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.

该实验室的最终目标是了解 骨适应机械力。 这种适应性可能导致 适得其反的骨骼损失，可能导致骨折，例如 在长时间的固定期间，太空旅行或应力屏蔽 植入物。 在这些条件下，希望预防或 减少骨质流失，甚至减少骨质。 合理设计 这种干预需要完全了解链接 在骨骼的机械负荷和所得的细胞之间 回答。响应改变机械载荷的重塑会影响 矿化细胞外基质的宏观结构 机械力，但重塑的效应子是骨头 细胞。 这些单元对局部机械的变化作用 环境。 蛋白质合成的程序改变是一个中心 重塑期间发生的细胞活动的组成部分 过程，这种蛋白质合成模式取决于非常大的 细胞中主动转录因子的频谱部分。这是 因此，我们的假设是骨细胞感知机械刺激 作为触发一系列特定转录的适当信号 事件。 因此，骨头最早，最重要的反应之一 到机械输入的细胞必须是激活和/或灭活 一组特定的转录因子。 识别此 特定的转录因子集是至关重要的一步 了解整个骨头的负载与 单个细胞的最终反应。 作为本提案的基础，我们认为研究 对机械改变反应的基因的启动子反应 加载将使我们能够确定转录因素 调解这些反应。 为此，我们选择了一组 基因启动子特别是因为已知它们受到调节 机械刺激骨细胞或骨细胞，因为有 证据表明其基因产物在骨骼的反应中起作用 进行机械输入。 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beta1整合素。 虽然这些启动子已经 在其他系统中部分表征，控制的元素 他们对负载的反应仍然未知。 我们建议通过这些特定目的解决这个关键问题：（1） 建立一个组织培养模型以研究骨细胞中的基因表达 在机械活跃的环境中生长。 （2）筛选发起人 已知对机械输入反应的几种基因 确定负载响应的顺式作用调节区域。 （3）确定 与这些元素相互作用的反式作用因素。 未来的研究超出了拟议工作的结论将使用 从这些候选基因分析到 确定常见的上游监管事件。确认角色 细胞对机械反应的特定转录因子 加载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 在药理上或通过过表达或灭活，使用 反义，基因敲除或跨域负面方法。