Cytochrome P450 regulation by hepatocyte nuclear factor 4 in human hepatocytes:: A study using adenovirus-mediated antisense targeting
Cytochrome P450 regulation by hepatocyte nuclear factor 4 in human hepatocytes:: A study using adenovirus-mediated antisense targeting
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DOI：
10.1053/jhep.2001.22176
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发表时间：
2001-03-01
期刊：
影响因子：
13.5
通讯作者：
Castell, JV
中科院分区：
文献类型：
--
作者：
Jover, R;Bort, R;Castell, JV
肝细胞核因子4（HNF 4）是核受体超家族的成员，对几个物种的特定细胞色素P450（CYP 450）启动子具有激活作用。然而，其在肝脏中人类CYP调节中的作用仍然知之甚少，因为尚未在人类相关模型中进行全面研究。在本研究中，我们研究了HNF 4是否在原代培养的人肝细胞中的7个主要转录因子基因的表达中起普遍作用。为此，我们开发了一种腺病毒载体，用于高效表达HNF 4反义RNA。用重组腺病毒转导人肝细胞导致反义转录物的时间依赖性增加，随后伴随载脂蛋白C III mRNA（HNF 4的靶基因）的减少。通过显示HNF 4反义RNA水平的增加导致HNF 4蛋白的减少来证实特异性，而视黄酸X受体-α（RXR α）（核受体超家族的最接近同源成员）不受影响。对转染HNF 4反义RNA的人肝细胞中CYP 3A 4、CYP 3A 5和CYP 2A 6基因表达的分析显示了独特的行为：（1）在阻断HNF 4翻译时，CYP 3A 4、CYP 3A 5和CYP 2A 6表现出重要的、剂量依赖性的下调;（2）观察到CYP 2B 6、CYP 2C 9和CYP 2D 6表达的中度抑制（40%-45%的减少）;（3）即使在没有这种转录因子的情况下，CYP 2 E1的水平也不受影响。总之，使用原始策略（有效的反义RNA表达载体），我们的研究表明，HNF 4是一种支持人肝细胞中主要药物代谢CYP表达的通用调节剂。
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 receptor-alpha (RXR alpha), 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.