Human Glucocorticoid Receptor Expression Research Articles

Epigenetic Clues to the Biological Embedding of Early Life Adversity

The concept of biological embedding has gained substantial traction as a framework for understanding the roots of complex multifactorial phenomena in health and disease. A body of research over several decades indicates that early life experiences have profound consequences for health in adulthood, including mental health, as a consequence of establishing long-term health gradients ( 1 Hertzman C. Boyce T. How experience gets under the skin to create gradients in developmental health. Annu Rev Public Health. 2010; 31: 329-347 Crossref PubMed Scopus (448) Google Scholar ). Early interventions have been proposed to have an enhanced impact on health trajectories in part because they act at a time of enhanced plasticity ( 2 Hanson M. Godfrey K.M. Lillycrop K.A. Burdge G.C. Gluckman P.D. Developmental plasticity and developmental origins of non-communicable disease: theoretical considerations and epigenetic mechanisms. Prog Biophys Mol Biol. 2010; 106: 272-280 Crossref Scopus (216) Google Scholar ). Early-life adversity in the form of physical and sexual abuse or severe neglect is well recognized to increase the risk of suicide ( 3 Mann J.J. Currier D.M. Stress, genetics and epigenetic effects on the neurobiology of suicidal behavior and depression. Eur Psychiatry. 2010; 25: 268-271 Crossref PubMed Scopus (128) Google Scholar ). It has been challenging, however, to elucidate biologic mechanisms that underlie long-term changes in brain and behavior that are associated with the increased risk. Differential Glucocorticoid Receptor Exon 1B, 1C, and 1H Expression and Methylation in Suicide Completers with a History of Childhood AbuseBiological PsychiatryVol. 72Issue 1PreviewChildhood abuse alters hypothalamic-pituitary-adrenal (HPA) function and increases the risk of suicide. Hippocampal glucocorticoid receptor (GR) activation regulates HPA activity, and human GR expression (hGR) is reduced in the hippocampus of suicide completers with a history of childhood abuse compared with controls. The abuse-related decrease in hGR expression associates with increased DNA methylation of the promoter of the hGR1F variant in the hippocampus. Full-Text PDF

Differential Glucocorticoid Receptor Exon 1B, 1C, and 1H Expression and Methylation in Suicide Completers with a History of Childhood Abuse

Childhood abuse alters hypothalamic-pituitary-adrenal (HPA) function and increases the risk of suicide. Hippocampal glucocorticoid receptor (GR) activation regulates HPA activity, and human GR expression (hGR) is reduced in the hippocampus of suicide completers with a history of childhood abuse compared with controls. The abuse-related decrease in hGR expression associates with increased DNA methylation of the promoter of the hGR(1F) variant in the hippocampus. In this study, we investigated the expression and methylation levels of other hGR splice variants in the hippocampus and anterior cingulate gyrus in suicide completers with and without a history of childhood abuse and in controls. Expression levels were quantified using quantitative reverse-transcriptase polymerase chain reaction and promoter methylation was assessed by pyrosequencing. In the hippocampus, the expression of total hGR and variants 1(B), 1(C), and 1(H) was decreased in suicide completers with histories of abuse compared with suicides with no histories of abuse and with control subjects. In the anterior cingulate gyrus, however, no group differences in hGR total or variant expression were found. Site-specific methylation in hGR1(B) and 1(C) promoter sequences were negatively correlated with total hGR messenger RNA, as well as with hGR1(B) and 1(C) expression. Luciferase assay showed that methylation in hGR promoter decreases transcriptional activity. In contrast, total and site-specific methylation in the hGR1(H) promoter was positively correlated with total hGR messenger RNA and hGR1(H) expression. These findings suggest that early-life events alter the expression of several hGR variants in the hippocampus of suicide completers through effects on promoter DNA methylation.

