Role and Regulation of INrf2

INrf2的作用和调节

• 批准号: 6930063
• 负责人: Anil Kumar Jaiswal
• 金额: $ 35.63万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 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/6930063
• 关键词: antioxidants; carcinogenesis; cytoprotection; environmental exposure; gene induction /repression; genetic regulatory element; genetic susceptibility; genetically modified animals; immunoprecipitation; laboratory mouse; liver; mass spectrometry; oxidation reduction reaction; oxidative stress; phosphorylation; skin; transcription factor; transfection; western blottings

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调节是介导的表达和一系列抗氧化剂基因的诱导。已知这些基因的产物在细胞保护抗氧化应激和肿瘤中起关键作用。最近，我们克隆并测序了指定为INRF2的NRF2的胞质抑制剂。 INRF2保留在细胞质中的NRF2。抗氧化剂拮抗NRF2的INRF2保留。 NRF2从INRF2释放。 NRF2在细胞核中易位，结合为IS并激活抗氧化基因的表达。初步研究表明，InRF2在S104处磷酸化，突变体InRF2S104A未能显着抑制使用野生型InRF2观察到的基因表达。研究还表明，在肝癌细胞中调节INRF2基因，并响应抗氧化剂而诱导。 该提案的目标是：1）阐明INRF2在INRF2同构化中的磷酸化/去磷酸化和/或氧化还原修饰的作用，NRF2在基本条件下降解以及抗氧化剂诱导的NRF2释放； 2）产生条件敲除小鼠，这些小鼠在肝脏和皮肤表皮中不表达InRF2，并研究INRF2在NRF2信号传导中的体内作用和对致癌物的敏感性； 3）表征调节基础表达的顺式元素和跨作用因子，降低肝癌细胞中INRF2的调节，并响应抗氧化剂的诱导。为此，我们将使用转染，直磷酸标记，免疫沉淀和西方分析，以及磷酸化和磷酸化特异性抗体和氧化还原调节剂的抑制剂。这将确定在基本条件下和/或氧化氧化物调制的磷酸化/或氧化还原是否在基本条件下和/或抗氧化剂响应的抗氧化剂调节和/或NRF2降解和/或从INRF2中释放NRF2的抗氧化剂。质量分光光度法将用于精确绘制INRF2中的修改位点。我们将使用Cre-Lox系统在小鼠模型中从内源性INRF2基因中删除外显子2-4。这将产生条件敲除小鼠，这些小鼠不会在肝脏和皮肤表皮中表达INRF2基因。与野生型小鼠相比，将对这些INRF2在NRF2信号传导中的作用及其对苯并（a）pyrene诱导的皮肤和肝肿瘤的敏感性进行分析。 PCR将用于构建INRF2缺失，内部缺失和突变，包括OCT-1，C/EBP和元素。转染和带/超级偏移测定法将用于识别控制肝肿瘤细胞中INRF2基因的下调和抗氧化剂诱导的下调元素和跨作用因子。