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Natural variants of the ?? isoform of the human glucocorticoid receptor do not alter sensitivity to glucocorticoids
Abstract
The beta isoform of the human glucocorticoid receptor, hGRbeta, is a product of alternative splicing of the hGR gene. The physiological function of this isoform is unknown up to now. Recent data are contradictory in that they either favor or argue against a role of hGRbeta as a repressor of the functional hGRalpha isoform. In the present study hGRbeta did not inhibit transcriptional activation of the MMTV-driven luciferase reporter gene by dexamethasone-activated hGRalpha in COS-1 cells. In addition, two naturally occurring variants of the hGRbeta isoform associated with altered sensitivity to glucocorticoids, termed hGRbeta-R23K and hGRbeta-N363S, did not repress hGRalpha, even when overexpressed 10-fold. We conclude that the hGRbeta isoform, as well as two of its natural variants, do not act as dominant negative inhibitors of hGRalpha function and that the beta isoform does not appear to play a role in the regulation of glucocorticoid sensitivity.
... [13][14][15][34][35][36] and that under hyper-TG or hyperglycaemic conditions adherence of leukocytes to the endothelium is increased due to activation of endothelial cells. 12,20,37 Monocytes from diabetic patients showed increased superoxide production upon ex vivo stimulation, suggestive of increased responsiveness of these cells. However, this activation was related to plasma TG only and not to elevated glucose or glyHb. ...
... This may be due to the relative tight glucose control of the diabetic patients we studied. In addition, leukocytes could become activated indirectly via triggering of endothelial cells by TG or glucose, 12,20,37 by unstable leukocyte-enriched atherosclerotic plaques, 41 or by endothelial injury in general, since coronary angioplasty has been shown to induce leukocyte activation. 42 We also have to underline that, as has been shown by others, the leukocytes we obtained by peripheral sampling are most certainly less activated than the cells contributing to local inflammatory processes, since activated leukocytes will probably adhere to the endothelium in vivo. ...
... 36 However, despite a significant number of studies reporting it, controversy regarding the negative effects of this variant does still exist, since several other in vivo and in vitro studies found no effects of the hGR variant, and the mechanisms responsible for the dominant negative effect are largely unknown. 13,19,[37][38][39] In the present study, levels of hGR mRNA were much lower than those of the other variants. This does not exclude a role for this variant in causing glucocorticoid resistance, since similar results have been obtained in previous studies reporting quantitative hGR mRNA levels, and it might have several explanations. ...
The serotonin syndrome is a complex of symptoms that are thought to be largely attributable to changes in sensitivity in the serotonin receptor systems in the brainstem and the spinal cord due to drugs. Severe cases are almost always caused by a combination of two or more 'serotonergic' drugs, of which at least one is a selective serotonin reuptake inhibitor or a monoamine oxidase inhibitor. Usually, the syndrome heals spontaneously after withdrawal of the medication. Cessation of 'serotonergic' medication is the preferred treatment as well as supportive care.
... Despite data that the dominant negative function may indeed be the mechanism of action for GR (14,32), three reports (29)(30)(31) argue against this concept. de Lange and colleagues demonstrated that human GR did not exert a dominant negative effect, but only a nonspecific repression of transcriptional activity in general (31). ...
... Despite data that the dominant negative function may indeed be the mechanism of action for GR (14,32), three reports (29)(30)(31) argue against this concept. de Lange and colleagues demonstrated that human GR did not exert a dominant negative effect, but only a nonspecific repression of transcriptional activity in general (31). Hecht and colleagues illustrated that GR levels are much lower than GR␣, and that GR does not repress GR␣-mediated activation of the MMTV promoter of the COS-7 cells (30). ...
As peripheral blood mononuclear cells from patients with nocturnal asthma (NA) exhibit reduced steroid responsiveness at 4:00 A.M. as compared with 4:00 P.M., we hypothesized that NA is associated with increased nocturnal airway cell expression of GRbeta, an endogenous inhibitor of steroid action. Ten subjects with NA and seven subjects with nonnocturnal asthma (NNA) underwent bronchoscopy with bronchoalveolar lavage (BAL) at 4:00 P.M. and 4:00 A.M. BAL lymphocytes and macrophages were incubated with dexamethasone (DEX) at 10(-5) to 10(-8) M. DEX suppressed proliferation of BAL lymphocytes similarly at 4:00 P.M. and 4:00 A.M. in both groups. However, BAL macrophages from NA exhibited less suppression of IL-8 and TNF-alpha production by DEX at 4:00 A.M. as compared with 4:00 P.M. (p = 0.0001), whereas in the NNA group DEX suppressed IL-8 and TNF-alpha production equally at both time points. GRbeta expression was increased at night only in NA, primarily due to significantly increased expression by BAL macrophages (p = 0.008). IL-13 mRNA expression was increased at night, but only in the NA group and addition of neutralizing antibodies to IL-13 reduced GRbeta expression by BAL macrophages. We conclude that the airway macrophage may be the airway inflammatory cell driving the reduction in steroid responsiveness at night in NA, and this function is modulated by IL-13.
... [13][14][15][34][35][36] and that under hyper-TG or hyperglycaemic conditions adherence of leukocytes to the endothelium is increased due to activation of endothelial cells. 12,20,37 Monocytes from diabetic patients showed increased superoxide production upon ex vivo stimulation, suggestive of increased responsiveness of these cells. However, this activation was related to plasma TG only and not to elevated glucose or glyHb. ...
... 36 However, despite a significant number of studies reporting it, controversy regarding the negative effects of this variant does still exist, since several other in vivo and in vitro studies found no effects of the hGR  variant, and the mechanisms responsible for the dominant negative effect are largely unknown. 13,19,[37][38][39] In the present study, levels of hGR  mRNA were much lower than those of the other variants. This does not exclude a role for this variant in causing glucocorticoid resistance, since similar results have been obtained in previous studies reporting quantitative hGR mRNA levels, and it might have several explanations. ...
In primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) significant therapeutic effects of glucocorticoids have not been documented. The most important clinical problem in patients with these diseases is fatigue, which is occasionally invalidating. Abnormalities in the hypothalamo-pituitary-adrenal axis have been suggested as a cause of fatigue. Most effects of glucocorticoids are mediated by the glucocorticoid receptor (hGR alpha). Recently a causative role for a splicing variant of the glucocorticoid receptor (hGR beta) has been proposed in glucocorticoid resistance in asthma and ulcerative colitis, whereas another splicing variant (hGR P) might be associated with glucocorticoid-resistant haematological malignancies. The aims of the present pilot study were to assess abnormalities in glucocorticoid receptor expression and to relate these abnormalities to the development of fatigue and to disease activity and severity in autoimmune cholestatic liver disease.
Five fatigued and five nonfatigued patients with PBC or PSC were included, and the results were compared with healthy controls.
The expression of hGR P was not different from controls, but hGR beta mRNA was significantly increased (p=0.02) and hGR alpha mRNA decreased (p=0.015). There were no significant differences between fatigued and nonfatigued patients. A significant negative correlation between the serum activity of alkaline phosphatase and hGR alpha and hGR P mRNA was found.
Although there was no relation with fatigue, abnormalities in hGR expression appear to occur in patients with these diseases, and may play a role in its pathophysiology and the poor response to glucocorticoid treatment.
... However, the inhibitory role of GRb on GRa activity has been subject of great controversy (Table 4). For example, several authors failed to detect any dominantnegative inhibition of GRb on GRa activity (88,89,91,98). Such discrepancies have been attributed to differences in the cell types and/or experimental approaches used, and also to non-specific squelching of transcriptional coactivators by GRb. ...
... Transfection studies • When overexpressed with respect to GRa, GRb is a dominant-negative inhibitor of GRa-mediated transactivation (17,(92)(93)(94) • GRb is not a dominant-negative inhibitor of GRa-mediated transactivation (88,89,91,98) • Little effect of GRb on GRa-mediated transrepression (89,91,93,94) Expression and regulation • High GRb immunoreactivity, mainly localized in inflammatory cells (68,84,95,(104)(105)(106)(107) • Very low levels of GRb mRNA (GRa/GRb > 500/1) and protein in most cells and tissues (17, 20, 53, 74, 85-88, 91, 93, 99-103) • GRb protein half-life is twice that of GRa (20) • No downregulation of GRb protein by GCs in GRb-transfected cells (20) and in • GRb is not differently regulated by GCs than is GRa in several cell types (67,74,101) skin biopsies from asthmatic patients (68) • No upregulation of GRb by IL-2 and IL-4 in PBMCs (109) • Upregulation of GRb by proinflammatory stimuli in different cell types (20,84,85,95,104,110) Expression in inflammatory diseases • Increased GRb immunoreactivity in PBMCs, T cells and macrophages from GC-resistant asthmatics (68,104,105) • Very low levels of GRb mRNA and no detection of GRb protein in PBMCs from GC-insensitive asthmatics (86,109) • Increased GRb immunoreactivity in inflammatory cells from fatal asthma (106) and nocturnal asthma (112) • Very low levels of GRb mRNA in nasal polyps (87, Pujols*) ...
Inhaled and intranasal glucocorticoids are the most common and effective drugs for controlling symptoms and airway inflammation in respiratory diseases such as asthma, allergic rhinitis, and nasal polyposis. The last few years have seen a growing understanding of the mechanisms of glucocorticoid action and, in particular, the receptor that mediates glucocorticoid actions, the glucocorticoid receptor (GR). In this revision we present an update on the GR gene, the expression and regulation of its gene products, namely GRalpha and GRbeta, as well as their alterations in pathological states. GRalpha is responsible for the induction and repression of target genes, it is expressed in virtually all human cells and tissues, and its expression is known to be downregulated by glucocorticoids. GRbeta has been found to act as a dominant negative inhibitor of GRalpha-mediated transactivation in in vitro studies with transfected cells, but it does not appear to have a significant inhibitory effect on GRalpha-mediated transrepression. In addition, for most tissues the expression of GRbeta, at least at the mRNA level, is extremely low compared with that of GRalpha. Some pro-inflammatory cytokines appear to upregulate the expression of GRbeta, and increased GRbeta expression has been reported in diseases associated with glucocorticoid resistance or insensitivity, such as bronchial asthma, nasal polyposis, and ulcerative colitis. However, the possible role of GRbeta in modulating glucocorticoid sensitivity and/or resistance in vivo has been highly debated and it is not yet clear.
... CR is thought to have a dominant role as a negative inhibitor of CR [21,22]. However, there are conflicting data refuting this role [23,24]. The insertion of arginine in the DNA-binding domain at exon 4 results in another receptor splice variant CR, which has decreased transactivation activity [25]. ...
... The effects of corticosteroids are mediated by the CR receptor, whilst CR has been shown by some investigators to have a dominant inhibitory role (see above). There is, however, some controversy over the inhibitory functional role of CR on CR [23,24]. Nevertheless, whilst in transfection experiments overexpression of CR reduces the effects of corticosteroids, the physiological relevance of this mechanism in vivo in humans, or indeed in other animal species, still remains to be proven. ...
... Transfection studies have revealed the ability of GRb to act as a dominant negative inhibitor of GRa activity (5,8) through a mechanism that involves the formation of transcriptionally impaired GRa-GRb heterodimers (8). However, other studies have challenged this concept (7,9,10). ...
... Cotransfection studies have shown that when GRb is more abundant than GRa, GRb acts as a dominant negative inhibitor of GRa activity (5) through a mechanism which mostly involves the formation of transcriptionally impaired GRa^GRb heterodimers (8). However, other researches found no evidence of a speci¢c dominant negative e¡ect of GRb on GRa activity (7,9,10). Although certain cell types are known to contain GRb, further studies analyzing the GRa/GRb ratio are needed. ...
