Excessive Copper Levels Disrupt Hepatic Nuclear Receptor Function

过量的铜水平会破坏肝核受体功能

• 批准号: 8205047
• 负责人: Clavia Ruth Wooton-Kee
• 金额: $ 1.77万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-25 至 2012-01-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8205047
• 关键词: ATP phosphohydrolase; Address; Age of Onset; Animal Model; Bile Acid Biosynthesis Pathway; Bile Acids; Bile fluid; Biliary; Bilirubin; Binding; Biological; Biological Assay; Cessation of life; Characteristics; Cholestasis; Cholesterol; Chromatin; Chronic; Chronic Disease; Cirrhosis; Coenzyme A; Complex; Copper; Cysteine; DNA Binding; DNA Binding Domain; Detection; Development; Dietary Zinc; Disease; Disease model; Dose; Estrogen Receptor alpha; Event; Excretory function; Gene Expression; Gene Targeting; Glutathione; Hepatic; Hepatocyte; Hepatolenticular Degeneration; Homeostasis; Human; Inbred LEC Rats; Incidence; Ligands; Live Birth; Liver; Liver Dysfunction; Liver Failure; Liver diseases; Measures; Mediating; Metabolic; Metabolic Pathway; Mixed Function Oxygenases; Molecular; Mus; Mutation; Neurologic Dysfunctions; Nuclear; Nuclear Receptors; Oxidoreductase; Pathology; Patients; Physiological; Pump; Regimen; Regulation; Research; Resolution; Risk; Signal Pathway; Staging; Structure; Supplementation; Symptoms; Testing; Therapeutic; Therapeutic Intervention; Triethylenetetramine; Zinc; bile salts; chelation; chromatin immunoprecipitation; human disease; improved; insight; liver function; liver transplantation; loss of function mutation; mRNA Expression; multidrug resistance-associated protein 2; novel; polypeptide; prevent; promoter; public health relevance; receptor; receptor binding; receptor expression; receptor function; response; tetrathiomolybdate

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.

描述（由申请人提供）：我们的长期目标是确定铜对威尔逊病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。威尔逊氏病是一种常染色体隐性遗传疾病，由于铜转运 P 型 ATP 酶 ATP7b 突变，导致肝脏铜过度积累，并与多种症状相关，包括脂肪变性、胆汁淤积、肝硬化和肝衰竭如神经功能障碍。威尔逊氏病是一种慢性严重的肝脏疾病，如果不治疗可能致命。在严重肝功能障碍出现之前开始螯合和/或锌疗法已被证明可以控制威尔逊病的症状。威尔逊病动物模型（长埃文斯肉桂 (LEC) 大鼠和 Atp7b-/- 小鼠）的多种代谢途径相关基因的表达降低，包括胆汁酸合成（胆固醇 71-羟化酶 (Cyp7a1)）、胆固醇合成（3-羟基-3-甲基-戊二酰辅酶A还原酶（HMG-CoA还原酶））、胆汁胆汁酸（胆汁盐输出泵（Bsep、Abcb11）和谷胱甘肽缀合复合转运（多药耐药相关蛋白 2（Mrp2、Abcc2））。这些基因的表达受核受体调节，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破坏对维持肝脏代谢稳态至关重要的信号通路。通过染色质免疫沉淀 (ChIP) 测定确定核受体 DNA 结合活性，并测量肝功能参数，例如胆汁流量、胆汁铜分泌、胆汁酸和胆红素。我们将测试以下具体目标：(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染色质共激活剂招募的影响 (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。这一具体目标将解决锌能否恢复核受体的 DNA 结合活性和靶基因 mRNA 表达并改善肝功能的问题。此外，还将向 Atp7b-/- 小鼠施用 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 Mrp2 介导的胆汁铜排泄。这些研究将深入了解与威尔逊病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氏病进展的分子机制以及可用药物、锌和 n-乙酰半胱氨酸的治疗干预来直接推进肝病的研究。 公共健康相关性：我们将测量威尔逊氏病小鼠模型中核受体的 DNA 结合活性，威尔逊氏病是一种慢性疾病，其特征是肝铜积累过多和严重的肝功能障碍，如果不治疗会导致死亡。我们的研究将确定因铜水平过高而破坏的分子机制，以及恢复肝基因表达的核受体调节从而改善肝功能的治疗方案。这项研究专门研究了与人类肝脏病理学和威尔逊氏病相关的生物学机制。