Hippo-YAP in podocyte health and disease

项目概要 对临床相关信号通路的了解有限，限制了治疗药物的发展 人类肾小球疾病的药物。我们的长期目标是加强假定的治疗药物的研发管线 通过阐明细节和功能意义，可用于解决人类肾小球疾病的目标 调节足细胞损伤和存活的关键信号通路。我们的初步数据已经确定了 YAP， Hippo 信号通路的关键效应器，作为足细胞存活的重要调节因子。亚普 足细胞失活导致小鼠 FSGS，YAP 表达减少与 人类肾小球疾病的发生和进展。我们检测到细胞内钙增加 YAP 沉默足细胞中钙门控钾通道表达的摄取和显着上调。 本申请的总体目标是确定 YAP 调节足细胞存活的机制 并测试其作为潜在治疗靶点的作用。我们的中心假设是 YAP 在足细胞中失活 在 Hippo 激酶 LATS 的影响下通过经典磷酸化和细胞质隔离。 YAP 的表达和功能也可以在遗传和转录水平上进行调节。 YAP 减少 信号传导增强足细胞中嘌呤能受体介导的钙摄取和钙门控钾 通道激活导致肌动蛋白细胞骨架的破坏。拟议研究的理由 是定义调节 YAP 功能的基本机制将促进对 肾小球疾病进展以及寻找可供临床使用的新治疗靶点。我们的 假设将通过追求两个具体目标来检验：目标 1 将探索 YAP 的功能意义 足细胞存活中的磷酸化和核质穿梭。我们将确定细胞质是否 足细胞中 YAP 表达增强损伤易感性，增强核 YAP 信号传导具有保护作用 在蛋白尿肾病中。我们还将开发一种新型 YAP 激动剂并测试其保护作用 足细胞免受损伤。在目标 2 中，我们将确定关键的信号传导途径和细胞结构变化 由 YAP 失活诱导。我们的创新方法采用最先进的微加工 3D 芯片， 电生理学和原子力显微镜量化 YAP 期间足细胞的生物物理特性 正常和疾病条件下的抑制和激活。通过同源建模，我们将生成新颖的 小分子 YAP 激动剂可能对蛋白尿肾病具有保护作用。这些贡献是 意义重大，因为它们不仅有可能促进对疾病发病机制的理解 肾小球疾病，但可以帮助确定新的治疗靶点。

Project Summary A limited understanding of clinically relevant signaling pathways has limited the development of therapeutic agents for human glomerular disease. Our long-term goal is to enhance the pipeline of putative therapeutic targets available to tackle human glomerular disease by elucidating the details and functional significance of key signaling pathways that regulate podocyte injury and survival. Our preliminary data have identified YAP, the key effector of the Hippo signaling pathway, as an important regulator of podocyte survival. YAP inactivation in podocytes causes FSGS in mice and decreased YAP expression is associated with the development and progression of human glomerular disease. We have detected increased intracellular calcium uptake and marked upregulation of calcium-gated potassium channel expression in YAP silenced podocytes. The overall objective of this application is to define the mechanism by which YAP regulates podocyte survival and test its role as a potential therapeutic target. Our central hypothesis is that YAP is inactivated in podocytes by canonical phosphorylation and cytoplasmic sequestration under the influence of the Hippo kinase LATS. YAP expression and function can also be regulated at the genetic and transcriptional level. Decreased YAP signaling enhances purinergic receptor-mediated calcium uptake in podocytes and calcium-gated potassium channel activation contributing to disruption of the actin cytoskeleton. The rationale for the proposed research is that defining the underlying mechanisms that regulate YAP function will advance understanding of glomerular disease progression as well as the quest for novel therapeutic targets available for clinical use. Our hypothesis will be tested by pursuing two specific aims: Aim 1 will explore the functional significance of YAP phosphorylation and nuclear-cytoplasmic shuttling in podocyte survival. We will determine whether cytoplasmic YAP expression in podocytes enhances injury susceptibility and enhancing nuclear YAP signaling is protective in proteinuric kidney disease. We will also develop a novel YAP agonist and test its role in protecting podocytes from injury. In Aim 2 we will determine the key signaling pathways and cellular structural changes induced by YAP inactivation. Our innovative approach utilizes state of the art microfabricated 3-D chips, electrophysiology and atomic force microscopy to quantify the biophysical properties of podocytes during YAP inhibition and activation under normal and disease conditions. By homology modeling, we will generate novel small molecule YAP agonists that could be protective in proteinuric kidney disease. These contributions are significant because they have the potential to not only advance understanding of the pathogenesis of glomerular disease but could help identify novel therapeutic targets.