GRα Expression Research Articles

Expression of eight glucocorticoid receptor isoforms in the human preterm placenta vary with fetal sex and birthweight

IntroductionAdministration of betamethasone to women at risk of preterm delivery is known to be associated with reduced fetal growth via alterations in placental function and possibly direct effects on the fetus. The placental glucocorticoid receptor (GR) is central to this response and recent evidence suggests there are numerous isoforms for GR in term placentae. In this study we have questioned whether GR isoform expression varies in preterm placentae in relation to betamethasone exposure, fetal sex and birthweight. MethodsPreterm (24–36 completed weeks of gestation, n = 55) and term placentae (>37 completed weeks of gestation, n = 56) were collected at delivery. Placental GR expression was examined using Western Blot and analysed in relation to gestational age at delivery, fetal sex, birthweight and betamethasone exposure. Data was analysed using non-parametric tests. ResultsEight known isoforms of the GR were detected in the preterm placenta and include GRα (94 kDa), GRβ (91 kDa), GRα C (81 kDa) GR P (74 kDa) GR A (65 kDa), GRα D1–3 (50–55 kDa). Expression varied between preterm and term placentae with a greater expression of GRα C in preterm placentae relative to term placentae. The only sex differences in preterm placentae was that GRα D2 expression was higher in males than females. There were no alterations in preterm placental GR expression in association with betamethasone exposure. DiscussionGRα C is the isoform involved in glucocorticoid induced apoptosis and suggests that its predominance in preterm placentae may contribute to the pathophysiology of preterm birth.

Effects of maternal dexamethasone treatment early in pregnancy on glucocorticoid receptors in the ovine placenta.

The effects of endogenous cortisol on binucleate cells (BNCs), which promote fetal growth, may be mediated by glucocorticoid receptors (GRs), and exposure to dexamethasone (DEX) in early pregnancy stages of placental development might modify this response. In this article, we have investigated the expression of GR as a determinant of these responses. Pregnant ewes carrying singleton fetuses (n = 119) were randomized to control (2 mL saline/ewe) or DEX-treated groups (intramuscular injections of 0.14 mg/kg ewe weight per 12 hours) at 40 to 41 days of gestation (dG). Placental tissue was collected at 50, 100, 125, and 140 dG. Total glucocorticoid receptor protein (GRt) was increased significantly by DEX at 50 and 125 dG in females only, but decreased in males at 125 dG as compared to controls. Glucocorticoid receptor α (GRα) protein was not changed after DEX treatment. Three BNC phenotypes were detected regarding GRα expression (++, +-, --), DEX increased the proportion of (++) and decreased (--) BNC at 140 dG. Effects were sex- and cell type dependent, modifying the responsiveness of the placenta to endogenous cortisol. We speculate that 3 maturational stages of BNCs exist and that the overall activity of BNCs is determined by the distribution of these 3 cell types, which may become altered through early pregnancy exposure to elevated glucocorticoids.

Hypermethylation of glucocorticoid receptor gene promoter results in glucocorticoid receptor gene low expression in peripheral blood mononuclear cells of patients with systemic lupus erythematosus.

Our aim was to investigate the relationship between the DNA methylation status of glucocorticoid receptor (GR) gene promoter and mRNA expression level of GRα gene of peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE). Fifteen newly emerging SLE patients and fifteen healthy controls were enrolled in this study. DNA and total RNA were extracted from the PBMCs of the SLE patients and healthy controls. The DNA methylation status of GR gene promoter 1 of PBMCs was detected through bisulfite-sequencing PCR. The mRNA expression of GRα, DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) and growth arrest, and DNA damage-induced 45α (GADD45α) of PBMCs was detected using the quantitative real-time polymerase chain reaction method. The mRNA expression of GRα was significantly declined in SLE patients, and the mRNA expression of DNMT1 and GADD45α was significantly elevated in SLE patients. The global methylation status of PBMCs in SLE patients was obviously lower than healthy controls. There were 38, 25, 30, and 49 CpG islands in amplified fragment of GR promoter 1D, 1E, 1F, and 1H, respectively. The overall mean methylation status of the 152 CpG islands of the four promoters was significantly elevated in SLE patients. There was a negative correlation between hypermethylation of GR promoter and GRα mRNA expression in SLE patients. This study demonstrated that hypermethylation of GRα promoter may result in GRα gene low expression in PBMCs of patients with SLE. This study also found that the global methylation status of PBMCs in SLE patients was obviously lower than healthy controls, and it was related to the elevated GADD45α mRNA expression in SLE patients. These conclusions have to be certified by larger-scale clinical studies.

