Wild-type Glucocorticoid Receptor Research Articles

Reduced steroid activation of elephant shark GR and MR after inserting four amino acids from the DNA-binding domain of lamprey corticoid receptor-1.

Atlantic sea lamprey contains two corticoid receptors (CRs), CR1 and CR2, that have identical amino acid sequences, except for a four amino acid insert (Thr-Arg-Gln-Gly) in the CR1 DNA-binding domain (DBD). Steroids are stronger transcriptional activators of CR2 than of CR1 suggesting that the insert reduces the transcriptional response of lamprey CR1 to steroids. The DBD in elephant shark mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), which are descended from a CR, lack these four amino acids, suggesting that a CR2 is their common ancestor. To determine if, similar to lamprey CR1, the presence of this insert in elephant shark MR and GR decreases transcriptional activation by corticosteroids, we inserted these four CR1-specific residues into the DBD of elephant shark MR and GR. Compared to steroid activation of wild-type elephant shark MR and GR, cortisol, corticosterone, aldosterone, 11-deoxycorticosterone and 11-deoxycortisol had lower transcriptional activation of these mutant MR and GR receptors, indicating that the absence of this four-residue segment in the DBD in wild-type elephant shark MR and GR increases transcriptional activation by corticosteroids.

Adrenal suppression in patients with chronic obstructive pulmonary disease treated with glucocorticoids: Role of specific glucocorticoid receptor polymorphisms.

Single-nucleotide polymorphisms (SNPs) of the glucocorticoid receptor (GR) gene NR3C1 have been associated with an altered sensitivity to glucocorticoids, and thus may alter the therapeutic effects of glucocorticoids. We investigated the prevalence of adrenal suppression after treatment with glucocorticoids and evaluated whether GR SNPs were associated with altered risks of adrenal suppression and metabolic disorders in patients with chronic obstructive pulmonary disease (COPD). In an observational prospective cohort study, we recruited 78 patients with severe COPD receiving 5 days glucocorticoid treatment for an exacerbation of COPD. In total, 55% of these patients were also receiving regular inhaled corticosteroids (ICS). Adrenal function was evaluated with a corticotropin test 30 days after the exacerbation. Patients were genotyped for Bcl1, N363S, ER22/23EK, and 9β SNPs. The prevalence of adrenal suppression (corticotropin-stimulated plasma-cortisol ≤ 420 nmol/L) 30 days after glucocorticoid treatment was 4/78 (5%). There was no difference between carriers and non-carriers of the polymorphisms (Bcl1, 9β, ER22/23K, and N363S) in corticotropin stimulated plasma-cortisol concentrations. In the haplotype analyses, we included the 50 patients who had a high-sensitivity (76%), a low-sensitivity (4%), or a wild-type (20%) GR haplotype. There was no difference in the frequency of adrenal suppression or metabolic disorders between the two stratified groups: (a) high-sensitivity (Bcl1 and/or N363S) haplotypes vs. (b) low-sensitivity (9β and/or ER22/23K) plus wild-type haplotypes (p > 0.05). Carriers of the high-sensitivity GR gene haplotype exhibited a steeper decline in stimulated P-cortisol with increased ICS dose (slope, -1.35 vs. 0.94; p = 0.17), compared to the group with low-sensitivity or wild-type haplotypes, respectively. In total, 5% of patients exhibited insufficient adrenal function. The Bcl1 and N363S polymorphisms did not seem to increase the risk of glucocorticoid suppression or metabolic disorders in adults treated with glucocorticoids for COPD exacerbations.

Effect of 3 NR3C1 Mutations in the Pathogenesis of Pituitary ACTH Adenoma.

