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关键发育过程。了解控制未成熟和增殖的上皮细胞转化为成熟分化细胞类型的机制，对于我们理解肾脏的发育以及损伤后的修复和再生机制具有重要意义。肿瘤抑制蛋白P53因其在细胞周期调控和转录调控中的双重作用，在终末分化调控中占有独特的地位。在这项建议中要检验的总体假设是，P53在哺乳动物肾脏发育中对末端上皮细胞分化的转录控制中发挥关键作用。第一个目的是验证P53刺激肾上皮细胞生化分化的假设；通过凝胶移位和瞬时转染实验，在体外检测P53与肾功能基因启动子和其他末端分化基因的结合和激活。染色质免疫沉淀分析将检测在体内终末分化过程中，是否选择性地激活了与靶启动子结合的P53。免疫组织化学研究将检测内源性P53和靶末端分化基因的细胞共定位。第二个目的是研究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发育中的p53基因缺失小鼠将被评估末端分化标志物的时空异常表达、不受控制的上皮细胞增殖、肾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/原位杂交技术识别导致肾发育不全的形态发生事件。为了进一步证实P53在肾细胞分化中的作用，我们将确定在肾脏特异性启动子的控制下，是否可以通过转基因过表达显性负性突变体P53来重现P53缺失的肾脏表型。此外，还将通过诱导肾缺血/再灌注损伤来对p53基因缺失的小鼠施加压力，并检测肾上皮细胞的修复和再生能力。第三个目的是阐明P53在终末分化命运中的作用。在选定的末端分化基因的启动子中含有野生型或突变型P53结合位点的报告转基因小鼠将在体内测试适当的转基因表达的时空规范。这些结果将促进我们对上皮细胞分化转录程序的理解，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.