GRα Expression Research Articles

Honokiol Exerts Antidepressant Effects in Rats Exposed to Chronic Unpredictable Mild Stress by Regulating Brain Derived Neurotrophic Factor Level and Hypothalamus-Pituitary-Adrenal Axis Activity.

Honokiol (HNK), the main active component of Magnolia officinalis, has shown a variety of pharmacological activities. In the present study, we measured the antidepressant-like effects of HNK in a rat model of chronic unpredictable mild stress (CUMS) and explored its possible mechanisms. The antidepressant-like effects of HNK were assessed in rats by an open field test (OFT), sucrose preference test (SPT) and forced swimming test (FST). Then, serum levels of corticotrophin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and corticosterone (CORT) and hippocampal brain-derived neurotrophic factor (BDNF) and glucocorticoid receptor α (GRα) levels were assessed to explore the possible mechanisms. We identified that HNK treatment (2, 4, and 8mg/kg) alleviated the CUMS-induced behavioural deficits. Treatment with HNK also normalized the CUMS-induced hyperactivity of the limbic hypothalamic-pituitary-adrenal (HPA) axis, as indicated by reduced CRH, ACTH and CORT serum levels. In addition, HNK increased the expression of GRα (mRNA and protein) and BDNF (mRNA and protein) in the hippocampus. These data confirmed the antidepressant-like effects of HNK, which may be related to its normalizing the function of the HPA axis and increasing the BDNF level in the hippocampus.

Inhibition of Osteoblast Function by Brucella abortus is Reversed by Dehydroepiandrosterone and Involves ERK1/2 and Estrogen Receptor.

Brucella abortus induces an inflammatory response that stimulates the endocrine system resulting in the secretion of cortisol and dehydroepiandrosterone (DHEA). Osteoarticular brucellosis is the most common presentation of the active disease in humans, and we have previously demonstrated that B. abortus infection inhibits osteoblast function. We aimed to evaluate the role of cortisol and DHEA on osteoblast during B. abortus infection. B. abortus infection induces apoptosis and inhibits osteoblast function. DHEA treatment reversed the effect of B. abortus infection on osteoblast by increasing their proliferation, inhibiting osteoblast apoptosis, and reversing the inhibitory effect of B. abortus on osteoblast differentiation and function. By contrast, cortisol increased the effect of B. abortus infection. Cortisol regulates target genes by binding to the glucocorticoid receptor (GR). B. abortus infection inhibited GRα expression. Cell responses to cortisol not only depend on GR expression but also on its intracellular bioavailability, that is, dependent on the activity of the isoenzymes 11β-hydroxysteroid dehydrogenase (HSD) type-1, 11β-HSD2 (which convert cortisone to cortisol and vice versa, respectively). Alterations in the expression of these isoenzymes in bone cells are associated with bone loss. B. abortus infection increased 11β-HSD1 expression but had no effect on 11β-HSD2. DHEA reversed the inhibitory effect induced by B. abortus infection on osteoblast matrix deposition in an estrogen receptor- and ERK1/2-dependent manner. We conclude that DHEA intervention improves osteoblast function during B. abortus infection making it a potential candidate to ameliorate the osteoarticular symptoms of brucellosis.

Icariin inhibits LPS-induced cell inflammatory response by promoting GRα nuclear translocation and upregulating GRα expression

AimsIcariin (ICA) is a flavonoid isolated from certain plant species in the genus Epimedium, especially Epimedium brevicornum. Previous studies indicated that ICA has certain regulatory effects on some inflammatory diseases, and that ICA regulates the activity of glucocorticoid receptor (GR) and NF-κB. But the causal link between GR and NF-κB and other downstream pathways in effects of ICA remained elusive, therefore here we have investigated whether ICA could promote GR function, in turn, to regulate NF-κB and/or other factors to achieve its anti-inflammatory effect. Main methodsInflammatory cell models were induced by lipopolysaccharide (LPS) in RAW 264.7 and HeLa cell line. Observation of GRα nuclear translocation by confocal laser scanning microscopy. GRα and inflammatory cytokines expression was detected by RT-qPCR, Western Blotting and ELISA. Co-immunoprecipitation technique was used to detect the binding of GRα to downstream transcription factors. GRα activity was blocked by GRα antagonist RU486, and GR downstream transcription factors including NF-κB, c-Jun, and Stat3 were silenced by corresponding RNA interference. Key findingsIn both inflammatory cell models, ICA decreased LPS-induced production of inflammatory cytokines (IL-6 and TNF-α). While ICA up-regulated the amount of GRα and promoted its nucleus translocation. The increased GRα in the nucleus by ICA bound more NF-κB, c-Jun, and Stat3. Blockade GRα and silence of NF-κB, c-Jun, and Stat3 expression partially abolished the anti-inflammatory effects of ICA. SignificancePromoted GR function and the consequent inhibition of pro-inflammatory transcription factors contribute a main mechanism by which ICA exerts its anti-inflammatory effect.

