Radjendirane V, Jaiswal A K
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA.
Biochem Pharmacol. 1999 Aug 15;58(4):597-603. doi: 10.1016/s0006-2952(99)00143-4.
Xenobiotics and antioxidants induce expression of detoxifying enzymes including NAD(P)H: quinone oxidoreductase (NQO1), NRH:quinone oxidoreductase (NQO2), and glutathione S-transferase Ya (GST Ya), presumably to provide protection to cells against electrophilic and oxidative stress. Antioxidant response elements (AREs) have been found in the promoter regions of the various detoxifying enzyme genes. An ARE is required for basal expression and induction of the various detoxifying enzyme genes in response to xenobiotics and antioxidants. In this study, we demonstrated that exposure of cells to xenobiotics [e.g. beta-naphthoflavone (beta-NF)] and antioxidants [e.g. tert-butyl hydroquinone (t-BHQ)] also induced the expression of the proto-oncogene c-jun. The induction of c-jun gene expression followed kinetics similar to the induction of NQO1 and NQO2 genes with respect to the level and time of exposure. Sequence analysis of the c-jun gene promoter revealed the presence of an ARE between nucleotides -538 and -514. The c-jun ARE was highly homologous to the AREs from genes encoding NQO1, NQO2, and GST Ya. Constructs containing the c-jun ARE and 1.7 and 4.5 kb of the c-jun promoter ligated to the chloramphenicol acetyltransferase (CAT) gene, upon transfection in human hepatoblastoma (Hep-G2) cells, expressed the CAT gene, which was inducible with beta-NF and t-BHQ. Band shift assays indicated binding of two specific nuclear protein complexes with the c-jun gene ARE. The faster running c-jun gene ARE-nuclear protein complex was specifically competed out by unlabeled NQO1 and GST Ya gene AREs. These results suggest that c-jun gene expression is coordinately induced and regulated with detoxifying enzyme genes in response to xenobiotics and antioxidants. The results also suggest involvement of an ARE-mediated mechanism of induction of c-jun gene expression. However, a comparison of fold induction of endogenous c-jun gene and transfected c-jun promoter/ARE-CAT constructs indicated involvement of another ARE upstream of the 4.5-kb promoter and/or additional mechanisms such as stabilization of c-Jun RNA in response to exposure to xenobiotics and antioxidants.
外源性物质和抗氧化剂可诱导包括NAD(P)H:醌氧化还原酶（NQO1）、NRH:醌氧化还原酶（NQO2）和谷胱甘肽S-转移酶Ya（GST Ya）在内的解毒酶的表达，推测这是为了保护细胞免受亲电试剂和氧化应激的影响。在各种解毒酶基因的启动子区域发现了抗氧化反应元件（AREs）。AREs是各种解毒酶基因基础表达以及对外源性物质和抗氧化剂产生诱导反应所必需的。在本研究中，我们证明细胞暴露于外源性物质[如β-萘黄酮（β-NF）]和抗氧化剂[如叔丁基对苯二酚（t-BHQ）]也会诱导原癌基因c-jun的表达。c-jun基因表达的诱导在暴露水平和时间方面遵循与NQO1和NQO2基因诱导相似的动力学。对c-jun基因启动子的序列分析显示在核苷酸-538至-514之间存在一个ARE。c-jun的ARE与编码NQO1、NQO2和GST Ya的基因的ARE高度同源。包含c-jun的ARE以及1.7 kb和4.5 kb的c-jun启动子并与氯霉素乙酰转移酶（CAT）基因连接的构建体，在转染到人肝癌细胞系（Hep-G2）后，可表达CAT基因，该基因可被β-NF和t-BHQ诱导。凝胶迁移实验表明有两种特异性核蛋白复合物与c-jun基因的ARE结合。迁移速度较快的c-jun基因ARE-核蛋白复合物可被未标记的NQO1和GST Ya基因的ARE特异性竞争取代。这些结果表明，c-jun基因表达与解毒酶基因在对外源性物质和抗氧化剂的反应中是协同诱导和调控的。结果还提示存在一种ARE介导的c-jun基因表达诱导机制。然而，内源性c-jun基因与转染的c-jun启动子/ARE-CAT构建体的诱导倍数比较表明，在4.5 kb启动子上游存在另一个ARE以及/或者存在其他机制，如对外源性物质和抗氧化剂暴露反应中c-Jun RNA的稳定化。
