MEKK1 down-regulates PKD1 promoter by a novel mechanism

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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.