Investigating the Role of Wnt3a and Wnt5a as Factors of Hepatic Stellate Cell Activation in Alcohol-Related Liver Fibrosis - PROJECT SUMMARY Alcohol-associated Liver Disease (ALD)-related mortality rates have increased 23% in the United States since 2019. While there remain concurrent increases in both the prevalence and mortality from alcohol misuse, patients have no therapeutic options outside from liver transplantation. Despite what is known about the pathogenesis of ALD, the mechanisms underlying the acceleration across the spectrum of liver disease remain poorly understood. It is known that patients with progressive ALD have progressive scarring of the liver and is the main predictor of patient outcomes. Excessive tissue scarring is characterized by highly cross-linked extracellular matrix (ECM) in response to injury. Hepatic stellate cells (HSC), the tissue resident fibroblast, play a key role in the initiation, progression, and resolution of liver fibrosis by transdifferentiating into activated myofibroblasts that secrete ECM in response to chronic injury, yet the mechanisms involved in HSC transdifferentiation are not well characterized. The goal of this proposal is to elucidate novel mechanisms of alcohol-induced fibrotic gene reprogramming in the liver and identify specific factors amplifying HSC activation in the diseased liver. While the uncontrolled regulation of Wnt/-catenin signaling is a hallmark of various hepatic pathologies including liver cancer, there are limited studies investigating alcohol exposure on canonical -catenin activation in specifically HSCs. Importantly, in canonical -catenin-dependent pathways, the association and release of the yes- associated protein-1 (YAP)/transcriptional coactivator (TAZ) (YAP/TAZ) from the -catenin destruction complex occurs in human embryonic kidney cells, yet it remains unknown if the convergence of these pathways occur in HSCs to drive their transdifferentiation in models of ALD. Our preliminary in vitro data show increased expression of -catenin and the transducers of Wnts, Frizzled (Fzd) receptors 1, 2, and 7, during spontaneous activation in primary mouse HSCs. Despite this knowledge, the exact role of Wnts driving the pathogenesis of fibrosis in models of ALD remain unclear. Our working hypothesis is that alcohol increases the expression of Wnts, leading to Wnt-dependent canonical -catenin activation, via YAP/TAZ, to promote hepatic stellate cell activation and production of ECM during the early pathogenesis of liver fibrosis in ALD. To test this, in Aim 1 we will determine if canonical Wnt ligands promote HSC transdifferentiation, in ex vivo models of alcohol exposure. Following treatment with ethanol and exogenous Wnt ligands, we will characterize HSC activation and fibrotic gene expression. We will then define if ethanol exposure impacts the sequestration and association of YAP/TAZ in the -catenin destruction complex to promote HSC transdifferentiation. In Aim 2, we will characterize the role of canonical Wnt signaling in HSCs using wild-type (C57BL/6J) and LRP6 knockout mice after ethanol exposure to measure primary outcomes related to hepatic fibrosis by gene and protein expression.
