Glucocorticoid Receptor Splice Research Articles

A preferential switch between placental GR exon 1 promoter variants in the presence of maternal asthma or inflammation upregulates GRα D isoforms

IntroductionThe human placenta expresses multiple glucocorticoid receptor (GR) isoforms that may be partially regulated by the untranslated 5’ exon 1 GR gene promoter region which consists of 9 different promoters and 13 splice variants. The objective of this study was to determine which GR exon 1 variants are expressed in the human placenta and relate these findings to GR mRNA and protein expression. MethodsPlacental extracts from pregnancies with or without the complication of maternal asthma and trophoblast cells exposed to an inflammatory challenge in vitro were examined using PCR and Western blot to measure GR exon 1 variants, GR splice variant mRNA and GR protein isoforms, respectively. ResultsAll 9 GR exon 1 variants were detectable in the human placenta and included GR exons 1A, 1B, 1C, 1D, 1E, 1F, 1H, 1I and 1J. In the presence of maternal asthma and a male fetus there was preferential expression of GR exon 1B, 1C, IF and 1J (KW-ANOVA, P < 0.05) which were positively correlated with GRα D3 protein isoform. In female placentae from pregnancies complicated by asthma there was no upregulation of any exon 1 variant (KW-ANOVA, P < 0.05). Exposure of BeWo trophoblast cell line to an inflammatory challenge, lipopolysaccharide, in vitro, resulted in preferential expression of GR exon 1B, 1D, 1E and 1H and associated with GRα-D1 protein upregulation. DiscussionThe preferential expression of different GR exon 1 promoters drive the upregulation of GRα D isoforms and contribute to glucocorticoid resistance observed in male placentae of pregnancies complicated by asthma.

Kinetics of the in vivo expression of glucocorticoid receptor splice variants during prednisone treatment in childhood acute lymphoblastic leukaemia

The in vivo glucocorticoid response in childhood acute lymphoblastic leukaemia (ALL) correlates with the response to multi-agent chemotherapy. However, it is still unclear, whether the expression levels of glucocorticoid receptor (GR) splice variants facilitate the escape from glucocorticoid-induced apoptosis and hence contribute to glucocorticoid resistance. In the present study, the initial in vivo expression of the common GR (cGR) and its splice variants GR-alpha, GR-gamma and GR-P was determined using a quantitative RT-PCR approach. Two cohorts of glucocorticoid sensitive (prednisone good responder, PGR) and resistant (prednisone poor responder, PPR) patients were compared. The kinetics of GR splice variant expression was measured during 36 hr following the first use of glucocorticoids in seven patients. The GR splice variant GR-gamma showed the most pronounced differential regulation comparing PPR and PGR patients in both cohorts. GR-alpha and GR-gamma were upregulated faster and to a higher level in PGR as compared to PPR in the in vivo stimulation cohort. Here as well, the most pronounced effect was observed for GR-gamma. Differential regulation of the cGR and its splice variants under glucocorticoid treatment rather than the expression level at diagnosis is associated with glucocorticoid response.

Expression of Glucocorticoid Receptor Messenger Ribonucleic Acid Transcripts in the Human Placenta at Term

