Role of heat shock protein 90 in alcoholic liver disease

热休克蛋白 90 在酒精性肝病中的作用

• 批准号: 7741184
• 负责人: Pranoti Mandrekar
• 金额: $ 38.95万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7741184
• 关键词: 17-(Allylamino)-17-demethoxygeldanamycin; 17-(Dimethylaminoethylamino)-17-Demethoxygeldanamycin; Ablation; Acetylation; Affect; Alcohol consumption; Alcoholic Liver Diseases; Alcohols; Binding; Chronic; Cirrhosis; Development; Drug Delivery Systems; Drug Design; Exposure to; Fatty Liver; Functional disorder; HSF1; Health; Heat Stress Disorders; Heat shock proteins; Heat-Shock Proteins 90; Hepatic; Inflammation; Inflammatory; Inflammatory Response; Injury; Injury to Liver; Knowledge; Kupffer Cells; Link; Liver; Macrophage Activation; Malignant Neoplasms; Measures; Mediating; Molecular Chaperones; Morbidity - disease rate; Mus; NADPH Oxidase; Oxidation-Reduction; Oxidative Stress; Pathogenesis; Pathway interactions; Phosphotransferases; Play; Predisposition; Primary carcinoma of the liver cells; Production; Role; STAT1 gene; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; Sp1 Transcription Factor; Steatohepatitis; Stream; Stress; TLR4 gene; Testing; alcohol exposure; base; cell injury; chromatin immunoprecipitation; chromatin remodeling; cytokine; dimer; feeding; heat shock transcription factor; human IRAK1 protein; inhibitor/antagonist; macrophage; mortality; novel; problem drinker; promoter; public health relevance; transcription factor

DESCRIPTION (provided by applicant): Activation of liver resident macrophages and increased pro-inflammatory cytokine production is a hallmark in the pathogenesis of alcoholic liver disease (ALD). Alcohol-induced oxidative stress plays an important part in macrophage activation. Heat shock proteins are induced by oxidative stress and function as molecular chaperones. Heat shock protein 90 (hsp90) and Grp94/gp96 chaperone signaling molecules of the TLR4 pathway to regulate inflammatory cytokines. Thus, hsp90 and gp96 could link stress and inflammatory pathways and play an important role in development of ALD. Our preliminary studies show that chronic alcohol feeding in mice increases hsp90 and gp96 in isolated Kupffer cells (KCs), compared to pair-fed controls. Hsp90 from chronic alcohol-exposed macrophages associates with IKKb kinase, a pivotal kinase in NFkB activation and TNFa production. Chronic alcohol exposure also increases IKKb kinase activity in macrophages. We hypothesize that chronic alcohol exposure modulates hsp90 and gp96 in macrophages and regulates TLR4 induced NFkB activation and TNFa production, contributing to liver injury. Thus, hsp90 and gp96 play an important role in the pathophysiology of ALD. The specific aims are as follows: 1) To determine the activation and function of hsp90 in alcoholic liver injury by: A) Measuring chaperone activity, dimer formation, and acetylation of hsp90 in whole liver and isolated Kupffer cells from alcohol-fed mice. B) Evaluating the effect of hsp90 inhibitors, 17-AAG and/or 17-DMAG in induction of pro-inflammatory cytokine production, and alleviation of alcoholic liver injury. 2) To examine the role of hsp90 in alcohol-induced sensitization to LPS-induced inflammatory cytokine production by: A) Studying the interactions of MyD88- dependent down-stream signaling molecules, IRAK-1 and IKK with co-chaperone cdc37 and hsp90 in alcoholic Kupffer cells and whole livers. B) Examining whether hsp90 is required in chronic alcohol mediated LPS-induced, MyD88 independent signaling. C) Evaluating the interaction of Gp96, an ER form of hsp90, with TLR4 in alcohol exposed hepatic macrophages/KCs. 3) To assess the mechanisms by which chronic alcohol induces hsp90 in macrophages by: A) Characterization of the binding of HSF-1, NFkB, stimulatory protein-1 (Sp1) and STAT1 by chromatin immunoprecipitation analysis. B) Delineating the effect of HSF-1 deficiency and HSF-1 inhibition using siRNA on induction of hsp90. C) Determining the role of ROS dependent HSF1 activation on induction of hsp90 by evaluating effect of Rac1 and NADPH oxidase in alcohol-exposed macrophages. PUBLIC HEALTH RELEVANCE: Alcoholic liver disease continues to be a major health problem with respect to morbidity and mortality. Oxidative stress and TLR-induced pro-inflammatory cytokines play an important role in pathogenesis of liver injury by alcohol. Heat shock proteins induced by oxidative stress play an important role in inflammatory responses. Particularly hsp90 regulates and maintains stability of key kinases involved in the LPS-signaling pathway. We hypothesize that hsp90 plays a pivotal role in alcoholic liver disease by maintaining function of kinases that induce pro-inflammatory cytokines. Commercially available hsp90 inhibitors could be used to reduce alcoholic liver disease by ablation of inflammatory responses and these inhibitors will be tested. The significance of HSF-1, a transcription factor that induces hsp90, will provide further knowledge of the essential role of HSF-1 in induction of hsp90 in alcoholic liver injury. Understanding these pathways will provide novel mechanisms and thus extend our horizons to identify potential new drug targets for alcoholic liver disease.

