Project description:GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase) a key enzyme of gluconeogenesis, and suppress the immune system which makes them one of the most important therapeutic agents in the treatment of allergic, autoimmune and inflammatory diseases. The biologic actions of circulating glucocorticoids are transmitted to the cells nucleus by the glucocorticoid receptor (GR). The nuclear liver X receptors (LXRs) bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR), and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. The aim of this study is to evaluate the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats and suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. Mechanistically, and in vitro chromatin immunoprecipitation assay, we found that LXRα/RXRα bound GREs and inhibited GR binding to these DNA sequences in a gene-specific fashion. These novel results were further confirmed in in vivo binding assays, and in gel mobility shift assays, where recombinant LXRα/RXRα proteins were used to examine their interaction with classic or G6Pase GREs. We propose that administration of LXR agonists may be beneficial in glucocorticoid treatment- or stress-associated dysmetabolic states by directly attenuating the transcriptional activity of the GR on glucose and/or lipid metabolism.

Project description:GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase) a key enzyme of gluconeogenesis, and suppress the immune system which makes them one of the most important therapeutic agents in the treatment of allergic, autoimmune and inflammatory diseases. The biologic actions of circulating glucocorticoids are transmitted to the cells nucleus by the glucocorticoid receptor (GR). The nuclear liver X receptors (LXRs) bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR), and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. The aim of this study is to evaluate the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats and suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. Mechanistically, and in vitro chromatin immunoprecipitation assay, we found that LXRÎ±/RXRÎ± bound GREs and inhibited GR binding to these DNA sequences in a gene-specific fashion. These novel results were further confirmed in in vivo binding assays, and in gel mobility shift assays, where recombinant LXRÎ±/RXRÎ± proteins were used to examine their interaction with classic or G6Pase GREs. We propose that administration of LXR agonists may be beneficial in glucocorticoid treatment- or stress-associated dysmetabolic states by directly attenuating the transcriptional activity of the GR on glucose and/or lipid metabolism. 4 Sprague-Dawley rats (180-200g) were orally gavaged for three days with GW3965 prepared in 1% Tween 80, 0.5% hydroxypropyl methylcellulose, 20 mM Na2HPO4 and 20 mM NaH2PO4 (50 mg/kg animal). Another set of animals were also orally gavaged with the same volume of the vehicle control (1% Tween 80, 0.5% hydroxypropyl methylcellulose, 20 mM Na2HPO4 and 20 mM NaH2PO4). On the third day, GW3965 or its vehicle control was administered first to the animals followed by an intra-muscularly injection of dexamethasone (1.5 mg/kg) or physiologic saline in two animals from each group. Twenty-four hours after the injection, levels of blood glucose were measured in these rats using the Freestyle GlucoMeter (Abbott Laboratories, Abbott Park, IL) by sampling blood from their tails. The animals were then euthanized using CO2, and their livers were harvested for extraction and purification of total RNA and proteins. Five Î¼g of total RNA purified from rat livers was used for producing probes with the One-Cycle Target Labeling and Control Reagents Kit (Affymetrix, Inc., Santa Clara, CA). [Rat230_2] Affymetrix Rat Genome 230 2.0 Arrays (Affymetrix Inc.) were then labeled with the prepared probes.

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Differential repression of TLR responses by PPARg and LXRs

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