Transcriptional and metabolic effects of glucocorticoid receptor α and β signaling in zebrafish.

In humans and zebrafish, 2 glucocorticoid (GC) receptor (GR) splice variants exist: the canonical GR α-isoform (GRα), and the GRβ. In the present study, we have used the zebrafish model system in order to reveal genes affected by each of these 2 receptor isoforms. By injecting zebrafish embryos with different splice-blocking morpholinos, we could knock down both GR isoforms or could target the alternative splicing of the GR pre-mRNA in favor of the GRβ. In addition, specific GRβ overexpression was achieved by injecting mRNA. Embryos were treated with the synthetic GC dexamethasone, and transcriptome analysis was performed. Two distinct gene clusters were found that were regulated by GRα: one that was regulated by GRα under basal conditions (presence of endogenous cortisol only), and one that was regulated upon increased activation of GRα (using a pharmacological dose of dexamathasone). GRβ may act as a dominant-negative inhibitor of GRα when GRβ is overexpressed and the GRα expression level is knocked down simultaneously. However, without GRα knockdown, no evidence for this activity was found. In addition, the data indicate regulation of gene transcription through other mechanisms of action by GRβ. We also investigated the concentrations of several metabolites using nuclear magnetic resonance spectroscopy. We found that dexamethasone treatment and knockdown of GRα together with overexpression of GRβ had opposite effects on glucose, amino acid, and fatty acid levels. Thus, we have shed new light on the molecular mechanisms of GC-induced effects on metabolism, which are known to increase the risk of obesity, hyperglycemia, and diabetes.

Interleukin-1β regulates the expression of glucocorticoid receptor isoforms in nasal polyps in vitro via p38 MAPK and JNK signal transduction pathways.

BackgroundTo explore the upstream signal transduction mechanisms responsible for the imbalanced expression of glucocorticoid receptor (GR) isoforms in chronic rhinosinusitis (CRS) mucosa.MethodsAn in vitro model of Glucocorticoid resistance was established by inducing nasal polyp tissue with IL-1β. Changes in the protein and mRNA expression of GRα, GRβ and the key enzymes in the p38 MAPK and JNK signal pathways were measured, respectively, before and after being induced with different doses of IL-1β and specific inhibitors of p38 MAPK, JNK, ERK, PI3K and PKC. The Glucocorticoid sensitivity was measured using in vitro Glucocorticoid binding assay. Analysis of variance (ANOVA) was used to analyze the data.ResultsThe mRNA and protein expression levels of GRα, GRβ and key enzymes of the p38 MAPK and JNK pathways increased both in time- and concentration-dependent manners in IL-1β-induced nasal polyp tissue. The expression of GRβ increased more significantly than that of GRα, and the GRα/GRβ ratio decreased in time- and concentration-dependent manners. Statistically significant differences were found in the GRα/GRβ ratio and the mRNA expression of phospho-p38 MAPK and phospho-JNK between the IL-1β-induced groups and the control groups (P < 0.05). Either a specific inhibitor of the p38 MAPK pathway or a specific inhibitor of the JNK pathway increased the GRα/GRβ ratio and the Glucocorticoid affinity. None of the specific inhibitors of ERK, PI3K or PKC had any influence on the expression of GR isoforms.ConclusionOur results demonstrated that the imbalanced expression of GR isoforms in nasal polyp tissue induced by IL-1β in vitro is mediated through the p38 MAPK and JNK signal pathways.

