The Role of Reactive Oxygen Species in Osteocyte Mechano-Transduction and Sclerostin Regulation

摘要 由于衰老、瘫痪而停用或长时间的太空飞行影响而导致的骨量减少 各个年龄段的许多人。目前的治疗方法旨在减缓骨量的丧失，但目前还没有一种疗法针对 在这些情况下，缺乏机械负荷会导致骨量下降。我们最近描述了一种 NADPH氧化酶2(NOX2)在体外产生活性氧的新途径 开始的流体剪应力，进而激活瞬时受体电位香草素4(TRPV4)通道，以允许 用于钙离子内流。这最终导致钙/钙调蛋白依赖的激酶II(CaMKII)激活，并 硬化素降解，解除对成骨细胞的抑制，以允许骨形成。尽管机械装置- 已描述了在体外培养的骨细胞中控制硬化素丰度的转导级联，保真度和 这一信号通路对硬化素调节和骨量的影响还没有在体内进行检测。 这项提案将研究NOX2的损失--机械转导级联反应中必要的早期步骤， 在活体动物模型中，在非刺激和机械刺激条件下影响骨质量。 在体外和体内初步数据的支持下，我们假设机械转导途径 在体外控制硬化素在体内是保守的，干扰依赖于NOX2的ROS的产生将 影响基础骨性能和损害骨获得对机械负荷的反应。我们将解决这一问题 一个目标的假设和三个独立的实验：(1)检查骨细胞中NOX2的缺失 在体内影响基础骨特性和力学反应。考虑到我们之前工作的体外性质， 这一途径，这一机械转导途径对体内骨力学反应的贡献 仍然悬而未决。为了扩展这些体外发现，这项提议将使用体外和体内模型， 特别是条件删除模型，以解决几个知识差距，包括细胞自治角色 NOX2在骨细胞中的表达以及NOX2介导的ROS对骨骼生理和功能的贡献。这 该提案将验证新的治疗靶点，例如ROS的产生，这些靶点可以用来模拟负荷 在传统机械负载不可能实现的人群中。

ABSTRACT The development of low bone mass caused by aging, disuse due to paralysis, or extended space flights impacts many individuals of all ages. Current therapeutics aim to slow the loss of bone mass, but none currently target the lack of mechanical loading that leads to declining bone mass in these situations. We have recently described a novel pathway in vitro in which NADPH Oxidase 2 (NOX2) produces reactive oxygen species following the onset of fluid shear stress that, in turn, activates transient receptor potential vanilloid 4 (TRPV4) channels to allow for calcium influx. This ultimately results in calcium/calmodulin-dependent kinase II (CaMKII) activation and sclerostin degradation, lifting the inhibition on osteoblasts to allow for bone formation. Though the mechano- transduction cascade controlling sclerostin abundance has been described in in vitro osteocytes, the fidelity and impact of this signaling pathway's impact on sclerostin regulation and bone mass has not been examined in vivo. This proposal will examine how the loss of NOX2, a necessary early step in the mechano-transduction cascade, affects bone quality, both in unstimulated and mechanically-stimulated conditions, in an in vivo animal model. Supported by in vitro and in vivo preliminary data, we hypothesize that the mechano-transduction pathway controlling sclerostin in vitro is conserved in vivo and that disrupting NOX2-dependent ROS production will affect basal bone properties and impair bone acquisition in response to mechanical load. We will address this hypothesis in one aim with three independent experiments: (1) Examine how NOX2 deletion in osteocytes impacts basal bone properties and mechano-responsiveness in vivo. Given the in vitro nature of our prior work on this pathway, the contribution of this mechano-transduction pathway to in vivo bone mechano-responsiveness remained unresolved. To extend these in vitro findings, this proposal will use in vitro and in vivo models, specifically a conditional deletion model, to address several knowledge gaps, including the cell autonomous role of NOX2 in osteocytes and the contribution of NOX2-mediated ROS to skeletal physiology and function. This proposal will validate novel therapeutic targets, such as ROS production, which can be exploited to mimic load in populations where traditional mechanical load is not possible.