Glucocorticoid Receptor Levels Research Articles

Abstract LB-A10: ORIC-101 overcomes glucocorticoid-driven resistance to enzalutamide in castration-resistant prostate cancer

Abstract Prostate cancer is the second leading cause of cancer-related death in men. Androgen deprivation is commonly used to treat hormone-sensitive prostate cancer, while second-generation antiandrogens, including enzalutamide and abiraterone, benefit patients with castration-resistant prostate cancer (CRPC). New therapeutic strategies are needed for patients who relapse with metastatic CRPC. The glucocorticoid receptor (GR) has been implicated as a major bypass mechanism to escape androgen blockade in patients with CRPC, indicating that GR-targeted therapies may provide an avenue to overcome therapeutic resistance in this clinical setting. We discovered a novel, selective, oral GR antagonist, ORIC-101, which fully reverses GR-driven resistance to enzalutamide in a dose-dependent manner in preclinical models. Specifically, we show that GR is widely expressed in prostate cancer cell lines, organoids, and tumor tissue, and that GR levels are upregulated upon enzalutamide treatment. We demonstrate in preclinical studies that physiological levels of glucocorticoids promote tumor cell growth, stimulate androgen-regulated gene expression, and drive resistance to enzalutamide. Importantly, these effects were completely reversed by ORIC-101, suggesting ORIC-101 overcomes GR-driven resistance to enzalutamide. To inform the clinical development of ORIC-101 in combination with enzalutamide, we identified FKBP5 and KLK3 as direct transcriptional targets of both androgen receptor and GR. We subsequently validated that secreted PSA, a product of the KLK3 gene, can be induced by glucocorticoids and this induction is fully inhibited by ORIC-101. These findings confirm that targeting GR may help overcome resistance to enzalutamide in CRPC, and that FKBP5 and KLK3 are rational PD biomarkers that may be used in clinical trials of ORIC-101, along with PSA. Citation Format: Haiying Zhou, Shravani Barkund, Aleksandr Pankov, Sharvani Sinha, Dena Sutimantanapi, Lori S. Friedman, Omar Kabbarah. ORIC-101 overcomes glucocorticoid-driven resistance to enzalutamide in castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A10. doi:10.1158/1535-7163.TARG-19-LB-A10

Prenatal Glucocorticoid Exposure Results in Changes in Gene Transcription and DNA Methylation in the Female Juvenile Guinea Pig Hippocampus Across Three Generations

Synthetic glucocorticoids (sGC) are administered to women at risk for pre-term delivery, to mature the fetal lung and decrease neonatal morbidity. sGC also profoundly affect the fetal brain. The hippocampus expresses high levels of glucocorticoid (GR) and mineralocorticoid receptor (MR), and its development is affected by elevated fetal glucocorticoid levels. Antenatal sGC results in neuroendocrine and behavioral changes that persist in three generations of female guinea pig offspring of the paternal lineage. We hypothesized that antenatal sGC results in transgenerational changes in gene expression that correlate with changes in DNA methylation. We used RNASeq and capture probe bisulfite sequencing to investigate the transcriptomic and epigenomic effects of antenatal sGC exposure in the hippocampus of three generations of juvenile female offspring from the paternal lineage. Antenatal sGC exposure (F0 pregnancy) resulted in generation-specific changes in hippocampal gene transcription and DNA methylation. Significant changes in individual CpG methylation occurred in RNApol II binding regions of small non-coding RNA (snRNA) genes, which implicates alternative splicing as a mechanism involved in transgenerational transmission of the effects of antenatal sGC. This study provides novel perspectives on the mechanisms involved in transgenerational transmission and highlights the importance of human studies to determine the longer-term effects of antenatal sGC on hippocampal-related function.

Quercetin, caffeic acid and resveratrol regulate circadian clock genes and aging-related genes in young and old human lung fibroblast cells.

The circadian timing system of mammals is synchronized in concert with a central clock, but is also influenced by additional stimuli, including nutrients. However, little research has been done on polyphenols other than resveratrol and there seem to be no studies on their influence on young and old cells. The purpose of this study was to analyse the potential effects of quercetin, caffeic acid, and resveratrol on young and old fibroblast cells in the expressions of different clock genes and aging-related genes, and further investigate the mechanism. The mRNA expression of different clock genes and aging-related genes was assessed by quantitative real-time PCR. The protein levels of clock genes (BMAL1, PER1 and SIRT1) and glucocorticoid receptor α (GRα) were assessed by ELISA. Quercetin and caffeic acid in old fibroblast cells showed higher clock gene expression than resveratrol, quercetin increased Sirt1 expression, and caffeic acid increased Sirt6 expression indicating the possibility of an anti-aging effect. Also, quercetin and caffeic acid showed higher clock-controlled gene (Sirt1 and NR1D1) expression than resveratrol in young fibroblast cells. It appears that caffeic acid acts on NRF2 expression, and in turn to the actions of GRα, GDF11, Sirt1, and Sirt6. Regarding the increased expression of Per1, the activation effect on NR1D1 was confirmed only for caffeic acid in young fibroblast cells. Our results have confirmed the interplay of the circadian clock genes and cellular aging.

