The Role of SPARC in Procollagen Processing in the Periodontal Ligament

SPARC 在牙周膜原胶原加工中的作用

• 批准号: 8598808
• 负责人: Jessica Marie Trombetta-eSilva
• 金额: $ 4.8万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-05 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598808
• 关键词: Address; Adenoviruses; Affect; Age; Alveolar Bone Loss; Binding; Binding Proteins; Binding Sites; Biological Assay; Caliber; Cell Surface Receptors; Cell surface; Collagen; Collagen Fiber; Collagen Fibril; Collagen Receptors; Collagen Type I; Connective Tissue; Cysteine; Data; Dental Cementum; Deposition; Disease; Electron Microscopy; Extracellular Matrix; Fiber; Fibroblasts; Fibrosis; Homeostasis; Immunoblot Analysis; Immunofluorescence Immunologic; In Vitro; Infection; Integrins; Knockout Mice; Label; Lipopolysaccharides; Measures; Mediating; Metabolic; Methods; Modeling; N-terminal; Natural regeneration; Null Lymphocytes; Organ Culture Techniques; Osteonectin; Outcome; Periodontal Diseases; Periodontal Ligament; Process; Procollagen; Proteins; Publishing; Regulation; Reporting; Role; Scientist; Techniques; Testing; Thick; Tissues; Tooth structure; Training; Wild Type Mouse; Work; alveolar bone; bone; career; discoidin domain receptor 2; knock-down; mutant; novel; prevent; public health relevance; receptor; repaired; response; therapeutic target; uptake

DESCRIPTION (provided by applicant): The collagen fibers that span the periodontal ligament (PDL) connect teeth to the bone socket by weaving through the cementum of each tooth and into the alveolar bone. PDL has high rates of extracellular matrix turnover, as compared to other collagen rich tissues, characterized by procollagen synthesis, processing, and deposition in addition to uptake of insoluble collagen. Thus PDL provides an excellent tissue milieu for investigating mechanisms of procollagen processing. SPARC, a collagen-binding protein, has been identified as a key factor in collagen ECM deposition. Accordingly, we reported that SPARC-null mice had significantly less collagen in PDL as compared to age-matched wild type (WT) PDL. Interestingly, the PDL collagen fibers in SPARC-null mice were also significantly thinner than WT PDL fibers. When challenged with lipopolysaccharide to induce periodontal disease, SPARC-null mice had more severe disease as measured by loss of alveolar bone and PDL collagen. To address cellular mechanisms of decreased homeostatic collagen content and response to disease in the absence of SPARC, we cultured PDL fibroblasts and used a novel organ culture model. Our preliminary results demonstrated, both in vitro and ex vivo, that SPARC-null PDL fibroblasts had a deficiency in procollagen processing, as indicated by an accumulation of procollagen in null cells and tissue that was not evident in WT conditions. Furthermore, increases in procollagen were found associated with cell surfaces in the absence of SPARC. These results suggested that SPARC acts to decrease collagen interaction with cell surface receptors and that collagen engagement by cell surface receptors hampered efficient procollagen processing. These Specific Aims will test the hypothesis that SPARC mediates collagen homeostasis in PDL fibroblasts by competing with collagen receptor engagement, an activity required for efficient procollagen processing and subsequent collagen fibril incorporation. Mutant SPARC lacking collagen binding will be transfected into SPARC-null PDL fibroblasts to determine whether the collagen binding capacity of SPARC is required to modulate procollagen processing. In addition, adenovirus constructs will be used to knockdown two collagen cellular receptors known to share collagen binding sites with SPARC, integrin 2 and Discoidin Domain Receptor (DDR) 2, in both WT and SPARC-null PDL. We predict that knockdown of integrin 2 and DDR2 will promote procollagen processing in WT and SPARC-null PDL, thereby decreasing levels of procollagen and increasing levels of fully mature collagen 1. Furthermore, we will investigate the outcome of DDR2 and integrin 2 receptors knockdown on collagen fibril assembly using our ex vivo model. Thus, this project will determine the role of SPARC, DDR2, and integrin 2 on procollagen processing and collagen fibril formation. The training plan proposed here will test the above hypothesis and prepare me for a career as an academic scientist.

描述（由申请人提供）：跨越牙周韧带（PDL）的胶原纤维通过穿过每颗牙齿的牙骨质并进入牙槽骨，将牙齿连接到骨槽。与其他富含胶原蛋白的组织相比，PDL 具有较高的细胞外基质周转率，其特征是除了不溶性胶原蛋白的摄取之外还具有原胶原合成、加工和沉积。因此，PDL 为研究前胶原加工机制提供了极好的组织环境。 SPARC 是一种胶原蛋白结合蛋白，已被确定为胶原蛋白 ECM 沉积的关键因素。因此，我们报告说，与年龄匹配的野生型 (WT) PDL 相比，SPARC 缺失小鼠的 PDL 中胶原蛋白显着减少。有趣的是，SPARC null 小鼠的 PDL 胶原纤维也明显比 WT PDL 纤维更细。当用脂多糖诱导牙周病时，SPARC 缺失小鼠患有更严重的疾病（通过牙槽骨和 PDL 胶原蛋白的损失来衡量）。为了解决在缺乏 SPARC 的情况下稳态胶原含量降低和对疾病反应的细胞机制，我们培养了 PDL 成纤维细胞并使用了一种新型器官培养模型。我们的初步结果表明，无论是体外还是离体，SPARC-null PDL 成纤维细胞在原胶原加工方面存在缺陷，正如原胶原在无效细胞和组织中的积累所表明的那样，这在 WT 条件下并不明显。此外，在没有 SPARC 的情况下，发现原胶原的增加与细胞表面相关。这些结果表明，SPARC 可以减少胶原蛋白与细胞表面受体的相互作用，并且细胞表面受体与胶原蛋白的结合阻碍了有效的前胶原加工。这些具体目标将检验 SPARC 通过与胶原蛋白受体参与竞争来介导 PDL 成纤维细胞中胶原蛋白稳态的假设，胶原蛋白受体参与是有效前胶原加工和随后的胶原原纤维掺入所需的活动。将缺乏胶原蛋白结合的突变体 SPARC 转染至 SPARC 无效的 PDL 成纤维细胞中，以确定调节原胶原加工是否需要 SPARC 的胶原蛋白结合能力。此外，在 WT 和 SPARC-null PDL 中，腺病毒构建体将用于敲低两种已知与 SPARC 共享胶原蛋白结合位点的胶原蛋白细胞受体，即整联蛋白 2 和盘状蛋白结构域受体 (DDR) 2。我们预测整合素 2 和 DDR2 的敲低将促进 WT 和 SPARC-null PDL 中的前胶原加工，从而降低前胶原水平并增加完全成熟的胶原 1 的水平。此外，我们将使用我们的离体模型研究 DDR2 和整合素 2 受体敲低对胶原纤维组装的结果。因此，该项目将确定 SPARC、DDR2 和整合素 2 在前胶原加工和胶原纤维形成中的作用。这里提出的培训计划将检验上述假设，并为我作为一名学术科学家的职业生涯做好准备。