The lower sensitivity of the inflamed nasal mucosa to glucocorticoids might be related to an increased expression of the glucocorticoid receptor (GR) beta isoform. We investigated GRalpha and GRbeta mRNA expression in epithelial cells from nasal mucosa and nasal polyps. GRalpha mRNA was at least 1000 times more expressed than GRbeta mRNA in both tissues. GRbeta expression (mean+/-SEM of 10(3) cDNA copies/microg of total RNA) was higher in nasal polyps (1.15+/-0.19; n=27; P<0.01) than in nasal mucosa (0.62+/-0.10; n=32). Nasal polyps with > 3% of inflammatory cells had higher GRbeta levels (1.40+/-0.29; n=16) than both nasal mucosa (P<0.01) and polyps with < or = 3% of inflammatory cells (0.80+/-0.18; n=11; P<0.05). No difference in GRbeta expression was found between nasal mucosa and polyps with < or = 3% of inflammatory cells. GRbeta expression correlated with the inflammatory cell number, especially with mast cells (r=0.50, P<0.0001). There was no difference in GRalpha mRNA expression between nasal mucosa and nasal polyps. In summary, GRalpha is far more expressed than GRbeta in both tissues. The increased expression of GRbeta may be related to the presence of inflammatory cells.
... Whether the two known GR polymorphisms, i.e. N363S and R23K, affect GC sensitivity is still controversial (Koper et al. 1997, Huizenga et al. 1998, De Lange et al. 1999). ...
... De Lange et al. (2001) found little, if any, expression of GR-in various hematopoietic tumors. In concert with reports that failed to discover dominant negative activity of GR-(Hecht et al. 1997, De Lange et al. 1999), this argued against a critical role of the GR-isoform in GC resistance in leukemia. Two further GR variants have been described: the splice variant GR-, with an additional arginine in position 450A and about 50% less transactivation ability than GR-(Rivers et al. 1999), and GR-B that lacks the N-terminal 16 amino acids because of alternative translation initiation and which is nearly twice as effective as the longer GR-species in gene transactivation , but not in transrepression (Yudt & Cidlowski 2001). ...
Glucocorticoid (GC) resistance is a phenomenon of major significance in a number of clinical situations, including the therapy of lymphoid malignancies. Resistance may concern all, or just selected, GC effects, it may be absolute or just reflect a state of reduced sensitivity and, clinically relevant, be reversible or irreversible. Numerous molecular mechanisms can be envisaged acting either 'upstream' in the GC-triggered signaling pathway, i.e. at the level of the GC receptor (GR), or 'downstream' at the level of the GC-regulated genes responsible for individual GC effects. In lymphoid malignancies, GCs have anti-leukemic effects through the induction of apoptosis and/or cell cycle arrest. In this condition evidence for only a small number of mechanisms for GC resistance has been provided, mostly at the level of the GR. Herein, we review reports and hypotheses regarding 'upstream' and 'downstream' mechanisms for GC resistance in lymphoblastic leukemia and present an in vitro GC resistance model that might allow identification of resistance mechanisms.
... Transfection studies revealed the ability of GR- to act as a dominant negative inhibitor of GR-␣ activity (2,32,33) through a mechanism that involves the formation of transcriptionally impaired GR-␣-GR- heterodimers (32). However, other investigators have challenged this concept (5,14,22). The expression of GR-, both at the mRNA and protein level, seems to be much lower than that of GR-␣ (10,22,24,33,35). ...
... In cotransfection studies, it has been shown that, when GR- is more abundant than GR-␣, GR- acts as a dominant negative inhibitor of GR-␣ activity (2, 33) through a mechanism that mostly involves the formation of transcriptionally impaired GR-␣-GR- heterodimers (32). However, other investigators (5,14,22) found no evidence for a specific dominant negative effect of GR- on GR-␣ activity. It has been argued that the ability of GR- to regulate GR-␣ activity in vivo would depend on its expression level relative to that of GR-␣ and the strength of its association with heat shock protein (hsp) 90 (32). ...
Alternative splicing of the human glucocorticoid receptor (GR) primary transcript generates two protein isoforms: GR-alpha and GR-beta. We investigated the expression of both GR isoforms in healthy human cells and tissues. GR-alpha mRNA abundance (x10(6) cDNA copies/microg total RNA) was as follows: brain (3.83 +/- 0.80) > skeletal muscle > macrophages > lung > kidney > liver > heart > eosinophils > peripheral blood mononuclear cells (PBMCs) > nasal mucosa > neutrophils > colon (0.33 +/- 0.04). GR-beta mRNA was much less expressed than GR-alpha mRNA. Its abundance (x10(3) cDNA copies/microg total RNA) was as follows: eosinophils (1.55 +/- 0.58) > PBMCs > liver > or = skeletal muscle > kidney > macrophages > lung > neutrophils > brain > or = nasal mucosa > heart (0.15 +/- 0.08). GR-beta mRNA was not found in colon. While GR-alpha protein was detected in all cells and tissues, GR-beta was not detected in any specimen. Our results suggest that, in physiological conditions, the default splicing pathway is the one leading to GR-alpha. The alternative splicing event leading to GR-beta is minimally activated.
... The ability of overexpressed GRβ to alter transcription from GC-inducible, AP-1-inducible or NF-κB-inducible promoters has been examined using transient transfection and reporter gene assays. The initial observation of a dominant negative effect of GRβ on GRα-mediated transactivation in COS-7 [12] and HeLa cells [13] was not reproduced in Jurkat T cells [23], COS-7 cells [14, 24] and COS-1 cells [25] . Our results suggest that the dominant negative effect of GRβ on transactivation is cell type specific. ...
... For example, the promoter used to overexpress GRβ, the relative amounts of reporter gene construct, and GRα or GRβ expression vectors were not the same in every study. Moreover, in many studies [13,14,23,25,26], the reporter gene assay did not include a constitutive reporter gene to correct values for variations in transfection efficiency. This control is of crucial importance because efficiency of transient transfection may vary. ...
Glucocorticoids (GCs) are routinely used as anti-inflammatory drugs in the treatment of asthma. They act through binding to glucocorticoid receptor alpha (GRalpha), which represses numerous genes encoding pro-inflammatory mediators. A hormone binding deficient GR isoform named GRbeta has been isolated in humans. When overexpressed by transfection, GRbeta may function as a dominant negative modulator of GRalpha. However, to act as such, GRbeta has to be more abundant than GRalpha, and conflicting data have been obtained concerning the relative levels of the two isoforms in cell lines and freshly isolated cells. Moreover, the dominant negative effect was not confirmed by independent laboratories. In GC-resistant asthmatics, GRbeta was expressed by an increased number of peripheral blood mononuclear cells (PBMCs), airway T cells, and cells found in skin biopsies of tuberculin responses. However, the relative amounts of GRalpha and GRbeta in these cells were not determined. In GC-dependent asthmatics, PBMCs expressed GRalpha predominantly. No cells containing higher levels of GRbeta than GRalpha have yet been reported in asthmatics. Even if the existence of such cells is demonstrated, the role of GRbeta in asthma will remain a matter of controversy because functional studies have given discrepant data.
... However, some issues still remain unresolved. Several researchers could not reproduce the dominant-negative activity of hGRβ in vitro (Hecht et al., 1997; de Lange et al., 1999). In addition, the high hGRβ expression levels at which the dominant-negative activity in vitro is observed are in sharp contrast with its low expression levels in vivo (Oakley et al., 1996), which makes the relevance of the in vitro results questionable. ...
Glucocorticoids regulate a plethora of physiological processes, and are widely used clinically as anti-inflammatory drugs. Their effects are mediated by the glucocorticoid receptor (GR), a ligand-activated transcription factor. Currently, zebrafish embryos are being developed into a model system for GR research, since they are easy to manipulate genetically and their phenotype can easily be visualized because of their transparent bodies. In addition, the zebrafish GR gene shows a relatively high level of similarity with its human equivalent. First, both the zebrafish and the human genome contain only a single gene encoding the GR. In all other fish species studied thus far, two GR genes have been found. Second, the zebrafish contains a C-terminal GR splice variant with high similarity to the human GRbeta, which has been shown to be a dominant-negative inhibitor of the canonical GRalpha and may be involved in glucocorticoid resistance. Thus, zebrafish embryos are potentially a useful model system for glucocorticoid receptor research, but currently only a limited number of tools is available. In this review, we discuss which tools are available and which need to be developed, in order to exploit the full potential of the zebrafish as a model system for GR research.
... However, some debate exists as to the role of GRb within different tissues, including the brain. GRb is normally expressed at low levels in the brain, particularly in comparison with the GRa isoform [44,45], and it has been reported that the presence of GRb alone does not significantly decrease GRa activity [48,49]. Nevertheless, higher expression of GRb is present in the nucleus, and the GRb isoform is capable of dimerization with GRa, supporting the notion of its role in negative regulation of GRa activity [45,50]. ...
Glucocorticoid receptors (GRs) are ubiquitous transcription factors widely studied for their role in controlling events related to inflammation, stress and homeostasis. Recently, GRs have reemerged as crucial targets of investigation in neurological disorders, with a focus on pharmacological strategies to direct complex mechanistic GR regulation and improve therapy. In the brain, GRs control functions necessary for neurovascular integrity, including responses to stress, neurological changes mediated by the hypothalamic-pituitary-adrenal axis and brain-specific responses to corticosteroids. Therefore, this review will examine GR regulation at the neurovascular interface in normal and pathological conditions, pharmacological GR modulation and glucocorticoid insensitivity in neurological disorders.
... Transfection experiments with either cultured cells or reporter constructs in a variety of studies suggest that the beta isoform acts as a dominant negative regulator of GRα, inhibiting its ability to modulate transcription [19][20][21][22][23][24][25][26][27]. However, other reports have failed to document inhibition of GRα transcriptional activity by GRβ [28][29][30][31]. Increased relative levels of GRβ at both the mRNA and protein levels have been reported in many steroid resistant states and some autoimmune diseases [15,22,[32][33][34][35]. ...
Glucocorticoids are important regulators of metabolism and immune function. Synthetic glucocorticoids are extensively used for immunosuppression/anti-inflammatory therapy. Since the glucocorticoid receptor (GR) is central to most hormone effects; its in vivo regulation will influence hormone/drug action. An alternative splice variant, GRβ, is present in humans and may function as a dominant negative regulator of GR transcriptional activity. Recently, a similar splice variant was reported in mouse, although the mechanism of alternative splicing differs from that in humans. We present evidence that a splice variant of GR with an alternative C-terminus also occurs in the rat by a mechanism of intron inclusion. A highly quantitative qRT-PCR assay for the simultaneous measurement of both splice variants in a single sample was developed in order to accurately measure their regulation. We used this assay to assess the tissue specific expression of both mRNAs, and demonstrate that GRα is predominant in all tissues. In addition, the regulation of both GRα and GRβ mRNA by various physiological factors in rat liver was assessed. GRα showed a robust circadian rhythm, which was entrained with the circadian oscillation of the endogenous hormone. Time series experiments showed that both corticosteroids and LPS but not insulin dosing resulted in the transient down-regulation of GRα mRNA. LPS treatment also resulted in down-regulation of GRβ expression. A modest up-regulation in GRβ expression was observed only in animals having chronically elevated plasma insulin concentrations. However the expression of GRβ was significantly lower than that of GRα in all cases.
... Resistance to GCS is the extreme form of all possible reactions to these drugs (6). There are two forms of GCS resistance: type 1, induced by cytokines (acquired, reversible reduction of GCS binding potential of T lymphocytes) and type 2, mutations and polymorphisms of glucocorticoid receptor gene (mutations and polymorphisms of the h-GR/NR3C1 gene, or genes modulating the functions of GCR, GCR ligand-binding defects, decrease in the number of GCR) (11)(12)(13)(14)(15)(16)(17)(18). As evidenced from recent research, non-receptor transcriptional factors such as: [activator protein-1 (aP-1) and nuclear factor (nk)-κB protein] play an important role in the development of GCS resistance (6,9,10,19). ...
