The Role of SPARC in Procollagen Processing in the Periodontal Ligament

描述（由申请人提供）：跨牙周膜（PDL）的胶原纤维通过编织穿过每颗牙齿的牙骨质并进入牙槽骨，将牙齿连接到骨窝。与其他富含胶原蛋白的组织相比，PDL具有高细胞外基质更新率，其特征在于除了摄取不溶性胶原蛋白之外，还具有前胶原蛋白合成、加工和沉积。因此，PDL提供了一个很好的组织环境，研究机制的前胶原加工。胶原结合蛋白β 1已被鉴定为胶原ECM沉积的关键因子。因此，我们报道了与年龄匹配的野生型（WT）PDL相比，SPARC-敲除小鼠PDL中的胶原蛋白显著减少。有趣的是，在SPARC基因敲除小鼠中的PDL胶原纤维也比WT PDL纤维显著更细。当用脂多糖刺激诱发牙周病时，SPARC基因敲除小鼠的牙槽骨和PDL胶原的损失更严重。为了解决细胞机制的减少稳态胶原蛋白含量和疾病的反应，在没有胶原蛋白的情况下，我们培养PDL成纤维细胞，并使用一种新的器官培养模型。我们的初步结果表明，无论是在体外和离体，SPARC无效PDL成纤维细胞有一个缺陷，在前胶原处理，如由无效细胞和组织中的前胶原的积累，这是不明显的WT条件。此外，发现在没有胶原蛋白的情况下，前胶原蛋白的增加与细胞表面相关。这些结果表明，胶原蛋白的作用，以减少胶原蛋白与细胞表面受体的相互作用，并通过细胞表面受体的胶原蛋白的参与阻碍了有效的前胶原蛋白的加工。这些特定目的将检验以下假设：胶原蛋白通过与胶原蛋白受体结合竞争介导PDL成纤维细胞中的胶原蛋白稳态，胶原蛋白受体结合是有效的前胶原蛋白加工和随后的胶原蛋白原纤维掺入所需的活性。将缺乏胶原结合的突变体β 2受体转染到SPARC-无效PDL成纤维细胞中，以确定β 2受体的胶原结合能力是否是调节前胶原加工所必需的。此外，腺病毒构建体将用于敲低WT和无SPARC的PDL中已知与β-胶原共享胶原结合位点的两种胶原细胞受体，整联蛋白2和盘状结构域受体（DDR）2。我们预测，敲低整合素2和DDR2将促进WT和无SPARC 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，本项目将确定β 2，DDR2和整合素2对前胶原加工和胶原纤维形成的作用。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。 公共卫生相关性：本项目的重点是牙周膜中的前胶原加工和纤维组装的机制。由于牙周膜在纤维化组织中高度表达，因此了解牙周膜作用的机制可以确定纤维化治疗以及牙周膜修复的潜在靶点，理想情况下，可以确定这一重要组织的再生。

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.