Roundabout4 signaling in endothelial cells

摘要 在过去的四十年里，内衬血管壁的内皮细胞已成为临床研究的热门课题。 因为易于获得允许有效的药物靶向。血管生长失调是 与广泛的疾病状态相关，例如糖尿病视网膜病变、湿性年龄相关性黄斑变性， 退化和肿瘤生长。肿瘤生长依赖于血管生成。肿瘤的第一步 血管生成是内皮细胞活化和内皮细胞向肿瘤的定向迁移。 因此，了解定向迁移的机制对于开发治疗药物至关重要 它可以选择性地靶向肿瘤内皮细胞。这个实验室的目标是 鉴定和研究在肿瘤脉管系统中选择性上调的候选基因。Robo 4是一个优秀的 因为它选择性地在肿瘤血管中表达，并且对于内皮细胞的定向迁移至关重要。 细胞此外，Robo 4的细胞表面表达允许容易地获得潜在的药物。重要的是，具体 Robo 4上的信号复合体组装，如果被识别为负责定向迁移， 针对性地以前，我们已经表明，robo 4是必不可少的血管生成在体内斑马鱼和激活 体外内皮细胞中的Rho GTP酶。初步结果表明：（A）Robo 1和Robo 4相互作用， 在细胞表面和细胞内水平共享共同的信号分子;（B）蛋白质复合物 包括IRSp 53、Mena、Cdc 42-GTP和Vilse在内皮细胞中响应Robo 4一起组装; (C)Sry相关的高迁移率族（Sox）家族成员与Robo 4的胞质尾相互作用。我们 假设：“内皮细胞表面受体Robo 1和Robo 4与Slit 2相互作用， 通过细胞内信号蛋白（Cdc 42-GTP，Vilse， IRSp53，Mena，Sox）。“为了在目标1中验证这一假设，我们将调查Robo 1和Robo 4是否共同合作。 可操作地与Slit 2相互作用并指导Vilse和Mena在内皮细胞中的信号复合物组装 使用分子、生物化学和成像方法的组合。在目标2中，我们将确定 IRSp 53、Mena、Cdc 42-GTP、Vilse和Sox与Robo 1和Robo 4的胞质尾在一个细胞中的组装 负责指导内皮细胞迁移的信号传导复合物， 斑马鱼的生物化学和体内功能测定。我们预计，通过实现目标1和2， 将了解Robo 1和Robo 4如何通过以下方式支配内皮尖端细胞在复杂环境中导航： 揭示了两种分子在触发内皮细胞信号传导方面的相似性， 不同的信号分子在Robo 1和Robo 4的细胞质尾部组装， 响应，并整合不同的信号成分，形成丝状伪足。每一个机械步骤都是 假定的靶点和靶向这些步骤的药物将有益于以过度血管生成为特征的疾病 和血管渗漏

ABSTRACT Over the past four decades, endothelial cells lining the vessel wall have become an intense subject of clinical interest since ease of accessibility allows for effective drug targeting. Dysregulation of vessel growth is associated with wide range of disease states such as diabetic retinopathy, wet form of age related macular degeneration, and tumor growth. Tumor growth is angiogenesis dependent. The first step in tumor angiogenesis is endothelial cell activation and directional migration of endothelial cells towards the tumor. Therefore, understanding the mechanisms of directional migration is critical for development of therapeutics that can selectively target tumor endothelial cells from taking this first step. The goal of this laboratory is to identify and study candidate genes that are selectively upregulated in tumor vasculature. Robo4 is an excellent target since it is expressed selectively in tumor vessels and is critical for directional migration of endothelial cells. Further, the cell surface expression of Robo4 allows easy access to potential drugs. Importantly, specific signaling complex assembly on Robo4, if identified to be responsible for directional migration can also be targeted. Previously, we have shown that robo4 is essential for angiogenesis in vivo in zebrafish and activates Rho GTPases in endothelial cells in vitro. Preliminary results suggest that: (A) Robo1 and Robo4 interact and share common signaling molecules at both the cell surface and intracellular level; (B) A complex of proteins including IRSp53, Mena, Cdc42-GTP, and Vilse together assemble in endothelial cells in response to Robo4; (C) Members of the Sry-related high-mobility-group (Sox) family interact with Robo4's cytoplasmic tail. We hypothesize that: "Endothelial cell surface receptors Robo1 and Robo4 co-operatively interact with Slit2 to direct cell migration via a specific complex assembly of intracellular signaling proteins (Cdc42-GTP, Vilse, IRSp53, Mena, Sox)." To test this hypothesis in aim 1, we will investigate whether Robo1 and Robo4 co- operatively interact with Slit2 and direct signaling complex assembly of Vilse and Mena in endothelial cells using a combination of molecular, biochemical and imaging methods. In aim 2, we will identify the mechanism of assembly of IRSp53, Mena, Cdc42-GTP, Vilse and Sox with Robo1 and Robo4's cytoplasmic tail in a signaling complex responsible for directing endothelial cell migration using a combination of in vitro biochemical and in vivo functional assays in zebrafish. We anticipate that by accomplishing aims 1 and 2, we will understand how Robo1 and Robo4 dictate the endothelial tip cell to navigate through complex milieu by unraveling the similarities between the two molecules in triggering signaling in endothelial cells, the order in which the different signaling molecules assemble on Robo1 and Robo4's cytoplasmic tail, the cues they respond to, and the integration of different signaling components to form filopodia. Each mechanistic step is a putative target and drugs targeting these steps will benefit diseases characterized by excessive angiogenesis and vascular leakage.