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：
8497550
负责人：
Pranoti Mandrekar
金额：
$ 34.57万
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 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8497550
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 Design Drug Targeting Exposure to Fatty Liver Functional disorder HSF1 Health Heat Stress Disorders Heat shock proteins Heat-Shock Proteins 90 Hepatic Inflammation Inflammatory Inflammatory Response 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 TNF gene Testing alcohol exposure base cell injury chromatin immunoprecipitation chromatin remodeling cytokine dimer feeding heat shock transcription factor human IRAK1 protein inhibitor/antagonist liver injury macrophage mortality novel problem drinker promoter transcription factor

项目摘要

. ABSTRACT 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- depepndent 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.

。抽象的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）的发病机制。酒精引起的氧化应激在巨噬细胞激活。热休克蛋白由氧化应激诱导，并作为分子伴侣。 TLR4 的热休克蛋白 90 (hsp90) 和 Grp94/gp96 伴侣信号分子调节炎症细胞因子的途径。因此，hsp90 和 gp96 可能将压力和炎症联系起来途径并在 ALD 的发展中发挥重要作用。我们的初步研究表明，长期饮酒与配对喂养对照相比，喂养小鼠会增加分离的库普弗细胞 (KC) 中的 hsp90 和 gp96。来自慢性酒精暴露巨噬细胞的 Hsp90 与 IKKb 激酶（NFkB 中的关键激酶）相关激活和 TNFa 产生。长期接触酒精也会增加 IKKb 激酶活性巨噬细胞。我们假设慢性酒精暴露会调节巨噬细胞中的 hsp90 和 gp96 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）确定通过以下方式确定 hsp90 在酒精性肝损伤中的激活和功能： A) 测量伴侣活性、二聚体整个肝脏中 hsp90 的形成和乙酰化以及从酒精喂养的小鼠中分离出的 Kupffer 细胞。二）评估 hsp90 抑制剂、17-AAG 和/或 17-DMAG 在诱导促炎细胞因子中的作用产生和减轻酒精性肝损伤。 2) 研究hsp90在酒精诱导的酒精中毒中的作用通过以下方式对 LPS 诱导的炎症细胞因子产生敏感：A) 研究 MyD88- 的相互作用依赖下游信号分子，IRAK-1 和 IKK 以及共伴侣 cdc37 和 hsp90 酒精库普弗细胞和整个肝脏。 B) 检查慢性酒精介导的过程中是否需要 hsp90 LPS 诱导的、MyD88 独立的信号传导。 C) 评估 Gp96（hsp90 的 ER 形式）与酒精暴露的肝巨噬细胞/KC 中的 TLR4。 3) 评估长期酗酒的机制通过以下方式在巨噬细胞中诱导 hsp90：A) HSF-1、NFkB、刺激蛋白 1 结合的表征 (Sp1) 和 STAT1，通过染色质免疫沉淀分析。 B) 描述 HSF-1 缺乏的影响和使用 siRNA 抑制 HSF-1 诱导 hsp90。 C) 确定 ROS 依赖性 HSF1 的作用通过评估酒精暴露中 Rac1 和 NADPH 氧化酶的作用来激活 hsp90 的诱导巨噬细胞。