Corticosterone administration targeting a hypo-reactive HPA axis rescues a socially-avoidant phenotype in scarcity-adversity reared rats

It is well-established that children from low-income, under-resourced families are at increased risk of altered social development. However, the biological mechanisms by which poverty-related adversities can “get under the skin” to influence social behavior are poorly understood and cannot be easily ascertained using human research alone. This study utilized a rodent model of “scarcity-adversity,” which encompasses material resource deprivation (scarcity) and reduced caregiving quality (adversity), to explore how early-life scarcity-adversity causally influences social behavior via disruption of developing stress physiology. Results showed that early-life scarcity-adversity exposure increased social avoidance when offspring were tested in a social approach test in peri-adolescence. Furthermore, early-life scarcity-adversity led to blunted hypothalamic-pituitary-adrenal (HPA) axis activity as measured via adrenocorticotropic hormone (ACTH) and corticosterone (CORT) reactivity following the social approach test. Western blot analysis of brain tissue revealed that glucocorticoid receptor levels in the dorsal (but not ventral) hippocampus and medial prefrontal cortex were significantly elevated in scarcity-adversity reared rats following the social approach test. Finally, pharmacological repletion of CORT in scarcity-adversity reared peri-adolescents rescued social behavior. Our findings provide causal support that early-life scarcity-adversity exposure negatively impacts social development via a hypocorticosteronism-dependent mechanism, which can be targeted via CORT administration to rescue social behavior.

Effects of dietary energy level on appetite and central adenosine monophosphate-activated protein kinase (AMPK) in broilers.

Adenosine monophosphate-activated protein kinase (AMPK) acts as a sensor of cellular energy changes and is involved in the control of food intake. A total of 216 1-d-old broilers were randomly allotted into 3 treatments with 6 replicates per treatment and 12 broilers in each cage. The dietary treatments included 1) high-energy (HE) diet (3,500 kcal/kg), 2) normal-energy (NE) diet (3,200 kcal/kg), and 3) low-energy (LE) diet (2,900 kcal/kg). The present study was conducted to investigate the effects of dietary energy level on appetite and the central AMPK signal pathway. The results showed that a HE diet increased average daily gain (ADG), whereas a LE diet had the opposite effect (P < 0.05, N = 6). The average daily feed intake (ADFI) of the chickens fed the LE diet was significantly higher than that of the control (P < 0.05, N = 6). Overall, the feed conversion rate gradually decreased with increasing dietary energy level (P < 0.05, N = 6). Moreover, the chickens fed the LE and HE diets demonstrated markedly improved urea content compared with the control group (P < 0.0001, N = 8). The triglyceride (TG) content in the LE group was obviously higher than that in the HE group but showed no change compared with the control (P = 0.0678, N = 8). The abdominal fat rate gradually increased with increased dietary energy level (P = 0.0927, N = 8). The HE group showed downregulated gene expression levels of liver kinase B1 (LKB1), neuropeptide Y (NPY), cholecystokinin (CCK), and glucocorticoid receptor (GR) in the hypothalamus compared with the control group (P < 0.05, N = 8). However, LE treatment significantly increased the mRNA level of AMP-activated protein kinase α2 (AMPKα2) compared with other groups (P = 0.0110, N = 8). In conclusion, a HE diet inhibited appetite and central AMPK signaling. In contrast, a LE diet activated central AMPK and appetite. Overall, the central AMPK signal pathway and appetite were modulated in accordance with the energy level in the diet to regulate nutritional status and maintain energy homeostasis in birds.

Phosphodiesterase-2 Inhibitor Bay 60-7550 Ameliorates Aβ-Induced Cognitive and Memory Impairment via Regulation of the HPA Axis.

