Organelle Dysfunction and Apoptosis in Liver Epithelia

描述(由申请人提供)：本提案的总体目标是确定最终导致健康和转化的肝细胞致命性损伤的细胞机制。由于肿瘤坏死因子-α相关的凋亡诱导配体(TRAIL)在人类肝脏病理生物学和癌症治疗中的重要性，我们的计划正在研究该死亡配体导致肝损伤的细胞和亚细胞机制。具体地说，我们的长期目标是了解TRAIL介导的细胞死亡的机制。基于大量的初步数据，我们提出了一个新的中心假设，即TRAIL通过触发细胞死亡的溶酶体途径来传递细胞死亡信号。一种细胞毒性途径，其特征是将溶酶体蛋白水解酶组织蛋白酶B释放到胞浆中，导致组织蛋白酶B介导的线粒体功能障碍和细胞死亡。我们现在将使用现有的和互补的、分子、生化和细胞生物学的方法来确定TRAIL是如何触发这一未知的凋亡途径的。我们的建议有三个具体目标。首先，我们将直接验证TRAIL信号转导溶酶体通透性的假设：a)通过激活Bcl2蛋白家族中的促凋亡成员，特别是激活Bax；以及b)Bax易位到溶酶体膜内并在其内寡聚，导致组织蛋白酶B释放到胞浆中。其次，我们将检验这样一种假设，即通常在肝癌中过表达的抗凋亡蛋白cFLIP可以阻止TRAIL介导的溶酶体通透性：a)通过将激酶RIP1招募到TRAIL受体复合体，进而激活ERK1/2；以及b)通过ERK1/2介导的BID磷酸化，这是一种失活的翻译后修饰。最后，我们将验证TRAIL介导的损伤在胆管结扎小鼠中发生的假设：a)依赖于肝脏中TRAIL的主要细胞来源自然杀伤(NK)和自然杀伤-T(NKT)细胞的机制；以及b)肝细胞溶酶体的通透性。该提案在技术和概念上都是创新的，因为它使用尖端技术测试了试验细胞毒性的新概念。产生的信息的意义在于，它将为潜在的开发新的治疗策略提供一个框架，有效地促进人类肝癌的TRAIL治疗，并减轻TRAIL介导的人类肝损伤。拨款申请研究了TRAIL的细胞机制，TRAIL是一种正在开发的用于癌症治疗的自然死亡诱导蛋白，它导致细胞死亡。我们认为TRAIL通过破坏被称为溶酶体的关键细胞器来诱导细胞死亡。溶酶体的破坏会导致有毒化合物释放到细胞内，最终导致细胞死亡。这些研究的结果与肝损伤的机制和TRAIL作为化疗药物的使用是密切相关的。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to define the cellular mechanisms culminating in lethal injury of healthy and transformed liver cells. Because of the importance of tumor necrosis factor-alpha related apoptosis inducing ligand (TRAIL) in human liver pathobiology and cancer therapy, our program is working on the cellular and subcellular mechanisms causing liver injury by this death ligand. Specifically, our long term objectives are to understand the mechanisms involved in TRAIL-mediated cell death. Based on extensive preliminary data, we propose the novel CENTRAL HYPOTHESIS that TRAIL signals cell death by triggering the lysosomal pathway of cell death. A cytotoxic pathway characterized by release of the lysosomal protease cathepsin B into the cytosol which results in cathepsin B-mediated mitochondrial dysfunction and cell death. We will now employ current and complementary, molecular, biochemical and cell biological approaches to ascertain how TRAIL triggers this unexplored pathway of apoptosis. Our proposal has three SPECIFIC AIMS. FIRST, we will directly test the hypothesis that TRAIL signals lysosomal permeabilization: a) by activating proapoptotic members of the Bcl-2 family of proteins, specifically tBid activation of Bax; and b) Bax translocation to and oligomerization within lysosomal membranes causing release of cathepsin B into the cytosol. SECOND, we will test the hypothesis that the antiapoptotic protein cFLIP, commonly overexpressed in liver cancers, prevents TRAIL-mediated lysosomal permeabilization: a) by recruiting the kinase RIP1 to the TRAIL receptor complex which in turn activates ERK1/2; and b) by ERK1/2-mediated Bid phosphorylation, an inactivating post- translational modification. FINALLY, we will test the hypothesis that TRAIL-mediated injury in the bile duct ligated mouse occurs: a) by a mechanism dependent upon natural killer (NK) and natural killer-T (NKT) cells, the major cellular sources of TRAIL in the liver; and b) by hepatocyte lysosomal permeabilization. The proposal is innovative technically and conceptually as it tests new concepts for TRAIL cytotoxicity using sophisticated technologies. The significance of the information generated is that it will provide a framework for the potential development of novel therapeutic strategies effective for both promoting TRAIL therapy of human liver cancers and attenuating TRAIL-mediated human liver injury. The grant application examines the cellular mechanisms by which TRAIL, a natural death inducing protein being developed for cancer therapy, causes cell death. We propose that TRAIL induces cell death by disrupting a key cellular organelle, referred to as the lysosome. Lysosome disruption results in the release of toxic compounds into the cell culminating in cell demise. The results of these studies are germane both to mechanisms of liver injury and the use of TRAIL as a chemotherapeutic agent.