Liu Yanxia, Kern Jonathan T, Walker John R, Johnson Jeffrey A, Schultz Peter G, Luesch Hendrik
Department of Medicinal Chemistry, University of Florida, 1600 Southwest Archer Road, Gainesville, FL 32610, USA.
Proc Natl Acad Sci U S A. 2007 Mar 20;104(12):5205-10. doi: 10.1073/pnas.0700898104. Epub 2007 Mar 12.
The antioxidant response element (ARE) is a cis-acting regulatory enhancer element found in the 5' flanking region of many phase II detoxification enzymes. Up-regulation of ARE-dependent target genes is known to have neuroprotective effects; yet, the mechanism of activation is largely unknown. By screening an arrayed collection of approximately 15,000 full-length expression cDNAs in the human neuroblastoma cell line IMR-32 with an ARE-luciferase reporter, we have identified several cDNAs not previously associated with ARE activation. A subset of cDNAs, encoding sequestosome 1 (SQSTM1) and dipeptidylpeptidase 3 (DPP3), activated the ARE in primary mouse-derived cortical neurons. Overexpression of SQSTM1 and DPP3 in IMR-32 cells stimulated NF-E2-related factor 2 (NRF2) nuclear translocation and led to increased levels of NAD(P)H:quinone oxidoreductase 1, a protein which is transcriptionally regulated by the ARE. When transfected into IMR-32 neuroblastoma cells that were depleted of transcription factor NRF2 by RNA interference, SQSTM1 and DPP3 were unable to activate the ARE or induce NAD(P)H:quinone oxidoreductase 1 expression, indicating that the ARE activation upon ectopic expression of these cDNAs is mediated by NRF2. Studies with pharmacological inhibitors indicated that 1-phosphatidylinositol 3-kinase and protein kinase C signaling are essential for activity. Overexpression of these cDNAs conferred partial resistance to hydrogen peroxide or rotenone-induced toxicity, consistent with the induction of antioxidant and phase II detoxification enzymes, which can protect from oxidative stress. This work and other such studies may provide mechanisms for activating the ARE in the absence of general oxidative stress and a yet-unexploited therapeutic approach to degenerative diseases and aging.
抗氧化反应元件（ARE）是一种顺式作用调控增强子元件，存在于许多II相解毒酶的5'侧翼区域。已知ARE依赖性靶基因的上调具有神经保护作用；然而，其激活机制在很大程度上尚不清楚。通过用ARE - 荧光素酶报告基因筛选人神经母细胞瘤细胞系IMR - 32中约15,000个全长表达cDNA的阵列文库，我们鉴定出了几种先前与ARE激活无关的cDNA。其中一部分编码聚集体蛋白1（SQSTM1）和二肽基肽酶3（DPP3）的cDNA，在原代小鼠来源的皮质神经元中激活了ARE。在IMR - 32细胞中过表达SQSTM1和DPP3可刺激NF - E2相关因子2（NRF2）核转位，并导致NAD(P)H：醌氧化还原酶1水平升高，该蛋白受ARE转录调控。当将SQSTM1和DPP3转染到通过RNA干扰耗尽转录因子NRF2的IMR - 32神经母细胞瘤细胞中时，它们无法激活ARE或诱导NAD(P)H：醌氧化还原酶1表达，这表明这些cDNA异位表达时对ARE的激活是由NRF2介导的。药理学抑制剂研究表明，1 - 磷脂酰肌醇3 - 激酶和蛋白激酶C信号传导对活性至关重要。这些cDNA的过表达赋予了对过氧化氢或鱼藤酮诱导的毒性的部分抗性，这与抗氧化剂和II相解毒酶的诱导一致，这些酶可以保护细胞免受氧化应激。这项工作以及其他此类研究可能为在没有一般氧化应激的情况下激活ARE提供机制，并为退行性疾病和衰老提供一种尚未开发的治疗方法。