The dysfunction of the hypothalamus–pituitary–adrenal (HPA) axis is often seen in Alzheimer’s disease (AD) patients with cognitive deficits. Selective inhibition of phosphodiesterase (PDE) 4 and 5 has already proven to be effective in reducing beta-amyloid 1–42 (Aβ1–42)-mediated pathology by regulating corticotropin-releasing factor (CRF) and glucocorticoid receptor (GR) expression, suggesting that PDE-dependent signaling is involved in Aβ1–42-induced HPA axis dysfunction. However, nausea and vomiting are the side effects of some PDE4 inhibitors, which turn our attention to other PDEs. PDE2 are highly expressed in the hippocampus and cortex, which associate with learning and memory, but not in the area postrema that would cause vomiting. The present study suggested that microinjection of Aβ1–42 to the intracerebroventricle induced learning and memory impairments and dysregulation of the HPA axis by increased expression of CRF and GR. However, the PDE2 inhibitor Bay 60-7550 significantly ameliorated the learning and memory impairment in the Morris water maze (MWM) and step-down passive avoidance tests. The Aβ1–42-induced increased CRF and GR levels were also reversed by the treatment with Bay 60-7550. These Bay 60-7550’s effects were prevented by pretreatment with the PKG inhibitor KT5823. Moreover, the Bay 60-7550-induced downstream phosphorylation of cyclic AMP response element binding (pCREB) and brain-derived neurotrophic factor (BDNF) expression was also prevented (or partially prevented) by KT5823 or the PKA inhibitor H89. These results may lead to the discovery of novel strategies for the treatment of age-related cognitive disorders, such as AD, which affects approximately 44 million people worldwide.

Increased glucocorticoid receptor activity and proliferation in metastatic colon cancer

Metastasis is regarded as the fatal hallmark for colon cancer, but molecular mechanisms responsible for it have remained poorly defined. Glucocorticoid receptor (GR) within the tumor microenvironment mediates the effects of stress hormones which are used in clinics for their inflammation-modulatory and immunosuppressive properties. Further, epigenetic activation of GR promotes tumor heterogeneity and metastasis. Here, we sought to investigate the correlation between GR activation and proliferation and invasion in metastatic colon cancer microenvironment. We used proliferation/invasion assays, western blot, RT-qPCR, immunofluorescence staining and quantitative methylation to study glucocorticoid-GR signaling, including the involvement of CDK1, in human colon adenocarcinoma cell lines HT29 and T84 (a representative metastatic cell line). Nuclear expression levels of GR were significantly upregulated in metastatic T84 cells, and glucocorticoid derivative, dexamethasone (DEX) treatment caused increased proliferation and invasion in T84 cell, compared to HT29 cell. DEX treatment induced CDK1 expression which was accompanied by reduced CDK1 methylation, indicating epigenetic regulation. Depletion of GR suppressed proliferation of metastatic colon carcinoma cells and depletion of CDK1 had similar suppressing effects on proliferation as well as invasion of metastatic cells. Our study suggests that glucocorticoid-GR-CDK1 signaling induces proliferation and invasion of colon cancer cells and therapies involving the use of glucocorticoids need to exercise caution and re-evaluation.

Both insufficient and excessive glucocorticoid receptor-mediated signaling impair neuronal migration

Glucocorticoids (GCs) are a class of steroid hormones that regulate numerous physiological events in the human body. Clinically, glucocorticoids are used for anti-inflammatory and immunosuppressive actions via binding with glucocorticoid receptors (GRs). Emerging evidence has also indicated that inappropriate GC and GR levels are detrimental for brain development and eventually lead to severe neurological diseases. However, the roles of GC/GR signaling in brain development are not fully understood. Here, we showed that stable GR expression levels were critical for brain development, because both GR knockdown and overexpression severely impaired neuronal migration. Further studies showed that the multipolar-bipolar transition and leading process development were interrupted in GR-knockdown and GR-overexpressing neurons. To elucidate the underlying mechanism, we screened the protein levels of downstream molecules and identified RhoA as a factor negatively regulated by the GR. Restoration of the RhoA protein level partially rescued the neuronal migration defects in the GR-knockdown and GR-overexpressing neurons, indicating that RhoA played a major role in GR-mediated neuronal migration. These data suggest that an appropriate level of GC/GR signaling is essential for precise control of neuronal migration.

Glucocorticoid receptor upregulation increases radioresistance and triggers androgen independence of prostate cancer.

