ORGANIZATION OF MECHANICAL SIGNALS VIA MEMBRANE SCAFFOLD

通过膜支架组织机械信号

• 批准号: 6986682
• 负责人: Janet E Rubin
• 金额: $ 2.58万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6986682
• 关键词: biological signal transduction; biomechanics; bone metabolism; caveolins; enzyme activity; guanine nucleotide binding protein; histology; intracellular; isozymes; laboratory mouse; mitogen activated protein kinase; nitric oxide synthase; osteoclasts; osteoprotegerin; skeletal stress; small interfering RNA; stromal cells; tissue /cell culture; tumor necrosis factor alpha

DESCRIPTION (provided by applicant): Biophysical factors generated during loading inhibit osteoclastogenesis through regulation of two molecules, RANKL and eNOS. This response requires activation of ERK1/2, indicating that strain must initiate proximal events in the MARK signaling cascade. Our study of upstream molecules has revealed that low-magnitude strains activate a single isoform of Ras, H-Ras; and further we show that RNA silencing of H-Ras prevents strain regulated gene expression. The selective Ras activation offers clues to the nature of the mechanotransducer: Ras isoforms have specific spatial distributions within the membrane. H-Ras is located within 'signaling centers' associated with organized membrane (lipid rafts or caveolae). Thus, for H-Ras, the membrane serves as a platform where MARK signaling events are regulated. Our data further shows that disruption of lipid rafts prevents strain activation of H-Ras. The requirement for an intact organized membrane leads to our hypothsis that membrane organization of signaling molecules is required for mechanical regulation of RANKL and eNOS expression and that the organized membrane may serve as the mechanotransducer. We propose to focus on the proximal requirements for converting a mechanical signal into an intracellular chemical signal. In SA#1 we will examine the requirement for H-Ras in the cellular strain response resulting in distal changes in RANKL and eNOS. SA#2 examines the association of H-Ras with the organized membrane, including the requirement for caveolin-1 in mechanotransduction. These aims utilize 1 degree stromal murine cells challenged with substrate strain and measurements of specific Ras activation, RANKL and eNOS response, and silencing of key molecules through siRNA. SA#3 asks whether membrane organization is relevant for application of load to mice in vivo. We will apply load to tibia of wt C57/B6 as well as to H-Ras null and caveolin-1 null mice. Local expression of genes (RANKL, eNOS, Ras isoforms, caveolin) and bone histomorphometry will be analyzed after loading. With this work we will not only define the mechanisms involved in strain regulation of bone remodeling but also generate a new paradigm for a general mechanotransducer that converts mechanical information into intracellular signals through perturbation of the organized plasma membrane.

描述（由申请人提供）：加载期间产生的生物物理因子通过调节两个分子RANKL和ENOS来抑制破骨细胞生成。这种响应需要激活ERK1/2，表明应变必须启动标记信号级联中的近端事件。我们对上游分子的研究表明，低稳定性菌株激活了Ras，H-Ras的单一同工型。此外，我们表明H-RAS的RNA沉默可防止菌株调节的基因表达。选择性RAS激活为机械转换器的性质提供了线索：RAS同工型在膜内具有特定的空间分布。 H-RAS位于与有组织的膜（脂质筏或小窝）相关的“信号中心”内。因此，对于H-RAS，膜充当标记信号事件的平台。我们的数据进一步表明，脂质筏的破坏可防止H-RAS的应变激活。完整有组织的膜的需求导致我们的假设，即信号分子的膜组织对RANKL和ENOS表达的机械调节需要，并且有组织的膜可以用作机械传感器。我们建议专注于将机械信号转换为细胞内化学信号的近端要求。在SA＃1中，我们将检查细胞应变响应中H-RAS的需求，从而导致RANKL和ENOS的远端变化。 SA＃2检查了H-RAS与有组织的膜的关联，包括对机械转导的小窝蛋白-1的要求。这些目的利用1度基质鼠细胞受到底物应变的挑战，并测量了特定的RAS激活，RANKL和ENOS响应，以及通过siRNA对关键分子的沉默。 SA＃3询问膜组织是否与体内的老鼠的负载相关。我们将向WT C57/B6的胫骨以及H-RAS NULL和Caveolin-1 Null小鼠施加载荷。加载后，将分析基因（RANKL，ENOS，RAS同工型，小窝蛋白）和骨骼组态法的局部表达。通过这项工作，我们不仅将定义骨骼重塑应变的机制，而且还为通用机械转换器产生了新的范式，该机制将机械信息通过有组织的质膜扰动将机械信息转换为细胞内信号。