Itoh Masahiro, Nakajima Miki, Higashi Eriko, Yoshida Ryoko, Nagata Kiyoshi, Yamazoe Yasushi, Yokoi Tsuyoshi
Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
J Pharmacol Exp Ther. 2006 Nov;319(2):693-702. doi: 10.1124/jpet.106.107573. Epub 2006 Jul 20.
CYP2A6 plays important roles in the metabolism of nicotine and some clinically used drugs. Interindividual variability in the CYP2A6 expression level in human liver might be caused by an inducible property, but the molecular mechanism of induction is unclear. Rifampicin, phenobarbital, and 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime, which are activators of pregnane X receptor (PXR) and constitutive androstane receptor (CAR), induced CYP2A6 mRNA in human hepatocytes. We identified three direct repeat separated by four nucleotides (DR4)-like elements at -6698, -5476, and -4618 in the CYP2A6 gene, to which PXR and CAR could bind after dimerization with retinoid X receptor (RXR)-alpha. In luciferase assays, overexpression of PXR or CAR could not activate the transcriptional activity of CYP2A6 promoter constructs (-6754 to -1) in HepG2 cells. Cotransfection of hepatocyte nuclear factor-4alpha did not affect the transcriptional activities in the absence or presence of PXR or CAR. Interestingly, cotransfection of peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) as well as PXR significantly enhanced the transcriptional activity (3.9-fold of control). By the deletion of a possible suppresser region (-4533 to -185), the effects of PXR/PGC-1alpha on the transcriptional activity were increased (6.9-fold of control). Deletion or mutation analyses revealed that two DR4-like elements at -5476 and -4618 are essential for transactivation by PXR/PGC-1alpha. Chromatin immunoprecipitation assay revealed that PXR and PGC-1alpha bind to CYP2A6 chromatin. In conclusion, we found that CYP2A6 is induced via PXR and PGC-1alpha through the DR4-like element at the distal response region. This is the first study to report the molecular mechanism of the induction of CYP2A6.
细胞色素P450 2A6（CYP2A6）在尼古丁及某些临床用药的代谢过程中发挥着重要作用。人类肝脏中CYP2A6表达水平的个体差异可能是由其可诱导性引起的，但诱导的分子机制尚不清楚。利福平、苯巴比妥以及6-（4-氯苯基）咪唑并[2,1-b][1,3]噻唑-5-甲醛O-（3,4-二氯苄基）肟，这些孕烷X受体（PXR）和组成型雄烷受体（CAR）的激活剂，可在人肝细胞中诱导CYP2A6信使核糖核酸（mRNA）的产生。我们在CYP2A6基因的-6698、-5476和-4618位点鉴定出三个由四个核苷酸分隔的类似直接重复序列4（DR4）的元件，PXR和CAR与视黄酸X受体（RXR）-α二聚化后可与之结合。在荧光素酶检测中，过表达PXR或CAR并不能激活HepG2细胞中CYP2A6启动子构建体（-6754至-1）的转录活性。在不存在或存在PXR或CAR的情况下，共转染肝细胞核因子-4α并不影响转录活性。有趣的是，共转染过氧化物酶体增殖物激活受体-γ共激活因子1α（PGC-1α）以及PXR可显著增强转录活性（为对照的3.9倍）。通过缺失一个可能的抑制区域（-4533至-185），PXR/PGC-1α对转录活性的影响增强（为对照的6.9倍）。缺失或突变分析表明，-5476和-4618位点的两个类似DR4的元件对于PXR/PGC-1α的反式激活至关重要。染色质免疫沉淀检测表明，PXR和PGC-1α与CYP2A6染色质结合。总之，我们发现CYP2A6是通过PXR和PGC-1α经由远端反应区域的类似DR4的元件被诱导的。这是首篇报道CYP2A6诱导分子机制的研究。
