CUX-1 AND CELL CYCLE REGULATION IN PKD

这项建议的总体目标是确定同源异型盒基因Cux-1在多囊肾病细胞周期调节中的作用。Cux-1是果蝇基因Cut的小鼠同源物，它是昆虫排泄物和渗透压调节器官马氏管正常发育所必需的。哺乳动物CUT同源基因在多种细胞系中作为末端分化基因的转录抑制因子发挥作用。CuX-1抑制细胞周期蛋白激酶抑制物(CKI)p21在S期的表达，是控制G1-S转换网络的一部分。Cux-1还抑制CKI p27的表达，在转基因小鼠中异位表达Cux-1通过异常下调p27kip1的表达而导致多器官增生。我们最近的研究表明，Cux-1在PKD1缺失的肾脏中异位表达，无论是在囊性肾小管上皮细胞还是在正常肾小管上皮细胞中。此外，p27在 PKD1缺失型肾脏。在细胞周期中，Cux-1被组织蛋白L的核亚型降解，将Cux-1从抑制p27的全长蛋白转化为具有明显DNA结合活性的截短蛋白。最近的研究表明，与正常的人肾细胞相比，组织蛋白酶L在人多囊肾细胞的核提取液中减少，这与全长Cux-1蛋白的水平增加有关。此外，携带Cux-1DCR1组织蛋白L位点缺失的CPK小鼠的囊性肾脏明显大于CPK小鼠单独的囊性肾脏。拟议的研究将检验这样的假设，即多囊肾病中观察到的增殖性缺陷需要放松对Cux-1的调控，以及Cux-1表达和/或功能的变化改变了疾病的严重程度。我们将使用遗传学方法将功能丧失的Cux-1突变引入多囊肾病的肾脏特异性PKD1缺失的小鼠模型中，以确定是否需要Cux-1来发展囊性病变。我们将分析从这些小鼠分离的细胞，以确定Cux-1在调节PKD细胞周期中的功能作用。最后，我们将分析细胞周期调节的Cux-1的蛋白分解过程，以确定在PKD中减少的Cux-1的处理是否有助于解除调控的细胞增殖。这些研究将为多囊肾病的细胞增殖机制提供新的见解。

The overall aim of this proposal is to determine the role of the homeobox gene Cux-1 in cell cycle regulation in polycystic kidney disease. Cux-1 is the murine homologue of the Drosophila gene Cut, which is required for the proper development of the Malpighian tubules, the insect excretory and osmoregulatory organs. Mammalian Cut homologues function as transcriptional repressers of genes specifying terminal differentiation in multiple cell lineages. Cux-1 represses the expression of the cyclin kinase inhibitor (CKI) p21 in S phase and is part of the network controlling G1-S transition. Cux-1 also represses the CKI p27, and ectopic expression of Cux-1 in transgenic mice results in multiorgan hyperplasia from the aberrant down regulation of p27kip1 expression. Our recent studies demonstrate that Cux-1 is ectopically expressed in Pkd1 null kidneys, both in cystic and in normal tubule epithelial cells. Moreover, p27 is down regulated in Pkd1 null kidneys. Cux-1 is proteolytically processed during the cell cycle by a nuclear isoform of Cathepsin L, converting Cux-1 from a full length protein that represses p27, to a truncated protein with a distinct DNA binding activity. Recent studies show that Cathepsin L is reduced in nuclear extracts of human ADPKD cells, compared to normal human kidney cells, and this is associated with increased levels of the full length Cux-1 protein. Moreover, cpk mice bearing a deletion of one Cathepsin L site in Cux-1, called Cux-1 DCR1, exhibit cystic kidneys significantly larger than cystic kidneys of cpk mice alone. The proposed studies will test the hypotheses that deregulation of Cux-1 is required for the proliferative defects observed in polycystic kidney disease and that changes in Cux-1 expression and/or function modify the severity of the disease. We will use a genetic approach to introduce a loss-of-function Cux-1 mutation into kidney specific Pkd1 null murine models of polycystic kidney disease to determine whether Cux-1 is required to develop cysts. We will analyze cells isolated from these mice to determine the functional role of Cux-1 in regulating the cell cycle in PKD. Finally, we will analyze cell cycle regulated proteolytic processing of Cux-1 to determine whether reduced processing of Cux-1 in PKD contributes to deregulated cell proliferation. These studies will provide novel insights into the mechanisms of cell proliferation in polycystic kidney disease.