MEKK1 down-regulates PKD1 promoter by a novel mechanism

描述（由申请人提供）：人类多囊肾病-1 （PKD1）基因突变是绝大多数（85- 90%）常染色体显性多囊肾病（ADPKD）病例的原因。关于ADPKD是由PKD1基因表达异常减少还是异常增加引起的，目前有相互矛盾的观察结果。我们的主要目标是了解各种因子如何调节PKD1基因的转录。我们最近发现该启动子是b-catenin/TCF通路的靶标(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，在凝胶移位试验中结合蛋白质，但它不是由phorbol酯或其下游靶标组成活性MEKK1 （CAM）诱导的。相反，启动子片段被两者强烈抑制。然而，当细胞同时转染CAM和过量的c-jun、c-fos或两者时，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）和MEKK1的未知途径（下调该启动子）之间存在竞争。通过缺失分析发现，启动子中近200 bp的片段对CAM下调有反应。CAM效应不是通过在这200 bp的启动子中发现的Ets或Sp1介导的。由于之前没有报道表明MEKK1在非诱导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启动子的影响将是有趣的。在本提案中，我们提出完成以下三个具体目标：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启动子是否通过新的途径介导；目标3：研究CAM或MEKK1对内源性PKD1启动子的影响。我们将使用各种生物化学，细胞和分子生物学方法。这些研究不仅将提高我们对PKD1基因表达的认识，而且可能为MEKK1通路提供新的靶点和/或机制。因此，所寻求的答案既应具有根本意义，又应提供导致治疗方法的信息。此外，该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。

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.