Bronchial asthma is a disease of multifactorial etiology. The natural variability of the DNA sequence within the h-GR/NR3C1 gene affects both the conformation and the activity of glucocorticoid receptors. There are 2 major types of resistance to glucocorticoids (GCS)-resistant asthma failing to respond to treatment with high doses of inhaled and oral glucocorticoids. Type I GCS-resistant asthma is cytokine-induced or acquired. Type II GCS resistance involves generalized primary cortisol resistance, which affects all tissues and is likely to be associated with a mutation in the glucocorticoid receptor (GCR) gene or in genes that modulate GCR function. There are clear examples of glucocorticoid gene h-GR/NR3C1 polymorphisms that can influence responses and sensitivity to glucocorticosteroids. Among the numerous polymorphisms observed within this gene, N363S and I559N single nucleotide polymorphisms (SNPs) may play an important role in the development of bronchial asthma and in the alteration of sensitivity to GCS in severe bronchial asthma. The aim of this research project was to study the correlation between the N363S and I559N polymorphisms of the h-GR/NR3C1 gene and the occurrence of asthma in a population of Polish asthmatics. Peripheral blood was obtained from 210 healthy volunteers and 234 asthma patients. Structuralized anamnesis, spirometry and allergy skin prick tests were performed in all participants. Genotyping was carried out using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and PCR-HRM methods. In the healthy, non-atopic population, the GG variant of the N363S polymorphism was found with a 5.7% frequency. In asthma patients, GG SNP of N363S occurred with the frequency of 6.4%. In the groups of patients with uncontrolled moderate asthma and uncontrolled severe disease, the genotype distribution for the investigated polymorphisms were as follows: N363S, AA, AG, GG occurring with 0.8750/0.0834/0.0416 frequency and I559N, TT, TA, AA occurring with 1.000/0.000/0.000 frequency. The analysis demonstrated a significantly higher frequency of the A and G variants of the N363S polymorphisms in uncontrolled moderate asthma and uncontrolled severe disease than in the healthy population. No variant-related differences in the frequency of the studied I559N polymorphism were demonstrated in healthy controls and asthma patients. In conclusion, the N363S polymorphism of the h-GR/NR3C1 gene is significantly associated with an increased sensitivity to glucococorticoids in vivo and susceptibility to the development of a moderate to severe form of uncontrolled bronchial asthma in the Polish population. This observation needs to be confirmed in a larger group of subjects.
... [9]. In view of contemporary research concerning the role of gene expression in bronchial asthma, the studies investigating the functional significance of glucocorticoid receptor gene (NR3C1 nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)) in steroid-resistant or difficult-to-treat forms of the disease are particularly interesting91011121314. Complete resistance to glucocorticoids is very rare—it has been estimated to affect less than 1:1000 bronchial asthma patients. ...
N363S and ER22/23EK polymorphisms observed within glucocorticoid receptor gene (NR3C1) may play an important role in the development of bronchial asthma. NR3C1 gene is associated with an altered sensitivity to GCs. The aim of the research project was to study the correlation between this NR3C1 gene polymorphisms and occurrence of asthma in the population of Polish asthmatics. Peripheral blood was obtained from 207 healthy volunteers and 221 asthma patients. Genotyping was carried out with PCR-RFLP method. In the groups of patients with uncontrolled moderate asthma and uncontrolled severe disease, the genotype distribution for the investigated polymorphisms was as follows: N363S-AA, AG, GG occurring with 0.881/0.073/0.046 frequency and ER22/23EK-GG, GA, AA occurring with 0.963/0.037/0.000 frequency. Chi-square analysis revealed a significantly different (P < 0.05) distribution between cases and controls for the N363S polymorphisms. The N363S polymorphism of NR3C1 gene is significantly associated with bronchial asthma, susceptibility to the development of moderate to severe form of uncontrolled bronchial asthma.
... Supposedly, disturbances at the level of AP-1 affinity to GR are one of the molecular pathways of cellular resistance to the effect of GCS. Transcriptional factors, and among them AP-1 (activator protein-1) and nuclear factor kappa B (NF-kB) in particular, are the modulators of activity of numerous genes responsible for the development of inflammation, which, binding to GR block GCS-GR binding with DNA sequences of specific response elements (GRE)3435363738. ...
Bcl I in the promoter polymorphism observed within h-GR/NR3C1 gene may play an important role in the development of bronchial asthma and resistance to GCs in the severe bronchial asthma. The aim of the investigation was to study the correlation between this h-GR/NR3C1 gene polymorphism and occurrence of asthma in the population of Polish asthmatics. Peripheral blood was obtained from 70 healthy volunteers and 59 asthma patients. Structuralized anamnesis, spirometry and allergy skin prick tests were performed in all participants. Genotyping was carried out with PCR-RFLP method. In healthy, non-atopic population variants of Bcl I: GG, GC, CC were found with frequency 0.129/0.471/0.400, respectively. In asthma patients Bcl I: GG, GC, CC occurred with respective frequencies of 0.410/0.462/0.128. Chi-square analysis revealed a significantly different (P<0.05) distribution between cases and controls for the Bcl I polymorphism. The Bcl I polymorphism of h-GR/NR3C1 gene is significantly associated with bronchial asthma, susceptibility to the development of severe form and resistance to GCs in Polish population.
... That GRβ can interfere with GRα transcriptional activity when over-expressed in transfected cells is not disputed (Gougat et al., 2002;Oakley et al., 1999;Oakley & Cidlowski, 2013). However, the pathophysiological relevance of these findings have been questioned (de Lange et al., 1999). Furthermore, findings by one group have often not been reproduced by another. ...
... D'autres formes tronquées du GR ont été retrouvées dans des cellules de myélome et ont été associées au développement de la résistance aux glucocorticoïdes de ces cellules (de Lange et al., 1999, Sanchez-Vega et al., 2006. Outre ces variations d'épissage, le GR peut subir des modifications post-traductionnelles qui vont moduler son activité (Zhou et Cidlowski, 2005). ...
Mitochondria are integrators of intracellular signaling (adjusting its functioning to cellular energy demand) and initaitors of retrograde pathways (triggering cellular response to variations of functional status of mitochondria). This work focus on oxidative mitochondrial metabolism and signaling pathways, in HepG2 cells, in response of two energetic stresses : mitochondrial uncoupling and glucocorticoids treatement. Mitochondrial uncoupling triggers an increase in oxidative metabolism without any change in glycolysis (notably by a stimulation of nuclear transcription of genes encoding mitonchondrial proteins). Mitochondria are also one of targets of glucocorticoids, homones tht induce short term and long term effects. Rapid effects (modification of respiratory chain complexes I, II and III activities) involve dexamethasone binding on a membrane glucocorticoid receptor. These effects are mediated by calcium dependent activation of p38MAPK. Long term genomic effects (increase in respiratory chain capacity) implicate the classical intracellular glucocorticoid receptor. Modifications of the respiratory chain functioning induced by glucocorticoids involve the gradual recruitement of glucocorticoid binding sites (located in plasma membrane or in cytosol).
... The mRNA expression levels reported for GR-β vary from virtually zero (41)(42)(43)(44) to levels comparable with that of the GR-α mRNA (32,45). Immuno-histochemical detection of the GR-β protein produced very mixed results (46)(47)(48)(49)(50)(51), and in transient transfection experiments some authors found considerable dominant negative effects of co-transfected GR-β (32,45,49,52,53), while others found none (48,50,54,55). A summary of the literature dealing with this type of experiment is presented in Table 3. ...
Synthetic glucocorticoids are used therapeutically for numerous indications. However, due to their broad physiological effects across many systems, side effects of GC therapy can be extensive and limit the clinical utility of GCs as a drug. One of the main urgent questions at this moment is to develop insights into the cause of the differences in the response between individuals to therapeutically applied GCs. Some patients respond to low doses, with or without side effects, while others do not respond at all. This thesis discusses a number of possible explanations for these differences in GC sensitivity and is focused on genetic, but also on transcriptional and translational aspects of the GR gene. Furthermore, it is also described how glucocorticoid sensitivity disorders can be characterized clinically and biochemically.
An important tool in these studies has been a newly developed bioassay, measuring cellular GC sensitivity ex vivo, based on GR action at the transcriptional level by studying GC-regulated mRNA expression (chapter 2). Various polymorphism in the GR gene (N363S, ER22/23EK, and 9beta) are shown to affect GC sensitivity in vivo and in/ex vitro and result in a wide variety of phenotypic signs (chapters 3, 4, and 5). Furthermore, studies described in chapter 4, 5, 6, and 7 have demonstrated that also transcriptional and translational variants of the GR and the use of different promoters could modulate GC sensitivity. These factors modulating inter- individual sensitivity to GCs may have consequences for the use of GCs in a clinical setting. When treating patients with GCs, they need an individually determined optimal dose to obtain a balance between beneficial and adverse effects.
... Our preliminary studies show that CR is overexpressed in SR RA patients . There is, however, some controversy over the functional role of CR in antagonising CR (Hecht et al. 1997, de Lange et al. 1999. Whilst in transfection experiments, overexpression of CR reduces the effects of CS, the physiological relevance of this mechanism in vivo remains to be proven. ...
Corticosteroids (CS) can modulate gene expression and are often used to treat a range of immunological and inflammatory diseases such as asthma, inflammatory bowel disease and rheumatoid arthritis. However, a proportion of patients fail to show an adequate response. On this basis patients have been subdivided into CS-sensitive (SS) and -resistant (SR) subgroups. The ability of CS to inhibit peripheral blood T cell proliferation in vitro has also been used similarly. In rheumatoid arthritis (RA), the in vitro-defined SS and SR subgroups correlate with the clinical responses to CS therapy. The mechanisms responsible for this observation are unknown but they appear to involve a number of known molecular events related to the described mechanisms of action of CS. These include alterations in the functional status of CS receptor-alpha, perturbations of the cytokine and hormonal milieu and intracellular signalling pathways. Peripheral blood mononuclear cells (MNCs) from SR significantly overexpress activated NF-kappaB. In vitro, CS fail to significantly inhibit concanavalin A (conA)-induced NF-kappaB activation in MNCs from SR RA patients. The alterations in the intracellular signalling pathways may explain in part our observations seen in SR RA subjects, CS fail to significantly inhibit conA-induced interleukin (IL)-2 and IL-4 secretion and lipopolysaccharide-induced IL-8 and IL-1beta secretion in vitro. CS therapy fails to reduce the circulating levels of IL-8 and IL-1beta in RA patients. In asthma, CS fail to induce L10 in SR asthma patients. Other molecular mechanisms such as enhanced AP-1 expression and alterations in the MAP kinase pathway are most likely to be involved too and we are currently investigating such possibilities. A full understanding of the molecular basis of SR will lead to the development of more rational therapeutic strategies.
... Some transfection studies showed a negative effect of GR over GR␣, 66,67 whereas others did not. 60,68,69 As the expression level of the beta isoform is very low compared to the alpha isoform, the relevance of the beta expression or resistance in childhood leukemia is unlikely. ...
Glucocorticoids (GC) are probably the most important drugs in the treatment of ALL. Despite the extensive use of GC for many years, little is known about the molecular mechanisms of sensitivity and resistance. This review summarizes the knowledge on GC cytotoxicity in leukemia. The relevance of polymorphisms, splice variants and the number and regulation of the GC receptor are discussed. The role of multidrug resistance proteins, glutathione and glutathione S-transferase is evaluated, as well as the influence of the different heat-shock chaperone (hsp 90 and 70) and co-chaperone proteins (BAG-1 and others) which form a complex together with the GC receptor. Finally, the transactivation and transrepression (via NF-kappa B and AP-1 binding) of a wide range of genes (like c-myc) which initiates the final apoptosis pathway are discussed and suggestions for future directions of research in ALL patients are given.
... Our preliminary studies show that CR is overexpressed in SR RA patients . There is, however, some controversy over the functional role of CR in antagonising CR (Hecht et al. 1997, de Lange et al. 1999. Whilst in transfection experiments, overexpression of CR reduces the effects of CS, the physiological relevance of this mechanism in vivo remains to be proven. ...