Despite the overall success of radiotherapy, a significant number of patients develop radioresistance, which leads to local regional recurrence and distant metastasis. We studied whether repeated radiation treatment promotes androgen-independent survival of prostate cancer (PCa) cells and their metastatic potential. We also studied whether glucocorticoid receptor (GR) increase in radioresistant cells is associated with acquisition of these aggressive characteristics. Radioresistant LNCaP (LNCaPR18) and C4-2 (C4-2R26) PCa sublines were developed by repeated radiation treatments of parental cells. Levels and activations of androgen receptor (AR) and GR in radioresistant PCa cells and respective parental cells were investigated in quantitative real-time polymerase chain reaction/Western blot analyses and immunofluorescence staining. Androgen-independent survival of radioresistant cells was tested in in vitro cell growth assays and the castration-resistant survival of these cell-derived tumors were investigated in mouse xenografts. Higher GR levels, but lower AR levels were detected in radioresistant cells than in parental cells. Radiation-induced GR upregulation was associated with increased intracellular cyclic adenosine monophosphate. As a consequence of GR activation, LNCaPR18 cells survived well in an androgen-depleted culture condition while parental cells could not. Results of in vivo mouse studies showed survival of LNCaPR18 cell-derived tumors in castrated mice while parental cell-derived tumors regressed. The growth of LNCaPR18 cell-derived tumors in castrated mice was impaired when treated with the anti-GR agent mifepristone. In experiments with C4-2/C4-2R26 cell sets, GR activation in C4-2R26 cells increased their metastatic potential. GR activation in radioresistant cells mediates androgen independence and facilitates PCa progression.

Expression of glucocorticoid and mineralocorticoid receptor genes co-varies with a stress-related colour signal in barn owls

Glucocorticoid hormones are important intermediates between an organism and its environment. They enable an organism to adjust its behavioural and physiological processes in response to environmental changes by binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) expressed in many tissues, including the integument. The regulation of glucocorticoids co-varies with melanin-based colouration in numerous species, an association that might result from pleiotropic effects of genes in the melanocortin system and evolve within a signalling context. Most studies have focused on the circulating levels of glucocorticoids disregarding the receptors that mediate their action, and that might partly account for the covariation between the regulation of stress and melanin-based colouration. We investigated the association of the expression levels of GR and MR genes with melanin-based colouration in the growing feathers of nestling barn owls (Tyto alba). We also explored the association between GR and MR expression levels and the expression of genes related to the melanocortin system and melanogenesis to better understand the origin of the link between the expression of receptors to which corticosterone binds and melanin-based colouration. Nestling barn owls displaying larger eumelanic black feather spots expressed GR and MR at lower levels than smaller-spotted individuals. However, we found that the expression of the GR and MR genes was positively rather than negatively correlated with the expression of genes involved in the deposition of melanin pigments at the time we sampled the nestlings. This provides mixed evidence of the association between melanin-based traits and MR and GR gene expression. The finding that the expression of GR and MR was positively associated with the expression of the PCSK2 gene (encoding one of the protein convertase responsible for the production of hormone peptide ACTH and α-MSH) suggests that the melanocortin system may be implicated in the establishment of the covariation between melanin-based colour and the expression of receptors to which glucocorticoids bind. However, further studies investigating the expression of melanin-based traits with stress-related endpoints at different time points of feather development will be necessary to understand better the proximate mechanism linking melanin-based traits with stress.

Mitochondrial signaling in inflammation-induced depressive behavior in female and male rats: The role of glucocorticoid receptor

Mitochondrial dysfunction can result from the interplay between elevated inflammatory markers and alterations in hypothalamic–pituitary–adrenal (HPA) axis, and can contribute to pathogenesis of major depression. Therefore, in this study we investigated whether the effects of lipopolysaccharide (LPS) on glucocorticoid receptor (GR) could be associated with alterations in mitochondrial apoptotic signaling in the prefrontal cortex of male and female Wistar rats with depressive-like behavior. To that end, we measured LPS-induced alterations in the extrinsic and intrinsic apoptotic pathways in mitochondria and cytosol of PFC of female and male rats, as well as the levels of cleaved cytosolic PARP-1. We also measured the mitochondrial levels of GR and its phosphoisoforms pGR232 and pGR246, as well as the mRNA levels of two GR-regulated mitochondrial genes, COX-1 and COX-3.We discovered that although seven-day LPS treatment evoked depressive-like behavior and induced apoptosis in the PFC of both sexes, it affected apoptotic cascades in both sexes differently. In females the treatment initiated both intrinsic and extrinsic apoptotic cascade, while in males only intrinsic cascade was engaged. Alterations in intrinsic apoptotic pathway were more associated with GR alterations in males, where LPS treatment decreased levels of mitochondrial GR and increased pGR232/pGR246 ratio. Alterations in mitochondrial GR could be associated with changes in expression of genes involved in oxidative metabolism in the PFC of this sex, and could, in combination with elevated levels of BCL-2 and decreased levels of BAX detected in this cell fraction, mitigate the detrimental effect of LPS on mitochondria in male PFC.

