Glucocorticoid Receptor In Cells Research Articles

High-Contrast Facile Imaging with Target-Directing Fluorescent Molecular Rotors, the N3-Modified Thioflavin T Derivatives.

Immunostaining methods have generally been used not only for biological studies but also for clinical diagnoses for decades. However, recently, for these methods, improved rapidity and simplicity have been required for relevant techniques in laboratory research and medical applications. To this end, we present here a novel approach for designing fluorescent molecular rotor probes, i.e., N3-modified thioflavin T (ThT) derivatives, which enabled specific detection of interesting protein targets with sensitive fluorescence turn-on. As an example, we synthesized N3-( d-desthiobiotinyl-PEGylated) thioflavin T (ThT-PD) and N3-(cortisolyl-PEGylated) thioflavin T (ThT-PC) that carried d-desthiobioin and cortisol, respectively, via PEG linkers. Compared to those of the probes without the targets, ThT-PD and ThT-PC exhibited around 27- and 8-fold fluorescence intensities, respectively, with the target streptavidin and anti-cortisol antibody in excess of saturation, enabling quantitative detection of the targets. Furthermore, we successfully demonstrated the feasibility of ligand-tethering N3-ThT derivatives by the rapid specific staining of glucocorticoid receptors in cells, which was completed within only several minutes using ThT-PC after cell fixation, whereas it took ∼24 h for immunostaining to capture the corresponding fluorescence images.

Glucocorticoids-Resistant Leukemic B-Cells Undergo a Phenotypic Change That Increases Sensitivity to SRC/ABL Inhibition

▪Glucocorticoids (GCs) have a crucial role in the treatment of B-cell acute lymphoblastic leukemia (B-ALL). GCs resistance is considered a strong prognostic marker of relapse, which occurs in about 20% of pediatric B-ALL patients. GCs exert therapeutic effects by inducing cell cycle arrest and eventually, cell death. However, their precise mechanisms of action and resistance are not fully understood. We have previously demonstrated the importance of developmental state in treatment failure in B-ALL (Good Z Nat Med 2018 and Bendall SC Cell 2014), specifically at the developmental transition between pro-B to pre-B cells. Using this developmental framework, we examined the effect of GCs on healthy and malignant early B cells progenitors to better understand mediators of GC resistance.To understand the transcriptional programs mediating early B-cell differentiation, we sorted three B-cell developmental populations (pre-pro-B, pro-B/pre-B I and pre-B II) and performed RNA-seq analysis. Ingenuity pathway analysis revealed a coordinated upregulation of B cell developmental genes and Glucocorticoid Receptor (GCR) pathway genes in healthy pre-B I cells, suggesting a role of GCs in healthy B-cell development. We confirmed expression of the GCR in healthy pre-B I cells by mass cytometry (CyTOF). Further, in vitro treatment of healthy B-cells with GCs (dexamethasone, dex 1 uM) demonstrated pre-B I cycling cells to be most sensitive to GC-induced cell death.Given the importance of GCs in B-ALL treatment, we investigated the GCR pathway and its relationship with B-cell development in 18 B-ALL primary samples (14 diagnosis, 4 relapse; including Ph+=1; MLL rearranged=2; TEL-AML translocated=3). Samples were profiled by CyTOF with a 40-antibody panel including surface markers, signaling molecules, transcription factors, apoptosis and cell cycle molecules. To better comprehend the relation of GCR pathway with BCR signaling, primary cells were analyzed after 48 hours of treatment with dex (1uM), SRC/ABL inhibitor dasatinib (das, 100 nM) or their combination.Similar to results obtained in healthy B-cells, we found the highest expression of GCR in the pre B-I cells in all patients (p<0.001). Interestingly, samples from relapse or diagnostic high-risk groups demonstrated the highest expression of GCR in this cell population. As expected, dex induced cell cycle arrest and cell death in almost all the samples (88%), while two samples were completely unresponsive. Moreover, combined treatment resulted in increased cell death in 29% of patients, compared to dex or das alone.Interestingly, we also observed consistent patterns of phenotypic modulation in dex-resistant cells and to understand how these were related to B-cell development we performed developmental classification (Good Z Nat Med 2018) of leukemic cells in dex-treated versus untreated conditions. This analysis revealed a large decrease of early B-cell progenitors and corresponding increase of more mature subsets of either B and non-B cells (false discovery rate, FDR<0.001), suggesting a differentiating effect of GCs. This phenomenon was even more prominent when we performed, for some primary samples (n=4), longer culture up to six days. Moreover, dex-resistant cells showed aberrant activation of prpS6 and pCREB that was blunted by the combination with das resulting in a reversion of the phenotype and killing of the majority of the cells.To determine if this effect was directly related to GCR expression, we compared results between a GCR+ cell line (NALM6) and GCR- cell line (REH). Consistent with the primary samples, the combination of dex with das more effectively killed NALM6 cells yet the REH were unresponsive to either treatment. However, overexpressing GCR in REH cells reversed dex-resistance, also becoming more sensitive to the combined treatment with das. Similar to primary cells, NALM6 and REH GCR+ cells showed a phenotypic change when treated with dex, that will be further investigated to clarify the relationship between the dex-induced phenotype and its apoptotic effect.In summary, these data suggest a dual role of glucocorticoids on early B-cells; mediating both apoptosis and differentiation in relationship to cell cycle status and GCR level. These findings may carry therapeutic implications and suggest cell types vulnerable to BCR signaling inhibition as a therapeutic intervention to overcome GCs resistance. DisclosuresNolan:Akoya Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Flightless-I mediates the repression of estrogen receptor α target gene expression by the glucocorticoid receptor in MCF-7 cells.