Corticosteroids (CS) can modulate gene expression and are often used to treat a range of immunological and inflammatory diseases such as asthma, inflammatory bowel disease and rheumatoid arthritis. However, a proportion of patients fail to show an adequate response. On this basis patients have been subdivided into CS-sensitive (SS) and -resistant (SR) subgroups. The ability of CS to inhibit peripheral blood T cell proliferation in vitro has also been used similarly. In rheumatoid arthritis (RA), the in vitro-defined SS and SR subgroups correlate with the clinical responses to CS therapy. The mechanisms responsible for this observation are unknown but they appear to involve a number of known molecular events related to the described mechanisms of action of CS. These include alterations in the functional status of CS receptor-alpha, perturbations of the cytokine and hormonal milieu and intracellular signalling pathways. Peripheral blood mononuclear cells (MNCs) from SR significantly overexpress activated NF-kappaB. In vitro, CS fail to significantly inhibit concanavalin A (conA)-induced NF-kappaB activation in MNCs from SR RA patients. The alterations in the intracellular signalling pathways may explain in part our observations seen in SR RA subjects, CS fail to significantly inhibit conA-induced interleukin (IL)-2 and IL-4 secretion and lipopolysaccharide-induced IL-8 and IL-1beta secretion in vitro. CS therapy fails to reduce the circulating levels of IL-8 and IL-1beta in RA patients. In asthma, CS fail to induce L10 in SR asthma patients. Other molecular mechanisms such as enhanced AP-1 expression and alterations in the MAP kinase pathway are most likely to be involved too and we are currently investigating such possibilities. A full understanding of the molecular basis of SR will lead to the development of more rational therapeutic strategies.
... There is evidence to suggest that one role of hGRß is to dimerize with hGRα, creating a heterodimer that has less transcriptional regulatory activity than a normal hGRα homodimer 39 , although some authors disagree on this. 40 Although the ligand-binding isoform is the better studied in the literature, polymorphic variation in either hGRα or hGRß may potentially exert an influence on glucocorticoid-mediated transcriptional regulation. ...
The actions of glucocorticoid hormones are mediated by an intracellular receptor, the glucocorticoid receptor (GR). The mechanism of action of this ligand-inducible transcription factor is discussed, focusing on mechanisms of glucocorticoid resistance. Three mechanisms are highlighted: ligand-induced down-regulation of the receptor, the dominant-negative inhibition by the beta-isoform of the receptor, and repression by the transcription factor NF-kappa B. It has been shown that these mechanisms can significantly inhibit glucocorticoid signaling, and could therefore seriously decrease the efficacy of glucocorticoids used clinically.
Glucocorticoid (GC) resistance can occur in a number of diseases. It can be either generalized (i.e., familial glucocorticoid resistance) or localized (i.e., asthma). In many cases, a reason for this resistance to steroids lies with mutations or polymorphisms present in the glucocorticoid receptor gene (GR/NR3C1) that belongs to a large family of nuclear receptors. A number of GC-resistant cell lines have been isolated in vitro, some of which arose or may have arisen in vivo. These and the mutations defined in them are included in this review as well as mutations engineered in plasmids and expressed in vitro. It also lists polymorphisms and the individual studies where association-related studies have been performed. NR3C1 is located on chromosome 5q31 and contains 10 exons that code for a 777 amino acid protein. There are two naturally occurring isoforms of the NR3C1, GRalpha (functional) and GRbeta (no hormone-binding ability). A total of 15 missense, three nonsense, three frameshift, one splice site, and two alternative spliced mutations have been reported in the NR3C1 gene associated with glucocorticoid resistance as well as 16 polymorphisms. Mutation and polymorphism data for NR3C1 will soon be found on the newly created locus-specific database.
The hippocampus is an important target for glucocorticoid hormones. Glucocorticoid receptor (GR) mediated feedback in this area is important for control of behavioural adaptation. An alternative splice variant, the GRbeta (GRbeta) isoform, does not bind ligand and has been proposed to inhibit classic GRalpha-mediated transactivation of target genes. Hence, an increased ratio of GRbeta to GRalpha may induce relative corticosteroid-resistance, as e.g. presumed to occur in major depression. To investigate whether GRbeta is involved in the human hippocampus, we studied GRalpha and GRbeta expression levels in postmortem hippocampal tissue of control subjects by quantitative PCR (Taqman RT-PCR) and immunocytochemistry. Taqman RT-PCR demonstrated a very low relative abundance of GRbeta in the human hippocampus (GRalpha:GRbeta ratio approximately 14,500:1). Immunohistochemical analysis confirmed the occurrence of isolated profiles indeed displaying nuclear staining in the main hippocampal subregions. Subsequent double immunofluorescent analysis revealed that >98% of these GRbeta positive cells were double positive for leucocyte common antigen, that identifies exclusively blood-derived cells of haematopoietic origin, including microglia. We conclude that GRbeta is present in very low amounts in the control human hippocampus, and that of these low numbers of cells, notably, almost all are derived from blood which is inevitably present in postmortem tissue. A functionally relevant role for the GRbeta in control of the human hippocampus is therefore not very likely. Whether this is altered in disease conditions awaits further research.
We have quantified the basal and glucocorticoid-regulated levels of different transcripts from the human glucocorticoid receptor (GR) gene in the T-cell acute lymphoblastic leukemia cell line, CEM-C7, and in the B lymphoblastoid cell line, IM-9. Highly specific quantitative, reverse transcription-polymerase chain reaction assays measured total GR transcripts, transcripts encoding the isoforms glucocorticoid receptor alpha (GRalpha) and glucocorticoid receptor beta (GRbeta), and transcripts containing different forms of exon 1: 1A1, 1A2, 1A3, 1B, and 1C. GRalpha and GRbeta transcripts are coordinately upregulated in CEM-C7 cells and coordinately downregulated in IM-9 cells by dexamethasone. The concentration of GRalpha mRNA is more than a 1000-fold higher than that for GRbeta mRNA. Transcripts with different exon 1 forms are all upregulated in CEM-C7 cells and all downregulated in IM-9 cells by dexamethasone, but transcripts containing exons 1A1, 1A2, or 1A3 are regulated to a higher degree than transcripts containing exon 1B or exon 1C. However, exon 1B- and exon 1C-containing transcripts are substantially more abundant than exon 1A-containing transcripts, with exon 1A3-containing transcripts more abundant than exon 1A1- or exon 1A2-containing transcripts. Analysis using models for glucocorticoid receptor autoregulation kinetics suggests that the minor 1A3-containing transcript component could be important for GR protein upregulation, and hence apoptosis, in CEM-C7 cells. These studies suggest that GRalpha transcripts containing exons 1A3, 1B, and 1C contribute most to the intracellular level of GR mRNA and may be the most relevant for steroid-mediated apoptosis in T-lymphoblasts.
Early treatment response is a strong predictor for treatment outcome in childhood acute lymphoblastic leukemia (ALL), treated within the protocols of the Berlin-Frankfurt-Münster (BFM) study group. In the ALL-BFM trials, early treatment response is assessed by in vivo response to glucocorticoids (prednisone response), the molecular background of which is unknown. Initial in vivo resistance to glucocorticoid (GC) treatment in childhood ALL (prednisone-poor response) is associated with a dramatically shorter event-free survival than that found in GC-sensitive patients (prednisone-good responders). The intracellular effects of glucocorticoids are mediated by the glucocorticoid receptor (GR). The protein expression of the GR has been linked to in vivo and in vitro GC resistance in various diseases treated with GC. However, existing data are conflicting.
We performed a case-control study for prednisone response to investigate the association of in vivo GC resistance and GR protein expression in childhood ALL. GR expression was assessed using Western blot technology.
The median relative GR protein expression of all patients was 0.87. Overall, we did not find different GR protein expression in PPR and PGR patients. GR protein expression was 0.91 in PGR patients versus 0.85 in PPR ones of in vivo GC resistance and GR expression.
We conclude that the expression of GR is of minor importance for in vivo GC resistance in childhood ALL.
Glucocorticoids (GC) control cell cycle progression and induce apoptosis in cells of the lymphoid lineage. Physiologically, these phenomena have been implicated in regulating immune functions and repertoire generation. Clinically, they form the basis of inclusion of GC in essentially all chemotherapy protocols for lymphoid malignancies. In spite of their significance, the molecular mechanisms underlying the anti-leukemic GC effects and the clinically important phenomenon of GC resistance are still unknown. This review summarizes recent findings related to GC-induced apoptosis, cell cycle arrest, and GC resistance with particular emphasis on acute lymphoblastic leukemia (ALL). We hypothesize that under conditions of physiological Bcl-2 expression, GC might induce classical programmed cell death by directly perturbing the Bcl-2 rheostat. In the presence of anti-apoptotic Bcl-2 proteins, cell death might result from accumulating catabolic and/or other detrimental GC effects driven by, and critically dependent on, GC receptor (GR) autoinduction. Although still controversial, there is increasing evidence for release of apoptogenic factors through pores in the outer mitochondrial membrane, rather than deltapsiloss-dependent membrane rupture, with maintenance of mitochondrial function at least in the early phase of the death response. GC-induced cell cycle arrest in ALL cells appears to be independent of apoptosis induction and vice versa, and critically depends on repression of both cyclin-D3 and c-myc followed by increased expression of the cyclin-dependent kinase inhibitor, p27Kip1. Since development of GC-resistant clones requires both cell cycle progression and survival, GC resistance might frequently result from structural or regulatory defects in GR expression, perhaps the most efficient means to target both pathways concurrently.
Sensitivity to glucocorticoids may be related to the concentration of glucocorticoid receptors alpha (GRalpha) and beta (GRbeta). A study was undertaken to assess GRalpha and GRbeta expression in steroid insensitive interstitial lung disease (idiopathic pulmonary fibrosis (IPF)) and steroid sensitive interstitial lung diseases (sarcoidosis and cryptogenic organising pneumonia (COP)).
Lung tissue was obtained from control subjects and from patients with IPF, sarcoidosis, and COP. Pulmonary function tests were carried out at the time of lung biopsy and every 3 months. GRalpha and GRbeta expression was evaluated by both competitive RT-PCR and immunohistochemistry. Data are presented as median and 25-75th percentile.
GRalpha mRNA expression (10(5) cDNA copies/ micro g total RNA) was higher in patients with steroid sensitive interstitial lung diseases (10.0; 7.8-14.9; n = 11) than in patients with IPF (4.4; 3.2-6.6; n = 19; p<0.001). GRbeta expression was at least 1000 times lower than that of GRalpha and did not differ between the three groups. A negative correlation was found between GRalpha mRNA levels and the fibrotic pathology score of the tissue (r = -0.484, p<0.01) and a positive correlation was found between GRalpha mRNA levels and improvement in forced vital capacity (r = 0.633; p<0.01) after treatment of patients with glucocorticoids. Immunoreactivity for GR protein was also higher in patients with sarcoidosis and COP than in those with IPF.
The variable response of some interstitial lung diseases to steroid treatment may be the result of differences in the expression of GRalpha.
Glucocorticoid (GC) resistance is a phenomenon of major significance in a number of clinical situations, including the therapy of lymphoid malignancies. Resistance may concern all, or just selected, GC effects, it may be absolute or just reflect a state of reduced sensitivity and, clinically relevant, be reversible or irreversible. Numerous molecular mechanisms can be envisaged acting either 'upstream' in the GC-triggered signaling pathway, i.e. at the level of the GC receptor (GR), or 'downstream' at the level of the GC-regulated genes responsible for individual GC effects. In lymphoid malignancies, GCs have anti-leukemic effects through the induction of apoptosis and/or cell cycle arrest. In this condition evidence for only a small number of mechanisms for GC resistance has been provided, mostly at the level of the GR. Herein, we review reports and hypotheses regarding 'upstream' and 'downstream' mechanisms for GC resistance in lymphoblastic leukemia and present an in vitro GC resistance model that might allow identification of resistance mechanisms.
The central nervous system (CNS) and immune systems regulate each other; the immune system is regulated by the CNS through hormonal signals and neuronal pathways and conversely the CNS is regulated by the immune system through cellular pathways and molecular signals including cytokines, chemokines, and interleukins (ILs). The CNS regulates the immune system through several routes: the hypothalamic-pituitary-adrenal (HPA) axis, the autonomic nervous system, and the peripheral nervous system. This neuroendocrine regulation of immune function through resultant glucocorticoid release is essential for survival from stress, infection, or inflammatory diseases. Glucocorticoids function through the glucocorticoid receptor (GR) to elicit multiple effects on immune cells and molecules. This chapter will mainly focus on the regulation of the immune response by the neuroendocrine system and the implications for inflammatory disease. Interruptions of this regulatory loop at multiple levels predispose and enhance inflammatory diseases and cause glucocorticoid resistance in a number of diseases.