Differential promoter use and alternative splicing generate a variety of glucocorticoid receptor (GR) mRNA transcripts, potentially altering the cortisol responsiveness of gestational tissues during pregnancy and labor. We examined GR mRNA transcript expression in term placentae before and after labor, in association with fetal sex and after glucocorticoid treatment. RNA from 34 placentae and from eight placental explants incubated with glucocorticoids were analyzed for the GR mRNA variants GR-alpha, GR-beta, GR-P, and GR-gamma and the untranslated exon one variants 1A1, 1A2, 1A3, 1B, and 1C by quantitative RT-PCR. mRNA expression was assessed. All GR mRNA variants examined were detected in the human placenta, with GR-alpha and GR-1C mRNA having the highest expression of GR splice variants and exon 1 variants, respectively. GR-P mRNA abundance decreased with spontaneous labor (P < 0.01). GR-1A3 mRNA abundance changed with fetal sex, with a higher level in placentae of male fetuses (P < 0.05). GR-1C was the preferential promoter for GR-alpha, GR-gamma, and GR-P mRNA. GR-beta mRNA was preferentially associated with GR-1A1. GR-P mRNA transcription switched to the GR-1A1 promoter after labor and to the GR-1A3 promoter in placentae from male fetuses. Glucocorticoid treatment significantly reduced transcription from promoters GR-1B and -1C and decreased GR-alpha and GR-P mRNA abundance. The human placenta expresses a variety of GR mRNA transcripts. GR-alpha mRNA transcribed from the 1C promoter generates the majority of placental GR. However, alterations in promoter use and alternative splicing may modulate responses to cortisol during stressful events.

Discovery of a Functional Glucocorticoid Receptor β-Isoform in Zebrafish

In humans, two glucocorticoid receptor (GR) splice variants exist: GRalpha and GRbeta, which are identical between amino acids 1-727 and then diverge. Whereas GRalpha (the canonical GR) acts as a ligand-activated transcription factor, GRbeta does not bind traditional glucocorticoid agonists, lacks GRalpha's transactivational activity, and acts as a dominant-negative inhibitor of GRalpha. It has been suggested that this receptor isoform is involved in the induction of glucocorticoid resistance in asthma patients. Unfortunately, a GR beta-isoform has been detected in only humans, and therefore, an animal model for studies on this isoform is lacking. In the present study, we demonstrate that in zebrafish a GR isoform exists that diverges from the canonical zebrafish GR at the same position as human GRbeta from human GRalpha. The zebrafish GR beta-isoform acts as a dominant-negative inhibitor in reporter assays, and the extent of inhibition and the effective GRalpha/GRbeta ratio is similar to studies performed with the human GR isoforms. In addition, the subcellular localization of zebrafish GRbeta is similar to its human equivalent. Finally, expression levels of GRalpha and GRbeta were determined in adult zebrafish tissues and at several developmental stages. Both receptor isoforms were detected throughout the body, and GRbeta mRNA levels were relatively low compared with GRalpha mRNA levels, as in humans. Thus, for the first time, a GR beta-isoform has been identified in a nonhuman animal species, shedding new light on the relevance of this GR splice variant and providing a versatile animal model for studies on the GR system.

Associations between promoter usage and alternative splicing of the glucocorticoid receptor gene

The glucocorticoid receptor (GR) is widely expressed in various tissues throughout the human body. At least three different 3'-splice variants of the GR have been reported: GR-alpha, which is functionally active; GR-beta, which is a dominant negative inhibitor of GR-alpha function; and GR-P, which is thought to activate the function of GR-alpha. At least seven different variants for exon 1 exist, 1A-1F and 1H, each with its own promoter. In this study, we explored if tissue-specific splicing of the 3'-end variants of the GR is influenced by alternative promoter usage. cDNAs of different tissues and cell lines were used to investigate which part of transcripts carrying each of the three major variants for exons 1, 1A, 1B, or 1C, encodes for the splice variants GR-alpha, GR-beta, and GR-P. Our data demonstrate that the expression of GR-alpha is preferentially regulated by promoter 1C and that for the expression of GR-P promoter 1B is predominantly used. This indicates that regulation of GR splice variants could partly occur through selective use of the multiple promoters, and that this is another way to sensitize cells and tissues to the different activities of the GR isoforms.

