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

目前正在努力开发新的策略来刺激骨折修复。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益处 给病人。当研究Cyp 24 a1缺陷小鼠的骨愈合时， 代谢产物24，25（OH）2D，我们已经测量了一个显着的，可重复的损害愈伤组织形成过程中， 骨折修复外源性24，25（OH）2D可纠正愈伤组织形成缺陷。我们 随后克隆了Fam 57 b2，其编码与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FAM 57 B2缺陷的小鼠表现出相同的受损骨痂 与Cyp 24 a1缺陷小鼠相比， FAM 57 B2含有一个结构域，表明在鞘脂合成酶中具有潜在的酶活性。 通路我们已经测量了FAM 57 B2的乳糖神经酰胺（LacCer）的24，25（OH）2D依赖性生产， 支持维生素D代谢物对FAM 57 B2酶活性的变构调节。 我们的研究结果有力地表明，FAM 57 B2是骨折过程中24，25（OH）2D介导的信号传导的效应子 修复.我们假设FAM 57 B2是一种乳糖神经酰胺合酶，其酶活性是 受24，25（OH）2D结合刺激，LacCer作为第二信使优化 骨折修复过程中的软骨内骨化。我们确定了以下具体目标，以测试 使用分子、生物化学和遗传学方法的组合提出的假设： 1.鉴定FAM 57 B2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.通过Cyp 24 a1的细胞类型特异性失活来确定骨折修复中24，25（OH）2D的来源。 4.表征和拯救软骨细胞特异性Fam 57 b缺陷小鼠的骨折修复表型。 我们将研究FAM 57 B2蛋白的结构-功能关系，并确认其变构调节作用 它的酶活性。将用LacCer处理软骨细胞，以检查对细胞反应的影响。 Cyp 24 a1将在软骨细胞、成骨细胞或巨噬细胞中失活，以确定 24，25（OH）2D的合成用于最佳的骨折愈合。拯救的愈伤组织形成缺陷表型的 通过施用LacCer的软骨细胞特异性Fam 57 b缺陷小鼠将证实它作为第二个 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，包括组织形态学、基因表达监测和生物力学测试。 本研究旨在阐明24，25（OH）2D及其效应子的生理作用 分子FAM 57 B2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的治疗管理。

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.