The Role of TNF alpha Converting Enzyme in Alcoholic Liver Disease

TNF α 转化酶在酒精性肝病中的作用

• 批准号: 8974372
• 负责人: Natalie J. Torok
• 金额: --
• 依托单位: VA NORTHERN CALIFORNIA HEALTH CARE SYS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8974372
• 关键词: Acetaldehyde; Acute; Address; Affect; Alcoholic Liver Diseases; Animal Model; Apoptosis; Apoptotic; Area; Attenuated; Biological Assay; Biosensor; CYP2E1 gene; Cell Death; Cell Nucleus; Cells; Ceramides; Cessation of life; Chromatin; Chronic; Cirrhosis; Coculture Techniques; Collagen; Complement; Complex; Data; Deoxyribonucleases; Development; Disease; Disease Progression; Down-Regulation; Enzymes; Event; Extracellular Matrix; FDA approved; Fibrosis; Future; Health; Hepatic Stellate Cell; Hepatocyte; Human; Hydrogen Peroxide; Inflammation; Inflammatory; Injury; Ligands; Lipid Peroxidation; Mediating; Membrane Microdomains; Mitochondria; Modeling; Morbidity - disease rate; Mus; Myofibroblast; NADPH Oxidase; Oxidative Stress; Pathogenesis; Pathway interactions; Patients; Play; Production; Regulation; Role; Signal Transduction; Source; Steatohepatitis; System; TIMP3 gene; TNF gene; TNF-alpha converting enzyme; Testing; Time; United States Department of Veterans Affairs; Veterans; Work; Xanthine Oxidase; apoptosis inducing factor; base; chronic liver disease; effective therapy; in vivo; inhibitor/antagonist; liver injury; liver transplantation; mortality; mutant; novel; paracrine; problem drinker; promoter; research study; transcription factor; treatment strategy

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.

描述（由申请人提供）： 项目摘要：慢性肝细胞凋亡和静止肝星状细胞 (HSC) 活化为产生细胞外基质的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 TNFα 的激活是 ALD 进展中的主要事件，然而 ROS 的来源以及信号事件如何整合最终导致活性 TNFα 的产生尚未得到很好的阐明。为了研究这些途径，我们进行了一些原始观察：我们证明NOX4诱导导致HSC中胶原蛋白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发挥作用。 NOX4 在 ALD 患者中表达上调；在 NOX4-/- 小鼠中，脂肪变性、TACE 活性、TNFα 水平和脂质过氧化均减弱。对于该提案特别重要的是，我们创建了细胞特定的 NOX4-/- 模型。因此，我们的中心假设是 NOX4 是酒精性肝损伤中的一种重要酶，在诱导 TNFα 转换酶（TACE 或 ADAM17）中发挥作用，从而激活潜在的 TNFα。我们提出了三个具体目标来解决主要假设产生的关键领域：我们的第一个目标是确定 NOX4 诱导导致肝星状细胞中 TACE 活性增加的途径。 a）我们提出实验来解决乙醛的机制； EtOH 的代谢物诱导 NOX4 启动子。确定酒精损伤期间控制 NOX4 的关键转录因子将产生重大影响。我们将进行 DNase 足迹分析和染色质 IP 分析，并针对相应区域生成启动子缺失突变体。 b) 我们将通过研究 Sirtuin1 和金属蛋白酶组织抑制剂 3 (TIMP3)（TACE 的天然抑制剂）的下调来探究 NOX4 诱导导致 TACE 激活的途径。我们将检验以下假设：低 TIMP3 活性会导致 TACE 激活以及胞外域裂解和 TNFα 激活。我们的第二个目标是研究 NOX4 诱导肝细胞损伤的机制，无论是由于肝细胞中直接 NOX4 诱导还是通过活性 HSC 的旁分泌作用。此目的的实验将解决：a) NOX4 在肝细胞中作为促凋亡酶的作用。我们将检验这样的假设：肝细胞中 NOX4 的激活导致细胞凋亡诱导因子 (AIF) 易位至细胞核，从而形成染色质降解复合物。或者，TNFα 激活死亡配体途径将导致脂筏形成以及 NOX4 的募集和神经酰胺的形成。 b) 我们将确定NOX4/H2O2在HSC/肝细胞串扰中的作用和相互影响；使用带有集成生物传感器的新型微图案共培养系统。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生成的肝细胞或 HSC NOX4-/- 小鼠来定义肝细胞和 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。这项工作预计将突出未来的发展途径 有效的治疗方案。因此，作为对上述研究的补充，我们建议在 ALD 的急性和慢性模型中使用新型口服 NOX4 抑制剂。