Phagocytosis of apoptotic cells: Signaling via GULP

描述(由申请人提供)：程序性细胞死亡发生在我们身体的许多组织中，贯穿一生。快速有效地去除死亡细胞是保护邻近细胞的关键步骤，也是组织重塑和伤口愈合的重要组成部分。未能清除凋亡小体被认为是某些类型的慢性炎症和自身免疫性疾病的原因之一。在哺乳动物中，巨噬细胞和树突状细胞以及许多其他类型的细胞可以吞噬凋亡的身体。虽然已经确定了几种参与细胞凋亡识别的表面受体，但对调节摄取过程本身及其下游后果的细胞内信号事件知之甚少。最近在模式生物秀丽线虫中克隆了吞噬基因，并在哺乳动物中鉴定了它们的同源物，这为更详细地描述这一过程提供了一个令人兴奋的机会。这一建议的主要焦点将是确定通过一种进化保守的吞噬蛋白GULP介导的信号事件，并从分子上更好地了解哺乳动物吞噬凋亡细胞的过程。 CED-6是线虫中参与吞噬细胞凋亡的候选胞质适配蛋白，Ced-6具有相同的功能遗传途径，包括编码膜蛋白的另外两个基因Ced-1和Ced-7。优雅的蠕虫遗传学研究表明，Ced-6需要在吞噬细胞中表达，而不是在凋亡细胞中表达，并且Ced-6在Ced-1下游发挥作用。我们的实验室已经克隆了Ced-6的小鼠同源物，表示为GULP，并对哺乳动物吞噬过程中的GULP蛋白进行了初步鉴定。在目标1中，我们将使用J774巨噬细胞和原代巨噬细胞对GULP进行结构和功能的研究，了解GULP在吞噬后抗炎细胞因子产生中的作用。一些证据表明CD91/LRP 1是一种吞咽相互作用蛋白，而LRP很可能代表哺乳动物中的CED-1同源物。AIM2的重点将是通过使用体外LRP缺陷细胞系和体内条件性靶向LRP缺陷小鼠来确定GULP：LRP相互作用的生物学意义。为了在整个动物水平上了解吞咽功能，在Aim3中，我们将产生吞咽敲除小鼠，以及LZ区域特异突变的敲入小鼠。在这些小鼠研究中，我们将确定吞噬缺失或吞噬功能中断对吞噬的影响，以及由于吞噬缺陷对自身免疫的任何潜在影响。

DESCRIPTION (provided by applicant): Programmed cell death occurs in many tissues of our bodies throughout life. The quick and efficient removal of dying cells is a critical step that protects neighboring cells, and is also an important part of tissue remodeling and wound healing. The failure to remove apoptotic bodies has been implicated as a cause for certain types of chronic inflammation and autoimmune disorders. In mammals, macrophages and dendritic cells, as well as many other cells types can carry out the engulfment of apoptotic corpses. While several surface receptors that participate in the recognition of apoptotic cells have been identified, relatively little is known about the intracellular signaling events that regulate the uptake process itself and its downstream consequences. Recent cloning of the engulfment genes in the model organism C. elegans, and the identification of their homologues in mammals, has provided an exciting opportunity to delineate this process in better detail. The major focus of this proposal will be to determine the signaling events mediated through one of the evolutionarily conserved engulfment proteins GULP and to gain a better molecular understanding of the process of engulfment of apoptotic cells in mammals. CED-6 was identified as a candidate cytoplasmic adapter protein involved in engulfment of apoptotic cells in C. elegans, ced-6 is in the same functional genetic pathway that includes two other genes, ced-1 and ced-7, both of which encode membrane proteins. Elegant worm genetic studies have shown that ced-6 expression is required in the engulfing cell and not in the apoptotic cell and that ced-6 functions downstream of ced-1. Our laboratory has cloned the murine homologue of ced-6, denoted gulp, and has performed initial characterization of GULP protein in mammalian engulfment. In Aim 1, we will use the J774 macrophages and primary macrophages to perform structure: function studies of GULP and understand the role of GULP in anti-inflammatory cytokine production following engulfment. Several lines of evidence suggested CD91/LRP 1 as a GULP interacting protein, and LRP likely represents the CED- 1 homologue in mammals. The focus of Aim2 will be to determine the biological significance of GULP: LRP interaction through the use of LRP deficient cells lines in vitro, and conditionally targeted LRP deficient mice in vivo. To understand GULP function at the whole animal level, in Aim3, we will generate GULP knockout mice, as well as knock-in mice in which the LZ region is specifically mutated. In these mouse studies, we will determine the effect on engulfment due to the absence of GULP, or disrupted GULP function, and any potential effects on autoimmunity due to defective engulfment.