PROJECT SUMMARY Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis driven by enhanced free fatty acid (FFA) uptake and lipid synthesis with reduced lipid clearance through mitochondrial fatty acid oxidation and lipoprotein export. Excessive hepatic lipid accumulation can cause lipotoxicity, which promotes local inflammation, hepatocyte ballooning, and fibrosis. This process leads to the development of nonalcoholic steatohepatitis (NASH), that can further progress to cirrhosis, liver failure, or hepatocellular carcinoma if left unchecked. Current treatments for NAFLD/NASH seek to either reduce hepatic steatosis or diminish the fibrosis response that drives NASH development. While these treatments have shown great promise in pre-clinical trials, they have yet to provide a clear therapeutic benefit. The Eph family of receptor tyrosine kinases, the largest family in the mammalian genome, interact with Eph receptor-interacting (ephrin) ligands on adjacent cells to mediate cell adhesion/repulsion signaling during axonal guidance, tissue patterning, leukocyte homing, and metastasis. Differential ligand-dependent and ligand- independent phosphorylation events appear to mediate these often-inverse functions of EphA2 signaling. EphA2 interactions with ephrinA1 on an adjacent cell promote tyrosine autophosphorylation (Y588, Y772) and intrinsic kinase activity, resulting in the inhibition of proliferation and migration. In the absence of ligand, EphA2 can become phosphorylated on S897, classically by Akt, resulting in enhanced proliferation and migration through largely unknown mechanisms. Work from our lab demonstrated EphA2 expression in multiple cell types and EphA2 deletion reduces atherosclerotic plaque formation. Our preliminary data show that EphA2 knockout mice show significantly reduced liver weight/body weight ratios following high fat diet feeding associated with diminished hepatosteatosis and reduced inflammation. Furthermore, C57Bl/6J mice treated with a high fat diet for 8 weeks show enhanced expression of the EphA2 ligand ephrinA1, along with enhanced markers of EphA2 ligand-dependent signaling. In contrast, both NASH patients and mouse models of NASH show decreased ephrinA1 expression and enhanced markers of EphA2 ligand-independent signaling. Therefore, we hypothesize that ligand-dependent EphA2 signaling serves as an initial protective mechanism in early NAFLD that weakens as the disease progresses. Using a combination of cell culture and mouse models, we will determine the role of EphA2/EphrinA1 expression and signaling in lipid homeostasis and metabolism (Aim 1), and determine the role of EphA2/EphrinA1 expression and signaling in NAFLD/NASH progression (Aim 2). If successful, these studies will elucidate a novel role for EphA2 signaling in NAFLD/NASH pathogenesis and may provide a novel therapeutic target to reduce liver damage in NAFLD/NASH.