Nuclear factor Nrf2 and antioxidant response element regulate NRH:quinone oxidoreductase 2 (NQO2) gene expression and antioxidant induction

Human NRH:quinone oxidoreductase 2 (NQO2) is a cytosolic protein that catalyzes the metabolic reduction of quinones and provides protection against myelogenous hyperplasia and chemical carcinogenesis. NQO2 gene expression is induced in response to antioxidant tert-butylhydroquinone (tBHQ). Sequence analysis revealed six putative antioxidant response elements (ARE1 through 6) in the human NQO2 gene promoter. Deletion mutagenesis and transfection studies suggested that the ARE region between nucleotides -1433 and -1424 is essential for basal expression and antioxidant induction of NQO2 gene expression. Mutation of this ARE from 3.8 kb NQO2 gene promoter significantly repressed expression and abrogated the induction in response to antioxidant in transfected cells. Band shift, supershift, and chromatin immunoprecipitation (ChIP) assays demonstrated binding of nuclear factors Nrf2 and JunD with human NQO2 gene ARE. Coimmunoprecipitation experiments revealed an association between Nrf2 and JunD. Overexpression of Nrf2 upregulated and overexpression of Nrf2 dominant-negative mutant downregulated ARE-mediated NQO2 gene expression. The treatment of Hep-G2 cells with Nrf2-specific RNAi significantly reduced Nrf2 and NQO2 gene expression and tBHQ induction. The results combined demonstrated that Nrf2 associates with JunD, binds to ARE at nucleotide -1433, and regulates human NQO2 gene expression and induction in response to antioxidants.     

Role of Galpha12 and Galpha13 as novel switches for the activity of Nrf2, a key antioxidative transcription factor

Galpha12 and Galpha13 function as molecular regulators responding to extracellular stimuli. NF-E2-related factor 2 (Nrf2) is involved in a protective adaptive response to oxidative stress. This study investigated the regulation of Nrf2 by Galpha12 and Galpha13. A deficiency of Galpha12, but not of Galpha13, enhanced Nrf2 activity and target gene transactivation in embryo fibroblasts. In mice, Galpha12 knockout activated Nrf2 and thereby facilitated heme catabolism to bilirubin and its glucuronosyl conjugations. An oligonucleotide microarray demonstrated the transactivation of Nrf2 target genes by Galpha12 gene knockout. Galpha12 deficiency reduced Jun N-terminal protein kinase (JNK)-dependent Nrf2 ubiquitination required for proteasomal degradation, and so did Galpha13 deficiency. The absence of Galpha12, but not of Galpha13, increased protein kinase C delta (PKC delta) activation and the PKC delta-mediated serine phosphorylation of Nrf2. Galpha13 gene knockout or knockdown abrogated the Nrf2 phosphorylation induced by Galpha12 deficiency, suggesting that relief from Galpha12 repression leads to the Galpha13-mediated activation of Nrf2. Constitutive activation of Galpha13 promoted Nrf2 activity and target gene induction via Rho-mediated PKC delta activation, corroborating positive regulation by Galpha13. In summary, Galpha12 and Galpha13 transmit a JNK-dependent signal for Nrf2 ubiquitination, whereas Galpha13 regulates Rho-PKC delta-mediated Nrf2 phosphorylation, which is negatively balanced by Galpha12.     

内皮细胞和平滑肌细胞氧化应激时Nrf2/ARE信号通路对抗氧化基因表达的调控:与动脉粥样硬化和先兆子痫的关系

动脉粥样硬化、糖尿病、慢性肾功能衰竭和先兆子痫等血管疾病时活性氧(reactive oxygen species,ROS)生成增加,容易导致内皮依赖性血管舒张功能的损害和血管损伤,而细胞可以诱导多种编码Ⅱ相解毒酶和抗氧化蛋白的基因表达,从而减轻ROS和亲电子物质介导的细胞损伤。一个被称为抗氧化反应元件(antioxidant response element,ARE)或亲电子反应元件(electrophile response element,EpRE)的顺式转录调控元件,可以介导诸如亚铁血红素加氧酶1、γ-谷氨酰半胱氨酸合成酶、硫氧还蛋白还原酶、谷胱甘肽-S转移酶和NAD(P)H:苯醌氧化还原酶等基因的转录。其他抗氧化酶,如超氧化物歧化酶、过氧化氢酶和非酶清除剂(如谷胱甘肽)等也参与ROS的清除。转录因子NF-E2相关因子2(nuclear factor-erythroid 2-related factor 2, Nrf2)是属于Cap‘n’Collar家族的转录因子,具有碱性亮氨酸拉链(basic region-leucine zipper,bZIP),它在ARE介导的抗氧化基因表达中起重要的作用。在正常情况下,Kelch样环氧氯丙烷相关蛋白-1(Kelch-like ECH-associated protein-1,Keapl)与Nrf2耦联,并与肌动蛋白细胞骨架结合被锚定于胞浆,但是在半胱氨酸残基发生氧化的情况下,Nrf2和Keapl解耦联,进入细胞核并与ARE结合,从而激活多种抗氧化基因和Ⅱ相解毒酶基因的转录。蛋白激酶C、丝裂原活化蛋白激酶和磷脂酰肌醇-3激酶参与Nrf2／ARE信号转导的调控。本文综述了有关Nrf2／ARE信号转导通路在血管稳态和动脉硬化、先兆子痫等疾病情况下内皮及平滑肌细胞对抗持续性氧化应激中起的作用。