Glucocorticoids act through the glucocorticoid receptor (GR) encoded by the nuclear receptor subfamily 3 group C member 1 (NR3C1) gene. This study aimed to examine the function of NR3C1 variants and their possible pathogenic role in Cushing disease (CD). Next-generation sequencing was conducted in 49 CD patients. Corticotroph tumor GR protein expression was examined by immunohistochemistry (IHC). Constructs harboring the 3 NR3C1-mutant and wild-type (WT) GR were transfected into the murine corticotropic adenoma cell line (AtT-20), and GR protein expression was quantified by Western blot. Translocation activity was assessed by immunofluorescence and effects of the GR mutants on corticotroph tumor proliferation, pro-opiomelanocortin (POMC) transcription, and ACTH secretion were tested. Clinical features were similar in patients harboring the NR3C1 mutations and WT GR. Recurrent adenomas showed higher GR IHC scores than nonrecurrent tumors. In vitro studies demonstrated that the p.R469X mutant generated a truncated GR protein, and the p.D590G and p.Y693D GR mutants resulted in lower GR expression. Dexamethasone (DEX) treatment of AtT-20 cells demonstrated decreased DEX-induced nuclear translocation, increased cell proliferation, and attenuated suppression of POMC transcription of 3 GR mutants. Interestingly, the p.R469X GR mutant resulted in increased murine corticotroph tumor ACTH secretion compared to WT GR. Our findings identify 3/49 (6.1%) consecutive human corticotroph tumors harboring GR mutations. Further findings demonstrate the role NR3C1 plays in CD pathogenesis and offer insights into a novel treatment approach in this patient subset.

Anthropometrics and Metabolic Syndrome in Relation to Glucocorticoid Receptor Polymorphisms in Corticosteroid Users

Introduction: Corticosteroids are widely prescribed and their use has been linked to adverse cardiometabolic outcomes. A pivotal role in the action of corticosteroids is reserved for the glucocorticoid receptor (GR). Here, we assessed the relationship of glucocorticoid sensitivity-altering GR polymorphisms with anthropometrics and metabolic syndrome (MetS) in corticosteroid users. Methods: In this population-based cohort study (Lifelines), we genotyped 10,621 adult participants for GR hypersensitive (1/2 copies BclI and/or N363S) and GR resistant (1/2 copies ER22/23EK and/or 9β) variants. We assessed the relationship between functional GR polymorphisms with BMI, waist circumference (WC), and MetS in users of corticosteroids. Results: Overall corticosteroid use was associated with a significantly higher BMI and WC in GR wild-type (WT) users (BMI, +0.63 kg/m² [0.09–1.16], p = 0.022; WC, +2.03 cm [0.61–3.44], p = 0.005) and GR hypersensitive (BMI, +0.66 kg/m² [95% CI, 0.31–1.01]; WC, +2.06 cm [1.13–2.98], both p < 0.001) but not in GR resistant users. Significantly higher WC in GR resistant carriers was observed only for inhaled corticosteroid users. With respect to MetS, again only GR WT users (odds ratio [OR] 1.44 [1.07–1.94], p = 0.017) and GR hypersensitives (OR 1.23 [95% CI, 1.00–1.50], p = 0.046) were more likely to have MetS; even more pronounced in only inhaled corticosteroid users (GR WT users, OR 1.64 [1.06–2.55], p = 0.027; GR hypersensitive users, OR 1.43 [1.08–1.91], p = 0.013). Conclusions: Polymorphisms associated with increased GR sensitivity and WT GR are related to increased BMI, WC, and an increased MetS presence in corticosteroid users, especially of the inhaled types, when compared to nonusers. The adverse effects of corticosteroid use are less pronounced in users harboring GR resistant polymorphisms.

