Hydroxynonenal induces glutathione synthesis through JNK

羟基壬烯醛通过 JNK 诱导谷胱甘肽合成

• 批准号: 7637852
• 负责人: HENRY Jay FORMAN
• 金额: $ 32.47万
• 依托单位: UNIVERSITY OF CALIFORNIA, MERCED
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-22 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7637852
• 关键词: 4 hydroxynonenal; Address; Affinity Chromatography; Air Pollutants; Anabolism; Antibodies; Antioxidants; Be++ element; Beryllium; Binding; Biological Assay; Catalytic Domain; Cell Extracts; Cells; Chromatin; Complex; DNA; Dominant-Negative Mutation; Elements; Enzyme Induction; Enzymes; Epithelial Cells; Excision; Exposure to; FarGo; GCLC gene; GCLM gene; Gene Expression; Genes; Genetic Transcription; Glutamate-Cysteine Ligase; Glutathione; Goals; Hour; Human; Hydrogen Peroxide; Immunoprecipitation; In Situ; Indium; Inflammation; Injury; Interphase Cell; Investigation; Ligase Gene; Lipid Peroxidation; Luciferases; Lung; Lung diseases; MAPK14 gene; MAPK8 gene; Mass Spectrum Analysis; Measurement; Measures; Membrane; Metabolism; Nuclear Protein; Nuclear Proteins; Oxidants; Oxidative Stress; Pathologic Processes; Pathway interactions; Peptides; Phosphotransferases; Physiological; Post-Translational Protein Processing; Proteins; Regulation; Reporter; Research Personnel; Rest; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Signal Transduction; Signaling Protein; Stress; System; Tissues; Transcription Factor AP-1; Transcription factor genes; Western Blotting; Xenobiotics; chromatin immunoprecipitation; design; human GCLC protein; inhibitor/antagonist; lung injury; overexpression; oxidation; oxidative damage; programs; protein folding; response; stress activated protein kinase; transcription factor

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.

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）生物合成，从而保护肺部免受氧化损伤。肺部对氧化应激的适应涉及细胞清除反应性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 的能力增强，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（例如 4-羟基壬烯醛 (HNE)）至关重要。 HNE 在暴露于任何类型的氧化应激时都会产生，但有趣的是，它是通过增加谷氨酸半胱氨酸连接酶 (GCL) 调节亚基和催化亚基转录的最强 GSH 合成诱导剂之一。 GCL 催化 GSH 合成的限速和第一步。有大量证据支持 TRE 和 EpRE 顺式元件在两个 GCL 基因的调节中的作用。我们发现，EpRE 和 TRE 元件激活的一个探索较少的模式是转录因子 (TF) 转换。我们的结果还表明，抑制应激激活蛋白激酶 JNK 会导致 HNE 完全抑制 GCL 诱导。因此，我们假设 HNE 诱导的两个 GCL 基因的转录涉及从非活性或抑制性 EpRE 和 TRE TF 复合物转变为转录活性复合物。我们还假设 JNK 信号通路的 HNE 激活对于 GCL 基因表达中的 EpRE 和 AP-1 激活至关重要。目的是：1) 确定与 EpRE 和 TRE 结合复合物结合的转录因子结合复合物响应 HNE 的变化如何导致 GCL 基因转录增加；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 测定将用于量化 TF 结合的变化，并且 TF 功能将通过沉默 TF 基因并测量 EpRE 和 TRE 驱动的荧光素酶报告基因来确定。在目标 2 中，将检查 JNK 抑制对与 EpRE 结合的 TF 的结合和功能的影响，同时确定 JNK 途径中的哪种蛋白与 HNE 结合。相关性：氧化损伤是炎症、其他呼吸道疾病和暴露于空气污染物期间肺损伤的主要组成部分。在适应亚致死氧化应激过程中，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，将研究 GCL 响应膜氧化有毒产物而增加的机制，希望有助于了解如何在不使用有毒试剂的情况下增加 GSH。