Glucocorticoid-inducible Genes Research Articles

The long-acting β2 -adrenoceptor agonist, indacaterol, enhances glucocorticoid receptor-mediated transcription in human airway epithelial cells in a gene- and agonist-dependent manner.

Inhaled glucocorticoid (ICS)/long-acting β2 -adrenoceptor agonist (LABA) combination therapy is a recommended treatment option for patients with moderate/severe asthma in whom adequate control cannot be achieved by an ICS alone. Previously, we discovered that LABAs can augment dexamethasone-inducible gene expression and proposed that this effect may explain how these two drugs interact to deliver superior clinical benefit. Herein, we extended that observation by analysing, pharmacodynamically, the effect of the LABA, indacaterol, on glucocorticoid receptor (GR)-mediated gene transcription induced by seven ligands with intrinsic activity values that span the spectrum of full agonism to antagonism. BEAS-2B human airway epithelial cells stably transfected with a 2× glucocorticoid response element luciferase reporter were used to model gene transcription together with an analysis of several glucocorticoid-inducible genes. Indacaterol augmented glucocorticoid-induced reporter activation in a manner that was positively related to the intrinsic activity of the GR agonist. This effect was demonstrated by an increase in response maxima without a change in GR agonist affinity or efficacy. Indacaterol also enhanced glucocorticoid-inducible gene expression. However, the magnitude of this effect was dependent on both the GR agonist and the gene of interest. These data suggest that indacaterol activates a molecular rheostat, which increases the transcriptional competency of GR in an agonist- and gene-dependent manner without apparently changing the relationship between fractional GR occupancy and response. These findings provide a platform to rationally design ICS/LABA combination therapy that is based on the generation of agonist-dependent gene expression profiles in target and off-target tissues.

An analysis of glucocorticoid receptor-mediated gene expression in BEAS-2B human airway epithelial cells identifies distinct, ligand-directed, transcription profiles with implications for asthma therapeutics.

International asthma guidelines recommend that inhaled glucocorticoids be used as a monotherapy in all patients with mild to moderate disease because of their ability to suppress airways inflammation. Current evidence suggests that the therapeutic benefit of glucocorticoids is due to the transactivation and transrepression of anti-inflammatory and pro-inflammatory genes respectively. However, the extent to which clinically relevant glucocorticoids are equivalent in their ability to modulate gene expression is unclear. A pharmacodynamics investigation of glucocorticoid receptor (GR)-mediated gene transactivation in BEAS-2B human airway epithelial cells was performed using a glucocorticoid response element luciferase reporter coupled with an analysis of glucocorticoid-inducible genes encoding proteins with anti-inflammatory and adverse-effect potential. Using transactivation as a functionally relevant output, a given glucocorticoid displayed a unique, gene expression 'fingerprint' where intrinsic efficacy and GR density were essential determinants. We showed that depending on the gene selected for analysis, a given glucocorticoid can behave as an antagonist, partial agonist, full agonist or even 'super agonist'. In the likely event that different, tissue-dependent gene expression profiles are reproduced in vivo, then the anti-inflammatory and adverse-effect potential of many glucocorticoids currently available as asthma therapeutics may not be equivalent. The generation of gene expression 'fingerprints' in target and off-target human tissues could assist the rational design of GR agonists with improved therapeutic ratios. This approach could identify compounds that are useful in the management of severe asthma and other inflammatory disorders where systemic exposure is desirable.

Expression of glucocorticoid inducible genes is associated with reductions in cornu ammonis and dentate gyrus volumes in patients with major depressive disorder.

