A Model of Glucocorticoid Receptor Interaction With Coregulators Predicts Transcriptional Regulation of Target Genes.

Regulatory factors that control gene transcription in multicellular organisms are assembled in multicomponent complexes by combinatorial interactions. In this context, nuclear receptors provide well-characterized and physiologically relevant systems to study ligand-induced transcription resulting from the integration of cellular and genomic information in a cell- and gene-specific manner. Here, we developed a mathematical model describing the interactions between the glucocorticoid receptor (GR) and other components of a multifactorial regulatory complex controlling the transcription of GR-target genes, such as coregulator peptides. We support the validity of the model in relation to gene-specific GR transactivation with gene transcription data from A549 cells and in vitro real time quantification of coregulator-GR interactions. The model accurately describes and helps to interpret ligand-specific and gene-specific transcriptional regulation by the GR. The comprehensive character of the model allows future insight into the function and relative contribution of the molecular species proposed in ligand- and gene-specific transcriptional regulation.

Functional analysis reveals no transcriptional role for the glucocorticoid receptor ß-isoform in zebrafish.

In humans, two splice variants of the glucocorticoid receptor (GR) exist: the canonical α-isoform, and the ß-isoform, which has been shown to have a dominant-negative effect on hGRα. Previously, we have established the occurrence of a GR ß-isoform in zebrafish, and in the present study we have investigated the functional role of the zebrafish GRß (zGRß). Reporter assays in COS-1 cells demonstrated a dominant-negative effect of zGRß but no such effect was observed in zebrafish PAC2 cells using induction of the fk506 binding protein 5 (fkbp5) gene as readout. Subsequently, we generated a transgenic fish line with inducible expression of zGRß. Transcriptome analysis suggested transcriptional regulation of genes by zGRß in this line, but further validation failed to confirm this role. Based on these results, its low expression level and its poor evolutionary conservation, we suggest that the zebrafish GR ß-isoform does not have a functional role in transcriptional regulation.

Glucocorticoid-Induced Attenuation of the Inflammatory Response in Zebrafish.

Glucocorticoids are steroid hormones that are secreted upon stress. Their effects are mediated by the glucocorticoid receptor, which acts as a transcription factor. Because the antiinflammatory activity of glucocorticoids has been well established, they are widely used clinically to treat many inflammatory and immune-related diseases. However, the exact specificity, mechanisms, and level of regulation of different inflammatory pathways have not been fully elucidated. In the present study, a tail fin amputation assay was used in 3-day-old zebrafish larvae to study the immunomodulatory effects of the synthetic glucocorticoid beclomethasone. First, a transcriptome analysis was performed, which showed that upon amputation mainly immune-related genes are regulated. This regulation was inhibited by beclomethasone for 86% of regulated genes. For two immune-related genes, tlr4bb and alox5ap, the amputation-induced increase was not attenuated by beclomethasone. Alox5ap is involved in eicosanoid biosynthesis, but the increase in leukotriene B4 concentration upon amputation was abolished, and lipoxin A4 levels were unaffected by beclomethasone. Furthermore, we studied the migration of neutrophils and macrophages toward the wound site. Our results show that amputation induced migration of both types of leukocytes and that this migration was dependent on de novo protein synthesis. Beclomethasone treatment attenuated the migratory behavior of neutrophils in a glucocorticoid receptor-dependent manner but left the migration of macrophages unaffected. In conclusion, beclomethasone has a dramatic inhibitory effect on the amputation-induced proinflammatory gene regulation, and this is reflected in an inhibition of the neutrophil migration but not the migration of macrophages, which are likely to be involved in inflammation resolution.

Transcriptional and metabolic effects of glucocorticoid receptor α and ß signaling in zebrafish.

In humans and zebrafish, 2 glucocorticoid (GC) receptor (GR) splice variants exist: the canonical GR α-isoform (GRα), and the GRß. In the present study, we have used the zebrafish model system in order to reveal genes affected by each of these 2 receptor isoforms. By injecting zebrafish embryos with different splice-blocking morpholinos, we could knock down both GR isoforms or could target the alternative splicing of the GR pre-mRNA in favor of the GRß. In addition, specific GRß overexpression was achieved by injecting mRNA. Embryos were treated with the synthetic GC dexamethasone, and transcriptome analysis was performed. Two distinct gene clusters were found that were regulated by GRα: one that was regulated by GRα under basal conditions (presence of endogenous cortisol only), and one that was regulated upon increased activation of GRα (using a pharmacological dose of dexamathasone). GRß may act as a dominant-negative inhibitor of GRα when GRß is overexpressed and the GRα expression level is knocked down simultaneously. However, without GRα knockdown, no evidence for this activity was found. In addition, the data indicate regulation of gene transcription through other mechanisms of action by GRß. We also investigated the concentrations of several metabolites using nuclear magnetic resonance spectroscopy. We found that dexamethasone treatment and knockdown of GRα together with overexpression of GRß had opposite effects on glucose, amino acid, and fatty acid levels. Thus, we have shed new light on the molecular mechanisms of GC-induced effects on metabolism, which are known to increase the risk of obesity, hyperglycemia, and diabetes.

The zebrafish as an in vivo model system for glucocorticoid resistance.

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.