Role and Regulation of INrf2

INrf2的作用和调节

基本信息

批准号：
7367816
负责人：
Anil Kumar Jaiswal
金额：
$ 33.1万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-04-08 至 2010-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7367816
关键词：
Actins Amino Acid Sequence Antioxidants Applications Grants BTB/POZ Domain Benzo(a)pyrene Binding Biological Assay C-terminal Carcinogen exposure Carcinogens Cell Nucleus Cells Condition Cysteine Cytoprotection Cytoskeleton Cytosol Deletion Mutation Development Down-Regulation Elements Enzymes Epidermis Event Exons Exposure to Gene Expression Gene Expression Regulation Genes Genotype Goals Hepatic Hepatocyte Homodimerization Immunoprecipitation Investigation Knockout Mice Label Laboratories Liver Liver neoplasms Luciferases Maps Mediating Modification Mus N-terminal NAD(P)H dehydrogenase (quinone) 1, human NQO1 gene Neoplasms Nuclear Orthophosphate Oxidation-Reduction Oxidative Stress Phospho-Specific Antibodies Phosphorylation Plasmids Play Polymerase Chain Reaction Predisposition Protein Dephosphorylation Proteins Reactive Oxygen Species Regulation Response Elements Role Serine Signal Transduction Site Site-Directed Mutagenesis Skin Spectrophotometry Stress System Testing Threonine Tissues Trans-Activators Transfection Tyrosine Wild Type Mouse Xenobiotics chemical carcinogenesis gel electrophoresis hepatoma cell in vivo inhibitor/antagonist mouse model mutant neoplastic cell response transcription factor tumor

项目摘要

DESCRIPTION (provided by applicant): Nuclear transcription factor Nrf2 regulates ARE-mediated expression and coordinated induction of a battery of antioxidant genes. The products of these genes are known to play critical roles in cellular protection against oxidative stress and neoplasia. Recently, we cloned and sequenced a cytosolic inhibitor of Nrf2 designated as INrf2. INrf2 retains Nrf2 in the cytosol. Antioxidants antagonize INrf2 retention of Nrf2. Nrf2 releases from INrf2. Nrf2 translocates in the nucleus, binds to ARE and activate ARE-mediated expression of antioxidant genes. Preliminary studies have shown that INrf2 is phosphorylated at S104 and that mutant INrf2S104A failed to significantly repress ARE-mediated gene expression as observed with wild type INrf2. The studies have also shown that INrf2 gene is ubiquitously expressed, down regulated in hepatoma cells and induced in response to antioxidants. The goals of this proposal are: 1) elucidate the role of phosphorylation/dephosphorylation and/or redox modification of INrf2 in homodimerization of INrf2, Nrf2 degradation in basic conditions and antioxidant-induced release of Nrf2; 2) generate conditional knockout mice that do not express INrf2 in liver and epidermis of skin and investigate in vivo roles of INrf2 in Nrf2 signaling and susceptibility to carcinogens; and 3) characterize the cis-elements and trans-acting factors that regulate basal expression, down regulation of INrf2 in hepatoma cells and induction in response to antioxidants. To this end, we will use transfection, orthophosphate labeling, immunoprecipitation and Western analysis along with inhibitors of phosphorylation and phospho-specific antibodies and redox modulators. This will determine if INrf2 is phosphorylated/dephosphorylated and/or redox modulated in basic conditions and in response to antioxidants that leads to homodimerization and/or Nrf2 degradation and/or release of Nrf2 from INrf2. Mass spectrophotometry will be used to precisely map the modified sites in INrf2. We will use Cre-Lox system to delete exon 2-4 from endogenous INrf2 gene in mouse models. This will generate conditional knockout mice that do not express the INrf2 gene in liver and epidermis of skin. These will be analyzed for the role of INrf2 in Nrf2 signaling and their susceptibility to benzo(a)pyrene induced skin and liver tumors as compared to wild type mice. PCR will be used to construct INrf2 deletions, internal deletions and mutations, including Oct-1, c/EBP and ARE elements. The transfection and band/super shift assays will be used to identify the cis-elements and trans-acting factors that control the down regulation of INrf2 gene in hepatic tumor cells and antioxidant induction.

描述（由申请人提供）：核转录因子 Nrf2 调节 ARE 介导的表达并协调诱导一系列抗氧化基因。已知这些基因的产物在针对氧化应激和肿瘤的细胞保护中发挥着关键作用。最近，我们克隆并测序了 Nrf2 的胞质抑制剂，命名为 INrf2。 INrf2 将 Nrf2 保留在细胞质中。抗氧化剂拮抗 INrf2 对 Nrf2 的保留。 Nrf2 从 INrf2 发布。 Nrf2 在细胞核中易位，与 ARE 结合并激活 ARE 介导的抗氧化基因表达。初步研究表明，INrf2 在 S104 位点被磷酸化，并且突变型 INrf2S104A 未能显着抑制 ARE 介导的基因表达，如野生型 INrf2 所观察到的。研究还表明，INrf2 基因普遍表达，在肝癌细胞中下调，并响应抗氧化剂而被诱导。该提案的目标是：1）阐明INrf2的磷酸化/去磷酸化和/或氧化还原修饰在INrf2同二聚化、碱性条件下Nrf2降解和抗氧化剂诱导的Nrf2释放中的作用； 2) 生成肝脏和皮肤表皮不表达INrf2的条件敲除小鼠，并研究INrf2在Nrf2信号传导和致癌物易感性中的体内作用； 3) 表征调节肝癌细胞中 INrf2 基础表达、下调以及诱导抗氧化剂反应的顺式元件和反式作用因子。为此，我们将使用转染、正磷酸盐标记、免疫沉淀和蛋白质印迹分析以及磷酸化抑制剂、磷酸化特异性抗体和氧化还原调节剂。这将确定 INrf2 在碱性条件下是否被磷酸化/去磷酸化和/或氧化还原调节，并响应导致同二聚化和/或 Nrf2 降解和/或从 INrf2 释放 Nrf2 的抗氧化剂。质谱法将用于精确定位 INrf2 中的修饰位点。我们将使用 Cre-Lox 系统删除小鼠模型中内源性 INrf2 基因的外显子 2-4。这将产生在肝脏和皮肤表皮中不表达INrf2基因的条件性基因敲除小鼠。将分析这些小鼠与野生型小鼠相比 INrf2 在 Nrf2 信号传导中的作用以及它们对苯并（a）芘诱导的皮肤和肝脏肿瘤的易感性。 PCR将用于构建INrf2缺失、内部缺失和突变，包括Oct-1、c/EBP和ARE元件。转染和带/超位移分析将用于鉴定控制肝肿瘤细胞中 INrf2 基因下调和抗氧化诱导的顺式元件和反式作用因子。