Roundabout4 signaling in endothelial cells

描述（由申请人提供）：在过去的四十年里，血管壁上的内皮细胞已经成为临床关注的热点，因为易于获取允许有效的药物靶向。血管生长失调与多种疾病状态有关，如糖尿病视网膜病变、湿型年龄相关性黄斑变性和肿瘤生长。肿瘤的生长依赖于血管生成。肿瘤血管生成的第一步是内皮细胞的激活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能够选择性靶向肿瘤内皮细胞的治疗方法至关重要。本实验室的目的是鉴定和研究在肿瘤脉管系统中选择性上调的候选基因。Robo4是一个很好的靶标，因为它在肿瘤血管中选择性表达，对内皮细胞的定向迁移至关重要。此外，Robo4的细胞表面表达使潜在的药物更容易获得。重要的是，如果确定Robo4上的特定信号复合物组装负责定向迁移，也可以作为目标。在此之前，我们已经证明了robo4对斑马鱼体内血管生成至关重要，并在体外激活内皮细胞中的Rho gtpase。初步结果表明：(A) Robo1和Robo4在细胞表面和细胞内水平上相互作用并共享共同的信号分子；(B) IRSp53、Mena、Cdc42-GTP和Vilse等蛋白复合物在内皮细胞中聚集，响应Robo4；(C) sry相关的高迁移群（Sox）家族成员与Robo4的细胞质尾部相互作用。我们假设：“内皮细胞表面受体Robo1和Robo4与Slit2协同作用，通过细胞内信号蛋白（Cdc42-GTP, Vilse, IRSp53, Mena, Sox）的特定复杂组装指导细胞迁移。”为了在aim 1中验证这一假设，我们将结合分子、生化和成像方法，研究Robo1和Robo4是否与内皮细胞中的Slit2和Vilse和Mena的直接信号复合物组装协同相互作用。在目标2中，我们将在斑马鱼中使用体外生化和体内功能分析相结合的方法，确定IRSp53、Mena、Cdc42-GTP、Vilse和Sox与Robo1和Robo4的细胞质尾部在负责指导内皮细胞迁移的信号复合体中的组装机制。我们预计，通过完成目标1和目标2，我们将通过揭示两种分子在内皮细胞中触发信号的相似性、不同信号分子在Robo1和Robo4的细胞质尾部聚集的顺序、它们响应的线索以及不同信号成分形成丝状伪足的整合，了解Robo1和Robo4如何指示内皮尖端细胞在复杂环境中导航。每个机制步骤都是一个假定的靶点，针对这些步骤的药物将有益于以过度血管生成和血管渗漏为特征的疾病。公共卫生相关性：肿瘤生长依赖于血管生成。我们感兴趣的是确定血管靶点，最好是肿瘤细胞与正常内皮细胞中差异表达的细胞表面分子。该提案研究了一个这样的目标，即Robo4。Robo4在肿瘤血管中高度表达。肿瘤血管渗漏，血管床常呈混沌状。肿瘤血管生成的第一步是内皮细胞的激活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能够选择性靶向肿瘤内皮细胞的治疗方法至关重要。该提案将确定机器人在介导内皮细胞定向迁移中利用的机制。我们提出了两个目标来研究机器人用于调解定向迁移的机制。实现这两个目标将确定Robo1和Robo4如何在内皮细胞内响应Slit2配体机械地组装信号复合物，从而协调内皮细胞的定向迁移。本研究将确定Robo1和Robo4信号装置共同的细胞内分子，这些分子可作为药物设计的靶标，有利于与肿瘤生长相关的内皮细胞迁移失调相关的条件。

DESCRIPTION (provided by applicant): 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 zebra fish 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 assembles 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 zebra fish. 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. PUBLIC HEALTH RELEVANCE: Tumor growth is angiogenesis dependent. We are interested in identifying vascular targets preferably cell surface molecules that are differentially expressed in tumor versus normal endothelial cells. This proposal studies one such target namely Robo4. Robo4 is highly expressed in tumor vessels. Tumor vessels are leaky, and the vascular bed often has a chaotic appearance. 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. This proposal will identify the mechanisms utilized by Robos in mediating directional migration in endothelial cells. We propose two aims to investigate mechanisms used by Robos to mediate directional migration. Accomplishing both aims will determine how Robo1 and Robo4 mechanistically assemble a signaling complex inside endothelial cells in response to Slit2 ligand, thereby coordinating directional migration of endothelial cells. This study will identify intracellular molecules common to the Robo1 and Robo4 signaling apparatus that serve as targets for drug design benefiting conditions associated with deregulated endothelial cell migration such as those associated with tumor growth.