Patients with glucocorticoid (CC)-dependent asthma present an ongoing inflammation of the airways despite chronic long-term treatment with oral GC. Interleukin (IL)-8 and granulocyte/macrophage colony-stimulating factor (GM-CSF) have been implicated in airway inflammation in severe asthma and their synthesis is normally repressed by GC. To further characterize the inflammatory process in GC-dependent asthma, we measured the release of IL-8 and CM-CSF by peripheral blood mononuclear cells (PBMC) of eight normal subjects, six untreated controlled asthmatics, six untreated uncontrolled asthmatics, and nine CC-dependent asthmatics. We show that PBMC from CC-dependent asthmatics released high amounts of these cytokines despite chronic in vivo exposure to CC (p < 0.001 versus normal subjects). In contrast, when untreated uncontrolled asthmatics were given a short course of oral CC, IL-8 and CM-CSF production was inhibited (p = 0.0078). Release of IL-8 and GM-CSF by PBMC of CC-dependent asthmatics was reduced after in vitro GC treatment (p < 0.002). We investigated whether the incapacity of CC to inhibit production of these cytokines in vivo was the result of a dysregulation of the glucocorticoid receptor (GR) in CC-dependent asthma. GR alpha and GR beta are, respectively, the functional receptor and a putative dominant negative form of the receptor. Western blot and polymerase chain reaction (PCR) analyses indicated that GR alpha was expressed at similar level in all groups and was largely predominant over GR beta. Thus, persistent release of IL-8 and CM-CSF in CC-dependent asthma is not associated with low expression of GR alpha or overexpression of GR beta.
Overweight is associated with the N363S variant in the glucocorticoid receptor (encoded by nuclear receptor subfamily 3, group C, member 1 gene: NR3C1). The present study examined whether the N363S polymorphism might also be associated with coronary artery disease (CAD). This involved 556 patients with CAD, of which 437 were analyzed, and 302 control subjects, all being of Anglo-Celtic descent residing in Sydney. An extensive range of phenotypic parameters was collected from the patients, and leukocyte DNA from all subjects was genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis for the A1218G (N363S) variant. Frequency of the S363 allele was 0.04 in healthy normal-weight control subjects but was 0.15 in patients with CAD (P=2.0x10(-5)) and was also elevated in subjects with CAD who were not overweight (0.14) (P=2.6x10(-5)), supporting a primary association with CAD. Frequency of S363 allele carriers in subjects with CAD who had angina was particularly high: unstable angina (0.45), stable angina (0.29), and no angina (0.26) (P for trend=0.016). Elevated cholesterol (P=0.027), triglycerides (P=0.005), and total cholesterol/HDL ratio (P=0.011), after Bonferroni, tracked with the S363 allele, consistent with accentuation of mechanisms that predispose to atheroma formation in coronary vessels. The data suggest a role for glucocorticoid receptor variation in the underlying cause of CAD.
Following exposure to stress, cortisol is secreted from the adrenal cortex under the control of the hypothalamic-pituitary-adrenal axis (HPA-axis). Central in the regulation of the HPA-axis is a two tied corticosteroid-receptor system, comprised of high and low affinity receptors, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), respectively. In addition, these corticosteroid receptors mediate the effects of cortisol during stress on both central and peripheral targets. Cortisol modulates gene-expression of corticosteroid-responsive genes, with the effect lasting from hours to days. Mutations in the GR-gene are being associated with corticosteroid resistance and haematological malignancies, although these mutations are relatively rare and probably not a common cause of these diseases. However, several GR-gene variants and single nucleotide polymorphisms (SNP) in the GR-gene have been identified which are relatively common in the human population. The GRbeta-variant, for example, has been proposed to influence corticosteroid-sensitivity and most evidence has been derived from the immune system and in particular asthma. With respect to polymorphisms, a BclI restriction fragment polymorphism and a Asp363Ser have been described, which not only influence the regulation of the HPA-axis, but are also associated with changes in metabolism and cardiovascular control. These associations of a GR-gene polymorphism with metabolism and cardivascular control, and also with the regulation of the HPA-axis, indicates an important underlying role of cortisol in the etiology of these complex disorders. Therefore, we propose that a common underlying defect in these complex disorders is a disregulation of the HPA-axis, especially during stress. The clinical implication is that the regulation of the HPA-axis should be envisioned as a primary target of new drugs for the treatment of stress-related disorders.
Glucocorticoids are steroid hormones that are secreted by the adrenal gland in a diurnal rhythm and after acute stress. They have diverse effects ranging from altering an organism’s metabolism and behavior to the function of its immune system. Clinically, they are used to treat a wide variety of diseases, including allergic and autoimmune diseases like asthma and rheumatoid arthritis. They are generally effective therapies against these pathologies because of their well-known anti-inflammatory effects. The actions of glucocorticoids are mediated by an intracellular receptor, the glucocorticoid receptor (GR). The GR is a member of the steroid receptor subfamily that includes the mineralocorticoid, progesterone, androgen, and estrogen receptors. In turn, the steroid receptors belong to the nuclear receptor superfamily, which includes the thyroid, retinoid, and vitamin-D receptors. Upon activation by their ligand, these receptors can act as transcription factors, i.e., they can activate or repress the transcription of specific target genes.
The ability of natural and synthetic glucocorticoids to elicit numerous and diverse physiological responses is remarkable. How the product of a single gene can participate in such a myriad of cell- and tissue-specific pathways has remained largely unknown. The last several years have seen increased description of glucocorticoid receptor (GR) protein isoforms. Here we review the current state of knowledge regarding naturally occurring GR isoforms and discuss how this array of receptor species generates the diversity associated with the glucocorticoid response. We propose that the multiplicity of receptor forms have unique tissue- specific actions on the downstream biology providing a mechanism to create GR signaling networks.
The human glucocorticoid receptor isoforms GRalpha and GRbeta are generated by alternative splicing. Upon hormone binding, GRalpha regulates positively or negatively transcription. In particular, it represses numerous genes encoding pro-inflammatory mediators by inhibiting the transcription factors activator protein (AP)-1 and nuclear factor (NF)-kappaB. The observation that GRbeta, which does not bind the hormone, may act as a dominant negative receptor is subject to controversy. Because GRbeta must be more abundant than GRalpha to act as such, we evaluated the relative amounts of GRalpha and GRbeta in COS-1, A549 and HeLa cells using a monoclonal antibody that recognises the two isoforms equally well on western blots. Messenger RNA levels of GRalpha and GRbeta were compared by reverse transcriptase polymerase chain reaction analysis. To gain insight into the possible function of GRbeta, we examined the ability of overexpressed GRbeta to alter transcription of glucocorticoid, AP-1 and NF-kappaB inducible reporter genes using transient transfection in COS-1 and A549 cells. Subcellular localisation of GRbeta was determined in A549 cells by immunofluoresence microscopy. Data indicate that GRalpha is the predominant endogenous isoform in A549 and HeLa cells. GRbeta became the major form after transfection with the corresponding expression vector and translocated into cell nuclei even in the absence of hormone. Overexpression of GRbeta inhibited glucocorticoid-induced transcription markedly in COS-1 cells but weakly in A549 cells. We found that GRbeta did not act as a dominant negative modulator of GRalpha for repression of AP-1 and NF-kappaB activities. In fact, both GRbeta and GRalpha inhibited hormone-independently these activities by 25-60%. This property was not shared by the closely related mineralocorticoid receptor. Our results suggest that overexpression of either GRalpha or GRbeta may have an anti-inflammatory effect.
In experimentally produced congenital diaphragmatic hernia (CDH), antenatal glucocorticoids have been shown to improve morphologic and biochemical lung immaturity and normalize the thickened pulmonary vascular wall. The action of glucocorticoids on a target tissue is mediated by glucocorticoid receptors (GRs), which have 2 isoforms; GRalpha binds glucocorticoids and acts as a ligand-dependent transcription factor, and GRbeta does not bind glucocorticoids and acts as an inhibitor of GRalpha. The aim of this study was to examine the expression of GR gene and its isoforms in the CDH lung.
RNA was extracted from archival lung tissue of 11 patients (mean age, 3.5 days) with CDH. Five age-matched newborns (mean age, 13.5 days) with sudden death syndrome served as control. Reverse transcription (RT) and polymerase chain reaction (PCR) was performed using primers specific to the common region of GR, GRalpha, and GRbeta. Soluble enzyme immunohistochemistry was carried out using polyclonal antibodies to GRalpha.
Relative mRNA levels of GR, GRalpha, and GRbeta as detected by RT-PCR were increased significantly in CDH lung compared with controls. GRalpha immunoreactivity confined only to the cytoplasm of the cells was markedly increased in the epithelium and interstitial cells in the CDH lung compared with controls.
The findings of increased mRNA expression of GR and particularly of its isoform GRalpha in the CDH lung suggests that GR may play an important role in regulating target cell responsiveness to glucocorticoids in the hypoplastic lung.
To study the occurrence and function of polymorphism in the human glucocorticoid receptor (hGR) gene in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).
We used single stranded conformation polymorphism (SSCP) and direct sequencing to study the hGR gene in 30 patients with RA, 40 with SLE, and 24 controls. A newly identified polymorphism was transfected in COS-1 cells and the stability of the mRNA containing the polymorphism was tested using real-time PCR.
A polymorphism in the hGR gene in exon9beta, in an "ATTTA" motif, was found to be significantly associated with RA. Introduction of this polymorphism in the hGRb mRNA was found to significantly increase stability in vitro compared to the wild-type sequence.
Our findings show an association between RA and a previously unreported polymorphism in the hGR gene. This polymorphism increased stability of hGRbeta mRNA, which could contribute to an altered glucocorticoid sensitivity since the hGRbeta is thought to function as an inhibitor of hGRalpha activity.
Glucocorticoids play an important role in the treatment of a number of hematological malignancies, such as multiple myeloma. The effects of glucocorticoids are mediated through the glucocorticoid receptor alpha, the abundance of which can be modulated by alternative splicing of the glucocorticoid receptor mRNA. Two splice variants of the glucocorticoid receptor mRNA have been described: glucocorticoid receptor beta, which reportedly has a dominant negative effect on the actions of the glucocorticoid receptor alpha, and glucocorticoid receptor P, of which the effects are unknown. In this study, we have investigated the expression levels of these two splice variants at the mRNA level in multiple myeloma cells and in a number of other hematological tumors. Although the glucocorticoid receptor beta mRNA was, if at all, expressed at very low levels, considerable amounts (up to 50% of the total glucocorticoid receptor mRNA) glucocorticoid receptor P mRNA was present in most hematological malignancies. In transient transfection studies in several cell types and in multiple myeloma cell lines, the glucocorticoid receptor P increased the activity of the glucocorticoid receptor alpha. These results suggest that the relative levels of the glucocorticoid receptor alpha and the glucocorticoid receptor P may play a role in the occurrence of glucocorticoid resistance in tumor cells during the treatment of hematological malignancies with glucocorticoids.
Patients with glucocorticoid (GC)-dependent asthma present an ongoing inflammation of the airways despite chronic long-term treatment with oral GC. Interleukin (IL)-8 and granulocyte/macrophage colony-stimulating factor (GM-CSF) have been implicated in airway inflammation in severe asthma and their synthesis is normally repressed by GC. To further characterize the inflammatory process in GC-dependent asthma, we measured the release of IL-8 and GM-CSF by peripheral blood mononuclear cells (PBMC) of eight normal subjects, six untreated controlled asthmatics, six untreated uncontrolled asthmatics, and nine GC-dependent asthmatics. We show that PBMC from GC-dependent asthmatics released high amounts of these cytokines despite chronic in vivo exposure to GC (p < 0.001 versus normal subjects). In contrast, when untreated uncontrolled asthmatics were given a short course of oral GC, IL-8 and GM-CSF production was inhibited (p = 0.0078). Release of IL-8 and GM-CSF by PBMC of GC-dependent asthmatics was reduced after in vitro GC treatment (p < 0.002). We investigated whether the incapacity of GC to inhibit production of these cytokines in vivo was the result of a dysregulation of the glucocorticoid receptor (GR) in GC-dependent asthma. GRalpha and GRbeta are, respectively, the functional receptor and a putative dominant negative form of the receptor. Western blot and polymerase chain reaction (PCR) analyses indicated that GRalpha was expressed at similar level in all groups and was largely predominant over GRbeta. Thus, persistent release of IL-8 and GM-CSF in GC-dependent asthma is not associated with low expression of GRalpha or overexpression of GRbeta.