The human homologue of flightless-I (FLII) belong to the gelsolin protein family and contain a gelsolin-like domain at the C-terminus and a leucine-rich repeat (LRR) domain at the N-terminus. FLII regulates estrogen receptor alpha (ERα) and glucocorticoid receptor (GR)-mediated transcription by direct interaction through different domains, suggestive of its potential role in the crosstalk between the ERα and GR signaling pathway. Here, we demonstrate that FLII plays a critical role in GR-mediated repression of ERα target gene expression. In FLII-depleted cells, the reduction in 17-β-estradiol (E2)-induced ERα occupancy following treatment with dexamethasone (Dex) at the estrogen responsive element (ERE) site of the ERα target gene was significantly inhibited. The ERE binding of GR by the cotreatment with E2 and Dex was significantly inhibited by FLII depletion, indicating that FLII is required for the recruitment of GR at the ERE sites of ERα target genes. In addition, the recruitment of ERα-induced FLII to ERE sites was significantly reduced by Dex treatment. In protein binding assays, GR inhibited the E2-induced interaction between ERα and FLII, suggesting that GR interferes with the binding of ERα and FLII at the ERα target genes, resulting in the release of ERα and FLII from EREs. Taken together, our data reveal an unknown mechanism by which the transcription coactivator FLII regulates the GR-mediated repression of ERα target gene expression in MCF-7 cells.

Modulation of glucocorticoid receptor in human epileptic endothelial cells impacts drug biotransformation in an in vitro blood–brain barrier model

