Hydroxynonenal induces glutathione synthesis through JNK

描述（由申请人提供）：该竞争性更新申请的重点是通过激活EpRE和TRE顺式元件以及JNK信号通路，通过脂质过氧化产物4-羟基-2-壬烯醛（HNE）诱导谷氨酸半胱氨酸连接酶（GCL）， GSH合成中的速率限制酶。亚致死暴露于氧化剂和亲电试剂的常见反应是谷胱甘肽浓度的增加，这对适应更大的压力至关重要。这种增加依赖于谷氨酸半胱氨酸连接酶（GCL，也称为-谷氨酰半胱氨酸合成酶）的催化和调节亚基的表达增加，GCL是重新合成谷胱甘肽的第一个酶。健康人血浆中的HNE为0.3-0.7微摩尔，但在炎症性疾病和暴露于空气污染物时增加十倍以上。虽然HNE在氧化应激下的组织中浓度可能是有毒的，但它是GSH、GCL亚基基因和其他II期酶的有效诱导性物质。我们最近发现转录因子转换似乎可以调节GCL，通过可渗透的显性负肽抑制JNK可以完全抑制HNE诱导的GCL，而ERK或p38MAPK抑制剂都无效。我们的假设是，HNE通过改变EpRE和AP-1结合复合物的组成来诱导两种人类GCL基因，从抑制转录的复合物到激活转录的复合物。我们还假设EpRE和AP-1结合复合物组成的改变是通过JNK信号通路的HNE激活介导的。使用人支气管上皮细胞（HBE1）进行所有研究，本提案的具体目的是：1-确定调节GCLC， GCLM， NQO1和GST4A4对HNE的反应的EpRE结合复合物的组成；2-确定HNE引起EpRE结合复合物组成改变的机制；3-确定AP-1复合物在HNE反应中的变化与GCL基因转录增加的关系；4-确定HNE如何激活JNK通路。方法包括DNA亲和层析，高效液相色谱串联质谱，免疫耗尽EMSA，染色质免疫沉淀测定，Western blotting，蛋白质易位，蛋白质磷酸化测量。长期目标是了解HNE诱导GCL的分子基础，以帮助开发具有较小潜在毒性的药物的合理设计。

DESCRIPTION (provided by applicant): This competing renewal application focuses on induction of glutamate cysteine ligase (GCL), the rate limiting enzyme in glutathione (GSH) synthesis, by the lipid peroxidation product, 4-hydroxy-2-nonenal (HNE) through activation of EpRE and TRE cis elements and the JNK signaling pathway. A common response to sublethal exposure to oxidants and electrophiles is an increase in GSH concentration, which is critical to adaptation to greater stress. This increase is dependent on the increased expression of the catalytic and modulatory subunits of glutamate cysteine ligase (GCL, also called (-glutamylcysteine synthetase), the first enzyme in de novo GSH synthesis. HNE in healthy human plasma is 0.3-0.7 micromolar, but increases more than tenfold in inflammatory diseases and exposure to air pollutants. Although HNE can be toxic, at concentrations found in tissues under oxidative stress, it is a potent inducer of GSH, both GCL subunit genes, and other Phase II enzymes. We recently found that transcription factor switching appears to regulate GCL and that inhibition of JNK by a permeable dominant negative peptide completely inhibited GCL induction by HNE while neither ERK or p38MAPK inhibitors were effective. Our hypothesis is that induction of both human GCL genes by HNE occurs through alteration of the compositions of the EpRE and AP-1 binding complexes from those that repress to those that activate transcription. We also hypothesize that the alteration of composition of the EpRE and AP-1 binding complexes is mediated through HNE activation of the JNK signaling pathway. Using human bronchial epithelial cells (HBE1) for all studies, the specific aims of this proposal are to: 1- determine the composition of the complexes binding to the EpRE that regulate GCLC, GCLM, NQO1, and GST4A4 in response to HNE; 2- determine the mechanism of HNE by which HNE causes changing composition of the EpRE binding complexes; 3- determine how the changes in AP-1 complexes in response to HNE relate to the increase of GCL gene transcription; and 4- determine how HNE activates the JNK pathway. The approaches include DNA affinity chromatography, HPLC tandem mass spectrometry, immunodepletion EMSA, chromatin immunoprecipitation assays, Western blotting, protein translocation, protein phosphorylation measurements. Understanding the molecular basis of HNE induction of GCL to assist development of rational design of a drug with a less potential toxicity is the long range goal.