Radiation-induced glucocorticoid receptor promotes CD44+ prostate cancer stem cell growth through activation of SGK1-Wnt/β-catenin signaling.

We observed cancer stem cell (CSC) population increase in radioresistant LNCaP (LNCaPR18) and C4-2 (C4-2R26) prostate cancer (PCa) cells compared with respective parental cells. Since the CD44 level increase was most significant in radioresistant PCa cells compared with parental cells among CSC markers tested, we isolated the CD44+ population from LNCaP/LNCaPR18 and C4-2/C4-2R26 cell sets via the immunomagnetic separation method and used them as CSC sources. We detected lower AR level, but higher glucocorticoid receptor (GR) level in CD44+ CSCs than CD44- non-CSCs. Higher GR level in CD44+ CSCs than CD44- cells was also detected when cells were isolated from mouse tumor tissues of LNCaPR18 cell and C4-2R26 cell-derived human xenografts and grown in culture. We then found blocking the GR signaling by adding the anti-GR agent mifepristone into the cell culture inhibited the CD44+ CSC growth while the addition of the anti-AR agent enzalutamide enhanced the CSC growth. In xenograft mouse studies in which tumors were developed from the injection of CD44+ CSCs of LNCaPR18 or C4-2R26 cell lines, retarded tumor growth in mifepristone-injected mice was observed compared with vehicle-treated mice. We next discovered the GR regulation of Wnt/β-catenin signaling pathway. We further found that the serum/glucocorticoid regulated kinase 1 (SGK1) is the GR downstream molecule that mediates Wnt/β-catenin signaling activation. Therefore, inhibition of either SGK1 or Wnt/β-catenin signaling impaired the in vitro CD44+ CSC growth. From these results, we suggest that blocking GR signaling or its downstream SGK1-Wnt/β-catenin signaling axis may suppress the radiation-induced CSC increase in PCa. KEY MESSAGES: Higher CSC population exists in radioresistant PCa cells than parental cells. Higher GR levels (and lower AR level) in CD44+ CSCs than CD44- non-CSCs. Use of anti-GR agent blocked the growth of CD44+ CSCs in in vitro/in vivo tests. GR downstream SGK1-Wnt/β-catenin signaling axis mediates the CSC increase. Targeting this signaling axis may enhance the radiotherapy efficacy in treating PCa.

Anxiolytic impact of Apelin-13 in a rat model of Alzheimer's disease: Involvement of glucocorticoid receptor and FKBP5

Apelin-13 is known to be one of the predominant neuropeptides with marked protective role in circuits involved in mood disturbances. The most putative hypothesis in pathophysiology of Alzheimer's disease (AD) is Amyloid beta (Aβ) aggregation which interrupt proper function of hypothalamic–pituitary–adrenal (HPA) axis and are associated with anxiety. Here, we assessed the potential anxiolytic effect of Apelin-13 in a rodent cognitive impairment model induced by intrahippocampal Aβ 25-35 administration. We evaluated the memory impairment and anxiogenic behavior using shuttle box and Elevated plus maze apparatuses. We also measured the glucocorticoid receptor (GR) and FK506 binding protein 51 (FKBP5) expression as important markers showing the proper feedback mechanism within the HPA axis.Our findings showed that Aβ 25-35 administration induced memory impairment and anxiety behaviors. Apelin-13 exerted the anxiolytic effects and provided protection against Aβ 25-35 -induced passive avoidance memory impairment. Moreover, Apelin-13 caused an increase in GR and a decrease in FKBP5 expression levels in Aβ 25-35 treated animals.Taken together, these findings showed the anxiolytic effect of Apelin-13. This effect at least in part, may be mediated through the regulation of GR and FKBP5 expression levels which have a pivotal role in the appropriate negative feedback mechanism within the HPA axis. These data suggest that Apelin-13 might be considered as a potential neuropeptide defense that reduces anxiety along with neuroprotective effect against the Aβ 25-35 -induced injury.

