Role of heat shock protein 90 in alcoholic liver disease

描述（由申请人提供）：肝脏巨噬细胞的激活和促炎细胞因子的增加是酒精性肝病（ALD）发病机制的一个标志。酒精诱导的氧化应激在巨噬细胞活化中起重要作用。热休克蛋白是由氧化应激诱导产生的，具有分子伴侣的功能。TLR4通路的热休克蛋白90 （hsp90）和Grp94/gp96伴侣信号分子调控炎症细胞因子。因此，hsp90和gp96可能连接应激和炎症通路，在ALD的发生发展中发挥重要作用。我们的初步研究表明，与成对喂养的对照组相比，小鼠慢性酒精喂养增加了分离的KCs中的hsp90和gp96。慢性酒精暴露的巨噬细胞Hsp90与IKKb激酶相关，IKKb激酶是NFkB激活和TNFa产生的关键激酶。慢性酒精暴露也增加巨噬细胞的IKKb激酶活性。我们假设慢性酒精暴露可调节巨噬细胞中的hsp90和gp96，并调节TLR4诱导的NFkB激活和TNFa产生，从而导致肝损伤。因此，hsp90和gp96在ALD的病理生理中起着重要的作用。具体目的如下：1)通过测定酒精性肝损伤小鼠全肝和离体Kupffer细胞中hsp90的伴侣活性、二聚体形成和乙酰化程度，确定hsp90在酒精性肝损伤中的活化和功能。B)评价hsp90抑制剂、17-AAG和/或17-DMAG在诱导促炎细胞因子产生和减轻酒精性肝损伤中的作用。2)通过研究MyD88依赖的下游信号分子IRAK-1和IKK与酒精性Kupffer细胞和全肝中的共伴侣cdc37和hsp90的相互作用，探讨hsp90在酒精诱导的脂多糖诱导炎症细胞因子产生中的作用。B)检查慢性酒精介导的脂多糖诱导的MyD88独立信号传导是否需要hsp90。C)评估酒精暴露的肝巨噬细胞/KCs中Gp96 （hsp90的ER形式）与TLR4的相互作用。3)探讨慢性酒精诱导巨噬细胞hsp90的机制：A)通过染色质免疫沉淀分析表征HSF-1、NFkB、刺激蛋白-1 （Sp1）和STAT1的结合。B)描述HSF-1缺乏和使用siRNA抑制HSF-1对hsp90诱导的影响。C)通过评估酒精暴露巨噬细胞中Rac1和NADPH氧化酶的作用，确定ROS依赖性HSF1激活对hsp90诱导的作用。公共卫生相关性：酒精性肝病在发病率和死亡率方面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hsp90调节和维持参与lps信号通路的关键激酶的稳定性。我们假设hsp90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hsp90抑制剂可通过消炎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hsp90的转录因子，其意义将进一步了解HSF-1在酒精性肝损伤中诱导hsp90的重要作用。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物靶点。

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.