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.

摘要 人体在柠檬酸盐的可获得性和排除性之间保持适当的平衡，这取决于 生理需要，并由饮食、肾脏清除、细胞代谢和骨重建决定。柠檬酸盐是 被所有好氧生物用来产生可用的化学能，在骨骼中的含量高得惊人 浓度(1-5wt%)。事实上，两项使用高分辨率核磁共振对柠檬酸盐进行建模的独立研究 磷灰石晶体中的分子表明柠檬酸盐的掺入程度以及它的空间取向 在矿物结构内，是保持良好的生物力学性能的关键。这些观察结果 提出几个构成这一提议基础的基本问题：1)柠檬酸的作用机制是什么 向骨骼输送？2)柠檬酸在骨骼中的分配如何影响全局柠檬酸盐的处理？ 这种分区受监管吗？ 在初步研究中，我们展示了细胞膜外Na+/柠檬酸的功能表达。 共转运蛋白，溶质载体家族13成员5(SLC13a5)，在成骨细胞矿化中的作用。的干扰 SLC13A5介导的柠檬酸转运，无论是遗传上还是药物上，都破坏了成骨细胞介导的 矿物成核作用。缺乏SLc13a5的小鼠表现出血和尿柠檬酸水平升高，骨量减少 以及牙釉质的质量和缺陷，这些病理特征类似于人类的突变 在SLC13A5中。有趣的是，代谢流量分析显示13C-葡萄糖衍生的13C-柠檬酸盐显著升高 (M+2)在SLc13a5缺失成骨细胞沉积的磷灰石中，分配给增加的线粒体柠檬酸 生产和出口。此外，我们还发现slc13a5的表达受到亲钙性的强烈调控。 甲状旁腺素(PTH)。这些发现表明存在一种成骨细胞特有的机制， 控制柠檬酸盐的生产和向骨骼的输送，以及全身柠檬酸盐的可利用性。具体来说， 我们推测膜柠檬酸转运体SLC13A5感知细胞外柠檬酸浓度 并使成骨细胞能够在细胞外柠檬酸水平时调整其内源性柠檬酸盐的产生 下降或对钙调节激素如甲状旁腺素的反应。 开发了三个目标来评估我们在人类中SLC13A5下游的新代谢途径 使用来自SLC13A5疾病患者的HiPSCs和乳牙建立疾病模型，并定义 SLC13A5在生理条件下或甲状旁腺素反应中柠檬酸在血与骨之间分配中的作用。 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 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 计划是通过坚实的指导、协作和培训机会为这一目标量身定做的。在……里面 结合机构的支持，我相信我在申请表中概述的学习/活动将有所帮助 在我现有技能的基础上，促进我向独立调查员的转变。