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与人类胆汁淤滞性肝病（如原发性硬化性胆管炎）相关的模型中最终导致胆管细胞和肝细胞致命损伤的细胞机制。肿瘤坏死因子相关凋亡诱导配体（Tumor necrosis factor-related apoptosis inducing ligand， TRAIL）在与这些肝脏疾病相关的模型中参与了胆管细胞和肝细胞的损伤。因此，我们的长期目标是了解这种死亡配体引起胆管细胞和肝细胞损伤的细胞和亚细胞机制。我们独特的初步数据表明，磷酸氟酸簇分选-2 （PACS-2）蛋白是trail通过溶酶体细胞死亡途径诱导的细胞毒性的关键调节因子。细胞凋亡抑制剂-1 （cIAP-1）通过控制PACS-2蛋白水平调控溶酶体细胞死亡途径。在体内，胆管细胞似乎对通过这一途径杀死TRAIL具有内在敏感性，而在体内，肝细胞仅在胆汁淤积时才对TRAIL毒性敏感。基于这些初步数据，我们提出了新的中央假设，即TRAIL通过依赖于PACS-2的溶酶体途径诱导肝细胞死亡，而在体内，比肝细胞表达更少的cIAP-1的胆管细胞对这一途径优先敏感。我们现在将采用当前和互补的分子、生化和细胞生物学方法来确定TRAIL如何触发这种基于细胞器的凋亡途径。我们的建议有三个具体目标。首先，我们将通过以下方式直接验证PACS-2在TRAIL细胞毒性过程中介导溶酶体膜渗透的假设：a)结合bh3蛋白Bim，该蛋白招募并激活Bcl-2家族中有效的促凋亡蛋白Bax；b)促进溶酶体膜内Bax寡聚化，导致溶酶体断裂。其次，我们将直接验证E3连接酶cIAP-1通过以下途径调节TRAIL细胞毒性的假设：a)通过泛素化依赖机制调节PACS- 2蛋白水平，导致其蛋白酶体降解；b)通过控制PACS-2蛋白水平调节溶酶体细胞死亡途径。最后，我们将直接验证以下假设：PACS-2缺失对胆汁淤积性肝损伤相关模型有益：a)在原发性硬化性胆管炎模型中减少trail介导的体内胆管细胞损伤；b)通过减少胆管结扎小鼠梗阻性胆汁淤积时的肝细胞损伤。该提案具有创新性，因为它使用复杂的技术测试了TRAIL细胞毒性的新概念。所产生的信息将为开发有效减轻胆汁淤滞期间trail介导的人肝损伤的新型治疗策略提供框架。

DESCRIPTION (provided by applicant): The OVERALL OBJECTIVE of this proposal is to define the cellular mechanisms culminating in lethal injury of cholangiocytes and hepatocytes in models relevant to human cholestatic liver diseases (e.g., primary sclerosing cholangitis). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) contributes to both cholangiocyte and hepatocyte injury in models relevant to these liver diseases. Thus, our long term objectives are to understand the cellular and subcellular mechanisms causing cholangiocyte and hepatocyte injury by this death ligand. Our distinctive preliminary data implicate phosphofurin acidic cluster sorting-2 (PACS-2) protein as a critical regulator of TRAIL-induced cytotoxicity by a lysosomal cell death pathway. Cellular inhibitor of apoptosis-1 (cIAP-1) regulates the lysosomal cell death pathway by controlling PACS-2 protein levels. Cholangiocytes appear to be intrinsically sensitive to TRAIL killing via this pathway in vivo, whereas hepatocytes in vivo only become sensitive to TRAIL toxicity during cholestasis. Based upon this preliminary data, we propose the novel CENTRAL HYPOTHESIS that TRAIL induces liver cell death by a lysosomal pathway dependent upon PACS-2, and cholangiocytes, which express less cIAP-1 than hepatocytes, are preferentially sensitive to this pathway in vivo. We will now employ current and complementary molecular, biochemical and cell biological approaches to ascertain how TRAIL triggers this organelle-based pathway of apoptosis. Our proposal has three SPECIFIC AIMS. FIRST, we will directly test the hypothesis that PACS-2 mediates lysosomal membrane permeabilization during TRAIL cytotoxicity by: a) binding Bim, a BH3-only protein, which recruits and activates Bax, a potent proapoptotic protein of the Bcl-2 family; and b) facilitating Bax oligomerization within lysosomal membranes which causes lysosomal disruption. SECOND, we will directly test the hypothesis that cIAP-1, an E3 ligase, modulates TRAIL cytotoxicity by: a) regulating PACS- 2 protein levels via an ubiquitination-dependent mechanism resulting in its proteasomal degradation; and b) by regulating the lysosomal cell death pathway through control of PACS-2 protein levels. FINALLY, we will directly test the hypothesis that: PACS-2 deletion is salutary in models relevant to cholestatic liver injury by: a) reducing TRAIL-mediated cholangiocyte injury in vivo in a model reminiscent of primary sclerosing cholangitis; and b) by reducing hepatocyte injury in vivo during obstructive cholestasis in the bile duct ligated mouse. The proposal is innovative as it tests new concepts for TRAIL cytotoxicity using sophisticated technologies. The information generated will provide a framework for the development of novel therapeutic strategies effective for attenuating TRAIL-mediated human liver injury during cholestasis. PUBLIC HEALTH RELEVANCE: The application examines the cellular mechanisms by which TRAIL, a natural protein, 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 cellular demise. The results of these studies are germane to mechanisms of liver injury in a variety of human diseases.