Glucocorticoid Receptor Homodimer Research Articles

Molecular View on the Dissociation Pathways and Transactivation Regulation Mechanism of Nonsteroidal GR Ligands.

As a major drug target for anti-inflammatory therapy, the glucocorticoid receptor (GR) regulates a wide range of physiological processes through transactivation (TA) or transrepression. GR TA is involved in many adverse effects of GR-targeting drugs, and therefore, the discovery of novel GR ligands with lower TA activity and longer residence time is quite urgent. Undoubtedly, understanding the ligand dissociation mechanisms and the structural basis of the TA regulation is crucial for the development of novel GR-targeting drugs. Here, we used random accelerated molecular dynamics (RAMD) and funnel metadynamics (FM) simulations to explore the dissociation mechanisms of 5 classic glucocorticoids and 6 nonsteroidal GR ligands. Multiple ligand dissociation pathways were discovered. The classic glucocorticoids exhibit a strong preference for Path I, and most nonsteroidal ligands tend to dissociate along mixed pathways. We also find that the distinct unbinding preferences for AZD2906 and AZD9567, two representative nonsteroidal ligands with similar scaffolds but different TA activities, are primarily determined by their different polar interactions with the surrounding residues. Notably, the binding of AZD9567 poses a substantial impact on the conformation of the GR homodimer interface, which provides a valuable clue to understand the mechanisms of the TA-related side effects induced by the adjustments of the homodimerization process. These findings are critical for the structure-based rational design of novel GR ligands with more potent anti-inflammatory potency and reduced side effects.

Relationship Between Homodimeric Glucocorticoid Receptor and Transcriptional Regulation Assessed via an In Vitro Fluorescence Correlation Spectroscopy-Microwell System

Glucocorticoid receptor (GR) is a hormone-activated transcription regulatory protein involved in metabolism as well as adrenocortical responses to psychosocial stress. Ligand-activated GR localizes to the nucleus, where GR homodimers regulate gene transcription via direct binding to glucocorticoid response elements (GREs). The role of GR homodimers in transcriptional activation has not yet been elucidated. In this study, we determined the concentration of GR homodimer, and its dissociation constant (Kd), at the single-cell level, by using fluorescence correlation spectroscopy (FCS) combined with a microwell system. Results from dissociation constant analysis and diffusion analysis suggested that GR forms complexes with other proteins as well as homodimers. We determined the relationship between the concentration of GR homodimer and transcriptional activity using a triple-color FCS-microwell system-based fluorescent reporter assay. The binding affinity of GR to GREs was analyzed via fluorescence cross-correlation spectroscopy (FCCS). Our findings indicate that the GR homodimer is essential for activating target gene transcription.

