Glucocorticoid receptor haploinsufficiency causes hypertension and attenuates hypothalamic-pituitary-adrenal axis and blood pressure adaptions to high-fat diet

Glucocorticoid hormones are critical to respond and adapt to stress. Genetic variations in the glucocorticoid receptor (GR) gene alter hypothalamic-pituitary-adrenal (HPA) axis activity and associate with hypertension and susceptibility to metabolic disease. Here we test the hypothesis that reduced GR density alters blood pressure and glucose and lipid homeostasis and limits adaption to obesogenic diet. Heterozygous GR βgeo/+ mice were generated from embryonic stem (ES) cells with a gene trap integration of a β-galactosidase-neomycin phosphotransferase (βgeo) cassette into the GR gene creating a transcriptionally inactive GR fusion protein. Although GRβgeo/+ mice have 50% less functional GR, they have normal lipid and glucose homeostasis due to compensatory HPA axis activation but are hypertensive due to activation of the renin-angiotensin- aldosterone system (RAAS). When challenged with a high-fat diet, weight gain, adiposity, and glucose intolerance were similarly increased in control and GRβgeo/+ mice, suggesting preserved control of intermediary metabolism and energy balance. However, whereas a high-fat diet caused HPA activation and increased blood pressure in control mice, these adaptions were attenuated or abolished in GRβgeo/+ mice. Thus, reduced GR density balanced by HPA activation leaves glucocorticoid functions unaffected but mineralocorticoid functions increased, causing hypertension. Importantly, reduced GR limits HPA and blood pressure adaptions to obesogenic diet.

Targeting glucocorticoid receptors that promote resilience in the treatment of addiction

There is strong evidence to suggest that the combination of alcohol and chronic repetitive stress leads to long-lasting effects on brain function, specifically areas associated with stress, motivation and decision-making such as the amygdala, nucleus accumbens and prefrontal cortex. Alcohol and stress together facilitate the imprinting of long-lasting memories. The molecular mechanisms and circuits involved are being studied but are not fully understood. Current evidence suggests that corticosterone (animals) or cortisol (humans), in addition to direct transcriptional effects on the genome, can directly regulate pre- and postsynaptic synaptic transmission through membrane bound glucocorticoid receptors (GR). Indeed, corticosterone-sensitive synaptic receptors may be critical sites for stress regulation of synaptic responses. Direct modulation of synaptic transmission by corticosterone may contribute to the regulation of synaptic plasticity and memory during stress (Johnson et al., 2005; Prager et al., 2010). Specifically, previous data has shown that long term alcohol (1) increases the expression of NR2Bcontaining NMDA receptors at glutamate synapses, (2) changes receptor density, and (3) changes morphology of dendritic spines (Prendergast and Mulholland; 2012). During alcohol withdrawal these changes are associated with increased glucocorticoid signalling and increased neuronal excitability. It has therefore been proposed that these synapse changes lead to the anxiety and alcohol craving associated with withdrawal (Prendergast and Mulholland; 2012). My lab is targeting this receptor system and the amygdala in order to understand the effect of combining alcohol and stress on these pathways. Lastly, we are testing GR specific compounds as potential new medications to promote the development of resilience to developing addiction.

An N-ethyl-n-nitrosourea induced corticotropin-releasing hormone promoter mutation provides a mouse model for endogenous glucocorticoid excess

Cushing's syndrome, which is characterized by excessive circulating glucocorticoid concentrations, maybe due to ACTH-dependent or -independent causes that include anterior pituitary and adrenal cortical tumors, respectively. ACTH secretion is stimulated by CRH, and we report a mouse model for Cushing's syndrome due to an N-ethyl-N-nitrosourea (ENU) induced Crh mutation at -120 bp of the promoter region, which significantly increased luciferase reporter activity and was thus a gain-of-function mutation. Crh -120/+ mice, when compared with wild-type littermates, had obesity, muscle wasting, thin skin, hair loss, and elevated plasma and urinary concentrations of corticosterone. In addition, Crh-120/+ mice had hyperglycemia, hyperfructosaminemia, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hyperleptinemia but normal adiponectin. Crh -120/+ mice also had low bone mineral density, hypercalcemia, hypercalciuria, and decreased concentrations of plasma PTH and osteocalcin. Bone histomorphometry revealed Crh-120/+ mice to have significant reductions in mineralizing surface area, mineral apposition, bone formation rates, osteoblast number, and the percentage of corticoendosteal bone covered by osteoblasts, which was accompanied by an increase in adipocytes in the bone marrow. Thus, a mouse model for Cushing's syndrome has been established, and this will help in further elucidating the pathophysiological effects of glucocorticoid excess and in evaluating treatments for corticosteroid-induced osteoporosis.