MicroRNA-124 regulates glucocorticoid sensitivity by targeting phosphodiesterase 4B in diffuse large B cell lymphoma

Glucocorticoids (GCs) are chemotherapeutic drugs commonly used to treat hematological malignancies. However, a significant fraction of patients develop resistance to GCs during treatment. A better insight into how GC resistance develops is therefore needed. It was previously shown that cyclic AMP (cAMP) induces sensitivity to GCs by inhibiting the AKT/mTOR/MCL1 signaling, while high levels of phosphodiesterase 4B (PDE4B) reverse the effect of cAMP on GC responses in B-cell lymphoma. Here, we show that miR-124 influences GC-induced apoptosis by directly targeting PDE4B. Stable expression of miR-124 in diffuse large B cell lymphoma (DLBCL) cell lines diminished PDE4B expression. This was associated with increased cAMP levels, inhibition of the AKT/mTOR/MCL1 survival pathway, upregulation of GRα expression, and improved sensitivity to GCs in the presence of forskolin, an activator of adenylyl cyclase. Interestingly, miR-124 did not affect GC sensitivity in the absence of forskolin, indicating that the effect of this miRNA is accomplished via downregulation of PDE4B expression. Further, restoration of PDE4B expression in miR-124 cells rescued the phenotypic effect of this miRNA, demonstrating the critical role of PDE4B in miR-124-mediated regulation of the GC response. Our study supports the notion that miR-124 could be an attractive therapeutic target for overcoming GC resistance in DLBCL.

The prognostic value of glucocorticoid receptors for adult acute lymphoblastic leukemia.

BackgroundTherapeutic protocols used in adult acute lymphoblastic leukemia (ALL) are widely variable, and glucocorticoids (GCs) are essential components in ALL treatment. Therefore, this study aimed to evaluate the distribution of prominent glucocorticoid receptor (GR) gene polymorphic variants among adult ALL patients. We also investigated the association between GR messenger ribonucleic acid (mRNA) isoform expressions and the response to chemotherapy.MethodsFifty-two newly diagnosed Philadelphia-negative adult ALL patients and 30 healthy control subjects were enrolled in this study. Genotyping was carried out using a polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis. GR mRNA isoform expressions were assayed by quantitative real-time PCR.ResultsALL patients in this study had a median age of 34 years (range, 18-75). GRα expression was associated with complete remission (P=0.03), while GRγ mRNA expression was significantly higher in GC resistant patients (P=0.032) and in non-responders (P=0.019). However, there were no significant associations with GC resistance. The BclI polymorphic variant of the GR gene was the most frequent in adult ALL patients and was not associated with the GC response. Both higher GRα expression and lower GRγ expression were associated with achievement of complete remission, while higher GRγ expression was associated with GC-resistance.ConclusionOur data suggest that the level of GR isoform expression may be useful in predicting GC response, achievement of complete remission, and better event-free survival in ALL patients. However, further evaluation with a larger cohort of patients is warranted.

Differences in leukocyte profile, gene expression, and metabolite status of dairy cows with or without sole ulcers

Sole ulcers are one of the most severe pathologies causing lameness in dairy cows and are associated with abnormal behavior and impaired production performance. However, little is known about how or whether lameness caused by sole ulcers affects the cow systemically. This study compared hematology profile, leukocyte gene expression, and physiological responses [metabolite, cortisol, the endogenous steroid hormone dehydroepiandrosterone (DHEA), and haptoglobin concentrations] of cows with sole ulcers and healthy cows. Twelve clinically lame cows (lame) were identified as having at least one sole ulcer and no other disorder, and matched with a cow that had good locomotion and no disorders (sound), using days in milk, liveweight, body condition score, and diet. Blood samples were taken from all 24 cows within 24h of sole ulcer diagnosis. Leukocyte counts were obtained using an automated cell counter, cortisol and DHEA concentration by ELISA, and plasma haptoglobin, urea, total protein, creatine kinase, and glucose were analyzed on an Olympus analyzer. Expression of 16 genes associated with lameness or stress were estimated using reverse transcription-PCR. Data were analyzed using the MIXED procedure in SAS software (version 9.3; SAS Institute Inc., Cary, NC). Lame cows had a higher neutrophil percentage, a numerically lower lymphocyte percentage, and tended to have a higher neutrophil:lymphocyte ratio than sound cows. Serum cortisol and DHEA concentrations were higher in lame than in sound cows. Lame cows also tended to have higher haptoglobin and glucose levels than sound, as well as higher protein yet lower urea levels. Sound cows tended to have higher relative expression of the gene coding for colony-stimulating factor 2 than lame, but in all other cases where differences were detected in cytokine gene expression (IL-1α, IL-1β, CXCL8, and IL-10), relative gene expression in sound cows tended to be, or was, lower than in lame. Relative expression of MMP-13, GR-α, Fas, haptoglobin, and CD62L were, or tended to be, higher in lame than sound cows. A high neutrophil:lymphocyte ratio in combination with higher cortisol levels in cows with ulcers is indicative of physiological stress. Moreover, increased DHEA and a higher cortisol:DHEA ratio, as well as a tendency for higher haptoglobin levels and increased haptoglobin mRNA expression, are indicative of systemic inflammation. Increased cytokine mRNA expression indicates activation of the immune system compared with healthy cows. Increased expression of MMP-13 mRNA has been found in cows with impaired locomotion and thus could be implicated in development of claw horn disorders.

