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: 6857697
• 负责人: M. Rafiq Islam
• 金额: $ 9.14万
• 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 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6857697
• 关键词: cadherins; gel mobility shift assay; gene expression; gene mutation; genetic promoter element; genetic regulation; genetic transcription; mitogen activated protein kinase; oligonucleotides; polycystic kidney; polymerase chain reaction; site directed mutagenesis; transcription factor; transfection; western blottings

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(申请人提供)：人类多囊肾病-1(PKD1)基因突变是绝大多数(85-90%)常染色体显性遗传性多囊肾病(ADPKD)病例的原因。关于ADPKD是由PKD1基因的异常降低还是异常升高引起的，有相互矛盾的观察结果。我们的主要目标是了解各种因素是如何调控PKD1基因的转录的。我们最近发现该启动子是b-连环蛋白/TCF途径的靶点(1)。在初步研究中，尽管启动子的3.3kb区域含有凝胶漂移实验中结合蛋白质的远端AP-1位点，但它不被佛波酯或其下游靶标--具有结构性活性的MEKK1(CAM)诱导。相反，启动子片段被两者强烈抑制。然而，当CAM与过量的c-jun、c-fos或两者共同转染细胞时，启动子上的AP-1位点被激活，这表明激活AP-1的经典的MEKK1 MAP-1通路与下调该启动子的未知MEKK1通路之间存在竞争。缺失分析发现，该启动子近端的200bP片段对CAM的下调有反应。CAM效应不是通过启动子这200个碱基上的Ets或Sp1介导的。由于先前没有报道表明MEKK1在未诱导的细胞中具有转录抑制作用，因此研究MEKK1的这种新的下调机制及其对内源性PKD1启动子的影响将是很有意义的。在这个方案中，我们建议完成以下三个特定的目标：目标1：确定受CAM下调的PKD1启动子中的靶序列，目标2：研究CAM下调PKD1启动子的作用是否通过一种新的途径，以及目标3：研究CAM或MEKK1对内源性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。这些研究不仅将加深我们对PKD1基因表达的理解，还可能为MEKK1通路提供一个新的靶点和/或机制。因此，寻求的答案应该既具有基本意义，又应该提供有助于治疗方法的信息。此外，这项由地区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。