Circulating Glucocorticoid Bioactivity and Serum Glucocorticoid-Responsive Biomarkers during Steroid Therapy in Children and Adolescents with Inflammatory Bowel Disease

Objective: Glucocorticoid therapy is used worldwide to treat various inflammatory and immune conditions, including inflammatory bowel disease (IBD). In IBD, 80% of the patients obtain a positive response to the therapy; however the development of glucocorticoid-related side-effects is common. Our aim was therefore to study the possibility of optimizing glucocorticoid therapy in children and adolescents with IBD by measuring circulating glucocorticoid bioactivity (GBA) and serum glucocorticoid-responsive biomarkers in patients receiving steroid treatment for active disease. Methods: A total of sixty-nine paediatric IBD patients from the Paediatric Outpatient Clinics of the University Hospitals of Helsinki and Tampere participated in the studies. Control patients included 101 non-IBD patients and 41 disease controls in remission. In patients with active disease, blood samples were withdrawn before the glucocorticoid therapy was started, at 2-4 weeks after the initiation of the steroid and at 1-month intervals thereafter. Clinical response to glucocorticoid treatment and the development of steroid adverse events was carefully registered. GBA was analyzed with a COS-1 cell bioassay. The measured glucocorticoid therapy-responsive biomarkers included adipocyte-derived adiponectin and leptin, bone turnover-related collagen markers amino-terminal type I procollagen propeptide (PINP) and carboxyterminal telopeptide of type I collagen (ICTP) as well as insulin-like growth factor 1 (IGF-1) and sex hormone-binding globulin (SHBG), and inflammatory marker high-sensitivity C-reactive protein (hs-CRP). Results: The most promising marker for glucocorticoid sensitivity was serum adiponectin that associated with steroid therapy–related adverse events. Serum leptin indicated a similar trend. In contrast, circulating GBA rose in all subjects receiving glucocorticoid treatment but did not associate with the clinical response to steroids or with glucocorticoid therapy-related side-effects. Of notice, young patients (<10 years) showed similar GBA levels than older patients, despite receiving higher weight-adjusted doses of glucocorticoid. Markers of bone formation were lower in children with active IBD than in the control patients, probably reflecting the suppressive effect of the active inflammation. The onset of the glucocorticoid therapy further suppressed bone turnover. Inflammatory marker hs-CRP decreased readily after the initiation of the steroid, however the decrease did not associate with the clinical response to glucocorticoids. Conclusions: This is the first study to show that adipocyte-derived adiponectin associates with steroid therapy-induced side-effects. Further studies are needed, but it is possible that the adiponectin measurement could aid the recognition of glucocorticoid-sensitive patients in the future. GBA and the other markers reflecting glucocorticoid activity in different tissues changed during the treatment, however their change did not correlate with the therapeutic response to steroids or with the development of glucocorticoid-related side effects and therefore cannot guide the therapy in these patients. Studies such as as the present one that combine clinical data with newly developed biomolecular technology are needed to step-by-step build a general picture of the glucocorticoid actions in different tissues.

Interferon-gamma- and glucocorticoid-mediated pathways synergize to enhance death of CD4(+) CD8(+) thymocytes during Salmonella enterica serovar Typhimurium infection

Thymic atrophy is known to occur during infections; however, there is limited understanding of its causes and of the cross-talk between different pathways. This study investigates mechanisms involved in thymic atrophy during a model of oral infection by Salmonella enterica serovar Typhimurium (S.typhimurium). Significant death of CD4+CD8+ thymocytes, but not of single-positive thymocytes or peripheral lymphocytes, is observed at later stages during infection with live, but not heat-killed, bacteria. The death of CD4+CD8+ thymocytes is Fas-independent as shown by infection studies with lpr mice. However, apoptosis occurs with lowering of mitochondrial potential and higher caspase-3 activity. The amounts of cortisol, a glucocorticoid, and interferon- (IFN-), an inflammatory cytokine, increase upon infection. To investigate the functional roles of these molecules, studies were performed using Ifn/ mice together with RU486, a glucocorticoid receptor antagonist. Treatment of C57BL/6 mice with RU486 does not affect colony-forming units (CFU), amounts of IFN- and mouse survival; however, there is partial rescue in thymocyte death. Upon infection, Ifn/ mice display higher CFU and lower survival but more surviving thymocytes are recovered. However, there is no difference in cortisol amounts in C57BL/6 and Ifn/ mice. Importantly, the number of CD4+CD8+ thymocytes is significantly higher in Ifn/ mice treated with RU486 along with lower caspase-3 activity and mitochondrial damage. Hence, endogenous glucocorticoid and IFN--mediated pathways are parallel but synergize in an additive manner to induce death of CD4+CD8+ thymocytes during S.typhimurium infection. The implications of this study for host responses during infection are discussed.