Strategies for the Characterization of Disorders in Cortisol Sensitivity

The clinical presentation of abnormalities in glucocorticoid (GC) sensitivity is diverse, and therefore it is difficult to diagnose this condition. The objective of the study was to develop strategies for the characterization of GC sensitivity disorders. The study was conducted in an outpatient clinic. Nine patients with GC sensitivity disorders participated. Sequence analysis of the GC receptor (GR), determination of GR number per cell, GR ligand-binding affinity, and GR splice regulation were performed in freshly prepared peripheral blood mononuclear lymphocytes and Epstein-Barr virus-transformed lymphoblasts. Cellular GC sensitivity was determined ex vivo by measuring the effect of dexamethasone on GC-induced leucine-zipper and IL-2 mRNA levels and on cell proliferation. Differences in GR number per cell, GR affinity, GR splice variants, and effects on transactivation or transrepression of GC-sensitive genes were observed between patients and controls. Epstein-Barr virus transformation of lymphoblasts had no influence on GR affinity but increased the GR number 5-fold in healthy controls. In patients diagnosed as cortisol resistant, however, GR number after transformation was increased significantly less than 5-fold, whereas a higher GR number was observed in a patient suspected of cortisol hypersensitivity. This study illustrates several strategies to define abnormalities in GC sensitivity by describing nine patients with affected GC sensitivity, all with a unique clinical course and background.

Expression of the Human Glucocorticoid Receptor Splice Variants α, β, and P in Peripheral Blood Mononuclear Leukocytes in Healthy Controls and in Patients with Hyper- and Hypocortisolism

The effects of cortisol are mediated by the alpha-isoform of the glucocorticoid receptor (GR). GR-alpha levels and activity are modulated by alternative splicing of the common pre-mRNA into mRNAs for the GR-beta and GR-P isoforms. The objective of this study was to investigate whether chronic hypercortisolism, chronic hypocortisolism, or acute, relative hypocortisolism influences the expression levels of the GR splice variants in mononuclear leukocytes. This was a case-control study. The study was performed at a university hospital. Eighteen patients with Cushing's syndrome, five patients with hypocortisolemia, seven patients undergoing metyrapone testing, and 14 controls were studied. The main outcome measures were mRNA levels, GR affinity, and number per cell. All three GR mRNA isoforms were detected in participants from all groups at relative levels of alpha/P/beta = 1:0.25:0.001. There was a significant correlation between the expression levels of the three splice variants and between the mRNA levels and the number of receptors per cell. The GR in Cushing patients had an increased Kd (P < 0.05) preoperatively. GR number was not significantly different. Postoperatively, the Kd decreased. GR-beta mRNA expression was increased compared with controls (P < 0.05) and was decreased after surgery (P < 0.05). In patients with chronic hypocortisolism, GR-alpha mRNA expression was increased, and receptor numbers were increased (P < 0.05), whereas GR affinity was normal. No changes were observed in patients undergoing a metyrapone test. Cushing's syndrome is accompanied by a reversible decrease in GR affinity, possibly related to an increased GR-beta expression, which may be a compensatory mechanism to GC excess. In chronic hypocortisolism, adaptive changes in GR status seem to occur at the level of GR number.

5′UTR sequences of the glucocorticoid receptor 1A transcript encode a peptide associated with translational regulation of the glucocorticoid receptor

