Excessive Copper Levels Disrupt Hepatic Nuclear Receptor Function

描述(申请人提供)：我们的长期目标是确定铜对肝豆状核变性小鼠模型中肝细胞核受体的DNA结合活性和结构以及肝细胞核受体靶基因表达的影响。肝豆状核变性是一种常染色体隐性遗传病，由于铜转运P型ATP7B基因突变导致肝脏铜蓄积过多，并与各种症状有关，包括脂肪变性、胆汁淤积、肝硬变和肝功能衰竭以及神经功能障碍。威尔逊病是一种慢性严重的肝脏疾病，如果不治疗就会致命。螯合和/或锌治疗在严重肝功能障碍发作之前就开始了，已经被证明可以控制肝豆状核变性的症状。威尔逊病动物模型(Long Evans Cinnamon(Long Evans Cinnamon(Long Evans Cinnamon，Long Evans Cinnamon，Long Evans Cinnamon(Long Evans Cinnamon，Long Evans Cinnamon，Long Evans Cinnamon(Long Evans Cinnamon，Long Evans Cinnamon，Long Evans Cinnamon(Long Evans Cinnamon，Long Evans Cinnamon，Long Evans Cinnamon(Long Evans Cinnamon，大鼠和ATP7B-/-小鼠))减少了多种代谢途径相关基因的表达，包括胆汁酸合成(胆固醇71-这些基因的表达受核受体的调节，包括法尼醇X受体(FXR)和肝X受体(LXR)。我们的假设是，过量的铜会使核受体失活，并扰乱对维持肝脏代谢动态平衡至关重要的信号通路。我们将利用ATP7B-/-小鼠通过染色质免疫沉淀(CHIP)试验来确定核受体DNA结合活性，并测量肝功能参数，如胆汁流量、胆汁铜分泌、胆汁酸和胆红素。我们将测试以下特定目标：(1)确定过量铜对配体介导的靶基因表达和核受体以及染色质辅助激活因子募集的影响(2)确定铜对核受体结构的影响(3)确定锌和/或N-乙酰半胱氨酸对野生型和ATP7B-/-小鼠核受体DNA结合活性和肝功能的影响。这一特定的目的将解决锌是否恢复核受体的DNA结合活性和靶基因mRNA的表达，并改善肝功能。此外，将向ATP7B-/-小鼠注射N-乙酰半胱氨酸和锌，以确定增加肝脏谷胱甘肽水平是否会增加MRP2介导的胆汁铜排泄。这些研究将深入了解与肝豆状核变性相关的肝功能障碍的分子机制。因此，我们的发现将通过阐明肝豆状核变性进展的分子机制，以及现有的药理药物锌和N-乙酰半胱氨酸的治疗干预，直接推动肝病的研究。 公共卫生相关性：我们将在肝豆状核变性的小鼠动物模型中测量核受体的DNA结合活性。肝豆状核变性是一种慢性疾病，其特征是肝脏铜过度堆积和严重的肝功能障碍，如果不治疗就会导致死亡。我们的研究将确定过量铜水平和治疗方案扰乱的分子机制，以恢复肝脏基因表达的核受体调节，从而改善肝功能。这项研究专门针对与人类肝脏病理和肝豆状核变性相关的生物学机制。

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.