Alterations of the glucocorticoid system and of hippocampal volumes have consistently been reported in patients with major depressive disorders (MDD). The aim of the present study was to investigate whether the messenger RNA (mRNA) expression of glucocorticoid inducible genes is associated with changes in the cornu ammonis (CA) and dentate gyrus subfields. Forty-three patients with MDD and 43 healthy controls were recruited and investigated with high resolution magnetic resonance imaging. Hippocampal subfields were measured using freesurfer. Measurement of whole blood mRNA expression of glucocorticoid inducible genes serum and glucocorticoid-regulated kinase 1 (SGK1), FK506 binding protein 5 (FKBP5), and glucocorticoid induced leucine zipper (GILZ) was performed. Patients with MDD had significantly smaller volumes of CA1, CA2/3, CA4/DG, and subiculum compared to healthy controls. In the regression analysis, the factor diagnosis had a significant moderating effect on the association of SGK1 and hippocampal volumes. Patients with low expression of SGK1 had significantly smaller CA2/3 and CA4/DG volumes compared to patients with high expression of SGK1 mRNA and to healthy controls with low/high expression of SGK1, respectively. Therefore, a lack of mRNA expression of glucocorticoid inducible genes in patients with MDD that seems to correspond to a blunted cortisol response is associated with smaller hippocampal CA and dentate gyrus volumes. SGK1 seems to be particularly relevant for stress-related mental disorders.

105. Enhanced glucocorticoid sensitivity coupled with reduced microglial activation in a genetically predisposed animal model of depression

The Wistar-Kyoto (WKY) rat exhibits a genetic predisposition to a stress-sensitive depression-related phenotype. The aim of this investigation was to assess stress-related glucocorticoid sensitivity and immune responses in the strain so as to elucidate putative neuro-immune interactions. Circulating corticosterone concentrations were lower in WKY rats relative to the comparator Wistar strain at baseline but increased in both strains in response to stress. Both WKY and Wistar strains exhibited a stress-related increase in the expression of the glucocorticoid-inducible genes GILZ, SGK1 and p11 in the brain (frontal cortex, hippocampus and hypothalamus), the response being greater in the WKY strain. GR expression was suppressed in response to stress in the WKY strain only. Similarly, expression of the monocyte/microglial markers, CD-11B and CD74, were attenuated in the WKY relative to the Wistar strain following stress exposure. This was observed in tandem with a stress-related increase in IL-6 expression in the WKY rat only. Both strains exhibited a stress-related increase in the expression of FKBP5 and TDO in the liver, the response being greater in the WKY rat. Measurement of hepatic TDO activity revealed that conversion of tryptophan to kynurenine was greater in the WKY rat relative to Wistar controls following stress exposure indicative of kynurenine pathway induction. The observed interaction between the stress-induced endocrine and immune axes may account for the depressive-related phenotype characteristic of this strain.

EPA-0743 – Stress-gene interactions on brain structure and function in major depression

Major depressive disorder (MDD) is a complex disorder and recent research suggests that muliple genetic and environmental factors seem to underly MDD criteria as well as seem to influence brain structure and function. Aim of the study was to investigate whether epigenetic regulation and mRNA expression of glucocorticoid inducible genes (SGK-1, GILZ) are associated with structural alterations in hippocampal subfields and functional brain changes. Moreover, we investigated whether there are interactive effects between the genetic variant of the BICC1 gene and ELA. Patients with MDD and healthy controls were investigated using structural magnetic resonance imaging (MRI) and functional MRI with an emotion inhibition task. Analysis of a single nucleotide polymorphism in the BICC1-1 (rs999845) gene as well as mRNA expression and methylation of DNA was performed. Hippocampal subfield volumes of dendate gyrus and CA2 were reduced in patients with MDD. These were associated with reduced mRNA expression of glucocorticoid inducible genes and with DNA methylation. Moreover, MDD patients with a history of ELA, who carry the protective T-allele, had smaller hippocampal head volumes compared to MDD patients without ELA. FMRI showed that patients and controls carrying the protective T-allele of BICC1 activate the emotion regulation system significantly more compared to those participants homozygous for the major C-allele (p<0.05, family wise error corrected). These results are suggestive for environment x gene interactions in MDD and for the impact of epigenetic regulation on brain structure and function.

