broad field of future directions aimed at evaluating the mechanisms involved in the transmission across generations of the digestive features of IBS, including, for example, on the role of gut microbiota changes in vertical transmission or epigenetic modifications of intestinal mast cells and the junctional region of intestinal epithelial cells in vertical transfer.

Mannotriose regulates learning and memory signal transduction in the hippocampus.

Rehmannia is a commonly used Chinese herb, which improves learning and memory. However, the crucial components of the signal transduction pathway associated with this effect remain elusive. Pri-mary hippocampal neurons were cultured in vitro, insulted with high-concentration (1 × 10−4 mol/L) cor-ticosterone, and treated with 1 × 10−4 mol/L mannotriose. Thiazolyl blue tetrazolium bromide assay and western blot analysis showed that hippocampal neuron survival rates and protein levels of glucocorti-coid receptor, serum and glucocorticoid-regulated protein kinase, and brain-derived neurotrophic factor were all dramatically decreased after high-concentration corticosterone-induced injury. This effect was reversed by mannotriose, to a similar level as RU38486 and donepezil. Our findings indicate that mannotriose could protect hippocampal neurons from high-concentration corticosterone-induced injury. The mechanism by which this occurred was associated with levels of glucocorticoid receptor protein, serum and glucocorticoid-regulated protein kinase, and brain-derived neurotrophic factor.

Deletions In TBL1XR1 Results In Glucocorticoid Resistance By Decreasing Glucocorticoid Signaling In Childhood B-Lymphoblastic Leukemia

IntroductionThe prognosis for children with acute lymphoblastic leukemia who relapse is poor and discovery of the underlying mechanisms that lead to drug resistance is a top priority. Relapsed blasts have intrinsic chemoresistance compared to diagnosis blasts especially to glucocorticoids (Klumper et. al, 1995). Furthermore, resistance to glucocorticoids is associated with a poor prognosis in childhood ALL (Dördelmann M et. al, Blood 1999, Schmiegelow K et. al, Leukemia 2001, Tissing WJ et. al, Leukemia 2003). We have previously identified recurrent deletions with concordant decreased gene expression in TBL1XR1 in 10.7% of patients at relapse (Hogan et. al, 2011). TBL1XR1 codes for the TBLR1 protein which is responsible for the dismissal and degradation of nuclear corepressor (N-CoR) complex proteins including N-CoR1, SMRT, GPS2, and histone deacetylases (HDAC) (Perissi V et. al, Cell 2004). We hypothesized that TBL1XR1 deletions may result in resistance to glucocorticoid agonist, prednisolone through up-regulation of N-CoR complex proteins. MethodsB-precursor ALL cell lines Reh, and RS4;11 were transduced with lentiviral constructs containing control and TBL1XR1 targeting shRNAs. Knockdown was confirmed by RT-PCR and western blotting. Stable cell lines were treated with prednisolone, doxorubicin, 6-thioguanine, or etoposide for 24-48 hours. Cell viability and apoptosis were measured by cell titer glo luminescence assay (promega) and annexin V-PE and 7-Amino-actinomycin D (7AAD) staining (Annexin V-PE Apoptosis Detection Kit, BD Pharmingen, San Diego, CA, USA) respectively. To determine changes in global gene expression by TBL1XR1 knockdown, stable Reh cell lines were treated with prednisolone or vehicle for 8 hours and then collected for RNA extraction (Qiagen, RNeasy mini kit) and microarray analysis. Microarray data was validated by RT-PCR. To elucidate the mechanism of resistance we performed small-scale biochemical fractionation and chromatin immunoprecipitation (ChIP) detecting levels of glucocorticoid receptor (GR), TBLR1, N-CoR1, and HDAC3 residing on the chromatin as well as gene specific glucocorticoid response elements (GREs). ResultsIn this study, we demonstrate that knockdown of TBL1XR1 results in resistance to the glucocorticoid agonist prednisolone but not other classes of chemotherapeutic agents. We discovered that 51 of the 117 genes induced by prednisolone in control cells had decreased induction of at least 50%. We validated a subset of prednisolone induced genes including, GILZ, TXNIP, ZEB1, ST6GALNAC3, IL21R, and CCPG1 by RT-PCR. To explore the mechanism of TBL1XR1 mediated decrease in GR signaling we determined the effect of TBL1XR1 depletion of GR recruitment to total bulk chromatin. In TBL1XR1 knockdown cells, no GR was detected in the chromatin associated fractions in vehicle or prednisolone treatment conditions, despite similar levels of GR protein between control and TBL1XR1 knock down lines. We show that the decreased GR levels is associated with an increased level of NCoR1 detected in the chromatin fraction of TBLR1 depleted cells; however no change in HDAC3 levels were observed. We confirmed these results by interrogating the gene regulatory regions of GILZ and TXNIP by ChIP. In TBL1XR1 depleted lines a decrease in GR occupancy in prednisolone stimulated cells was observed compared to control lines. We also observed increased levels of N-CoR1, and HDAC3 occupying these GREs. To interrogate the functional relationship between increased NCoR1 and HDAC3 levels on the gene regulatory region as a result of TBL1XR1 knockdown we depleted NCoR1 or inhibited HDAC3 using a pan HDAC inhibitor SAHA and examined the impact of prednisolone treatment on cell viability and induction of GILZ. We found that upon NCoR1 depletion or HDAC inhibition, TBL1XR1 knockdown line was no longer resistant to prednisolone and the induction of GILZ was restored.ConclusionsReduction of TBL1XR1 results in prednisolone resistance in ALL by decreasing GR occupancy on gene regulatory regions through the upregulation of the NCoR co-repressor complex at these sites. Our work and others has provided insight into the importance of transcription regulatory complexes in steroid resistance in ALL (and perhaps other malignancies) as well as opportunities for novel therapeutic approaches. Disclosures:No relevant conflicts of interest to declare.