Glucocorticoids remain the first-line treatment for a range of autoimmune and allergic diseases. However, 30% of patients fail to achieve disease control at tolerable systemic doses and continue to have an increased immune response with poor clinical outcome. This steroid refractory (SR) phenotype has previously been attributed to enhanced expression of inactive glucocorticoid receptor isoforms and cytokine-mediated suppression of glucocorticoid (GC) signaling, in particular by interleukin-2. These mechanisms are discussed, with emphasis on recent evidence for the role of the CD4(+)CD25(int) and GC-induced T regulatory cell subsets in perpetrating SR disease.
Inhaled and intranasal glucocorticoids (GCs) are the most common and effective drugs for controlling symptoms and airway inflammation in respiratory diseases such as allergic rhinitis, chronic rhinosinusitis with/without nasal polyps, and asthma, and the respiratory epithelium is a primary target of GC anti-inflammatory actions. GC effects are mediated through the GC receptor (GR). In humans, one single GR gene gives rise to two main GR products, namely GRalpha and GRbeta, which are subject to translational and posttranslational modifications. GRalpha is expressed in virtually all human cells and tissues, including respiratory epithelial cells, and - at least in vitro - is downregulated by GC. GRalpha mediates the anti-inflammatory actions of GC by activating transcription of anti-inflammatory genes through binding of GRalpha to glucocorticoid response elements (GRE) located in the promoter region of target genes, repressing transcription of proinflammatory genes through direct interaction between GRalpha and proinflammatory transcription factors, such as AP-1 and NF-kappaB (transrepression), and also by destabilizing the mRNA of proinflammatory mediators. GRbeta acts as a dominant negative inhibitor of GRalpha-mediated transactivation and transrepression in certain in vitro studies with transfected cells. The GRbeta message is expressed at low levels in numerous tissues and its protein is mainly expressed in inflammatory cells, although it has also been detected in airway epithelial cells. Increased GRbeta expression has been reported in bronchial asthma and nasal polyposis, and after incubation of cells with certain proinflammatory stimuli. However, the role of GRbeta in modulating GC sensitivity in vivo has been highly debated and is as yet unclear.
Glucocorticoids (GCs) are the most common and effective drugs for treating inflammatory airway diseases, but some patients respond poorly to them. GC effects are mediated through the glucocorticoid receptor (GR). We present an update on the GR gene, the GR alpha and GR beta splicing variants, their translational and post-translational modifications, as well as their alterations in disease. GR alpha is ubiquitously expressed and is responsible for the induction and repression of target genes. GR beta acts as a dominant negative inhibitor of GR alpha-mediated transactivation and transrepression in certain cell types. The GR beta message is expressed at low levels in numerous tissues and its protein is only expressed in specific cell types. Increased GR beta expression has been reported in bronchial asthma, nasal polyposis and inflammatory bowel diseases (IBD), and after incubation of cells with certain proinflammatory stimuli. In addition to GR beta, other mechanisms explaining GC resistance include alterations in GR binding to ligand, nuclear translocation, and binding to GRE, and/or a defective cross-talk with transcription factors and cofactors.
TNF is a Janus-faced protein. It possesses impressive anti-tumor activities, but it is also one of the strongest known pro-inflammatory cytokines, which hampers its use as a systemic anti-cancer agent. TNF has been shown to play a detrimental role in inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Glucocorticoids are strongly anti-inflammatory and exert their therapeutic effects through binding to their receptor, the glucocorticoid receptor. Therefore, glucocorticoids have been used for over half a century for the treatment of inflammatory diseases. However, many patients are or become resistant to the therapeutic effects of glucocorticoids. Inflammatory cytokines have been suggested to play an important role in this steroid insensitivity or glucocorticoid resistance. This review aims to highlight the mechanisms of mutual inhibition between TNF and GR signaling pathways.
Glucocorticoids regulate a wide range of systems in vertebrate organisms, and their effects are mediated by the glucocorticoid receptor (GR). The responsiveness to glucocorticoids differs largely between individuals. Resistance to glucocorticoids is an important medical problem, since it limits the efficacy of glucocorticoids when they are used to treat immune-related diseases like asthma and rheumatoid arthritis. Glucocorticoid resistance also contributes to the pathogenesis of other diseases, like major depression because of the decreased negative feedback on the hypothalamic pituitary adrenal axis. In this review, we present the zebrafish as an excellent in vivo model system to study glucocorticoid resistance. First, the zebrafish is the only non-primate animal model in which a beta-isoform of GR occurs, which is a splice variant with dominant-negative activity. Zebrafish are easily genetically modified, so the expression of GRbeta can be varied, creating an in vivo model for GRbeta-induced glucocorticoid resistance. Second, by performing a forward-genetic screen using the glucocorticoid-induced decrease in POMC expression in the pituitary gland as a readout, several zebrafish mutants have been obtained which appear to be resistant to glucocorticoid treatment. We present here two types of in vivo models for studying glucocorticoid resistance, that will be used to study the molecular mechanism of glucocorticoid signaling and resistance. Finally these models will be used to screen for small molecules that can alleviate glucocorticoid resistance.
To investigate the expression and quantity of glucocorticoid receptor-alpha and -beta in polyp tissues taken from the patients treated were subsequently treated with topical glucocorticoid (GC).
Eighty patients with nasal polyps were initially enrolled in the study. All polyp specimens were obtained prior to treatment. Patients then received daily topical GC spray treatment for one month. Polyp specimens were tested for glucocorticoid receptor (GR) GR-alpha and GR-beta mRNA expression using fluorescent quantitative-reverse transcription-polymerase chain reaction (FQ-RT-PCR). Thirty healthy nasal mucosa tissue samples were tested at the same time.
Forty patients finished the study and were divided into two groups: GC-sensitive (n=26) and GC-insensitive (n=14), according to treatment results. GR-beta mRNA expression in the nasal polyp tissues of the GC-insensitive group (5.72+/-0.58x10(2) copies/microg) was higher than that in the GC-sensitive group (4.82+/-0.28x10(2) copies/microg, P < 0.05) and in the normal nasal mucosa group (4.44+/-0.35x10(2) copies/microg, P < 0.01). There was also a difference in the relative expression of GR-alpha and GR-beta between the GC-sensitive group (GR-alpha/GR-beta= 829.42+/-67.36) and the GC-insensitive group (535.7+/-89) (P < 0.01).
GR-beta mRNA was highly expressed in patients with nasal polyps. Down- regulation of GR-alpha mRNA suggests the existence of glucocorticoid insensitivity. Expression of GR-beta may plays an important role in the evaluation of the glucocorticoid therapeutic effect in patients with nasal polyps.
Limited proteolysis of in vitro produced human androgen receptor was used to probe the different conformations of the receptor after binding of androgens
and several antiandrogens. The results provide evidence for five different conformations of the receptor, as detected by the
formation of proteolysis resisting fragments: 1) an initial conformation of the unoccupied receptor not resisting proteolytic
attack; and receptor conformations characterized by 2) a 35-kDa proteolysis resisting fragment spanning the ligand binding
domain and part of the hinge region, obtained with most antagonists, and in an initial step after agonist binding; 3) a 29-kDa
proteolysis resisting fragment spanning the ligand binding domain, obtained in the presence of agonists after an activation
process; 4 and 5) 30- and 25-kDa fragments, derived from 2 and 3, but missing part of the C terminus, obtained with RU486
(RU486 has antiandrogenic properties, besides its effects as an antiprogestagen/antiglucocorticoid). Concomitantly with the
change from 2 to 3 (and of 4 to 5 for RU486), dissociation of the 8 S complex of receptor with associated proteins occurred.
With a mutant receptor (LNCaP cell mutation in C-terminal region), some antagonists activated transcription analogous to agonists,
and induced the activated receptor conformation 3. A mutant lacking the C-terminal 12 amino acids bound RU486 but not androgens,
and formed with RU486 conformation 5. These data imply that, after the initial rapid binding of ligand, androgens induce a
conformational change of the receptor, a process that also involves release of associated proteins. RU486 induces an inappropriate
conformation of the C-terminal end, similar as found for its effect on the progesterone receptor. In contrast, the other antiandrogens
act at a different step in the mechanism of action: they do not induce an abnormal conformation, but act earlier and prevent
a conformation change by stabilizing a complex with associated proteins.
Glucocorticoid resistance due to mutations in the gene for the glucocorticoid receptor has been suggested to be more common
than is thought at present, owing to the relative mildness of its symptoms and the difficulty of its diagnosis. To investigate
the prevalence of mutations in the glucocorticoid receptor gene responsible for relative insensitivity to glucocorticoids,
we carried out polymerase chain reaction/single-strand conformation analysis of the glucocorticoid receptor gene in a group
of 20, otherwise healthy, persons with a reduced response in a dexamethasone suppression test and in 20 controls. We did not
find mutations or polymorphisms associated with a reduced sensitivity to glucocorticoids. However, we identified five novel
polymorphisms in the gene for the human glucocorticoid receptor, which may be useful in analyzing whether loss of (part of)
the glucocorticoid receptor gene plays a role in glucocorticoid-resistant malignancies. Although relative resistance to glucocorticoids
seems to be rather frequent in otherwise healthy persons, it is not usually associated with mutations or polymorphisms in
the glucocorticoid receptor gene.
Familial glucocorticoid resistance is a hypertensive, hyperandrogenic disorder characterized by increased serum cortisol concentrations in the absence of stigmata of Cushing's syndrome. Our previous studies of the first reported kindred showed a two- to threefold reduction in glucocorticoid receptor-ligand binding affinity in the propositus, and a lesser reduction in affinity in his mildly affected son and nephew. Glucocorticoid receptor cDNA from these three patients was amplified by polymerase chain reaction and sequenced. The cDNA nucleotide sequence was normal, except for nucleotide 2054, which substituted valine for aspartic acid at amino acid residue 641. The propositus was homozygous while the other relatives were heterozygous for the mutation. COS-7 monkey kidney cells were cotransfected with expression vectors for either wild type or Val 641-mutant receptors, together with the reporter plasmid pMMTV-CAT. Dexamethasone increased chloramphenicol acetyltransferase activity in cells expressing wild type receptor, but had no effect in cells expressing Val 641-mutant receptors, despite similar receptor concentrations, as indicated by Western blotting. The binding affinity for dexamethasone of the Val 641-mutant receptor was threefold lower than that of the wild type receptor. These results suggest that glucocorticoid resistance in this family is due to a point mutation in the steroid-binding domain of the glucocorticoid receptor.
A 29-year-old woman with moderately elevated blood pressure and signs of hyperandrogenism (hirsutism and acne) but without typical Cushing's syndrome symptoms has been followed for almost 6 years. Steroid and glucocorticoid receptor studies indicated a primary glucocorticoid receptor defect. Elevated androgen values were of special interest. Clinical manifestation of hyperandrogenism seemed not to be proportional to the biochemical findings. Therefore, the possibility of a partial androgen receptor defect should also be considered.
The precise molecular abnormalities that cause primary cortisol resistance have not been completely described. In a subject with primary cortisol resistance we have observed glucocorticoid receptors (hGR) with a decreased affinity for dexamethasone. We hypothesize that a mutation of the hGR glucocorticoid-binding domain is the cause of cortisol resistance. Total RNA isolated from the index subject's mononuclear leukocytes was used to produce first strand hGR cDNAs, and the entire hGR cDNA was amplified in segments and sequenced. At nucleotide 2,317 we identified a homozygous A for G point mutation that predicts an isoleucine (ATT) for valine (GTT) substitution at amino acid 729. When the wild-type hGR and hGR-Ile 729 were expressed in COS-1 cells and assayed for [3H]-Dexamethasone binding, the dissociation constants were 0.799 +/- 0.068 and 1.54 +/- 0.06 nM (mean +/- SEM) (P < 0.01), respectively. When the wild-type hGR and hGR-Ile 729 were expressed in CV-1 cells that were cotransfected with the mouse mammary tumor virus long terminal repeat fused to the chloramphenicol acetyl transferase (CAT) gene, the hGR-Ile 729 conferred a fourfold decrease in apparent potency on dexamethasone stimulation of CAT activity. The isoleucine for valine substitution at amino acid 729 impairs the function of the hGR and is the likely cause of primary cortisol resistance in this subject.
