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.
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