Role of Surfactant Protein D in Surfactant Homeostasis

描述（由申请人提供）：本提案旨在继续资助HL-63329，以确定表面活性蛋白D（SPD）调节表面活性磷脂稳态的机制。 SP-D是在脊椎动物肺的细支气管和肺泡上皮细胞中表达的多肽聚集素家族的43 kd成员。 虽然SP-D在介导先天宿主对病毒、细菌和真菌病原体的防御中起重要作用，但我们在SP-D基因靶向小鼠中的发现表明，SP-D在1）正常表面活性剂池大小，2）管状髓鞘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产生，（4）维持发育过程中正常的肺泡结构。 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、catalysts和/或再循环的模型。 因此，本发明的目的旨在辨别SP-D调节表面活性剂稳态的机制。 具体目的旨在检验两个备择假设：1）SP-D有助于产生独特的表面活性剂形式，其摄取催化剂和路由降解受到干扰，导致表面活性剂磷脂在气隙和II型细胞中蓄积，和2）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的备择和/或重叠假设。 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。 将评估存在或不存在SP-D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催化的影响。 将使用SP-D在肺中条件性表达的SP-D（-/-）小鼠。 在目的2中，将评估嵌合突变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突变蛋白将与表面活性剂磷脂重构，以识别介导SP-D对表面活性剂结构及其体外代谢/催化作用的精确结构域。 将在SP-D中测试位点特异性SP-D突变蛋白的功能 （-/-）小鼠，其中表达突变SP-D蛋白。 最后，在具体目标3中，我们将利用肺和分离的SP-D（-/-）小鼠的II型细胞的微阵列分析，以确定在存在和不存在SP-D的情况下改变脂质池大小的基因组反应。 SP-D在表面活性物质稳态和肺防御中起关键作用。 阐明SP-D对表面活性物质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）的发病机制的理解。 预期的研究将为SP-D在肺保护中的作用以及确定表面活性剂稳态的基本机制提供基本见解。

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.