SummaryObjectiveNuclear receptors and cytochrome P450 (CYP) regulate hepatic metabolism of several drugs. Nuclear receptors are expressed at the neurovascular unit of patients with drug‐resistant epilepsy. We studied whether glucocorticoid receptor (GR) silencing or inhibition in human epileptic brain endothelial cells (EPI‐ECs) functionally impacts drug bioavailability across an in vitro model of the blood–brain barrier (BBB) by CYP‐multidrug transporter (multidrug resistance protein 1, MDR1) mechanisms.MethodsSurgically resected brain specimens from patients with drug‐resistant epilepsy, primary EPI‐ECs, and control human brain microvascular endothelial cells (HBMECs) were used. Expression of GR, pregnane X receptor, CYP3A4, and MDR1 was analyzed pre‐ and post‐GR silencing in EPI‐ECs. Endothelial cells were co‐cultured with astrocytes and seeded in an in vitro flow‐based BBB model (DIV‐BBB). Alternatively, the GR inhibitor mifepristone was added to the EPI‐EC DIV‐BBB. Integrity of the BBB was monitored by measuring transendothelial electrical resistance. Cell viability was assessed by glucose‐lactate levels. Permeability of [3H]sucrose and [14C]phenytoin was quantified. CYP function was determined by measuring resorufin formation and oxcarbazepine (OXC) metabolism.ResultsSilencing and inhibition of GR in EPI‐ECs resulted in decreased pregnane X receptor, CYP3A4, and MDR1 expression. GR silencing or inhibition did not affect BBB properties in vitro, as transendothelial electrical resistance and Psucrose were unaltered, and glucose metabolism was maintained. GR EPI‐EC silencing or inhibition led to (1) increased Pphenytoin BBB permeability as compared to control; (2) decreased CYP function, indirectly evaluated by resorufin formation; (3) improved OXC bioavailability with increased abluminal (brain‐side) OXC levels as compared to control.SignificanceOur results suggest that modulating GR expression in EPI‐ECs at the BBB modifies drug metabolism and penetration by a mechanism encompassing P450 and efflux transporters. The latter could be exploited for future drug design and to overcome pharmacoresistance.

Glucocorticoids and PD-1

Signaling by the glucocorticoid receptor in NK cells induces expression of the checkpoint inhibitor PD-1 to prevent immunopathology.

Glucocorticoid receptor in stromal cells is essential for glucocorticoid-mediated suppression of inflammation in arthritis

BackgroundGlucocorticoid (GC) therapy is frequently used to treat rheumatoid arthritis due to potent anti-inflammatory actions of GCs. Direct actions of GCs on immune cells were suggested to suppress inflammation.ObjectivesDefine the...

Programming for increased expression of hippocampal GAD67 mediated the hypersensitivity of the hypothalamic\u2013pituitary\u2013adrenal axis in male offspring rats with prenatal ethanol exposure

An imbalance of excitatory and inhibitory signals in the brain has been proposed to be one of the main pathological features of various diseases related to hypothalamic–pituitary–adrenal axis (HPAA) dysfunction. Excessive glutamate release induces neuronal excitotoxicity, while glutamic acid decarboxylase (GAD) 67 promotes the transformation of excessive glutamate to γ-aminobutyric acid (GABA). Our previous studies demonstrated that prenatal ethanol exposure (PEE) causes foetal over-exposure to maternal corticosterone and hypersensitivity of the HPAA after birth, but its intrauterine programming mechanism is unknown. In this study, PEE was shown to lead to an enhanced potential excitatory ability of the hypothalamus and hypersensitivity of the HPAA, as well as mild abnormal hippocampal morphology, demethylation of the -1019 to -691-bp region in the hippocampal GAD67 promoter and upregulation of GAD67 expression accompanied by a reduction in glutamatergic neurons and increase in GABAergic neurons in PEE male offspring. Similar changes were also found in PEE male foetal rats. Furthermore, corticosterone increased the expression of the glucocorticoid receptor (GR) and GAD67 in foetal hippocampal H19-7 cells in a concentration-dependent manner, accompanied by demethylation of the GAD67 promoter, a decrease in glutamatergic neurons and increase in GABAergic neurons. The GR inhibitor, mifepristone, reversed the effects of corticosterone on H19-7 cells. These results suggested that PEE-induced excessive corticosterone can lead to upregulation of GAD67 through epigenetic modification mediated by the GR in the male foetal hippocampus, thereby weakening the negative regulation of the HPAA by the hippocampus and increasing the potential excitatory ability of the hypothalamus. These changes persisted until after birth, resulting in hypersensitivity of the HPAA. However, gender differences were observed in the hippocampal development, morphology and GAD67 expression associated with PEE. Programming for the increased expression of hippocampal GAD67 is a potential mechanism responsible for the hypersensitivity of the HPAA in PEE male rats.