Chlamydophila pneumoniae inhibits corticosteroid-induced suppression of metalloproteinase-9 and tissue inhibitor metalloproteinase-1 secretion by human peripheral blood mononuclear cells

Chlamydophila pneumoniae infection has been suggested to be associated with severe asthma characterized by persistent airway limitation, which may be related to airway remodelling. We investigated whether C. pneumoniae infection affected the secretion of metalloproteinase-9 (MMP9) and tissue inhibitor metalloproteinase-1 (TIMP1), and altered the responsiveness of inflammatory cells to corticosteroids. Human peripheral blood mononuclear cells (PBMCs) were cultured in vitro in the presence or absence of C. pneumoniae. Secretion of both MMP9 and TIMP1 was strongly suppressed by dexamethasone treatment in uninfected cells. MMP9 secretion was also significantly inhibited by dexamethasone in C. pneumoniae-infected cells, but TIMP1 secretion was not; hence the MMP9 to TIMP1 ratio decreased. Interestingly, expression of human glucocorticoid receptor β, which is believed to confer resistance to corticosteroids, was enhanced by dexamethasone treatment in C. pneumoniae-infected PBMCs. We conclude that C. pneumoniae infection may promote airway remodelling by decreasing the ratio of MMP9 to TIMP1 secreted by inflammatory cells, and by altering cellular responsiveness to corticosteroids.

Expression of Human Glucocorticoid Receptor in T Lymphocytes in Acute-on-Chronic Hepatitis B Liver Failure

Glucocorticoid receptor (GR) participates in the pathogenesis of liver inflammation. However, the potential role of GR in acute-on-chronic hepatitis B liver failure (ACHBLF) is still obscure. This present study was aimed to determine peripheral GR expression in ACHBLF patients. Forty patients with ACHBLF, 20 patients with chronic hepatitis B (CHB) and 16 healthy controls were included in this retrospectively study. Flow cytometry was used to determine the peripheral expression of GR+T lymphocytes. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed for assessing relative mRNA levels of GR alpha and beta isoforms in peripheral blood mononuclear cells. Serum cortisol level was evaluated using radioimmunoassay. The serum cortisol level and the percentage of GR+T lymphocytes in ACHBLF patients were significantly decreased compared with CHB patients and healthy controls. However, there were no significant differences in mean fluorescence intensity (MFI) of GR+T lymphocytes within three groups. The relative GR alpha mRNA expression in ACHBLF patients was significant decreased compared with healthy controls. However, the relative GR beta mRNA expression in ACHBLF patients was significantly increased compared with CHB patients and healthy controls. In ACHBLF patients, the percentage of GR+T lymphocyte was significantly positively associated with relative GR alpha expression, prothrombin activity, and HBV DNA level, but negatively correlated with serum cortisol level and MELD score. Furthermore, the percentage of GR+T lymphocytes was also obviously elevated in survivors than non-survivors. It was strongly suggested that GR play an important role in the pathogenesis of ACHBLF.

Identification and characterization of glucocorticoid receptor-binding sites in the human genome

Glucocorticoids regulate gene expression via binding of the ligand-activated glucocorticoid receptor (GR) to glucocorticoid-responsive elements (GRE). To identify GR-binding sites, we developed a modified yeast one-hybrid system which enables rapid and efficient identification of genomic targets for DNA-binding proteins. The human GR expression vector was transformed into yeast cells containing a library of human genomic fragments cloned upstream of the reporter gene URA3. The genomic fragments with GR-binding sites were identified by growth of yeast clones in media lacking uracil but containing dexamethasone. DNA fragments were recovered by colony-direct PCR and GRE sequences were predicted by in silico analysis. Using electrophoretic mobility shift assay and fluorescence correlation spectroscopy, we demonstrated that 314 predicted GREs could directly interact with recombinant human GR proteins. In addition, when the genomic fragments were inserted in front of the heterologous SV40 promoter, at least 150 fragments could function as GREs in HEK293 cells. Furthermore, we identified four functional regulatory polymorphisms which may influence individual variation in sensitivity to glucocorticoids. These results provide insights into the molecular mechanisms underlying the physiological and pathological actions of glucocorticoid.

Expression of human glucocorticoid receptor in lymphocytes of patients with autoimmune hepatitis.