We have recently reported that glucocorticoid receptor (GR) transcript 1A, one of the five mouse GR splice variants (1A–1E), encodes membrane GR (mGR), which subsequently participates in mediating the apoptotic effects of glucocorticoids (GCs); all transcripts vary at their 5′UTR. Computer analysis of the entire1026 bp comprising the 5′UTR of transcript 1A identified five putative translation start sites at positions 85, 217, 478, 628, and 892 with the potential to encode peptides of 33, 93, 6, 18, and 41 amino acids, respectively. We then separately generated point mutations at these five upstream AUG codons of the GR 1A cDNA and performed in vitro transcription/translation experiments to investigate the regulatory effects of these sites on GR synthesis. GR translation products were immuno-captured with BUGR-2 antibody (Ab), then subjected to Western blot analysis. Mutation of the uAUG codon-2 completely inhibited GR synthesis, while mutations at the other four uAUG codons had no significant effect on the translation of transcript 1A. Antibodies (Abs) against the uORF-2 and uORF-5 protein products were used to perform Western blot analysis on cytosolic proteins from S-49 cells (which express GR transcript 1A), U937 cells transfected with GR 1A cDNA, or in vitro translation products from this cDNA. This assay identified an intense immunoreactive band of ∼8.5 kDa recognized only with Ab to the uORF-2 peptide; this size is consistent with the computer-predicted size of the uORF-2 product, suggesting that the uORF-2 product is indeed synthesized in cells. No peptide was identified with Ab to uORF-5 peptide. Indirect fluorescent Ab staining, confocal microscopy and FACS analysis all showed that the ORF-2 peptide is localized both in the interior of the cell and at the plasma membrane. Using Ab to ORF-2 peptide for immunoadsorption we then asked whether cellular factors interact with the product of uORF-2. Immuno-captured uORF-2 peptide levels correlated with the concentrations of several salt-wash-sensitive cellular proteins, suggesting that protein–protein interactions occur between this upstream open reading frame (uORF) product and other factors. The uORF-2 product, however, does not appear to directly interact with GR, since there was no reciprocal immuno-capture between these two proteins. In summary, our results show that cells can synthesize the uORF-2 peptide, blocking of the synthesis of the uORF-2 peptide product abolishes translation of GR from the GR 1A transcript, and the peptide product of uORF-2 interacts with other cellular factors which might be involved in translation of GR. J. Cell. Biochem. 81:149–161, 2001. © 2001 Wiley-Liss, Inc.

5?UTR sequences of the glucocorticoid receptor 1A transcript encode a peptide associated with translational regulation of the glucocorticoid receptor

We have recently reported that glucocorticoid receptor (GR) transcript 1A, one of the five mouse GR splice variants (1A-1E), encodes membrane GR (mGR), which subsequently participates in mediating the apoptotic effects of glucocorticoids (GCs); all transcripts vary at their 5'UTR. Computer analysis of the entire1026 bp comprising the 5'UTR of transcript 1A identified five putative translation start sites at positions 85, 217, 478, 628, and 892 with the potential to encode peptides of 33, 93, 6, 18, and 41 amino acids, respectively. We then separately generated point mutations at these five upstream AUG codons of the GR 1A cDNA and performed in vitro transcription/translation experiments to investigate the regulatory effects of these sites on GR synthesis. GR translation products were immuno-captured with BUGR-2 antibody (Ab), then subjected to Western blot analysis. Mutation of the uAUG codon-2 completely inhibited GR synthesis, while mutations at the other four uAUG codons had no significant effect on the translation of transcript 1A. Antibodies (Abs) against the uORF-2 and uORF-5 protein products were used to perform Western blot analysis on cytosolic proteins from S-49 cells (which express GR transcript 1A), U937 cells transfected with GR 1A cDNA, or in vitro translation products from this cDNA. This assay identified an intense immunoreactive band of approximately 8.5 kDa recognized only with Ab to the uORF-2 peptide; this size is consistent with the computer-predicted size of the uORF-2 product, suggesting that the uORF-2 product is indeed synthesized in cells. No peptide was identified with Ab to uORF-5 peptide. Indirect fluorescent Ab staining, confocal microscopy and FACS analysis all showed that the ORF-2 peptide is localized both in the interior of the cell and at the plasma membrane. Using Ab to ORF-2 peptide for immunoadsorption we then asked whether cellular factors interact with the product of uORF-2. Immuno-captured uORF-2 peptide levels correlated with the concentrations of several salt-wash-sensitive cellular proteins, suggesting that protein-protein interactions occur between this upstream open reading frame (uORF) product and other factors. The uORF-2 product, however, does not appear to directly interact with GR, since there was no reciprocal immuno-capture between these two proteins. In summary, our results show that cells can synthesize the uORF-2 peptide, blocking of the synthesis of the uORF-2 peptide product abolishes translation of GR from the GR 1A transcript, and the peptide product of uORF-2 interacts with other cellular factors which might be involved in translation of GR.