Role of heat shock protein 90 in alcoholic liver disease

描述(由申请人提供)：肝脏内滞留巨噬细胞的激活和促炎细胞因子的产生是酒精性肝病(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的，具有分子伴侣的功能。热休克蛋白90(HSP90)和Grp94/gp96是TLR4途径的伴侣信号分子，调节炎症细胞因子。因此，HSP90和gp96在ALD的发生发展过程中起着重要的作用。我们的初步研究表明，与配对喂养的对照组相比，小鼠长期饮酒增加了分离的Kupffer细胞(KCs)中HSP90和gp96的表达。慢性酒精暴露的巨噬细胞产生的HSP90与IKKB激酶有关，IKKB是NFkB激活和TNFa产生的关键激酶。长期酒精暴露还会增加巨噬细胞中IKKB激酶的活性。我们假设慢性酒精暴露调节巨噬细胞中的HSP90和gp96，并调节TLR4诱导的NFkB激活和TNFa的产生，从而导致肝损伤。因此，HSP90和gp96在ALD的病理生理过程中起重要作用。1)通过检测酒精喂养小鼠全肝和分离的枯否细胞中HSP90的伴侣活性、二聚体形成和乙酰化，以确定HSP90在酒精性肝损伤中的激活和功能。B)评估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方面的作用。2)研究HSP90在酒精诱导的酒精性Kupffer细胞和全肝细胞中对内毒素诱导的炎性细胞因子产生的增敏作用：a)研究依赖于MyD88的下游信号分子IRAK-1和IKK与辅伴侣cdc37和HSP90在酒精诱导的Kupffer细胞和全肝细胞中的相互作用。B)检测慢性酒精介导的内毒素诱导的MyD88非依赖信号中是否需要HSP90。C)评估HSP90的内质网形式gp96与TLR4在酒精暴露的肝巨噬细胞/KCs中的相互作用。3)通过染色质免疫沉淀分析HSF-1、NFkB、刺激蛋白-1(Sp1)和STAT1的结合，探讨慢性酒精诱导巨噬细胞HSP90的机制。B)阐明HSF-1缺乏和使用siRNA抑制HSF-1对HSP90诱导的影响。C)通过评估ROS依赖的HSF1激活在酒精暴露的巨噬细胞中的作用，确定ROS依赖的HSF1在HSP90诱导中的作用。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。尤其是HSP90调节和维持参与内毒素信号通路的关键蛋白的稳定性。我们假设HSP90在酒精性肝病中通过维持诱导促炎细胞因子的激酶的功能而发挥关键作用。商业上可获得的HSP90抑制剂可以通过消融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HSP90的转录因子，其意义将为进一步了解HSF-1在诱导HSP90在酒精性肝损伤中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，以确定潜在的治疗酒精性肝病的新药靶点。

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.