Excessive Copper Levels Disrupt Hepatic Nuclear Receptor Function

描述（由申请人提供）：我们的长期目标是确定铜对肝豆状核变性小鼠动物模型中肝核受体的DNA结合活性和结构以及肝核受体靶基因表达的影响。威尔逊病是一种常染色体隐性遗传疾病，由于铜转运P型ATP酶ATP 7 b突变导致肝脏铜过度蓄积，并与多种症状相关，包括脂肪变性、胆汁淤积、肝硬化和肝功能衰竭以及神经功能障碍。威尔逊氏病是一种慢性和严重的肝脏疾病，如果不治疗是致命的。螯合和/或锌治疗在严重肝功能障碍发作之前开始已被证明可以控制威尔逊病的症状。肝豆状核变性动物模型（Long Evans Cinnamon（LEC）大鼠和Atp 7 b-/-小鼠）降低了参与几种代谢途径（包括胆汁酸合成）的基因表达（胆固醇71-羟化酶（Cyp 7a 1）），胆固醇合成（3-羟基-3-甲基-戊二酰-CoA还原酶（HMG-CoA还原酶）），胆汁胆汁酸（胆盐输出泵（Bsep，Abcb 11）和谷胱甘肽缀合的化合物转运（多药耐药相关蛋白2（Mrp 2，Abcc 2））。这些基因的表达受到核受体的调节，包括法尼醇X受体（FXR）和肝脏X受体（LXR）。我们的假设是过量的铜使核受体失活，并破坏了维持肝脏代谢稳态的关键信号通路。我们将利用Atp 7 b-/-小鼠通过染色质免疫沉淀（ChIP）测定核受体DNA结合活性，并测量肝功能参数，如胆汁流量、胆汁分泌铜、胆汁酸和胆红素。我们将测试以下特定目的：（1）确定过量铜对配体介导的靶基因表达诱导以及核受体和共激活因子募集至染色质的影响（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Atp 7 b-/-小鼠核受体DNA结合活性和肝功能的影响。该特定目标将解决锌是否恢复核受体和靶基因mRNA表达的DNA结合活性并改善肝功能。此外，将对Atp 7 b-/-小鼠给予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强Mrp 2介导的胆汁铜排泄。这些研究将提供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，我们的研究结果将通过阐明威尔逊病进展中涉及的分子机制和可用药理学药物锌和n-乙酰半胱氨酸的治疗干预来直接推进肝病的研究。 公共卫生相关性：我们将在威尔逊病的小鼠动物模型中测量核受体的DNA结合活性，威尔逊病是一种慢性疾病，其特征是肝脏铜过度积累和严重肝功能障碍，如果不治疗会导致死亡。我们的研究将确定被过量铜水平和治疗方案破坏的分子机制，以恢复肝细胞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学和威尔逊病相关的生物学机制。

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.