The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR.

Region-specific alterations in glucocorticoid receptor expression in the postmortem brain of teenage suicide victims

Abnormal function of the hypothalamic-pituitary-adrenal (HPA) axis has been implicated in the pathophysiology of depression and suicide. The purpose of this study was to test the hypothesis that the reported dysregulation of the HPA axis in suicide may be related to a disturbed feedback inhibition caused by decreased corticoid receptors in the brain. We therefore determined the protein and gene expression of glucocorticoid (GR) and mineralocorticoid receptors (MR) in the postmortem brain of teenage suicide victims and matched normal controls. Protein and mRNA expression of GR (GR-α and GR-β) and MR and the mRNA expression of glucocorticoid-induced leucine zipper (GILZ), a target gene for GR were determined by immunolabeling using Western blot technique and the real-time RT-polymerase chain reaction (qPCR) technique in the prefrontal cortex (PFC), hippocampus, subiculum, and amygdala obtained from 24 teenage suicide victims and 24 teenage control subjects. We observed that protein and gene expression of GR-α was significantly decreased in the PFC and amygdala, but not in the hippocampus or subiculum, of teenage suicide victims compared with normal control subjects. Also, the mRNA levels of GR inducible target gene GILZ was significantly decreased in PFC and amygdaloid nuclei but not in hippocampus compared with controls. In contrast, no significant differences were observed in protein or gene expression of MR in any of the areas studied between teenage suicide victims and normal control subjects. There was no difference in the expression of GR-β in the PFC between suicide victims and normal controls. These results suggested that the observed dysregulation of the HPA axis in suicide may be related to a decreased expression of GR-α and GR inducible genes in the PFC and amygdala of teenage suicide victims. The reason why GR receptors are not dysregulated in the hippocampus or subiculum, presumably two sites of stress action, are not clear at this time.

Phosphodiesterase 4 Inhibitors Augment the Ability of Formoterol to Enhance Glucocorticoid-Dependent Gene Transcription in Human Airway Epithelial Cells: A Novel Mechanism for the Clinical Efficacy of Roflumilast in Severe Chronic Obstructive Pulmonary Disease

Post-hoc analysis of two phase III clinical studies found that the phosphodiesterase 4 (PDE4) inhibitor, roflumilast, reduced exacerbation frequency in patients with severe chronic obstructive pulmonary disease (COPD) who were taking inhaled corticosteroids (ICS) concomitantly, whereas patients not taking ICS derived no such benefit. In contrast, in two different trials also performed in patients with severe COPD, roflumilast reduced exacerbation rates in the absence of ICS, indicating that PDE4 inhibition alone is sufficient for therapeutic activity to be realized. Given that roflumilast is recommended as an "add-on" medication to patients with severe disease who will inevitably be taking a long-acting β2-adrenoceptor agonist (LABA)/ICS combination therapy, we tested the hypothesis that roflumilast augments the ability of glucocorticoids to induce genes with anti-inflammatory activity. Using a glucocorticoid response element (GRE) luciferase reporter transfected into human airway epithelial cells [both bronchial epithelium + adenovirus 12 - SV40 hybrid (BEAS-2B) cells and primary cultures], roflumilast enhanced fluticasone propionate-induced GRE-dependent transcription. Roflumilast also produced a sinistral displacement of the concentration-response curves that described the augmentation of GRE-dependent transcription by the LABA formoterol. In BEAS-2B cells and primary airway epithelia, roflumilast interacted with formoterol in a positive cooperative manner to enhance the expression of several glucocorticoid-inducible genes that have anti-inflammatory potential. We suggest that the ability of roflumilast and formoterol to interact in this way supports the concept that these drugs together may impart clinical benefit beyond that achievable by an ICS alone, a PDE4 inhibitor alone, or an ICS/LABA combination therapy. Roflumilast may, therefore, be especially effective in patients with severe COPD.

