Cellular Mechanisms of Hepatotoxicity.

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肝毒性的小鼠模型具有高度可重复性，并广泛用于阐明药物和毒素引起的肝损伤机制。最近，一个主要的作用，信号转导通路在调节毒性下游的APAP代谢已被确定使用该模型。已发现c-jun-N-末端激酶（JNK）的激活及其对线粒体的作用在导致APAP诱导的坏死的信号通路中是关键的。该提案的目的是阐明APAP诱导的MAPK激酶活化的机制，以及活化的JNK靶向线粒体在肝细胞中实施APAP致死效应的意义、靶点和后果。其目标如下：（1）确定P-JNK与特定线粒体靶蛋白结合的作用和意义，所述特定线粒体靶蛋白已在APAP肝毒性的发病机制中被鉴定：结合配偶体将通过免疫沉淀和蛋白质组学方法鉴定，线粒体JNK靶的重要性将通过腺病毒-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的早期和持续活化的信号转导途径：各种上游途径如GSK 32、Ras/Raf-1、MLK-3、MEKK-1、PKC 1、Akt 1+2将使用反义寡核苷酸沉默和/或敲除小鼠进行检查，以阐明随后的ASK-1所需的JNK的初始ASK-1非依赖性激活。（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GRP 78的敲除或过表达来评估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坏死的信号转导途径中起关键作用，这将使用RIP-1的特异性抑制剂和反义沉默来评估。这四个目标的阐明将导致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反应是美国急性肝衰竭的主要原因，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（杀死肝细胞）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。

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.