Comparing the DNA‐Binding Specificity of Glucocorticoid and Androgen Receptors

Prostate cancer is the second most common cancer among men, with an estimated 175,000 diagnoses expected in 2019. Androgen hormones fuel prostate tumor growth through the actions of the androgen receptor (AR), a member of the nuclear hormone family of transcription factors. Therapies for prostate cancer include surgery and inhibition of AR with antagonists. Patients, however, often relapse with castration‐resistant prostate cancer (CRPC) in which the cancer grows in the absence of AR ligand action. In some cases, the need for androgens is subverted by overexpression of the AR‐related glucocorticoid receptor (GR). GR effectively replaces AR by regulating genes that drive prostate growth and survival. We have previously used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method along with the SelexGLM computational algorithm to demonstrate that GR and AR have similar, but distinct DNA sequence specificities. Upon overexpression, GR is able to bind genomic loci once preferentially bound by AR and regulate associated genes. To better understand differences in AR and GR DNA recognition, we are now measuring DNA sequence preferences of both AR and GR using the No Read Left Behind (NRLB) method. NRLB yields high resolution models of specificity after just one round of selection, allowing rapid screening of both wildtype and hybrid AR and GR DNA binding domains. We have over‐expressed and purified both the AR and GR DNA binding domains, and are designing hybrid mutants. Exploring the similarities and differences between AR, GR and the hybrid molecules should help us understand how GR might be playing a role in CRPC.Support or Funding Information• Grant: NSF:CAREER (MCB‐1552862) to M.A. Pufall

Functional characterization of canine wild type glucocorticoid receptor and an insertional mutation in a dog

BackgroundGlucocorticoids, among the most widely utilized drugs in veterinary medicine, are employed to treat a wide variety of diseases; however, their use often induces adverse events in dogs. The efficacy of glucocorticoids usually depends on dosage, although differences in sensitivity to glucocorticoids in individual animals have been reported. Glucocorticoids bind to the cytoplasmic glucocorticoid receptor (GR), which is expressed in almost all cells. These receptors are key factors in determining individual sensitivity to glucocorticoids. This study examined individual differences in glucocorticoid sensitivity in dogs, focusing on reactivity of the GR to prednisolone.ResultsWe first molecularly cloned the GR gene from a healthy dog. We discovered a mutant GR in a dog suspected to have iatrogenic Cushing syndrome. The mutant GR had extra nucleotides between exons 6 and 7, resulting in a truncated form of GR that was 98 amino acids shorter than the wild-type dog GR. The truncated GR exhibited very low reactivity to prednisolone, irrespective of concentration.ConclusionsWe have identified the truncated form of canine GR in a dog with iatrogenic Cushing syndrome. This truncated form showed the very less sensitivity to glucocorticoid in vitro, unfortunately, we could not elucidate its clinical significance. However, our data is a first report about the function of canine GR, and will facilitate the analysis of canine glucocorticoid sensitivity.

OR26-6 Glucocorticoid Receptor Quaternary Structure Drives Chromatin Occupancy and Transcriptional Outcome

The multimeric structure adopted by transcription factor complexes at their genomic sites of interaction is a longstanding problem in transcription biology. Information bearing on this question is often inferred from indirect data sources. Most transcription factors, including nuclear receptors, are widely modeled as binding regulatory elements as monomers, homodimers, or heterodimers. Glucocorticoid receptor (GR)’s dimeric/monomeric status is widely believed to drive pharmacological output of synthetic glucocorticoids. A widely discussed model suggests that dimeric GR regulates unfavorable metabolic pathways, while monomeric GR is responsible for anti-inflammatory activities. However, recent studies have indicated that dimeric GR is equally active in anti-inflammatory activities. Furthermore, quantitative fluorescence microscopy techniques in live cells show that the GR likely forms tetramers on enhancers during gene activation. These results suggest that higher oligomerization states are important for the gene regulatory responses of GR. To unveil the relationship between GR’s oligomerization and transcriptional outcome, we developed a GR null cell system to study how wild type GR and three different receptor oligomerization mutants bind chromatin and affect transcription at the genomic level. All mutants display unique quaternary structure characteristics; DNA-binding domain dimerization deficient (GRdim, A465T), monomeric (GRmon, A465T/I634A), and tetrameric (GRtetra, P481R) receptor. GRtetra mimics the DNA-bound conformation and forms tetramers independent of DNA binding. Integration of ChIP-seq, ATAC-seq, and RNA-seq data indicates that GR must form dimers to bind even pre-accessible chromatin, as monomeric GR is essentially a non-functional receptor. In comparison, GRtetra, acts as a super receptor by binding glucocorticoid response elements not accessible to the wild type receptor. This super receptor behavior can be observed in two distinct cell types. The sites occupied only by GRtetra in a given cell line are bound by the wild type GR in numerous other cell types, such as liver, macrophages, and pre-adipose cells. This suggest that the sites are authentic response elements. In addition, GRtetra both induces and, more importantly, represses more genes in response to hormone than the wild type GR. These results argue that DNA binding induces a structural transition to the tetrameric state, forming a transient higher order structure that drives both the activating and repressive actions of glucocorticoids. This has important implications for the therapeutic uses of steroid hormones and the goal of finding selective anti-inflammatory drugs that do not create unwanted side-effects.