T25: Selective glucocorticoid receptor agonists as novel anti-cancer agents

Glucocorticoids (GCs) are widely used in treatment of many cancer types due to its ability to induce apoptosis in malignant cells in blood cancer therapy, and to prevent nausea, emesis and chemotherapy-associated hepatotoxicity in case of solid tumors. However, severe dose-limiting side effects occur, including osteoporosis, diabetes and other metabolic complications. Moreover, in therapy of solid tumors GCs strongly affect microenvironment which could be associated with poor prognosis, risk of metastasis and high frequency of relapses. Biological response to GCs is mediated by glucocorticoid receptor (GR), a well-characterized transcription factor. GR controls gene expression via (1) transactivation, which requires binding of GR homodimers to glucocorticoid-responsive elements (GRE) in gene promoters and enhancers, and (2) dimerization-independent transrepression mediated via negative interaction between GR and other transcription factors including major effectors of inflammation and proliferation. Transrepression plays an important role in anti-inflammatory and anti-cancer effects of GR, including normalizing influence on microenvironment, while side effects are associated with GR transactivation. In particular, GCs induce insulin resistance in adipocytes, a major component of the mammary microenvironment, which secrete pro-inflammatory cytokines and growth factors, implicated in tumor progression. Selective GR agonists (SEGRA) that preferentially activate GR transrepression could be a better option for treatment of cancer. Dozens of candidate SEGRAs were identified, synthesized and tested by industry and academia, with some having reached clinical trials. One of the novel GR modulators is 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride, or CpdA, synthetic analogue of aziridine precursor isolated from Namibian shrub Salsola tuberculatiformis Botschantzev. It was shown that CpdA acts as “dissociated” GR ligand: it competes with GCs for GR binding and efficiently induces GR transrepression but not transactivation. We and other authors reported recently that CpdA inhibits survival of prostate cancer cells as well as blood cancer cells in GR-dependent fashion. Furthermore, primary leukemia cells from T-ALL patients appeared to be equally sensitive to GCs and CpdA. Our further studies were concentrated on three directions: (1) GC/SEGRA-based chemotherapy. We screened biological effect of CpdA in combination with traditional agents (doxorubicin, vincristine) and newer therapeutics (Bortezomib, Carfilzomib, MLN-4924, Rapamycin). Pretreatment of lymphoma cells with proteasome inhibitor Bortezomib resulted in GR accumulation and enhanced ligand properties of CpdA. We also revealed remarkable GR-dependent cooperation between CpdA and Bortezomib in suppressing survival of lymphoma cells in vitro and in vivo. Also surprising findings were substantial cooperation in anti-cancer effect of immunosuppressant Rapamycin and CpdA in vitro, and unexpected “dissociated” effect of Rapamycin on GR signaling realized through down -regulation of REDD1, mTORC1 inhibitor. These data suggested high clinical potential of Rapamycin/GC combination in cancer treatment. (2) SEGRA list extention We used two approaches to extend SEGRA list: (1) synthesis of CpdA enantiomers and (2) its chemical derivatives. Chemical analogues of CpdA were designed by appending of bulky substituent into benzene ring, alkylation of carbon atom adjacent to chlorine atom or appending of substituents to nitrogen atom. Evaluation of biological properties of enantiomers revealed higher GR-dependent anti-cancer potential of S-CpdA. Cytotoxic and proapoptotic effects of CpdA analogues were comparable with precursor. (3) Selection of tumor types acceptable for SEGRA treatment. CpdA was selected for NCI-60 in Vitro Cell Line Screening Project providing direct support to anticancer drug discovery program. It was shown that CpdA affect viability of some adherent cancer cell lines. We demonstrated that CpdA unlike GCs did not modify microenvironment and disintegrate tight junctions between cells decreasing risk of metastasis in case of solid tumors. It demonstrates reasonability of further investigations. Overall, our data provide the rationale for novel therapy of cancer based on combination of non-steroidal GR modulators with classic and modern chemotherapeutics. Approaches to obtain more SEGRAs were elaborated.

Competitive and compensatory effects of androgen signaling and glucocorticoid signaling

Androgens and glucocorticoids have competitive and compensatory effects in several physiological and pathophysiological processes. Although blood androgen levels affect blood glucocorticoid levels and vice versa, it does not fully explain the relationship between the effects of androgens and glucocorticoids. Androgens and glucocorticoids exert their functions through binding to androgen receptor (AR) and glucocorticoid receptor (GR), respectively. AR homodimer and GR homodimer bind to the androgen response element (ARE) and glucocorticoid response element (GRE), respectively, where they positively or negatively regulate transcription. AR/GR heterodimer can also form but whether it has a physiological role is unclear. Notably, some ARE/GRE sites are recognized by both AR and GR. This review focuses on the functional interventions between androgen signaling and glucocorticoid signaling in target cells that are involved in muscle atrophy, lipid metabolism in adipocytes and hepatocytes, and pancreatic β-cell death. Androgens and glucocorticoids exert opposite effects by differentially regulating key genes ( e.g. , insulin-like growth factor-1, atrogin-1, and thioredoxin-interacting protein) involved in these physiological processes. We also review functional compensation between these steroids in the development of castration-resistant prostate cancer in which glucocorticoids compensate for the castration-induced loss of AR function by activating key genes ( e.g. , serum/glucocorticoid-regulated kinase 1). The gene expressions regulated by androgens and glucocorticoids are regulated through at least three different mechanisms in target cells: (i) regulation of applicable ligand levels by modulation of steroid metabolite enzyme levels, (ii) regulation of each other's receptor levels, and (iii) competitive binding between AR and GR on ARE/GRE sites. Recent findings shed light on the complicated relationship between androgen signaling and glucocorticoid signaling in various cellular processes.

Genomic redistribution of GR monomers and dimers mediates transcriptional response to exogenous glucocorticoid in vivo.