Spatial memory deficits in maternal iron deficiency paradigms are associated with altered glucocorticoid levels

``The goal of this study was to examine the effect of maternal iron deficiency on the developing hippocampus in order to define a developmental window for this effect, and to see whether iron deficiency causes changes in glucocorticoid levels. The study was carried out using pre-natal, post-natal, and pre + post-natal iron deficiency paradigm. Iron deficient pregnant dams and their pups displayed elevated corticosterone which, in turn, differentially affected glucocorticoid receptor (GR) expression in the CA1 and the dentate gyrus. Brain Derived Neurotrophic Factor (BDNF) was reduced in the hippocampi of pups following elevated corticosterone levels. Reduced neurogenesis at P7 was seen in pups born to iron deficient mothers, and these pups had reduced numbers of hippocampal pyramidal and granule cells as adults. Hippocampal subdivision volumes also were altered. The structural and molecular defects in the pups were correlated with radial arm maze performance; reference memory function was especially affected. Pups from dams that were iron deficient throughout pregnancy and lactation displayed the complete spectrum of defects, while pups from dams that were iron deficient only during pregnancy or during lactation displayed subsets of defects. These findings show that maternal iron deficiency is associated with altered levels of corticosterone and GR expression, and with spatial memory deficits in their pups.'' (C) 2013 Elsevier Inc. All rights reserved.

Corticosterone targets distinct steps of synaptic transmission via concentration specific activation of mineralocorticoid and glucocorticoid receptors

Hippocampal neurons are affected by chronic stress and have a high density of cytoplasmic mineralocorticoid and glucocorticoid receptors (MR and GR). Detailed studies on the genomic effects of the stress hormone corticosterone at physiologically relevant concentrations on different steps in synaptic transmission are limited. In this study, we tried to delineate how activation of MR and GR by different concentrations of corticosterone affects synaptic transmission at various levels. The effect of 3-h corticosterone (25, 50, and 100nM) treatment on depolarization-mediated calcium influx, vesicular release and properties of miniature excitatory post-synaptic currents (mEPSCs) were studied in cultured hippocampal neurons. Activation of MR with 25nM corticosterone treatment resulted in enhanced depolarization-mediated calcium influx via a transcription-dependent process and increased frequency of mEPSCs with larger amplitude. On the other hand, activation of GR upon 100nM corticosterone treatment resulted in increase in the rate of vesicular release via the genomic actions of GR. Furthermore, GR activation led to significant increase in the frequency of mEPSCs with larger amplitude and faster decay. Our studies indicate that differential activation of the dual receptor system of MR and GR by corticosterone targets the steps in synaptic transmission differently.

Glucocorticoid receptor and protein/RNA synthesis-dependent mechanisms underlie the control of synaptic plasticity by stress

Learning and memory are exquisitely sensitive to behavioral stress, but the underlying mechanisms are still poorly understood. Because activity-dependent persistent changes in synaptic strength are believed to mediate memory processes in brain areas such as the hippocampus we have examined the means by which stress affects synaptic plasticity in the CA1 region of the hippocampus of anesthetized rats, Inescapable behavioral stress (placement on an elevated platform for 30 min) switched the direction of plasticity, favoring low frequency stimulation-induced decreases in synaptic transmission (long-term depression, LTD), and opposing the induction of long-term potentiation by high frequency stimulation, We have discovered that glucocorticoid receptor activation mediates these effects of stress on LTD and longterm potentiation in a protein synthesis-dependent manner because they were prevented by the glucocorticoid receptor antagonist RU 38486 and the protein synthesis inhibitor emetine. Consistent with this, the ability of exogenously applied corticosterone in non-stressed rats to mimic the effects of stress on synaptic plasticity was also blocked by these agents, The enablement of low frequency stimulation-induced LTD by both stress and exogenous corticosterone was also blocked by the transcription inhibitor actinomycin D, Thus, naturally occurring synaptic plasticity is liable to be reversed in stressful situations via glucocorticoid receptor activation and mechanisms dependent on the synthesis of new protein and RNA, This indicates that the modulation of hippocampus-mediated learning by acute inescapable stress requires glucocorticoid receptor-dependent initiation of transcription and translation.

