Role of Surfactant Protein D in Surfactant Homeostasis

描述(申请人提供)：这项提案寻求继续资助HL-63329，以确定表面活性蛋白D(Spd)调节表面活性物质磷脂稳态的机制。SP-D是集合素多肽家族中43kd的成员，表达于脊椎动物肺的细支气管和肺泡上皮细胞。虽然SP-D在调节宿主对病毒、细菌和真菌病原体的天然防御中发挥了重要作用，但我们在SP-D基因靶向的小鼠中的发现表明，SP-D在1)正常表面活性物质池大小的产生、2)管状髓鞘和其他肺泡脂的结构、3)巨噬细胞激活、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中发挥关键作用。我们的初步数据有力地支持了SP-D调节肺内第二型上皮细胞表面活性物质代谢、分解代谢和/或循环的模型。因此，目前的目的是为了弄清SP-D调节表面活性物质动态平衡的机制。具体目的是检验两种替代假设：1)SP-D有助于产生独特的表面活性物质形式，其摄取、分解代谢和路径降解受到干扰，导致表面活性物质磷脂在空气和II型细胞中积累；2)替代和/或重叠的假设，即SP-D直接与II型上皮细胞相互作用，改变表面活性物质的动态平衡。在具体目标1中，我们将在体内和体外修复SP-D的实验中，确定SP-D对表面活性物质结构的影响。有或没有SP-D对第二类细胞的超微结构、大聚集/小聚集比率以及对表面活性物质颗粒和表面活性物质包被珠的摄取或分解代谢的影响将被评估。在肺中有条件表达SP-D的SP-D(-/-)小鼠将被利用。在目标2中，将评估嵌合突变SP-D分子的结构和功能。将在体外和体内产生SP-D和突变型SP-D分子。SP-D/SP-A突变蛋白将与表面活性物质磷脂重组，以识别介导SP-D对表面活性物质结构及其体外代谢/分解代谢的影响的精确结构域。将在表达突变SP-D蛋白的SP-D(-/-)小鼠身上测试特定位置的SP-D突变蛋白的功能。最后，在特定的目标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确定在存在和不存在SP-D的情况下对脂池大小改变的基因组反应。SP-D在维持肺表面活性物质的动态平衡和保护肺功能方面发挥着重要作用。阐明SP-D在表面活性物质动态平衡和宿主防御中的关键作用将有助于我们对囊性纤维化、急性细菌感染和ARDS等一系列急、慢性肺部疾病的发病机制的理解。这些预期的研究将为SP-D在肺保护中的作用以及决定表面活性物质动态平衡的基本机制提供基本的见解。

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.