The Role and Regulation of the 11 Beta-Hydroxysteroid Dehydrogenase Enzyme System in Patients with Inflammatory Bowel Disease.

Glucocorticoids are known to modulate a number of immunological responses including counteracting inflammation. Within tissues expressing the glucocorticoid and mineralocorticoid receptors including the colon, glucocorticoid metabolism is regulated by the isoenzymes of 11ß-hydroxysteroid dehydrogenase (11β-HSD). 11β-HSD1 acts as an oxidoreductase converting inactive cortisone into active cortisol, while 11β-HSD2 acts as a dehydrogenase converting active cortisol to inactive cortisone. Hexose-6 phosphate dehydrogenase (H6PDH) is a key regulator of 11β-HSD1 activity via its generation of NADPH. Variations in the 11β-HSD enzyme system in relation to levels of expression and regulation may have a role in IBD. The aim of this study was to investigate possible abnormalities of 11β-HSD enzyme system in the colon of patients with IBD. By using quantitative real-time PCR, we investigated the transcription levels of 11β-HSD1 and 2 in colonic tissue from IBD patients and healthy controls undergoing a colonoscopy for disease assessment. Disease activity was recorded using clinical (Mayo Score/Harvey-Bradshaw Index), Biochemical (C-reactive protein), histological, and endoscopic parameters. In addition, transcription levels of H6PDH and the glucocorticoid receptor alpha (GR-α) as well as key pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, Rela (subunit for NF Kappa B)) were later examined among this group, and results were correlated with 11β-HSD2 gene expression. Results and patient demographics were expressed as a mean (and SD), and differences between IBD patients and control groups were analyzed using a Student's t test or Mann-Whitney U test as appropriate, with a p value of ≤0.05 considered significant. Results were controlled for disease activity as outlined above. Results have demonstrated a significant downregulation in 11β-HSD2 expression in IBD patients compared with controls (13.8±17.1 au vs. 318.4±521.1au, p=0.01), whereas levels of 11β-HSD1 did not appear to vary across the two groups. Among IBD patients, there was a trend toward higher 11β-HSD1 expression in inflamed tissue compared with matched non-inflamed tissue (422.1±944 au vs. 102.2±103.9, P=0.09). Levels of H6PDH and the GR-α expression did not appear to vary among active inflamed IBD tissue and controls. As a result, we examined the association between pro-inflammatory cytokines and levels of 11β-HSD2 expression. Results showed an upregulation of key pro-inflammatory cytokine mRNA expression (TNF-α, IL-1β, IL-6) during inflammation with an associated downregulation of 11β-HSD2 mRNA expression when compared to controls. Dysregulation in this pathway could have a potential role in IBD pathogenesis and may account for exogenous glucocorticoid resistance in IBD. Further work assessing the role of the 11β-HSD enzyme system in steroid-resistant subjects is warranted.

Increased methylation of glucocorticoid receptor gene promoter 1F in peripheral blood of patients with generalized anxiety disorder

Previous findings on the dysfunction of hypothalamic-pituitary-adrenal (HPA) axis in generalized anxiety disorder (GAD) are controversial, and the molecular mechanisms underlying such dysfunction remain unclear. We analyzed the methylation status of the NR3C1 1F promoter and the expression of glucocorticoid receptor-α isoform (GRα) in peripheral blood mononuclear cells (PMBCs), the basal cortisol level in serum, and a functional neuroendocrine marker for GR sensitivity in the PMBCs in 64 patients with current GAD and 85 healthy controls. We found that patients with GAD had significantly elevated levels of morning basal serum cortisol (P < 0.0001) and diminished GR sensitivity in the PBMCs (P < 0.0001) compared with healthy controls. The overall methylation levels across NR3C1 1F promoter (P < 0.0001) and percent methylation at each of the 5 CpG sites including CpG12, 21, 30, 31, and 32 (P < 0.001) significantly increased. Accordingly, the mRNA levels of GRα significantly decreased (P < 0.0001) in the PBMCs in patients with GAD compared with healthy controls, with the effects specific in patients without childhood traumatic experience. Moreover, both serum basal cortisol levels and GR sensitivity in the PBMCs were negatively correlated with the overall methylation levels of the NR3C1 1F promoter (P < 0.0001) and positively correlated with GRα mRNA levels (P = 0.007) in the PBMCs. In sum, our study revealed the increased activity of the HPA axis and diminished peripheral glucocorticoid responsiveness of GR underlying episodes of GAD. Furthermore, such dysfunction of the HPA axis is associated with both increased DNA methylation of NR3C1 1F promoter and decreased GRα expression.

