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态肝星状细胞（HSC）对产生肌纤维细胞的细胞外基质的激活对于酒精性肝病（ALD）的发展至关重要。反应性氧化应激（ROS）介导的损伤和TNFALPHA的激活是ALD进展中的主要事件，但是ROS的来源以及如何整合活跃TNFALPHA产生的信号传导事件。为了研究这些途径，我们进行了几个原始观察结果：我们已经证明，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凋亡中起着作用。 NOX4在患有ALD的人类中被上调。在NOX4 - / - 小鼠脂肪变性中，TACE活性，TNFALPHA水平和脂质过氧化。对于此提案，我们创建了特定于单元格的NOX4 - / - 模型，这一点尤其重要。因此，我们的中心假设是NOX4是酒精性肝损伤的重要酶，在诱导Tnfalpha转化酶（TACE或ADAM17）中发挥作用，从而激活潜在的TNFALPHA。我们提出了三个特定的目的，以解决主要假设产生的关键领域：我们的第一个目的是确定NOX4诱导导致肝星状细胞中TACE活性增加的途径。 a）我们提出了实验来解决乙醛的机制； ETOH的代谢产物诱导NOX4启动子。确定在酒精损伤期间控制NOX4的关键转录因子将产生重大影响。我们将执行DNase足迹分析和染色质IP分析，并且将生成针对相应区域的启动子缺失突变体。 b）我们将通过研究sirtuin1和金属蛋白酶3（timp3）的sirtuin1和组织抑制剂的下调（TACE的天然抑制剂）的下调，从而询问NOX4诱导的途径，从而导致TACE激活。我们将测试较低的TIMP3活性导致TACE激活和tnfalpha的激活的假设。我们的第二个目的是研究NOX4可以诱导肝细胞损伤的机制，这是由于肝细胞的直接NOX4诱导或活性HSC的旁分泌作用而导致的。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。我们将检验以下假设：肝细胞中Nox4的激活导致凋亡诱导因子（AIF）转移至细胞核，从而形成染色质质量降解复合物。另外，tnfalpha对死亡配体途径的激活将导致脂质筏的形成以及NOX4和神经酰胺形成的募集。 b）我们将确定NOX4/H2O2在HSC/肝细胞串扰和相互效应中的作用；使用与集成生物传感器的新型微图案共培养系统。在第三个目的中，我们将研究NOX4介导的氧化应激是否是体内ALD期间的关键事件。在机械研究中，我们将使用肝细胞或HSC NOX4 - / - 小鼠定义肝细胞和HSC对酒精肝损伤的特定贡献。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的途径 有效的治疗选择。因此，在上述研究中，我们建议在ALD的急性和慢性模型中使用新型的口服NOX4抑制剂。