Background and aim: Alternative splicing of human glucocorticoid receptor (hGR) premessenger RNA (mRNA) generates two highly homogenous isoforms, termed hGRalpha and hGRbeta. hGRalpha is a ligand-activated transcription factor, which, in the hormone-bound state, modulates the expression of glucocorticoid-responsive genes by binding to specific glucocorticoid response element DNA sequences. In contrast, hGRbeta may be an endogenous inhibitor of glucocorticoid action and transcriptionally inactive. hGRbeta protein has been known to correlate with the development of glucocorticoid resistance. Glucocorticoids can effectively relieve autoimmune hepatitis (AIH), but some patients with this disease are refractory even when glucocorticoids are administered. The aim of this study was to determine the incidence of hGRbeta mRNA in patients with AIH by reverse transcription polymerase chain reaction (RT-PCR), and to compare the clinical characteristics of hGRbeta-positive and -negative patients with AIH. Materials and methods: RNA was obtained from peripheral blood mononuclear cells (PBMCs) of 62 patients, consisting of 26 with AIH, 10 with primary biliary cirrhosis (PBC), seven with chronic viral hepatitis (CVH), 10 with ulcerative colitis (UC), six with pemphigus, and three with systemic lupus erythematosus (SLE), and 10 healthy volunteers. The total RNA obtained was reverse transcribed, the resulting complementary DNA amplified using specific primers for hGRalpha and hGRbeta. Results: The hGRalpha mRNA was detected in RNA from PBMCs of all patients and healthy volunteers. The hGRbeta mRNA was detected in 15 (57.6%) patients with AIH. This incidence was significantly higher than that for patients with PBC (0%) or CVH (28.6%) or for healthy volunteers (20.0%) ( [Formula: see text] ). Of the hGRbeta-positive and -negative groups, serum ALT and total bilirubin (TB) levels were significantly higher in the positive group ( [Formula: see text] ). The total dose of glucocorticoid was higher in the positive group, but the difference was not statistically significant. However, the average monthly dose was significantly higher in the positive group ( [Formula: see text] ). The rate of relapse of AIH was significantly higher in the positive group (60.0%) than in the negative (10.0%) ( [Formula: see text] ). The rate of usage of immunosuppressive drugs was higher in the positive group (33.3%) than in the negative (18.2%), but the difference was not statistically significant. Conclusions: These data show that hGRbeta expression in PBMCs of patients with AIH assessed by RT-PCR is closely associated with resistance to glucocorticoids which affects the outcome of therapy with this drug.

Expression of human glucocorticoid receptor in lymphocytes of patients with autoimmune hepatitis

Expression of human glucocorticoid receptor in lymphocytes of patients with autoimmune hepatitis

Transactivation Assay for Determination of Glucocorticoid Bioactivity in Human Serum

We have developed a mammalian cell (COS-1) bioassay, which measures glucocorticoid bioactivity (GBA) directly from a small amount of human serum. The assay is based on the expression of human glucocorticoid receptor (GR) together with a coactivator protein and reporter plasmid containing GR response elements upstream of the luciferase gene. Ten microliters of human serum, in duplicate, are added directly to the cell culture medium, and GBA is derived from reporter gene activity. The assay differentiates between biopotencies of synthetic steroids, and importantly, mifepristone (RU486) is able to block glucocorticoid-induced response. The assay is sensitive (<15.6 nm cortisol in fetal calf serum) and precise, with the within- and between-assay coefficients of variation less than 8% and 10%, respectively. We measured serum GBA (bioassay) and cortisol (RIA) levels in 34 asthmatic children (age range, 5.7–14.2 yr) at baseline and after treatment with either inhaled budesonide (800 μg/d, n = 14), fluticasone propionate (500 μg/d, n = 14), or cromones (control group, n = 6). Pretreatment serum GBA and cortisol levels correlated strongly (r = 0.90, P < 0.0001, n = 34). Two months of treatment with inhaled budesonide resulted in excess GBA in circulation, which was not attributable to endogenous cortisol (P < 0.001). In the fluticasone propionate group, the presence of serum excess GBA was at the borderline of statistical significance (P < 0.08) after 2 months of inhalation therapy, and no excess GBA was detected in the cromone group. In conclusion, our bioassay enables measurement of mammalian cell response to bioactive glucocorticoids in circulation and provides a novel means to investigate patients receiving drugs acting through the GR.