Active Ingredients of Epimedii Folium and Ligustri Lucidi Fructus Balanced GR/HSP90 to Improve the Sensitivity of Asthmatic Rats to Budesonide

This study aimed to investigate the possible molecular mechanisms of active ingredients of Epimedii Folium (EF) and Ligustri Lucidi Fructus (LLF) combined with Budesonide (Bun) in asthmatic rats. Rats were divided into 5 groups, including normal group, asthma model group, Bun group, group of active ingredients of EL and LLF (EL), and group of coadministration of Bun with EL (Bun&EL). The asthmatic model was prepared by ovalbumin sensitizing and challenging. Lymphocyte apoptosis, GR protein and binding, and the protein and mRNA of GRα, GRβ, and HSP90 were tested. The results showed that Bun&EL ① markedly increased lymphocyte apoptosis, GR and HSP90 protein, and GR binding in BALF and ② enhanced the expressions of GRα and HSP90 and the ratio of GRα to GRβ or to HSP90 both in protein and in mRNA levels in lung, ③ while decrease occurred in GRβ mRNA and the mRNA ratio of GRβ to HSP90 compared with asthma or Bun group. Moreover, there was a significant correlation between GRα and GRβ in protein level, or between GRα and HSP90 both in protein and in mRNA levels. EL may effectively enhance the sensitivity of asthmatic rats to Bun via balancing GR/HSP90. And these findings will be beneficial for the treatment of asthma in the future.

Determinants of altered intracellular endocrine-immune interplay in Bosnian war refugees suffering from PTSD

Posttraumatic Stress Disorder (PTSD) has been repeatedly linked to changes in glucocorticoid (GC) sensitivity. To increase our understanding of this phenomenon and its potential relevance for PTSD development and treatment, the current study investigates the interplay between two key moderators, glucocorticoid receptor (GRα) and GR co-chaperone FKBP5, and their relation to GC sensitivity.A GC sensitivity assay was performed in 52 Bosnian war refugees (19m; 40.8±8.7 years) clinically diagnosed with PTSD to divide the patient group into a high (HS) and a low (LS) GC sensitivity group. Expression of GRα and FKBP5 mRNA was quantified by real-time RT-PCR.Links between gene expression and GC sensitivity were driven by the HS group of PTSD patients, which also showed increased expression of GRα but not FKBP5 compared to the LS group. Further, expressions of FKBP5 and GRα were strongly correlated in the HS patient group, while this association was missing in the LS PTSD group.Our findings suggest that PTSD phenotypes may be characterized by differences in intracellular signaling transduction processes. The associations of expression of GRα and FKBP5 in the high-sensitive PTSD subgroup may thereby reflect physiological adaptation to preserve immune-relevant GC signaling. Further research is needed to understand the role and consequences of GRα-FKBP5 dissociation in low GC sensitivity PTSD patients.

Glucocorticoid receptor beta increases migration of human bladder cancer cells.

Bladder cancer is observed worldwide having been associated with a host of environmental and lifestyle risk factors. Recent investigations on anti-inflammatory glucocorticoid signaling point to a pathway that may impact bladder cancer. Here we show an inverse effect on the glucocorticoid receptor (GR) isoform signaling that may lead to bladder cancer. We found similar GRα expression levels in the transitional uroepithelial cancer cell lines T24 and UMUC-3. However, the T24 cells showed a significant (p < 0.05) increased expression of GRβ compared to UMUC-3, which also correlated with higher migration rates. Knockdown of GRβ in the T24 cells resulted in a decreased migration rate. Mutational analysis of the 3′ untranslated region (UTR) of human GRβ revealed that miR144 might positively regulate expression. Indeed, overexpression of miR144 increased GRβ by 3.8 fold. In addition, miR144 and GRβ were upregulated during migration. We used a peptide nucleic acid conjugated to a cell penetrating-peptide (Sweet-P) to block the binding site for miR144 in the 3′UTR of GRβ. Sweet-P effectively prevented miR144 actions and decreased GRβ expression, as well as the migration of the T24 human bladder cancer cells. Therefore, GRβ may have a significant role in bladder cancer, and possibly serve as a therapeutic target for the disease.