Reduced expression of glucocorticoid-inducible genes GILZ and SGK-1: high IL-6 levels are associated with reduced hippocampal volumes in major depressive disorder.

Neuroplasticity may have a core role in the pathophysiology of major depressive disorder (MDD), a concept supported by experimental studies that found that excessive cortisol secretion and/or excessive production of inflammatory cytokines impairs neuronal plasticity and neurogenesis in the hippocampus. The objective of this study was to examine how changes in the glucocorticoid and inflammatory systems may affect hippocampal volumes in MDD. A multimodal approach with structural neuroimaging of hippocampus and amygdala, measurement of peripheral inflammatory proteins interleukin (IL)-6 and C-reactive protein (CRP), glucocorticoid receptor (GR) mRNA expression, and expression of glucocorticoid-inducible genes (glucocorticoid-inducible genes Leucin Zipper (GILZ) and glucocorticoid-inducible kinase-1 (SGK-1)) was used in 40 patients with MDD and 43 healthy controls (HC). Patients with MDD showed smaller hippocampal volumes and increased inflammatory proteins IL-6 and CRP compared with HC. Childhood maltreatment was associated with increased CRP. Patients with MDD, who had less expression of the glucocorticoid-inducible genes GILZ or SGK-1 had smaller hippocampal volumes. Regression analysis showed a strong positive effect of GILZ and SGK-1 mRNA expression, and further inverse effects of IL-6 concentration, on hippocampal volumes. These findings suggest that childhood maltreatment, peripheral inflammatory and glucocorticoid markers and hippocampal volume are interrelated factors in the pathophysiology of MDD. Glucocorticoid-inducible genes GILZ and SGK-1 might be promising candidate markers for hippocampal volume changes relevant for diseases like MDD. Further studies need to explore the possible clinical usefulness of such a blood biomarker, for example, for diagnosis or prediction of therapy response.

Dexamethasone and RU24858 Induce Survival and Growth Factor Receptor Bound Protein 2, Leukotriene B4 Receptor 1 and Annexin-1 Expression in Primary Human Neutrophils.

Glucocorticoids are widely used anti-inflammatory medication in diseases like asthma and chronic obstructive pulmonary disease. Glucocorticoids can either activate (transactivation) or inhibit (transrepression) transcription. RU24858 was introduced as a “dissociated” glucocorticoid and it has been reported to transrepress but not to transactivate. The aim of this study was to compare the effects of RU24858 and dexamethasone in human neutrophils. RU24858 delayed spontaneous neutrophil apoptosis and further enhanced GM-CSF- induced neutrophil survival to a similar extent as dexamethasone. Like dexamethasone RU24858 also reduced CXCL8 and MIP-1α. Unexpectedly however, RU24858 increased the expression of the glucocorticoid-inducible genes BLT-1, Annexin-1 and Grb-2 in neutrophils to a similar level as seen with dexamethasone. We have shown here that dexamethasone and RU24858 both increase Grb-2, BLT1 and Annexin-1 expression and inhibit CXCL8 and MIP-1α production. This suggests that RU24858 was not able to dissociate between transactivation and transrepression in human neutrophils but enhanced neutrophil survival.

Pharmacological interference with the glucocorticoid system influences symptoms and lifespan in a mouse model of Rett syndrome

Rett syndrome (RTT) is caused by loss-of-function mutations in the X-linked gene MECP2 coding for methyl CpG-binding protein 2 (MeCP2). This protein can act as transcriptional repressor, and we showed in a previous study that glucocorticoid-inducible genes are up-regulated in an RTT mouse model and that these genes are direct MeCP2 targets. Here, we report that pharmacological intervention with the glucocorticoid system has an impact on the symptoms and lifespan in an RTT mouse model. Our data support a functional implication of the stress hormone system in RTT and suggest this hormone system as potential therapeutic target.

