Liver X Receptors Regulate the Transcriptional Activity of the Glucocorticoid Receptor: Implications for the Carbohydrate Metabolism

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 through the glucocorticoid receptor (GR). The liver X receptors (LXRs), on the other hand, 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. Since the actions of these receptors overlap with each other, we evaluated 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. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXRα/RXRα binding to GREs. Endogenous LXRα/RXRα bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that administration of LXR agonists may be beneficial in glucocorticoid treatment- or stress-associated dysmetabolic states by directly and gene-specifically attenuating the transcriptional activity of the GR on glucose and/or lipid metabolism

Liver X Receptors Regulate the Transcriptional Activity of the Glucocorticoid Receptor: Implications for the Carbohydrate Metabolism

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 through the glucocorticoid receptor (GR). The liver X receptors (LXRs), on the other hand, 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. Since the actions of these receptors overlap with each other, we evaluated 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. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXR alpha/RXR alpha binding to GREs. Endogenous LXR alpha/RXR alpha bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that administration of LXR agonists may be beneficial in glucocorticoid treatment- or stress-associated dysmetabolic states by directly and gene-specifically attenuating the transcriptional activity of the GR on glucose and/or lipid metabolism

Peripheral CLOCK regulates target-tissue glucocorticoid receptor transcriptional activity in a circadian fashion in man.

Circulating cortisol fluctuates diurnally under the control of the "master" circadian CLOCK, while the peripheral "slave" counterpart of the latter regulates the transcriptional activity of the glucocorticoid receptor (GR) at local glucocorticoid target tissues through acetylation. In this manuscript, we studied the effect of CLOCK-mediated GR acetylation on the sensitivity of peripheral tissues to glucocorticoids in humans.We examined GR acetylation and mRNA expression of GR, CLOCK-related and glucocorticoid-responsive genes in peripheral blood mononuclear cells (PBMCs) obtained at 8 am and 8 pm from 10 healthy subjects, as well as in PBMCs obtained in the morning and cultured for 24 hours with exposure to 3-hour hydrocortisone pulses every 6 hours. We used EBV-transformed lymphocytes (EBVLs) as non-synchronized controls.GR acetylation was higher in the morning than in the evening in PBMCs, mirroring the fluctuations of circulating cortisol in reverse phase. All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone in non-synchronized EBVLs, however, some of these genes did not show the expected diurnal mRNA fluctuations in PBMCs in vivo. Instead, their mRNA oscillated in a Clock- and a GR acetylation-dependent fashion in naturally synchronized PBMCs cultured ex vivo in the absence of the endogenous glucocorticoid, suggesting that circulating cortisol might prevent circadian GR acetylation-dependent effects in some glucocorticoid-responsive genes in vivo.Peripheral CLOCK-mediated circadian acetylation of the human GR may function as a target-tissue, gene-specific counter regulatory mechanism to the actions of diurnally fluctuating cortisol, effectively decreasing tissue sensitivity to glucocorticoids in the morning and increasing it at night

Viral Infection Increases Glucocorticoid-Induced Interleukin-10 Production through ERK-Mediated Phosphorylation of the Glucocorticoid Receptor in Dendritic Cells: Potential Clinical Implications

<div><p>The hypothalamic-pituitary-adrenal axis plays a central role in the adaptive response to stress including infection of pathogens through glucocorticoids. Physical and/or mental stress alter susceptibility to viral infection possibly by affecting this regulatory system, thus we explored potential cellular targets and mechanisms that underlie this phenomenon in key immune components dendritic cells (DCs). Dexamethasone (DEX) treatment and subsequent Newcastle disease virus (NDV) infection most significantly and cooperatively stimulated mRNA expression of the interleukin (IL)-10 in murine bone marrow-derived DCs among 89 genes involved in the Toll-like receptor signaling pathways. NDV increased DEX-induced IL-10 mRNA and protein expression by 7- and 3-fold, respectively, which was observed from 3 hours after infection. Conventional DCs (cDCs), but not plasmacytoid DCs (pDCs) were major sources of IL-10 in bone marrow-derived DCs treated with DEX and/or infected with NDV. Murine cytomegalovirus and DEX increased serum IL-10 cooperatively in female mice. Pre-treatment of DCs with the extracellular signal-regulated kinase (ERK) inhibitor U0126 abolished cooperative induction of IL-10 by DEX and NDV. Further, ERK overexpression increased IL-10 promoter activity stimulated by wild-type human GR but not by its mutant defective in serine 203, whereas ERK knockdown abolished NDV/DEX cooperation on IL-10 mRNA and phosphorylation of the mouse GR at serine 213. NDV also increased DEX-induced mRNA expression of three known glucocorticoid-responsive genes unrelated to the Toll-like receptor signaling pathways in DCs. These results indicate that virus and glucocorticoids cooperatively increase production of anti-inflammatory cytokine IL-10 by potentiating the transcriptional activity of GR in DCs, through which virus appears to facilitate its own propagation in infected hosts. The results may further underlie in part known exacerbation of IL-10/T helper-2-related allergic disorders by stress and viral infection.</p></div

NDV increases DEX-induced expression of several glucocorticoid-responsive genes through phosphorylation of GR.

