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至关重要。焦磷酸盐（PPi）是一种直接 羟基磷灰石晶体生长的抑制剂，从而抑制矿化。PPi的主要来源是ATP，即 被外核苷酸焦磷酸酶磷酸二酯酶（ENPP）水解，包括ENPP 1。ENPP 1是 在骨中沿着进行性关节强直蛋白（ANK）表达，ANK将细胞内PPi转运至 细胞外间隙虽然ANK和ENPP 1增加细胞外PPi，组织非特异性碱性磷酸酶， 磷酸酶（TNAP，由ALPL编码）将PPi水解成无机磷酸盐（Pi），从而降低PPi在 细胞外间隙尽管ANK、ENPP 1和TNAP之间的直接或间接相互作用的具体细节 这些因素中的任何一个的缺失对矿化组织都有显著的影响。建议的研究 本文旨在阐明焦磷酸调节剂的功能，其最终目标是 开发针对矿化障碍的疗法。 异常的PPi调节（如ENPP 1、ANK或ALPL突变个体所表现的）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。 改变包括过度牙骨质形成（ENPP 1，ANK）或牙骨质形成不足，导致 过早的牙齿脱落（ALPL），未经治疗的牙周病的不良后遗症。牙周 疾病是一种全球性负担，具有重大的心理社会和经济后果，其特点是破坏了 牙周组织现有的再生疗法是不可预测的，并且不针对牙骨质再生。到 为了研究Ank和Enpp 1在牙骨质形成中作用，我沿着我的同事开发了Ank， Enpp 1双突变小鼠。在初步的研究中，我们注意到一个更显着的高牙骨质表型， 8周龄Ank，Enpp 1双突变体与单突变体。此外，我们注意到， PDL区域/牙骨质内的SIBLING蛋白的基因/蛋白表达中的单突变体和双突变体， 已知的矿化过程调节剂。制定了三个目标来定义ANK和ENPP 1的作用 在牙周发育和维护中，确定ANK和ENPP 1 调节牙骨质形成（例如，关注调节SIBLING的直接和间接能力），并确定 Enpp 1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，提供科学见解， 牙周病的发病机制，并将这些知识转化为有形的临床应用（例如， 诊断/预后测试、预防策略和治疗干预）。我的职业发展计划 已经为实现这一目标量身定制了坚实的指导，合作和研究培训机会 和临床环境。在机构的支持下，我有信心在我的研究/活动概述 申请将有助于建立在我现有的技能，并促进我过渡到一个独立的调查员。

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.