Chronic hepatocyte apoptosis and the activation of the quiescent hepatic stellate cells (HSC) to extracellular matrix-producing myofibroblasts are central to the development of alcoholic liver disease (ALD). Reactive oxidative stress (ROS)-mediated injury and activation of TNFalpha are major events in the progression of ALD, however the sources of ROS and how the signaling events are integrated culminating in active TNFalpha production are not well elucidated. To study these pathways, we have made several original observations: we have demonstrated that NOX4 induction results in a direct activation of the collagen I promoter in HSC, and in hepatocytes it plays a role in death ligand-induced apoptosis. NOX4 is upregulated in humans with ALD; and in the NOX4-/- mice steatosis, TACE activity, TNFalpha levels and lipid peroxidation were attenuated. Of particular importance for this proposal we created cell-specific NOX4-/- models. Thus our CENTRAL HYPOTHESIS is that NOX4 is an important enzyme in alcoholic liver injury playing a role in the induction of the TNFalpha converting enzyme (TACE, or ADAM17) and thereby activating latent TNFalpha. We propose three SPECIFIC AIMS to address the key areas generated by the main hypothesis:
Our first aim i s to determine the pathways by which NOX4 induction results in an increase in TACE activity in hepatic stellate cells. a) We propose experiments to address the mechanism by which acetaldehyde; the metabolite of EtOH induces the NOX4 promoter. Identifying the key transcription factors controlling NOX4 during alcoholic injury will have a major impact. We will perform DNase footprint analysis and chromatin IP assays and promoter deletion mutants will be generated targeting the corresponding areas. b) We will interrogate the pathways of NOX4 induction leading to the activation of TACE by studying the downregulation of Sirtuin1 and the tissue inhibitor of metalloproteinases 3 (TIMP3), the natural inhibitor of TACE. We will test the hypothesis that the low TIMP3 activity results in TACE activation and ectodomain cleavage and activation of TNFalpha.
Our second aim i s to investigate the mechanism by which NOX4 can induce hepatocyte injury, either as a result of direct NOX4 induction in hepatocytes or by paracrine effects from active HSC. The experiments in this aim will address: a) the role of NOX4 in hepatocytes as a proapoptotic enzyme. We will test the hypothesis that the activation of NOX4 in hepatocytes results in the translocation of the apoptosis inducing factor (AIF) to the nucleus resulting in the formation of a chromatin degrading complex. Alternatively, activation of the death ligand pathway by TNFalpha will result in lipid raft formation and the recruitment of NOX4 and ceramide formation. b) We will determine the role of NOX4/H2O2 in the HSC/hepatocyte crosstalk and reciprocal effects; using a novel micropatterned co-culture system with integrated biosensors. In the third aim we will study if NOX4-mediated oxidative stress is a key event during ALD in vivo. In mechanistic studies we will define the specific contribution of hepatocytes and HSC to alcoholic liver injury using hepatocyte or HSC NOX4-/- mice that we have recently generated. We will study the effects on both acute and chronic progressive alcoholic liver injury. This work is expected to highlight future avenues in developing effective treatment options. Thus complementing the above studies, we propose to use a novel orally available NOX4 inhibitor in the acute and chronic models of ALD.
Alcoholic liver disease is one of the most common of chronic liver diseases, affecting an increasing number of patients at the Veterans Administrations, leading to significant morbidity and mortality. Currently there are no FDA-approved treatments for alcoholic liver disease. Liver transplantation is the only curative approach; and is only available for a small proportion of patients. Thus there is an urgent need to better understand the pathogenesis of inflammation and fibrosis in this disease and to develop rational treatment approaches. This proposal is aimed at elucidating the role of NOX4-mediated oxidative stress in promoting TNFalpha production, inflammation and fibrosis. Based on the data generated we propose to use a novel NOX4 inhibitor as a new treatment strategy to halt disease progression in ALD.