Selective Prostacyclin Receptor Agonism Augments Glucocorticoid-Induced Gene Expression in Human Bronchial Epithelial Cells

Prostacyclin receptor (IP-receptor) agonists display anti-inflammatory and antiviral activity in cell-based assays and in preclinical models of asthma and chronic obstructive pulmonary disease. In this study, we have extended these observations by demonstrating that IP-receptor activation also can enhance the ability of glucocorticoids to induce genes with anti-inflammatory activity. BEAS-2B bronchial epithelial cells stably transfected with a glucocorticoid response element (GRE) luciferase reporter were activated in a concentration-dependent manner by the glucocorticoid dexamethasone. An IP-receptor agonist, taprostene, increased cAMP in these cells and augmented luciferase expression at all concentrations of dexamethasone examined. Analysis of the concentration-response relationship that described this effect showed that taprostene increased the magnitude of transcription without affecting the potency of dexamethasone and was, thus, steroid-sparing in this simple system. RO3244794, an IP-receptor antagonist, and oligonucleotides that selectively silenced the IP-receptor gene, PTGIR, abolished these effects of taprostene. Infection of BEAS-2B GRE reporter cells with an adenovirus vector encoding a highly selective inhibitor of cAMP-dependent protein kinase (PKA) also prevented taprostene from enhancing GRE-dependent transcription. In BEAS-2B cells and primary cultures of human airway epithelial cells, taprostene and dexamethasone interacted either additively or cooperatively in the expression of three glucocorticoid-inducible genes (GILZ, MKP-1, and p57(kip2)) that have anti-inflammatory potential. Collectively, these data show that IP-receptor agonists can augment the ability of glucocorticoids to induce anti-inflammatory genes in human airway epithelial cells by activating a cAMP/PKA-dependent mechanism. This observation may have clinical relevance in the treatment of airway inflammatory diseases that are either refractory or respond suboptimally to glucocorticoids.

Analysis of the Dissociated Steroid RU24858 Does Not Exclude a Role for Inducible Genes in the Anti-Inflammatory Actions of Glucocorticoids

Although repression of inflammatory gene expression makes glucocorticoids powerful anti-inflammatory agents, side effects limit usage and drive the search for improved glucocorticoid receptor (GR) ligands. In A549 pulmonary cells, dexamethasone and the prototypical dissociated ligand RU24858 (Mol Endocrinol 11:1245-1255, 1997) repress interleukin (IL)-1beta-induced expression of cyclooxygenase (COX)-2 and IL-8. Although RU24858 is a weaker GR ligand, both glucocorticoids showed similar efficacies on transrepression of nuclear factor kappaB (NF-kappaB)-dependent transcription, whereas RU24858 yielded less than 12% of the response to dexamethasone on a classic glucocorticoid response element (GRE) reporter (transactivation). Modest NF-kappaB-dependent transrepression ( approximately 40%), along with analysis of IL-8 transcription rate and the accumulation of unspliced nuclear RNA, indicates that transrepression does not fully account for the repression of genes such as IL-8. This was confirmed by the finding that mRNA degradation is increased by both dexamethasone and RU24858. Analysis of IL-1beta-induced steady-state mRNA levels for IL-8 and COX-2 show that dexamethasone- and RU24858-dependent repression of these genes is attenuated by inhibitors of transcription and protein synthesis. Because similar effects were observed with respect to COX-2 and IL-8 protein expression, we conclude that glucocorticoid-dependent gene expression is necessary for repression by both glucocorticoids. Despite RU24858 being defective at classic GRE-dependent transactivation, both dexamethasone and RU24858 induced the expression of potentially anti-inflammatory genes and metabolic genes, suggesting the importance of nontraditional glucocorticoid-dependent gene expression. Thus, classic transactivation- and transrepressionbased screens for anti-inflammatory "dissociated" GR ligands may be flawed because they may not reflect the effects on real glucocorticoid-inducible genes.

