FKBP5 in the pathogenesis of alcohol-associated liver disease

项目摘要 酒精性肝病是一种复杂的疾病，其发病机制是一个多步骤的过程。 通过一系列的组织病理学变化而进展。FK506结合蛋白-51(FKBP51，由 FKBP5基因，又称FKBP5)，属于免疫亲和素FKBP家族。FKBP5是一个重要的 参与调节许多关键细胞信号级联反应的蛋白质。我们观察到了一个双重的 酒精性肝硬变患者肝脏FKBP5基因表达上调 控制。在酒精喂养的动物中，FKBP5在转录本和蛋白水平的表达也增加 老鼠。有趣的是，FKBP5的缺失对酒精性肝损伤有保护作用。我们的首要目标是 进一步了解FKBP5在酒精性肝损伤中的作用机制。要实现 为了达到这个目标，我们将首先确定酒精是如何诱导FKBP5表达的。我们的初步数据显示 酒精性肝硬变患者FKBP5基因启动子甲基化水平显著下调 区域。在第一个具体目标中，我们将仔细剖析乙醇如何诱导FKBP5的上游途径 通过检验假设降低位于其CpG岛上的FKBP5的甲基化水平来表达 乙醇诱导的5‘UTR启动子区域的转录和蛋白表达增加。接下来，我们将 确定酒精-FKBP5轴在酒精性肝损伤中的下游分子机制。 我们的数据表明，在喂食乙醇的野生型小鼠中，河马途径被关闭，如下所示 YAP的去磷酸化(可能是通过减少其上游的p-MST1/2)导致YAP 核移位；在饲喂乙醇的FKBP5-/-小鼠中被取消的观察。我们会小心地 确定FKBP5和YAP磷酸化的机制作用，作为探索 酒精-FKBP5轴在酒精性肝损伤中的下游分子机制最后， 公开的单细胞RNAseq数据表明FKBP5在肝细胞中的差异表达 和其他非肝实质细胞，特别是库普弗细胞。要解决特定细胞类型的作用 FKBP5在ALD发病机制中的作用，我们成功地建立了一种新的FKBP5 fl/fl小鼠模型。 CHRISPR/CAS9战略。我们的初步数据表明，乙醇可以诱导FKBP5在脑内的表达 乙醇对Kupffer细胞和肝细胞均有显著诱导作用 肝细胞的表达。我们将在我们的全球范围内检验FKBP5对ALD的保护作用 基因敲除模型主要由Kupffer细胞驱动，使用Kupffer细胞特异性的FKBP5-/-小鼠模型。已被占用 我们一起开发了动物和细胞模型，以机械方式检查上行和下行 FKBP5在ALD发病机制中的作用途径这项建议具有重要意义，它可能导致 通过靶向FKBP5确定ALD患者的潜在治疗干预措施。

Project Summary Alcohol-associated liver disease (ALD) is a complex disorder; its pathogenesis is a multi-step process that progresses through a spectrum of histopathological changes. FK506-binding protein-51 (FKBP51, encoded by the FKBP5 gene, also called FKBP5) belongs to the FKBP family of immunophilins. FKBP5 is an important protein involved in the regulation of many key cellular signaling cascades. We observed a two-fold upregulation of FKBP5 mRNA expression in the liver of patients with alcoholic cirrhosis compared to healthy controls. The increase in Fkbp5 expression at the transcript and protein level was also observed in ethanol-fed mice. Interestingly, loss of Fkbp5 protected against alcohol-induced liver injury. Our overarching goal is to further understanding the mechanistic action of FKBP5 in mediating alcohol-induced liver injury. To achieve this goal, we will first determine how alcohol induces FKBP5 expression. Our preliminary data suggested that patients with alcoholic cirrhosis had significant downregulation in FKBP5 methylation levels at its promoter region. In the first specific aim, we will carefully dissect the upstream pathway of how ethanol induces FKBP5 expression by testing the hypothesis that reduced methylation levels of Fkbp5 at the CpG island located at its 5’ UTR promoter region by ethanol lead to an increase in its transcript and protein expression. Next, we will determine the downstream molecular mechanism of the alcohol-FKBP5 axis in alcohol-induced liver injury. Our data suggested that in wild-type mice fed with ethanol, the Hippo pathway was turned off as indicated by dephosphorylation of YAP (likely through the reduction in p-MST1/2, its upstream kinase) leading to YAP nuclear translocation; the observation which was abrogated in Fkbp5-/- mice fed with ethanol. We will carefully determine the mechanistic role of Fkbp5 and Yap phosphorylation, as an essential step in exploring the downstream molecular mechanism of the alcohol-FKBP5 axis in alcohol-induced liver injury. Lastly, the publicly available single-cell RNASeq data suggested the differential expression of FKBP5 in the hepatocytes and other non-hepatic parenchymal cells, notably Kupffer cells. To address the role of cell-type-specific FKBP5 in the pathogenesis of ALD, we successfully generated a novel Fkbp5 fl/fl mouse model using the CHRISPR/Cas9 strategy. Our preliminary data illustrated that ethanol can induce the expression of Fkbp5 in both hepatocytes and Kupffer cells with a significant induction in Kupffer cells treated with ethanol compared to that of hepatocytes. We will test the hypothesis that the protective effect of Fkbp5 against ALD in our global knockout model is driven primarily by Kupffer cells using Kupffer cell-specific Fkbp5-/- mouse model. Taken together, we have developed animal and cellular models to mechanistically examine both up- and downstream pathways on the role of FKBP5 in ALD pathogenesis. This proposal is of significance and it may lead to the identification of potential therapeutic interventions in patients with ALD by targeting FKBP5.