Structural Determinants of Aldosterone Binding Selectivity in the Mineralocorticoid Receptor

The structural determinants of aldosterone binding specificity in the mineralocorticoid receptor (MR) have not been determined. The MR has greatest sequence identity with the better characterized glucocorticoid receptor (GR), which is reflected in their overlapping ligand binding specificities. There must be subtle sequence differences that can account for the MR-specific binding of aldosterone and the shared binding of cortisol. To characterize ligand binding specificity, chimeras were made between the human MR and GR ligand-binding domains (LBDs). Three points were chosen as break points to generate a total of 16 different constructs. These chimeric LBDs were placed in a human GR expression vector containing the GR DNA-binding and N-terminal domains and assayed by co-transfection into CV-1 cells with the mouse mammary tumor virus-luciferase reporter plasmid. Binding of [(3)H]aldosterone and [(3)H]dexamethasone was also measured. All of the constructs that are potently activated by aldosterone contain amino acids 804-874 of the MR. The results of the ligand binding experiments using [(3)H]aldosterone were consistent with the transactivation assay. Cortisol activation of the chimeras was surprisingly complex. Constructs that are activated by cortisol contain either amino acids 804-874 and 932-984 of the MR or amino acids 598-668 and 726-777 of the GR. However, all of the chimeras retained the ability to bind the synthetic glucocorticoid [(3)H]dexamethasone, and cortisol was able to displace [(3)H]dexamethasone binding, suggesting that the differential effects of cortisol on transcriptional activation are caused by an effect that occurs downstream of ligand binding. These results identify a subregion of the MR LBD that confers specificity of aldosterone binding, which contrasts with cortisol binding where differential effects between chimeras appear to be mediated by interactions distal to ligand binding.

Interleukin-1 regulation of corticotropin-releasing factor (CRF), glucocorticoid receptor, c-fos and c-jun messenger RNA in the NPLC-KC cell line

The NPLC-KC human hepatoma cell line expresses corticotropin-releasing factor (CRF) and it has been demonstrated that CRF secretion and synthesis in this cell line increases in response to activators of the protein kinase A (PKA) and C (PKC) pathways as well as interleukin-1 (IL1). CRF expression with all three agents can be inhibited with the synthetic steroid-dexamethasone (DEX). In this report, we have examined the effect of IL1 ( β form) in the presence and absence of DEX on CRF mRNA (mRNA) expression as well as the expression of human glucocorticoid receptor (GR) mRNA and the mRNA of the proto-oncogenes (c-jun and c-fos) that have been implicated in CRF regulation. NPLC-KC cells were incubated with picomolar concentrations of IL1. Following this total RNA was extracted from the cells and Northern Blots were probed with 32P-labelled human DNA probes for the CRF, GR, c-jun and c-fos genes. Levels of mRNA expression were measured using a PhosphoImager and were normalized to mRNA levels of control probe glyceraldehyde-3-phosphate dehydrogenase (GAPD). CRF mRNA was significantly increased with IL1 treatment in a time and concentration dependent manner. CRF mRNA expression increased with increasing concentrations of IL1 over the range of 1–100 pM; expression of CRF mRNA also peaked after 24 h of 100 pM IL1 treatment and reached a level of expression approximately seven times higher than control. This pattern of expression was significantly inhibited in the presence of 100 nM DEX. Levels of the GR, c-fos and c-jun mRNAs were also significantly increased in the presence of IL1 and inhibited when DEX was co-incubated with IL1. The results reveal that IL1 stimulation of CRF mRNA expression by IL1 in the NPLC-KC cell line is accompanied by activation of GR mRNA as well as the mRNA of the immediate early genes—c-fos and c-jun. The results also demonstrate that this cell line may serve as a model system for the molecular mechanisms by which IL1 regulates CRF in central nervous system (CNS) neurons.