Backbone 1H, 13C, and 15N resonance assignments of the ligand binding domain of the human wildtype glucocorticoid receptor and the F602S mutant variant

The glucocorticoid receptor (GR) is a nuclear hormone receptor that regulates key genes controlling development, metabolism, and the immune response. GR agonists are efficacious for treatment of inflammatory, allergic, and immunological disorders. Steroid hormone binding to the ligand-binding domain (LBD) of GR is known to change the structural and dynamical properties of the receptor, which in turn control its interactions with DNA and various co-regulators and drive the pharmacological response. Previous biophysical studies of the GR LBD have required the use of mutant forms to overcome issues with limited protein stability and high aggregation propensity. However, these mutant variants are known to also influence the functional response of the receptor. Here we report a successful protocol for protein expression, purification, and NMR characterization of the wildtype human GR LBD. We achieved chemical shift assignments for 90% of the LBD backbone resonances, with 216 out of 240 non-proline residues assigned in the 1H–15N TROSY spectrum. These advancements form the basis for future investigations of allosteric effects in GR signaling.

Loss of the podocyte glucocorticoid receptor exacerbates proteinuria after injury

Nephrotic syndrome is a common disorder in adults and children whose etiology is largely unknown. Glucocorticoids remain the mainstay of therapy in most cases, though their mechanism of action remains poorly understood. Emerging evidence suggests that immunomodulatory therapies used in nephrotic syndrome directly target the podocytes. To study how steroids directly affect the podocytes in the treatment of proteinuria, we created a mouse model with podocyte-specific deletion of the glucocorticoid receptor. The podocyte-specific glucocorticoid receptor (GR) knockout mice had similar renal function and protein excretion compared to wild type. However, after glomerular injury induced by either LPS or nephrotoxic serum, the podocyte GR knockout mice demonstrated worsened proteinuria compared to wild type. Ultrastructural examination of podocytes confirmed more robust foot process effacement in the knockout animals. Expression of several key slit diaphragm protein was down regulated in pGR KO mice. Primary podocytes isolated from wild type and podocyte GR knockout mice showed similar actin stress fiber staining patterns in unstimulated conditions. Yet, when exposed to LPS, GR knockout podocytes demonstrated fewer stress fibers and impaired migration compared to wild type podocytes. We conclude that the podocyte glucocorticoid receptor is important for limiting proteinuria in settings of podocyte injury.

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 Polymorphisms and Outcomes in Pediatric Septic Shock.

Polymorphisms of the glucocorticoid receptor gene are associated with outcome and corticosteroid responsiveness among patients with inflammatory disorders. We conducted a candidate gene association study to test the hypothesis that these polymorphisms are associated with outcome and corticosteroid responsiveness among children with septic shock. We genotyped 482 children with septic shock for the presence of two glucocorticoid receptor polymorphisms (rs56149945 and rs41423247) associated with increased sensitivity and one glucocorticoid receptor polymorphism (rs6198) associated with decreased sensitivity to corticosteroids. The primary outcome variable was complicated course, defined as 28-day mortality or the persistence of two or more organ failures 7 days after a septic shock diagnosis. We used logistic regression to test for an association between corticosteroid exposure and outcome, within genotype group, and adjusted for illness severity. Multiple PICUs in the United States. Standard care. There were no differences in outcome when comparing the various genotype groups. Among patients homozygous for the wild-type glucocorticoid receptor allele, corticosteroids were independently associated with increased odds of complicated course (odds ratio, 2.30; 95% CI, 1.01-5.21; p = 0.047). Based on these glucocorticoid receptor polymorphisms, we could not detect a beneficial effect of corticosteroids among any genotype group. Among children homozygous for the wild-type allele, corticosteroids were independently associated with increased odds of poor outcome.

