Sugatani Junko, Nishitani Shinichi, Yamakawa Kasumi, Yoshinari Kouichi, Sueyoshi Tatsuya, Negishi Masahiko, Miwa Masao
Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan.
Mol Pharmacol. 2005 Mar;67(3):845-55. doi: 10.1124/mol.104.007161. Epub 2004 Nov 22.
UDP-glucuronosyltransferase (UGT) 1A1 glucuronidates endogenous metabolites, such as bilirubin, and exogenous substances, and plays a critical role in their detoxification and excretion. In a previous article, we described the phenobarbital response activity to a 290-base pair (bp) distal enhancer sequence (-3499/-3210) of the human UGT1A1 gene that is activated by the constitutive androstane receptor (CAR). Here, we show that dexamethasone at submicromolar concentrations enhances the pregnane X receptor (PXR) activator-mediated expression of the UGT1A1 gene and protein in HepG2 cells. We investigated the molecular mechanism of UGT1A1 induction by glucocorticoids at submicromolar concentrations and PXR activators and the functional cross-talk between the glucocorticoid receptor (GR) and CAR/PXR. The glucocorticoid-response element (GRE) was characterized by cotransfection experiments, site-directed mutagenesis, and electrophoretic mobility shift assays. Analysis of the human UGT1A1 promoter revealed GREs at -3404/-3389 and -3251/-3236 close to the CAR/PXR response element gtNR1 (-3382/-3367). Furthermore, in an in vitro reporter gene assay, dexamethasone effectively enhanced CAR/PXR-mediated transactivation of the 290-bp distal enhancer module in HepG2 cells and CV-1 cells in the presence of exogenously expressed GR and glucocorticoid receptor-interacting protein 1 (GRIP1). In glutathione S-transferase pull-down experiments, CAR and PXR interacted with GRIP1. Together, these results demonstrate a rational mechanistic basis for UGT1A1 induction by glucocorticoids and PXR activators, showing that activated GR enhances CAR/PXR-mediated UGT1A1 regulation with the transcriptional cofactor GRIP1 and that GR may be involved synergistically in the xenobiotic-responsive regulation of UGT1A1 by CAR/PXR.
尿苷二磷酸葡萄糖醛酸基转移酶（UGT）1A1可使胆红素等内源性代谢产物及外源性物质发生葡萄糖醛酸化，并在这些物质的解毒和排泄过程中发挥关键作用。在之前的一篇文章中，我们描述了人类UGT1A1基因290个碱基对（bp）的远端增强子序列（-3499/-3210）对苯巴比妥的反应活性，该序列由组成型雄烷受体（CAR）激活。在此，我们表明亚微摩尔浓度的地塞米松可增强孕烷X受体（PXR）激活剂介导的HepG2细胞中UGT1A1基因和蛋白的表达。我们研究了亚微摩尔浓度的糖皮质激素和PXR激活剂诱导UGT1A1的分子机制，以及糖皮质激素受体（GR）与CAR/PXR之间的功能性相互作用。通过共转染实验、定点诱变和电泳迁移率变动分析对糖皮质激素反应元件（GRE）进行了表征。对人类UGT1A1启动子的分析显示，在靠近CAR/PXR反应元件gtNR1（-3382/-3367）的-3404/-3389和-3251/-3236处存在GRE。此外，在体外报告基因测定中，在地塞米松存在外源性表达的GR和糖皮质激素受体相互作用蛋白1（GRIP1）的情况下，地塞米松可有效增强CAR/PXR介导的HepG2细胞和CV-1细胞中290-bp远端增强子模块的反式激活。在谷胱甘肽S-转移酶下拉实验中，CAR和PXR与GRIP1相互作用。这些结果共同证明了糖皮质激素和PXR激活剂诱导UGT1A1的合理机制基础，表明激活的GR通过转录辅因子GRIP1增强CAR/PXR介导的UGT1A1调节，并且GR可能协同参与CAR/PXR对UGT1A1的外源性物质反应调节。
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