MRNA expression of glucocorticoid receptor and serological and virological markers of chronic hepatitis B.

Glucocorticoid receptor (GR) function is essential for glucocorticoid action on various effector cells. The aim of the present study was to investigate the mRNA expression profiles of GRα and GRβ in peripheral blood mononuclear cells (PBMC) and examine the association between the expression levels of the GR isoforms and the serological and virological hepatitis B virus (HBV) status in patients with chronic hepatitis B (CHB). A total of 29 CHB patients were examined in the present study, which were divided into subgroups according to serological and virological markers. The levels of GRα and GRβ in PBMCs, HBV viral loads, HBV surface antigen (HBsAg), HBV e antigen (HBeAg) and pre‑S1Ag were measured. A total of 43 healthy individuals served as controls. GRα was present in the PBMCs of all CHB patients and healthy controls, whereas GRβ‑specific products were present in only 93.1% of the CHB patients and 86.0% of the healthy controls. The GRα levels were positively correlated with the expression of GRβ in the CHB patients (r=0.419; P<0.05) and were significantly lower compared with those observed in the healthy controls (60.51 ± 23.73, vs. 100.00 ± 40.75; P<0.001). Compared with the healthy controls, significant differences were observed in the mRNA expression of GRα in the CHB patients when stratified according to the HBeAg, pre‑S1Ag and HBV viral load status (P<0.05), but not in the pre‑S1Ag‑positive patients. These data demonstrated that the mRNA expression profile of GRα differed between the CHB patients and the healthy controls. In addition, the HBV serological and virological markers were not associated with the mRNA levels of the GR isoforms in the CHB patients.

11β-Hydroxysteroid Dehydrogenase Type 1 is not Overexpressed in Cushing’s Syndrome Adipose Depots

Glucocorticoids have a major role in adipose tissue distribution, partially regulated at a pre-receptor level by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). 11β-HSD1 generates intracellular cortisol promoting adipocyte differentiation. The distinctive expression of 11βHSD1 and glucocorticoid receptor-α (GRα) may explain the preferential abdominal fat depots in Cushing’s syndrome (CS). Aim: To quantify and evaluate the effects of chronic exposure to hypercortisolism on the expression of 11β-HSD1 and GRα in adipose depots of patients with CS. Subjects and Methods: Samples of visceral (VAT) and subcutaneous adipose tissue (SAT) were obtained during elective abdominal surgery from female patients with CS (n=10), obese (n=15) and nonobese controls (n=10), in whom body mass index (BMI), abdominal circumference (AC) and salivary cortisol (SF) were previously determined. 11β-HSD1 and GRα expressions were quantified by real-time PCR. Results: 11β-HSD1 expressions in SAT and VAT of CS were not different from nonobese and were upregulated in obese (P<0.0001 and P<0.05, respectively). Additionally, GRα mRNA was downregulated in SAT of obese and CS (P<0.0001 for both). In the whole group, neither 11β-HSD1 nor GRα levels showed correlation with SF. However, 11β-HSD1 mRNA correlated positively with BMI and AC in SAT and VAT and GRα correlated negatively with both in SAT. Conclusions: Chronic hypercortisolism, as seen in CS, does not result in upregulation of 11β-HSD1 expression in adipose depots, in contrast with in vitro observations. This may suggest a protective mechanism, since 11β-HSD1 is emerging as a key component in homeostatic adaptation, rather than the cause of visceral obesity.

