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: 9334111
• 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/9334111
• 关键词: Acyl Coenzyme A; Allosteric Regulation; Animals; Annual Reports; 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; Hormones; Impaired wound healing; Impairment; Integral Membrane Protein; Laboratories; Lactosylceramides; Lead; Ligand Binding; Measures; Mediating; Membrane; Molecular; Mouse Strains; Mus; Operative Surgical Procedures; Osteoblasts; Osteotomy; Outcome; Pathway interactions; Patients; Phenotype; Physiologic Ossification; Physiological; Positioning Attribute; Pre-Clinical Model; Production; Property; Proteins; Public Health; Reporting; Reproducibility; 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; mechanical properties; novel; novel strategies; promoter; repaired; response; retinal rods; sample fixation; 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.

人们正在努力开发刺激骨折修复的新策略。一定比例 的骨折显示愈合受损，骨折治疗的任何改进都将带来相当大的好处 给患者。研究 Cyp24a1 缺陷小鼠的骨愈合时，该小鼠无法合成维生素 D 代谢物 24,25(OH)2D，我们测量到愈伤组织形成过程中存在显着的、可重复的损伤 骨折修复。愈伤组织形成缺陷可以通过外源施用24,25(OH)2D来纠正。我们 随后克隆了 Fam57b2，编码与 24,25(OH)2D 特异性相互作用的跨膜蛋白。 我们的初步结果表明，软骨细胞中缺乏 FAM57B2 的小鼠表现出同样受损的愈伤组织 与 Cyp24a1 缺陷小鼠相比，骨折修复过程中的形成率更高。 FAM57B2 包含一个结构域，表明鞘脂合成物中具有潜在的酶活性 途径。我们测量了 FAM57B2 依赖于 24,25(OH)2D 的乳糖神经酰胺 (LacCer) 生产， 支持维生素 D 代谢物对 FAM57B2 酶活性的变构调节。 我们的结果强烈表明 FAM57B2 是骨折期间 24,25(OH)2D 介导的信号传导的效应器 维修。我们假设 FAM57B2 是一种乳糖神经酰胺合酶，其酶活性为 由 24,25(OH)2D 结合刺激，LacCer 作为第二信使进行优化 骨折修复过程中软骨内骨化。我们确定了以下具体目标来测试 结合分子、生化和遗传学方法提出的假设： 1. 识别 FAM57B2 的配体结合口袋和酶部分。 2. 检查软骨细胞对 LacCer 治疗的反应。 3. 使用 Cyp24a1 的细胞类型特异性失活确定骨折修复中 24,25(OH)2D 的来源。 4. 表征并挽救软骨细胞特异性 Fam57b 缺陷小鼠的骨折修复表型。 我们将检查 FAM57B2 蛋白的结构-功能关系并确认变构调节 其酶活性。将用 LacCer 处理软骨细胞以检查对细胞反应的影响。 Cyp24a1 将在软骨细胞、成骨细胞或巨噬细胞中失活，以确定相关位点 合成 24,25(OH)2D 以实现最佳骨折愈合。愈伤组织形成缺陷表型的拯救 通过给予 LacCer 软骨细胞特异性 Fam57b 缺陷小鼠将证实它作为第二个 转导 24,25(OH)2D 信号的信使。骨折修复期间的骨愈合将使用 一系列技术，包括组织形态测量、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 及其效应子的生理作用 分子 FAM57B2 在骨折修复中的作用。我们的工作可能导致使用 24,25(OH)2D、合适的类似物，或 乳糖神经酰胺在骨折和手术截骨愈合治疗中的应用。