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，从而保护肺免受氧化损伤。肺对氧化应激的适应包括细胞清除活性分子和有毒分子的能力的增强。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对于适应是必不可少的，因为它在清除过氧化氢和有毒的脂质过氧化产物，如4-羟基壬烯醛(HNE)方面至关重要。HNE是在任何类型的氧化应激下产生的，但有趣的是，它是GSH合成的最强诱导剂之一，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。GCL催化GSH合成的限速和第一步反应。有大量证据支持tre和EpRE顺式元件在调控这两个GCL基因中的作用。我们发现，EPRE和TrE元件的激活模式很少探索是转录因子(TF)的切换。我们的结果还表明，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基因的转录涉及从失活或抑制EpRE和TrE TF复合体到转录活性复合体的转换。我们还假设，JNK信号通路的HNE激活对于EpRE和AP-1在GCL基因表达中的激活都是关键的。其目的是：1)确定转录因子结合复合体与EpRE和TrE结合复合体对HNE的反应是如何引起两种GCL基因转录增加的；2)确定HNE激活JNK的机制。正常的人支气管上皮细胞(NHBE)和HBE1细胞都将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分析来鉴定潜在的TF，然后使用染色质免疫沉淀(ChIP)技术来原位鉴定整个GCL基因背景下的EpRE和tre TF复合体。“NoShift”和Shift-Western分析将被用来量化TF结合的变化，而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来确定。在目标2中，将检测JNK抑制对与EpRE结合的TF的结合和功能的影响，并确定JNK途径中的哪种蛋白与HNE结合。相关性：氧化损伤是炎症、其他呼吸系统疾病和暴露在空气污染物中的肺损伤的主要组成部分。在亚致死性氧化应激的适应过程中，内源性抗氧化剂谷胱甘肽通过GCL酶的诱导而增加。在这项研究中，我们将研究GCL对膜氧化有毒产物的反应机制，希望能帮助我们理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.