A silencer element in the first intron of the glutamine synthetase gene and its role in hepatocellular expression

The enzyme glutamine synthetase (GS) ranks as one of the most remarkable glucocorticoid-inducible mammalian genes. In many cells the synthetic glucocorticoid dexamethasone (Dex) increases GS expression severalfold. The response is mediated by a cellular glucocorticoid receptor (GR) that interacts with glucocorticoid responsive elements (GREs). In the rat gene it was demonstrated that two GREs are responsible for the effect observed in cell lines from lung and muscle, whereas the response to glucocorticoids in cell lines of hepatocellular origin is mediated by an element located in the first intron. Surprisingly, although some cell lines derived from the liver respond to the presence of Dex with an enhanced expression of GS, primary hepatocytes do not. Performing transfection experiments with different cell lines a sequence was detected downstream of the GRE that works as a silencer element, repressing the response to glucocorticoids. Gel mobility shift assays demonstrate the binding of an as yet unknown protein to the element. Interestingly, the silencer is also able to repress the potent 5‘-enhancer of the GS gene. Therefore, it is the first negative regulatory element within the GS gene that may contribute to the well known and still unexplained phenomenon of restricted expression of GS within the liver.

A silencer element in the first intron of the glutamine synthetase gene represses induction by glucocorticoids.

The enzyme glutamine synthetase (GS) ranks as one of the most remarkable glucocorticoid-inducible mammalian genes. In many tissues and cell lines, the synthetic glucocorticoid dexamethasone alone increases GS expression several fold. The direct response is mainly mediated by a cellular glucocorticoid receptor that, upon binding of the hormone, interacts with glucocorticoid responsive elements (GREs) of the gene. In cells of hepatocellular origin the response is mediated by a GRE located in the first intron of the gene. Surprisingly, hepatocytes do not respond to glucocorticoids with enhanced GS expression, despite the presence of an intact glucocorticoid receptor, which, in the same cells, stimulates expression of other genes such as tyrosine amino transferase. Reporter gene assays identified a sequence element downstream from the intronic GRE that inhibits the enhancement of expression by glucocorticoids. This silencer was designated GS silencer element of the rat. Gel mobility shift assays demonstrate the binding of a factor in hepatocyte nuclear extract. This yet unknown factor was designated GS silencer-binding protein. It is absent in FAO cells that respond to glucocorticoids with enhanced expression of GS and present in HepG2 cells that do not respond.

Nucleocytoplasmic shuttling of lgl2 is developmentally regulated in fetal lung.

To investigate molecular mechanisms of lung organogenesis, we searched for glucocorticoid-inducible genes in developing lung. We cloned LGL2, a developmentally and hormonally regulated gene in fetal lung (Zhang, C., N. B. Sweezey, S. Gagnon, B. Muskat, D. Koehler, M. Post, and F. Kaplan. 2000. A novel karyopherin-beta homolog is developmentally and hormonally regulated in fetal lung. Am. J. Respir. Cell Mol. Biol. 22:451-459). A comparison of lgl2 protein to sequences in the genome database suggested that lgl2 is a nuclear transport receptor. We report on the functional characterization of lgl2 as an importin beta protein and on the developmental regulation of its nucleocytoplasmic shuttling in fetal lung. We investigated the subcellular localization and Ran-binding properties of lgl2 and its N- and C-terminal regions. We used fluorescence recovery after photobleaching and fluorescence loss in photobleaching to study nucleocytoplasmic shuttling of lgl2. We showed that N-terminal lgl2 supports shuttling at a reduced rate. We showed that the nucleocytoplasmic distribution of lgl2 favors the nucleus in fetal lung and that lgl2 enters the nucleus much more rapidly at fetal Day 18 than at Day 21. Total nuclear recovery of lgl2 was dramatically different at the two time points. Early in development, nuclear import of transcription factors in response to hormones and growth agonists regulates prominent signal transduction pathways that govern lung organogenesis. We speculate that lgl2 may be one important modulator of this process.

