Cellular Mechanisms of Hepatotoxicity.

描述(由申请人提供)：扑热息痛(APAP)毒性是急性肝功能衰竭的主要原因。APAP小鼠肝毒性模型重复性好，被广泛用于阐明药物和毒素所致肝损伤的机制。最近，利用这个模型已经确定了信号转导通路在APAP代谢下游的毒性调节中的主要作用。已发现c-Jun-N末端激酶(JNK)的激活及其对线粒体的影响在APAP诱导的坏死的信号通路中起关键作用。本研究的目的是阐明APAP诱导MAPK激酶活化的机制，以及激活的JNK靶向线粒体在APAP对肝细胞的致死作用中的意义、靶点和后果。目的如下：(1)确定P-JNK与特定的线粒体靶蛋白结合在APAP肝毒性发病机制中的作用和意义：结合伙伴将通过免疫沉淀和蛋白质组学方法确定，线粒体JNK靶点的重要性将通过腺病毒shRNA敲除来确定；纯激活的JNK对线粒体功能和完整性的影响将被评估：(2)确定在APAP毒性中导致JNK早期和持续激活的信号转导通路：各种上游通路，如GSK32、RAS/Raf-1、MLK-3、Mekk-1、PKC1、Akt1+2的相互作用，将通过反义寡核苷酸沉默和/或基因敲除小鼠来阐明随后ASK-1依赖的JNK持续激活所需的初始ASK-1独立激活；(3)确定内质网应激在APAP毒性中的作用：将通过敲除或过度表达关键的内质网应激感受器GRP78来评估内质网应激对导致线粒体崩溃的信号转导通路的可能贡献；(4)确定RIP激酶在APAP诱导的坏死中的作用及其与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，这将使用特定的抑制剂和RIP-1的反义沉默来评估。阐明这四个目标将有助于更好地理解信号转导途径和线粒体在肝毒性中的作用和相互作用，并将发现新的治疗靶点。 公共卫生相关性：醋氨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，是药物开发的主要绊脚石。这项建议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，以防止扑热息痛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.