Reduced Peripheral Expression of the Glucocorticoid Receptor α Isoform in Individuals with Posttraumatic Stress Disorder: A Cumulative Effect of Trauma Burden

BackgroundPosttraumatic stress disorder (PTSD) is a serious psychiatric condition that was found to be associated with altered functioning of the hypothalamic-pituitary-adrenal (HPA) axis and changes in glucocorticoid (GC) responsiveness. The physiological actions of GCs are primarily mediated through GC receptors (GR) of which isoforms with different biological activities exist. This study aimed to investigate whether trauma-experience and/or PTSD are associated with altered expression of GR splice variants.MethodsGRα and GRβ mRNA expression levels were determined by real-time quantitative PCR in whole blood samples of individuals with chronic and severe forms of PTSD (n = 42) as well as in ethnically matched reference subjects (non-PTSD, n = 35).ResultsIndividuals suffering from PTSD exhibited significantly lower expression of the predominant and functionally active GRα isoform compared to non-PTSD subjects. This effect remained significant when accounting for gender, smoking, psychotropic medication or comorbid depression. Moreover, the GRα expression level was significantly negatively correlated with the number of traumatic event types experienced, both in the whole sample and within the PTSD patient group. Expression of the less abundant and non-ligand binding GRβ isoform was comparable between patient and reference groups.ConclusionsReduced expression of the functionally active GRα isoform in peripheral blood cells of individuals with PTSD seems to be a cumulative effect of trauma burden rather than a specific feature of PTSD since non-PTSD subjects with high trauma load showed an intermediate phenotype between PTSD patients and individuals with no or few traumatic experiences.

Inflammatory activation is associated with a reduced glucocorticoid receptor alpha/beta expression ratio in monocytes of inpatients with melancholic major depressive disorder

In this study, we used new technology to investigate whether a coherent pattern of enhanced expression of inflammatory and other immune activation genes in circulating monocytes is found in patients with major depression. Since a high inflammatory state of monocytes might be related to glucocorticoid resistance, we also included the genes for the two isoforms of the glucocorticoid receptor. For this study, we aimed at finding a similar coherent pattern of inflammatory and immune activation genes in monocytes of patients with MDD and recruited 47 medication-free melancholic MDD inpatients and 42 healthy controls. A quantitative-polymerase chain reaction (Q-PCR) monocyte gene expression analysis was performed using a panel of inflammatory-related genes previously identified as abnormally regulated in mood disorder patients. Selected serum cytokines/chemokines were assessed using a cytometric bead array. Depressive symptoms were analysed using Hamilton depression scores (HAMD). Thirty-four of the 47 monocyte inflammatory-related genes were significantly upregulated and 2 were significantly downregulated as compared to controls, the latter including the gene for the active GRα in particular in those with a high HAMD score. The reduced GRα expression correlated strongly to the upregulation of the inflammatory genes in monocytes. Serum levels of IL6, IL8, CCL2 and VEGF were significantly increased in patients compared to controls. Our data show the deregulation of two interrelated homoeostatic systems, that is, the immune system and the glucocorticoid system, co-occurring in major depression.

Glucocorticoid receptor gene expression and promoter CpG modifications throughout the human brain

Glucocorticoids and the glucocorticoid (GR) and mineralocorticoid (MR) receptors have been implicated in many processes, particularly in negative feedback regulation of the hypothalamic-pituitary-adrenal axis. Epigenetically programmed GR alternative promoter usage underlies transcriptional control of GR levels, generation of GR 3′ splice variants, and the overall GC response in the brain. No detailed analysis of GR first exons or GR transcript variants throughout the human brain has been reported. Therefore we investigated post mortem tissues from 28 brain regions of 5 individuals. GR first exons were expressed throughout the healthy human brain with no region-specific usage patterns. First exon levels were highly inter-correlated suggesting that they are co-regulated. GR 3′ splice variants (GRα and GR-P) were equally distributed in all regions, and GRβ expression was always low. GR/MR ratios showed significant differences between the 28 tissues with the highest ratio in the pituitary gland. Modification levels of individual CpG dinucleotides, including 5-mC and 5-hmC, in promoters 1D, 1E, 1F, and 1H were low, and diffusely clustered; despite significant heterogeneity between the donors. In agreement with this clustering, sum modification levels rather than individual CpG modifications correlated with GR expression. Two-way ANOVA showed that this sum modification was both promoter and brain region specific, but that there was however no promoter*tissue interaction. The heterogeneity between donors may however hide such an interaction. In both promoters 1F and 1H modification levels correlated with GRα expression suggesting that 5-mC and 5-hmC play an important role in fine tuning GR expression levels throughout the brain.