Relationship between expression of glucocorticoid receptor isoforms and glucocorticoid resistance in immune thrombocytopenia

Background: Glucocorticoids (GCs) play an important role in the treatment of several hematological malignancies, such as immune thrombocytopenia (ITP). The glucocorticoid receptor (GR) mediates the effects of GCs. Five isoforms of GR mRNA were described: GRα, GRβ, GRγ, GRP, and GRA. GR levels are regulated by alternative splicing of GR mRNA. Several studies demonstrated that a lower GR expression was associated with poor GC response.Purpose: This study investigated the expression of GR isoforms and the relationship between GC resistance in ITP.Methods: This study determined GRα/β/γ/P mRNA and GRα/β protein expression levels using SYBR Green Real-time PCR and Western blot, respectively, in 49 newly diagnosed ITP patients and 31 controls.Results: The expression of GR isoform mRNA in ITP and controls showed the following trend: GRα > GRP > GRγ > GRβ. The expression of GRα, β mRNA and the total frequency of the four GR isoforms in ITP was significantly higher than in controls (P < 0.05). The expression of GRα mRNA and protein in the GC-resistant group was significantly lower than that in the GC-sensitive group and controls (P < 0.05). GRβ could not be detected at the protein level in our experimental conditions.Conclusion: GRα and GRP were the main GR isoforms responsible for the effects of GC, and GRα and GRP exhibited synergistic effects. The down-regulation of GRα levels may play an important role in GC resistance in ITP. The effects of GCs in ITP were not associated with changes in GRβ and GRγ.

The pro-social neurohormone oxytocin reverses the actions of the stress hormone cortisol in human ovarian carcinoma cells in vitro.

The journey patients with ovarian cancer travel from non-specific symptoms causing delayed diagnosis through surgery and chemotherapy, culminating in a 5-year survival rate of 43%, must have a profound and detrimental psychological impact on patients. Emerging studies link higher levels of oxytocin (OT) and increased social support, an independent prognostic factor in cancer, with a moderating effect on stress. In contrast, there is a known association of tumour cell proliferation with elevated cortisol (stress hormone) levels. We hypothesise therefore that there is cross-talk between cortisol and oxytocin at a molecular level. Three ovarian cancer cell lines, used as in vitro models, were treated with cortisol at concentrations mimicking physiological stress in vivo in the presence or absence of OT. OT reduced cell proliferation and migration, induced apoptosis and autophagy for all three cell lines, partially reversing the effects of cortisol. Quantitative RT-PCR of tissue taken from ovarian cancer patients revealed that the glucocorticoid receptor (splice variant GR-P) and OT receptor (OTR) were significantly upregulated compared to controls. Tissue microarray revealed that the expression of GRα was lower in the ovarian cancer samples compared to normal tissue. OT is also shown to drive alternative splicing of the GR gene and cortisol-induced OTR expression. OT was able to transactivate GR in the presence of cortisol, thus providing further evidence of cross-talk in vitro. These data provide explanations for why social support might help distressed ovarian cancer patients and help define novel hypotheses regarding potential therapeutic interventions in socially isolated patients.

Chronic stress accelerates ligature-induced periodontitis by suppressing glucocorticoid receptor-α signaling.

