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; 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 身体在柠檬酸盐的可用性和消除之间保持足够的平衡，具体取决于 生理需求，并由饮食，肾脏清除，细胞代谢和骨骼重塑确定。柠檬酸盐是 所有有氧生物都用来产生可用的化学能，并以惊人的高度存在于骨骼中 浓度（1-5 wt％）。实际上，两项使用高分辨率NMR对柠檬酸盐建模的独立研究 磷灰石晶体内的分子表明，柠檬酸含量及其空间取向 在矿物结构中，对于维持有利的生物力学特性至关重要。这些观察 提示几个基本问​​题，这些问题构成了该建议的基础：1）柠檬酸盐的机制是什么 传递到骨头？ 2）在骨骼中柠檬酸盐的分配如何影响全球柠檬酸盐处理？ 3）如何 这个分区受到监管吗？ 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 共转运蛋白，溶质载体家族13成员5（SLC13A5），矿化成骨细胞。干扰 SLC13A5介导的柠檬酸盐转运，无论是遗传学还是药理，都会破坏成骨细胞介导 矿物成核。缺乏SLC13A5的小鼠显示血清和柠檬酸尿液水平增加，骨体积降低 和质量以及牙齿搪瓷的缺陷，病理特征类似于人类在突变中看到的特征 在SLC13A5中。有趣的是，代谢通量分析显示13c-葡萄糖衍生的13C-柠檬酸盐的高程 （M+2）在SLC13A5无效成骨细胞沉积的磷灰石中，该细胞分配给了增加的线粒体柠檬酸盐 生产和出口。此外，我们发现SLC13A5表达受到钙化的强烈调节 甲状旁腺激素（PTH）。这些发现表明存在成骨细胞特定机制 控制柠檬酸盐向骨骼以及全身柠檬酸盐的可用性控制。具体来说， 我们假设柠檬酸膜转运蛋白转运蛋白SLC13A5感受到细胞外柠檬酸盐的浓度 并使成骨细胞在细胞外柠檬酸盐水平时调整其内源柠檬酸盐的产生 下降或响应调节激素（例如PTH）的钙。 开发了三个目标来评估人类SLC13A5下游的新代谢途径 使用HIPSC和源自SLC13A5疾病患者的原发性牙齿的疾病模型，并定义 SLC13A5在生理条件下或对PTH响应的血液和骨骼之间在柠檬酸盐分配中的作用。 作为一名年轻科学家，我的最终目标是进行生产研究，以提供科学见解 骨骼矿化和这些机制在一般生理学中的整合。我的职业发展 计划通过可靠的指导，合作和培训机会量身定制了这一目标。在 与机构支持的结合，我相信我的申请中概述的研究/活动将有所帮助 建立在我现有的技能基础上，并促进我向独立调查员的过渡。