Glucocorticoids (GCs) are commonly prescribed drugs, but their anti-inflammatory benefits are mitigated by metabolic side effects. Their transcriptional effects, including tissue-specific gene activation and repression, are mediated by the glucocorticoid receptor (GR), which is known to bind as a homodimer to a palindromic DNA sequence. Using ChIP-exo in mouse liver under endogenous corticosterone exposure, we report here that monomeric GR interaction with a half-site motif is more prevalent than homodimer binding. Monomers colocalize with lineage-determining transcription factors in both liver and primary macrophages, and the GR half-site motif drives transcription, suggesting that monomeric binding is fundamental to GR's tissue-specific functions. In response to exogenous GC in vivo, GR dimers assemble on chromatin near ligand-activated genes, concomitant with monomer evacuation of sites near repressed genes. Thus, pharmacological GCs mediate gene expression by favoring GR homodimer occupancy at classic palindromic sites at the expense of monomeric binding. The findings have important implications for improving therapies that target GR.

3P171 蛍光相関分光法を用いた単一細胞由来のグルココルチコイドレセプター二量体形成と転写活性の定量(細胞生物的課題,ポスター,第52回日本生物物理学会年会(2014年度))

3P171 蛍光相関分光法を用いた単一細胞由来のグルココルチコイドレセプター二量体形成と転写活性の定量(細胞生物的課題,ポスター,第52回日本生物物理学会年会(2014年度))

Selective modulation through the glucocorticoid receptor ameliorates muscle pathology inmdxmice

The over-expression of NF-κB signalling in both muscle and immune cells contribute to the pathology in dystrophic muscle. The anti-inflammatory properties of glucocorticoids, mediated predominantly through monomeric glucocorticoid receptor inhibition of transcription factors such as NF-κB (transrepression), are postulated to be an important mechanism for their beneficial effects in Duchenne muscular dystrophy. Chronic glucocorticoid therapy is associated with adverse effects on metabolism, growth, bone mineral density and the maintenance of muscle mass. These detrimental effects result from direct glucocorticoid receptor homodimer interactions with glucocorticoid response elements of the relevant genes. Compound A, a non-steroidal selective glucocorticoid receptor modulator, is capable of transrepression without transactivation. We confirm the in vitro NF-κB inhibitory activity of compound A in H-2K(b) -tsA58 mdx myoblasts and myotubes, and demonstrate improvements in disease phenotype of dystrophin deficient mdx mice. Compound A treatment in mdx mice from 18 days of post-natal age to 8 weeks of age increased the absolute and normalized forelimb and hindlimb grip strength, attenuated cathepsin-B enzyme activity (a surrogate marker for inflammation) in forelimb and hindlimb muscles, decreased serum creatine kinase levels and reduced IL-6, CCL2, IFNγ, TNF and IL-12p70 cytokine levels in gastrocnemius (GA) muscles. Compared with compound A, treatment with prednisolone, a classical glucocorticoid, in both wild-type and mdx mice was associated with reduced body weight, reduced GA, tibialis anterior and extensor digitorum longus muscle mass and shorter tibial lengths. Prednisolone increased osteopontin (Spp1) gene expression and osteopontin protein levels in the GA muscles of mdx mice and had less favourable effects on the expression of Foxo1, Foxo3, Fbxo32, Trim63, Mstn and Igf1 in GA muscles, as well as hepatic Igf1 in wild-type mice. In conclusion, selective glucocorticoid receptor modulation by compound A represents a potential therapeutic strategy to improve dystrophic pathology.

Combination of a selective activator of the glucocorticoid receptor Compound A with a proteasome inhibitor as a novel strategy for chemotherapy of hematologic malignancies

