Jover R, Bort R, Gómez-Lechón M J, Castell J V
Unidad de Hepatología Experimental, Centro de Investigación, Hospital Universitario La Fe, SVS, Valencia, Spain.
Hepatology. 2001 Mar;33(3):668-75. doi: 10.1053/jhep.2001.22176.
Hepatocyte nuclear factor 4 (HNF4) is a member of the nuclear receptor super-family that has shown activating effects on particular cytochrome P450 (CYP) promoters from several species. However, its role in the regulation of human CYPs in the liver is still poorly understood, as no comprehensive studies in human-relevant models have been performed. In the present study, we have investigated whether HNF4 plays a general role in the expression of 7 major CYP genes in primary cultured human hepatocytes. To this end, we developed an adenoviral vector for efficient expression of HNF4 antisense RNA. Transduction of human hepatocytes with the recombinant adenovirus resulted in a time-dependent increase in the antisense transcript, followed by a concomitant decrease in apolipoprotein C III mRNA (a target gene of HNF4). Specificity was confirmed by showing that increasing levels of HNF4 antisense RNA resulted in the reduction of HNF4 protein, whereas retinoic X receptoralpha-(RXRalpha), the closest homologous member of the nuclear receptor super-family, was unaffected. Analysis of CYP gene expression in human hepatocytes transfected with HNF4 antisense RNA revealed singular behaviors: (1) CYP3A4, CYP3A5, and CYP2A6 showed an important, dose-dependent down-regulation on blockage of HNF4 translation; (2) a moderate inhibition of CYP2B6, CYP2C9, and CYP2D6 expression was observed (40%-45% reduction); (3) the levels of CYP2E1 were not affected even in the absence of this transcription factor. In conclusion, using an original strategy (efficient antisense RNA expression vector), our study shows that HNF4 is a general regulator supporting the expression of major drug-metabolizing CYPs in human hepatocytes.
肝细胞核因子4（HNF4）是核受体超家族的成员之一，已被证明对来自多个物种的特定细胞色素P450（CYP）启动子具有激活作用。然而，由于尚未在与人类相关的模型中进行全面研究，其在肝脏中对人类CYPs的调节作用仍知之甚少。在本研究中，我们调查了HNF4在原代培养的人肝细胞中7种主要CYP基因表达中是否发挥普遍作用。为此，我们构建了一种用于高效表达HNF4反义RNA的腺病毒载体。用重组腺病毒转导人肝细胞导致反义转录本随时间增加，随后载脂蛋白C III mRNA（HNF4的一个靶基因）随之减少。通过显示HNF4反义RNA水平的增加导致HNF4蛋白减少，而核受体超家族中最接近的同源成员视黄酸X受体α（RXRα）不受影响，证实了其特异性。对转染了HNF4反义RNA的人肝细胞中CYP基因表达的分析揭示了独特的行为：（1）CYP3A4、CYP3A5和CYP2A6在HNF4翻译受阻时表现出重要的、剂量依赖性下调；（2）观察到CYP2B6、CYP2C9和CYP2D6表达受到中度抑制（降低40%-45%）；（3）即使在没有这种转录因子的情况下，CYP2E1的水平也不受影响。总之，通过使用一种原始策略（高效反义RNA表达载体），我们的研究表明HNF4是支持人肝细胞中主要药物代谢CYPs表达的普遍调节因子。
