TERMINAL DIFFERENTIATION OF THE RENAL EPITHELIUM

描述（申请人提供）：终末分化是细胞周期退出与生理功能表达相协调的重要发育过程。了解控制未成熟和增殖上皮细胞向成熟分化细胞类型转化的机制对于我们理解肾脏发育以及损伤后修复和再生机制非常重要。肿瘤抑制蛋白p53由于具有细胞周期调控和转录调控的双重作用，在终末分化调控因子中占有独特的地位。本研究要验证的总体假设是，p53在哺乳动物肾脏发育过程中，在末端上皮细胞分化的转录控制中起关键作用。第一个目的将检验p53刺激肾上皮生化分化的假设；将使用凝胶转移和瞬时转染检测P53与肾功能基因启动子和其他终端分化基因的体外结合和激活。染色质免疫沉淀法将检测p53与目标启动子的结合是否在体内终末分化过程中被选择性激活。免疫组织化学研究将检查内源性p53和靶末端分化基因的细胞共定位。第二个目标将检查p53在肾脏形态和功能分化中的作用。在不同遗传背景下培育的p53缺失小鼠将被评估终端分化标志物的时空异常表达、上皮细胞增殖不受控制、小管发育不良和肾功能受损的迹象。将使用发育阶段特异性标记物和免疫组织化学/原位杂交技术确定导致肾脏发育不良的形态发生事件。为了进一步证实p53在肾细胞分化中的作用，我们将在肾特异性启动子的控制下，通过转基因过表达显性阴性突变体p53来确定p53的无肾表型是否可以再现。此外，p53缺失小鼠将被诱导肾缺血再灌注损伤，并检测肾上皮的修复和再生能力。第三个目标将阐明p53在终分化命运规范中的作用。在选定的末端分化基因启动子中含有野生型或突变型p53结合位点的报告基因转基因小鼠将在体内进行适当的时空规范转基因表达测试。这一结果将促进我们对上皮细胞分化的转录程序的理解，并阐明p53在调节肾脏上皮细胞分化中的新功能。

DESCRIPTION (provided by applicant): Terminal differentiation is a crucial developmental process in which cell cycle exit is coordinated with the expression of physiological functions. Understanding mechanisms that control the transformation of immature and proliferating epithelial cells into mature differentiating cell types is important for our understanding of renal development as well as mechanisms of repair and regeneration after injury. The tumor suppressor protein, p53, occupies a unique position among regulators of terminal differentiation due to its dual roles in cell cycle control and transcriptional regulation. The overall hypothesis to be tested in this proposal is that p53 plays a key role in the transcriptional control of terminal epithelial cell differentiation in the developing mammalian kidney. The First Aim will test the hypothesis that p53 stimulates the biochemical differentiation of the renal epithelium; p53 will be examined for in vitro binding to and activation of promoters of renal function genes and other terminal differentiation genes using gel shift and transient transfection assays. Chromatin immunoprecipitation assays will test if p53 binding to target promoters is selectively activated during the process of terminal differentiation in vivo. Immunohistochemical studies will examine the cellular co-localization of endogenous p53 and target terminal differentiation genes. The Second Aim will examine the role of p53 in the morphological and functional differentiation of the kidney. Developing p53 null mice bred on various genetic backgrounds will be assessed for signs of aberrant spatiotemporal expression of terminal differentiation markers, uncontrolled epithelial cell proliferation, tubular dysgenesis, and impaired renal function. The morphogenetic events leading to the renal dysgenesis will be identified using developmental stage-specific markers and immunohistochemistry/in situ hybridization techniques. To further confirm the role of p53 in renal cell differentiation, we will determine whether the p53 null renal phenotype can be recapitulated by transgenic over expression of dominant negative mutant p53 under the control of kidney-specific promoters. In addition, p53 null mice will be stressed by induction of renal ischemia/reperfusion injury and the capacity of the renal epithelium to undergo repair and regeneration will be examined. The Third Aim will elucidate the role of p53 in specification of terminal differentiation fate. Reporter transgenic mice harboring wild-type or mutant p53-binding sites in the promoter of a selected terminal differentiation gene will be tested for appropriate spatio-temporal specification of transgene expression in vivo. The results should advance our understanding of the transcriptional program of epithelial cell differentiation and elucidate a novel function for p53 in regulating epithelial cell differentiation in the kidney.