Nuclear receptors constitutive androstane receptor and pregnane X receptor activate a drug-responsive enhancer of the murine 5-aminolevulinic acid synthase gene
Nuclear receptors constitutive androstane receptor and pregnane X receptor activate a drug-responsive enhancer of the murine 5-aminolevulinic acid synthase gene
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DOI：
10.1074/jbc.m306148200
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发表时间：
2003-10-10
影响因子：
4.8
通讯作者：
Meyer, UA
中科院分区：
文献类型：
--
作者：
Fraser, DJ;Zumsteg, A;Meyer, UA
核受体参与了越来越多的基因表达的转录调控，包括细胞色素P450和5-氨基乙酰酸合成酶（ALAS1），这是血红素生物合成途径中的第一个也是限速酶。虽然诱导细胞色素P450的药物也能诱导ALAS1，但控制这些途径的调节机制尚未完全阐明。我们已经在小鼠ALAS1基因中发现了一个药物反应增强子。该序列介导多种化合物的转录激活，包括典型的细胞色素P450泛诱导剂苯巴比妥和美替拉酮，以及孕烷X受体和组成型雄烷受体的特异性激活剂。通过瞬时转染leghorn男性肝癌细胞系报告基因构建物，鉴定了ALAS1药物反应增强子序列。使用NUBIScan算法，在元件中确定了DR4核受体结合位点，并通过定点诱变证实了它们在介导ALAS1转录激活中的作用。电泳迁移位移试验表明小鼠妊娠X受体和组成型雄烷受体在ADRES上有明显的相互作用。CV-1细胞的反激活实验表明，核受体是ALAS1转录激活的主要贡献者。此外，敲除动物的体内研究证实，ALAS1的诱导至少部分是由核受体介导的。这些研究首次通过哺乳动物ALAS1的药物反应元件解释药物诱导。
Nuclear receptors have been implicated in the transcriptional regulation of expression of a growing number of genes, including cytochromes P450 and 5-aminolevulinate synthase (ALAS1), the first and rate-limiting enzyme in the heme biosynthesis pathway. Although drugs that induce cytochromes P450 also induce ALAS1, the regulatory mechanisms governing these pathways have not been fully elucidated. We have identified a drug-responsive enhancer in the murine ALAS1 gene. This sequence mediates transcriptional activation by a wide range of compounds including typical cytochrome P450 pan-inducers phenobarbital and metyrapone, as well as specific activators of the pregnane X receptor and the constitutive androstane receptor. ALAS1 drug-responsive enhancer sequences were identified by transient transfection of reporter gene constructs in the drug-responsive leghorn male hepatoma cell line. Using the NUBIScan algorithm, DR4 nuclear receptor binding sites were identified within the elements and their roles in mediating transcriptional activation of ALAS1 were confirmed by site-directed mutagenesis. Electrophoretic mobility shift assays demonstrate clear interactions of mouse pregnane X receptor and constitutive androstane receptor on the ADRES. Transactivation assays in CV-1 cells implicate the nuclear receptors as major contributors to transcriptional activation of ALAS1. Moreover, in vivo studies in knock-out animals confirm the induction of ALAS1 is mediated at least in part by nuclear receptors. These studies are the first to explain drug induction via drug response elements for mammalian ALAS1.