C-terminal Hsp90-inhibitors restore glucocorticoid sensitivity in Cushing's disease

Cushing's disease is a severe neuroendocrine condition caused by partially glucocorticoid (Gc) resistant corticotroph adenomas of the anterior pituitary which express a functional glucocorticoid receptor (GR). The adenomas excessively secrete adrenocorticotropin (ACTH) into the bloodstream, and the patients suffer from chronic hypercortisolism. The pathomechanism of the partial Gc-resistance in Cushing's disease is scarcely understood and no safe and efficacious pharmacologic treatment to restore Gc-sensitivity in corticotroph adenomas exists. The molecular chaperone Hsp90 is essential for the function of the GR, and aberrant expression levels of Hsp90 have a negative impact on GR-signaling. We show by immunohistochemical staining that the inducible Hsp90α-isoform is strongly overexpressed in biopsy specimens of corticotroph adenomas from patients with Cushing's disease as compared to normal pituitary tissue. To investigate the role of Hsp90 in the GR-signaling of corticotroph cells, we pharmacologically inhibited Hsp90 with small-molecule compounds. The N-terminal Hsp90-inhibitor 17-AAG induces GR protein degradation and abolishes all aspects of GR-function. In sharp contrast, Novobiocin and the recently identified C-terminal Hsp90-inhibitor MPP-482 potentiate the activity of the agonist-bound GR. This effect correlates with the dissociation of the GR::Hsp90-complex by the C-terminal Hsp90-inhibitors at the biochemical level, while the GR protein level and the Hsp90-dependent nucleocytoplasmatic transport of the agonist-bound GR remain unaffected. Finally, MPP-482 enhances the suppression of ACTH-production by the GR in primary cultures of human corticotroph adenomas, restoring Gc-sensitivity. We show here that C-terminal Hsp90-inhibitors potentially reverse the Gc-resistance in human corticotroph adenoma cells by a novel mechanism of action. This work presents the proof-of-concept for the treatment of patients with Cushing's disease with the clinically safe Hsp90-inhibitor MPP-482 in the future.

Analysis of Glucocorticoid Receptors and Their Apoptotic Response to Dexamethasone in Male Murine B Cells During Development

Glucocorticoids have an important role in the resolution of inflammation and clinically they are routinely used to treat allergies, asthma, sepsis, and autoimmune diseases. In addition, glucocorticoids are well recognized to negatively impact the development and function of T cells in the immune system by inducing apoptosis. Less is known however about glucocorticoid function in B lymphocytes. Herein, we demonstrate that the glucocorticoid receptor (GR) is present in B-cell populations isolated from both the spleen and the bone marrow. B-cell populations were found to express more GR than non-B-cell populations from both the spleen and the bone marrow. GR protein was found in all B-cell (B220+) developmental subsets (Mature IgM+IgD+, Immature IgM+IgD-, and Pro/Pre IgM-IgD-) isolated from spleen. GR staining intensity was varied among the B-cell developmental subsets and was found to be higher in B cells isolated from the spleen (secondary lymphoid organ) versus the bone marrow (primary lymphoid organ). Ex vivo cell culture of murine splenocytes and bone marrow lymphocytes indicated that dexamethasone stimulated apoptosis in all B-cell developmental subsets demonstrating glucocorticoid responsiveness. Furthermore, in vivo administration of dexamethasone to adrenalectomized mice reduced B-cell numbers in both spleen and bone marrow. These data suggest that glucocorticoid signaling has an important understudied role in B-cell life-or-death decisions.

