Impaired expression of glutathione synthetic enzyme genes in mice with targeted deletion of the Nrf2 basic-leucine zipper protein
Impaired expression of glutathione synthetic enzyme genes in mice with targeted deletion of the Nrf2 basic-leucine zipper protein
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DOI：
10.1016/s0167-4781(00)00238-4
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发表时间：
2000-12-15
期刊：
影响因子：
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通讯作者：
Kwong, M
中科院分区：
文献类型：
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作者：
Chan, JY;Kwong, M
在外源生物的解毒和对氧化应激的防御中起作用的基因的转录激活部分是由抗氧化反应元件(ARE)介导的。例如，研究表明，重链和轻链谷氨酰半胱氨酸合成酶(GCS(H)和GCS(L))基因的启动子都需要ARE。Cnc-bZIP因子与小的Maf蛋白一起，被证明以异源二聚体的形式与NF-E2/AP-1元件结合，这与ARE的共同序列相似。Nrf1和Nrf2是两个广泛表达的Cnc-bZIP因子，参与了氧化应激反应相关基因的调控。在这项研究中，我们研究了Nrf2突变对谷胱甘肽合成相关基因表达的影响。我们观察到GCS(H)和GCS(L)基因在NRF2(-/-)成纤维细胞和肝脏中的转录减少。相应地，在Nrf2缺乏的肝脏和成纤维细胞中，谷胱甘肽水平降低。通过对NRF2(-/-)成纤维细胞的瞬时转染研究，我们发现携带人GCS(L)启动子的报告结构的转录激活以及GCS(H)启动子的功能区域都需要激活蛋白NRF2。经凝胶迁移率改变分析，重组NRF2与GCS(L)启动子和GCS(H)启动子的ARE结合。NRF2基因的过表达恢复了NRF2(-/-)成纤维细胞的谷胱甘肽水平，这与GCS(H)和GCS(L)转录本的稳态水平升高相关。这些结果建立了Nrf2转录因子和GSH生物合成之间的联系。(C)2000 Elsevier Science B.V.保留所有权利。
Transcriptional activation of genes that play a role in detoxification of xenobiotics and defense against oxidative stress is mediated in part by the antioxidant response element (ARE). For example, it has been shown that the promoters for both the heavy and light chain gamma -glutamylcysteine synthetase (GCS(H) and GCS(L)) genes require the ARE. CNC-bZIP factors, together with small Maf proteins, have been shown to bind as heterodimers to the NF-E2/AP-1 element, which is similar to the consensus sequence for the ARE. Nrf1 and Nrf2, two widely expressed CNC-bZIP factors, have been implicated in the regulation of genes involved in oxidative stress response. In this study, we examined the effect of nrf2 mutation on the expression of genes involved in glutathione synthesis. We observed that transcripts for gcs(H) and gcs(L) genes were decreased in nrf2(-/-) fibroblasts and livers. Correspondingly, glutathione levels were decreased in Nrf2 deficient livers and fibroblasts. By transient transfection studies in nrf2(-/-) fibroblasts, we show that transcriptional activation of reporter constructs bearing the human GCS(L) promoter, as well as the functional ARE of GCS(H) promoter, required the activator protein Nrf2. By electrophoretic mobility shift assay, recombinant Nrf2 binds the ARE of the GCS(L) and GCS(H) promoters. Overexpression of Nrf2 cDNA restored glutathione (GSH) levels in nrf2(-/-) fibroblasts, which correlated with increased steady state levels of gcs(H) and gcs(L) transcripts. These results establish a link between Nrf2 transcription factor and GSH biosynthesis. (C) 2000 Elsevier Science B.V. All rights reserved.