Glucocorticoids are widely used for the treatment of hematological malignancies; however, their chronic use results in numerous metabolic side effects. Thus, the development of selective glucocorticoid receptor (GR) activators (SEGRA) with improved therapeutic index is important. GR regulates gene expression via (1) transactivation that requires GR homodimer binding to gene promoters and is linked to side effects and (2) transrepression-mediated via negative GR interaction with other transcription factors. Novel GR modulator Compound A (CpdA) prevents GR dimerization, retains glucocorticoid anti-inflammatory activity and has fewer side effects compared with glucocorticoids in vivo. Here we tested CpdA anticancer activity in human T- and B-lymphoma and multiple myeloma cells expressing GR and their counterparts with silenced GR. We found that CpdA in GR-dependent manner strongly inhibited growth and viability of human T-, B-lymphoma and multiple myeloma cells. Furthermore, primary leukemia cell cultures from T-ALL patients appeared to be equally sensitive to glucocorticoid dexamethasone and CpdA. It is known that GR expression is controlled by proteasome. We showed that pretreatment of lymphoma CEM and NCEB cells with proteasome-inhibitor Bortezomib resulted in GR accumulation and enhanced ligand properties of CpdA, shifting GR activity toward transrepression evaluated by inhibition of NFкB and AP-1 transcription factors. We also revealed remarkable GR-dependent cooperation between CpdA and Bortezomib in suppressing growth and survival of T- and B-lymphoma and multiple myeloma MM.1S cells. Overall, our data provide the rationale for novel GR-based therapy for hematological malignancies based on combination of SEGRA with proteasome inhibitors.

Abstract 3814: Combination of selective modular of the glucocorticoid receptor (SEGRA) with proteasome inhibitors as a novel strategy for chemotherapy of hematologic malignancies

Abstract Glucocorticoids are widely used for the treatment of hematological malignancies; however chronic treatment with steroids results in numerous adverse effects. Glucocorticoid receptor (GR) regulates gene expression via transactivation that requires GR homodimer binding to gene promoters; and transrepression mediated via negative interaction between GR and other transcription factors. GR transactivation is linked to metabolic side effects, while GR transrepression underlies glucocorticoid therapeutic action. One of the novel GR modulators is 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride also called CpdA. CpdA is a synthetic analogue of a aziridine precursor isolated from the Namibian shrub Salsola tuberculatiformis Botschantzev. Using representative human T cell (CEM) and B cell (NCEB) lymphoma cell lines expressing functional GR, and their counterparts with blocked GR expression, we showed that CpdA indeed acted as dissociated GR ligand, and inhibited growth and survival of these cells via GR. Sensitivity to therapeutic effects of GCs directly depends on the amount of functional GR. The 26S protesome controls GR protein stability, and is responsible for desensitization to GCs via hormone-induced GR degradation and following development of glucocorticoid resistance in patients. Thus, the use of proteasome inhibitors represents a pharmacological approach to prevent GR down-regulation and to elevate the level of GR in cells. Proteasome inhibitor Bortezomib (BZ) was approved by FDA for the treatment of patients with multiple myeloma and mantle cell lymphoma. Since proteasome inhibitors stabilize GR, we hypothesized that BZ will augment CpdA effects as a selective GR modulator and enhance its chemotherapeutic activity. We showed that pretreatment of CEM and NCEB cells with proteasome inhibitor Bortezomib resulted in GR accumulation, and enhanced CpdA ligand properties further shifting GR activity towards transrepression evaluated by inhibiton of NF-kB and AP-1 factors. We also revealed remarkable GR-dependent synergy between CpdA and BZ in suppressing growth and survival of T and B lymphoma cells. Overall, our data provide the rationale for novel GR-based therapy for lymphoma and leukemia based on the combination of non-steroidal GR modulators with proteasome inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3814. doi:1538-7445.AM2012-3814

Corticosteroid effects on calcium signaling in limbic neurons

Corticosteroid hormones, which are released in high amounts after stress, easily pass the blood–brain-barrier. In the brain they bind to intracellular receptors which act as transcriptional regulators. These receptors are highly expressed in neurons of the hippocampal formation and the amygdala, areas that play a role in (emotional) memory formation. Voltage gated Ca2+ channels are among the most prominent targets of corticosteroid hormones. When the levels of corticosterone - the prevalent corticosteroid in rats and mice- are low, L-type Ca2+ currents of CA1 hippocampal cells are small. However, when hormone levels rise e.g. after stress, the amplitude of L-type Ca2+ currents will be slowly enhanced, through a process requiring DNA binding of glucocorticoid receptor homodimers. Kinetic properties and voltage dependency of the currents remain unchanged. Neurons in the basolateral amygdala respond in a comparable fashion, but Ca2+ currents of neurons in the dentate gyrus are unaffected by corticosteroids. The stress-induced increase in Ca2+ influx has considerable functional consequences in health and disease. At the short term, i.e. 1–4h after stress, the enhanced Ca2+ influx contributes to stronger firing frequency accommodation and a higher threshold for the induction of long-term potentiation. This helps to normalize neuronal activity after stress and presumably protects earlier encoded, stress-related information. At the longer term, though, increased Ca2+ load may impose a risk, increasing the vulnerability of limbic cells to additional challenges e.g. during epileptic or ischemic episodes.