描述（由申请人提供）：肝脏驻留巨噬细胞的激活和促炎细胞因子产生的增加是酒精性肝病（ALD）发病机制的标志。酒精诱导的氧化应激在巨噬细胞活化中发挥重要作用。热休克蛋白是由氧化应激诱导的，具有分子伴侣的功能。热休克蛋白90（hsp 90）和Grp 94/gp 96伴侣信号分子的TLR 4途径，以调节炎症细胞因子。因此，hsp 90和gp 96可能是应激和炎症通路的联系，在ALD的发生发展中起重要作用。我们的初步研究表明，慢性酒精喂养小鼠增加热休克蛋白90和gp 96在孤立的枯否细胞（KCs），与配对喂养的控制。来自慢性酒精暴露的巨噬细胞的Hsp 90与IKKb激酶相关，IKKb激酶是NFkB活化和TNF α产生中的关键激酶。慢性酒精暴露也增加巨噬细胞中IKKb激酶的活性。我们推测慢性酒精暴露调节巨噬细胞中的hsp 90和gp 96，并调节TLR 4诱导的NF κ B活化和TNF α产生，从而导致肝损伤。因此，hsp 90和gp 96在ALD的病理生理学中起重要作用。具体目的如下：1）通过以下方法确定hsp 90在酒精性肝损伤中的活化和功能：A）测量来自酒精喂养的小鼠的整个肝脏和分离的Kupffer细胞中的hsp 90的伴侣活性、二聚体形成和乙酰化。B）评估hsp 90抑制剂、17-AAG和/或17-DMAG在诱导促炎性细胞因子产生和减轻酒精性肝损伤中的作用。2)通过以下方式检查hsp 90在酒精诱导的对LPS诱导的炎性细胞因子产生的敏化中的作用：A）研究MyD 88依赖性下游信号传导分子IRAK-1和IKK与共伴侣cdc 37和hsp 90在酒精性枯否细胞和整个肝脏中的相互作用。B）检查在慢性酒精介导的LPS诱导的、MyD 88非依赖性信号传导中是否需要hsp 90。C）评估Gp 96（hsp 90的ER形式）与暴露于酒精的肝巨噬细胞/KC中的TLR 4的相互作用。3)为了评估慢性酒精诱导巨噬细胞中hsp 90的机制，通过：A）通过染色质免疫沉淀分析表征HSF-1、NF κ B、刺激蛋白-1（Sp1）和STAT 1的结合。B）描述HSF-1缺陷和使用siRNA的HSF-1抑制对hsp 90诱导的影响。C）通过评估Rac 1和NADPH氧化酶在暴露于酒精的巨噬细胞中的作用来确定ROS依赖性HSF 1活化对hsp 90诱导的作用。公共卫生相关性：就发病率和死亡率而言，酒精性肝病仍然是一个主要的健康问题。氧化应激和TLR诱导的促炎细胞因子在酒精性肝损伤的发病机制中起重要作用。氧化应激诱导的热休克蛋白在炎症反应中起重要作用。特别是hsp 90调节和维持LPS信号通路中涉及的关键激酶的稳定性。我们推测热休克蛋白90通过维持诱导促炎细胞因子的激酶的功能在酒精性肝病中起关键作用。市售的热休克蛋白90抑制剂可用于通过消除炎症反应来减少酒精性肝病，这些抑制剂将进行测试。HSF-1是一种诱导hsp 90的转录因子，其意义将为进一步了解HSF-1在酒精性肝损伤中诱导hsp 90的重要作用提供依据。了解这些途径将提供新的机制，从而扩大我们的视野，以确定酒精性肝病的潜在新药靶点。