CUX-1 AND CELL CYCLE REGULATION IN PKD

该提案的总体目标是确定同源异型盒基因Cux-1在多囊肾疾病细胞周期调控中的作用。Cux-1是果蝇基因Cut的鼠同源物，其是马氏管、昆虫排泄和排泄调节器官的正常发育所必需的。哺乳动物切割同源物在多个细胞谱系中作为指定终末分化的基因的转录抑制物起作用。Cux-1抑制细胞周期蛋白激酶抑制剂（CKI）p21在S期的表达，并且是控制G1-S转换的网络的一部分。Cux-1还抑制CKI p27，并且Cux-1在转基因小鼠中的异位表达导致来自p27 kip 1表达的异常下调的多器官增生。我们最近的研究表明，Cux-1异位表达在Pkd 1无效的肾脏，无论是在囊性和正常肾小管上皮细胞。此外，p27下调， PKD 1无效肾。Cux-1在细胞周期期间通过组织蛋白酶L的核同种型进行蛋白水解加工，将Cux-1从抑制p27的全长蛋白转化为具有独特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ADPKD细胞的核提取物中减少，这与全长Cux-1蛋白水平的增加有关。此外，携带Cux-1中的一个组织蛋白酶L位点缺失的cpk小鼠（称为Cux-1DCR 1）表现出比单独的cpk小鼠的囊性肾显著更大的囊性肾。拟议的研究将测试这样的假设，即在多囊肾疾病中观察到的增殖缺陷需要Cux-1的失调，并且Cux-1表达和/或功能的变化改变疾病的严重程度。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特异性Pkd 1无效小鼠模型中，以确定Cux-1是否需要发展囊肿。我们将分析从这些小鼠中分离的细胞，以确定Cux-1在PKD中调节细胞周期的功能作用。最后，我们将分析细胞周期调节的Cux-1蛋白水解加工，以确定是否减少加工的Cux-1在PKD的细胞增殖失调。这些研究将为多囊肾疾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.