Dexamethasone Accumulation in Dexamethasone Sensitive and Resistant Acute Lymphoblastic Leukemia

Introduction:Glucocorticoids (GC) have been at the forefront of acute lymphoblastic leukemia (ALL) treatment for a number of decades. However, there is heterogeneity of response, both in terms of GC-related toxicity and leukemia cell sensitivity. Contributing factors include marked pharmacokinetic variability observed in children (Yang et al. JCO, 2008; Jackson et al.AACR annual meeting abstract CT115, 2016) and a several hundred fold range of GC cellular response. Cellular resistance can be caused by deletion of the glucocorticoid receptor (GR) but is more commonly downstream of the GR. One neglected upstream parameter relates to GC accumulation, which may be an important factor in ALL GC response, given the evidence for drug transporters in ALL cells. Therefore, this study aimed to determine whether variation in intracellular dexamethasone (dex) levels is a determinant of dex sensitivity in an ALL setting.Methods:A number of cell lines including PreB697, GC resistant PreB697 sub-lines and REH cells, along with primagraft material from 9 patients and 6 primary patient samples (5 presentation and 1 relapse) were studied. The relative sensitivity of cells to dex was assessed using Alamar Blue drug sensitivity assay. Two methods were developed to assess intracellular dex accumulation; a liquid-chromatography mass spectrometry (LC/MS) method and a flow cytometry method, using dex conjugated to the fluorochrome FITC analysed on a FACSCalibur flow cytometry machine. GR status of the cells was confirmed by western blotting.Results:Dex GI50 values (concentration giving 50% growth inhibition) ranged from 37nM in PreB697 cells to >1000nM in GC resistant sub-lines and REH cells. Dex GI50 values in patient ALL cells ranged from 2 to >1000nM. Dex resistant cells were defined as having a dex GI50 of >500nM. The mean GI50 of the dex sensitive cells was 3.8nM. Western blotting suggested wildtype GR status in all samples, with R3D11 and REH serving as hemizygous deleted and GR negative controls, respectively.The mean dex accumulation was measured in cells using an LC/MS assay developed from a fully validated assay measuring plasma dex concentrations (Jackson et al. NCRI annual meeting abstract BACR9, 2014). Dex was stable in RF10 media for at least 8 hours and there was no matrix effect of RF10 media on dex chromatograms compared to dex in plasma. An incubation concentration of 500nM was chosen as this is the observed median value of dex cell exposure clinically. Dex concentrations were quantifiable in cell numbers of 1 x 106after incubation with 500nM dex, allowing measurement of patient samples where limited numbers of cells are available.Dex accumulation in cell lines after incubation with 500nM dex for 4h was 2.1 and 1.8 pmol/million cells in PreB697 and dex resistant sub-lines, respectively (range for resistant subclones 1.2 - 2.1pmol/million cells). There was greater variability in patient cells with a 40-fold range seen, but dex accumulation was not significantly different between sensitive (mean, 1.0 pmol/million; range, 0.1-2.3) and resistant cells (1.4 pmol/million; range, 0.4-4.4) (unpaired students t-test, p=0.17).To assess intra-leukemia heterogeneity in terms of dex accumulation, a flow based assay was established using dex-FITC. Incubation conditions of 500nM dex-FITC at 37°C for 45 minutes were optimal. Dex-FITC accumulation did not differ significantly between sensitive and resistant cells; mean fluorescence intensity of 4.2 (range 1.5-5.9) versus 4.1 (range 2.0 - 9.1) in sensitive and resistant cells, respectively (p=0.97, unpaired students t test). Dex-FITC accumulation appeared uniform within the ALL samples examined.Conclusions:These data suggest that variations in dex accumulation are unlikely to play a role in dex resistance in ALL, at least in vitro. Advancement of the flow-based dex accumulation assay to include leukaemia-associated immunophenotype markers will allow measurement in dex-resistant MRD in vivo. Given that 35% of patients do not achieve plasma concentrations of 200nM dex (Jackson et al. AACR annual meeting abstract CT115, 2016), a combined approach incorporating pharmacokinetic assessments, drug accumulation and cellular response in ALL cells, may allow a comprehensive understanding of dex pharmacology in order to optimise its clinical utility. DisclosuresNo relevant conflicts of interest to declare.

