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

摘要 由于衰老、瘫痪或长期太空飞行影响而导致的低骨量发展 所有年龄段的许多人。目前的治疗方法旨在减缓骨量丢失，但目前没有一种针对骨丢失的治疗方法。 在这些情况下，缺乏导致骨量下降的机械负荷。我们最近描述了一个 NADPH氧化酶2（NOX 2）在体外产生活性氧的新途径 流体剪切应力的开始，进而激活瞬时受体电位香草酸4（TRPV 4）通道， 钙离子流入。这最终导致钙/钙调蛋白依赖性激酶II（CaMKII）活化， 硬化蛋白降解，解除对成骨细胞的抑制以允许骨形成。虽然机械- 已经在体外骨细胞中描述了控制硬化蛋白丰度的转导级联， 该信号传导途径对硬化蛋白调节和骨质量的影响尚未在体内检测。 这项提案将研究如何损失的NOX 2，一个必要的早期步骤，在机械转导级联， 在体内动物模型中，在非刺激和机械刺激条件下影响骨质量。 在体外和体内的初步数据的支持下，我们假设， 在体外控制sclerostin在体内是保守的，并且破坏NOX 2依赖的ROS产生将 影响基础骨特性并损害骨获得以响应机械负荷。我们会解决这个问题 三个独立实验的一个目的的假设：（1）检查骨细胞中的NOX 2缺失如何 影响体内的基础骨特性和机械反应性。考虑到我们之前的体外研究 这一途径，这一机械传导途径的贡献，在体内骨机械反应 仍然没有解决。为了扩展这些体外研究结果，本提案将使用体外和体内模型， 特别是一个条件删除模型，以解决几个知识差距，包括细胞自主作用 骨细胞中的NOX 2以及NOX 2介导的ROS对骨骼生理和功能的贡献。这 一项提案将验证新的治疗靶点，如ROS的产生，这可以用来模拟负载 在传统的机械负荷是不可能的人群中。

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.