Role of Surfactant Protein D in Surfactant Homeostasis

表面活性剂蛋白 D 在表面活性剂稳态中的作用

• 批准号: 6685448
• 负责人: MACHIKO IKEGAMI
• 金额: $ 37.25万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6685448
• 关键词: RNA; alveolar macrophages; confocal scanning microscopy; enzyme linked immunosorbent assay; flow cytometry; genetically modified animals; immunocytochemistry; laboratory mouse; lipid metabolism; lung; lung alveolus; microarray technology; phosphatidylcholines; phospholipids; protein structure function; pulmonary surfactants; respiratory epithelium; tissue /cell culture

DESCRIPTION (provided by applicant): This proposal seeks to continue funding of HL-63329 to determine the mechanisms by which surfactant protein D (SPD) regulates surfactant phospholipid homeostasis. SP-D is a 43 kd member of the collectin family of polypeptides that is expressed in bronchiolar and alveolar epithelial cells of the vertebrate lung. While SP-D plays an important rote in the mediation of innate host defenses against viral, bacterial and fungal pathogens, our findings in SP-D gene targeted mice demonstrated that SP-D played a critical role in the generation of 1) normal surfactant pool sizes, 2) the structure of tubular myelin and other alveolar lipids, 3) macrophage activation, oxidant and metalloproteinase production, and 4) maintenance of normal alveolar structure during development. Our preliminary data strongly support a model in which SP-D regulates surfactant metabolism, catabolism, and/or recycling by type II epithelial cells in the lung. The present aims are therefore designed to discern the mechanisms by which SP-D regulates surfactant homeostasis. The specific aims are designed to test two alternative hypothesis: 1) that SP-D contributes to the generation of unique surfactant forms whose uptake catabolism and routing degradation are perturbed leading to the accumulation of surfactant phospholipids in the airspaces and in type II ceils, and 2) the alternative and/or overlapping hypothesis that SP-D interacts directly with type II epithelial cells to alter surfactant homeostasis. In Specific Aim 1 we will determine the effects of SP-D on surfactant structure in experiments in which SP-D is restored in vivo and in vitro. Effects of the presence or absence of SP-D on ultrastructure, large aggregate/small aggregate ratios and uptake or catabolism of surfactant particles and surfactant-coated beads by type II cells will be assessed. SP-D(-/-) mice in which SP-D is conditionally expressed in the lung will be utilized. In Aim 2, the structure and function of chimeric mutant SP-D molecules will be assessed. SP-D and mutant SP-D molecules will be produced in vitro and in vivo. SP-D/SP-A mutant proteins will be reconstituted with surfactant phospholipids to discern the precise structural domains mediating the effects of SP-D on surfactant structure and its metabolism/catabolism in vitro. Function of site-specific SP-D mutant proteins will be tested in SP-D (-/-) mice in which the mutant SP-D proteins are expressed. Finally, in Specific Aim 3 we will utilize microarray analyses of lung and isolated type II cells from SP-D (-/-) mice to define the genomic responses to altered lipid pool sizes in the presence and absence of SP-D. SP-D plays a critical role in surfactant homeostasis and in defense of the lung. Elucidation of the critical roles of SP-D on surfactant homeostasis and host defense will enhance our understanding of the pathogenesis of a number of acute and chronic lung disorders including cystic fibrosis, acute bacterial infection and ARDS. The intended studies will provide fundamental insights into the role of SP-D in the protection of the lung, as well as into the basic mechanisms determining surfactant homeostasis.

描述（由申请人提供）：该提案寻求继续资助 HL-63329，以确定表面活性剂蛋白 D (SPD) 调节表面活性剂磷脂稳态的机制。 SP-D 是多肽集合素家族的 43 kd 成员，在脊椎动物肺的细支气管和肺泡上皮细胞中表达。 虽然 SP-D 在介导宿主对病毒、细菌和真菌病原体的先天防御中发挥着重要作用，但我们在 SP-D 基因靶向小鼠中的研究结果表明，SP-D 在 1) 正常表面活性剂池的生成中发挥着关键作用大小，2) 肾小管髓磷脂和其他肺泡脂质的结构，3) 巨噬细胞活化，氧化剂和金属蛋白酶的产生，以及 4) 发育过程中正常肺泡结构的维持。 我们的初步数据强烈支持 SP-D 调节肺中 II 型上皮细胞的表面活性剂代谢、分解代谢和/或再循环的模型。 因此，本发明的目的是探究 SP-D 调节表面活性剂稳态的机制。 具体目标旨在检验两个替代假设：1）SP-D 有助于产生独特的表面活性剂形式，其吸收分解代谢和路径降解受到干扰，导致表面活性剂磷脂在空气空间和 II 型细胞中积聚，以及2) 另一种和/或重叠的假设，即 SP-D 直接与 II 型上皮细胞相互作用以改变表面活性剂稳态。 在具体目标 1 中，我们将在体内和体外恢复 SP-D 的实验中确定 SP-D 对表面活性剂结构的影响。 将评估 SP-D 的存在或不存在对超微结构、大聚集体/小聚集体比率以及 II 型细胞对表面活性剂颗粒和表面活性剂包被珠的摄取或分解代谢的影响。 将使用其中SP-D在肺中条件性表达的SP-D(-/-)小鼠。 在目标 2 中，将评估嵌合突变体 SP-D 分子的结构和功能。 SP-D 和突变 SP-D 分子将在体外和体内产生。 SP-D/SP-A 突变蛋白将用表面活性剂磷脂重构，以辨别介导 SP-D 对表面活性剂结构及其体外代谢/分解代谢影响的精确结构域。 位点特异性 SP-D 突变蛋白的功能将在表达突变 SP-D 蛋白的 SP-D (-/-) 小鼠中进行测试。 最后，在特定目标 3 中，我们将利用来自 SP-D (-/-) 小鼠的肺和分离的 II 型细胞的微阵列分析来定义在存在和不存在 SP-D 的情况下对改变的脂质库大小的基因组反应。 SP-D 在表面活性剂稳态和肺部防御中发挥着关键作用。 阐明 SP-D 对表面活性剂稳态和宿主防御的关键作用将增强我们对许多急性和慢性肺部疾病（包括囊性纤维化、急性细菌感染和 ARDS）发病机制的理解。 预期的研究将为 SP-D 在保护肺中的作用以及决定表面活性剂稳态的基本机制提供基本见解。