Cortisol resistance and the guinea pig glucocorticoid receptor

The guinea pig has been employed as a model to study the structure/function relationships of the glucocorticoid receptor (GR), and to determine the regions of the receptor important for binding hormone and antihormone. Guinea pigs have high levels of circulating cortisol and GR with a ∼ 20-fold lower affinity for dexamethasone than mouse GR. Cloning and sequencing of guinea pig GR has identified 24 amino acid changes in the ligand-binding domain (LBD) compared to the human GR. By substituting the guinea pig GR LBD for the human LBD in a human GR expression vector we have shown in cotransfection studies that guinea pig GR LBD confers glucocorticoid resistance as observed in vivo. In initial studies guinea pig GR LBD appeared constitutively active; in subsequent studies to determine which of the 24 amino acid changes present in the guinea pig GR LBD conferred resistance, it became apparent that the guinea pig LBD (LBDΔ), amplifed by PCR for subcloning into the human GR expression vector, contained a single adenine deletion in the hinge region within ten bases of the PCR primer. This single base deletion resulted in a frameshift bringing a stop codon into frame one codon after the deletion. While this now clearly accounts for the observed constitutive activity, since it is known that C-terminally truncated steroid receptors exhibit constitutive activation such a truncation is more difficult to reconcile with the repeatedly demonstrable hormone dose-response curves obtained with this guinea pig GR LBDΔ. Studies with the correct guinea pig GR LBD show hormone resistance, with half-maximal activity at 3 nM dexamethasone (compared to 0.8 nM with the human GR), but not constitutive activity. Secondly, it has recently been shown that the protein kinase A activator, 8-bromo-cAMP, is able to enhance the response of the mouse GR to dexamethasone, and in addition elicits an agonist response from the glucocorticoid antagonist RU 486. We have examined whether this also holds for the human GR, and whether changes in the LBD alter the response for guinea pig GR. For guinea pig GR LBDΔ constitutive activity is enhanced 3-fold in the presence of 8-bromo-cAMP alone; in contrast with the results for mouse GR, RU 486 uniquely antagonizes the action of 8-bromo-cAMP. Both wild-type guinea pig GR LBD and human GR respond similarly to mouse GR in this context. Third, guinea pig GR have tryptophan at residue 632, which in all other species examined to date is the cysteine, shown to be the site of covalent attachment for the affinity label dexamethasone 21-mesylate. Mutation of cysteine to tryptophan in human GR had no effect on the dose response curve, and the reciprocal mutation of tryptophan to cysteine in the guinea pig GR LBD was also without effect. Despite the predicted importance of this residue, it would thus appear not to be critical for receptor response to steroid.

Stable overproduction of intact glucocorticoid receptors in mammalian cells using a selectable glucocorticoid responsive dihydrofolate reductase gene.

We have generated several mammalian cell lines that stably express high levels of intact glucocorticoid receptor. These cells were created by cotransfecting a glucocorticoid-dependent dihydrofolate reductase (DHFR) gene into DHFR-deficient Chinese hamster ovary (CHO) cells together with a plasmid directing the expression of human glucocorticoid receptor. Using this approach, transfection frequencies indicate that the inclusion of glucocorticoid receptor cDNA increased the efficiency of DHFR transformation greater than 10-fold over nonreceptor control DNA. When a stably cotransfected line (designated MG/hGR) was subjected to short term growth in cytotoxic concentrations of the antifolate methotrexate, these cells strongly resisted growth inhibition when dexamethasone was present in the medium. This effect was steroid specific and was inhibited by the glucocorticoid antagonist RU38486. In an effort to exploit the methotrexate-induced coamplification properties of the DHFR gene as a means of creating cell lines having increased levels of glucocorticoid receptor, MG/hGR cells were chronically exposed to a relatively low concentration of methotrexate (50 nM). After this treatment a resistant line was isolated (MG/hGR/MTX50) that displayed complete dependence on exogenous glucocorticoid for growth. To investigate the molecular basis for the enhanced ability of MG/hGR/MTX50 cells to resist the cytotoxic effects of methotrexate in the presence of dexamethasone, glucocorticoid receptor protein in these cells was characterized and compared to parental CHO cells and methotrexate sensitive MG/hGR cells. Affinity labeling with [3H]dexamethasone mesylate and Western blot analysis with antiglucocorticoid receptor antiserum revealed that nontransfected CHO cells have virtually undetectable levels of glucocorticoid receptor protein whereas cotransfected MG/hGR cells contain at least 3 times more intact monomeric receptor protein of Mr 94,000. Correspondingly, analysis of receptor protein in MG/hGR/MTX50 cells indicated that these cells contain 8 to 10 times more glucocorticoid receptor than nontransfected CHO cells. Scatchard analysis of steroid binding curves revealed that these increases correspond to 6,600, 22,000 and 63,000 dexamethasone binding sites per cell for nontransfected CHO cells, cotransfected MG/hGR cells, and MG/hGR/MTX50 cells, respectively. Sedimentation profiles of native receptor in transfected and methotrexate-resistant cells further support the progressive increase in receptor content and demonstrate that glucocorticoid receptor exists in cotransfected cels as an oligomeric complex under hypotonic conditions (9S complex in the presence of 20 mM sodium molybdate, 7S in the absence of molybdate), which dissociates to a monomeric 4S species in the presence of 0.4 M KCl. These physicochemical properties are indistinguishable from those observed for the endogenous hamster glucocorticoid receptor and suggest that stably transfected human glucocort