SIGNALS FOR RENAL ENDOTHELIAL CAPILLARY MORPHOGENESIS

肾内皮毛细血管形态发生的信号

• 批准号: 2628901
• 负责人: TOM DANIEL
• 金额: $ 18.93万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2628901
• 关键词: angiogenesis; biological signal transduction; capillary; cell cell interaction; cell line; cell migration; chimeric proteins; cytoplasm; gene expression; human tissue; integrins; tissue /cell culture; vascular endothelium; yeast two hybrid system

DESCRIPTION (Adapted from Investigator's Abstract): Vasculogenesis and angiogenic remodeling are basic requirements for organogenesis and wound healing. For vasculogenesis to occur, the endothelial cell progenitors must assemble properly. The current proposal will further define the molecular processes that direct renal microvascular endothelial targeting and assembly. The proposal hypothesizes that renal and other microvascular endothelial cells discriminate appropriate partners for vascular structure assembly based on signals they receive upon juxtacrine contact during directed migration. In this regard, previous studies by this investigator have shown that the ELK subclass of Eph family of receptors are critical in cultured human renal microvascular endothelial cells (HRMEC) attachment and assembly behavior. Further, the investigator has shown that the signals of the ELK receptor depend upon juxtacrine presentation of their ligands in variably oligomerized states on the surfaces of the contacting cells. Three aims will be addressed. The experimental approach will involve traditional biochemical affinity studies as well as yeast-two hybrid system and use of dominant negative protein interactions. The first specific aim, to define the molecular basis for LERK-2 oligomerization, will be accomplished using two complementary approaches. The first will employ a yeast two-hybrid screen utilizing the cytoplasmic domain of LERK-2 as bait to screen a murine embryonic library. This study anticipates finding proteins that interact with the cytoplasmic domain of LERK-2. The complementary approach will involve standard cell culture labeling experiments to provide information about the cellular distribution about LERK-2 in stimulated and unstimulated cells. Subsequently, other experiments will utilize a LERK-2 cytoplasmic domain created as a GST fusion protein to allow binding to GSH-beads. This fusion protein will then be used to capture potential interacting proteins from the cytosol of stimulated cells. Finally, a series of chemical cross-linking studies and gel filtration electrophoresis experiments will be employed to investigate the state of oligomerization of LERK-2 following stimulation of endothelial cells. The second specific aim will focus on ELK signaling and, specifically, will investigate the molecular basis for the interaction between ELK and LMP-PTP, which was demonstrated by previous studies in this investigator's laboratory. In this regard, this laboratory has accumulated a wide range of experimental tools to address this interaction and will use the yeast two-hybrid system and baculovirus expression system to address these issues in addition to transient transfection assays and a dominant negative approach to examine these interactions. The third specific aim is a descriptive study that will determine the type of integrin present on cell lines used in this laboratory, using standard FACS analysis. In addition, transfected cell lines will be established to examine the interaction among integrins and signaling by ELK-LERK-2 interactions. This is a proposal which seeks a further 5 years of funding for the study of capillary morphogenesis by renal microvascular endothelial cells. The project is now in its fourth year, and has provided novel insights into cell-cell recognition events. In brief, it was learned that the ELK-LERK-2 ligand-receptor pair is present in renal endothelial cells during development, and that LERK-2, when presented as tetramers to ELK can stimulate precapillary cord formation in vitro. In contrast, dimeric LERK-2 only stimulates cell adhesion and migration in a matrix-specific fashion. Proteins that associate with the cytoplasmic tail of activated ELK have been identified, and while Grb-2 and Grb-10 associate with ELK stimulated by dimeric LERK-2, a tyrosine phosphatase LMW-PTP only associates with the receptor when it binds clustered, tetrameric LERK-2. It is therefore proposed that ELK and LERK-2 are counter-receptors on renal microvascular endothelial cells which cluster during cell-cell contact, and that both, ELK and LERK-2 serve to signal intracellular events which stimulate capillary cord formation. The approaches proposed for next term of the project are to use the yeast two hybrid system extensively, and to study in vitro interactions with GST fusions of ELK and LERK-2 (and mutants thereof) with the aim of identifying proteins that interact with the cytoplasmic tail of LERK-2 (aim 1) and ELK (aim 2). For ELK, this approach has been partially successful and aim 2 therefore seeks to further define signaling events that stimulate cell assembly by studying specific mutants of ELK and its partners, their mutual interactions, and their potential effects on cell assembly. Since dimeric LERK-2 stimulates cell adhesion and migration as well as ELK tyrosine phosphorylation, and since Grb-2, Grb-4, and Grb-10 have been found to associate with ELK upon stimulation with dimeric and tetrameric LERK-2, aim three is designed to assess the influence of LERK-2-ELK on integrin function, the involvement of small G-proteins in stimulating adhesion, cytoskeletal reorganization and migration, and activation of the JNK/SAP kinase pathway.

