Cux1 and cell cycle regulation in kidney development and disease

该提案的总体目标是确定Cux1调节表达的机制， 细胞周期蛋白激酶抑制剂p27在肾脏发育和多囊肾病中的作用Cux1在大肠杆菌中高表达， 在正常肾脏发育过程中，肾细胞在生肾区表达，但当肾单位经历 终末分化Cux1在人ADPKD细胞和小鼠ADPKD模型中异位表达。 多囊肾病，包括Pkd 1 null和cpk小鼠。异位表达Cux 1的转基因小鼠 发生由p27的异常抑制引起的肾增生。然而，对Cux 1的研究 转基因小鼠证明了Cux1单独的异位表达不足以引起囊肿的发展。 相反，Cux1表达水平的增加似乎是囊肿进展所需的。我们的初步结果 表明降低多囊肾病中Cux1水平导致p27表达增加， 细胞周期停滞和减少囊肿生长。基于这些结果，我们假设， p27通过Cux1导致在PKD中观察到的增殖缺陷。因此，在具体目标1中，我们将描述 从携带Pkd 1和Cux 1靶向缺失的小鼠分离的集合管细胞中的细胞增殖， 并确定是否需要Cux1在p27启动子上形成阻遏物复合物。我们的初步 结果表明，Cux1与Grg 4相互作用，募集组蛋白脱乙酰酶HDAC 1和HDAC 3， 调节p27表达。因此，在具体目标2中，我们将定义Cux 1、Grg 4和 p27启动子，并使用染色质构象捕获分析来表征形成的阻遏复合物 p27启动子最后，用HDAC抑制剂处理Pkd 1缺失胚胎减缓了囊肿进展。 因此，在具体目标3中，我们将确定HDAC抑制是否导致p27基因表达增加， 以及PKD中Cux1对p27的调节是否依赖于HDAC活性。这些研究将提供新的 了解肾脏发育和多囊肾疾病过程中细胞增殖的机制。

The overall aim of this proposal is to determine the mechanism by which Cux1 regulates the expression of the cyclin kinase inhibitor p27 in kidney development and polycystic kidney disease. Cux1 is highly expressed in the nephrogenic zone during normal kidney development, but is sharply downregulated when nephrons undergo terminal differentiation. Cux1 is ectopically expressed in human ADPKD cells, and in mouse models of polycystic kidney disease, including Pkd1 null and cpk mice. Transgenic mice that ectopically express Cux1 develop renal hyperplasia resulting from the aberrant repression of p27. However, studies on the Cux1 transgenic mice demonstrate that ectopic expression of Cux1 alone is insufficient to cause cyst development. Rather, increased levels of Cux1 expression appear to be required for cyst progression. Our preliminary results suggest that reducing Cux1 levels in polycystic kidney disease results in increased expression of p27, leading to cell cycle arrest and decreased cyst growth. Based on these results, we hypothesize that the downregulation of p27 by Cux1 results in the proliferation defects observed in PKD. Thus, in specific aim 1, we will characterize cell proliferation in collecting duct cells isolated from mice carrying targeted deletions of both Pkd1 and Cux1, and determine whether Cux1 is required to form a repressor complex on the p27 promoter. Our preliminary results suggest that Cux1 interacts with Grg4 to recruit the histone deacetylases HDAC1 and HDAC3 to regulate p27 expression. Thus, in specific aim 2, we will define the interaction between Cux1, Grg4, and the p27 promoter, and use the chromatin conformation capture assay to characterize the repression complex formed on the p27 promoter. Finally, treatment of Pkd1 null embryos with HDAC inhibitors slows cyst progression. Thus, in specific aim 3, we will determine whether HDAC inhibition results in increased p27 gene expression, and whether Cux1 regulation of p27 in PKD is dependent on HDAC activity. These studies will provide novel insights into the mechanisms of cell proliferation during kidney development and polycystic kidney disease.