Moehlenkamp J D, Johnson J A
Department of Pharmacology, Toxicology, and Therapeutics, Mental Retardation Research Center and Center for Environmental and Occupational Health, University of Kansas Medical Center, Kansas City 66160-7417, USA.
Arch Biochem Biophys. 1999 Mar 1;363(1):98-106. doi: 10.1006/abbi.1998.1046.
The present investigation demonstrates distinct patterns of activation for antioxidant/electrophile-responsive elements (ARE/EpREs) in cells of neuronal versus hepatic origin suggesting the possibility of cell-/tissue-specific signaling pathways and/or transcription factors required for ARE/EpRE activation. The ARE/EpRE is a cis-acting regulatory element found in 5'-flanking regions of numerous genes including NAD(P)H:quinone oxidoreductase (QR) and glutathione S-transferases. Insomuch as ARE/EpRE activation has been studied primarily in hepatoma cell lines there is little information on how these responsive elements and corresponding genes are regulated in brain. ARE/EpRE-reporter constructs were transiently transfected into IMR-32 human neuroblastoma cells. Activation of ARE/EpRE sequences by tert-butylhydroquinone (tBHQ), a redox-cycling compound, in IMR-32 cells (20- to 30-fold) is dramatically different from the minimal response seen in HepG2 human hepatoma cells (2- to 3-fold). beta-napthoflavone, an ARE/EpRE inducer in HepG2 cells, as well as the oxidants hydrogen peroxide and tert-butyl hydroperoxide did not induce the ARE/EpRE in IMR-32 cells. In addition, we show that the core sequence containing a complete 5' palindrome is necessary for maximal activation of the ARE/EpRE in IMR-32 cells. Mutations within this palindromic sequence decrease basal level expression and block induction by tBHQ but not phorbol 12-myristate 13-acetate. Furthermore, activation of the hQR-ARE/EpRE by tBHQ correlates with induction of endogenous QR activity in IMR-32 neuroblastoma cells (15-fold). Thus, elucidating the mechanism of ARE/EpRE activation in this human neuroblastoma cell line may identify unknown transcription factors or signal transduction cascades regulating ARE/EpRE-driven gene expression.
本研究表明，神经元来源和肝脏来源的细胞中抗氧化剂/亲电试剂反应元件（ARE/EpREs）的激活模式不同，这表明存在细胞/组织特异性信号通路和/或ARE/EpRE激活所需的转录因子。ARE/EpRE是一种顺式作用调节元件，存在于许多基因的5'侧翼区域，包括NAD(P)H:醌氧化还原酶（QR）和谷胱甘肽S-转移酶。由于ARE/EpRE激活主要在肝癌细胞系中进行研究，关于这些反应元件和相应基因在脑中如何调节的信息很少。将ARE/EpRE报告基因构建体瞬时转染到IMR-32人神经母细胞瘤细胞中。氧化还原循环化合物叔丁基对苯二酚（tBHQ）对IMR-32细胞中ARE/EpRE序列的激活（20至30倍）与在HepG2人肝癌细胞中所见的最小反应（2至3倍）有显著差异。β-萘黄酮是HepG2细胞中的ARE/EpRE诱导剂，以及氧化剂过氧化氢和叔丁基过氧化氢在IMR-32细胞中均未诱导ARE/EpRE。此外，我们表明含有完整5'回文的核心序列对于IMR-32细胞中ARE/EpRE的最大激活是必需的。该回文序列内的突变降低基础水平表达并阻断tBHQ的诱导，但不阻断佛波醇12-肉豆蔻酸酯13-乙酸酯的诱导。此外，tBHQ对hQR-ARE/EpRE的激活与IMR-32神经母细胞瘤细胞中内源性QR活性的诱导（15倍）相关。因此，阐明该人神经母细胞瘤细胞系中ARE/EpRE激活的机制可能会识别调节ARE/EpRE驱动基因表达的未知转录因子或信号转导级联反应。