Morphine conditioned place preference depends on glucocorticoid receptors in both hippocampus and nucleus accumbens

Learned association between drugs of abuse and context is essential for the formation of drug conditioned place preference (CPP), which is believed to engage many brain regions including hippocampus, and nucleus accumbens (NAc). The underlying mechanisms

The role of glucocorticoid-induced tumour necrosis factor receptor in developing mouse sympathetic neurons

Hereditary sensory autonomic neuropathy IV (HSAN IV) is an autosomal recessive disorder characterised by inability to feel pain and anhidrosis and is a consequence of defective NGF/TrkA signalling and growth of sensory and sympathetic neurons. Glucocortiocoid-induced tumour necrosis factors receptor (GITR), a transmembrane protein, activated by its specific ligand, GITRL, is well known for its role in the regulation of innate and acquired immune system responses. Recently, GITR was found to be required for NGF-dependant and extracellular signal-related kinase 1/2 (ERK1/2)-induced neurite growth and target innervation in the developing sympathetic nervous system (SNS). Given this novel role of GITR, it is possible that strategies targeting GITR have potential therapeutic benefit in promoting neurite growth in autonomic neuropathies such as HSAN IV. Using P1 mouse SCG neurons as a model, in addition to various SCG cell treatments, knock down models and transfection methods, we investigated whether GITR increases the sensitivity of sympathetic neurons to NGF; the region of GITR required for the enhancement of NGF-promoted growth, the signalling pathways downstream of GITR and how extensively GITR is involved in regulating peripheral innervation of the SNS. Results indicate that the region responsible for the growth promoting effects of GITR lies in its juxtamembrane intracellular region (here termed the growth promoting domain (GPD)) of GITR. The GPD of GITR activates ERK1/2 and inhibits nuclear factor kappa B (NF-κB) in an inverse fashion to provide an optimal cellular growth environment for P1 SCG neurons. While deleting the GPD of GITR had no effect on TrkA expression, constitutive phosphorylation of specific sites in the GPD reduced TrkA expression indicating a possible role for GITR in increasing the sensitivity of SCG neurons to NGF by the regulation of these sites, TrkA expression and subsequent NGF/TrkA binding. GITR appears to be heterogeneously required for NGF-promoted target innervation of SCG neurons in some organs, implying additional factors are involved in extensive NGF-target innervation of the SNS. In conclusion, this study answers basic biological questions regarding the molecular mechanism behind the role of GITR in the development of the SNS, and provides a basis for future research if GITR modulation is to be developed as a strategy for promoting axonal growth.

Characterization of Glucocorticoid Receptor Promoter Methylation in Breast Cancer

Epidemiological studies have identified psychological stress as a significant risk factor in breast cancer. The stress response is regulated by the HPA axis in the brain and is mediated by glucocorticoid receptor (GR) signalling. It has been found that early life events can affect epigenetic programming of GR, and methylation of the GR promoter has been reported in colorectal tumourigenesis. Decreased GR expression has also been observed in breast cancer. In addition, it has been previously demonstrated that unliganded GR can serve as a direct activator of the BRCA1 promoter in mammary epithelial cells. We propose a model whereby methylation of the GR promoter in the breast significantly lowers GR expression, resulting in insufficient BRCA1 promoter activation and an increased risk of developing cancer. Antibody-based methylated DNA enrichment was followed by qPCR analysis (MeDIP-qPCR) in a novel assay developed to detect locus-specific methylation levels. It was found that 13% of primary breast tumours were hypermethylated at the GR proximal promoter whereas no methylation was detected in normal tissue. RT-PCR and 5’ RACE analysis identified exon 1B as the predominant alternative first exon in the breast. Tumours methylated near exon 1B had decreased GR expression compared to unmethylated samples, suggesting that this region is important for transcriptional regulation of GR. It was also determined that GR and BRCA1 expression was decreased in breast tumour compared to normal tissue. Furthermore, the relative expression of GR and BRCA1 measured by qRT-PCR was correlated in normal tissue but this association was not found in tumour tissue. From this, it appears that lower GR levels with associated decreased BRCA1 expression in tissues may be a predisposing factor for breast cancer. Based on these results we propose a role for GR as a potential tumour suppressor gene in the breast due to its association with BRCA1, also a tumour suppressor gene, as well as its consistently decreased expression in breast tumours and methylation of its proximal promoter in a subset of cancer patients.