Alternative splicing of the human glucocorticoid receptor (hGR) pre-mRNA generates two highly homologous isoforms, termed hGR alpha and hGR beta. hGR alpha 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 (GRE) DNA sequences. In contrast, hGR beta does not bind glucocorticoids and is transcriptionally inactive. We demonstrate here that hGR beta is able to inhibit the effects of hormone-activated hGR alpha on a glucocorticoid-responsive reporter gene in a concentration-dependent manner. [3H]-Dexamethasone binding studies indicate that hGR beta does not alter the affinity of hGR alpha for its hormonal ligand. The presence of hGR beta in nuclear extracts and its ability to bind to a radiolabeled GRE oligonucleotide suggest that its inhibitory effect may be due to competition for GRE target sites. Reverse transcription-PCR analysis shows expression of hGR beta mRNA in multiple human tissues. These results indicate that hGR beta may be a physiologically and pathophysiologically relevant endogenous inhibitor of glucocorticoid action, which may participate in defining the sensitivity of target tissues to glucocorticoids. They also underline the importance of distinguishing between the two receptor isoforms in all future studies of hGR function and the need to revisit old data.
Alternative splicing of the transcripts of the human glucocorticoid receptor gene results in two mutually exclusive products, the classic, ligand-binding glucocorticoid receptor (hGR alpha), and a dominant negative non-ligand-binding isoform, hGR beta.
We examined the existence of and quantified both hGR alpha and hGR beta isoforms in a panel of human tissues, as well as in intact and fractionated HeLa cells, using specific quantitative Western blots and/or immunocytochemistry. We studied the potential interactions of hGR beta with heat shock protein (hsp) 90 and/or hGR alpha using cross-immunoadsorption/precipitation procedures followed by Western blots.
For the first time, we demonstrated the natural existence of the hGR beta protein, which was widely expressed in human tissues. The ratio of immunoreactive hGR alpha to hGR beta varied from 0.2 to 1.0 among different tissues, and was approximately 0.2 in HeLa cells. In the latter, both isoforms were distributed in the cytoplasm and nucleus in the absence of the hormonal ligand, and translocated into the nucleus after addition of dexamethasone. The cytosolic and nuclear hGR alpha-to-hGR beta ratio remained the same before and after dexamethasone exposure, suggesting that upon activation the two isoforms translocated into the nucleus in equal proportions. hGR alpha- and hGR beta-specific antibodies cross-adsorbed and precipitated cytosolic and nuclear glucocorticoid hGR alpha and hGR beta, respectively, as well as hsp90, suggesting that hGR alpha and hGR beta are in complex with hsp90 and/or each other.
The hGR beta protein is widely expressed throughout the human body and present mostly in the cytoplasm of human cells, in complex with hsp90 and other proteins. In the presence of glucocorticoid, hGR beta probably heterodimerizes with ligand-bound hGR alpha and translocates into the nucleus to act as a dominant negative inhibitor of the classic receptor.
The molecular mechanisms underlying primary glucocorticoid resistance or hypersensitivity are not well understood. Using transfected COS-1 cells as a model system, we studied gene regulation by naturally occurring mutants of the glucocorticoid receptor (GR) with single-point mutations in the regions encoding the ligand-binding domain or the N-terminal domain reflecting different phenotypic expression. We analyzed the capacity of these GR variants to regulate transcription from different promoters, either by binding directly to positive or negative glucocorticoid-response elements on the DNA or by interfering with protein-protein interactions. Decreased dexamethasone (DEX) binding to GR variants carrying mutations in the ligand-binding domain correlated well with decreased capacity to activate transcription from the mouse mammary tumor virus (MMTV) promoter. One variant, D641V, which suboptimally activated MMTV promoter-mediated transcription, repressed a PRL promoter element containing a negative glucocorticoid-response element with wild type activity. DEX-induced repression of transcription from elements of the intercellular adhesion molecule-1 promoter via nuclear factor-kappaB by the D641V variant was even more efficient compared with the wild type GR. We observed a general DEX-responsive AP-1-mediated transcriptional repression of the collagenase-1 promoter, even when receptor variants did not activate transcription from the MMTV promoter. Our findings indicate that different point mutations in the GR can affect separate pathways of gene regulation in a differential fashion, which can explain the various phenotypes observed.
Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript generates two receptor isoforms, hGRalpha and hGRbeta, with different carboxyl termini diverging at amino acid 727. By reverse transcriptase-polymerase chain reactions it was previously demonstrated that the hGRbeta message had a widespread tissue distribution. To demonstrate the presence of hGRbeta as protein we produced specific rabbit antisera to hGRbeta, as well as a hGRbeta-specific mouse monoclonal IgM antibody, by peptide immunizations. By SDS-polyacrylamide gel electrophoresis and Western immunoblotting we showed that hGRbeta is endogenously expressed at the protein level in HeLa cells and human lymphatic leukemia cells. Using an antibody directed against an epitope shared by both isoforms we showed a relatively lower expression of the hGRbeta form. We also showed that hGRbeta bound to hsp90 by immunoprecipitation of in vitro translated hGRbeta in reticulocyte lysate with hsp90-specific antibodies, a coprecipitation occurring also in the presence of dexamethasone. We could not demonstrate that hGRbeta inhibited the effects of dexamethasone-activated hGRalpha on a glucocorticoid-responsive reporter gene. In conclusion, low hGRbeta expression levels and hGRbeta-hsp90 interaction maintained in the presence of ligand and lack of inhibition of hormone-activated hGRalpha effects challenge the concept of the hGRbeta isoform as a proposed dominant negative inhibitor of hGRalpha activity.
We investigated whether a polymorphism at nucleotide position 1220, resulting in an asparagine-to-serine change at codon 363 in the glucocorticoid receptor (GR) gene is associated with an altered sensitivity to glucocorticoids. In a group of 216 elderly persons, 13 heterozygotes for the N363S polymorphism were identified by PCR/single strand conformation polymorphism analysis. In 2 dexamethasone (DEX) suppression tests (DSTs), using 1 and 0.25 mg DEX, the circulating cortisol and insulin concentrations were compared between N363S carriers and controls. In the 1-mg DST, there were no differences between N363S carriers and controls, with respect to adrenal suppression, but there was a significantly higher (P < 0.05) insulin response in N363S carriers. In the 0.25-mg DST, a significantly larger (P < 0.05) cortisol suppression and higher (P < 0.05) insulin response were seen in N363S carriers. Comparison of blood pressure, body mass index (BMI), and bone mineral density (BMD) between the N363S carriers and controls showed that N363S carriers had a higher (P < 0.05) BMI but normal blood pressure. There was an obvious trend towards lower age-, BMI-, and sex-adjusted BMD in the lumbar spine in N363S carriers. GR characteristics measured in 41 controls and 9 N363S carriers in peripheral mononuclear leucocytes showed no differences between N363S carriers and controls, with respect to GR number and ligand binding affinity. However, there was a trend towards greater sensitivity to DEX in the carriers' lymphocytes, in a mitogen-induced cell proliferation assay. In transfection assays, the capacity of the codon 363 variant to activate mouse mammary tumor virus promotor-mediated transcription in COS-1 cells was unaltered, when compared with the wild-type GR. We conclude that in 6.0% of our study population, a polymorphism in codon 363 of the GR gene was found. Individuals carrying this polymorphism seemed healthy at clinical examination but had a higher sensitivity to exogenously administered glucocorticoids, with respect to both cortisol suppression and insulin response. Life-long exposure to the mutated allele may be accompanied by an increased BMI and a lowered BMD in the lumbar spine but does not affect blood pressure.
Glucocorticoid resistance results from the partial, albeit apparently generalized, inability of glucocorticoids to exert their effects on target tissues. The coids to exert their effects on target tissues. The condition is associated with compensatory increases in circulating pituitary corticotropin and cortisol, with the former causing excess secretion of both adrenal androgens and adrenal steroid biosynthesis intermediates with salt-retaining activity
Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two highly homologous protein isoforms, termed hGRa and hGRb, that differ at their carboxy-termini. In contrast to the well characterized hGRa isoform, which modulates gene expression in a hormone-dependent fashion, the biological sig- nificance of hGRb has only recently begun to emerge. We and others have shown that the hGRb messenger RNA transcript is widely ex- pressed in human tissues and that the hGRb protein functions as a dominant negative inhibitor of hGRa in transfected cells. Unfortu- nately, these initial studies did not determine whether the hGRb protein was made in vivo. Such analyses are hindered because avail- able anti-hGR antibodies cannot discriminate between the similarly sized hGRa and hGRb proteins. Therefore, to investigate the expres- sion of the hGRb protein, we have produced an antipeptide, hGRb- specific antibody termed BShGR. This antibody was made against the unique 15-amino acid peptide at the carboxy-terminus of hGRb and recognizes both the native and denatured conformations of hGRb, but does not cross-react with hGRa. Using BShGR on Western blots and in immunoprecipitation experiments, we detected the hGRb protein in a variety of human cell lines and tissues. Immunocytochemistry was then performed with BShGR on HeLa S3 and CEM-C7 cells and on tissue sections prepared from lung, thymus, and liver to assess the cellular and subcellular distribution of hGRb. In all immunopositive cells, hGRb was found in the nucleus independent of glucocorticoid treatment. Within tissues, the hGRb protein was expressed most abundantly in the epithelial cells lining the terminal bronchiole of the lung, forming the outer layer of Hassall's corpuscle in the thymus, and lining the bile duct in the liver. As a potential in vivo inhibitor of hGRa activity, expression of hGRb may be an important factor regulating target cell responsiveness to glucocorticoids. (Endocrinology 138: 5028 -5038, 1997)
Primary (partial) cortisol receptor resistance was previously reported in a total of 7 patients and 14 asymptomatic family members. Its occurrence is considered to be extremely rare. In the present study we report on 6 patients (2 males and 4 females) with the syndrome. The first male patient presented with mild hypertension. Hydrochlorothiazide therapy resulted in life-threatening hypokalemia. The second male patient had slight hypertension without hypokalemia. All four female patients presented between the age of 20-30 yr with acne, hirsutism, and irregular menstruations. Low dose dexamethasone therapy (1-1.5 mg/day) was of clinical benefit in these patients. All patients showed insufficient suppression of serum cortisol concentrations in the overnight 1-mg dexamethasone test. The diurnal rhythm of ACTH and cortisol was intact, albeit at an elevated level. There was a normal increase in ACTH, cortisol, and GH (except in one obese patient) in response to insulin-induced hypoglycemia, while cortisol production was elevated in three patients. Circulating adrenal androgen levels were increased in all patients. Glucocorticoid receptors were investigated in a whole cell dexamethasone binding assay in mononuclear leukocytes. In the first male patient, the number of receptors was very low, while the affinity was lower than that in controls. A lowered affinity to dexamethasone was found in one female patient, while a lowered number of receptors was found in three patients. In the second male patient, no abnormalities were found. As a bioassay for glucocorticoid action we also measured dexamethasone suppressibility of mitogen-stimulated incorporation of [3H]thymidine in mononuclear leukocytes. In the male patient with normal receptor status, dexamethasone suppressibility of [3H]thymidine incorporation was significantly lower than that in healthy controls with respect to both maximal suppression and IC50. Partial cortisol receptor resistance might be less rare than previously thought. In the six patients presented, at least three different forms can be recognized. Therapy with dexamethasone was successful in female patients with acne and hirsutism, as the secondary increase in the production of adrenal androgens was effectively controlled.
A retrospective survey was accomplished on 420 consecutive patients who had undergone dexamethasone suppression tests between 1975-1988 due to suspected adrenal disorders. We found 7 patients in whom glucocorticoid resistance was apparent. They showed 4-6 abnormalities of the 7 investigations used: insensitivity to dexamethasone inhibition (n = 7), increased urinary cortisol (n = 3), glucocorticoid receptor (GR) thermolability (n = 4), decreased number of glucocorticoid receptors (n = 4), abnormal ligand affinity of GR (n = 4), abnormal basal GR mRNA expression (n = 4), and abnormal down-regulation of basal GR mRNA levels by dexamethasone (n = 1). The four patients with GR thermolability also showed increased basal GR mRNA levels. In the other patients the number of GR per cell was decreased without an up-regulation of GR mRNA. It is concluded that the syndromes of glucocorticoid resistance vary notably, clinically as well as biochemically; in patients evaluated for adrenocortical disorders the syndrome is apparently encountered in 1-2% of the patients.
