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致死性肝细胞损伤的细胞机制。由于肿瘤坏死因子- α (tnf - α)在人肝损伤中的重要性，我们的项目正在研究这种细胞因子引起肝损伤的细胞和亚细胞机制。具体来说，我们的长期目标是了解tnf - α介导的肝细胞凋亡的机制。基于广泛的初步数据，我们提出了新的中心假设，即tnf - α信号选择性溶酶体渗透导致组织蛋白酶B释放到细胞质中，在那里它启动线粒体功能障碍导致细胞凋亡和肝损伤。我们现在将采用现有的和互补的分子、生化和细胞生物学方法来确定tnf - α是如何触发这种未被探索的凋亡途径的。我们的建议有三个具体目标。首先，我们将直接检验tnf - α信号溶酶体通透性的假设：a)通过连接蛋白FAN（与中性鞘磷脂酶相关的因子）激活中性鞘磷脂酶，随后生成鞘磷脂；b)依赖于溶酶体内组织蛋白酶b的过程（即组织蛋白酶b依赖性组织蛋白酶b释放）。其次，我们将检验细胞质组织蛋白酶B介导细胞凋亡的假设：a)通过激活细胞凋亡的线粒体途径；b)切割Bcl-2家族的促凋亡成员Bid，导致其激活并易位到线粒体，并诱导细胞色素c的释放。最后，我们将验证组织蛋白酶B介导肝损伤的假设：a)在胆管结扎小鼠内源性刺激tnf - α后，组织蛋白酶B敲除小鼠对肝损伤具有抗性；b)组织蛋白酶b基因敲除小鼠胆管结扎后肝纤维化参数降低。该提案在技术和概念上都是创新的，因为它使用复杂的技术测试了tnf - α介导的肝损伤和细胞凋亡的新概念。所产生的信息的意义在于，它将为有效减轻人类肝损伤的新型治疗策略的潜在发展提供一个框架。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to define the cellular mechanisms culminating in lethal liver cell injury. Because of the importance of tumor necrosis factor-alpha (TNF-alpha) in human liver injury, our program is working on the cellular and subcellular mechanisms causing liver injury by this cytokine. Specifically, our long-term objectives are to understand the mechanisms involved in TNF-alpha-mediated hepatocyte apoptosis. Based on extensive preliminary data, we propose the novel CENTRAL HYPOTHESIS that TNF-alpha signals selective lysosomal permeabilization resulting in cathepsin B release into the cytosol, where it initiates mitochondrial dysfunction resulting in apoptosis and liver injury. We will now employ current and complementary, molecular, biochemical and cell biological approaches to ascertain how TNF-alpha triggers this unexplored pathway of apoptosis. Our proposal has three SPECIFIC AIMS. FIRST, we will directly test the hypothesis that TNF-alpha signal lysosomal permeabilization: a) by activating neutral sphingomyelinases through the adaptor protein FAN (factor associated with neutral sphingomyelinase) with subsequent sphingosine generation; and b) by a process dependent upon intra-lysosomal cathepsin B (i.e., cathepsin B-dependent cathepsin B release). SECOND, we will test the hypothesis that cytosolic cathepsin B mediates apoptosis: a) by activating the mitochondrial pathway of apoptosis; and b) by cleaving the proapoptotic member of the Bcl-2 family Bid, resulting in its activation and translocation to mitochondria where it induces cytochrome c release. FINALLY, we will test the hypothesis that cathepsin B mediates liver injury such that: a) cathepsin B knockout mice are resistant to liver injury following endogenous stimulation of TNF-alpha in the bile duct-ligated mouse; and b) cathepsin B knockout mice have reduced parameters of liver fibrosis following bile duct ligation. The proposal is innovative, technically and conceptually, as it tests new concepts for TNF-alpha mediated liver injury and apoptosis in general 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 in attenuating human liver injury.