Cellular Mechanisms of Hepatotoxicity.

肝毒性的细胞机制。

基本信息

批准号：
8101550
负责人：
NEIL KAPLOWITZ
金额：
$ 40.75万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-08-15 至 2015-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8101550
关键词：
Acetaminophen Acute Liver Failure Adenoviruses Adverse drug effect Antisense Oligonucleotides Binding Bioenergetics Cessation of life Complex Data Event Failure GRP78 gene Glycogen Synthase Kinases Goals Hepatocyte Hepatotoxicity Immunoprecipitation Injury Knock-out Knockout Mice Laboratories Lead Liver MAP3K1 gene MEKKs Mediating Membrane Metabolism Mitochondria Mitogen-Activated Protein Kinase Kinases Modeling Molecular Molecular Chaperones Mus N-acetyl-4-benzoquinoneimine N-terminal Necrosis Pathogenesis Pathology Pathway interactions Pharmaceutical Preparations Phosphotransferases Play Process Proteins Proteomics Ras/Raf Research Role Series Signal Pathway Signal Transduction Signal Transduction Pathway Source Stress Toxic effect Toxicology Toxin United States Work biological adaptation to stress drug development hepatic necrosis improved in vivo inhibitor/antagonist insight killings kinase inhibitor mitochondrial permeability transition pore mouse model new therapeutic target overexpression prevent programs sensor small hairpin RNA stress-activated protein kinase 1

项目摘要

DESCRIPTION (provided by applicant): Acetaminophen (APAP) toxicity is the leading cause of acute liver failure. The mouse model of APAP hepatotoxicity is highly reproducible and widely utilized to elucidate the mechanisms of liver injury, due to drugs and toxins. Recently, a major role for signal transduction pathways in modulating toxicity downstream of APAP metabolism has been identified using this model. The activation of c-jun-N-terminal kinase (JNK) and its effects on mitochondria have been found to be critical in the signaling pathway leading to APAP-induced necrosis. The goal of this proposal is to elucidate the mechanism of APAP-induced activation of MAPK kinases and the significance, targets, and consequences of the targeting of activated JNK to mitochondria in carrying out the lethal effects of APAP in hepatocytes. The aims are as follows: (1) Determine the role and significance of binding of P-JNK to specific mitochondrial target protein which has been identified in the pathogenesis of APAP hepatotoxicity: Binding partners will be identified by immunoprecipitation and proteomic approaches and the importance of the mitochondrial JNK target will be determined by adenovirus-shRNA knockdown; the effects of pure activated JNK on mitochondrial function and integrity will be assessed: (2) Determine the signal transduction pathways that lead to both early and sustained activation of JNK in APAP toxicity: the interplay of various upstream pathways such as GSK32, Ras/Raf-1, MLK-3, MEKK-1, PKC1, Akt 1+2, will be examined using antisense oligonucleotide silencing and/or knockout mice to elucidate the initial ASK-1 independent activation of JNK required for subsequent ASK-1 dependent sustained JNK activation; (3) Determine the role of ER stress in APAP toxicity: the plausible contribution of ER stress to the signal transduction pathways leading to mitochondrial collapse will be assessed by exploiting knockout or over expression of the key ER stress sensor, GRP78; (4) Determine the role of RIP kinases in APAP-induced necrosis and the relationship to the GSK/JNK/Sab pathway: RIP may play a key role in signal transduction pathways leading to APAP necrosis which will be assessed using specific inhibitors and antisense silencing of RIP-1. The elucidation of these four aims will lead to an improved understanding of the role and interplay of signal transduction pathways and mitochondria in hepatotoxicity and which will identify new therapeutic targets. PUBLIC HEALTH RELEVANCE: Adverse effects of drugs such as acetaminophen on the liver, which is the leading cause of acute liver failure in the United States, are a major stumbling block in drug development. This proposal is aimed at developing a detailed understanding of the molecular pathways in the liver that lead to injury (killing of liver cells) and identifying new therapeutic targets to prevent injury form acetaminophen or other causes.

描述（由申请人提供）：对乙酰氨基酚（APAP）毒性是急性肝衰竭的主要原因。 APAP 肝毒性小鼠模型具有高度重复性，广泛用于阐明药物和毒素引起的肝损伤机制。最近，使用该模型确定了信号转导途径在调节 APAP 代谢下游毒性中的主要作用。已发现 c-jun-N 末端激酶 (JNK) 的激活及其对线粒体的影响在导致 APAP 诱导坏死的信号通路中至关重要。本提案的目的是阐明 APAP 诱导的 MAPK 激酶激活机制，以及在肝细胞中发挥 APAP 致死作用时，激活的 JNK 靶向线粒体的意义、靶点和后果。目的如下： (1) 确定 P-JNK 与特定线粒体靶蛋白结合的作用和意义，该蛋白在 APAP 肝毒性的发病机制中已被鉴定：结合伙伴将通过免疫沉淀和蛋白质组学方法进行鉴定，以及结合伙伴的重要性线粒体JNK靶标将通过腺病毒-shRNA敲低来确定；将评估纯活化的 JNK 对线粒体功能和完整性的影响：（2）确定导致 APAP 毒性中 JNK 早期和持续活化的信号转导途径：各种上游途径（例如 GSK32、Ras/Raf）的相互作用-1、MLK-3、MEKK-1、PKC1、Akt 1+2，将使用反义寡核苷酸沉默和/或敲除小鼠进行检查，以阐明最初的ASK-1 独立激活 JNK 是后续 ASK-1 依赖性持续 JNK 激活所必需的； (3) 确定 ER 应激在 APAP 毒性中的作用：通过利用关键 ER 应激传感器 GRP78 的敲除或过度表达，评估 ER 应激对导致线粒体崩溃的信号转导途径的合理贡献； (4)确定RIP激酶在APAP诱导的坏死中的作用以及与GSK/JNK/Sab通路的关系：RIP可能在导致APAP坏死的信号转导通路中发挥关键作用，将使用特异性抑制剂和反义沉默来评估RIP激酶的作用RIP-1 的。这四个目标的阐明将有助于更好地了解信号转导途径和线粒体在肝毒性中的作用和相互作用，并将确定新的治疗靶点。公共卫生相关性：对乙酰氨基酚等药物对肝脏的不良影响是美国急性肝功能衰竭的主要原因，也是药物开发的主要障碍。该提案旨在详细了解肝脏中导致损伤（肝细胞死亡）的分子途径，并确定新的治疗靶点以防止对乙酰氨基酚或其他原因造成的损伤。