Muscle sparing in muscle RING finger 1 null mice: response to synthetic glucocorticoids

Skeletal muscle atrophy occurs under a variety of conditions and can result from alterations in both protein synthesis and protein degradation. The muscle-specific E3 ubiquitin ligases, MuRF1 and MAFbx, are excellent markers of muscle atrophy and increase under divergent atrophy-inducing conditions such as denervation and glucocorticoid treatment. While deletion of MuRF1 or MAFbx has been reported to spare muscle mass following 14 days of denervation, their role in other atrophy-inducing conditions is unclear. The goal of this study was to determine whether deletion of MuRF1 or MAFbx attenuates muscle atrophy after 2 weeks of treatment with the synthetic glucocorticoid dexamethasone (DEX). The response of the triceps surae (TS) and tibialis anterior (TA) muscles to 14 days of DEX treatment (3 mg kg(-1) day(-1)) was examined in 4 month-old male and female wild type (WT) and MuRF1 or MAFbx knock out (KO) mice. Following 14 days of DEX treatment, muscle wet weight was significantly decreased in the TS and TA of WT mice. Comparison of WT and KO mice following DEX treatment revealed significant sparing of mass in both sexes of the MuRF1 KO mice, but no muscle sparing in MAFbx KO mice. Further analysis of the MuRF1 KO mice showed significant sparing of fibre cross-sectional area and tension output in the gastrocnemius (GA) after DEX treatment. Muscle sparing in the MuRF1 KO mice was related to maintenance of protein synthesis, with no observed increases in protein degradation in either WT or MuRF1 KO mice. These results demonstrate that MuRF1 and MAFbx do not function similarly under all atrophy models, and that the primary role of MuRF1 may extend beyond controlling protein degradation via the ubiquitin proteasome system.

Abstract 669: Anticancer effects of novel non steroidal glucocorticoid receptor ligand, Compound A, in T-cell leukemia lines

Abstract Glucocorticoids (GCs) are widely used in chemotherapy of hematologic malignancies, including T cell leukemia. The biological effects of GCs are mediated by the glucocorticoid receptor (GR), a well known transcription factor. Therapeutic effects of GCs are mainly mediated via GR transrepression that involves negative interaction between GR and other transcription factors. In contrast, side effects of glucocorticoids are mediated by GR transactivation that requires binding of GR homodimer to hormone-responsive elements in gene promoters. Selective GR activators (SEGRA) that shift GR functions towards transrepression have improved therapeutic index and hold a great potential for the GR-targeted chemotherapies. Recently we and others characterized a novel non-steroidal GR ligand, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride also called Compound A (CpdA). CpdA prevents GR dimerization and transactivation, but strongly enhances GR transrepression. We also revealed that CpdA possesses anti-cancer activity: it inhibited growth and viability of prostate cancer cells in GR-dependent fashion. Here we analyzed CpdA effects in K562 and CEM T-cell leukemia lines. We found that both cell lines express functional GR. CpdA at 10-6-10-5M concentration range exerted cytostatic and apoptotic effects in both cell lines. CEM cells were more sensitive to CpdA than K562 that correlated with the higher amount of GR protein in these cells. We found that in contrast to GCs, CpdA did not induce GR transactivation evaluated by RT-PCR of known GR-target genes such as FKBP51. At the same time, Luciferase assay showed that CpdA efficiently activated transrepression of NF-kB and AP-1 factors. Currently, GCs are used for the treatment of blood cancers in combined chemotherapy with different anti-cancer drugs including proteasome inhibitor Bortezomib (BZ). It is known that proteasome inhibitors increase GR stability. Thus, we expected that BZ will maximize ligand properties of CpdA as GR modulator and will increase its anti-cancer effects. We found that BZ induced caspase-dependent apoptosis in both T-cell leukemia cell lines. The effect of BZ on GR expression and function and on apoptosis induced by CpdA is under study. In addition, we have performed Ames test and Topoisomerase I assay as first steps towards the evaluation of toxicological profile of CpdA. We have not revealed mutagenic effect of CpdA either in frameshift or in base-substitution indicator strains at the wide range of concentrations (10-9-10-4M). CpdA also did not inhibit eukaryotic topoisomerase I activity. Overall, our data suggest that novel GR modulator CpdA has a high chemotherapeutic potential for the treatment of T-cell leukemia. The detailed mechanisms of its anti-cancer effects in transformed T-cells are under investigation. Work is supported by grants: RFBR 10-04-00979, RO1CA118890, ACS IL 160185, UICC ICRETT-09-137. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 669. doi:10.1158/1538-7445.AM2011-669