High concentrations of dexamethasone suppresses the proliferation but not the differentiation of further maturation of human osteoblast precursor in vitro: relevance to glucocorticoid-induced osteoporosis

Objective. The use of glucocorticoids (GCs) in the treatment of RA is a frequent cause of bone loss. In vitro, however, this same class of steroids has been shown to promote the recruitment and/or maturation of primitive osteogenic precursors present in the colony forming unit-fibroblastic (CFU-F) fraction of human bone and marrow. In an effort to reconcile these conflicting observations, we investigated the effects of the synthetic GC dexamethasone (Dx) on parameters of growth and osteogenic differentiation in cultures of bone marrow stromal cells derived from a large cohort of adult human donors (n=30). Methods. Marrow suspensions were cultured in the absence and presence of Dx at concentrations between 10 pm and 1 µm. After 28 days we determined the number and diameter of colonies formed, the total number of cells, the surface expression of receptors for selected growth factors and extracellular matrix proteins and, based on the expression of the developmental markers alkaline phosphatase (AP) and the antigen recognized by the STRO-1 monoclonal antibody, the proportion of cells undergoing osteogenic differentiation and their extent of maturation. Results. At a physiologically equivalent concentration, Dx had no effect on the adhesion of CFU-F or on their subsequent proliferation, but did promote their osteogenic differentiation and further maturation. These effects were independent of changes in the expression of the receptors for fibroblast growth factors, insulin-like growth factor 1, nerve growth factor, platelet-derived growth factors and parathyroid hormone/parathyroid hormone-related protein, but were associated with changes in the number of cells expressing the 2 and 4, but not ß1, integrin subunits. At supraphysiological concentrations, the effects of Dx on the osteogenic recruitment and maturation of CFU-F and their progeny were maintained but at the expense of a decrease in cell number. Conclusions. A decrease in the proliferation of osteogenic precursors, but not in their differentiation or maturation, is likely to be a key factor in the genesis of GC-induced bone loss.

Glucocorticoid induced muscle atrophy is associated with upregulation of myostatin gene expression

The mechanisms by which excessive glucocorticoids cause muscular atrophy remain unclear. We previously demonstrated that dexamethasone increases the expression of myostatin, a negative regulator of skeletal muscle mass, in vitro. In the present study, we tested the hypothesis that dexamethasone-induced muscle loss is associated with increased myostatin expression in vivo. Daily administration (60, 600, 1,200 micro g/kg body wt) of dexamethasone for 5 days resulted in rapid, dose-dependent loss of body weight (-4.0, -13.4, -17.2%, respectively, P <0.05 for each comparison), and muscle atrophy (6.3, 15.0, 16.6% below controls, respectively). These changes were associated with dose-dependent, marked induction of intramuscular myostatin mRNA (66.3, 450, 527.6% increase above controls, P <0.05 for each comparison) and protein expression (0.0, 260.5, 318.4% increase above controls, P <0.05). We found that the effect of dexamethasone on body weight and muscle loss and upregulation of intramuscular myostatin expression was time dependent. When dexamethasone treatment (600 micro g. kg-1. day-1) was extended from 5 to 10 days, the rate of body weight loss was markedly reduced to approximately 2% within this extended period. The concentrations of intramuscular myosin heavy chain type II in dexamethasone-treated rats were significantly lower (-43% after 5-day treatment, -14% after 10-day treatment) than their respective corresponding controls. The intramuscular myostatin concentration in rats treated with dexamethasone for 10 days returned to basal level. Concurrent treatment with RU-486 blocked dexamethasone-induced myostatin expression and significantly attenuated body loss and muscle atrophy. We propose that dexamethasone-induced muscle loss is mediated, at least in part, by the upregulation of myostatin expression through a glucocorticoid receptor-mediated pathway.