Periodontitis is a common chronic inflammatory disease. Recent studies have shown that chronic stress (CS) might modulate periodontal disease, but there are few models of CS-induced periodontitis, and the underlying mechanisms are unclear. The present study established a rat model of periodontitis associated with CS induced by nylon thread ligatures. The severity of periodontitis was evaluated in this model by radiographic and pathological examination. The inflammatory reaction indicated by the elevated serum levels of interleukin (IL)-1β, IL-6 and IL-8 was assessed by enzyme-linked immunosorbent assay. Toll-like receptor-4 (TLR4) and glucocorticoid receptor-α (GR-α) expressions were detected by reverse transcriptase-PCR and western blotting. Open-field tests and serum corticosterone were used to evaluate CS. The results showed that CS induced behavioral changes and increased corticosterone levels of the animals with periodontitis. CS stimulation markedly increased alveolar bone loss, periodontal pocket depth and the number of plaques. It also enhanced the inflammatory reaction. These results suggest that CS accelerated the ligature-induced pathological changes associated with periodontitis. Further analysis of the mechanisms involved showed that GR-α expression was significantly downregulated in periodontal tissues of the animals undergoing CS. Blocking GR-α signaling in lipopolysaccharide and corticosteroid-treated human periodontal ligament fibroblast cells in vitro significantly upregulated the expression of p-Akt (protein kinase B) and TLR4, promoted nuclear factor-κB activity and increased levels of IL-1β, IL-6 and IL-8. This research suggests that CS might accelerate the pathological progression of periodontitis by a GR-α signaling-mediated inflammatory response and that this may be a potential therapeutic target for the treatment of periodontal disease, particularly in patients with CS.

Down-regulation of GRα expression and inhibition of its nuclear translocation by hypoxia

AimsGlucocorticoids are the most effective anti-inflammatory agent in treating pulmonary diseases typically accompanied by hypoxia. Our previous study has demonstrated that glucocorticoid receptor α (GRα) expression is reduced in hypoxia but the underlying mechanism remains elusive. In this study we aim to identify the signaling pathway involved in hypoxia-induced down-regulation of GRα, and whether hypoxia affects nuclear translocation of GRα. Main methodsFemale C57BL/6 mice were sensitized with saline or ovalbumin (OVA) as the in vivo model. Mice were divided into control and OVA groups, and their lung histology and the expression of hypoxia inducible factor (HIF-1) and GRα were examined. A549 cells were exposed to chemical hypoxia as the in vitro model, where mitogen-activated protein kinases (MAPKs) were inhibited specifically by SB203580. Next, under normal or hypoxic conditions, the expression of GRα, MAPKs and HIF-1 signal protein were determined by Western blot analysis, and GRα translocation were observed through live-cell imaging. Key findingsIn OVA challenged mice the expression of GRα was down-regulated whereas HIF-1 was up-regulated. Hypoxia caused a time-dependent decrease of GRα expression, and activated multiple signaling pathways including MAPKs and HIF-1. Moreover, GRα expression increased with MAPK inhibition. Interestingly, only MAPK inhibitor SB203580, but not JNK inhibitor SP600125 or ERK inhibitor U0126 improved the expression of GRα under hypoxic condition. GRα nuclear translocation was also significantly inhibited by hypoxia. SignificanceHypoxia down-regulated the expression of GRα through p38 signaling pathway, as well as inhibited GRα nuclear translocation significantly.

Expression of Glucocorticoid Receptors GRα and GRβ in Bullous Pemphigoid.

First-line treatments of bullous pemphigoid (BP) are topical and systemic glucocorticoids (GC). The actions of GC are mediated by glucocorticoid receptors (GR), which exist in several isoforms, of which GRα and GRβ are the most important. In many inflammatory diseases, up-regulation of GRβ is associated with GC insensitivity. The aims of this study were to determine the expression of GRα and GRβ in patients with BP and to investigate the effect of prednisolone treatment on the expression of GR isoforms in BP. Quantitative real-time PCR (qPCR) analysis demonstrated that GR isoform mRNAs are expressed in peripheral blood mononuclear cells (PBMC) from patients with BP. Expression of GRα and GRβ protein was confirmed by immunohistochemical staining of BP skin biopsies and by Western blot analysis and flow cytometric analysis of PBMCs. During prednisolone treatment, GRα and GRβ expression varied markedly, but changes were not suitable as a clinical marker of GC sensitivity in patients with BP.

