Influence of Genetic Background on Bone Anabolic Response to Mechanical Loading

总结 骨质疏松症是一种低骨量和骨折风险增加的遗传性疾病。药物可用于 治疗低骨量，但它们有风险，适应症有限。因此，仍然需要新的 预防和治疗骨质疏松症。机械负荷可以建立和维持骨量， 在动物中是一种有效的骨合成代谢刺激物。对小鼠骨骼负荷的研究提供了一个 在基因、细胞和组织水平上研究骨锚定的强大平台。然而，迄今为止， 由于缺乏遗传多样性，大多数小鼠骨负荷研究都是使用近交系进行的。 C57 B1/6（B6）小鼠，以及少数使用多个品系的小鼠，使用遗传多样性有限的小鼠。因此，在本发明中， 关于遗传背景对骨对机械刺激的合成代谢反应的贡献， 加载中我们的目标是检验骨对机械负荷的反应是 可遗传的（即，取决于基因背景）。我们将用来自八个基因不同的小鼠来测试这个 近交系小鼠品系，包括五种常见的实验室品系和三种野生衍生品系。这些菌株 占小鼠基因组变异的89%。在目标1中，我们将应用应变匹配的胫骨载荷 8个近交系的成年小鼠，并确定皮质骨形成反应。我们将 包括雌性和雄性小鼠，以允许评估基因型和性别主效应，以及性别-基因型 交互.然后，我们将对鉴定为低应答和高应答的小鼠品系进行RNA-seq分析 以探索可能驱动差异加载响应的转录组差异。骨骼 负载驱动骨细胞腔隙小管网络（LCN）中的流体流动，并且人们普遍认为， 骨细胞吸收这种流体流动刺激并调节对负荷的合成代谢反应。更改 LCN的发生与骨质疏松和衰老有关，可能改变骨细胞的微力学环境， 将合成代谢反应转变为负荷。在目标2中，我们建议从以下方面表征骨中的LCN形态： 八个近交系，并测试这些性状是否在品系之间变化。我们将使用亚微米 分辨率X射线显微镜和共聚焦成像来表征骨细胞LCN。我们将探索 LCN形态的菌株间变化是否与对以下物质的合成代谢反应的变化相关： 菌株之间的负载（来自目标1）。本提案适合作为R21探索性/开发性 格兰特.目标1是发展性的，它将确定骨骼对负荷的合成代谢反应是否是可遗传的。如果 因此，它将为未来使用遗传多样性小鼠来发现基因的大规模研究提供理论基础。 影响骨骼对负荷的反应。目的2：评估骨细胞LCN形态的遗传度， 这也可能促使未来的研究确定致病基因。它还将探索一个基本的 骨力学生物学中的一个问题-骨对负荷的反应是否取决于骨细胞LCN 形态学

SUMMARY Osteoporosis is a heritable disease of low bone mass and increased fracture risk. Drugs are available to treat low bone mass, yet they carry risks and have limited indications. Thus, there remains a need for new approaches to prevent and treat osteoporosis. Mechanical loading can build and maintain bone mass in humans, and is a potent bone anabolic stimulus in animals. Studies of skeletal loading in mice provide a powerful platform to study bone anabolism at the gene, cell and tissue levels. Yet studies to date have been limited by a lack of genetic diversity – most murine bone loading studies have been done using inbred C57Bl/6 (B6) mice, and the few done with multiple strains used mice with limited genetic diversity. Thus, little is known about the contribution of genetic background to bone's anabolic response to mechanical loading. Our goal is to test the overall hypothesis that the response of bone to mechanical loading is heritable (i.e., depends on genetic background). We will test this using mice from eight genetically diverse inbred mouse strains, including five common laboratory strains and three wild-derived strains. These strains account for 89% of the variation in the mouse genome. In Aim 1, we will apply strain-matched tibial loading to young-adult mice of the eight inbred strains, and determine cortical bone formation responses. We will include female and male mice, to allow assessment of genotype and sex main effects, and sex-genotype interactions. We will then perform RNA-seq analysis on mouse strains identified as low and high responders in order to explore the transcriptomic differences that may drive the differential loading responses. Skeletal loading drives fluid flow in the osteocyte lacunocanalicular network (LCN), and it is widely believed that osteocytes transduce this fluid flow stimulus and regulate the anabolic response to loading. Changes to the LCN occur with osteoporosis and aging, and may alter the osteocyte micromechanical environment, and in turn the anabolic response to loading. In Aim 2, we propose to characterize LCN morphology in bones from the eight inbred strains, and test whether these traits vary between strains. We will use sub-micron resolution X-ray microscopy and confocal imaging to characterize the osteocyte LCN. We will then explore whether inter-strain variations in LCN morphology are associated with variations in anabolic response to loading between strains (from Aim 1). This proposal is appropriate as an R21 Exploratory/ Developmental grant. Aim 1 is developmental; it will establish whether bone's anabolic response to loading is heritable. If so, it will provide rationale for large-scale future studies using genetically diverse mice to discover genes that influence bone's response to loading. Aim 2 will assess the heritability of osteocyte LCN morphology, which may likewise motivate future studies to identify causal genes. It will also explore a fundamental question in bone mechanobiology – whether bone's response to loading depends on osteocyte LCN morphology.