The glucocorticoid receptor in recipient cells keeps cytokine secretion in acute graft-versus-host disease at bay.

Graft-versus-host disease (GvHD) is a life-threatening complication of hematopoietic stem cell transplantation (HSCT), which is caused by allogeneic T cells recognizing molecules of the recipient as foreign. Endogenous glucocorticoids (GC) released from the adrenal gland are crucial in regulating such inflammatory diseases. Here we demonstrate that genetically engineered mice, that are largely unresponsive to GC, suffer from aggravated clinical symptoms and increased mortality after HSCT, effects that could be tempered by neutralization of IL-6. Interestingly, selective ablation of the GC receptor (GR) in recipient myeloid cells resulted in fulminant disease as well. While histopathological analysis of the jejunum failed to reveal any differences between sick mice of both genotypes, systemic IL-6 and TNFα secretion was strongly increased in transplanted mice lacking the GR in myeloid cells briefly before the majority of them succumbed to the disease. Collectively, our findings reveal an important role of the GR in recipient cells in limiting the cytokine storm caused by GvHD induction.

Impaired resolution of DSS-induced colitis in mice lacking the glucocorticoid receptor in myeloid cells.

Inflammatory bowel disease (IBD) is a highly prevalent intestinal disorder for which no cure exists. Currently, the standard first-line treatment of IBD consists of systemic glucocorticoid (GC) application, even though therapy can be complicated by unresponsiveness or adverse effects. In view of the importance of macrophages and neutrophils for the pathogenesis of IBD we set out to define the relevance of these cell types as targets of GC using the mouse model of DSS-induced colitis. We found that the disease did not resolve in GRlysM mice lacking the GC receptor (GR) in myeloid cells after removal of the chemical insult. While clinical symptoms and tissue damage in the colon ameliorated again in GRflox mice, the disease further aggravated in GRlysM littermates. The observed difference coincided with an increased abundance of macrophages in inflammatory infiltrates in the colon of mutant mice whereas neutrophil and T cell numbers were similar. Concomitantly, systemic IL-6 secretion and mRNA levels of pro-inflammatory cytokines in the colon were elevated in GRlysM mice and gene expression of scavenger receptors and IL-10 was diminished. Taken together, our results reveal an important role of myeloid cells as targets of GC in DSS-induced colitis and probably in IBD in humans as well.

Phosphorylation and Ubiquitination Regulate Protein Phosphatase 5 Activity and Its Prosurvival Role in Kidney Cancer

The serine/threonine protein phosphatase 5 (PP5) regulates multiple cellular signaling networks. A number of cellular factors, including heat shock protein 90 (Hsp90), promote the activation of PP5. However, it is unclear whether post-translational modifications also influence PP5 phosphatase activity. Here, we show an "on/off switch" mechanism for PP5 regulation. The casein kinase 1δ (CK1δ) phosphorylates T362 in the catalytic domain of PP5, which activates and enhances phosphatase activity independent of Hsp90. Overexpression of the phosphomimetic T362E-PP5 mutant hyper-dephosphorylates substrates such as the co-chaperone Cdc37 and glucocorticoid receptor in cells. Our proteomic approach revealed that the tumor suppressor von Hippel-Lindau protein (VHL) interacts with and ubiquitinates K185/K199-PP5 for proteasomal degradation in a hypoxia- and prolyl-hydroxylation-independent manner. Finally, VHL-deficient clear cell renal cell carcinoma (ccRCC) cell lines and patient tumors exhibit elevated PP5 levels. Downregulation of PP5 causes ccRCC cells to undergo apoptosis, suggesting a prosurvival role for PP5 in kidney cancer.

