Nakamura Akiko, Nakajima Miki, Higashi Eriko, Yamanaka Hiroyuki, Yokoi Tsuyoshi
Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-Machi, Kanazawa, Japan.
Pharmacogenet Genomics. 2008 Aug;18(8):709-20. doi: 10.1097/FPC.0b013e32830500c9.
Human uridine diphosphate-glucuronosyltransferase 2B7 (UGT2B7) plays important roles in the metabolism of some clinical drugs, carcinogens, and steroid hormones. The molecular mechanisms of the inducible expression of UGT2B7 in response to xenobiotics have not been fully clarified. We sought to investigate whether the UGT2B7 is under the control of NF-E2 p45-related factor 2 (Nrf2), a key transcriptional factor regulating the expression of cytoprotective enzymes.
HepG2, HuH7, HLE, and Caco-2 cells were treated with sulforaphane (SFN), and the UGT2B7 mRNA levels were determined by real-time reverse transcriptase PCR. These cells were genotyped for the UGT2B7*2 (H268Y) allele using the PCR-restriction fragment length polymorphism method. Luciferase analyses and gel shift analyses were performed to identify the responsive regions for Nrf2 signaling.
The UGT2B7 mRNA was induced by SFN in HepG2 and HuH7 genotyped as UGT2B71/1, but not in HLE and Caco-2 cells genotyped as UGT2B72/2. In HepG2 cells, the UGT2B7 protein level and morphine glucuronosyltransferase activity were also significantly induced by SFN. The induction was prominently decreased with small interfering RNA for Nrf2. In the 5'-flanking region (-2.5 kb) of the UGT2B72 allele, a 324-base pair insertion at -2067 and 12 single nucleotide polymorphisms simultaneously existed. Luciferase analyses and gel shift analyses revealed that an antioxidant responsive element at -1170 was responsible for the transactivation by Nrf2. In addition, a region from -990 to -858 on the UGT2B71 allele was also responsible for the transactivation by Nrf2. Abrogation of the Nrf2-dependent transactivation of the UGT2B7*2 allele was owing to the single nucleotide polymorphism -900A>G.
UGT2B7 is transcriptionally regulated by Nrf2, but the mechanism is hindered by polymorphisms in the promoter region of UGT2B7*2. The allele-specific mechanism may cause variability of the glucuronidation in response to oxidative stress.
人尿苷二磷酸葡萄糖醛酸基转移酶2B7（UGT2B7）在某些临床药物、致癌物和甾体激素的代谢中发挥重要作用。UGT2B7对外源物质诱导表达的分子机制尚未完全阐明。我们试图研究UGT2B7是否受核因子E2 p45相关因子2（Nrf2）的调控，Nrf2是一种调节细胞保护酶表达的关键转录因子。
用萝卜硫素（SFN）处理HepG2、HuH7、HLE和Caco-2细胞，通过实时逆转录PCR测定UGT2B7 mRNA水平。采用PCR-限制性片段长度多态性方法对这些细胞的UGT2B7*2（H268Y）等位基因进行基因分型。进行荧光素酶分析和凝胶迁移分析以鉴定Nrf2信号的反应区域。
SFN诱导UGT2B71/1基因型的HepG2和HuH7细胞中UGT2B7 mRNA表达，但不诱导UGT2B72/2基因型的HLE和Caco-2细胞中UGT2B7 mRNA表达。在HepG2细胞中，SFN也显著诱导UGT2B7蛋白水平和吗啡葡萄糖醛酸基转移酶活性。用Nrf2小干扰RNA处理后，诱导作用明显降低。在UGT2B72等位基因的5'侧翼区（-2.5 kb），-2067处有一个324碱基对的插入片段，同时存在12个单核苷酸多态性。荧光素酶分析和凝胶迁移分析显示，-1170处的抗氧化反应元件负责Nrf2的反式激活。此外，UGT2B71等位基因上-990至-858区域也负责Nrf2的反式激活。UGT2B7*2等位基因Nrf2依赖性反式激活的消除是由于单核苷酸多态性-900A>G。
UGT2B7受Nrf2转录调控，但该机制因UGT2B7*2启动子区域的多态性而受阻。等位基因特异性机制可能导致葡萄糖醛酸化在氧化应激反应中的变异性。
