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: 8878032
• 负责人: Jessica Marie Trombetta-eSilva
• 金额: $ 4.85万
• 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 2016-07-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8878032
• 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水泥并进入牙槽骨，将牙齿连接到骨窝。与其他富含胶原蛋白的组织相比，PDL具有较高的细胞外基质更新率，其特征是胶原蛋白的合成，加工和沉积，除了吸收不溶性胶原蛋白。因此，PDL提供了一个出色的组织环境，用于研究procollagen加工的机制。 SPARC是一种胶原蛋白结合蛋白，已被确定为胶原ECM沉积中的关键因素。因此，我们报道说，与年龄匹配的野生型（WT）PDL相比，PDL的SPARC-NULL小鼠的胶原蛋白明显较小。有趣的是，SPARC-NULL小鼠中的PDL胶原纤维也比WT PDL纤维明显薄。当受到脂多糖诱导牙周疾病的挑战时，Sparc-null小鼠患有更严重的疾病，如肺泡骨和PDL胶原蛋白的损失所衡量。为了解决稳态胶原蛋白含量降低的细胞机制和在没有SPARC的情况下对疾病的反应，我们培养了PDL成纤维细胞并使用了一种新型的器官培养模型。我们的初步结果表明，在体外和离体中，SPARC-NULL PDL成纤维细胞在Procollagen加工方面缺乏，如在零细胞和组织中储原氮的积累所表明的那样，在WT条件下并不明显。此外，在没有SPARC的情况下，发现了与细胞表面相关的Procollagen的增加。这些结果表明，SPARC的作用可减少与细胞表面受体的胶原蛋白相互作用，并且细胞表面受体的胶原蛋白接合阻碍了有效的procollagen加工。这些具体目的将检验以下假设，即SPARC通过与胶原蛋白受体参与竞争，在PDL成纤维细胞中介导胶原蛋白稳态，这是有效的Procollagen加工所需的活动以及随后的胶原原纤维掺入。缺乏胶原蛋白结合的突变体SPARC将被转染到SPARC-NULL PDL成纤维细胞中，以确定是否需要SPARC的胶原蛋白结合能力来调节Procollagen加工。此外，在WT和SPARC-NULL PDL中，腺病毒构建体将用于敲低两个已知可与SPARC，Integin 2和盘状蛋白结构域受体（DDR）2共享胶原蛋白结合位点的胶原蛋白细胞受体。我们预测，整联蛋白2和DDR2的敲低将促进WT和SPARC-NULL PDL中的Procollagen加工，从而降低了胶原蛋白的胶原蛋白水平，并增加了完全成熟的胶原蛋白的水平。此外，我们将研究DDR2和Integin 2受Exdibril组装模型的DDR2和Integin 2受体的结果。因此，该项目将确定SPARC，DDR2和整联蛋白2对胶原蛋白纤维形成的作用。这里提出的培训计划将检验上述假设，并为我做好学术科学家的职业做好准备。