Hydroxynonenal induces glutathione synthesis through JNK

描述（由申请人提供）：我们的长期目标是了解如何调节谷胱甘肽（GSH）的生物合成，从而保护肺部免受氧化损伤。肺对氧化应激的适应涉及细胞去除活性和有毒分子的能力的增加。也许最常见的机制是合成内源性抗氧化剂GSH的能力增加，GSH对于适应至关重要，因为它对于去除氢过氧化物和有毒脂质过氧化产物（如4-羟基壬烯醛（HNE））至关重要。HNE在暴露于任何种类的氧化应激时产生，但有趣的是，它是通过增加谷氨酸半胱氨酸连接酶（GCL）的调节和催化亚基的转录来合成谷胱甘肽的最强诱导剂之一。GCL催化谷胱甘肽合成的限速和第一步。有很多证据支持TRE和EpRE - cis元件在两个GCL基因的调控中的作用。我们发现EpRE和TRE元件的激活模式是转录因子（TF）开关。我们的研究结果还表明，抑制应激激活蛋白激酶（JNK）可以完全抑制HNE诱导的GCL。因此，我们假设HNE诱导的两个GCL基因的转录涉及从无活性或抑制EpRE和TRE TF复合物到转录活性复合物的转换。我们还假设，JNK信号通路的HNE激活对于GCL基因表达中EpRE和AP-1的激活至关重要。目的是：1)确定HNE引起的与EpRE和TRE结合复合物结合的转录因子结合复合物的变化如何导致GCL基因的转录增加；2)确定HNE激活JNK的机制。正常人支气管上皮细胞（NHBE）和HBE1细胞将被用于暴露于亚毒性浓度的HNE。在目标1中，我们将通过DNA亲和层析和LC-MS/MS分析鉴定潜在的TF，然后使用染色质免疫沉淀（ChiP）测定在整个GCL基因的背景下原位鉴定EpRE和TRE TF复合物。“NoShift”和Shift-Western试验将用于量化TF结合的变化，TF功能将通过沉默TF基因和测量EpRE和TRE驱动的荧光素酶报告基因来确定。在Aim 2中，将研究JNK抑制对与EpRE结合的tf的结合和功能的影响，并确定JNK途径中的哪个蛋白质与HNE结合。相关性：氧化损伤是炎症、其他呼吸系统疾病和暴露于空气污染物时肺损伤的一个主要组成部分。内源性抗氧化剂谷胱甘肽通过诱导酶GCL在适应亚致死氧化应激过程中增加。在这项研究中，将研究GSH在膜氧化毒性产物反应中增加的机制，希望能使人们了解如何在不使用有毒物质的情况下增加GSH。

DESCRIPTION (provided by applicant): Our long range goal is to understand how to regulate glutathione (GSH) biosynthesis and thereby protect lungs from oxidative damage. Adaptation to oxidative stress in the lung involves an increase in the ability of cells to remove reactive and toxic molecules. Perhaps the most common mechanism for this is an increase in the ability to synthesize the endogenous antioxidant, GSH, which is essential for adaptation as it is critical in removal of hydroperoxides and toxic lipid peroxidation products, such as 4-hydroxynonenal (HNE). HNE is produced during exposure to any kind of oxidative stress but, interestingly, is one of the strongest inducers of GSH synthesis through increased transcription of both the regulatory and catalytic subunits of glutamate cysteine ligase (GCL). GCL catalyzes the rate limiting and first step in GSH synthesis. There is much evidence supporting roles for TRE and EpRE cis elements in regulation of the two GCL genes. We found that a little explored mode of activation of the EpRE and TRE elements is transcription factor (TF) switching. Our results also indicated that inhibition of the stress activated protein kinase, JNK, results in complete suppression of GCL induction by HNE. Thus, we hypothesize that HNE induced transcription of the two GCL genes involves switching from inactive or suppressing EpRE and TRE TF complexes to transcriptionally active complexes. We also hypothesize that HNE activation of the JNK signaling pathway is critical to both EpRE and AP-1 activation in GCL gene expression. The aims are: 1) to determine how the changes in the transcription factor binding complexes that bind to EpRE and TRE binding complexes in response to HNE cause increased transcription of both GCL genes and 2) to determine the mechanism of JNK activation by HNE. Both normal human bronchial epithelial cells (NHBE) and HBE1 cells will be used in exposures to subtoxic concentrations of HNE. In Aim 1, we will identify potential TFs by DNA affinity chromatography and LC-MS/MS analysis and then use chromatin immunoprecipitation (ChiP) assays to identify the EpRE and TRE TF complexes in the context of the whole GCL genes in situ. "NoShift" and Shift-Western assays will be used to quantify changes in TF binding and TF functionality will be determined by silencing TF genes and measuring EpRE and TRE driven luciferase reporters. In Aim 2, the effect of JNK inhibition on binding and function of TFs bound to EpRE will be examined along with a determination of which protein in the JNK pathway binds HNE. Relevance: Oxidative damage is a major component of lung injury during inflammation, other respiratory diseases and in exposure to air pollutants. The endogenous antioxidant glutathione increases during adaptation to sublethal oxidative stress through induction of the enzyme, GCL. In this investigation, the mechanism through which GCL increases in response to a toxic product of membrane oxidation will be investigated, hopefully leading to understanding of how to increase GSH without the use of toxic agents.