<p><b>A</b>. NDV increased DEX-induced mRNA expression of 3 well-known glucocorticoid-responsive genes in DCs. DCs were pre-treated with DEX (10⁻⁶M) for 30 min, and were infected with NDV (MOI = 10) for 6 hours. mRNA levels of <i>DUSP1</i>, <i>GILZ</i> and <i>PER1</i> were measured in the real-time qPCR. Bars represent means and standard errors of fold changes of their mRNA expression obtained in three independent experiments. **: p<0.01, compared the 2 conditions indicated. <b>B</b>: ERK2 increased DEX-induced transcriptional activity of the GILZ promoter in the presence of wild type human GR in HCT116 cells, while it lost the effect in the presence of the mutant GR harboring S203A replacement. HCT116 cells were transfected with ERK2 D319N-expressing plasmid in the presence of wild type human GR or its mutant with S203A replacement, together with the pGILZ-luc reporter and the pGL4.73[<i>hRluc</i>/SV40] control plasmid. Cells were subsequently treated with DEX (10⁻⁶ M) for 24 hours. Bars represent means and standard errors of the firefly luciferase activity corrected for renilla luciferase activity. **: p<0.01, n.s.: not significant, compared the 2 conditions indicated.</p

GR mediated DEX pre-treatment- and NDV infection-induced cooperative IL-10 mRNA expression in DCs.

<p><b>A</b>. RU486 suppressed DEX-induced increase of IL-10 mRNA and protein expression in DCs. DCs were pre-treated with RU486 (10⁻⁵ M) and/or DEX (10⁻⁶ M) for 30 min, and were infected with NDV (MOI = 10) for 6 hours. IL-10 mRNA and protein expression are shown. Bars represent means and standard errors of fold changes of the IL-10 mRNA expression and protein concentrations in culture media obtained from three independent experiments. **: p<0.01, compared to the 2 conditions indicated. <b>B & C</b>. DEX pre-treatment and NDV infection did not change GR mRNA and protein expression in DCs. DCs were pre-treated with DEX (10⁻⁶ M) for 30 min, and were infected with NDV (MOI = 10) for 6 hours. Bars in panel <b>A</b> and <b>B</b> represent means and standard errors of fold changes of the mRNA expression obtained in three independent experiments. Whole cell extracts obtained from DCs were run on SDS-PAGE gels, and GR and control β-actin were visualized with their specific antibodies in Western blots in panel <b>C</b>.</p

NDV infection increased IL-10 expression by phosphorylating GR at serine 203 (human) and 213 (mouse).

<p><b>A</b>. ERK increased GR-induced transcriptional activity of the IL-10 promoter in HCT116 cells. HCT116 cells were transfected with wild type ERK1-, 2- or its constitutively active mutant ERK2 D319N-expressing plasmid in the presence of wild type human GR, together with the pGL3 reporter carrying the IL-10 promoter and the pGL4.73[<i>hRluc</i>/SV40] control plasmid. Cells were subsequently treated with DEX (10⁻⁶ M) for 24 hours. Bars represent means and standard errors of the firefly luciferase activity corrected for renilla luciferase activity. ***: p<0.001, compared the 2 conditions indicated. <b>B & C</b>. NDV infection increased DEX-induced IL-10 mRNA expression though ERK1/2 in RAW264.7 cells. RAW264.7 cells were transfected with ERK2 D319N-expressing plasmids and/or ERK1/2 siRNAs and treated/infected with DEX (10⁻⁶ M) and/or NDV for 24 hours. The effect of ERK1/2 siRNA on the mRNA expression of ERK1 and 2 was shown in panel <b>C</b>. Bars represent means and standard errors of fold changes of the IL-10, ERK1 and ERK2 mRNA. **: p<0.01, compared the 2 conditions indicated. <b>D</b>. ERK2 increased DEX-induced IL-10 promoter activity in the presence of wild type human GR but not in the presence of its mutant defective in serine 203 in HCT116 cells. HCT116 cells were transfected with wild type ERK2-expressing plasmid in the presence of wild type human GR or its mutant with S203A replacement, together with the pGL3 reporter carrying the IL-10 promoter and the pGL4.73[<i>hRluc</i>/SV40] control plasmid. Cells were subsequently treated with DEX (10⁻⁶ M) for 24 hours. Bars represent means and standard errors of the firefly luciferase activity corrected for renilla luciferase activity. **: p<0.01, n.s.: not significant, compared the 2 conditions indicated. <b>E</b>. ERK2 D319N increased DEX-induced phosphorylation of wild type human GR at serine 203, while it had no effect on the mutant GR with S203A replacement in HCT116 cells. HCT116 cells were transfected with ERK2 D319N-expressing plasmid together with wild type GR- or GR S203A mutant-expressing plasmid, and were treated with DEX (10⁻⁶ M) for 30 min. Whole cell extracts obtained from these cells were run on SDS-PAGE gels and the GR phosphorylated at serine 203 and its entire fraction were visualized with their specific antibodies in Western blots. <b>F</b>. NDV infection increased DEX-induced phosphorylation of mouse GR at serine 213, while ERK1/2 knockdown abolished the NDV effect in RAW264.7 cells. RAW264.7 cells were transfected with ERK1/2 siRNAs and treated/infected with DEX (10⁻⁶ M) and/or NDV for 30 min. Total cell lysates obtained from these cells were run on SDS-PAGE gels and the mouse GR phosphorylated at serine 213 and its entire fraction were visualized with their specific antibodies in Western blots.</p

