Comprehensive Analysis of Polycystin Related Endothelial Cell Signaling Pathways

描述(申请人提供)：常染色体显性遗传性多囊肾病(ADPKD)是最常见的遗传性肾功能衰竭，由两个基因突变引起，即PKD1和PKD2。根据动脉瘤与ADPKD的相关性，推测多囊蛋白(PKD蛋白)在维持血管完整性方面起着重要作用。这种表型与内皮细胞缺陷有关，因为PKD缺失的动物表现为水肿和出血。尽管这个问题与此相关，但关于多囊藻毒素在血管内皮细胞中所起的作用知之甚少。我们最近使用Cre-Lox技术在小鼠体内诱导内皮细胞特异性敲除PKD1或PKD2。我们发现，内皮细胞中任一基因的选择性失活都会产生所描述的血管缺陷的一个子集 在PKD缺陷型胚胎中，围产儿死亡率约30-40%，偶见出血、羊水过多和胎盘异常。值得注意的是，这些胚胎缺乏水肿，这是所有目标PKD零等位基因的普遍特征。这些研究证实了多囊蛋白在内皮细胞室中的功能作用，并促使我们假设功能多囊蛋白的缺失会导致内皮细胞相关信号通路的失调。在这一应用中，我们提出了三个具体的目标，试图从机制上理解多囊蛋白在细胞和整个动物水平上在内皮细胞中所起的功能作用。在Aim1中，我们将使用PKD零等位基因和条件等位基因来探索PKD1/PKD2突变动物的水肿形成的基础。目的2是基于我们观察到的多囊蛋白耗竭的内皮细胞表现出缺陷的细胞迁移。我们将深入开展 使用延时成像和生化方法对这种表型进行表征。此外，我们将使用新的细胞生物传感器来探测与细胞迁移有关的明确定义的信号通路。在目标3中，我们将使用从不同妊娠时间点的胚胎分离的内皮细胞的综合微阵列研究来推断在体内PKD突变的内皮细胞中被破坏的调节网络。在这些研究的结论中，我们将获得关于多囊蛋白如何适应血管发育的新见解。人们希望这些基本范式将对定义多囊蛋白功能产生更广泛的影响，尽管进行了多年的密集研究，但对这个问题仍然知之甚少。

DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of inherited renal failure and is caused by mutations two genes, PKD1 and PKD2. Based on the association of aneurysms and ADPKD it has been speculated that polycystins (PKD proteins) play an important role in maintaining blood vessel integrity. An endothelial cell defect has been implicated in this phenotype since Pkd null animals exhibit edema and hemorrhage. Despite the relevance of this problem, there is little known about the role that polycystins play in endothelial cells. We recently used Cre-Lox technology to induce an endothelial cell specific knock out of Pkd1 or Pkd2 in mice. We found that selective inactivation of either gene in endothelial cells yielded a subset of the vascular defects described in Pkd null embryos including ~30-40% perinatal lethality, occasional hemorrhage, polyhydramnios and placental abnormalities. Remarkably, these embryos lacked edema, which is a universal feature of all targeted Pkd null alleles. These studies demonstrated a functional role for polycystins in the endothelial cell compartment and prompted us to hypothesize that loss of functional polycystins results in dysregulation of endothelial cell related signaling pathways. n this application, we propose three specific aims that seek to develop a mechanistic understanding of the functional role that polycystins play in endothelial cells at both the cellula and whole animal level. In Aim1 we will use Pkd null and conditional alleles to explore the basis of edema formation in Pkd1/Pkd2 mutant animals. Aim 2 is based on our observation that polycystin depleted endothelial cells exhibit defective cell migration. We will conduct an in depth characterization of this phenotype using time-lapse imaging and biochemical methods. In addition we will use novel cellular biosensors to probe well-defined signaling pathways implicated in cell migration. In Aim 3 we will employ comprehensive microarray studies of endothelial cells isolated from embryos at different gestational time points to infer the regulator networks that are disrupted in Pkd mutant endothelial cells in vivo. At the conclusion of these studies, we will have gained novel insights into how polycystins fit into the context of vascular development. The hope is that these fundamental paradigms will have broader implications for defining polycystin function, a subject that remains vaguely understood despite many years of intense investigation.