Regulation of Tendon Extracellular Matrix Assembly by the Fibroblast Surfaceome

描述(申请人提供)：这项建议将集中在肌腱成纤维细胞表面(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。纤维形成或装配的失调与肌肉骨骼疾病有关，如肌腱形成不当、肌腱炎症和损伤后肌腱愈合受损。这一建议的一般假设是，肌腱成纤维细胞表面体随着发育而变化，表达的不同蛋白质谱调控肌腱形成的不同阶段。对于AIMS 1和AIMS 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。V型胶原(Col V)和XI型胶原(Col XI)在肌腱成纤维细胞表面表达，并在胶原纤维组装的起始处相互作用。目的1是确定Col V和Col XI与成纤维细胞膜结合的机制，以及哪些特定蛋白负责将这些胶原连接到成纤维细胞膜上。整合素和蛋白多糖可能参与了胶原V和XI与肌腱成纤维细胞表面的连接。实验设计包括Brij洗涤剂提取肌腱中的蛋白质，免疫沉淀(IP)Col V和XI及其相关蛋白质，以及这些蛋白质的蛋白质组学鉴定。这些候选蛋白将通过(Western)blotts在肌腱提取液中的定位以及通过免疫组织化学(IHC)在成纤维细胞表面的定位来证实。目的2是确定组成肌腱成纤维细胞-细胞表面和表面相关(基质)谱系的蛋白质，并在肌腱功能调节中发挥作用。表面体还可以包括信号转导(如成纤维细胞生长因子受体)、细胞锚定(如黏附分子)、细胞迁移和基质转换(如金属蛋白酶)所必需的分子。肌腱中的蛋白质将用Brij提取，并通过蛋白质组学进行鉴定。在目标2而不是目标1中确定的蛋白质将通过斑点杂交定位于提取液中，并通过IHC定位于肌腱中。目的3是确定AIMS 1和AIMS 2中确定的调控成纤维细胞表面蛋白的发育差异。将使用1个月(生长)、3个月(成熟)和18个月(衰老)的野生型动物。AIMS 1和AIMS 2的候选蛋白将通过斑点定位于这些动物的肌腱提取液中，并通过IHC定位于肌腱中。了解发育中肌腱的调节蛋白及其与年龄相关的变化，可以更好地利用未成熟肌腱的已知再生能力。这将允许更好的临床治疗，甚至逆转那些有遗传性肌腱缺陷或年老或受伤的人虚弱的肌腱状况。

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.