Biophysical Regulation of Bone Remodeling

描述（由申请人提供）：骨的机械负荷启动抗分解代谢和合成代谢细胞反应，促进结构能力骨骼的形成。在竞争性更新中提出的工作将通过检查基因调控的新时间序列和破译这种合成代谢过程的编排是否通过单个启动信号级联产生，从而推进我们对骨骼负载反应的研究。我们的数据显示，机械应变调节由典型Wnt应答者组成的早期集群，随后是由Runx2、osterix （Osx）和eNOS代表的合成代谢基因集群。这种应变响应模式反映了基因对剪切力的响应，表明存在典型的生物力学响应。一个共同的信号通路涉及HRas/ERK1/2被假设来调节那些构成聚集反应的基因。这将在SA1中进行研究，比较应变和振荡剪切后的候选响应。我们的数据进一步表明了加载响应的时间模式：典型的¿-catenin靶响应在4小时时很强烈，但在18小时后恢复到基础水平，而Runx2和osterix的变化直到加载后18小时才可测量。Caveolin-1是脂质筏中的一种结构分子，通过限制catenin接近诱导其核易位的信号来调节catenin的活性。在施加应变的4小时内，沉默成骨细胞中的caveolin-1会加速负载诱导的Runx2和Osx的增加，我们认为这是通过增强¿-catenin信号传导来实现的。这表明-连环蛋白可能对后来的机械效应很重要；早期（-catenin靶点）和晚期（需要HRas/ERK1/2激活）细胞对机械刺激的反应之间的因果关系是SA 2的主题。在这个目的中，我们还跟踪了野生型和caveolin-1缺失小鼠体内负荷后骨骼中的基因和细胞靶点，以验证这些反应在骨骼中的作用。最后，SA3将在时间微阵列中比较应变和剪切之间的全局基因反应，以阐明两种力之间的不同机械信号，无论是在对照细胞中，还是在那些假定的早期反应（通过¿-catenin）被改变的细胞中。这将使我们能够识别新的信号目标，并验证那些对加载响应至关重要的目标。这项工作将利用应变和振荡剪切力对小鼠原代基质细胞和成骨细胞系的体外作用，以及小鼠的体内负荷。我们已经掌握了必要的细胞和分子工具，以及一个小洞蛋白-1空小鼠。总之，我们的实验室处于一个强有力的位置，为理解负载在骨细胞中产生抗分解代谢和促合成代谢反应的机制带来新的见解。公共卫生相关性：运动对生成功能充足的骨骼的作用涉及到控制成骨细胞谱系间充质干细胞的分化。骨细胞机械刺激引发的信号级联反应赋予细胞抗分解代谢和促合成代谢的表型。本文提出的工作旨在了解导致这种表型的负载诱导信号和反应，从而为产生负载反应的机制带来新的见解。

DESCRIPTION (provided by applicant): Mechanical loading of bone initiates an anti-catabolic and anabolic cellular response that promotes formation of a structurally competent skeleton. The work proposed in this competitive renewal will advance our study of the loaded response of the skeleton by examining a novel temporal sequence of gene regulation and deciphering whether orchestration of this anabolic process arises through a single initiating signal cascade. Our data reveal that mechanical strain regulates an early cluster consisting of canonical Wnt responders followed by a late cluster of anabolic genes, represented by Runx2, osterix (Osx) and eNOS. This pattern of strain response is mirrored by gene response to shear force suggesting that there is a prototypical biomechanical response. A common signaling pathway involving HRas/ERK1/2 is hypothesized to regulate those genes comprising the clustered response. This will be studied in SA1, comparing these candidate responses after strain and oscillatory shear. Our data further suggests a temporal pattern to the loading response: the canonical ¿-catenin target response is vigorous at 4 h but returns to basal levels by 18 h while alterations in Runx2 and osterix are not measurable until 18 h after application of loading. Caveolin-1, a structural molecule in the lipid raft, regulates ¿-catenin activity by limiting ¿-catenin accessibility to signals that induce its nuclear translocation. Silencing caveolin-1 in osteoblasts accelerates load induced increase in Runx2 and Osx to within 4 hours of applying strain, an effect we propose occurs through enhancement of ¿-catenin signaling. This suggests that ¿-catenin may be important for later mechanical effects; causal relationships between early (¿-catenin targets) and late (requiring HRas/ERK1/2 activation) cell responses to mechanical stimulation are the subject of SA 2. In this aim we also track gene and cellular targets in bone after in vivo loading of both wild-type and caveolin-1 null mice to verify that these responses in the skeleton. Finally, SA3 will compare the global gene response between strain and shear in a temporal microarray to elucidate differential mechanical signals between the two forces, both in control cells, and in those where the putative early response (via ¿-catenin) is altered. This will allow us to identify new signaling targets and verify those critical to the loaded response. The work proposed will utilize strain and oscillatory shear force applied to primary murine stromal cells and an osteoblast cell line in vitro, as well as in vivo loading of mice. Necessary cellular and molecular tools, and a caveolin-1 null mouse are in hand. In summary, our laboratory is in a strong position to bring novel insights into understanding the mechanisms by which loading generates an anti-catabolic and pro-anabolic response in bone cells. PUBLIC HEALTH RELEVANCE: The role of exercise to generate a functionally sufficient skeleton involves control of the differentiation of mesenchymal stem cells along the osteoblast lineage. The signaling cascades initiated by mechanical stimulation of bone cells confer a cellular phenotype that is both anti- catabolic and pro-anabolic. Work proposed here seeks to understand the loading induced signals and responses that result in this phenotype, thereby bringing novel insights into the mechanisms by which the loaded response is generated.