Exposure to environmental levels of waterborne cadmium impacts corticosteroidogenic and metabolic capacities, and compromises secondary stressor performance in rainbow trout

The physiological responses to waterborne cadmium exposure have been well documented; however, few studies have examined animal performances at low exposure concentrations of this metal. We tested the hypothesis that longer-term exposure to low levels of cadmium will compromise the steroidogenic and metabolic capacities, and reduce the cortisol response to a secondary stressor in fish. To test this, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to 0 (control), 0.75 or 2.0μg/L waterborne cadmium in a flow-through system and were sampled at 1, 7 and 28 d of exposure. There were only very slight disturbances in basal plasma cortisol, lactate or glucose levels in response to cadmium exposure over the 28 d period. Chronic cadmium exposure significantly affected key genes involved in corticosteroidogenesis, including melanocortin 2 receptor, steroidogenic acute regulatory protein and cytochrome P450 side chain cleavage enzyme. At 28 d, the high cadmium exposure group showed a significant drop in the glucocorticoid receptor and mineralocorticoid receptor protein expressions in the liver and brain, respectively. There were also perturbations in the metabolic capacities in the liver and gill of cadmium-exposed trout. Subjecting these fish to a secondary handling disturbance led to a significant attenuation of the stressor-induced plasma cortisol, glucose and lactate levels in the cadmium groups. Collectively, although trout appears to adjust to subchronic exposure to low levels of cadmium, it may be at the cost of impaired interrenal steroidogenic and tissue-specific metabolic capacities, leading to a compromised secondary stress performance in rainbow trout.

Maternal Undernutrition Programs Tissue-Specific Epigenetic Changes in the Glucocorticoid Receptor in Adult Offspring

Epidemiological data indicate that an adverse maternal environment during pregnancy predisposes offspring to metabolic syndrome with increased obesity, and type 2 diabetes. The mechanisms are still unclear although epigenetic modifications are implicated and the hypothalamus is a likely target. We hypothesized that maternal undernutrition (UN) around conception in sheep would lead to epigenetic changes in hypothalamic neurons regulating energy balance in the offspring, up to 5 years after the maternal insult. We found striking evidence of decreased glucocorticoid receptor (GR) promoter methylation, decreased histone lysine 27 trimethylation, and increased histone H3 lysine 9 acetylation in hypothalami from male and female adult offspring of UN mothers. These findings are entirely compatible with the increased GR mRNA and protein observed in the hypothalami. The increased GR predicted the decreased hypothalamic proopiomelanocortin expression and increased obesity that we observed in the 5-year-old adult males. The epigenetic and expression changes in GR were specific to the hypothalamus. Hippocampal GR mRNA and protein were decreased in UN offspring, whereas pituitary GR was altered in a sex-specific manner. In peripheral polymorphonuclear leukocytes there were no changes in GR methylation or protein, indicating that this epigenetic analysis did not predict changes in the brain. Overall, these results suggest that moderate changes in maternal nutrition, around the time of conception, signal life-long and tissue-specific epigenetic alterations in a key gene regulating energy balance in the hypothalamus.

Influence of chronic stress on brain corticosteroid receptors and HPA axis activity