The evolution, structure and function of the ray finned fish (Actinopterygii) glucocorticoid receptors

Basal ray-finned fish (Actinopterygii) possess a single glucocorticoid receptor (GR) and when compared to the lobe-finned vertebrate (Sarcopterygii) GR possess nine additional amino acids between the zinc-finger of the DNA binding domain. A whole genome duplication event which occurred between 320 and 350MYA in the teleost lineage following the split from the basal ray-finned fish resulted in 2 GRs: one GR group, GR1, has retained the 9 amino acids insert whereas the other group, GR2, has not. The exception to this is the zebrafish, that have lost one of the GRs, but they do possess 2 GRs with a splice variant that lacks the C-terminal portion of the GR to form GRβ which acts as a dominant-repressor of the wildtype GR. Another splice variant sees the basal ray-finned GR and teleost GR1 without the 9 amino acids insert. The molecular basis for GRs retention is beginning to be unravelled. In Pantadon buchholzi, rainbow trout, carp, marine and Japanese medaka GR2 is more sensitive to glucocorticoids (GC), thus potentially playing a more significant role in regulating gene expression at basal circulatory GC concentrations. However, this division in GC sensitivity is not seen in other species. The few studies to evaluate the significance of the 9 amino acid insert have shown that it affect maximal transactivational activity the extent to which is dependent on the number of glucocorticoid response elements (GREs) present in the reporter plasmid. The retention of these GRs would suggest there was an evolutionary advantage, which saw the development of a complex regulatory process to mediate the actions of the glucocorticoids.

Role of Pro-637 and Gln-642 in human glucocorticoid receptors and Ser-843 and Leu-848 in mineralocorticoid receptors in their differential responses to cortisol and aldosterone

Mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) are descended from a common ancestral corticoid receptor. The basis for specificities of human MR for aldosterone and human GR for glucocorticoids, such as cortisol, bearing 17α-hydroxyl-groups, is incompletely understood. Differences in MR at S843 and L848 and GR at the corresponding P637 and Q642 have been proposed as important in their different responses to glucocorticoids with 17α-hydroxyl-groups. We investigated the impact of these residues on binding affinity (Ki) and transcriptional activation (EC50) of mutants MR-S843P, MR-L848Q and MR-S843P/L848Q and mutants GR-P637S, GR-Q642L and GR-P637S/Q642L in the presence of different corticosteroids. Aldosterone, cortisol and corticosterone had similar affinities for wild-type MR and all mutants, while dexamethasone had increased affinity for the three mutants. However, transactivation of MR-S843P and MR-S843P/L848Q by all four steroids was significantly lower than for wild-type MR. In contrast, transactivation of MR-L848Q tended to be 3-fold higher for cortisol and corticosterone and increased 7-fold for dexamethasone, indicating that MR-L848Q has an increased response to glucocorticoids, while retaining a strong response to aldosterone. Compared to wild-type GR, GR-P637S and GR-Q642L had increased affinities and significantly increased transcriptional activity with aldosterone and corticosterone, and GR-P637S had similar transcriptional activity with cortisol and dexamethasone, while GR-Q642L and GR-P637S/Q642L had a significant decrease in transcriptional activity with cortisol and dexamethasone. 3D-models of these MR and GR mutants revealed that dexamethasone and aldosterone, respectively, fit nicely into the steroid-binding pocket, consistent with the affinity of dexamethasone for MR mutants and aldosterone for GR mutants.