Glucocorticoid receptors GRα and GRβ are expressed in inflammatory dermatoses

Glucocorticoids (GC) are the most commonly used anti-inflammatory drugs in dermatology. The actions of GCs are mediated by the glucocorticoid receptor (GR). Alternative splicing of GR mRNA gives rise to different isoforms, GRα and GRβ being the most important. GRβ antagonizes the activity of GRα and its up-regulation has been associated with glucocorticoid insensitivity in several non-cutaneous inflammatory diseases. Using immunohistochemical stainings, we analyzed the expression of GRα and GRβ in lesional skin samples of patients with atopic dermatitis, lichen ruber planus, eczema nummulare and lichen simplex chronicus. We also conducted a study of 13 severe atopic patients to investigate the effect of prednisolone treatment on the expression of GR isoforms using quantitative PCR, western blot and immunohistochemical analysis. GRα and GRβ were expressed in atopic dermatitis, lichen ruber planus, eczema nummulare and lichen simplex chronicus. Our novel finding was that GRβ is abundant in keratinocytes and cutaneous neutrophils. Nuclear staining of both GRα and GRβ was strongest in keratinocytes of patients with lichen ruber planus, whereas the least nuclear positivity was detected in keratinocytes of patients with atopic dermatitis. In severe atopic dermatitis GRα and GRβ were expressed in both peripheral blood mononuclear cells and the skin. The expression of GRα and GRβ varied during prednisolone therapy but the changes were not related to treatment response or GC insensitivity. GRα and GRβ are expressed in inflammatory dermatoses. In severe atopic dermatitis the increased expression of GRβ mRNA is not connected to insensitivity against prednisolone treatment.

Glucocorticoid Regulation of Erythropoiesis in Humans: A Study of Patients with Cushing's Disease

Cushing's disease (CD) is a rare endocrine disorder (1.2-2.4/million/year) characterized by chronic excess endogenous glucocorticoids (GC) due to an adrenocorticotropic hormone-secreting pituitary adenoma. Untreated CD results in increased mortality and multiple morbidities (obesity, diabetes, hypertension, cardiovascular disease) and, in one case report, erythrocytosis (Gursoy et al, J End Invest. 2006;29:742). The effects of chronic GC exposure on erythropoiesis in a CD cohort have not yet been studied.We prospectively quantified hematocrit (Hct), hemoglobin (Hb) and platelets (ptl) values in CD patients before (v1) and after surgical remission (v2, mean time since surgery=14.5 months) and in matched healthy controls (HC). Frequency, antigenic profiling and erythroid (Ery) expansion potential of circulating hematopoietic progenitor cells (HPC) in the three cohorts were also evaluated. The subjects analyzed included 28 v1 [6 males, 22 females, mean age=41 years; body mass index (BMI)=33.3 kg/m2 ] and 13 HC [2 males, 11 females, mean age=41 years; BMI=30.7 kg/m2 (range 26.7-34.7, p=0.073). Eleven patients were analyzed over time in both v1 and v2.Mean cortisol in v1 (22.2±6.3 ug/dL) was higher than in HC (8.5±3.8 ug/dL, p=3.4E-07) and decreased in v2 (17.6±5.1% vs. 8.7±3.9%, N= 11, p=0.0006). Hct was higher in v1 than in HC (39.8±4.7%, vs. 38.8±2.7%, p = 0.045). In the 11 patients analyzed over time, hct decreased in v2 vs. v1 (39.2±4.7% vs. 42.3±4.4%, p=0.011). Hb in v1 was not different than in HC (13.10±1.6 vs. 13.12±3.8 g/dL, p =0.225) but decreased in patients studied both in v1 and V2 (14.1±1.5 g/dL vs. 13.2±1.8 g/dL, p=0.009). Similarly, plts were not different in v1 and HC (272.3±81.4 vs. 240.8±76.0 K/uL, p=0.274) but decreased in v2 (307.7±112.1 vs. 270.6±74.5 K/uL, p=0.021). In v1, Hct did not correlate with serum (R = 0.34, p = 0.33) or 24h urine (R = 0.072, p = 0.73) cortisol concentrations.There was no difference in frequency of HPC among v1, v2 and HC [2.6±3.0, 0.34±0.28 and 1.26±0.67% of CD34+ cells and 30.2±27.2, 23.7±13.2 and 16.5±11.5 CFC/105 mononuclear cells (MNC) in v1, v2 and HC]. CD34pos cells from all groups expressed similar levels of cKIT, IL-3Rβ and prominin1, but a greater proportion of those from v1 and v2 expressed thrombospondin and thrombopoietin (Mpl) receptors than those from HC (2 vs 0.4%), suggesting that CD HPC are biased toward erythro-megakaryocytopoiesis. Consistently, in cultures without the synthetic GC dexamethasone (Dex), MNC from v1 (12±6 %) and v2 (19%) generated in 10 days a greater proportion of Erys than MNC from HC (2±1% ). However, in cultures with Dex, MNC from v1 (48±25), but not those from v2 (70±23), generated less Erys than MNC from HC (83±26 , p=0.03), suggesting that Erys from v1 HPC respond poorly to Dex. This was tested by comparing the ability of Dex to induce biochemical (GRα phosphorylation at S211 and cell-surface expression of CXCR4/calreticulin) and biological (proliferation in synergy with growth factors, GFs) responses in Erys from CD and HC. Erys from v1, v2 and HC expressed equivalent levels of GRα but those from v1 Erys contained lower levels of pGRαS211/S203 than those from v2 or HC (Fig 1A). In contrast with HC Erys, Dex decreased GRα and did not induce pGRαS211 in v1 Erys (Fig 1B). Moreover, Dex increased cell-surface expression of CXCR4 (MFI from 580 to 700) and calreticulin (MFI from 300 to 700) and proliferation (by 30%) in HC Erys but not in those from v1 s (CXCR4 MFI from 278 to 154, calreticulin MFI from 136 to 152; proliferation increases by 6%).These results indicate that chronic GC excess increases Hct values but may also activate a post-transcriptional mechanism that reduces GRα expression inducing desensitization of erythroid cells to GC. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Glucocorticoid Receptor-β Is Downregulated by Vitamin D Treatment in Children with Low Vitamin D Levels but Not in Allergic Asthma

