Role of aberrant regulation of organelle stress responses in alcohol-induced live

描述（申请人提供）：酒精性肝损伤的发病机制非常复杂，无疑涉及多种机制和途径的相互作用、细胞器功能障碍以及内在因素和环境因素的相互作用。长期摄入酒精导致细胞内和细胞间损伤和修复过程更加复杂。我们的研究重点是线粒体和ER应激反应在酒精诱导的肝脏疾病演变中的作用。我们已经获得了大量的数据表明，线粒体和ER都被慢性酒精消耗所损伤，并且延长的线粒体和ER应激反应有助于肝损伤，包括肝坏死性炎症和细胞死亡以及导致纤维化和肝硬化的严重脂肪肝。酒精如何在遗传学和表观遗传学上调节这两种细胞器的应激反应基因尚不清楚。为了确定酒精消耗破坏线粒体和ER保护反应进入损伤促进过程的机制，我们假设酒精导致线粒体和ER应激反应基因启动子上转录因子的异常募集和表观遗传变化，从而导致肝损伤。我们建议测试一下可能性。具体目标是：1.使用定量PCR（qPCR）和ChIP分析，我们将研究启动子特异性转录因子的募集。（XBP-1，CHOP，ATF 6，ATF 4，CREB，TORC 3，PGC-1a），一般转录因子（Pol II，NF-Y，NFR，Sp1，TBP和p300）和表观遗传标记（H3-K4和H3-K79的甲基化以及H3和H4的乙酰化）（Grp 78、Grp 94、PDI、钙网蛋白、HSP 10和HSP 60）通过用相应的线粒体和ER应激诱导剂处理原代小鼠肝细胞; 2.我们将进行体内研究，并通过给小鼠喂食酒精与配对喂食对照相比，用qPCR和ChIP测定法测量与目标1中所述相同的参数; 3.我们将利用Lox-Cre系统以及Dox-Tet先进系统来创建和表征肝脏特异性Grp 78基因敲除小鼠; 4.我们将确定Grp 78缺失对酒精小鼠肝脏中两个细胞器基因启动子的转录因子募集和表观遗传变化的影响，将ER应激与线粒体功能障碍结合起来，并评估ER应激对酒精诱导的肝损伤的具体贡献。我们的总体目标是揭示转录和表观遗传异常调节的两个细胞器的压力反应，酒精与酒精性肝病的发病机制。我们预计这项工作将导致预防或治疗酒精性肝病的新方法，并将广泛适用于其他类型的肝病。 公共卫生相关性：慢性过量饮酒导致严重的脂肪肝和损伤，是美国肝脏相关死亡和移植的主要原因。我们已经在动物模型中确定了肝脏疾病与酒精引起的线粒体和ER应激反应延长之间的联系。这项研究旨在了解酒精如何促进这两种细胞器的损伤过程，以及细胞器损伤如何导致肝脏疾病，这将为预防和治疗肝脏疾病开辟新的途径。

DESCRIPTION (provided by applicant): The pathogenesis of alcohol-induced liver injury is very complex and undoubtedly involves the interplay of multiple mechanisms and pathways, dysfunction of cellular organelles, and interactions of intrinsic and environmental factors. The chronic feeding of alcohol leads to even more complexity as these processes of intracellular and intercellular injury and repair progress. We have focused our research on role of mitochondrial and ER stress responses in the evolution of alcohol-induced liver disease. We have obtained considerable data indicating that both mitochondria and ER are damaged by chronic alcohol consumption and that prolonged mitochondrial and ER stress responses contribute to liver injury including hepatic necroinflammation and cell death and severe fatty liver leading to fibrosis and cirrhosis. How alcohol regulates genetically and epigenetically the stress response genes of the two organelles is not known. To define the mechanisms by which alcohol consumption derails the mitochondrial and ER protective responses into injury promoting processes, we hypothesize that alcohol causes aberrant recruitment of transcription factors and epigenetic changes on the mitochondrial and ER stress response gene promoters which lead to liver injury. We propose to test the possibilities. The specific aims are: 1. Using quantitative PCR (qPCR) and ChIP assays, we will study the recruitment of promoter specific transcription factors (XBP-1, CHOP, ATF6, ATF4, CREB, TORC3, PGC-1a), general transcription factors (Pol II, NF-Y, NFR, Sp1, TBP and p300) and epigenetic marks (methylation of H3-K4 and H3-K79 and acetylation of H3 and H4) in promoters of mitochondrial and ER stress response genes (Grp78, Grp94, PDI, Calreticulin, HSP10, and HSP 60) by treating primary mouse hepatocytes with respective mitochondria and ER stress inducing agents; 2. We will perform in vivo study and measure the same parameters as described in Aim 1 with qPCR and ChIP assays by feeding mice alcohol compared to pair-fed control; 3. We will utilize Lox-Cre system as well as Dox-Tet advanced system to create and characterize liver specific Grp78 gene knockout mice; 4. We will determine effects of Grp78 deletion on the transcription factor recruitment and epigenetic changes on the two organelle gene promoters in the liver of mice fed alcohol, couple ER stress to mitochondrial dysfunction, and assess specific contribution of ER stress to alcohol-induced liver injury. Our overall goal is to reveal transcriptional and epigenetic abnormal regulation of the two organelle stress responses by alcohol in relation to the pathogenesis of alcoholic liver disease. We anticipate that this work will lead to new approaches to prevent or treat alcoholic liver disease and will be widely applicable in other types of liver disease. PUBLIC HEALTH RELEVANCE: Chronic excessive alcohol use leads to severe fatty liver and injury and is a leading cause of liver-related death and transplantation in the United States. We have identified links between liver disease and prolonged mitochondrial and ER stress responses caused by alcohol in animal models. This research is to understand how alcohol promotes injury processes in the two organelles and how organelle damages contribute to liver disease which will open new avenues for preventing and treating liver disease.