The Role of SPARC in Procollagen Processing in the Periodontal Ligament

描述（由申请人提供）：跨越牙周韧带（PDL）的胶原纤维通过每颗牙齿的牙骨质编织进入牙槽骨，将牙齿与骨窝连接起来。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，其特点是前胶原蛋白的合成、加工和沉积，以及不溶性胶原蛋白的吸收。因此，PDL为研究前胶原加工机制提供了良好的组织环境。SPARC是一种胶原结合蛋白，被认为是胶原ECM沉积的关键因子。因此，我们报道了SPARC-null小鼠与年龄匹配的野生型（WT） PDL相比，PDL中的胶原蛋白明显减少。有趣的是，SPARC-null小鼠的PDL胶原纤维也明显比WT的PDL纤维更薄。当用脂多糖诱导牙周病时，通过测量牙槽骨和PDL胶原蛋白的损失，SPARC-null小鼠的疾病更严重。为了研究在缺乏SPARC的情况下，稳态胶原含量下降和对疾病反应的细胞机制，我们培养了PDL成纤维细胞，并使用了一种新的器官培养模型。我们的初步结果表明，在体外和离体实验中，SPARC-null的PDL成纤维细胞在前胶原加工方面存在缺陷，正如在WT条件下不明显的前胶原在空细胞和组织中的积累所表明的那样。此外，在缺乏SPARC的情况下，前胶原蛋白的增加与细胞表面有关。这些结果表明，SPARC可以减少胶原与细胞表面受体的相互作用，并且胶原与细胞表面受体的结合阻碍了前胶原的有效加工。这些特定目标将验证SPARC通过与胶原受体参与竞争来介导PDL成纤维细胞胶原稳态的假设，胶原受体参与是有效的前胶原加工和随后的胶原原纤维结合所必需的活动。将缺乏胶原结合的突变型SPARC转染到SPARC缺失的PDL成纤维细胞中，以确定SPARC的胶原结合能力是否需要调节前胶原加工。此外，腺病毒构建体将用于敲除两种已知与SPARC共享胶原结合位点的胶原细胞受体，整合素2和盘状蛋白结构域受体（DDR） 2，在WT和SPARC-null PDL中。我们预测，整合素2和DDR2的敲低会促进WT和SPARC-null PDL的前胶原加工，从而降低前胶原水平，增加完全成熟胶原1的水平。此外，我们将使用离体模型研究DDR2和整合素2受体敲低对胶原原纤维组装的影响。因此，本项目将确定SPARC、DDR2和整合素2在前胶原加工和胶原纤维形成中的作用。这里提出的培养计划将检验上述假设，为我成为一名学术科学家做好准备。

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.