Regulation of Tendon Extracellular Matrix Assembly by the Fibroblast Surfaceome

成纤维细胞表面组对肌腱细胞外基质组装的调节

• 批准号: 7915344
• 负责人: Simone M-L Smith
• 金额: $ 5.05万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7915344
• 关键词: Address; Adhesions; Age-Months; Animals; Binding; Binding Sites; Cell Adhesion; Cell Adhesion Molecules; Cell membrane; Cell surface; Cells; Clinical Treatment; Collagen; Collagen Fibril; Complex; Cytoskeleton; Data; Defect; Detergents; Development; Environment; Experimental Designs; Extracellular Matrix; Fiber; Fibroblast Growth Factor Receptors; Fibroblasts; Fibronectins; Flexor; Genetic; Goals; Growth Factor; Growth Factor Receptors; Healed; Heparan Sulfate Proteoglycan; Heparin Binding; Immunohistochemistry; Immunoprecipitation; Inflammation; Injury; Integrins; Link; Matrix Metalloproteinases; Membrane; Membrane Proteins; Metalloproteases; Mus; Musculoskeletal Diseases; Proteins; Proteoglycan; Proteomics; Protocols documentation; Receptor Cell; Recruitment Activity; Regulation; Role; Signal Transduction; Site; Staging; Stimulus; Structure; Surface; Tendinitis; Tendon Injuries; Tendon structure; Weight-Bearing state; Western Blotting; age related; aged; animal extract; biglycan; cell motility; decorin; extracellular; fibrillogenesis; fibromodulin; genetic regulatory protein; healing; injured; lumican; migration; mouse model; regenerative; syndecan

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.

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组（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。原纤维形成或组装的失调与肌肉骨骼疾病有关，例如肌腱形成不当、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组随着发育而变化，并且表达的不同蛋白质库调节肌腱形成的阶段。对于目标1和2，将使用来自野生型PI小鼠的肌腱。在PI，肌腱主要处于形成的最早阶段（成核），其中成纤维细胞表面至关重要。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起始中在那里相互作用。目的1是确定Col V和Col XI与成纤维细胞膜相关的机制，以及哪些特异性蛋白负责将这些胶原连接到成纤维细胞膜。整合素和蛋白聚糖可能参与了胶原V和XI与肌腱成纤维细胞表面的连接。实验设计包括从肌腱中提取蛋白质的Brij去污剂，Col V和XI及其相关蛋白质的免疫沉淀（IP），以及这些蛋白质的蛋白质组学鉴定。将通过（Western）印迹法在肌腱提取物中定位这些候选蛋白以及通过免疫组织化学（IHC）在成纤维细胞表面定位这些候选蛋白来确认数据。目的2是鉴定组成肌腱成纤维细胞表面和表面相关（基质）库的蛋白质，这些蛋白质在调节肌腱功能中具有作用。表面组还可以包括信号转导（如FGF受体）、细胞锚定（如粘附分子）和细胞迁移和基质转换（如金属蛋白酶）中必需的分子。肌腱中的蛋白质将用Brij提取并通过蛋白质组学鉴定。在Aim 2中而不是Aim 1中鉴定的蛋白质将通过印迹定位在提取物中，并通过IHC定位在肌腱中。目的3是确定目的1和2中鉴定的调节性成纤维细胞表面组蛋白的发育差异。将使用1个月（生长）、3个月（成熟）和18个月（老化）的野生型动物。将通过印迹法在这些动物的肌腱提取物中以及通过IHC在肌腱中定位来自目的1和2的候选蛋白。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，甚至逆转遗传性肌腱缺陷或老年人或受伤者的衰弱肌腱状况。