BackgroundDisruption of the glucocorticoid negative feedback system evoked in animals by chronic stress can be induced by downregulation of glucocorticoid receptors (GRs) in several brain regions. In the present study, the dynamics of the changes in GRs, in brain structures involved in stress reactions, prefrontal cortex, hippocampus and hypothalamus was compared with the peripheral hypothalamo-pituitary-adrenocortical (HPA) axis hormones response to chronic stress. MethodsRats were exposed to 10min restraint or restrained twice a day for 3, 7or 14 days, and 24h after the last stress session exposed to homotypic stress for 10min. Control rats were not restrained. After rapid decapitation at 0, 1, 2, and 3h after stress termination, trunk blood for plasma adrenocorticotropic hormone (ACTH) and corticosterone determinations was collected and prefrontal cortex, hippocampus and hypothalamus were excised and frozen. Plasma hormones were determined using commercially available kits and glucocorticoids and mineralocorticoids protein levels in brain structure samples were determined by western blot procedure. ResultsRestraint stress alone significantly decreased glucocorticoid receptor (GR) level in prefrontal cortex and hippocampus, and increased mineralocorticoid receptor (MR) level in hypothalamus. Prior repeated stress for 3 days significantly increased GR protein level in hippocampus and diminished that level in hypothalamus in 7 days stressed rats. Acute stress-induced strong increase in plasma ACTH and corticosterone levels decreased to control level after 1 or 2h, respectively. Prior repeated stress for 3 days markedly diminished the fall in plasma ACTH level and repeated stress for 7 days moderately deepened this decrease. Plasma ACTH level induced by homotypic stress in rats exposed to restraint for 3, 7, and 14 days did not markedly differ from its control level, whereas plasma corticosterone response was significantly diminished. The fast decrease of stress-induced high plasma ACTH and corticosterone levels was accompanied by a parallel decline of GR level only in prefrontal cortex but not in the hippocampus or hypothalamus. ConclusionsComparison of the dynamics of changes in plasma ACTH and corticosterone level with respective alterations in GR and MR in brain structures suggests that the buffering effect of repeated stress depends on the period of habituation to stress and the brain structure involved in regulation of these stress response.

The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR.

Rapamycin Sensitizes Glucocorticoid Resistant Acute Lymphoblastic Leukemia CEM-C1 Cells to Dexamethasone Induced Apoptosis through both mTOR Suppression and Up-Regulation and Activation of Glucocorticoid Receptor

ObjectiveTo explore the role of glucocorticoid (GC) receptor (GR) in rapamycin's reversion of GC resistance in human GC-resistant T-acute lymphoblastic leukemia (ALL) CEM-C1 cells. MethodsCEM-C1 cells were cultured in vitro and treated with rapamycin at different concentrations with or without 1 μmol/L dexamethasone (Dex). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test was performed to assess cell proliferation. The cell cycle and cell apoptosis were analyzed by flow cytometry. The expression of GRα mRNA was determined by real-time quantitative RT-PCR. The expression of GR, p-70S6K, Mcl-1, and Bim proteins was detected by Western blot. ResultsWhen incubated with rapamycin at different concentrations, CEM-C1 cells showed significant growth inhibition in a time- and concentration-dependent manner. The growth inhibition was synergistically increased when CEM-C1 cells were treated with rapamycin plus 1 μmol/L Dex. CEM-C1 cells treated with rapamycin alone showed no apparent apoptosis, and were arrested at G0/G1 phase. After the treatment with Dex plus rapamycin, CEM-C1 cells demonstrated apparent apoptosis and increased the cell cycle arrested at G0/G1 phase. Rapamycin combined with Dex up-regulated GRα, phosphorylated GR(p-GR), and pro-apoptotic protein Bim-EL in CEM-C1 cells, but inhibited the expression of p-p70S6K, a downstream target protein of mTOR (mammalian target of rapamycin). ConclusionAfter the treatment with rapamycin plus Dex, Dex resistant CEM-C1 cells induce growth inhibition and apoptosis. The underlying mechanism may involve inhibition of the mTOR signaling pathway and also be associated with up-regulation of GR expression and activation of GC-GR signaling pathway.

Protective mechanisms of ulinastatin and dexamethasone against acute lung injury induced by lipopolysaccharide endotoxin in rats