Structural Analysis on the Pathologic Mutant Glucocorticoid Receptor Ligand-Binding Domains.

Glucocorticoid receptor (GR) gene mutations may cause familial or sporadic generalized glucocorticoid resistance syndrome. Most of the missense forms distribute in the ligand-binding domain and impair its ligand-binding activity and formation of the activation function (AF)-2 that binds LXXLL motif-containing coactivators. We performed molecular dynamics simulations to ligand-binding domain of pathologic GR mutants to reveal their structural defects. Several calculated parameters including interaction energy for dexamethasone or the LXXLL peptide indicate that destruction of ligand-binding pocket (LBP) is a primary character. Their LBP defects are driven primarily by loss/reduction of the electrostatic interaction formed by R611 and T739 of the receptor to dexamethasone and a subsequent conformational mismatch, which deacylcortivazol resolves with its large phenylpyrazole moiety and efficiently stimulates transcriptional activity of the mutant receptors with LBP defect. Reduced affinity of the LXXLL peptide to AF-2 is caused mainly by disruption of the electrostatic bonds to the noncore leucine residues of this peptide that determine the peptide's specificity to GR, as well as by reduced noncovalent interaction against core leucines and subsequent exposure of the AF-2 surface to solvent. The results reveal molecular defects of pathologic mutant receptors and provide important insights to the actions of wild-type GR.

Electrophilic lipid mediator 15-deoxy-Δ12,14-prostaglandin j2 modifies glucocorticoid signaling via receptor SUMOylation.

Cortisol, the central stress hormone in humans, activates the glucocorticoid receptor (GR). Anti-inflammatory effects are the most important pharmaceutical effects mediated by the GR. Inasmuch as electrophilic cyclopentenone prostaglandin 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) has potent anti-inflammatory properties and activates the SUMOylation pathway, we have investigated the effect of 15d-PGJ2 on glucocorticoid signaling and receptor SUMOylation. To this end, we studied isogenic HEK293 cells expressing either wild-type GR or SUMOylation-defective GR. Interestingly, 15d-PGJ2 triggered SUMO-2 and -3 (SUMO-2/3) modification in the primary SUMOylation sites of the GR. Gene expression profiling and pathway analyses indicate that 15d-PGJ2 inhibits GR signaling in a genome-wide fashion that is significantly dependent on the GR SUMOylation sites. Chromatin immunoprecipitation assays showed that the repressive effect of 15d-PGJ2 on GR target gene expression occurs in parallel with the inhibition of receptor binding to the target gene chromatin. Furthermore, depletion of UBC9, the sole SUMO E2 conjugase, from HEK293 cells confirmed the involvement of active SUMOylation in the regulatory process. Taken together, our data indicate that GR SUMOylation modulates the glucocorticoid signaling during acute cell stress. Our data also suggest that GR SUMOylation modulates cross talk of the glucocorticoid signaling with other transcription factors that are responsive to cell stress.

Glucocorticoid receptor in prostate epithelia is not required for corticosteroid-induced epithelial hyperproliferation in the mouse prostate.

