Organelle Dysfunction and Apoptosis in Liver Epithelia

描述（由申请人提供）：本提案的总体目的是定义在与人类胆汁淤积性肝病相关的模型中最终导致胆管细胞和肝细胞致死性损伤的细胞机制（例如，原发性硬化性胆管炎）。肿瘤坏死因子相关凋亡诱导配体（TRAIL）有助于胆管细胞和肝细胞损伤的相关模型，这些肝脏疾病。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暗示磷酸弗林酸性簇分选-2（PACS-2）蛋白作为一个关键的调节TRAIL诱导的细胞毒性的溶酶体细胞死亡途径。细胞凋亡抑制因子-1（cIAP-1）通过控制PACS-2蛋白水平调节溶酶体细胞死亡途径。在体内，胆管细胞似乎对通过该途径的TRAIL杀伤具有内在敏感性，而在体内，肝细胞仅在胆汁淤积期间对TRAIL毒性敏感。基于这些初步数据，我们提出了一个新的中枢假说，即TRAIL通过依赖于PACS-2的溶酶体途径诱导肝细胞死亡，而表达比肝细胞少的cIAP-1的胆管细胞在体内优先对该途径敏感。我们现在将采用当前和互补的分子，生物化学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测试以下假设：a）结合Bim（一种仅含BH 3的蛋白质），其募集并激活Bax（一种Bcl-2家族的有效促凋亡蛋白）;和b）促进溶酶体膜内的Bax寡聚化，导致溶酶体破坏。B）促进溶酶体膜内的Bax寡聚化。第二，我们将直接检验cIAP-1（一种E3连接酶）通过以下方式调节TRAIL细胞毒性的假设：a）通过泛素化依赖性机制调节PACS- 2蛋白水平，导致其蛋白酶体降解;和B）通过控制PACS-2蛋白水平调节溶酶体细胞死亡途径。最后，我们将直接检验以下假设：PACS-2缺失在胆汁淤积性肝损伤相关模型中是有益的：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;和B）在胆管结扎小鼠阻塞性胆汁淤积期间减少体内肝细胞损伤。该提案是创新的，因为它使用复杂的技术测试TRAIL细胞毒性的新概念。所产生的信息将提供一个框架，为发展新的治疗策略，有效地减弱TRAIL介导的人类肝损伤胆汁淤积。

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.