Levels of inflammatory cytokines, adrenal steroids, and mRNA for GRα, GRβ and 11βHSD1 in TB pleurisy

Our previous work on the immune-endocrine features of patients with pulmonary tuberculosis (TB) showed markedly decreased plasma levels of dehydroepiandrosterone (DHEA) together with augmented concentrations of Cortisol and pro- and anti-inflammatory cytokines. Studies in peripheral blood mononuclear cells (PBMC) indicated a lower mRNA α/β ratio of glucocorticoid receptors -GR- together with a higher 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) mRNA expression in cases with severe pulmonary TB. Since Pleural TB (PLTB) is a rather benign manifestation of TB, we now analyzed the systemic and local immune-endocrine profile as well as the GRα, GRβ, 11βHSD1 and 11βHSD2 transcripts in PBMC and pleural effusion mononuclear cells (PEMC) of patients with PLTB. PLTB patients had increased levels of IL-1β, IL-6 and IFNγ together with reduced Cortisol and DHEA concentrations in pleural fluids. Also, a significantly increased expression of 11βHSD1 and GRα was found in PEMC compared to PBMC. Findings point out to an appropriate immune response and a substantial inflammatory reaction, wherein the low Cortisol concentrations may be equally effective, because of the increased expression of GRα and 11βHSD1 transcripts which may optimize the immunomodulatory properties of Cortisol.

Region-specific alterations in glucocorticoid receptor expression in the postmortem brain of teenage suicide victims

Abnormal function of the hypothalamic-pituitary-adrenal (HPA) axis has been implicated in the pathophysiology of depression and suicide. The purpose of this study was to test the hypothesis that the reported dysregulation of the HPA axis in suicide may be related to a disturbed feedback inhibition caused by decreased corticoid receptors in the brain. We therefore determined the protein and gene expression of glucocorticoid (GR) and mineralocorticoid receptors (MR) in the postmortem brain of teenage suicide victims and matched normal controls. Protein and mRNA expression of GR (GR-α and GR-β) and MR and the mRNA expression of glucocorticoid-induced leucine zipper (GILZ), a target gene for GR were determined by immunolabeling using Western blot technique and the real-time RT-polymerase chain reaction (qPCR) technique in the prefrontal cortex (PFC), hippocampus, subiculum, and amygdala obtained from 24 teenage suicide victims and 24 teenage control subjects. We observed that protein and gene expression of GR-α was significantly decreased in the PFC and amygdala, but not in the hippocampus or subiculum, of teenage suicide victims compared with normal control subjects. Also, the mRNA levels of GR inducible target gene GILZ was significantly decreased in PFC and amygdaloid nuclei but not in hippocampus compared with controls. In contrast, no significant differences were observed in protein or gene expression of MR in any of the areas studied between teenage suicide victims and normal control subjects. There was no difference in the expression of GR-β in the PFC between suicide victims and normal controls. These results suggested that the observed dysregulation of the HPA axis in suicide may be related to a decreased expression of GR-α and GR inducible genes in the PFC and amygdala of teenage suicide victims. The reason why GR receptors are not dysregulated in the hippocampus or subiculum, presumably two sites of stress action, are not clear at this time.

The protective effect of different doses of dexamethasone on acute kidney injury induced by sepsis in mice