Glucocorticoids are used as a last resort treatment for prostate cancer but the cell-specific glucocorticoid receptor (GR) mediated actions and the role of endogenous glucocorticoids in prostate are not understood. We evaluated the influence of prostate epithelial GR mediated actions of glucocorticoids in prostate structural development by comparing the intact wild-type (WT) and prostate epithelia selective GR knockout (peGRKO) males at 8, 20, and 35 weeks of age. We also determined the cell-specific role of GR on corticosterone treatment induced prostate abnormalities by treating peGRKO and WT male mice with corticosterone depot pellets or placebo for 4 weeks. GR was not expressed in the epithelial cells of peGRKO prostate unlike WT but was expressed in stromal of both peGRKO and WT mice. Nevertheless, prostate weights, histological appearance, and secretory protein probasin expression in peGRKO were no different from WT. Despite lacking epithelial GR, the peGRKO prostate demonstrated corticosterone treatment induced hyperplasia similar to WT suggesting that stromal rather than epithelial GR mediates the hyperproliferative mouse prostate response to corticosterone. As circulating androgen levels were not affected by corticosterone treatment, this effect is likely to be mediated directly via prostate GR. Sustained administration of corticosterone induces prostate hyperplasia, which is mediated via GR expressed predominantly in the stroma. Thus GR mediated actions in the prostate may have significant cell-specific effects that could be utilized for more rational therapeutic approaches in prostate cancer treatment. This also illustrates the paracrine hormonal mechanisms in prostate pathophysiology.

Wanted: A New Model for Glucocorticoid Receptor Transactivation and Transrepression

Wanted: A New Model for Glucocorticoid Receptor Transactivation and Transrepression

SUMOylation regulates the chromatin occupancy and anti-proliferative gene programs of glucocorticoid receptor

In addition to the glucocorticoids, the glucocorticoid receptor (GR) is regulated by post-translational modifications, including SUMOylation. We have analyzed how SUMOylation influences the activity of endogenous GR target genes and the receptor chromatin binding by using isogenic HEK293 cells expressing wild-type GR (wtGR) or SUMOylation-defective GR (GR3KR). Gene expression profiling revealed that both dexamethasone up- and downregulated genes are affected by the GR SUMOylation and that the affected genes are significantly associated with pathways of cellular proliferation and survival. The GR3KR-expressing cells proliferated more rapidly, and their anti-proliferative response to dexamethasone was less pronounced than in the wtGR-expressing cells. ChIP-seq analyses indicated that the SUMOylation modulates the chromatin occupancy of GR on several loci associated with cellular growth in a fashion that parallels with their differential dexamethasone-regulated expression between the two cell lines. Moreover, chromatin SUMO-2/3 marks, which were associated with active GR-binding sites, showed markedly higher overlap with the wtGR cistrome than with the GR3KR cistrome. In sum, our results indicate that the SUMOylation does not simply repress the GR activity, but regulates the activity of the receptor in a target locus selective fashion, playing an important role in controlling the GR activity on genes influencing cell growth.

Impact of glucocorticoid receptor density on ligand-independent dimerization, cooperative ligand-binding and basal priming of transactivation: a cell culture model.

Glucocorticoid receptor (GR) levels vary between tissues and individuals and are altered by physiological and pharmacological effectors. However, the effects and implications of differences in GR concentration have not been fully elucidated. Using three statistically different GR concentrations in transiently transfected COS-1 cells, we demonstrate, using co-immunoprecipitation (CoIP) and fluorescent resonance energy transfer (FRET), that high levels of wild type GR (wtGR), but not of dimerization deficient GR (GRdim), display ligand-independent dimerization. Whole-cell saturation ligand-binding experiments furthermore establish that positive cooperative ligand-binding, with a concomitant increased ligand-binding affinity, is facilitated by ligand-independent dimerization at high concentrations of wtGR, but not GRdim. The down-stream consequences of ligand-independent dimerization at high concentrations of wtGR, but not GRdim, are shown to include basal priming of the system as witnessed by ligand-independent transactivation of both a GRE-containing promoter-reporter and the endogenous glucocorticoid (GC)-responsive gene, GILZ, as well as ligand-independent loading of GR onto the GILZ promoter. Pursuant to the basal priming of the system, addition of ligand results in a significantly greater modulation of transactivation potency than would be expected solely from the increase in ligand-binding affinity. Thus ligand-independent dimerization of the GR at high concentrations primes the system, through ligand-independent DNA loading and transactivation, which together with positive cooperative ligand-binding increases the potency of GR agonists and shifts the bio-character of partial GR agonists. Clearly GR-levels are a major factor in determining the sensitivity to GCs and a critical factor regulating transcriptional programs.