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

基本信息

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
项目类别：
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

项目摘要

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激活中起着重要作用。热休克蛋白由氧化应激诱导并充当分子伴侣。 TLR4 通路的热休克蛋白 90 (hsp90) 和 Grp94/gp96 分子伴侣信号分子调节炎症细胞因子。因此，hsp90和gp96可以将应激和炎症途径联系起来，并在ALD的发展中发挥重要作用。我们的初步研究表明，与配对喂养的对照组相比，长期饮酒的小鼠会增加分离的库普弗细胞 (KC) 中的 hsp90 和 gp96。来自长期暴露于酒精的巨噬细胞的 Hsp90 与 IKKb 激酶相关，IKKb 激酶是 NFkB 激活和 TNFa 产生的关键激酶。长期接触酒精也会增加巨噬细胞中 IKKb 激酶的活性。我们假设慢性酒精暴露会调节巨噬细胞中的 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的病理生理学中发挥重要作用。具体目标如下： 1) 通过以下方式确定 hsp90 在酒精性肝损伤中的激活和功能： A) 测量全肝和来自酒精喂养小鼠的分离的 Kupffer 细胞中 hsp90 的伴侣活性、二聚体形成和乙酰化。 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中的作用。 2) 通过以下方式检查 hsp90 在酒精诱导的对 LPS 诱导的炎症细胞因子产生的敏化中的作用： A) 研究 MyD88 依赖性下游信号分子、IRAK-1 和 IKK 与共伴侣 cdc37 和 hsp90 的相互作用酒精库普弗细胞和整个肝脏。 B) 检查慢性酒精介导的 LPS 诱导的、不依赖 MyD88 的信号传导是否需要 hsp90。 C) 评估 Gp96（hsp90 的 ER 形式）与酒精暴露的肝巨噬细胞/KC 中 TLR4 的相互作用。 3) 通过以下方式评估慢性酒精在巨噬细胞中诱导 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) 描述了 HSF-1 缺陷和使用 siRNA 抑制 HSF-1 对 hsp90 诱导的影响。 C) 通过评估暴露于酒精的巨噬细胞中 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发挥着重要作用。氧化应激诱导的热休克蛋白在炎症反应中发挥重要作用。特别是 hsp90 调节并维持 LPS 信号通路中涉及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功能，在酒精性肝病中发挥关键作用。市售的 hsp90 抑制剂可用于通过消除炎症反应来减少酒精性肝病，并且将对这些抑制剂进行测试。 HSF-1（一种诱导 hsp90 的转录因子）的重要性将为我们进一步了解 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。