Background: Vitamin D (VitD) enhances the anti-inflammatory effects of glucocorticoids (GC) in vitro. It was hypothesized that VitD3 (colecalciferol) treatment could have an impact on the expression of glucocorticoid receptors (GRs) in a group of children with low VitD levels, with or without asthma. Methods: Asthmatic children and healthy controls, all with low serum VitD levels (25-hydroxyvitamin D [25(OH)D] level <30 ng/mL) were recruited. VitD3 treatment at a dose of 300,000 IU was given orally. Blood samples were obtained at admission and 1 month after the treatment to examine serum 25(OH)D levels and the relative gene expression (RGE) of GR-α and -β in peripheral blood mononuclear cells. Results: Twenty-four children with asthma (Mage 11.1 ± 2.1 years) and 14 healthy controls (Mage 11.5 ± 1.7 years) were studied. The expression of GR-β was significantly higher in the control group at baseline compared with those with asthma (p = 0.006). With VitD3 treatment, there was a decrease in GRβ expression at...

Inhibition of microRNA-30a prevents puromycin aminonucleoside-induced podocytic apoptosis by upregulating the glucocorticoid receptor α

It is well‑established that steroid‑resistant nephrotic syndrome commonly involves podocytic injury, however, the underlying mechanism remains to be elucidated. Previous studies have demonstrated that glucocorticoids enhance podocytic recovery predominantly through the functional isoform, glucocorticoid receptor (GR)α. Our previous study demonstrated the reduced expression of GRα in podocytes from patients with steroid‑resistant nephrotic syndrome compared with those with steroid‑sensitive syndromes, which suggested that microRNAs (miRNAs) may be a potential therapeutic target in the modulation of steroid sensitivity. The present study screened miRNAs in murine injured podocytes by microarray and identified 10 miRNAs significantly upregulated, including miR‑30a, miR‑30d, miR‑100, miR181c, miR‑5099, miR‑3535, miR‑140‑3p, miR‑148‑3p and miR‑103‑3p. Bioinformatic target prediction indicated that the GR was a candidate target gene of miR‑30a. The data also indicated that miR‑30a negatively regulated GRα in normal and injured podocytes induced by puromycin aminonucleoside (PAN). In addition, the inhibition of miR‑30a prevented podocytic apoptosis induced by PAN. However, luciferase reporter assay data suggested an indirect effect on the transcriptional activity of GRα. The present study indicated that silencing of miR‑30a may improve steroid sensitivity in injured podocytes, although the mechanism cannot be explained by conventional means and remains to be elucidated.

Endothelial glucocorticoid receptor promoter methylation according to dexamethasone sensitivity.

