Mechanisms and impact of osteoblast "citration" on skeletal mineralization and global citrate homeostasis

成骨细胞“柠檬化”对骨骼矿化和整体柠檬酸盐稳态的机制和影响

• 批准号: 10448647
• 负责人: Naomi Dirckx
• 金额: $ 8.54万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10448647
• 关键词: Aerobic; Apatites; Biocompatible Materials; Biomechanics; Blood; Blood Circulation; Bone Resorption; Bone remodeling; Calcium; Cells; Cellular Metabolic Process; Chelating Agents; Chemicals; Citrates; Citric Acid Cycle; Collaborations; Collagen; Collagen Type I; Crystallization; Cues; DNA Sequence Alteration; Defect; Dental Enamel; Deposition; Development; Development Plans; Diabetes Mellitus; Diet; Disease; Disease model; Down-Regulation; Drops; Endocrine; Equilibrium; Family; Gatekeeping; Glucose; Goals; Homeostasis; Hormone Responsive; Hormones; Human; In Vitro; Intervention; Ions; Isotopes; Label; Link; Mediating; Membrane; Mentorship; Metabolic; Metabolic Diseases; Metabolic Pathway; Metabolism; Minerals; Mitochondria; Modeling; Mus; Mutation; Organism; Osteoblasts; Osteoporosis; PTH gene; Pathologic; Patients; Pharmacologic Substance; Pharmacology; Physiologic calcification; Physiological; Physiology; Production; Property; Radio; Regulation; Renal clearance function; Research; Research Personnel; Resolution; Role; Scientist; Secondary to; Serum; Signal Transduction; Skeleton; Solid; Space Perception; Structure; Testing; Tissues; Tooth structure; bone; bone mass; bone quality; career development; chelation; citrate carrier; deciduous tooth; extracellular; fracture risk; genetic manipulation; human disease; improved; in vivo; insight; mechanical properties; member; men; metabolic profile; mineralization; mouse model; nanocomposite; normal aging; novel; osteogenic; prevent; rare genetic disorder; response; skeletal; solid state nuclear magnetic resonance; solute; success; training opportunity; uptake; urinary

ABSTRACT The body maintains an adequate balance between citrate availability and elimination, depending on physiological needs and determined by diet, renal clearance, cell metabolism and bone remodeling. Citrate is used by all aerobic organisms to produce usable chemical energy and is present in bone at strikingly high concentrations (1-5 wt%). In fact, two independent studies using high resolution NMR to model the citrate molecule within the apatite crystal suggest that the degree of citrate incorporation, as well as its spatial orientation within the mineral structure, is critical for maintaining favorable biomechanical properties. These observations prompt several fundamental questions that form the basis for this proposal: 1) What is the mechanism for citrate delivery to bone?; 2) How does the partitioning of citrate in bone influence global citrate handling?; and 3) how is this partitioning regulated? In preliminary studies, we demonstrate functional expression of a membranous extracellular Na+/citrate cotransporter, Solute Carrier Family 13 Member 5 (Slc13a5), in mineralizing osteoblasts. Interference of SLC13A5-mediated citrate transport, either genetically or pharmacologically, disrupts osteoblast mediated mineral nucleation. Mice lacking Slc13a5 show increased serum and urinary citrate levels, reduced bone volume and quality, and defects in tooth enamel, pathological features similar to those seen in humans with mutations in SLC13A5. Intriguingly, metabolic flux analysis revealed striking elevations in 13C-Glucose-derived 13C-Citrate (m+2) in apatite deposited by Slc13a5 null osteoblasts which was allocated to increased mitochondrial citrate production and export. Moreover, we found that Slc13a5 expression was strongly regulated by the calciotropic parathyroid hormone (PTH). These findings suggest the existence of an osteoblast specific mechanism that controls both the production and delivery of citrate to bone as well as systemic citrate availability. Specifically, we postulate that the membrane citrate transporter SLC13A5 senses extracellular citrate concentrations and enables the osteoblast to adjust its endogenous citrate production when extracellular citrate levels drop or in response to calcium regulating hormones such as PTH. Three aims were developed to assess our new metabolic pathway downstream of SLC13A5 in a human disease model using hiPSCs and primary teeth derived from patients with SLC13A5 disease and to define the role of SLC13A5 in citrate partitioning between blood and bone in physiological conditions or in response to PTH. As a young scientist, my ultimate goal is to conduct productive research that provides scientific insights into skeletal mineralization and the integration of these mechanisms in general physiology. My career development plan has been tailored toward this goal with solid mentorship, collaborations, and training opportunities. In conjunction with institutional support, I am confident that the studies/activities outlined in my application will help build upon my existing skillset and facilitate my transition into an independent investigator.

抽象的 身体在柠檬酸盐的利用和消除之间保持适当的平衡，具体取决于 生理需求并由饮食、肾脏清除、细胞代谢和骨重塑决定。柠檬酸盐是 所有需氧生物都利用它来产生可用的化学能，并且在骨骼中的含量非常高 浓度（1-5 wt%）。事实上，两项独立研究使用高分辨率 NMR 来模拟柠檬酸盐 磷灰石晶体内的分子表明柠檬酸盐的结合程度及其空间取向 在矿物结构内，对于保持良好的生物力学性能至关重要。这些观察 提出了构成该提案基础的几个基本问​​题：1）柠檬酸盐的作用机制是什么 输送到骨头？ 2）柠檬酸盐在骨骼中的分配如何影响整体柠檬酸盐处理？； 3）如何 这种分区是否受到监管？ 在初步研究中，我们证明了膜细胞外 Na+/柠檬酸盐的功能表达 协同转运蛋白，溶质载体家族 13 成员 5 (Slc13a5)，在矿化成骨细胞中。的干扰 SLC13A5 介导的柠檬酸转运，无论是遗传上还是药理学上，都会破坏成骨细胞介导的柠檬酸转运 矿物成核。缺乏 Slc13a5 的小鼠血清和尿柠檬酸盐水平升高，骨量减少 牙釉质的质量和缺陷，病理特征与突变人类相似 在SLC13A5中。有趣的是，代谢流分析显示 13C-葡萄糖衍生的 13C-柠檬酸盐显着升高 (m+2) 由 Slc13a5 无效成骨细胞沉积的磷灰石，分配给增加的线粒体柠檬酸盐 生产和出口。此外，我们发现Slc13a5的表达受到促钙蛋白的强烈调节。 甲状旁腺激素（PTH）。这些发现表明存在成骨细胞特异性机制 控制柠檬酸盐的产生和向骨骼的输送以及全身柠檬酸盐的可用性。具体来说， 我们假设膜柠檬酸转运蛋白 SLC13A5 感知细胞外柠檬酸浓度 当细胞外柠檬酸盐水平升高时，使成骨细胞能够调整其内源性柠檬酸盐的产生 下降或对钙调节激素（如 PTH）做出反应。 制定了三个目标来评估人类 SLC13A5 下游的新代谢途径 使用来自 SLC13A5 疾病患者的 hiPSC 和乳牙建立疾病模型，并定义 生理条件下或响应 PTH 时，SLC13A5 在血液和骨骼之间柠檬酸盐分配中的作用。 作为一名年轻的科学家，我的最终目标是进行富有成效的研究，为以下问题提供科学见解 骨骼矿化以及这些机制在一般生理学中的整合。我的职业发展 该计划是通过坚实的指导、合作和培训机会来实现这一目标的。在 与机构支持相结合，我相信我的申请中概述的研究/活动将有所帮助 以我现有的技能为基础，促进我向独立调查员的转变。