Exploring the Molecular Basis of Action of the Passive Antiglucocorticoid 21-Hydroxy-6,19-epoxyprogesterone

21-Hydroxy-6,19-epoxyprogesterone (21OH-6,19OP) is a selective antiglucocorticoid that lacks the bulky substituent at C-11 found in active antagonists of the glucocorticoid receptor (GR). Ligand-free GR ligand-binding domain (LBD) and GR LBD complexed with 21OH-6,19OP or the agonist dexamethasone were simulated during 6 ns using molecular dynamics. Results suggest that the time fluctuation and average position adopted by the H1-H3 loop affect the ability of GR LBD-21OH-6,19OP complex to homodimerize, a necessary step in transcriptome assembly. A nuclear localization and a transactivation experiment showed that, although 21OH-6,19OP activates the translocation of the GR, the nuclear complex is unable to induce the transcription of a reporter driven by a promoter, that requires binding to a GR homodimer to be activated. These findings support the hypothesis that the passive antagonist mode of action of 21OH-6,19OP resides, at least in part, in the incapacity of the GR-21OH-6,19OP complex to dimerize.

Glucocorticoids Specifically Enhance L-Type Calcium Current Amplitude and Affect Calcium Channel Subunit Expression in the Mouse Hippocampus

Previous studies have shown that corticosterone enhances whole cell calcium currents in CA1 pyramidal neurons, through a pathway involving binding of glucocorticoid receptor homodimers to the DNA. We examined whether glucocorticoids show selectivity for L- over N-type of calcium currents. Moreover, we addressed the putative gene targets that eventually lead to the enhanced calcium currents. Electrophysiological recordings were performed in nucleated patches that allow excellent voltage control. Calcium currents in these patches almost exclusively involve N- and L-type channels. We found that L- but not N-type calcium currents were largely enhanced after treatment with a high dose of corticosterone sufficient to activate glucocorticoid receptors. Voltage dependency and kinetic properties of the currents were unaffected by the hormone. Nonstationary noise analysis suggests that the increased current is not caused by a larger unitary conductance, but rather to a doubling of the number of functional channels. Quantitative real-time PCR revealed that transcripts of the Ca(v)1 subunits encoding for the N- or L-type calcium channels are not upregulated in the mouse CA1 area; instead, a strong, direct, and consistent upregulation of the beta4 subunit was observed. This indicates that the corticosteroid-induced increase in number of L-type calcium channels is not caused by a simple transcriptional regulation of the pore-forming subunit of the channels.

Flanking Sequence Composition Differentially Affects the Binding and Functional Characteristics of Glucocorticoid Receptor Homo- and Heterodimers

The core binding sites for a multitude of transcription factors have been identified and characterized, but these sequences cannot fully account for the nuances of cell-specific and gene-specific control of gene transcription. Many factors may contribute to the precise responsiveness of a gene to a particular transcriptional regulatory protein, including the nucleotides in the proximity of the core binding site for that protein. Here, we examine two flanking sequences bordering a site in the gamma-fibrinogen gene regulatory region that binds a heterodimer of the Xenopus glucocorticoid receptor accessory factor (XGRAF) and the glucocorticoid receptor (GR). Mutation of the upstream flank results in a decrease in the level of XGRAF binding but little change in hormone induction. However, alteration of the downstream flank adjacent to the GR binding site causes a decrease in levels of both GR monomer binding and hormone induction. Conversion of the XGRAF-GR binding site to a full glucocorticoid response element (GRE) alters the role of the flanking sequences. A full GRE in this position requires the wild-type upstream flank to bind GR homodimer and induce transcription to maximal levels. In contrast, mutation of the downstream flank is not detrimental to either the binding or the function of the GR dimer. Thus, flanking sequence composition and dimer partner combine to influence GR function, underscoring the complexities involved in the identification of authentic transcription factor response elements.