Dissecting the genomic activity of a transcriptional regulator by the integrative analysis of omics data

In the study of genomic regulation, strategies to integrate the data produced by Next Generation Sequencing (NGS)-based technologies in a meaningful ensemble are eagerly awaited and must continuously evolve. Here, we describe an integrative strategy for the analysis of data generated by chromatin immunoprecipitation followed by NGS which combines algorithms for data overlap, normalization and epigenetic state analysis. The performance of our strategy is illustrated by presenting the analysis of data relative to the transcriptional regulator Estrogen Receptor alpha (ERα) in MCF-7 breast cancer cells and of Glucocorticoid Receptor (GR) in A549 lung cancer cells. We went through the definition of reference cistromes for different experimental contexts, the integration of data relative to co-regulators and the overlay of chromatin states as defined by epigenetic marks in MCF-7 cells. With our strategy, we identified novel features of estrogen-independent ERα activity, including FoxM1 interaction, eRNAs transcription and a peculiar ontology of connected genes.

Abstract 707: Personalization of dexamethasone in acute lymphoblastic leukemia

Abstract Synthetic glucocorticoids, such as dexamethasone (Dex), are pivotal in the treatment of childhood acute lymphoblastic leukemia (ALL) but are associated with significant variability, both in terms of toxicity and efficacy. We aimed to investigate three key variables to better understand how Dex personalization may be achieved: pharmacokinetics (PK), intracellular Dex accumulation, and cellular response, following Dex binding to the glucocorticoid receptor (GR) in ALL cells. For Dex PK studies, blood samples were collected post oral administration on one of the first three days of induction chemotherapy in 99 patients on the UKALL 2011 trial receiving either 6mg/m2 for28 days (standard arm) or 10mg/m2 for 14 days (short arm). Plasma Dex levels were analysed using a validated LC/MS method, and a non-compartmental pharmacokinetic analysis. To assess intracellular Dex levels, cell lines, primagraft (n=9) and primary patient samples (n=6) were studied. The plasma Dex LC/MS method was optimized to quantify Dex in ALL cell lysates. Dex accumulation was also assessed using flow cytometric analysis of Dex conjugated to FITC. Cellular Dex sensitivity was assessed using Alamar Blue assays. There was a wide Dex PK variability, with AUC0-12h, and Cmax significantly higher on the short compared to the standard arm; 564 (202-1606) versus 408 (142-1009), median (range), p=0.0003 and 0.0006, respectively. However there was substantial overlap between the two arms, with a number of patients on the standard arm exhibiting higher exposures than those on short therapy. Dex GI50 values ranged from 37 - &amp;gt; 1000 nM and 2 - &amp;gt; 1000 nM in cell lines and patient samples respectively. Western blotting indicated wildtype GR in all samples, with R3D11 and REH cell lines serving as hemizygous deleted and GR negative controls. Dex accumulation in cell lines was 2.1 and 1.8 (range 1.2 - 2.1) pmol/106 cells in PreB697 and Dex resistant sub-lines, respectively. While patient samples exhibited greater variability, Dex accumulation was not significantly different between sensitive and resistant cells; mean of 1.0 versus 1.4 (range 0.1-2.3, 0.4-4.4 pmol/ 106 cells, p=0.17). Flow cytometry Dex FITC accumulation confirmed these data, with a mean fluorescence intensity of 4.2 versus 4.1 (range 1.5-5.9, 2.0 - 9.1, respectively; p=0.97). These data suggest that while PK and cellular response are hugely variable, variations in drug accumulation do not appear to play a key role in Dex response in ALL cells. Importantly, 62% of patient cell samples had Dex GI50 values greater than plasma concentrations observed in any patient, on both arms on the UKALL 2011 trial. A combined approach incorporating PK assessments and cellular response in ALL cells should be further investigated, to allow a comprehensive understanding of Dex pharmacology with a view to optimizing its clinical utility. Citation Format: Rosanna K. Jackson, Ali Alhammer, Zach Dixon, Gareth J. Veal, Julie AE Irving. Personalization of dexamethasone in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 707. doi:10.1158/1538-7445.AM2017-707

