The Role of TNF alpha Converting Enzyme in Alcoholic Liver Disease

描述（由申请人提供）： 项目摘要：慢性肝细胞凋亡和静止的肝星状细胞（HSC）活化为细胞外基质产生肌成纤维细胞是酒精性肝病（ALD）发展的核心。反应性氧化应激（ROS）介导的损伤和TNF α的活化是ALD进展中的主要事件，然而ROS的来源以及信号传导事件如何整合最终导致活性TNF α产生还没有很好地阐明。为了研究这些途径，我们做了几个原始的观察：我们已经证明，NOX 4诱导导致HSC中胶原I启动子的直接激活，并且在肝细胞中，它在死亡配体诱导的细胞凋亡中起作用。在ALD患者中，NOX 4上调;在NOX 4-/-小鼠脂肪变性、TACE活性、TNF α水平和脂质过氧化作用减弱。对于该提议特别重要的是，我们创建了细胞特异性NOX 4-/-模型。因此，我们的中心假设是，NOX 4是酒精性肝损伤中的一种重要酶，在诱导TNF α转化酶（TACE，或ADAM 17）中起作用，从而激活潜伏的TNF α。我们提出了三个具体的目标，以解决主要假设产生的关键领域：我们的第一个目标是确定的途径，其中NOX 4诱导导致肝星状细胞TACE活性的增加。a）我们提出实验来解决乙醛（EtOH的代谢物）诱导NOX 4启动子的机制。确定酒精损伤过程中控制NOX 4的关键转录因子将产生重大影响。我们将进行DNA酶足迹分析和染色质IP测定，并将产生靶向相应区域的启动子缺失突变体。B）我们将通过研究Sirtuin 1和金属蛋白酶组织抑制剂3（TIMP 3）（TACE的天然抑制剂）的下调来探究导致TACE激活的NOX 4诱导途径。我们将检验低TIMP 3活性导致TACE激活和胞外域切割以及TNF α激活的假设。我们的第二个目的是研究NOX 4可以诱导肝细胞损伤的机制，无论是由于直接NOX 4诱导肝细胞或由活化HSC的旁分泌效应。在此目的的实验将解决：a）在肝细胞中作为促凋亡酶的NOX 4的作用。我们将测试的假设，在肝细胞中的活化的NOX 4的结果在易位的细胞凋亡诱导因子（AIF）的细胞核，导致形成的染色质降解复合物。或者，TNF α激活死亡配体途径将导致脂筏形成和N 0X 4的募集和神经酰胺形成。B）我们将确定NOX 4/H2 O2在HSC/肝细胞串扰和相互作用中的作用;使用具有集成生物传感器的新型微图案化共培养系统。在第三个目标中，我们将研究NOX 4介导的氧化应激是否是体内ALD过程中的关键事件。在机制研究中，我们将使用我们最近产生的肝细胞或HSC NOX 4-/-小鼠来确定肝细胞和HSC对酒精性肝损伤的具体贡献。我们将研究对急性和慢性进行性酒精性肝损伤的影响。预计这项工作将突出未来发展的途径， 有效的治疗选择。因此，补充上述研究，我们建议在ALD的急性和慢性模型中使用一种新的口服NOX 4抑制剂。

DESCRIPTION (provided by applicant): Project Summary: Chronic hepatocyte apoptosis and the activation of the quiescent hepatic stellate cells (HSC) to extracellular matrix-producing myofibroblasts are central to the development of alcoholic liver disease (ALD). Reactive oxidative stress (ROS)-mediated injury and activation of TNFalpha are major events in the progression of ALD, however the sources of ROS and how the signaling events are integrated culminating in active TNFalpha production are not well elucidated. To study these pathways, we have made several original observations: we have demonstrated that NOX4 induction results in a direct activation of the collagen I promoter in HSC, and in hepatocytes it plays a role in death ligand-induced apoptosis. NOX4 is upregulated in humans with ALD; and in the NOX4-/- mice steatosis, TACE activity, TNFalpha levels and lipid peroxidation were attenuated. Of particular importance for this proposal we created cell-specific NOX4-/- models. Thus our CENTRAL HYPOTHESIS is that NOX4 is an important enzyme in alcoholic liver injury playing a role in the induction of the TNFalpha converting enzyme (TACE, or ADAM17) and thereby activating latent TNFalpha. We propose three SPECIFIC AIMS to address the key areas generated by the main hypothesis: Our first aim is to determine the pathways by which NOX4 induction results in an increase in TACE activity in hepatic stellate cells. a) We propose experiments to address the mechanism by which acetaldehyde; the metabolite of EtOH induces the NOX4 promoter. Identifying the key transcription factors controlling NOX4 during alcoholic injury will have a major impact. We will perform DNase footprint analysis and chromatin IP assays and promoter deletion mutants will be generated targeting the corresponding areas. b) We will interrogate the pathways of NOX4 induction leading to the activation of TACE by studying the downregulation of Sirtuin1 and the tissue inhibitor of metalloproteinases 3 (TIMP3), the natural inhibitor of TACE. We will test the hypothesis that the low TIMP3 activity results in TACE activation and ectodomain cleavage and activation of TNFalpha. Our second aim is to investigate the mechanism by which NOX4 can induce hepatocyte injury, either as a result of direct NOX4 induction in hepatocytes or by paracrine effects from active HSC. The experiments in this aim will address: a) the role of NOX4 in hepatocytes as a proapoptotic enzyme. We will test the hypothesis that the activation of NOX4 in hepatocytes results in the translocation of the apoptosis inducing factor (AIF) to the nucleus resulting in the formation of a chromatin degrading complex. Alternatively, activation of the death ligand pathway by TNFalpha will result in lipid raft formation and the recruitment of NOX4 and ceramide formation. b) We will determine the role of NOX4/H2O2 in the HSC/hepatocyte crosstalk and reciprocal effects; using a novel micropatterned co-culture system with integrated biosensors. In the third aim we will study if NOX4-mediated oxidative stress is a key event during ALD in vivo. In mechanistic studies we will define the specific contribution of hepatocytes and HSC to alcoholic liver injury using hepatocyte or HSC NOX4-/- mice that we have recently generated. We will study the effects on both acute and chronic progressive alcoholic liver injury. This work is expected to highlight future avenues in developing effective treatment options. Thus complementing the above studies, we propose to use a novel orally available NOX4 inhibitor in the acute and chronic models of ALD.