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.

Project Narrative Alcohol-associated liver disease (ALD) is a major public health problem in the United States. This application examines the mechanisms by which the FK506-binding protein-51 (FKBP5) mediates the pathogenesis of alcohol-induced liver injury. The results of our application have the potential to identify a new therapeutic approach for ALD by targeting FKBP5.