Glucocorticoid receptor number and affinity differ between peripheral blood mononuclear cells and granulocytes in domestic pigs

The aim of the present study was to characterize the number and affinity of glucocorticoid receptors (GR) in peripheral blood mononuclear cells (PBMC) and granulocytes of domestic pigs because glucocorticoid signaling is considered important for animal health and welfare. To investigate GR binding characteristics in intact porcine immune cells, blood samples of 6 castrated male pigs were collected via indwelling vein catheters. Porcine PBMC and granulocytes were isolated using two-layer density gradients, followed by radioligand binding assays to determine the number of GR sites per cell and the dissociation constant Kd as a measure for GR binding affinity. The present study revealed a greater number of GR sites per cell (P = 0.039) in PBMC (mean ± SEM: 1,953 ± 207 sites/cell) compared to granulocytes (1,561 ± 159 sites/cell) in domestic pigs. Furthermore, porcine PBMC had a higher GR binding affinity than porcine granulocytes (P = 0.003) as the dissociation constant Kd of PBMC (1.8 ± 0.2 nM) was lower than that of granulocytes (3.5 ± 0.4 nM). Our results point to differences in underlying mechanisms of glucocorticoid signaling in different porcine leukocyte populations.

Anti-inflammatory and vasoconstrictive properties of Potentilla erecta – A traditional medicinal plant from the northern hemisphere

Ethnopharmacological relevancePotentilla erecta (L.) Raeusch is a medicinal plant of the Northern hemisphere belonging to the plant family of roses (Rosaceae). It has traditionally been used to treat inflammatory disorders of the skin and mucous membranes as well as chronic diarrhea. Aim of the studyIn the present study we analyzed the anti-inflammatory and vasoconstrictive effect of a Potentilla erecta extract (PE) and questioned if PE is similar effective as mild corticosteroids. Then we analyzed if PE acts in the skin via a similar mode of action as corticosteroids. Material and methodsThe anti-inflammatory effect of PE was analyzed in irradiated HaCaT keratinocytes by measuring the formation of IL-6 and PGE2. Additionally the effect of PE on TNF-α induced NF-κB activation was determined. As the anti-inflammatory effect of corticosteroids correlates with their vasoconstrictive properties we tested if PE displays also vasoconstriction. Therefore we performed an occlusive patch test and a collagen contraction assay. Furthermore the binding of PE to the glucocorticoid receptor was determined with stainings and reporter assays. The interaction of PE on the nitric oxide (NO) content was examined with radical scavenging and endothelial NO synthase (eNOS) reporter assays. ResultsIn irradiated or TNF-α stimulated HaCaT cells the formation of IL-6 and PGE2 or NF-κB activation was strongly reduced by PE. Furthermore PE showed a blanching effect comparable to hydrocortisone. However, in contrast to glucocorticoids, PE did not cause nuclear translocation of the glucocorticoid receptor in HaCaT cells. The blanching effect of PE was at least partly attributable to a scavenging effect of NO and inhibition of eNOS. ConclusionsPE displays anti-inflammatory and vasoconstrictive effects and might therefore be beneficial for the topical treatment of inflammatory skin disorders.

Difficult Family Relationships, Residential Greenspace, and Childhood Asthma.

Both the social environment and the physical environment are increasingly recognized as important to childhood diseases such as asthma. This study tested a novel hypothesis: that living in areas high in greenspace may help buffer the effects of difficult family relationships for children with asthma. A total of 150 children (ages 9-17), physician-diagnosed with asthma, participated in this study. To assess difficulties in parent-child relationships, parents and children completed measures of harsh/inconsistent parenting and parental hostility. Residential greenspace was calculated by using satellite-derived Normalized Difference Vegetation Index with a buffer of 250 m around the residential address. Outcomes included both clinical and biological measures: asthma control and functional limitations, as well as airway inflammation (fractional concentration of exhaled nitric oxide) and glucocorticoid receptor expression in T-helper cells. After controlling for potential confounding variables, including family income, child demographics, and child medical variables, few main effects were found. However, interactions between residential greenspace and difficult family relationships were found for asthma control (P = .02), asthma functional limitations (P = .04), airway inflammation (P = .007), and the abundance of glucocorticoid receptor in T-helper cells (P = .05). These interactions were all in a direction such that as the quality of parent-child relationships improved, greenspace became more strongly associated with better asthma outcomes. These findings suggest synergistic effects of positive environments across the physical and social domains. Children with asthma appear to benefit the most when they both live in high greenspace areas and have positive family relationships.

