Biophysical Regulation of Bone Remodeling

描述(申请人提供)：骨的机械负荷启动了一种抗分解代谢和合成代谢的细胞反应，促进了结构上合格骨骼的形成。这项竞争性更新中提出的工作将通过检查一个新的基因调控的时间序列并破译这一合成代谢过程的协调是否通过单一的启动信号级联来推进我们对骨骼负载反应的研究。我们的数据显示，机械应变调节由规范的Wnt响应者组成的早期簇，随后是以Runx2、Osterix(OSX)和eNOS为代表的合成代谢基因的晚期簇。这种应变反应模式反映在基因对剪切力的反应中，表明存在典型的生物力学反应。一条涉及HRAS/ERK1/2的共同信号通路被假设为调节组成聚集反应的那些基因。这将在SA1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：典型的连环蛋白靶反应在4h时强烈，但在18h时恢复到基础水平，而Runx2和Osterix的变化直到施加负荷后18h才可测量到。小窝蛋白-1是脂筏中的一种结构分子，通过限制连环蛋白对诱导其核转位的信号的可及性来调节连环蛋白的活性。沉默成骨细胞中的小窝蛋白-1可以加速负荷诱导的Runx2和OSx的增加，在施加应变后的4小时内，我们认为这种效应是通过增强连环蛋白信号而发生的。这表明？-catenin在以后的机械效应中可能是重要的；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aveolin-1缺失小鼠体内负载后骨骼中的基因和细胞靶点，以验证骨骼中的这些反应。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，以阐明这两种力之间的差异机械信号，这两种力都存在于对照细胞中，以及那些假定的早期反应(通过连接素)发生变化的细胞中。这将使我们能够识别新的信令目标，并验证那些对加载的响应至关重要的目标。这项拟议的工作将利用应变和振荡剪切力应用于体外培养的原代小鼠基质细胞和成骨细胞系，以及小鼠的体内负载。必要的细胞和分子工具，以及一个Caveolin-1零鼠标都在手中。总而言之，我们的实验室在理解负荷在骨细胞中产生抗分解代谢和促合成代谢反应的机制方面处于有利地位。公共卫生相关性：运动对生成功能充足的骨骼的作用涉及控制间充质干细胞沿成骨细胞谱系的分化。由骨细胞的机械刺激启动的信号级联赋予了一种既抗分解代谢又促进合成代谢的细胞表型。这里提出的工作试图理解导致这种表型的负荷诱导信号和反应，从而为产生负荷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.