Optimal bone fracture repair requires 24,25-dihydroxyvitamin D and its effector molecule, FAM57B2

最佳骨折修复需要 24,25-二羟基维生素 D 及其效应分子 FAM57B2

• 批准号: 9156097
• 负责人: Rene St-Arnaud
• 金额: $ 23.08万
• 依托单位: MCGILL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9156097
• 关键词: Acyl Coenzyme A; Allosteric Regulation; Animals; Binding; Biochemical Genetics; Biological; Biology; Biomechanics; Bone Injury; Bone Regeneration; Bone callus; Cartilage; Cell physiology; Ceramides; Chickens; Chondrocytes; Conflict (Psychology); Cytochrome P450; Data; Defect; Dihydroxycholecalciferols; Enzymes; Exhibits; Fracture; Fracture Healing; Gene Expression Profiling; Glucosylceramides; Glycosphingolipids; Goals; Healed; Hormones; Impaired wound healing; Impairment; Integral Membrane Protein; Laboratories; Lactosylceramides; Lead; Ligand Binding; Measures; Mechanics; Mediating; Membrane; Molecular; Mouse Strains; Mus; Operative Surgical Procedures; Osteoblasts; Osteotomy; Outcome; Pathway interactions; Patients; Phenotype; Physiological; Positioning Attribute; Pre-Clinical Model; Production; Property; Proteins; Public Health; Reporting; Research; Role; Second Messenger Systems; Serum; Signal Transduction; Signaling Molecule; Site; Skin; Source; Sphingolipids; Structure; Structure-Activity Relationship; Surgical Models; Techniques; Testing; Therapeutic; Tibial Fractures; Tissues; Transgenic Organisms; Variant; Vitamin D; Work; analog; bone healing; cell type; cohort; dihydroceramide desaturase; genetic approach; healing; human data; improved; macrophage; novel; novel strategies; promoter; repaired; response; sample fixation; second messenger; stem; tomography

There is an emerging effort to develop novel strategies to stimulate bone fracture repair. A certain percentage of fractures display impaired healing, and any improvement in fracture treatment would be of considerable benefit to patients. When studying bone healing in Cyp24a1-deficient mice, which cannot synthesize the vitamin D metabolite, 24,25(OH)2D, we have measured a significant, reproducible impairment in callus formation during fracture repair. The callus formation defect can be corrected by exogenous administration of 24,25(OH)2D. We subsequently cloned Fam57b2, encoding a transmembrane protein that specifically interacts with 24,25(OH)2D. Our preliminary results show that mice deficient for FAM57B2 in chondrocytes exhibit the same impaired callus formation during fracture repair than Cyp24a1-deficient mice. FAM57B2 contains a domain that suggests a potential enzymatic activity within the sphingolipid synthetic pathway. We have measured 24,25(OH)2D-dependent production of lactosylceramide (LacCer) by FAM57B2, supporting allosteric regulation of the enzymatic activity of FAM57B2 by the vitamin D metabolite. Our results strongly suggest that FAM57B2 is an effector of 24,25(OH)2D-mediated signaling during fracture repair. We hypothesize that FAM57B2 is a lactosylceramide synthase whose enzymatic activity is stimulated by 24,25(OH)2D binding and that LacCer acts as a second messenger to optimize endochondral ossification during fracture repair. We have identified the following specific aims to test the proposed hypothesis using a combination of molecular, biochemical, and genetic approaches: 1. Identify the ligand-binding pocket and the enzymatic moiety of FAM57B2. 2. Examine the response of chondrocytes to treatment with LacCer. 3. Ascertain the source of 24,25(OH)2D in fracture repair using cell-type specific inactivation of Cyp24a1. 4. Characterize and rescue the fracture repair phenotype of chondrocyte-specific Fam57b-deficient mice. We will examine structure-function relationships of the FAM57B2 protein and confirm the allosteric regulation of its enzymatic activity. Chondrocytes will be treated with LacCer to examine impact on cellular responses. Cyp24a1 will be inactivated in chondrocytes, osteoblasts, or macrophages to determine the relevant site of synthesis of 24,25(OH)2D for optimal fracture healing. Rescue of the callus formation defect phenotype of chondrocyte-specific Fam57b-deficient mice by administration of LacCer will confirm that it acts as a second messenger to transduce the 24,25(OH)2D signal. Bone healing during fracture repair will be assessed using an array of techniques including histomorphometry, gene expression monitoring, and biomechanical testing. The research that we propose will demonstrate the physiological role of 24,25(OH)2D and its effector molecule FAM57B2 in fracture repair. Our work could lead to the use of 24,25(OH)2D, suitable analogs, or lactosylceramide in the therapeutic management of fracture and surgical osteotomy healing.

有一种新的努力正在努力开发新的策略来刺激骨折修复。一定的百分比 骨折显示愈合不良，任何骨折治疗的改进都将是相当大的好处 对病人来说。在研究无法合成维生素D的CYP24A1缺陷小鼠的骨愈合时 代谢物，24，25(OH)2D，我们在愈伤组织形成过程中测量到了显著的、可重复的损害。 骨折修复。外源24，25(OH)2D可以纠正愈伤组织的形成缺陷。我们 随后克隆了Fam57b2，编码一种与24，25(OH)2D特异相互作用的跨膜蛋白。 我们的初步结果显示，软骨细胞中FAM57B2缺乏的小鼠表现出相同的受损骨痂 在骨折修复过程中形成的细胞比CYP24A1缺陷小鼠更多。 FAM57B2含有一个结构域，表明在鞘脂合成物中具有潜在的酶活性 路径。我们测量了FAM57B2对24，25(OH)2D依赖的乳糖基神经酰胺(LacCer)的产生。 支持维生素D代谢物对FAM57B2酶活性的变构调节。 我们的结果有力地表明，FAM57B2是24，25(OH)2D介导的骨折信号的效应者 修理。我们推测FAM57B2是一种乳糖基神经酰胺合成酶，其酶活性为 受24，25(OH)2D结合刺激，LacCer作为第二信使优化 骨折修复过程中软骨内骨化。我们已经确定了以下具体目标来测试 结合分子、生物化学和遗传学方法提出的假说： 1.鉴定FAM57B2的配体结合口袋和酶部分。 2.检测软骨细胞对LacCer处理的反应。 3.通过细胞类型特异性的CYP24A1失活，确定骨折修复中24，25(OH)2D的来源。 4.软骨细胞特异性Fam57b基因缺陷小鼠骨折修复表型的鉴定和挽救。 我们将研究FAM57B2蛋白的结构-功能关系，并确认变构调节 它的酶活性。软骨细胞将用LacCer处理，以检查对细胞反应的影响。 将在软骨细胞、成骨细胞或巨噬细胞中灭活细胞色素P24A1以确定相关位置 合成24，25(OH)2D以实现最佳的骨折愈合。愈伤组织形成缺陷表型的挽救 给予LacCer的软骨细胞特异性Fam57b缺陷小鼠将确认它作为第二个 信使来转换24，25(OH)2D信号。骨折修复过程中的骨愈合将使用 一系列技术，包括组织形态计量学、基因表达监测和生物力学测试。 我们提出的研究将证明24，25(OH)2D及其效应器的生理作用 FAM57B2分子与骨折修复我们的工作可能导致使用24，25(OH)2D，适当的类似物，或 乳糖基神经酰胺在骨折治疗和截骨术愈合中的应用。