To investigate the effect of different doses of dexamethasone (DEX) on sepsis induced acute kidney injury (AKI). One hundred and thirty healthy male Kunming mice were randomly divided into sham group, sepsis group, physiological-dose DEX group (0.12 mg/kg), stress-dose DEX group (1.2 mg/kg), and high-dose DEX group (12 mg/kg). The sepsis model was reproduced by cecal ligation and puncture (CLP) method. Histopathological changes in the kidney were observed at 24 hours and 48 hours after CLP. The expressions of glucocorticoid receptor-α (GR-α) in the kidney were detected by immunohistochemistry. The levels of GR-α mRNA and nuclear factor-ΚB (NF-ΚB) mRNA were determined by real-time polymerase chain reaction (PCR). The levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the plasma were measured by enzyme linked immunosorbent assay (ELISA). Compared with sham group mice, sepsis mice showed serious impairment in renal tubules. The mRNA and protein expression of GR-α were decreased, and NF-ΚB mRNA, plasma TNF-α and IL-1β were elevated. Compared with sepsis group, the histopathological changes in the kidney were mitigated with different levels in groups treated with different doses of DEX. GR-α protein and mRNA were up-regulated, NF-ΚB mRNA and plasma TNF-α, IL-1β were down-regulated obviously. The best effect could be seen in physiological DEX group [AKI score: 24 hours 1.480±0.334 vs. 3.040±0.517, 48 hours 1.840±0.167 vs. 3.400±0.400; GR-α protein (A value): 24 hours 0.102±0.009 vs. 0.088±0.005, 48 hours 0.103±0.008 vs. 0.085±0.006; GR-α mRNA: 24 hours 0.0400(0.0300, 0.0400) vs. 0.0100(0.0093, 0.0100), 48 hours 0.0350(0.0300, 0.0475) vs. 0.0100(0.0010, 0.0138); NF-ΚB mRNA: 24 hours 0.009±0.001 vs. 0.012±0.000,48 hours 0.011±0.000 vs. 0.013±0.001; TNF-α: 24 hours 105.84±3.84 ng/L vs. 135.52±4.49 ng/L, 48 hours 111.35±3.67 ng/L vs. 141.22±4.46 ng/L; IL-1β: 24 hours 45.71±2.93 ng/L vs. 64. 12±3.62 ng/L, 48 hours 57.04±3.04 ng/L vs. 74.87±3.67 ng/L; P<0.05 or P<0.01]. DEX given in physiological-dose could increase renal GR-α level and alleviate the sepsis induced kidney injury. The protective effect was much better than that of high dose DEX.

Low expression of glucocorticoid receptor alpha isoform in adult immune thrombocytopenia correlates with glucocorticoid resistance

The expression of glucocorticoid receptor (GR) isoforms has been linked to glucocorticoid (GC) resistance in various diseases treated with GC. However, existing data are conflicting in these diseases, and little information is available regarding immune thrombocytopenia (ITP). To further investigate the role of GR isoforms in GC resistance in adult ITP patients, we measured the mRNA expression of GR isoforms (GRα, GRβ, GRγ, GRp) in peripheral blood mononuclear cells (PBMC) from 54 newly diagnosed ITP patients, including GC-sensitive (GCS) and GC-resistant (GCR) patients and 35 healthy volunteers. The GRα and GRβ proteins in PBMC, nuclear factor-κB (NF-κB), and activator protein-1 (AP-1) in the nucleus were detected by Western blotting. Compared to normal subjects, both GRα and GRβ mRNAs were significantly increased in ITP patients (p < 0.05), while there was no significant difference in the mRNA expression of GRγ and GRp. Compared to GCR patients, the expressions of GRα mRNA and GRα protein were significantly higher in GCS patients (p < 0.05). Moreover, no significant difference in the mRNA expression of the GRβ, GRγ, and GRp isoforms was observed between GCS and GCR patients and the GRβ protein could not be detected. Compared to GCS group, the expression of p65/NF-κB was significantly higher in the GCR group (p < 0.05). Overall, we did not find differences in c-Jun/AP-1 protein expression between GCS and GCR patients. In summary, GC resistance in adult ITP patients is associated with a reduced expression of GRα, which may be related with increased NF-κB. GRβ was very low and may not be involved in GC resistance in adult ITP, warranting further exploration.

Persistent glucocorticoid resistance in systemic lupus erythematosus patients during clinical remission

