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: 7030206
• 负责人: M. Rafiq Islam
• 金额: $ 6.82万
• 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/7030206
• 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（由申请方提供）：人多囊肾病-1（PKD 1）基因突变导致绝大多数（85- 90%）常染色体显性多囊肾病（ADPKD）病例。关于ADPKD是由PKD 1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结果。我们的主要目标是了解各种因素如何调节PKD 1基因的转录。我们最近发现该启动子是b-连环蛋白/TCF途径的靶点（1）。在初步研究中，虽然3.3 kb的启动子区域包含一个远端AP-1位点，在凝胶迁移试验中结合蛋白质，它不是由佛波酯或其下游靶点，组成型活性MEKK 1（CAM）诱导。相反，启动子片段被两者强烈抑制。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，这表明激活AP-1的MEKK 1的经典MAP激酶途径与下调该启动子的MEKK 1的未知途径之间存在竞争。通过缺失分析发现启动子中的近端200 bp片段响应CAM下调。CAM效应不是通过Ets或Sp1介导的，在这200 bp的启动子中发现。由于没有先前的报道表明MEKK 1在未诱导的细胞中的转录抑制，因此研究MEKK 1的这种新的下调机制及其对内源性PKD 1启动子的作用将是有趣的。在这个提议中，我们提出了实现以下三个具体目标：目标1：确定受到CAM下调的PKD 1启动子中的靶序列，目标2：检查CAM对PKD 1启动子的下调是否由新途径介导，目标3：研究CAM或MEKK 1对内源性PKD 1启动子的影响。我们将使用各种生物化学、细胞和分子生物学方法。这些研究不仅有助于加深对PKD 1基因表达的理解，而且可能为MEKK 1通路提供新的靶点和/或机制。因此，所寻求的答案应具有根本意义，并应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州立大学和密苏里州科学、数学和计算学院的学生提供宝贵的研究经验。