The Role of SPARC in Procollagen Processing in the Periodontal Ligament

描述(申请人提供)：横跨牙周韧带(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窝。与其他富含胶原的组织相比，PDL具有较高的细胞外基质周转率，除了摄取不溶性胶原外，还具有前胶原合成、加工和沉积的特点。因此，PDL为研究前胶原蛋白的加工机制提供了良好的组织环境。SPARC是一种胶原结合蛋白，已被认为是胶原ECM沉积的关键因素。因此，我们报道，与年龄匹配的野生型(WT)PDL相比，SPARC基因缺失的小鼠PDL中的胶原蛋白显著减少。有趣的是，SPARC基因缺失小鼠的PDL胶原纤维也明显比WT PDL纤维薄。当用脂多糖诱导牙周疾病时，SPARC基因缺失的小鼠有更严重的疾病，通过牙槽骨和牙周胶原蛋白的丢失来衡量。为了解决在没有SPARC的情况下体内平衡胶原含量降低和疾病反应的细胞机制，我们培养了PDL成纤维细胞，并使用了一种新的器官培养模型。我们的初步结果表明，无论是在体外还是在体外，SPARC缺失的PDL成纤维细胞在前胶原处理方面都存在缺陷，这表明在空白细胞和组织中积累的前胶原在WT条件下不明显。此外，在没有SPARC的情况下，发现前胶原的增加与细胞表面有关。这些结果表明，SPARC减少了胶原与细胞表面受体的相互作用，细胞表面受体与胶原的结合阻碍了有效的前胶原加工。这些特定的目的将检验这一假说，即SPARC通过与胶原受体的参与竞争来介导PDL成纤维细胞中的胶原动态平衡，胶原受体参与是有效的前胶原蛋白加工和随后的胶原纤维掺入所必需的活动。缺乏胶原结合的突变体SPARC将被导入SPARC缺失的PDL成纤维细胞，以确定是否需要SPARC的胶原结合能力来调节前胶原的加工。此外，在WT和SPARC缺失的PDL中，腺病毒载体将被用来敲除两个已知与SPARC共享胶原结合位点的胶原细胞受体，整合素2和盘状结构域受体(DDR)2。我们预测，整合素2和DDR2的敲除将促进WT和SPARC缺失的PDL中前胶原的加工，从而降低前胶原水平，增加完全成熟的胶原1的水平。此外，我们将利用我们的体外模型来研究DDR2和整合素2受体敲除对胶原纤维组装的影响。因此，该项目将确定SPARC、DDR2和整合素2在前胶原蛋白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，并为我成为一名学术科学家做好准备。 公共卫生相关性：本项目重点研究SPARC在牙周韧带的前胶原加工和纤维组装中的作用机制。由于SPARC在纤维化组织中高度表达，了解SPARC的作用机制(S)可以确定治疗纤维化以及牙周韧带修复的潜在靶点，理想情况下，还可以使这一重要组织再生。

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. PUBLIC HEALTH RELEVANCE: This project focuses on the mechanism of SPARC in procollagen processing and fibril assembly in the periodontal ligament. As SPARC is highly expressed in fibrotic tissues, understanding the mechanism(s) of SPARC action could identify potential targets for therapeutic treatment of fibrosis as well as in periodontal ligament repair, and ideally, regeneration of this important tissue.