Glucocorticoid induced expression of glutamine synthetase in hepatoma cells

The enzyme glutamine synthetase (GS) ranks as one of the most remarkable glucocorticoid-inducible vertebrate genes. However, little is known about the responsible DNA elements and the mode of glucocorticoid action. This is especially the case for the induction of GS in hepatoma cells. In the work presented, the rat hepatoma cell line FAO was used as a model to study the induction of GS under the influence of glucocorticoids. FAO cells do not show GS activity in the absence of glucocorticoids and are strongly responding to their presence. Analyzing sequences of several thousand base pairs upstream and downstream from the transcriptional start point of the GS gene, a glucocorticoid responsible element was identified within the first intron of the gene. However, evidence is presented that aside from a primary effect on transcription glucocorticoids mediate their effect on the expression of GS also at the posttranscriptional level.

Positive Effects of Glucocorticoids on T Cell Function by Up-Regulation of IL-7 Receptor α

Despite the effects of glucocorticoids on immune function, relatively little is known about glucocorticoid-inducible genes and how their products may regulate lymphocyte function. Using DNA microarray technology to analyze gene expression in PBMC from healthy donors, we identified IL-7Ralpha as a glucocorticoid-inducible gene. This observation was confirmed at the mRNA and protein levels. Conversely, TCR signaling decreased IL-7Ralpha expression, and the relative strength of signaling between these two receptors determined the final IL-7Ralpha levels. The up-regulation of IL-7Ralpha by glucocorticoids was associated with enhanced IL-7-mediated signaling and function. Moreover, IL-7-mediated inhibition of apoptosis at increasing concentrations of glucocorticoids is consistent with enhanced cell sensitivity to IL-7 following glucocorticoid exposure. These observations provide a mechanism by which glucocorticoids may have a positive influence on T cell survival and function.

Glucocorticoid-inducible genes that regulate T-cell function.

Upon antigenic challenge, the immune system must detect and respond to subtle perturbations in local microenvironments. While many important transactions entail cell-to-cell contact, coordination between multiple cells and cell types is carried out through antigen-nonspecific soluble mediators, broadly classified as cytokines and hormones. Messages delivered by the cytokines are for the most part pro-inflammatory and/or immune activating, whereas those delivered by the glucocorticoid hormones are thought to be largely inhibitory. One of the major inhibitory effects of glucocorticoids is the suppression of the synthesis of a large number of cytokines (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, GM-CSF, TNF-α, and IFN-γ, Kunicka et al. 1993). When viewed individually, however, a number of effects of glucocorticoids appear to break this “rule,” with glucocorticoids acting in pro-inflammatory or immunostimulatory ways. For example, glucocorticoids can increase the expression of receptors for some of the pro-inflammatory cytokines that it suppresses (IL-1, IL-6, GM-CSF, and IFN-γ; Almawi et al. 1996). A more complicated example is the effect of glucocorticoids on lymphocyte viability. Whereas thymocytes, and to a lesser extent mature lymphocytes, are induced to undergo apoptosis in response to glucocorticoid receptor (GR) occupancy (immunosuppressive), glucocorticoids inhibit apoptosis elicited by T-cell receptor (TCR)-mediated activation (immunostimulatory). This phenomenon is due to the inhibition of activation-induced Fas ligand (FasL) upregulation (Yang et al. 1995), which in normal circumstances binds Fas and elicits a death response. This chapter deals with new information about two glucocorticoid-regulated genes that appear to have negative and positive effects on immune function: GILZ and the receptor for interleukin (IL)-7 (IL-7Rα).

Overexpression of the human glucocorticoid receptor alpha and beta isoforms inhibits AP-1 and NF-kappaB activities hormone independently.

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.