MEKK1 down-regulates PKD1 promoter by a novel mechanism

MEKK1 通过一种新机制下调 PKD1 启动子

• 批准号: 7250928
• 负责人: M. Rafiq Islam
• 金额: $ 3.51万
• 依托单位: NORTHWEST MISSOURI STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-15 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7250928
• 关键词: Academy; Aftercare; Autosomal Dominant Polycystic Kidney; Binding Proteins; Biochemical; Biological; Calcium Channel Blockers; Cell Line; Cells; Conflict (Psychology); Development; Distal; Down-Regulation; Electrophoretic Mobility Shift Assay; FOS gene; Funding; Gene Expression; Genes; Genetic Transcription; Goals; Human; JUN gene; MAP3K1 gene; Mediating; Missouri; Mitogen-Activated Protein Kinases; Molecular; Mutation; Numbers; Pathway interactions; Phorbol Esters; Polycystic Kidney Diseases; Promoter Regions; Protein Overexpression; Reporting; Repression; Research; Science; Signal Transduction; Site; Students; Therapeutic; Transcription Factor AP-1; Travel; Universities; deletion analysis; disease phenotype; experience; gene repression; improved; interest; novel; prevent; promoter; transcription factor

DESCRIPTION (provided by applicant): Mutations in the human polycystic kidney disease-1 (PKD1) gene are responsible for the vast majority (85- 90%) of autosomal dominant polycystic kidney disease (ADPKD) cases. There are conflicting observations as to whether ADPKD results from abnormally decreased or from abnormally increased expression of the PKD1 gene. Our broad goal is to understand how various factors regulate transcription of the PKD1 gene. We recently identified that this promoter is a target for the b-catenin/TCF pathway (1). In preliminary studies, although the 3.3 kb region of the promoter contains a distal AP-1 site that binds protein in gel shift assay, it was not induced by phorbol ester or its downstream target, a constitutively active MEKK1 (CAM). Rather, the promoter fragment was strongly repressed by both. The AP-1 site in the promoter, however, was activated when cells were co-transfected with CAM together with excessive amount of c-jun, c-fos or both, suggesting a competition between the classic MAP kinase pathway of MEKK1, which activates AP-1 and an unknown pathway of MEKK1, which down-regulates this promoter. A proximal 200 bp fragment in the promoter was found to be responsive to CAM down-regulation by deletion analysis. The CAM effect was not mediated through either Ets or Sp1 found in this 200 bp of the promoter. As no prior report has suggested transcription repression by MEKK1 in uninduced cells, it would be interesting to investigate this novel down- regulation mechanism of MEKK1 and its effect on the endogenous PKD1 promoter. In this proposal, we propose to accomplish the following three specific aims: Aim 1: Identify target sequence in the PKD1 promoter subject to down-regulation by CAM, Aim 2: Examine if the down-regulation of the PKD1 promoter by CAM is mediated by a novel pathway, and Aim 3: Investigate the effect of CAM or MEKK1 on the endogenous PKD1 promoter. We will use a variety of biochemical, and cellular and molecular biological approaches. These studies will not only improve our understanding of the PKD1 gene expression, but may also suggest a new target and/or mechanism of MEKK1 pathway. Thus, the answers sought should have both fundamental significances and should provide information leading to therapeutic approaches. Furthermore, this AREA-funded study will provide valuable research experience for students at Northwest Missouri State University and Missouri Academy of Science, Math and Computing.

描述（由申请人提供）：绝大多数（85-90%）常染色体显性多囊肾病（ADPKD）病例是由人类多囊肾病-1（PKD1）基因突变引起的。关于 ADPKD 是由 PKD1 基因表达异常减少还是异常增加引起的，存在相互矛盾的观察。我们的总体目标是了解各种因素如何调节 PKD1 基因的转录。我们最近发现该启动子是 b-连环蛋白/TCF 途径的靶标 (1)。在初步研究中，虽然启动子的 3.3 kb 区域包含一个远端 AP-1 位点，该位点在凝胶迁移测定中结合蛋白质，但它不是由佛波酯或其下游靶标（一种组成型活性 MEKK1 (CAM)）诱导的。相反，启动子片段受到两者的强烈抑制。然而，当细胞与 CAM 以及过量的 c-jun、c-fos 或两者共转染时，启动子中的 AP-1 位点被激活，这表明 MEKK1 的经典 MAP 激酶途径（激活 AP-1）与 MEKK1 的未知途径（下调该启动子）之间存在竞争。通过删除分析发现启动子中的近端 200 bp 片段对 CAM 下调有反应。 CAM 效应不是通过启动子的 200 bp 中发现的 Ets 或 Sp1 介导的。由于之前没有报道表明 MEKK1 在未诱导的细胞中抑制转录，因此研究 MEKK1 的这种新型下调机制及其对内源 PKD1 启动子的影响将是很有趣的。在本提案中，我们建议实现以下三个具体目标：目标 1：识别 PKD1 启动子中受 CAM 下调的靶序列，目标 2：检查 CAM 对 PKD1 启动子的下调是否是由新途径介导的，目标 3：研究 CAM 或 MEKK1 对内源 PKD1 启动子的影响。我们将使用各种生化、细胞和分子生物学方法。这些研究不仅将提高我们对 PKD1 基因表达的理解，而且还可能提出 MEKK1 通路的新靶点和/或机制。因此，所寻求的答案应该具有根本意义，并且应该提供导致治疗方法的信息。此外，这项由 AREA 资助的研究将为西北密苏里州立大学和密苏里科学、数学和计算学院的学生提供宝贵的研究经验。