Targeting proteotoxic stress responses in liver fibrosis

摘要 肝纤维化与慢性炎症和失调的伤口愈合反应有关， 暴露于各种类型的损伤，包括酒精、肥胖、NASH和病毒性肝炎。最近出现 旨在消除病原体的肝脏疗法在解决纤维化方面有希望。但要 设计抗纤维化治疗策略，我们需要更好地了解纤维化的发病机制。 细胞外基质的积聚是肝纤维化的标志。肝星状细胞（HSC）是 由促纤维化刺激激活，并且是肝脏中主要的ECM产生细胞。氧化的作用 应激和炎性细胞因子在HSC活化中的作用已经受到广泛的研究。靶向 应激途径是一种有吸引力的治疗策略，对有效治疗至关重要。 应激诱导的转录因子和蛋白毒性反应的主要调节因子，HSF 1介导 HSP 90和HSP 70的诱导，应激分子伴侣在炎症信号传导中很重要， 分别在HSC活化和逆转中的作用。我们的初步数据显示，HSF 1的缺乏， 不能诱导HSP 70促进HSC活化和纤维化，而且不能解决HSC 在纤维化恢复模型中的活化和纤维化。细胞质HSP 90 AA 1和内质网 内质网（ER）、HSP 90 B1/Grp 94/gp 96在纤维化肝组织中的表达可能参与了纤维化的发生。针对HSP 90， 使用特异性抑制剂17-DMAG，其诱导HSF 1活化和下游HSP 70，将描绘 HSF 1-HSP 70在肝星状细胞失活中的作用我们假设HSF 1的不活动和 在星状细胞活化过程中HSP 90的诱导有助于肝纤维化，而HSP 90的诱导有助于肝纤维化。 HSF 1和HSPA 1A/HSP 70有助于纤维化的消退。具体目标是：（1）解开 应激介导的转录因子HSF 1在肝纤维化调控中的作用：检测其作用 星状细胞特异性敲除HSF 1对纤维发生的影响;测试HSF 1-质粒纳米颗粒对 纤维化消退;评估HSF 1调控的纵向表达、活性和新靶基因 在纤维化和消退期间。2)为了评估是否靶向HSP 90 AA 1（胞质）和/或 HSP 90 B1/Grp 94/gp 96（ER）通过以下途径调节星状细胞活化和纤维化： 星状细胞活化过程中HSP 90 AA 1和gp 96的变化;研究HSP 90抑制剂17-DMAG对肝脏的影响 纤维化;测定星状细胞特异性抑制gp 96和HSP 90在体内对肝纤维化的作用。 3)通过以下方法研究HSF 1诱导的HSP 70在星状细胞功能中的重要性： Hsp 70及其对HSF 1的依赖性，在纤维化消退过程中;研究Hsp 70的过度表达是否 HSP 70灭活HSC并解决纤维化;在17-DMAG治疗期间评估HSF 1-HSP 70轴 HSC活化和纤维化。我们提出的研究将揭示HSF 1介导的应激途径的作用， 和伴侣蛋白在星状细胞活化和纤维化中的作用。

ABSTRACT Liver fibrosis is linked to inflammation and deregulated wound healing response, triggered by chronic exposure to various types of insults including alcohol, obesity, NASH and viral hepatitis. Recently emerging liver therapies directed to eliminate the pathogenic agent suggest promise in resolving fibrosis. However, to design anti-fibrotic therapeutic strategies we need a better understanding of the pathogenesis of fibrosis. Accumulation of extracellular matrix is the hallmark of hepatic fibrosis. Hepatic stellate cells (HSCs) are activated by pro-fibrogenic stimuli and are the main ECM producing cells in the liver. The role of oxidative stress and inflammatory cytokines in HSC activation has been under extensive investigation. Targeting stress pathways to inactivate HSCs is an attractive therapeutic strategy and crucial to effective treatment. Stress induced transcription factor and master regulator of proteotoxic responses, HSF1 mediates induction of HSP90 and HSP70, stress chaperones important in inflammatory signaling and likely to play a role in HSC activation and reversion, respectively. Our preliminary data show that deficiency of HSF1, unable to induce HSP70 promotes HSC activation and fibrosis and moreover, is unable to resolve HSC activation and fibrosis in a model of fibrosis recovery. Increased cytosolic HSP90AA1 and endoplasmic reticulum (ER), HSP90B1/Grp94/gp96 in fibrotic livers may contribute to fibrogenesis. Targeting HSP90, using a specific inhibitor, 17-DMAG, which induces HSF1 activation and downstream HSP70, will delineate the role of HSF1-HSP70 in inactivation of hepatic stellate cells. We hypothesize that HSF1 inactivity and HSP90 induction during stellate cell activation contributes to liver fibrosis, whereas induction of HSF1 and HSPA1A/HSP70 contributes to resolution of fibrosis. The Specific Aims are- 1) To unravel the role of stress mediated transcription factor, HSF1 on regulation of liver fibrosis by: Examining the effect of stellate cell specific knock-out of HSF1 on fibrogenesis; Testing effect of HSF1-plasmid nanoparticles on fibrosis resolution; Evaluating longitudinal expression, activity and novel target genes regulated by HSF1 during fibrosis and resolution. 2) To assess whether targeting HSP90AA1 (cytosolic) and/or HSP90B1/Grp94/gp96(ER) regulates stellate cell activation and fibrogenesis by: Evaluating the induction of HSP90AA1 and gp96 during stellate cell activation; Investigating effect of hsp90 inhibitor, 17-DMAG on liver fibrosis; Determining effect of stellate cell-specific inhibition of gp96 and HSP90 in vivo, on hepatic fibrosis. 3) To investigate importance of HSF1 induced HSP70 in stellate cell function by: Determining induction of Hsp70 and its dependence on HSF1, during resolution of fibrosis; Investigating whether overexpression of HSP70 inactivates HSC and resolves fibrosis; Evaluating HSF1-HSP70 axis during 17-DMAG treatment on HSC activation and fibrosis. Our proposed studies will unravel the role of HSF1 mediated stress pathways and chaperones in stellate cell activation and fibrosis.