Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan, Taiwan, ROC; Drug Discovery and Development Center, Chia Nan University of Pharmacy and Science, Tainan, Taiwan, ROC.
Department of Biological Sciences and Technology, National University of Tainan, Tainan, Taiwan, ROC.
Biochimie. 2018 May;148:99-106. doi: 10.1016/j.biochi.2018.03.001. Epub 2018 Mar 12.
Leucine-rich repeats and WD repeat domain-containing protein 1 (LRWD1) is implicated in the regulation of signal transduction, transcription, RNA processing and tumor development. However, LRWD1 transcriptional regulation is not fully understood. This study aimed to investigate the relationship between LRWD1 expression and reactive oxygen species (ROS) level in human embryonal carcinoma cell line, NT2/D1 cells, which will help in understanding the transcriptional regulatory role of ROS in cells. Results showed that the exposure of NT2/D1 cells to various concentrations of hydrogen peroxide (HO) and the nitric oxide (NO) donor sodium nitroprusside (SNP) caused a significant increase in the mRNA and protein expression of LRWD1. In addition, LRWD1 promoter luciferase reporter assay, and Chromatin Immunoprecipitation assay (CHIP assay) showed that nuclear factor erythroid-2-related factor (Nrf2) was involved in the regulation of LRWD1 expression in response to oxidative stress. The involvement of Nrf2 was confirmed by shRNA-mediated knockdown of Nrf2 in NT2/D1 cells, which caused a significant decrease in LRWD1 expression in response to oxidative stress. Similarly, LRWD1 knockdown resulted in the accumulation of HO and superoxide anion radical (O2-). Blocking ROS production by N-acetyl cysteine (NAC) protected NT2/D1 shLRWD1cells from HO-induced cell death. Collectively, oxidative stress increased LRWD1 expression through a Nrf2-dependent mechanism, which plays an important role in cellular adaptation to oxidative stress. These results highlight an evidence, on the molecular level, about LRWD1 transcriptional regulation under oxidative stress.
富含亮氨酸重复和 WD 重复域蛋白 1（LRWD1）参与信号转导、转录、RNA 加工和肿瘤发生的调节。然而，LRWD1 的转录调控尚未完全阐明。本研究旨在探讨人胚癌细胞系 NT2/D1 细胞中 LRWD1 表达与活性氧（ROS）水平之间的关系，这将有助于理解 ROS 在细胞中的转录调控作用。结果表明，NT2/D1 细胞暴露于不同浓度的过氧化氢（HO）和一氧化氮（NO）供体硝普酸钠（SNP）导致 LRWD1 的 mRNA 和蛋白表达显著增加。此外，LRWD1 启动子荧光素酶报告基因检测和染色质免疫沉淀（CHIP）assay 表明，核因子红细胞 2 相关因子（Nrf2）参与了氧化应激时 LRWD1 表达的调节。通过 NT2/D1 细胞中 Nrf2 的 shRNA 介导敲低证实了 Nrf2 的参与，这导致氧化应激时 LRWD1 表达显著降低。同样，LRWD1 的敲低导致 HO 和超氧阴离子自由基（O2-）的积累。N-乙酰半胱氨酸（NAC）阻断 ROS 的产生可保护 NT2/D1 shLRWD1 细胞免受 HO 诱导的细胞死亡。总之，氧化应激通过 Nrf2 依赖性机制增加 LRWD1 表达，这在细胞适应氧化应激中发挥重要作用。这些结果在分子水平上强调了氧化应激下 LRWD1 转录调控的证据。
