Heat shock protein 90 in alcoholic liver disease: targeting macrophage function

 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。应激诱导的热休克蛋白(HSPs)是巨噬细胞活化过程中重要的信号分子的分子伴侣。热休克蛋白90(HSP90)及其受体Grp94/gp96是TLR4途径中重要的信号分子伴侣，调节炎症细胞因子。HSP90和gp96共同连接应激和炎症通路，在ALD的发生发展中发挥重要作用。本项目上一期资助期间的研究证实：1)热休克蛋白90α(细胞质)和gp96/grp94(热休克蛋白90的内质网形式)在慢性酒精喂养的小鼠肝巨噬细胞、全肝以及人酒精性肝炎中增加，但不增加；2)热休克蛋白90α伴侣蛋白IKKβ和IRF3可促进TLR4信号转导2)用药理剂17-α在体内抑制热休克蛋白90可保护小鼠免受酒精性肝损伤，3)髓系特异性gp96缺乏可减轻酒精性肝损伤4)热休克蛋白1虽由酒精激活但不足以诱导热休克蛋白90 DNA的表达。另一方面，在损伤减少的过程中，HSP90抑制剂诱导的HSF1和HSP70在减轻肝脏炎症反应中起关键作用。我们推测胞浆Hsp90、α和ERgp96/grp94参与了肝巨噬细胞的活化，是治疗酒精性肝病的可能靶点。热休克蛋白90α增加促进巨噬细胞活化，未折叠蛋白反应基因gp96诱导Toll样受体4表面表达，高反应性参与酒精性肝病巨噬细胞的增敏作用。最后，我们认为HSF1和HSP70在抑制ALD HSP90的过程中发挥了抗炎作用。HSP90抑制剂的使用将有助于解开应激蛋白和炎症反应之间相互作用的创新途径，并将HSP90确立为ALD的新治疗靶点。1)研究热休克蛋白90α的抑制作用对急性酒精性肝病巨噬细胞活化的影响：a)利用热休克蛋白90αsiRNA靶向肝巨噬细胞和17-DMAG包裹的纳米颗粒。B)检测热休克蛋白90α抑制对肝脏巨噬细胞极化的影响。C)分析巨噬细胞特异性热休克蛋白90α抑制对酒精性脂肪变性的旁分泌作用。2)探讨髓系基因gp96在酒精致敏巨噬细胞中的作用：a)检测酒精性肝损伤时gp96表达的调节。B)研究gp96/TLR4相互作用和TLR4高反应性在ALD中的重要性。C)研究gp96在ALD时巨噬细胞UPR活化中的作用。3)研究HSP90抑制ALD时HSF1和HSP70的抗炎作用：A)探讨髓系特异性HSF1在巨噬细胞活化和ALD中的作用。B)研究HSF1在17-DMAG治疗和抑制ALD中的作用。C)确定HSP70在17-DMAG治疗期间的抗炎反应中的作用。

 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 role in macrophage activation. Stress induced heat shock proteins (hsps) function as molecular chaperones of signaling molecules important in macrophage activation. Heat shock protein 90 (hsp90) and its ER form, Grp94/gp96 are crucial chaperones of signaling molecules in the TLR4 pathway and thus regulate inflammatory cytokines. Together, hsp90 and gp96 can link stress and inflammatory pathways and play an important role in development of ALD. Studies during the last funding period of this project established that: 1) hsp90α (cytoplasmic) and gp96/grp94 (endoplasmic reticulum (ER) form of hsp90), but not hsp70 is increased in hepatic macrophages and whole livers of chronic alcohol-fed mice and in human alcoholic hepatitis livers, 2)Hsp90α chaperones IKKβ and IRF3 and facilitates TLR4 signaling 2) Inhibition of hsp90 in vivo using pharmacological agent 17-DMAG protects mice from alcoholic liver injury, 3) Myeloid-specific gp96 deficiency alleviates alcoholic liver injury 4) HSF1, although activated by alcohol is not sufficient for induction of hsp90α. On the other hand, HSF1 and hsp70 induced by hsp90 inhibitors during decreased injury is crucial for reduction of liver inflammatory responses. Here we hypothesize that cytoplasmic hsp90α and ER gp96/grp94 contribute to hepatic macrophage activation and are plausible therapeutic targets in ALD. Increased hsp90α promotes macrophage activation and gp96 (unfolded protein response (UPR) gene) induced toll-like receptor 4 surface expression and hyperresponsiveness contributes to macrophage sensitization in alcoholic liver disease. Finally we propose that HSF1 and hsp70 exert anti-inflammatory effects during hsp90 inhibition in ALD. Use of hsp90 inhibitors will help unravel innovative pathways of cross-talk between stress proteins and inflammatory responses and establish hsp90 as a new therapeutic target in ALD. The Specific Aims are as follows: 1) To study the effect of hsp90α inhibition on macrophage activation in ALD by: A) Using hsp90α siRNA to target liver macrophages and 17-DMAG packaged nanoparticles in ALD. B) Examining the effect of hsp90α inhibition on macrophage polarization in the liver. C) Analysis of paracrine effect of macrophage specific hsp90α inhibition on alcoholic steatosis. 2) To investigate the function of myeloid gp96, ER paralog of hsp90, in alcohol-induced sensitization of macrophages by: A) Determine the regulation of gp96 expression during alcoholic liver injury. B) Study importance of gp96/TLR4 interaction and TLR4 hyperresponsiveness during ALD. C) Characterize the role of gp96 in macrophage UPR activation during ALD. 3) To assess anti-inflammatory effects of HSF1 and hsp70 during hsp90 inhibition in ALD: A) Evaluate the role of myeloid-specific HSF1 in macrophage activation and ALD. B) Investigate the role of HSF1 during 17-DMAG treatment and inhibition of ALD. C) Determine the role of hsp70, in anti-inflammatory responses during 17- DMAG treatment.