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

 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信号分子的分子伴侣。热休克蛋白90（hsp 90）及其ER形式Grp 94/gp 96是TLR 4通路中信号分子的重要伴侣，从而调节炎性细胞因子。热休克蛋白90和糖蛋白96可以共同连接应激和炎症途径，并在ALD的发展中发挥重要作用。该项目上一个供资期间的研究表明：1）热休克蛋白90 α（细胞质）和gp 96/grp 94（内质网（ER）形式的hsp 90），但不是hsp 70增加在肝巨噬细胞和整个肝脏的慢性酒精喂养的小鼠和人类酒精性肝炎肝，2）Hsp 90 α陪伴IKKβ和IRF 3并促进TLR 4信号传导2）使用药理学试剂17-DMAG在体内抑制hsp 90保护小鼠免受酒精性肝损伤，3）骨髓特异性gp 96缺陷加重酒精性肝损伤。4）HSF 1虽然被酒精激活，但不足以诱导hsp 90 α。另一方面，在减少损伤期间由hsp 90抑制剂诱导的HSF 1和hsp 70对于减少肝脏炎症反应至关重要。在此，我们假设细胞质hsp 90 α和ER gp 96/grp 94有助于肝巨噬细胞活化，并且是ALD的合理治疗靶点。在酒精性肝病中，增加的hsp 90 α促进巨噬细胞活化和gp 96（未折叠蛋白反应（UPR）基因）诱导的toll样受体4表面表达和高反应性有助于巨噬细胞致敏。最后，我们提出，HSF 1和HSP 70发挥抗炎作用的过程中HSP 90抑制ALD。hsp 90抑制剂的使用将有助于解开应激蛋白和炎症反应之间的相互作用的创新途径，并将hsp 90确立为ALD的新治疗靶点。具体目的如下：1）通过以下方法研究hsp 90 α抑制剂对ALD中巨噬细胞活化的影响：A）使用hsp 90 α siRNA靶向肝巨噬细胞和17-DMAG包裹的纳米颗粒在ALD中。B）检查hsp 90 α抑制对肝脏中巨噬细胞极化的影响。C）巨噬细胞特异性hsp 90 α抑制对酒精性脂肪变性的旁分泌作用的分析。2)目的：探讨hsp 90的内质网蛋白gp 96在酒精诱导的巨噬细胞致敏中的作用。方法：（1）测定gp 96在酒精性肝损伤中的表达调控。B）研究ALD期间gp 96/TLR 4相互作用和TLR 4高反应性的重要性。C）表征ALD期间gp 96在巨噬细胞UPR活化中的作用。3)为了评估ALD中hsp 90抑制期间HSF 1和hsp 70的抗炎作用：A）评估骨髓特异性HSF 1在巨噬细胞活化和ALD中的作用。B）研究HSF 1在17-DMAG治疗和ALD抑制过程中的作用。C）确定hsp 70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.