Role of Surfactant Protein D in Surfactant Homeostasis

描述（由申请人提供）：本提案寻求继续资助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是一个43 kd的多肽集合家族成员，在脊椎动物肺细支气管和肺泡上皮细胞中表达。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着重要作用，但我们在SP-D基因靶向小鼠的研究结果表明，SP-D在1)正常表面活性剂池大小的产生、2)管状髓磷脂和其他肺泡脂质的结构、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着关键作用。我们的初步数据强烈支持SP-D调节肺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，目前的目的是为了辨别SP-D调节表面活性剂稳态的机制。具体目标旨在验证两个替代假设：1)SP-D有助于产生独特的表面活性剂形式，其摄取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型细胞中积累；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替代和/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SP-D的存在或不存在对II型细胞超微结构、大聚集体/小聚集体比率以及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。利用肺有条件表达SP-D的SP-D（-/-）小鼠。在第二阶段，我们将评估SP-D嵌合突变体分子的结构和功能。SP-D及其突变体SP-D分子将在体外和体内产生。用表面活性剂磷脂重组SP-D/SP-A突变蛋白，以确定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Specific Aim 3中，我们将利用微阵列分析SP-D（-/-）小鼠的肺和分离的II型细胞，以确定SP-D存在和不存在时脂质池大小改变的基因组反应。SP-D在表面活性剂稳态和肺防御中起关键作用。阐明SP-D在表面活性物质稳态和宿主防御中的关键作用，将有助于我们对包括囊性纤维化、急性细菌感染和ARDS在内的许多急慢性肺部疾病的发病机制的理解。预期的研究将为SP-D在肺保护中的作用以及决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.