DEX pre-treatment and NDV infection cooperatively increased IL-10 mRNA and protein expression in DCs.

<p>DCs were pre-treated with DEX (<b>A</b>: 10⁻⁶ M, <b>B</b>: 10⁻⁶, 10⁻⁷ or 10⁻⁸M) or CORT (<b>C</b>: 10⁻⁶ M) for 30 min, and were infected with NDV (MOI = 10) for 6 hours. Time-course of IL-10 mRNA (<b>A</b>, left panel) and protein (<b>A</b>, right panel) expression, and the effect of increasing concentrations of DEX on these parameters (<b>B</b>, left and right panel, respectively) are shown. Time-courses of IL-10 mRNA (left panel) and protein (right panel) expression obtained in the presence of CORT pre-treatment are also shown in panel <b>C</b>. Relative IL-10 mRNA expression (fold changes) was calculated by comparing to the baseline (the conditions at time “0″ or those obtained in the absence of DEX/CORT pre-treatment and NDV infection). Bars represent means and standard errors of fold changes of the IL-10 mRNA expression and protein concentrations in the culture media obtained from three independent experiments. *: p<0.05, **: p<0.01, ***: p<0.001, compared to the conditions obtained in the presence of DEX or CORT treatment alone. Ctl: control.</p

DEX pre-treatment and NDV infection cooperatively regulated mRNA expression of the four genes in DCs.

<p>Bone marrow-derived DCs were pre-treated with DEX (10⁻⁶M) for 30 min, and were infected with NDV (MOI = 10) for 6 hours. mRNA levels of top four genes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063587#pone.0063587.s003" target="_blank">Table S2</a> were measured in the real-time qPCR by using newly designed primers (panel <b>A</b>: <i>IL-10</i> and <i>CLEC4E</i>: genes whose mRNA expression was cooperatively enhanced by DEX pre-treatment and NDV infection, panel <b>B</b>: <i>PTGS2</i> and <i>IFNγ</i> : genes whose mRNA expression was stimulated by NDV infection but repressed by DEX pre-treatment). Their C_t values were normalized with those of <i>GAPDH</i>, and relative mRNA expression (fold changes) was calculated by comparing to the control obtained in the absence of DEX pre-treatment and NDV infection. Bars represent means and standard errors of fold changes of their mRNA expression obtained in three independent experiments. *: p<0.05, **: p<0.01, compared the 2 conditions indicated.</p

DEX pre-treatment and MCMV infection cooperatively induced IL-10 production <i>in vitro</i> and <i>in vivo</i>, and suppressed inflammation in the liver.

<p><b>A</b>. DEX pre-treatment and MCMV infection cooperatively increased IL-10 mRNA expression in DCs. DCs were pre-treated with DEX (10⁻⁷ or 10⁻⁸ M) for 30 min, and were infected with MCMV (1×10⁶ pfu) for 6 hours. Bars represent means and standard errors of fold changes of the IL-10 mRNA expression. **: p<0.01, compared to the 2 conditions indicated. <b>B & C</b>. DEX pre-treatment and MCMV infection cooperatively induced IL-10 production in mice and suppressed inflammation in their livers. Female mice were pre-treated with DEX (0.3 µg/g animal) twice (at 24 hours and 1 hour prior to viral injection), and were infected with MCMV (1×10⁶ pfu). Two days after the infection, sera and livers were collected. Panel <b>B</b> shows alternation in the serum IL-10 concentrations in mice, while panel <b>C</b> demonstrates representative images of the H&E staining of their livers (magnification 10×). The inset of the left lower panel shows an inflammatory site in the liver caused by MCMV infection (magnification 40×). Bars represent means and standard errors of the serum IL-10 concentrations obtained from three mice. ***: p<0.001, compared to virus infection alone.</p