SUN-008 The Glucocorticoid Receptor Is Essential For TGFβ and p38 MAPK Mediated Cancer Phenotypes in Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is the most metastatic and deadliest breast cancer (BC) subtype, accounting for 20-30% of all BCs. There is a critical need to identify molecular targets that could be exploited as new biomarkers of TNBC prognosis and for improving therapies. Although TNBC lacks estrogen and progesterone receptors, 15-40% of TNBC patients express the glucocorticoid receptor (GR). Women with TNBC that express high levels of GR have poor outcomes. We hypothesize that GR is a key mediator of advanced cancer phenotypes in TNBC. Specifically, we propose that GR acts as a “sensor” for stress signaling pathways commonly activated by soluble factors that are abundant within the tumor microenvironment (TME). Using TNBC models, we showed previously that GR is phosphorylated on Ser134 in response to cellular stress stimuli such as hypoxia. Herein, we show that pS134-GR is elevated in TNBC tumor tissue samples relative to non-TNBC tissues. In vitro studies in TNBC models demonstrate that GR Ser134 phosphorylation is promoted by cytokines (TGFbeta), and growth factors (HGF) and occurs in the absence of GR ligands such as Dexamethasone or cortisol. In response to stress signaling inputs, studies with kinase inhibitors confirmed that p38 MAPK is required for GR Ser134 (pS134-GR) phosphorylation. To evaluate the functional significance of pS134-GR, we created CRISPR models of MDA-MB-231 TNBC cells expressing either wt GR or phospho-mutant GR that cannot be phosphorylated at Ser134 (S134A). RNAseq studies were performed to identify pS134-GR target genes in TNBC models. Our transcriptome data demonstrated a requirement for pS134 GR in the expression of gene sets associated with TGFβ and p38 MAPK signaling. Pathway analysis revealed that pS134-GR target genes primarily regulate cancer cell migration. In vitro assays revealed that pS134-GR is essential for inducing cell migration and anchorage-independent growth in TNBC cells, even in the absence of exogenous GR ligands. Furthermore, using co-IP assays, we identified that upon phosphorylation at Ser134, GR interacts with the scaffolding protein 14-3-3zeta. Like pS134-GR, 14-3-3zeta is highly expressed in TNBC when compared to non-TNBC patients. We observed co-recruitment of both pS134-GR and 14-3-3zeta to known pS134-GR target genes (i.e. PTK6) in TNBC cells. These data prompted us to test the requirement for 14-3-3zeta in GR-mediated phenotypes. Short hairpin RNA knock-down experiments demonstrated that expression of 14-3-3zeta is required for serum and TGFbeta-induced TNBC cell migration. We conclude that the pS134-GR/14-3-3zeta complex is a key “sensor” of local stress signals within the TME (TGFbeta) and a potent mediator of cell migration in TNBC models. Further studies are aimed at exploring pS134 GR as a biomarker and therapeutic target in TNBC.

SUN-020 Analyses Of Clock Gene Expressions In Peripheral Leukocytes In Hypercortisolism

Background: Hypercortisolism causes metabolic disorders such as hyperglycemia and dyslipidemia. Recent studies have shown relationships between impairment of peripheral circadian clock function and the development of metabolic diseases. However, the association between phenotypes and peripheral circadian regulations in overt hypercortisolism and subclinical hypercortisolism clinically has not been elucidated. This study examined peripheral clock-related gene expressions of patients with hypercortisolism in regulation of involvement of glucocorticoid receptors. Methods: Blood samples were obtained from four patients with hypercortisolism including one patient of Cushing syndrome due to adrenal tumor, two patients of Cushing disease, and one patient due to ectopic ACTH producing tumor and five comparatively young volunteers as normal subjects at 08:00 and 20:00. Peripheral bone mononuclear cells (PBMCs) were extracted by Ficoll density gradient methods. Transcript levels of clock genes (Clock, Bmal1, Per1,Cry1, and ROR), glucocorticoid receptor (GR) and GILZ were determined by real-time quantitative PCR. Results: Serum cortisol levels were significantly higher in samples from 08:00 than 20:00 in normal subjects (p<0.05) and these levels from patients of hypercortisolism were significantly higher than normal subjects (p<0.01). Diurnal variation of cortisol levels in the patients was not observed. The expressions of Clock and Bmal1 mRNA in normal subjects showed higher at 20:00 than at 08:00 and Per1 mRNA was significantly lower at 20:00 than 08:00 (p<0.05), exhibiting rhythmicity in the PBMCs of 5 normal subjects. The expression levels of these clock-associated mRNAs and GR were significantly (p<0.01) lower in the patients than in normal subjects at 08:00 and 20:00. Moreover, the amplitudes of mRNA expression rhythms of clock-related genes tended to diminish in the patients. Multivariate analysis showed a positive correlation between Clock and Bmal1 in normal subjects and between Per1 and Cry1 significantly (p <0.01, p<0.01), and a same correlation was observed in hypercortisolem (P <0.01, p <0.001). Clock-associated genes and GR expressions showed a significant negative correlation with cortisol levels. Conclusions: These results suggest that rhythmic mRNA expression of clock genes is impaired in PBMCs of patients of hypercortisolism. Interestingly, molecules forming dimerization at the same phase in the circadian loops showed coordinated gene expressions in hypercortisolism. The expressions of the peripheral clock genes in the patients were markedly reduced, which was thought to be caused by a remarkable cortisol excess. Expressions of peripheral clock genes may be useful markers in predicting metabolic complications and therapeutic effects as well as leading to elucidate the pathology of metabolic disorders in subclinical hypercortisolism.

