Excessive Copper Levels Disrupt Hepatic Nuclear Receptor Function

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DESCRIPTION (provided by applicant): Our long-term objective is to determine the effect of copper on the DNA-binding activity and structure of hepatic nuclear receptors and expression of hepatic nuclear receptor target genes in a mouse animal model of Wilson's disease. Wilson's disease is an autosomal recessive disease that results in excessive hepatic copper accumulation due to mutations in the Cu-transporting P-type ATPase, ATP7b, and is associated with a variety of symptoms including steatosis, cholestasis, cirrhosis, and liver failure, as well as neurological dysfunction. Wilson's disease is a chronic and severe liver disorder that is fatal if not treated. Chelation and/or zinc therapy started before the onset of severe liver dysfunction has been shown to manage the symptoms of Wilson's disease. The Wilson's disease animal models (Long Evans Cinnamon (LEC) rat and Atp7b-/- mouse) have decreased expression of genes involved in several metabolic pathways, including bile acid synthesis (cholesterol 71-hydroxylase (Cyp7a1)), cholesterol synthesis (3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase)), biliary bile acid (bile salt export pump (Bsep, Abcb11) and glutathione conjugated- compound transport (multidrug resistance-associated protein 2 (Mrp2, Abcc2)). The expression of these genes is regulated by nuclear receptors, including the farnesoid X receptor (FXR) and liver X receptor (LXR). Our hypothesis is that excess copper inactivates nuclear receptors and disrupts signaling pathways critical for maintaining metabolic homeostasis in the liver. We will utilize the Atp7b-/- mouse to determine nuclear receptor DNA-binding activity via chromatin immunoprecipitation (ChIP) assays and to measure parameters of liver function, such as bile flow, biliary secretion of copper, bile acids, and bilirubin. We will test the following Specific Aims: (1) determine the effects of excess copper on ligand-mediated induction of target gene expression and nuclear receptor and coactivator recruitment to chromatin (2) determine the effect of copper on the structure of nuclear receptors (3) determine the effect of zinc and/or n-acetyl cysteine on nuclear receptor DNA-binding activity and liver function in wild-type and Atp7b-/- mice. This Specific Aim will address if zinc restores the DNA-binding activity of nuclear receptors and target gene mRNA expression and improves liver function. Additionally, n-acetyl cysteine and zinc will be administered to Atp7b-/- mice to determine if increasing hepatic glutathione levels enhance Mrp2-mediated biliary copper excretion. These studies will provide insight into the molecular mechanisms involved in liver dysfunction associated with Wilson's disease. Hence, our findings will directly advance the research of liver disease by elucidating molecular mechanisms involved in the progression of Wilson's disease and therapeutic interventions with available pharmacological agents, zinc and n-acetyl cysteine. PUBLIC HEALTH RELEVANCE: We will measure the DNA-binding activity of nuclear receptors in a mouse animal model for Wilson's disease, a chronic disease that is characterized by excessive hepatic copper accumulation and severe liver dysfunction that results in death if not treated. Our studies will determine molecular mechanisms that are disrupted by excessive copper levels and therapeutic regimens to restore nuclear receptor regulation of hepatic liver gene expression and hence improve liver function. This research specifically addresses the biological mechanisms relevant to human liver pathology and to Wilson's disease.