GENE TRANSCRIPTION IN MECHANICALLY LOADED BONE CELLS

机械负载骨细胞中的基因转录

• 批准号: 6171192
• 负责人: JEAN F WELTER
• 金额: $ 7.65万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6171192
• 关键词: DNA binding protein; DNA footprinting; gel mobility shift assay; gene expression; genetic promoter element; genetic regulatory element; genetic transcription; insulinlike growth factor; integrins; luciferin monooxygenase; mechanical stress; nitric oxide synthase; nucleic acid sequence; osteocytes; protooncogene; tissue /cell culture; transcription factor

The ultimate goal of this laboratory is to understand the ability of bone to adapt to mechanical forces. This adaptability can cause a counterproductive loss of bone mass which can result in fractures, e.g. during prolonged immobilization, space travel, or stress shielding by implants. Under these conditions it would be desirable to prevent or reduce bone loss, or even to increase bone mass. To rationally design this kind of intervention requires a complete understanding of the link between mechanical loading of the bone and the resulting cellular responses. Remodeling in response to altered mechanical loads affects the macrostructure of the mineralized extracellular matrix which carries the mechanical forces, but the effectors of the remodeling are bone cells. These cells act in response to changes in their local mechanical environment. An altered program of protein synthesis is a central component of the cellular activities which occur during the remodeling process, and this pattern of protein synthesis depends to a very large part on the spectrum of active transcription factors in the cell. It is therefore our hypothesis that bone cells perceive mechanical stimulation as an appropriate signal to trigger a series of specific transcriptional events. Thus, one of the earliest and most important responses of bone cells to mechanical input must be the activation and/or inactivation of a specific set of transcription factors. The identification of this specific set of transcription factors is a critically important step in understanding the link between loading of the whole bone, and the ultimate responses of the individual cells. As the basis for the present proposal, we argue that studying the promoter responses of genes that react to alterations in mechanical loading will enable us to identify the transcription factors that mediate these responses. For this purpose, we have selected a set of gene promoters specifically because they are known to be regulated by mechanical stimulation of bone or bone cells, and because there is evidence that their gene product plays a role in the response of bone to mechanical input. These are the transcription factor c-fos, insulin- like growth factor-1 (IGF-1), the inducible nitric oxide synthase (iNOS), and beta1 integrin. Although these promoters have been partially characterized in other systems, the elements which control their response to loads remain unknown. We propose to address this critical issue by these specific aims: (1) Establish a tissue culture model to study gene expression in bone cells growing in a mechanically active environment. (2) Screen the promoters of several genes which are known to respond to mechanical input, to identify load responsive cis-acting regulatory regions. (3) Identify the trans-acting factors which interact with these elements. Future studies beyond the conclusion of the proposed work would make use of the information gathered from analysis of these candidate genes to identify common upstream regulatory events. To confirm the role of specific transcription factors in the cellular response to mechanical loading, and to begin to design therapeutic interventions, we will proceed to directly manipulate the levels of these factors, either pharmacologically, or by overexpression or inactivation, using antisense, gene knockout, or transdominant negative approaches.

这个实验室的最终目标是了解 骨骼要适应机械力。这种适应性可能会导致 适得其反的骨量丢失，可能导致骨折，例如 在长时间的固定、太空旅行或应力屏蔽过程中 植入物。在这种情况下，最好是防止或 减少骨质流失，甚至增加骨量。合理设计 这种干预需要对其中的联系有充分的了解 在骨骼的机械载荷和由此产生的细胞之间 回应。机械负荷改变时的重塑影响 矿化细胞外基质的宏观结构 机械力，但重塑的执行者是骨骼 细胞。这些细胞对其局部机械结构的变化做出反应 环境。蛋白质合成的一个改变的程序是一个中心 重塑过程中发生的细胞活动的组成部分 而这种蛋白质合成模式依赖于一个非常大的 细胞中活跃的转录因子谱的一部分。它是 因此，我们关于骨细胞感知机械刺激的假设 作为一个适当的信号来触发一系列特定的转录 事件。因此，骨骼最早和最重要的反应之一 细胞的机械输入必须是激活和/或失活的 一组特定的转录因子。这一点的认定 一组特定的转录因子是在 了解整个骨骼的加载与 单个细胞的最终反应。 作为本建议的基础，我们认为研究 对机械性变化作出反应的基因的启动子反应 加载将使我们能够识别转录因子 调停这些反应。为此，我们选择了一套 基因启动子，特别是因为已知它们受 对骨或骨细胞的机械刺激，因为有 他们的基因产物在骨骼反应中发挥作用的证据 到机械输入。这些是转录因子c-fos，胰岛素- 与生长因子-1(IGF-1)一样，诱导型一氧化氮合酶 (INOS)和β1整合素。尽管这些推动者已经 在其他系统中具有部分特征的，控制 它们对负载的反应仍不清楚。 我们建议通过以下具体目标来解决这一关键问题：(1) 建立骨细胞基因表达的组织培养模型 在机械活动的环境中生长。(2)筛选发起人 在已知对机械输入有反应的几个基因中， 确定负荷响应型顺式作用监管区域。(3)确定 与这些元素相互作用的反式作用因子。 除了拟议工作的结论之外，今后的研究将利用 从这些候选基因的分析中收集的信息 确定常见的上游监管事件。确认…的作用 细胞对机械刺激反应中的特定转录因子 并开始设计治疗干预措施，我们将 继续直接操纵这些因素的水平，或者 药理上的，或通过过度表达或失活，使用 反义、基因敲除或易位显性否定方法。