Increased protein and mRNA expression of corticotropin-releasing factor (CRF), decreased CRF receptors and CRF binding protein in specific postmortem brain areas of teenage suicide subjects

Overactivity of hypothalamic-pituitary-adrenal (HPA) axis function has been implicated in depression and suicidal behavior. This is based on the observation of an abnormal dexamethasone (DEX) and DEX-adrenocorticotropic hormone (ACTH) test in patients with depression and suicidal behavior. Recently, some studies have also found abnormalities of glucocorticoid receptors (GR), mineralocorticoid receptors (MR), corticotropin releasing factor (CRF), CRF receptors (CRF-R) and CRF binding protein (CRF-BP) in depressed and suicidal patients. Some investigators have also observed increased levels of CRF in the cerebrospinal fluid (CSF) and altered levels of MR, GR and CRF in the postmortem brain of depressed and suicidal subjects. We have earlier reported decreased protein and mRNA expression of GR and GILZ, a chaperone protein, in the postmortem brain of teenage suicide subjects. We have further studied CRF and its receptors in different areas of the postmortem brain of suicide subjects, i.e., the prefrontal cortex (PFC), hippocampus (HIPPO), subiculum and amygdala (AMY) from teenage suicide subjects. The CRF and its receptors were determined in the PFC (Brodmann area 9), HIPPO, subiculum and different amygdaloid nuclei from 24 normal control subjects and 24 teenage suicide subjects. Protein expression of CRF, its receptors and CRF-BP was determined by immunolabeling using the Western blot technique and mRNA expression was determined by real-time PCR (qPCR) technique. We found that the mRNA levels of CRF were significantly increased in the PFC, in the central amygdaloid nucleus (CeAMY) and in the subiculum. mRNA levels of CRF-R1 and CRF-BP were significantly decreased in the PFC. We did not find any changes in the HIPPO of any of the CRF components we studied. When we compared the protein expression of CRF components we found that CRF was significantly increased and CRF-R1, CRF-R2 and CRF-BP significantly decreased in the PFC. On the other hand, there were no changes in the protein expression of CRF components in the HIPPO. Our results in the postmortem brain suggest that, as found by clinical studies in the CSF, there are significant alterations of CRF and its receptors in the postmortem brain of teenage suicide subjects. These alterations of CRF and its components were region-specific, as changes were not generally observed in the HIPPO.

SAT-005 Gene Expression and Behavioral Alterations in Mice with Conditional Knockout of GR, MR, or Both GR and MR in the Hippocampus

Glucocorticoids are primary stress hormones that regulate brain function. Aberrant levels of these steroids have been implicated in the pathogenesis of cognitive impairments and psychiatric disorders. Glucocorticoids exert their effects on cells via binding the glucocorticoid receptor (GR) and the closely related mineralocorticoid receptor (MR). A distinctive feature of the hippocampus is that it expresses high levels of both GR and MR. However, the specific roles played by these receptors in mediating the direct actions of glucocorticoids in the hippocampus are poorly understood. To elucidate the in vivo function of hippocampal GR and MR, we employed emx1-cre mice to ablate GR (hippocampal GRKO), MR (hippocampal MRKO), or both GR and MR (hippocampal GRMRdKO) in the hippocampus. The single and double knockout mice were born at the expected Mendelian ratio and survived normally through 12 months of age. Small reductions in body weight were observed for the hippocampal MRKO and hippocampal GRMRdKO mice, but not the hippocampal GRKO mice. RNA sequencing performed on hippocampal RNA revealed major differences in both the basal and glucocorticoid regulated transcriptome in the single and double knockout mice. In behavioral assays, all three mutant mice exhibited reduced anxiety in the marble burying test. However, only the hippocampal MRKO and hippocampal GRMRdKO mice showed decreased anxiety in the elevated plus maze and open field tests. A forced swim test revealed no genotype differences in depression-like behavior. In a conditioned fear test, the hippocampal GRKO mice showed a reduction in context-dependent learning whereas the hippocampal MRKO and hippocampal GRMRdKO mice showed an increase in cue-dependent learning. These findings demonstrate that GR and MR regulate both common and unique signaling pathways in the hippocampus that influence anxiety and learning and memory.