Glucocorticoids (GCs) are key drugs in the treatment of systemic lupus erythematosus (SLE). GC dose reduction during remission is related to disease activity, GC dose used, length of treatment, and individual GC sensitivity. We compared GC receptor α (GRα) isoform and nuclear factor kappaB (NF-κB) messenger RNA quantitation and in vivo GC sensitivity between SLE patients during remission and healthy controls. We performed a cross-sectional study of 19 women aged 22-49 years, including 9 SLE patients in clinical remission taking ≤5 mg prednisone and 10 matched controls. We evaluated GC sensitivity using 2 cortisol suppression tests: a very-low-dose intravenous dexamethasone suppression test (VLD-IV-DST) and a low-dose oral dexamethasone suppression test. GRα and NF-κB mRNA were quantified using real-time polymerase chain reaction. Although basal cortisol and adrenocorticotropic hormone levels were similar between the groups, the percentage of cortisol reduction after the VLD-IV-DST was 56% lower in SLE patients than in controls (P = 0.014). GRα and NF-κB gene expression levels were similar between the groups. The low-dose oral dexamethasone test caused intense cortisol suppression in all individuals, limiting the ability of this test to discriminate individual GC sensitivity. A positive correlation was found between the extent of cortisol suppression in vivo (VLD-IV-DST) and the number of days elapsed since the last flare of lupus activity. Despite clinical remission, SLE patients displayed partial GC resistance recognized by the VLD-IV-DST. The mechanism of this resistance is unrelated to altered GRα and NF-κB mRNA expression.

Phosphodiesterase‐4 inhibition improves corticosteroid insensitivity in pulmonary endothelial cells under oxidative stress

Several clinical studies have shown that smoking in asthmatics and chronic obstructive pulmonary disease patients is closely associated with corticosteroid refractoriness. In this work, we have analyzed glucocorticoid insensitivity in human pulmonary artery endothelial cells (HPAECs) under cigarette smoke extract (CSE) exposure as well as the possible additive effects of the combination therapy with a phosphodiesterase (PDE)-4 inhibitor. Interleukin (IL)-8 was measured in cell supernatants by ELISA. Histone deacetylase (HDAC), histone acetylase (HAT), and intracellular cAMP levels were measured by colorimetric assays and enzyme immunoassay, respectively. PDE4 isotypes and glucocorticoid receptor (GR)-α and β expression were measured by real-time RT-PCR. The PDE4 inhibitor rolipram dose dependently inhibited the IL-8 secretion induced by CSE 5%. In contrast, dexamethasone 1μM did not show inhibitory effect on IL-8 secretion. Combination of subeffective rolipram concentrations at 10nM increased the inhibitory effect of dexamethasone to ~45% of inhibition. Cigarette smoke extract 5% inhibited HDAC activity and increased HAT activity generating glucocorticoid insensitivity. Rolipram did not modify the HDAC activity, however partially inhibited the increase in HAT activity at 1μM. PDE4 isotypes were up-regulated by CSE 5% with the consequent cAMP down-regulation. Dexamethasone reduced all PDE4 isotypes expression and showed additive effects with rolipram enhancing cAMP levels. Furthermore, rolipram enhanced GR-α expression and inhibited the increase in GR-β induced by CSE. Combination of rolipram and dexamethasone shows additive properties in HPAECs under glucocorticoid insensitive conditions. These results may be of potential value in future anti-inflammatory therapies using combination of PDE4 inhibitors and glucocorticoids.

Expression of the cysteinyl leukotriene 1 receptor and glucocorticoid receptor-β in nasal polyps

The objective of this study was to analyze the expression of cysteinyl leukotriene 1 (CysLT1) receptor and glucocorticoid receptors (GRs) in nasal polyps, and to evaluate the relationship between the expression of CysLT1 receptors and that of GRs. Nasal polyps were taken from 32 patients of chronic rhinosinusitis with nasal polyposis. Samples of middle turbinate from seven healthy subjects were used as controls. Specimens were immunohistochemically stained for CysLT1 receptor, GR-α and GR-β receptor, and were quantified in the unit area of the tissues. Numbers of CysLT1 receptor-positive cells were much increased in nasal polyps than in middle turbinate (281 ± 67 vs. 157 ± 85 cells/mm(2), P = .01). There was no significant difference in the numbers of GR-α positive cells between nasal polyps and normal turbinate mucosa. GR-β positive cells were increased in nasal polyps as compared to normal turbinate mucosa (36 ± 8 vs. 19 ± 7 cells/mm(2), P = .03). A significant relationship was found between the expression of CysLT1 receptor and GR-β in nasal polyps (R = .525, P = .04), whereas there was no significant relationship between the expression of CysLT1 receptor and GR-α in nasal polyps. Our study shows that CysLT1 receptor expression predominates on GR-β over-expressed polyps. This may suggest the additional effect of CysLT1 receptor antagonist for the treatment of nasal polyposis resistant to steroid alone.