Objective To investigate the protective mechanisms of ulinastatin (UTI) and dexamethasone (DXM) against acute lung injury (ALI) induced by lipopolysaccharide (LPS) endotoxin in rats.Methods Twenty four male Sprague-Dawley rats were randomly divided into four groups with 6 mice each group:control group (Con group),LPS group,LPS plus ulinastatin group (UTI group),LPS plus dexamethasone group (DXM group).Rats were killed at 8 h time points,and the protein and mRNA expression levels of glucocorticoid receptor (GR) in the lungs were detected by Western blot and Real Time fluorescent quantitative PCR.The levels of tumor necrosis factor (TNF)-α and interleukin (IL)-10 were also tested by enzyme linked immunosorbent assay (ELISA) and at the same time we tested the lung wet/dry weight radio(W/D).The pathological changes in lung tissue were evaluated by HE staining.Results ① The protein expression levels of GR:LPS group (0.418±0.018) was significantly lower than that in control group (0.841±0.021)(P＜0.05).The protein expression levels of GR in UTI group (0.692±0.018) and DXM group (0.702± 0.024)were higher than that in LPS group (P＜0.05).No significant differences were found between UTI and DXM group in the protein expression levels of GR (P＞0.05).② The expression levels of GRmRNA:LPS group was significantly lower,but the expression level of GRmRNA in UTI group was slightly higher than that in LPS group.No significant differences were found between UTI and LPS group in the expression levels of GRmRNA (P＞0.05),but the expression level of GRmRNA in DXM group was significantly higher than that in UTI group,LPS group and Con group(P＜0.05).③ The expression level of TNF-α:LPS group(374±8) ng/L was significantly higher than that in control group (102±6) ng/L(P＜0.05),the expression level of TNF-α in UTI group(343± 6) ng/L and DXM group(282±8) ng/L fell in between control group and LPS group,but the expression level of TNF-α in DXM group were lower than that in UTI group and higher than those in LPS group,Con group (P＜0.05).④ The expression levels of IL-10:LPS group (264±10) ng/L was significantly higher than that in control group (43±3) ng/L (P＜0.05).In UTI group (369±10) ng/L,the expression level of IL-10 was higher than that in LPS group(P＜0.05).The expression level of IL-10 in DXM group (180±7) ng/L fell in between control group and LPS group (P＜0.05).⑤ The lung W/D:LPS group (5.58±0.28) was significantly higher than that in control group(4.33±0.27)(P＜0.05),the lung W/D in UTI group (5.15±0.18) and DXM group (4.79±0.21) fell in between control group and LPS group (P＜0.05),but the lung W/D in DXM group were lower than that in UTI group (P＜0.05).⑥ Histopathologic examination of lungs:destroyed lung structure,inflammatory cells infiltration,widened alveolar septum with bleeding and edema were seen in LPS group.However,pathological changes in UTI group were milder than those in LPS group and pathological changes in group DXM milder than those in UTI group.Conclusions The protective mechanisms of UTI and DXM against ALI induced by LPS endotoxin in rats are not the same.The protective effect of DXM against ALI is better than UTI. Key words: Acute lung injury; Glucocorticoid receptor; Lipopolysaccharide; Ulinastatin; Dexamethasone

Exogenous prenatal corticosterone exposure mimics the effects of prenatal stress on adult brain stress response systems and fear extinction behavior

Exposure to early-life stress is a risk factor for the development of cognitive and emotional disorders later in life. We previously demonstrated that prenatal stress (PNS) in rats results in long-term, stable changes in central stress-response systems and impairs the ability to extinguish conditioned fear responding, a component of post-traumatic stress disorder (PTSD). Maternal corticosterone (CORT), released during prenatal stress, is a possible mediator of these effects. The purpose of the present study was to investigate whether fetal exposure to CORT at levels induced by PNS is sufficient to alter the development of adult stress neurobiology and fear extinction behavior. Pregnant dams were subject to either PNS (60 min immobilization/day from ED 14-21) or a daily injection of CORT (10mg/kg), which approximated both fetal and maternal plasma CORT levels elicited during PNS. Control dams were given injections of oil vehicle. Male offspring were allowed to grow to adulthood undisturbed, at which point they were sacrificed and the medial prefrontal cortex (mPFC), hippocampus, hypothalamus, and a section of the rostral pons containing the locus coeruleus (LC) were dissected. PNS and prenatal CORT treatment decreased glucocorticoid receptor protein levels in the mPFC, hippocampus, and hypothalamus when compared to control offspring. Both treatments also decreased tyrosine hydroxylase levels in the LC. Finally, the effect of prenatal CORT exposure on fear extinction behavior was examined following chronic stress. Prenatal CORT impaired both acquisition and recall of cue-conditioned fear extinction. This effect was additive to the impairment induced by previous chronic stress. Thus, these data suggest that fetal exposure to high levels of maternal CORT is responsible for many of the lasting neurobiological consequences of PNS as they relate to the processes underlying extinction of learned fear. The data further suggest that adverse prenatal environments constitute a risk factor for PTSD-like symptomatology, especially when combined with chronic stressors later in life.