Division of Pulmonary, Allergy and Immunology, Cystic Fibrosis and Sleep, Department of Pediatrics, Emory School of Medicine, Emory University, 2015 Uppergate Drive #350, Atlanta, GA 30322, USA.
Cell Biol Toxicol. 2010 Dec;26(6):541-51. doi: 10.1007/s10565-010-9162-6. Epub 2010 Apr 29.
Tert-butylhydroquinone (tBHQ), the major metabolite of butylated hydroxyanisole, induces an antioxidant response through the redox-sensitive transcription factor, nuclear factor-E2-related factor-2 (Nrf2). However, the mechanism by which tBHQ induces Nrf2 activity is not entirely understood. Here, we show that tBHQ preferentially alters the redox status in the mitochondrial compartment in HeLa cells. HeLa cells treated with tBHQ showed a preferential oxidation of mitochondrial thioredoxin-2 (Trx2), while cellular glutathione and cytosolic thioredoxin-1 were not affected. Preferential mitochondrial oxidation by tBHQ was supported by detection of reactive oxygen species (ROS) specific to this compartment. To determine the role of Trx2 in regulating downstream effects of tBHQ, HeLa cells were transiently transfected with an empty, Trx2, or C93S (Cys93Ser) Trx2 dominant-negative mutant expression vector. Overexpression of Trx2 decreased basal mitochondrial ROS production, whereas expression of C93S Trx2 enhanced it. In addition, under untreated conditions, expression of C93S Trx2 led to an increase in the basal activities of Nrf2. With tBHQ treatments, Trx2 overexpression suppressed Nrf2 accumulation and activity, whereas expression of C93S Trx2 had no effect on the degree of inducibility or Nrf2 accumulation but did increase the overall activity of Nrf2. Quantitative polymerase chain reaction analysis of Nrf2-regulated gene expression corroborate Trx2 control of tBHQ-mediated Nrf2 activation. These data show a compartment-specific effect where tBHQ-induced Nrf2 signaling is mediated by Trx2 and suggest that antioxidant status in various compartments would provide different levels of control of redox signaling.
叔丁基对苯二酚（tBHQ）是丁基羟基茴香醚的主要代谢物，通过氧化还原敏感转录因子核因子-E2 相关因子-2（Nrf2）诱导抗氧化反应。然而，tBHQ 诱导 Nrf2 活性的机制尚不完全清楚。在这里，我们表明 tBHQ 优先改变 HeLa 细胞中线粒体区室的氧化还原状态。用 tBHQ 处理的 HeLa 细胞表现出线粒体硫氧还蛋白-2（Trx2）的优先氧化，而细胞谷胱甘肽和胞质硫氧还蛋白-1不受影响。tBHQ 对线粒体的优先氧化得到了该细胞器中活性氧物种（ROS）特异性检测的支持。为了确定 Trx2 在调节 tBHQ 下游效应中的作用，HeLa 细胞被瞬时转染为空载体、Trx2 或 C93S（Cys93Ser）Trx2 显性负突变体表达载体。Trx2 的过表达降低了基础线粒体 ROS 的产生，而 C93S Trx2 的表达增强了它。此外，在未处理条件下，C93S Trx2 的表达导致基础 Nrf2 活性增加。用 tBHQ 处理时，Trx2 的过表达抑制了 Nrf2 的积累和活性，而 C93S Trx2 的表达对诱导程度或 Nrf2 积累没有影响，但确实增加了 Nrf2 的整体活性。Nrf2 调节基因表达的定量聚合酶链反应分析证实了 Trx2 对 tBHQ 介导的 Nrf2 激活的控制。这些数据显示了一种区室特异性效应，其中 tBHQ 诱导的 Nrf2 信号通过 Trx2 介导，并表明不同区室中的抗氧化状态将提供不同水平的氧化还原信号控制。