The hormone-induced transformation process of the androgen receptor in the androgen-responsive human prostatic carcinoma cell line LNCaP was studied. Immunoprecipitation of the nontransformed cytosolic receptor (8S on sucrose gradients) with a specific monoclonal antibody (F39.4.1) resulted in coprecipitation of three heat-shock proteins (hsp90, hsp70, and hsp56). Upon incubation of the cells with the synthetic androgen R1881, the sedimentation value of the receptor complex decreased to an intermediate form of 6S, and an almost complete loss of coprecipitating heat-shock proteins was observed. After a 2-h incubation, the receptor was recovered in considerable part from the nuclear fraction (extraction with high salt; 4.6S form). By use of the bifunctional cross-linker dimethyl pimelimidate, dissociation of the 8S complex, but not of the 6S complex, was blocked. A newly developed monoclonal antibody (F52.24.4), directed against the C-terminal part of the DNA-binding domain of the androgen receptor, specifically recognized both the 4.6S and the 6S forms of the receptor but did not react with the nontransformed 8S form. It is concluded that the unoccupied androgen receptor is associated with several heat-shock proteins and that transformation of the receptor to the tight nuclear-binding form is a multistep process that involves the dissociation of heat-shock proteins from the receptor.
Antibodies against the N-terminal domain of the human androgen receptor (hAR) were prepared by two different approaches. Firstly, rabbits were immunized with a beta-galactosidase-hAR (amino acids (aa) 174-353) fusion protein. Secondly, two synthetic peptides corresponding to potentially antigenic sites located within this fragment (aa 201-222 and 301-320) were used as immunogens. The obtained antisera contained high titer anti-hAR antibodies as was established with several independent methods (e.g. sucrose gradient centrifugation, immunoprecipitation, Western blotting). The two anti-peptide antisera specifically stained nuclei of glandular epithelial cells in frozen sections of human prostate tissue. Progesterone, estradiol and glucocorticoid receptors were not immunoprecipitated with these antisera. The specific hAR antibodies provide new tools for the characterization of this steroid receptor as well as for diagnostic purposes in pathology of the human prostate and androgen resistance.
A 26-yr-old woman presented with hirsutism, male pattern scalp baldness ("geheimratsecken"), and menstrual irregularities. She had no hypertension or other signs and symptoms of Cushing's syndrome. Plasma cortisol levels were greatly elevated and did not suppress normally in response to dexamethasone. Cortisol binding to transcortin was normal. Plasma androstenedione and testosterone levels were also increased, but 17-hydroxyprogesterone and aldosterone levels were normal. Further studies revealed an increased cortisol production rate, increased 24-h urinary cortisol excretion, increased plasma ACTH levels, a normal diurnal rhythm of cortisol at an elevated level, and normal increments of plasma ACTH, cortisol, GH, and PRL in response to insulin-induced hypoglycemia. The father and two brothers also had increased plasma cortisol levels, which did not suppress normally in response to dexamethasone. Chronic therapy with dexamethasone (at first 1 and later 0.5 mg, three times daily) for more than 30 weeks resulted in decreased hirsutism, normalization of scalp hair and menstrual cyclicity, and normal plasma testosterone and androstenedione levels. No signs or symptoms of Cushing's syndrome developed, and the central regulation of secretion of ACTH, cortisol, GH, and PRL (insulin test, diurnal rhythm) remained qualitatively normal at a lower set-point. We conclude that this patient had autosomal dominantly inherited hereditary (partial) cortisol insensitivity, which had resulted in increased adrenocortical cortisol and androgen secretion. The latter had not resulted in clinical symptoms in the three afflicted male members of the family, but had in the propositus. The results also indicate the potential usefulness of the insulin test in distinguishing this disorder from Cushing's disease.
Glucocorticoid resistance results from the partial, albeit apparently generalized, inability of glucocorticoids to exert their effects on target tissues. The condition is associated with compensatory increases in circulating pituitary corticotropin and cortisol, with the former causing excess secretion of both adrenal androgens and adrenal steroid biosynthesis intermediates with salt-retaining activity. The manifestations of glucocorticoid resistance vary from chronic fatigue (perhaps a result of glucocorticoid deficiency in the central nervous system) to various degrees of hypertension with or without hypokalemic alkalosis or hyperandrogenism, or both, caused by increased cortisol and other salt-retaining steroids and adrenal androgens, respectively. In women, hyperandrogenism can result in acne, hirsutism, menstrual irregularities, oligoanovulation, and infertility; in men, it may lead to infertility and in children, to precocious puberty. Different molecular defects, such as point mutations or a microdeletion of the highly conserved glucocorticoid receptor gene, alter the functional characteristics or concentrations of the intracellular receptor and appear to cause glucocorticoid resistance. The extreme variability in the clinical manifestations of glucocorticoid resistance and its mimicry of many common diseases can be explained by the overall degree of glucocorticoid resistance, differing sensitivity of target tissues to mineralocorticoids or androgens or both, and perhaps different biochemical defects of the glucocorticoid receptor, with selective resistance of certain glucocorticoid responses in specific tissues. The various different symptoms of classic glucocorticoid resistance and the theoretical potential of this condition to appear surreptitiously emphasize the importance of the glucocorticoid receptor in the pathogenesis of human disease.
The clinical syndrome of generalized, compensated glucocorticoid resistance is characterized by increased cortisol secretion without clinical evidence of hyper- or hypocortisolism, and manifestations of androgen and/or mineralocorticoid excess. This condition results from partial failure of the glucocorticoid receptor (GR) to modulate transcription of its target genes. We studied the molecular mechanisms of this syndrome in a Dutch kindred, whose affected members had hypercortisolism and approximately half of normal GRs, and whose proband was a young woman with manifestations of hyperandrogenism. Using the polymerase chain reaction to amplify and sequence each of the nine exons of the GR gene alpha, along with their 5'- and 3'-flanking regions, we identified a 4-base deletion at the 3'-boundary of exon 6 in one GR allele (delta 4), which removed a donor splice site in all three affected members studied. In contrast, the sequence of exon 6 in the two unaffected siblings was normal. A single nucleotide substitution causing an amino acid substitution in the amino terminal domain of the GR (asparagine to serine, codon 363) was also discovered in exon 2 of the other allele (G1220) in the proband, in one of her affected brothers and in her unaffected sister. The functional importance of this mutation was tested in a cotransfection study using the recombinant expression vector pRShGR-Ser363 and the glucocorticoid responsive vector mouse mammary tumor virus-chloramphenicol transferase. This amino acid substitution did not alter the function of the glucocorticoid receptor. Using reverse transcription-polymerase chain reaction we could only identify messenger RNA transcripts of the G1220-allele but not of the delta 4-allele in the affected members of this family who were heterozygous for the G1220 mutation. This deletion in the glucocorticoid receptor gene was, thus, associated with the expression of only one allele and a decrease of GR protein by 50% in affected members of this glucocorticoid resistant family. The mutation identified in exon 2 did not segregate with the disease and appears to be of no functional significance. The presence of the null allele was apparently compensated for by increased cortisol production at the expense of concurrent hyperandrogenism.
Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two receptor isoforms, hGRalpha and hGRbeta, which differ at their carboxyl termini. The hGRalpha isoform conveys endocrine information to target tissues by altering patterns of gene expression in a hormone-dependent fashion. In contrast to hGRalpha, very little is known about the hGRbeta splice variant. Using hGRalpha- and hGRbeta-specific riboprobes on human multiple tissue Northern blots, we show that the hGRbeta message has a widespread tissue distribution. We also prove by reverse transcriptase-polymerase chain reaction that the alternative splicing event underlying the formation of the hGRbeta message occurs in these tissues. Because the hGRbeta protein differs from hGRalpha at the extreme COOH terminus, we investigated several of the biochemical properties of hGRbeta expressed in transfected cells. hGRbeta does not bind the glucocorticoid agonist dexamethasone nor the glucocorticoid antagonist RU38486 in vivo. Moreover, in contrast to hGRalpha, hGRbeta is located primarily in the nucleus of transfected cells independent of hormone administration. Finally, in the absence of hGRalpha, hGRbeta is transcriptionally inactive on a glucocorticoid-responsive enhancer. However, when both isoforms are expressed in the same cell, hGRbeta inhibits the hormone-induced, hGRalpha-mediated stimulation of gene expression. Thus, hGRbeta potentially functions as a dominant negative inhibitor of hGRalpha activity.
Generalized glucocorticoid resistance is associated with chronic hyperactivation of the hypothalamic-pituitary-adrenal axis, compensating for impaired glucocorticoid receptor function. We report a unique patient with sporadic generalized glucocorticoid resistance who, at age 33, presented with infertility and hypertension and, at 38, developed pituitary Cushing's disease. Leukocyte-binding studies revealed normal affinity of the glucocorticoid receptor but a reduction of binding sites by 50%. [3H]thymidine incorporation by this patient's lymphocytes was not suppressible by dexamethasone. He had a novel heterozygous missense mutation in the glucocorticoid receptor gene (isoleucine 559 to asparagine 559). The mutant receptor exhibited a strong dominant-negative effect on the ability of the wild-type receptor to induce gene transcription in vitro. The mutation was present in all of the patient's cultured lymphoblasts and fibroblasts as well as in 50% of his sperm, as demonstrated by single-cell polymerase chain reaction; it was not present in his parents and seven siblings. This novel mutation was thus both de novo and present in the germ line. Immunohistochemical staining of this patient's pituitary corticotropinoma revealed accumulation of p53 protein, indicating the presence of a putative somatic oncogenic mutation in the p53 gene in the tumor cells. Investigation of the lymphoblast and skin fibroblast cultures for p53 abnormalities did not show any aberration. Thus, a novel de novo germ line mutation of the glucocorticoid receptor with strong dominant-negative activity caused severe sporadic generalized glucocorticoid resistance, which preceded corticotroph adenoma formation. The latter probably was due to the combined effects of chronic corticotroph hyperstimulation, decreased glucocorticoid negative feedback, and at least one subsequent somatic defect in the control of the cell cycle.
Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two highly homologous protein isoforms, termed hGR alpha and hGRbeta, that differ at their carboxy-termini. In contrast to the well characterized hGR alpha isoform, which modulates gene expression in a hormone-dependent fashion, the biological significance of hGRbeta has only recently begun to emerge. We and others have shown that the hGRbeta messenger RNA transcript is widely expressed in human tissues and that the hGRbeta protein functions as a dominant negative inhibitor of hGR alpha in transfected cells. Unfortunately, these initial studies did not determine whether the hGRbeta protein was made in vivo. Such analyses are hindered because available anti-hGR antibodies cannot discriminate between the similarly sized hGR alpha and hGRbeta proteins. Therefore, to investigate the expression of the hGRbeta protein, we have produced an antipeptide, hGRbeta-specific antibody termed BShGR. This antibody was made against the unique 15-amino acid peptide at the carboxy-terminus of hGRbeta and recognizes both the native and denatured conformations of hGRbeta, but does not cross-react with hGR alpha. Using BShGR on Western blots and in immunoprecipitation experiments, we detected the hGRbeta protein in a variety of human cell lines and tissues. Immunocytochemistry was then performed with BShGR on HeLa S3 and CEM-C7 cells and on tissue sections prepared from lung, thymus, and liver to assess the cellular and subcellular distribution of hGRbeta. In all immunopositive cells, hGRbeta was found in the nucleus independent of glucocorticoid treatment. Within tissues, the hGRbeta protein was expressed most abundantly in the epithelial cells lining the terminal bronchiole of the lung, forming the outer layer of Hassall's corpuscle in the thymus, and lining the bile duct in the liver. As a potential in vivo inhibitor of hGR alpha activity, expression of hGRbeta may be an important factor regulating target cell responsiveness to glucocorticoids.