FKBP5 in the pathogenesis of alcohol-associated liver disease

项目摘要 酒精相关性肝病（ALD）是一种复杂的疾病;其发病机制是一个多步骤的过程， 通过一系列组织病理学变化进行。FK 506结合蛋白-51（FKBP 51，由 FKBP 5基因，也称为FKBP 5）属于亲免蛋白的FKBP家族。FKBP 5是一个重要的 参与许多关键细胞信号级联调节的蛋白质。我们观察到一个双重的 与健康人相比，酒精性肝硬化患者肝脏中FKBP 5 mRNA表达上调 对照Fkbp 5在转录和蛋白水平的表达增加也观察到乙醇喂养的 小鼠有趣的是，Fkbp 5的缺失可以保护酒精诱导的肝损伤。我们的首要目标是 进一步了解FKBP 5介导酒精诱导的肝损伤的机制。实现 为此，我们将首先确定酒精如何诱导FKBP 5表达。我们的初步数据显示， 酒精性肝硬化患者FKBP 5基因启动子甲基化水平显著下调 地区在第一个具体目标中，我们将仔细剖析乙醇如何诱导FKBP 5的上游途径。 通过检验位于其下游CpG岛的Fkbp 5甲基化水平降低的假设， 5' UTR启动子区经乙醇处理后，其转录和蛋白表达均增加。接下来我们就 确定酒精诱导的肝损伤中酒精-FKBP 5轴的下游分子机制。 我们的数据表明，在用乙醇喂养的野生型小鼠中，Hippo通路被关闭，如 雅普的去磷酸化（可能通过其上游激酶p-MST 1/2的减少）导致雅普 核易位;在用乙醇喂养的Fkbp 5-/-小鼠中消除的观察结果。我们将仔细 确定Fkbp 5和雅普磷酸化的机制作用，作为探索Fkbp 5和Yap磷酸化的重要步骤。 酒精诱导的肝损伤中酒精-FKBP 5轴的下游分子机制。最后 公开可用的单细胞RNASeq数据表明FKBP 5在肝细胞中的差异表达 和其它非肝实质细胞，特别是枯否细胞。为了解决细胞类型特异性的作用， FKBP 5在ALD发病机制中的作用，我们成功地利用FKBP 5 fl/fl小鼠模型， CHRISPR/Cas9策略。我们的初步数据表明，乙醇可以诱导Fkbp 5的表达， 肝细胞和枯否细胞，与对照组相比， 即肝细胞。我们将检验Fkbp 5在我们的全球范围内对ALD的保护作用的假设。 敲除模型主要由库普弗细胞驱动，使用库普弗细胞特异性Fkbp 5-/-小鼠模型。采取 我们一起开发了动物和细胞模型，以机械地检查上游和下游 FKBP 5在ALD发病机制中的作用。这一建议意义重大，可能会导致 通过靶向FKBP 5确定ALD患者的潜在治疗干预。

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.