Muscle-specific downregulation of GR levels inhibits adipogenesis in porcine intramuscular adipocyte tissue

Intramuscular adipose is conducive to good pork quality, whereas subcutaneous adipose is considered as waste in pig production. So uncovering the regulation differences between these two adiposes is helpful to tissue-specific control of fat deposition. In this study, we found the sensitivity to glucocorticoids (GCs) was lower in intramuscular adipocytes (IMA) compared with subcutaneous adipocytes (SA). Comparison of glucocorticoid receptor (GR) revealed that IMA had lower GR level which contributed to its reduced GCs sensitivity. Higher methylation levels of GR promotor 1-C and 1-H were detected in IMA compared with SA. GR expression decrease was also found in adipocytes when treated with muscle conditioned medium (MCM) in vitro, which resulted in significant inhibition of adipocytes proliferation and differentiation. Since abundant myostatin (MSTN) was detected in MCM by ELISA assay, we further investigated the effect of this myokine on adipocytes. MSTN treatment suppressed adipocytes GR expression, cell proliferation and differentiation, which mimicked the effects of MCM. The methylation levels of GR promotor 1-C and 1-H were also elevated after MSTN treatment. Our study reveals the role of GR in muscle fiber inhibition on intramuscular adipocytes, and identifies myostatin as a muscle-derived modulator for adipose GR level.

Genome-wide Regulatory Roles of the C2H2-type Zinc Finger Protein ZNF764 on the Glucocorticoid Receptor

The C2H2-type zinc finger protein ZNF764 acts as an enhancer for several steroid hormone receptors, and haploinsufficiency of this gene may be responsible for tissue resistance to multiple steroid hormones including glucocorticoids observed in a patient with 16p11.2 microdeletion. We examined genome-wide regulatory actions of ZNF764 on the glucocorticoid receptor (GR) in HeLa cells as a model system. ZNF764- and GR-binding sites demonstrated similar distribution in various genomic features. They positioned predominantly around 50–500 kbs from the transcription start sites of their nearby genes, and were closely localized with each other, overlapping in ~37% of them. ZNF764 demonstrated differential on/off effects on GR-binding and subsequent mRNA expression: some genes were highly dependent on the presence/absence of ZNF764, but others were not. Pathway analysis revealed that these 3 gene groups were involved in distinct cellular activities. ZNF764 physically interacted with GR at ligand-binding domain through its KRAB domain, and both its physical interaction to GR and zinc finger domain appear to be required for ZNF764 to regulate GR transcriptional activity. Thus, ZNF764 is a cofactor directing GR transcriptional activity toward specific biologic pathways by changing GR binding and transcriptional activity on the glucocorticoid-responsive genes.

Crosstalk between Aryl Hydrocarbon Receptor and Glucocorticoid Receptor in Human Retinal Pigment Epithelial Cells.

