Regulation of Tendon Extracellular Matrix Assembly by the Fibroblast Surfaceome

描述（由申请人提供）：该提案将重点关注肌腱成纤维细胞表面体（肌腱成纤维细胞表面的蛋白质）及其在调节胶原纤维形成和细胞外基质（ECM）组装中的作用。纤维形成或组装的失调与肌肉骨骼疾病有关，如肌腱形成不当、肌腱炎和损伤后肌腱愈合受损。该建议的一般假设是，肌腱成纤维细胞表面体随着发育而变化，表达的不同蛋白质库调节了肌腱形成的阶段。目的1和目的2将使用野生型PI小鼠的肌腱。在PI，肌腱主要处于形成的最早阶段（成核），成纤维细胞表面是至关重要的。胶原V （Col V）和XI （Col XI）在肌腱成纤维细胞表面表达，并在胶原原纤维组装的启动过程中相互作用。目的1是确定Col V和Col XI与成纤维细胞膜相关的机制，以及哪些特定蛋白负责将这些胶原系在成纤维细胞膜上。整合素和蛋白聚糖可能参与了Col V和XI与肌腱成纤维细胞表面的连接。实验设计包括用Brij清洗剂从肌腱中提取蛋白质，免疫沉淀（IP） Col V和XI及其相关蛋白，并对这些蛋白进行蛋白质组学鉴定。这些数据将通过（Western） blots在肌腱提取物中定位这些候选蛋白，并通过免疫组织化学（IHC）在成纤维细胞表面定位。目的2是鉴定构成肌腱成纤维细胞表面和表面相关（基质）库并在调节肌腱功能中起作用的蛋白质。表面小体还可能包括在信号转导（如FGF受体）、细胞锚定（如粘附分子）和细胞迁移和基质周转（如金属蛋白酶）中必不可少的分子。用Brij提取肌腱中的蛋白质并进行蛋白质组学鉴定。在Aim 2中发现的而不是Aim 1中的蛋白质将通过印迹定位在提取物中，通过免疫组化定位在肌腱中。目的3是确定目的1和目的2中发现的调节成纤维细胞表面体蛋白的发育差异。使用1个月（生长期）、3个月（成熟期）和18个月（衰老期）的野生型动物。Aims 1和Aims 2中的候选蛋白将通过免疫印迹和免疫组化在这些动物的肌腱提取物中定位。了解发育中的肌腱的调节蛋白及其与年龄相关的变化，可以更好地利用已知的未成熟肌腱的再生能力。这将允许更好的临床治疗，甚至逆转衰弱的肌腱条件，那些遗传肌腱缺陷或谁是年老或受伤。

DESCRIPTION (provided by applicant): This proposal will focus on the tendon fibroblast surfaceome (the proteins on the tendon fibroblast surface) and its role in regulating collagen fibrillogenesis and extracellular matrix (ECM) assembly. Dysregulation of fibrillogenesis or assembly is associated with musculoskeletal disorders such as improper tendon formation, tendonitis and impaired tendon healing after injury. The general hypothesis of this proposal is that the tendon fibroblast surfaceome changes with development and the differing repertoire of proteins expressed regulates the stages of tendon formation. For aims 1 and 2, tendons from wild type PI mice will be used. At PI, tendons are predominantly in the earliest stage of formation (nucleation), where the fibroblast surface is crucial. Collagen V (Col V) and XI (Col XI) are expressed at the tendon fibroblast surface and interact there in the initiation of collagen fibril assembly. Aim 1 is to define the mechanism by which Col V and Col XI are associated with the fibroblast membrane and which specific proteins are responsible for tethering these collagens to the fibroblast membrane. Integrins and proteoglycans may be involved in the linkage of Col V and XI to the tendon fibroblast surface. The experimental design includes a Brij detergent extraction of the proteins from tendons, immunoprecipitation (IP) of Col V and XI and their associated proteins, and proteomic identification of these proteins. The data will be confirmed by localization of these candidate proteins in tendon extracts by (Western) blots and on the fibroblast surface by immunohistochemistry (IHC). Aim 2 is to identify the proteins that comprise the tendon fibroblast cell-surface and surface-associated (matrix) repertoire and that have roles in regulating tendon function. The surfaceome may also include molecules essential in signal transduction (such as FGF receptors), cell anchoring (such as adhesion molecules) and cell migration and matrix turnover (such as metalloproteinases). The proteins in the tendons will be extracted with Brij and identified by proteomics. Proteins identified in Aim 2 but not Aim 1 will be localized in extracts by blots and in tendons by IHC. Aim 3 is to define the developmental differences in the regulatory fibroblast surfaceome proteins identified in aims 1 and 2. Wild-type animals at 1-month (growing), 3-months (mature) and 18-months (aging) will be used. The candidate proteins from Aims 1 and 2 will be localized in tendon extracts from these animals by blots and in tendons by IHC. Knowing the regulatory proteins of developing tendons and their age-related changes allows better exploitation of the known regenerative capabilities of immature tendons. This will allow better clinical treatment and even reversal of debilitating tendon conditions in those with genetic tendon defects or who are aged or injured.