Response of Glucocorticoid Receptor Alpha and Histone Deacetylase 2 to Glucocorticoid Treatment Predicts the Prognosis of Sudden Sensorineural Hearing Loss.

Objectives To investigate glucocorticoid receptor (GR) and histone deacetylase 2 (HDAC2) gene expression and protein levels in peripheral blood mononuclear cells (PBMCs) of patients with severe or profound sudden sensorineural hearing loss (SSNHL) and to explore the roles of GRs and HDAC2 in glucocorticoid (GC) insensitivity.Methods Fifty-five severe or profound SSNHL patients were enrolled in the study. According to hearing improvement after GC treatment, patients were assigned into two groups: GC-sensitive and GC-resistant. A normal reference group included 20 healthy volunteers without hearing loss. Quantitative real-time polymerase chain reaction and Western blot analyses were used to detect the relative expression of GRα, GRβ, and HDAC2 in PBMCs at the mRNA and protein levels.Results The protein levels of GRs and HDAC2 in PBMCs of SSNHL patients were lower than the normal reference values before GC treatment. Compared with the GC-resistant group, both the mRNA and protein levels of GRα and HDAC2 were significantly increased in the GC-sensitive group after GC treatment.Conclusion A lack of GRα and HDAC2 induction following steroid treatment in GC-resistant SSNHL patients may play a fundamental mechanistic role in GC insensitivity. Response of GRα and HDAC2 to steroid treatment may, thus, predict the prognosis of hearing improvement in SSNHL patients.

Corticosterone rather than ethanol epigenetic programmed testicular dysplasia caused by prenatal ethanol exposure in male offspring rats

ABSTRACTPrenatal ethanol exposure (PEE) could affect offspring’s testicular development. This study aimed to illuminate its intrauterine origin and the programming mechanism caused by PEE. Pregnant Wistar rats were given ethanol (4 g/kg.d) by gavage administration during gestational days (GD) 9–20. Serum samples and testes of male offspring rats were collected on GD20, postnatal week (PW) 6, and PW12. We found that PEE induced testicular morphological abnormality, low serum testosterone levels, expressive suppression of 3β-hydroxysteroid dehydrogenase (3β-HSD), and low acetylation levels of histone 3 lysine 14 (H3K14ac) of 3β-HSD before and after birth. In utero, when fetal rats were overexposed to corticosterone by PEE, the expression levels of testicular glucocorticoid receptor (GR) and histone deacetylase 2 (HDAC2) were increased, while that of steroidogenic factor 1 (SF1) was decreased. In vitro, corticosterone (rather than ethanol) at 500 to 2,000 nM concentration decreased testosterone production and 3β-HSD expression in a concentration-dependent manner. Moreover, corticosterone downregulated SF1 and upregulated HDAC2 via activating GR, accompanied by a low H3K14ac level of 3β-HSD; SF1 overexpression could reverse the increased HDAC2 expression, and knockdown of HDAC2 could partially reverse the inhibitory effects of corticosterone on H3K14ac level and 3β-HSD expression but not on SF1 expression. Taken together, PEE caused testicular dysplasia in male offspring rats, which was associated with corticosterone-induced low-functional programming of 3β-HSD through the GR/SF1/HDAC2/H3K14ac pathway. This study provides new academic perspectives to illuminate the theory of ‘Developmental Origins of Health and Disease.’

The Effect of Histone Deacetylase Inhibitor on the Expression Level of Glucococrticoid Receptor in Rat Forebrain under Hypoxia

The activity of the gene transcription depends to a large extent on the histone acetylation status and can be affected by different stimuli such as hypoxia. Histone deacetylase inhibitors promote gene transcription by facilitating histone acetylation and are thus considered as candidates for target therapy of posthypoxic states. In the present study, using immunohistochemistry, the effect of the histone deacetylase inhibitor trihostatin A (TSA) on the expression level of glucocorticoid receptor (GR) have been analyzed in the neocortex and hippocampus of rats in an original model of severe hypobaric hypoxia (SHH). It has been found that injections of TSA in rats facilitated GR expression after SHH in the neocortex and CA1 field but not in the dentate girus of hippocampus. GR overexpression enhances the toxicity of circulating glucocorticoid hormones on the hippocampal neurons. Stimulation of GR expression by TSA injections in the CA1 field of the hippocampus may lead to maladaptation, making possible damaging effects of unfavorable factors, such as hypoxia. The data obtained indicate that therapy using histone deacetylase inhibitors may have adverse side effects for vulnerable brain neurons.