The aryl hydrocarbon receptor (AHR) is known to mediate the cellular reaction involved in processing environmental contaminants and, ultimately, preventing accumulation of unfavorable extra lipids and proteins. Glucocorticoid receptor (GR) mediates the expression of genes associated with anti-inflammatory properties. Because AHR and GR are closely related in lipid metabolic dysregulation and inflammation, we speculate that AHR and GR may play a crucial role in AMD pathogenesis and focus on their crosstalk in human retinal pigment epithelial cells (ARPE-19). However, how AHR and GR regulate each other's signaling pathways is still poorly understood. In this research, we demonstrate that GR attenuates AHR-mediated gene expression by inhibition of nuclear translocation of AHR mediated by TCDD. Chromatin immunoprecipitation analysis demonstrated that GR repress AHR recruitment and chromatin accessibility response to TCDD + Dex treatment leading to repression of AHR target genes. In contrast, AHR facilitates GR-mediated expression in ARPE-19. AHR increases GR recruitment on GRE of GR target genes. Coimmunoprecipitation assay revealed that AHR is associated with GR in ARPE-19 cells and the interaction is enhanced by the addition of TCDD and Dex. Taken together, these studies provide a molecular mechanism of crosstalk between AHR and GR in target gene expression in ARPE-19 cells.

MicroRNAs 29b and 181a down-regulate the expression of the norepinephrine transporter and glucocorticoid receptors in PC12 cells.

MicroRNAs are short non-coding RNAs that provide global regulation of gene expression at the post-transcriptional level. Such regulation has been found to play a role in stress-induced epigenetic responses in the brain. The norepinephrine transporter (NET) and glucocorticoid receptors are closely related to the homeostatic integration and regulation after stress. Our previous studies demonstrated that NET mRNA and protein levels in rats are regulated by chronic stress and by administration of corticosterone, which is mediated through glucocorticoid receptors. Whether miRNAs are intermediaries in the regulation of these proteins remains to be elucidated. This study was undertaken to determine possible regulatory effects of miRNAs on the expression of NET and glucocorticoid receptors in the noradrenergic neuronal cell line. Using computational target prediction, we identified several candidate miRNAs potentially targeting NET and glucocorticoid receptors. Western blot results showed that over-expression of miR-181a and miR-29b significantly repressed protein levels of NET, which is accompanied by a reduced [3 H] norepinephrine uptake, and glucocorticoid receptors in PC12 cells. Luciferase reporter assays verified that both miR-181a and miR-29b bind the 3'UTR of mRNA of NET and glucocorticoid receptors. Furthermore, exposure of PC12 cells to corticosterone markedly reduced the endogenous levels of miR-29b, which was not reversed by the application of glucocorticoid receptor antagonist mifepristone. These observations indicate that miR-181a and miR-29b can function as the negative regulators of NET and glucocorticoid receptor translation invitro. This regulatory effect may be related to stress-induced up-regulation of the noradrenergic phenotype, a phenomenon observed in stress models and depressive patients. This study demonstrated that miR-29b and miR-181a, two short non-coding RNAs that provide global regulation of gene expression, markedly repressed protein levels of norepinephrine (NE) transporter and glucocorticoid receptor (GR), as well as NE uptake by binding the 3'UTR of their mRNAs in PC12 cells. Also, exposure of cells to corticosterone significantly reduced miR-29b levels through a GR-independent way.

DNA binding triggers tetramerization of the glucocorticoid receptor in live cells

Transcription factors dynamically bind to chromatin and are essential for the regulation of genes. Although a large percentage of these proteins appear to self-associate to form dimers or higher order oligomers, the stoichiometry of DNA-bound transcription factors has been poorly characterized in vivo. The glucocorticoid receptor (GR) is a ligand-regulated transcription factor widely believed to act as a dimer or a monomer. Using a unique set of imaging techniques coupled with a cell line containing an array of DNA binding elements, we show that GR is predominantly a tetramer when bound to its target DNA. We find that DNA binding triggers an interdomain allosteric regulation within the GR, leading to tetramerization. We therefore propose that dynamic changes in GR stoichiometry represent a previously unidentified level of regulation in steroid receptor activation. Quaternary structure analysis of other members of the steroid receptor family (estrogen, androgen, and progesterone receptors) reveals variation in oligomerization states among this family of transcription factors. Because GR's oligomerization state has been implicated in therapy outcome, our findings open new doors to the rational design of novel GR ligands and redefine the quaternary structure of steroid receptors.