描述（改编自研究者摘要）：血管生成和 血管生成重塑是器官发生和伤口的基本要求 康复。 为了发生血管生成，内皮细胞祖细胞必须 正确组装。 目前的提案将进一步定义分子 指导肾微血管内皮靶向的过程 集会。 该提案假设肾脏和其他微血管 内皮细胞区分血管结构的适当伙伴 根据它们在近分泌接触时收到的信号进行组装 定向迁移。 对此，本研究者之前的研究 已经表明 Eph 受体家族的 ELK 亚类在 培养的人肾微血管内皮细胞（HRMEC）附着和 装配行为。 此外，研究人员还表明，信号 ELK 受体依赖于其配体的近分泌呈递 接触细胞表面上的不同寡聚状态。 三 将解决目标。 实验方法将涉及传统的 生化亲和力研究以及酵母-两种杂交系统和使用 显性负蛋白相互作用。 第一个具体目标，定义 LERK-2 寡聚化的分子基础将使用以下方法完成 两种互补的方法。 第一个将采用酵母双杂交 利用 LERK-2 的细胞质结构域作为诱饵来筛选小鼠 胚胎文库。 这项研究预计会发现相互作用的蛋白质 具有 LERK-2 的细胞质结构域。 补充方法将 涉及标准细胞培养标记实验以提供信息 关于 LERK-2 在刺激和未刺激下的细胞分布 细胞。 随后，其他实验将利用 LERK-2 细胞质 作为 GST 融合蛋白创建的结构域，允许与 GSH 珠子结合。 这 然后融合蛋白将用于捕获潜在的相互作用蛋白 来自受刺激细胞的细胞质。 最后，一系列化学 交联研究和凝胶过滤电泳实验将 用于研究 LERK-2 的寡聚状态 刺激内皮细胞。 第二个具体目标将重点关注 ELK 信号传导，特别是 研究 ELK 和 LMP-PTP 之间相互作用的分子基础， 该研究者之前的研究已经证明了这一点 实验室。 在这方面，本实验室积累了广泛的经验 解决这种相互作用的实验工具将使用酵母 双杂交系统和杆状病毒表达系统来解决这些问题 除了瞬时转染测定和显性阴性 方法来检查这些相互作用。 第三个具体目标是一项描述性研究，以确定类型 本实验室使用的细胞系上存在的整合素，使用标准 流式细胞仪分析。 此外，将建立转染细胞系 通过 ELK-LERK-2 检查整合素和信号传导之间的相互作用 互动。 这是一项寻求进一步 5 年资助的提案 用于研究肾微血管内皮细胞的毛细血管形态发生 细胞。 该项目现已进入第四个年头，并提供了新颖的 对细胞间识别事件的见解。 简而言之，据悉 ELK-LERK-2 配体-受体对存在于肾内皮细胞中 在开发过程中，LERK-2 当以四聚体形式呈现给 ELK 时可以 刺激体外毛细血管前束的形成。 相比之下，二聚体 LERK-2 仅以基质特异性方式刺激细胞粘附和迁移。 与激活的 ELK 细胞质尾部相关的蛋白质已被 鉴定出，而 Grb-2 和 Grb-10 与 ELK 刺激相关 二聚体 LERK-2，一种酪氨酸磷酸酶 LMW-PTP，仅与 受体结合成簇的四聚体 LERK-2 时。 因此是 提出 ELK 和 LERK-2 是肾微血管的反受体 内皮细胞在细胞与细胞接触期间聚集，并且两者，ELK 和 LERK-2 用于发出刺激毛细血管的细胞内事件信号 绳索的形成。 为该项目的下一个学期提出的方法是： 广泛使用酵母二杂交系统，并进行体外研究 与 ELK 和 LERK-2（及其突变体）的 GST 融合体相互作用 鉴定与细胞质尾部相互作用的蛋白质的目的 LERK-2（目标 1）和 ELK（目标 2）。 对于 ELK 来说，这种方法已经部分被 因此，目标 2 旨在进一步定义信号事件： 通过研究 ELK 及其特定突变体来刺激细胞组装 伙伴、它们的相互作用以及它们对细胞的潜在影响 集会。 由于二聚体 LERK-2 刺激细胞粘附和迁移 以及 ELK 酪氨酸磷酸化，以及 Grb-2、Grb-4 和 Grb-10 已发现在二聚体和 ELK 刺激下与 ELK 相关联 四聚体 LERK-2，目标三旨在评估 LERK-2-ELK 对整合素功能的影响，小 G 蛋白参与 刺激粘附、细胞骨架重组和迁移，以及 JNK/SAP 激酶途径的激活。