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。快速有效地去除垂死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。凋亡小体的去除失败被认为是某些类型的慢性炎症和自身免疫性疾病的原因。在哺乳动物中，巨噬细胞和树突细胞以及许多其他类型的细胞可以吞噬凋亡的尸体。虽然已经确定了几种参与识别凋亡细胞的表面受体，但对调节摄取过程本身及其下游后果的细胞内信号传导事件知之甚少。模式生物C.秀丽线虫的发现以及它们在哺乳动物中的同源物的鉴定，为更详细地描述这一过程提供了一个令人兴奋的机会。该建议的主要重点将是确定通过进化上保守的吞噬蛋白GULP之一介导的信号事件，并获得更好的分子理解哺乳动物凋亡细胞的吞噬过程。 CED-6被鉴定为参与吞噬C.在线虫中，ced-6处于相同的功能性遗传途径中，该途径包括另外两个基因ced-1和ced-7，这两个基因都编码膜蛋白。线虫遗传学研究表明，ced-6的表达需要在吞噬细胞中，而不是在凋亡细胞中，ced-6的功能在ced-1的下游。我们的实验室已经克隆了ced-6的小鼠同源物，表示gulp，并进行了哺乳动物吞噬GULP蛋白的初步表征。在目的1中，我们将使用J774巨噬细胞和原代巨噬细胞进行GULP的结构：功能研究，并了解GULP在吞噬后抗炎细胞因子产生中的作用。一些证据表明CD 91/LRP 1是GULP相互作用蛋白，LRP可能代表哺乳动物中的CED- 1同源物。Aim 2的重点是通过使用体外LRP缺陷细胞系和体内条件靶向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理解GULP功能，在Aim 3中，我们将产生GULP敲除小鼠，以及LZ区域特异性突变的敲入小鼠。在这些小鼠研究中，我们将确定由于缺乏GULP或GULP功能中断而对吞噬的影响，以及由于吞噬缺陷而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.