We have previously shown that in vitro sensitivity to dexamethasone (DEX) stimulation in human endothelial cells is positively regulated by the glucocorticoid receptor (NR3C1, GR). The present study determined the role of differential GR transcriptional regulation in glucocorticoid sensitivity. We studied 25 human umbilical vein endothelial cells (HUVECs) that had been previously characterized as DEX-sensitive (n = 15), or resistant (n = 10). Real-time PCR analysis of GR 5′UTR mRNA isoforms showed that all HUVECs expressed isoforms 1B, 1C, 1D, 1F, and 1H, and isoforms 1B and 1C were predominantly expressed. DEX-resistant cells expressed higher basal levels of the 5′UTR mRNA isoforms 1C and 1D, but lower levels of the 5′UTR mRNA isoform 1F than DEX-sensitive cells. DEX treatment significantly decreased GRα and GR-1C mRNA isoform expression in DEX-resistant cells only. Reporter luciferase assays indicated that differential GR mRNA isoform expression was not due to differential promoter usage between DEX-sensitive and DEX-resistant cells. Analysis of promoter methylation, however, showed that DEX-sensitive cells have higher methylation levels of promoter 1D and lower methylation levels of promoter 1F than DEX-resistant cells. Treatment with 5-aza-2-deoxycytidine abolished the differential 5′UTR mRNA isoform expression between DEX-sensitive and DEX-resistant cells. Finally, both GRα overexpression and 5-aza-2-deoxycytidine treatment eliminated the differences between sensitivity groups to DEX-mediated downregulation of endothelial nitric oxide synthase (NOS3), and upregulation of plasminogen activator inhibitor 1 (SERPINE1). In sum, human endothelial GR 5′UTR mRNA expression is regulated by promoter methylation with DEX-sensitive and DEX-resistant cells having different GR promoter methylation patterns.

Role of the glucocorticoid receptor in the recurrence of primary nephrotic syndrome.

The present study aimed to investigate the changes in the expression levels of the glucocorticoid receptor (GR) and its subtypes in patients with recurrent renal syndrome. In addition, the effects of tumour necrosis factor α (TNF-α) and a TNF-α monoclonal antibody on these receptors in peripheral blood mononuclear cells (PBMCs) isolated from the patients was analysed. Furthermore, a new treatment method for recurrent renal syndrome was explored. The serum levels of TNF-α in the normal (A), stable renal syndrome (B) and renal syndrome recurrence (C) groups of patients were determined by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expression levels of GR, GRα and GRβ were determined by ELISA, western blot analysis and quantitative polymerase chain reaction in PBMC cultures from the three groups in the absence of intervention (blank control) and following stimulation with methylprednisolone, TNF-α and/or TNF-α monoclonal antibody. Group C exhibited higher expression levels of TNF-α and GRβ but a lower level of GRα expression (P<0.05) compared with the other groups. Regardless of methylprednisolone intervention, the expression levels of GR and GRβ in the three groups following stimulation by TNF-α were significantly higher compared with those in the respective blank control, whereas in group C, the GRα expression levels following TNF-α treatment were lower compared with those in the control group (P<0.05). The treatment of group C with TNF-α monoclonal antibodies resulted in higher GRα expression but lower GRβ expression compared with those in the blank control (P<0.05). The change in the ratios of the GR subtypes may be associated with renal syndrome recurrence. TNF-α may be involved in renal syndrome relapse by changing the levels of GR as well as the proportion of the GR subtypes. TNF-α monoclonal antibodies may mitigate the changes in the ratios of these subtypes.

Expression of glucocorticoid receptor-α in the epidermis of patients with psoriasis vulgaris.

Expression of glucocorticoid receptor (GR)-α is observed in almost all tissues and cells of the body; thus, investigating the expression of the receptor in the epidermis of patients with psoriasis vulgaris. The aim of the present study was to investigate GR-α expression in the epidermis of psoriasis vulgaris patients The study population consisted of 26 patients with psoriasis vulgaris and 10 normal control cases. None of the patients had received any prior treatment with glucocorticoids. Epithelial tissue samples were detected using a streptavidin peroxidation-enzymatic method for biopsy. Non-lesional tissue samples from within a range of 3 cm of lesions formed the non-lesional group, and lesional tissue samples formed the lesional group. Pathological image analysis system (named CMIAS) was used to convert image signals into numeric values, according to the optical density per unit area. GR-α expression was observed within the nucleus in the normal control group; however, cytoplasmic expression was observed in the lesions of the psoriatic group. The optical density values were significantly lower in the psoriatic group when compared with the normal control group, indicating a statistically significant difference in GR-α expression between the two groups (P<0.001), and this decreasing correlation was unaffected by the administration of steroids for 6 months. Therefore, decreased expression of GR-α may play an important role in the degeneration of keratinocytes in patients with psoriasis vulgaris.