Molecular mechanisms of corticosteroids in allergic diseases.

Molecular mechanisms of corticosteroids in allergic diseases.

High-yield purification of functionally active glucocorticoid receptor.

AbstractThe glucocorticoid receptor (GR) resides in the cytoplasm as a complex with chaperones. Upon ligand association, GR shuttles into the nucleus, and binds to its hormone response elements to control expression of particular genes (1-7). Transcriptional regulation by GR is thought to be a consequence of macromolecular assembly formation with proteins, including chromatin remodelers, histone modifiers, coactivators, and transcriptional machinery on a promoter (1-8-17). Nucleation of this large multisubunit complex by GR homodimers results in a chromatin transition (18-23). and preinitiation complex assembly. To date, the molecular details of the GR-induced macromolecular assembly and chromatin transition are poorly understood.KeywordsGlucocorticoid ReceptorLong Terminal RepeatColumn VolumeMouse Mammary Tumor VirusFast Protein Liquid ChromatographyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Free leucine dissociates homo- and heterodimers formed between proteins containing leucine heptad repeats

A highly specific method for the dissociation of protein dimers has been developed. The method involves exposure of the dimers to free leucine at a concentration ranging between 3 and 10 mM. Using this method it has been possible to dissociate goat uterine oestrogen receptor homodimers, heterodimers formed between the non-activated oestrogen receptor (naER) and the oestrogen receptor activation factor (E-RAF) of the goat uterus, c-jun homodimers derived from bovine bone marrow and also glucocorticoid receptor homodimers isolated from rat liver cytosol. The pattern of dimer dissociation by leucine clearly differentiates two classes of proteins. The first is represented by steroid hormone receptors where dimerization is apparently contributed by both coiled-coil dimerization interfaces and the conserved heptad repeats of leucine. The second is represented by oncoproteins like c-fos and c-jun which dimerize through the exclusive involvement of leucine zippers. The patterns of dissociation of these two groups of proteins from the concerned affinity columns are distinctly different. This indicates a possibility that the elution pattern may be used as a yardstick to determine whether two proteins dimerize through the exclusive involvement of leucine zippers or whether coiled-coil interfaces are also involved in the dimerization process.

Corticosteroid actions in hippocampus require DNA binding of glucocorticoid receptor homodimers.

Glucocorticoids are secreted from the adrenal gland in very high amounts after stress. In the brain, these stress hormones potently modulate ionic currents, monoaminergic transmission, synaptic plasticity and cellular viability, most notably in the hippocampus where corticosteroid receptors are highly enriched. Here we show that at least some of these actions require DNA binding of glucocorticoid receptor (GR) homodimers.

The glucocorticoid receptor is a key regulator of the decision between self-renewal and differentiation in erythroid progenitors.

During development and in regenerating tissues such as the bone marrow, progenitor cells constantly need to make decisions between proliferation and differentiation. We have used a model system, normal erythroid progenitors of the chicken, to determine the molecular players involved in making this decision. The molecules identified comprised receptor tyrosine kinases (c-Kit and c-ErbB) and members of the nuclear hormone receptor superfamily (thyroid hormone receptor and estrogen receptor). Here we identify the glucocorticoid receptor (GR) as a key regulator of erythroid progenitor self-renewal (i.e. continuous proliferation in the absence of differentiation). In media lacking a GR ligand or containing a GR antagonist, erythroid progenitors failed to self-renew, even if c-Kit, c-ErbB and the estrogen receptor were activated simultaneously. To induce self-renewal, the GR required the continuous presence of an activated receptor tyrosine kinase and had to cooperate with the estrogen receptor for full activity. Mutant analysis showed that DNA binding and a functional AF-2 transactivation domain are required for proliferation stimulation and differentiation arrest. c-myb was identified as a potential target gene of the GR in erythroblasts. It could be demonstrated that delta c-Myb, an activated c-Myb protein, can functionally replace the GR.