Biophysical Regulation of Bone Remodeling

描述（由申请方提供）：骨的机械负荷引发抗分解代谢和合成代谢细胞反应，促进结构活性骨架的形成。这项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并破译这种合成代谢过程的编排是否通过单一启动信号级联而产生，来推进我们对骨骼负载反应的研究。我们的数据表明，机械应变调节由典型Wnt响应者组成的早期簇，然后是以Runx 2、osterix（Osx）和eNOS为代表的合成代谢基因的晚期簇。这种应变响应模式反映了基因对剪切力的响应，表明存在原型生物力学响应。一个共同的信号通路，涉及HRas/ERK 1/2的假设，以调节这些基因组成的集群反应。这将在SA 1中进行研究，比较应变和振荡剪切后的这些候选响应。我们的数据进一步表明了负荷反应的时间模式：典型的连环蛋白靶向反应在4小时时是强烈的，但在18小时时恢复到基础水平，而Runx 2和osterix的改变直到施加负荷后18小时才可测量。小窝蛋白-1是脂筏中的一种结构分子，通过限制β-连环蛋白对诱导其核转位的信号的可及性来调节β-连环蛋白的活性。沉默成骨细胞中的小窝蛋白-1在施加应变的4小时内加速了Runx 2和Osx的负荷诱导的增加，我们认为这种作用是通过增强连环蛋白信号传导而发生的。这表明，β-连环蛋白可能对以后的机械效应很重要;早期（β-连环蛋白靶点）和晚期（需要HRas/ERK 1/2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。在这个目标中，我们还在野生型和小窝蛋白-1缺失小鼠的体内负载后追踪骨中的基因和细胞靶点，以验证骨骼中的这些反应。最后，SA 3将在时间微阵列中比较应变和剪切之间的全局基因响应，以阐明两种力之间的差异机械信号，无论是在对照细胞中，还是在那些假定的早期响应（通过β-连环蛋白）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。建议的工作将利用应变和振荡剪切力施加到原代鼠基质细胞和成骨细胞系在体外，以及在小鼠体内加载。必要的细胞和分子工具，以及一个caveolin-1无效的小鼠在手。总之，我们的实验室处于有利地位，可以为理解加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：运动产生功能足够的骨骼的作用涉及控制间充质干细胞沿着成骨细胞谱系的分化。由骨细胞的机械刺激引发的信号级联赋予抗分解代谢和促合成代谢的细胞表型。这里提出的工作旨在了解导致这种表型的负载诱导的信号和反应，从而带来新的见解的机制，负载的反应产生。

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.