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)激活为细胞外基质产生的肌成纤维细胞是酒精性肝病(ALD)发展的中心。反应氧化应激(ROS)介导的损伤和TNFpha的激活是ALD进展过程中的主要事件，然而ROS的来源以及这些信号事件如何整合最终导致活跃的TNFpha的产生还没有很好的阐明。为了研究这些途径，我们做了几个原创性的观察：我们已经证明了NOX4的诱导导致了HSC中I型胶原启动子的直接激活，并且在肝细胞中它在死亡配体诱导的细胞凋亡中发挥了作用。在患有ALD的人类中，NOX4表达上调；在NOX4-/-小鼠脂肪变性中，TACE活性、肿瘤坏死因子α水平和脂质过氧化作用减弱。对于这项提议，特别重要的是，我们创建了特定于细胞的NOX4-/-模型。因此，我们的中心假设是，NOX4在酒精性肝损伤中是一种重要的酶，在诱导肿瘤坏死因子α转换酶(TACE，或ADAM17)从而激活潜在的肿瘤坏死因子α方面发挥作用。我们提出了三个具体的目标来解决主要假设产生的关键区域：我们的第一个目标是确定NOX4诱导导致肝星状细胞TACE活性增加的途径。A)我们提出了实验来研究乙醛；乙醇的代谢产物诱导NOX4启动子的机制。确定在酒精损伤过程中控制NOX4的关键转录因子将具有重大影响。我们将进行DNA酶足迹分析和染色质IP分析，并针对相应区域产生启动子缺失突变体。B)我们将通过研究Sirtuin1和TACE天然抑制物金属蛋白酶组织抑制因子3(TIMP3)的下调来探讨NOX4诱导TACE激活的途径。我们将检验这样的假设，即低TIMP3活性导致TACE激活以及胞外结构域切割和激活TNFα。我们的第二个目的是研究NOX4直接诱导肝细胞损伤或通过激活的HSC的旁分泌作用而导致肝细胞损伤的机制。这一目标的实验将解决：a)NOX4作为促凋亡酶在肝细胞中的作用。我们将验证这样的假设，即肝细胞中NOX4的激活导致凋亡诱导因子(AIF)移位到细胞核，从而形成染色质降解复合体。另外，肿瘤坏死因子α激活死亡配体通路将导致脂筏的形成以及NOX4和神经酰胺的募集形成。B)我们将利用集成生物传感器的新型微图案共培养系统，确定NOX4/H_2O_2在HSC/肝细胞串扰和相互作用中的作用。在第三个目标中，我们将研究NOX4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培育的肝细胞或HSC NOX4-/-小鼠来确定肝细胞和HSC在酒精性肝损伤中的具体作用。我们将研究其对急性和慢性进行性酒精性肝损伤的影响。这项工作有望突出未来发展的途径 有效的治疗选择。因此，作为上述研究的补充，我们建议在ALD的急性和慢性模型中使用一种新的口服可用NOX4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.