Role of modulators of Pi/PPi in cementum formation and regeneration

摘要 适当的焦磷酸盐调节在矿化过程中是至关重要的。焦磷酸盐(PPI)是一种直接 抑制羟基磷灰石晶体生长，从而抑制矿化。PPI的一个主要来源是ATP，它是 被胞外核苷酸焦磷酸酶磷酸二酯酶(ENPP)水解，包括ENPP1。ENPP1是 与进行性强直蛋白(ANK)一起在骨骼中表达，ANK将细胞内的PPI转运到 细胞外空间。ANK和ENPP1增加细胞外PPI，组织非特异性碱性 磷酸酶(TNAP，由ALPL编码)将PPI水解成无机磷(PI)，从而降低PPI。 细胞外空间。尽管ANK、ENPP1和TNAP之间直接或间接相互作用的细节 这些因素中的任何一个的丧失对矿化组织都有显著的影响。这项研究建议 本文旨在阐明焦磷酸盐调节剂的功能，其最终目标是 开发针对矿化障碍的治疗方法。 PPI调节异常(如具有ENPP1、ANK或ALPL突变的个体所表现的)可导致 牙周组织(牙骨质、牙周韧带、牙槽骨)，特别是牙骨质的改变。 改变包括过多的牙骨质形成(ENPP1，ANK)或缺乏牙骨质形成导致 牙齿过早剥落(ALPL)，一种未经治疗的牙周病的不良后遗症。牙周 疾病是一种具有重大心理社会和经济后果的全球负担，其特点是 牙周组织。现有的再生疗法是不可预测的，并且不以牙骨质再生为目标。至 研究Ank和Enpp1在牙骨质形成中的作用，我和我的同事们开发了Ank， Enpp1双突变小鼠。在初步研究中，我们注意到一种更明显的骨水泥增高症表型 8周龄Ank，Enpp1双突变体与单突变体。此外，我们注意到两者之间的明显差异 兄弟蛋白的基因/蛋白表达在PDL区/牙骨质内的单突变和双突变， 已知的矿化过程的调节剂。制定了三个目标来定义ANK和ENPP1角色 在牙周发育和维护中，确定ANK和ENPP1的机械途径 调控牙骨质形成(例如，侧重于直接和间接调节兄弟姐妹的能力)，并确定 Enpp1抑制剂用于牙周组织再生的可行性。 作为一名临床科学家，我的最终目标是进行富有成效的研究，为 牙周病的发病机制，并将这一知识转化为切实的临床应用(例如 诊断/预后测试、预防战略和治疗干预)。我的职业发展计划 通过坚实的导师、协作和研究培训机会，为实现这一目标量身定做 和临床环境。在机构支持下，我有信心在我的 申请将有助于在我现有技能的基础上发展，并促进我向独立调查员的转变。

ABSTRACT Proper pyrophosphate regulation is critical in mineralization processes. Pyrophosphate (PPi) is a direct inhibitor of hydroxyapatite crystal growth, thus inhibiting mineralization. A main source of PPi is ATP, which is hydrolyzed by ectonucleotide pyrophosphatase phosphodiesterases (ENPP), including ENPP1. ENPP1 is expressed in bone along with progressive ankylosis protein (ANK), which transports intracellular PPi to extracellular spaces. While ANK and ENPP1 increase extracellular PPi, tissue non-specific alkaline phosphatase (TNAP, encoded by ALPL), hydrolyzes PPi into inorganic phosphate (Pi), thus decreasing PPi in extracellular spaces. Although the specifics of direct or indirect interactions between ANK, ENPP1, and TNAP are unknown, loss of any of these factors has dramatic effects on mineralized tissue. The studies proposed here were developed to elucidate functions of pyrophosphate regulators with the ultimate goal of developing therapies targeted toward mineralization disorders. Abnormal PPi regulation (as exhibited by individuals with ENPP1, ANK or ALPL mutations) can lead to alterations in the periodontium (cementum, periodontal ligament, alveolar bone), specifically cementum. Alterations include excessive cementum formation (ENPP1, ANK) or deficient cementum formation leading to premature tooth exfoliation (ALPL), an adverse sequelae of untreated periodontal disease. Periodontal disease, a global burden with significant psychosocial and financial consequences, features destruction of the periodontium. Existing regenerative therapies are unpredictable and do not target cementum regeneration. To investigate roles of Ank and Enpp1 during cementogenesis, I, along with my colleagues, developed Ank, Enpp1 double mutant mice. In preliminary studies, we noted a more dramatic hypercementosis phenotype in 8wk old Ank, Enpp1 double mutants versus single mutants. Additionally, we noted distinct differences between single and double mutants within the PDL region/cementum in gene/protein expression of SIBLING proteins, known modulators of the mineralization process. Three aims were developed to define ANK and ENPP1 roles in periodontal development and maintenance, identify mechanistic pathways in which ANK and ENPP1 regulate cementogenesis (e.g. focused on direct and indirect ability to modulate SIBLINGs), and determine the feasibility of using Enpp1 inhibitors to regenerate periodontaltissues. As a clinician scientist, my ultimate goal is to conduct productive research that provides scientific insights into periodontal disease pathogenesis and translate this knowledge into tangible clinical applications (e.g. diagnostic/prognostic tests, prevention strategies, and therapeutic interventions). My career development plan has been tailored toward this goal with solid mentorship, collaborations, and training opportunities in research and clinical settings. In conjunction with institutional support, I am confident studies/activities outlined in my application will help build upon my existing skillset and facilitate my transition into an independent investigator.