Molecular basis for rhythmic expression of CYP3A4 in serum-shocked HepG2 cells
Molecular basis for rhythmic expression of CYP3A4 in serum-shocked HepG2 cells
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DOI：
10.1097/fpc.0b013e3282f12a61
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发表时间：
2007-12
影响因子：
2.6
通讯作者：
Takako Takiguchi;Miho Tomita;Naoya Matsunaga;Hiroo Nakagawa;S. Koyanagi;S. Ohdo
中科院分区：
文献类型：
--
作者：
Takako Takiguchi;Miho Tomita;Naoya Matsunaga;Hiroo Nakagawa;S. Koyanagi;S. Ohdo
目的几种主要通过CYP3A4代谢消除的药物，其药代动力学随给药时间的不同而变化，但其变化机制尚不清楚。在本研究中，我们研究了肝细胞中CYP3A4 mRNA表达的24小时振荡是如何产生的。方法和结果将HepG2细胞短暂暴露于50%的血清中，可诱导时钟基因表达24小时振荡，以血清休克HepG2细胞为体外模型，研究人肝脏生物钟的分子机制。在血清休克的HepG2细胞中，CYP3A4的mRNA水平和代谢活性都有节律性波动，周期约为24 h。CYP3A4基因表达的振荡似乎是其代谢活性节律性变化的根本原因。荧光素酶报告基因分析和电泳迁移转移实验显示，昼夜节律转录因子d位点结合蛋白（DBP）通过结合转录起始位点上游附近的DNA序列激活CYP3A4基因的转录。DBP对CYP3A4基因的反激活被E4启动子结合蛋白-4 （E4BP4）抑制，E4BP4是生物钟的负组分。结论DBP和E4BP4可能存在一种往复机制，即DBP在白天DBP丰富的时间激活CYP3A4基因的转录，而E4BP4在白天其他时间抑制CYP3A4基因的转录。我们目前的发现提供了生物钟和异种代谢之间的分子联系。
Objective Although the pharmacokinetics of several drugs that are mainly eliminated by the CYP3A4 metabolism vary according to their dosing time, the mechanism of the variation remains poorly understood. In this study, we investigated how the 24-h oscillation in the expression of CYP3A4 mRNA was generated in hepatic cells. Methods and results As brief exposure of HepG2 cells to 50% serum induced the 24-h oscillation in the expression of clock genes, serum-shocked HepG2 cells were employed as an in-vitro model to study the molecular mechanism underlying the circadian clock in the human liver. Both mRNA levels and metabolic activity of CYP3A4 in serum-shocked HepG2 cells fluctuated rhythmically with a period length of about 24 h. The oscillation in the expression of the CYP3A4 gene seemed to be the underlying cause of the rhythmic change in its metabolic activity. Luciferase reporter gene analysis and electrophoretic mobility shift assay revealed that the circadian transcriptional factor, D-site-binding protein (DBP), activated the transcription of the CYP3A4 gene by binding to the DNA sequence near the upstream of the transcriptional start site. The transactivation of the CYP3A4 gene by DBP was repressed by the E4 promoter-binding protein-4 (E4BP4), a negative component of the circadian clock. Conclusions Results from this study suggest that DBP and E4BP4 might consist of a reciprocating mechanism in which DBP activates the transcription of the CYP3A4 gene during the time of day when DBP is abundant, and E4BP4 suppresses the transcription at other times of day. Our current findings provide a molecular link between the circadian clock and the xenobiotic metabolism.