The effect of GW3965 and dexamethasone on gene expression of rat livers

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 LXRa/RXRa 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 LXRa/RXRa 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.糖皮质激素（glucocorticoids）是一类甾体激素，可通过提升糖异生关键酶——葡萄糖-6-磷酸酶（glucose-6-phosphatase, G6Pase）的转录速率，强力调控碳水化合物中间代谢，同时还可抑制免疫系统，因此成为治疗变态反应性、自身免疫性与炎症性疾病的核心治疗药物之一。循环中的糖皮质激素需通过糖皮质激素受体（glucocorticoid receptor, GR）将生物信号传递至细胞核内。肝核X受体（liver X receptors, LXRs）可结合胆固醇代谢物，与类视黄醇X受体（retinoid X receptor, RXR）形成异二聚体，进而调控胆固醇周转、通过下调G6Pase表达调节肝脏葡萄糖代谢，并抑制免疫细胞内一组炎症基因的表达。本研究旨在探究GR与LXR介导的信号通路之间的串扰效应。本研究通过基于瞬时转染的报告基因实验，以及使用靶向LXRs的小干扰RNA（small interfering RNAs, siRNAs）进行基因沉默的方法，证实过表达或通过配体GW3965激活LXRs/RXRs，可以基因特异性方式抑制GR介导的部分糖皮质激素反应元件（glucocorticoid response element, GRE）驱动的启动子的反式激活活性。在大鼠体内，GW3965对LXRs的激活可削弱地塞米松诱导的循环血糖升高；在大鼠、小鼠及人肝癌HepG2细胞中，GW3965可抑制地塞米松诱导的肝脏葡萄糖-6-磷酸酶（G6Pase）的mRNA表达。对大鼠肝脏开展的微阵列转录组分析显示，GW3965可差异性调控约15%的内源性糖皮质激素响应基因的糖皮质激素诱导转录活性。机制层面，通过体外染色质免疫沉淀实验，我们发现LXRα/RXRα可结合GRE序列，并以基因特异性方式抑制GR与这些DNA序列的结合。上述全新发现进一步通过体内结合实验，以及使用重组LXRα/RXRα蛋白检测其与经典GRE或G6Pase GRE相互作用的凝胶迁移实验得到了验证。我们提出，应用LXR受体激动剂可通过直接抑制GR在葡萄糖和/或脂质代谢相关通路中的转录活性，从而改善糖皮质激素治疗或应激相关的代谢紊乱状态。