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，细胞细胞器的功能障碍，以及内在和环境因素的相互作用。随着这些细胞内和细胞间的损伤和修复过程的进展，长期的酒精喂养会导致更加复杂的过程。我们的研究重点是线粒体和内质网应激反应在酒精性肝病演变中的作用。我们已经获得了大量的数据，表明长期饮酒会损害线粒体和内质网，而长期的线粒体和内质网应激反应会导致肝脏损伤，包括肝脏坏死性炎症和细胞死亡，以及严重的脂肪肝，导致纤维化和肝硬变。酒精如何从遗传和表观上调节这两个细胞器的应激反应基因尚不清楚。为了确定饮酒使线粒体和内质网保护反应脱轨进入损伤促进过程的机制，我们假设酒精导致转录因子的异常募集和线粒体和内质网应激反应基因启动子的表观遗传改变，从而导致肝损伤。我们建议测试一下可能性。1.利用定量聚合酶链式反应(QPCR)和芯片分析技术，研究线粒体和内质网应激反应基因(GRP78、Grp94、PDI、Calreticlin、HSP10、HSP60)启动子中的启动子特异性转录因子(XBP-1、CHOP、ATF6、ATF4、CREB、TORC3、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)的募集；2.我们将进行体内研究，并通过qPCR和芯片分析，以酒精喂养的小鼠为对照，测量与目标1相同的参数；3.我们将利用Lox-CRE系统以及Dox-Tet高级系统来建立和鉴定肝脏特异的GRP78基因敲除小鼠；4.我们将确定GRP78缺失对饮酒小鼠肝脏两个细胞器基因启动子的转录因子募集和表观遗传学变化的影响，将ER应激与线粒体功能障碍偶联，并评估ER应激在酒精性肝损伤中的特异性贡献。我们的总体目标是揭示酒精对这两种细胞器应激反应的转录和表观遗传异常调节与酒精性肝病发病机制的关系。我们预计这项工作将带来预防或治疗酒精性肝病的新方法，并将广泛应用于其他类型的肝病。 公共卫生相关性：长期过量饮酒会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病与酒精引起的线粒体和内质网应激反应延长之间的联系。本研究旨在了解酒精如何促进这两个细胞器的损伤过程，以及细胞器损伤如何导致肝病，这将为预防和治疗肝病开辟新的途径。

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.