Burger H J, Schuetz J D, Schuetz E G, Guzelian P S
Department of Medicine, Medical College of Virginia, Richmond 23298.
Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2145-9. doi: 10.1073/pnas.89.6.2145.
The family 3A cytochromes P-450, among the most abundant members of this supergene family of microsomal hemoproteins expressed in animal and human liver, are inducible by glucocorticoids but also by such antiglucocorticoids as pregnenolone 16 alpha-carbonitrile (PCN). To investigate the mechanism for this nonclassical glucocorticoid effect, we analyzed the ability of 1.5 kilobases of DNA or of its successive subsegments isolated from the 5' flanking region of the rat CYP3A1 structural gene to modulate transcription of a reporter gene consisting of a viral promoter coupled to the chloramphenicol acetyltransferase (CAT) structural gene (expression vector pBLCAT2) and transiently expressed in a homologous cell system consisting of primary monolayer cultures of adult rat hepatocytes in which CYP3A1 mRNA and protein are inducible. The CAT activity measured after chimeric gene constructions were transferred into the cultured rat hepatocytes by lipofection increased as much as 7.2-fold if the cells were treated with dexamethasone (DEX). One CYP3A1 fragment (positions -220 to -56; 164 base pairs), which does not contain a traditional glucocorticoid responsive element, conferred dose-dependent DEX responsiveness independent of its orientation but not its position in pBLCAT2. This construction was activated by addition of PCN to the cultures and was synergistically induced by PCN plus DEX. In contrast, induction of CAT activity in cultures containing MMTVCAT, a plasmid containing the CAT gene controlled by the mouse mammary tumor virus long terminal repeat, was unaffected by PCN treatment, required lower concentrations of DEX for a maximal response, and was inhibited by treatment with DEX plus PCN. We conclude that a primary mechanism for induction of CYP3A1 is stimulated transcription through a pathway activated by steroid hormones.
3A 族细胞色素 P-450 是在动物和人类肝脏中表达的微粒体血红素蛋白超基因家族中最为丰富的成员之一，它不仅可被糖皮质激素诱导，还可被诸如孕烯醇酮 16α-腈（PCN）等抗糖皮质激素诱导。为了研究这种非经典糖皮质激素效应的机制，我们分析了从大鼠 CYP3A1 结构基因 5'侧翼区分离的 1.5 千碱基 DNA 或其连续亚片段调节报告基因转录的能力，该报告基因由与氯霉素乙酰转移酶（CAT）结构基因偶联的病毒启动子组成（表达载体 pBLCAT2），并在由成年大鼠肝细胞原代单层培养物组成的同源细胞系统中瞬时表达，其中 CYP3A1 mRNA 和蛋白质是可诱导的。通过脂质转染将嵌合基因构建体转移到培养的大鼠肝细胞后测量的 CAT 活性，如果细胞用 dexamethasone（DEX）处理，可增加多达 7.2 倍。一个 CYP3A1 片段（位置 -220 至 -56；164 个碱基对），它不包含传统的糖皮质激素反应元件，赋予了剂量依赖性的 DEX 反应性，与其在 pBLCAT2 中的方向无关，但与其位置无关。通过向培养物中添加 PCN 可激活该构建体，并且 PCN 加 DEX 可协同诱导。相反，在含有 MMTVCAT（一种含有由小鼠乳腺肿瘤病毒长末端重复序列控制的 CAT 基因的质粒）的培养物中 CAT 活性的诱导不受 PCN 处理的影响，达到最大反应需要较低浓度的 DEX，并且用 DEX 加 PCN 处理可抑制。我们得出结论，CYP3A1 诱导的主要机制是通过类固醇激素激活的途径刺激转录。
