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（由申请方提供）：加载过程中产生的生物物理因子通过调节两种分子RANKL和eNOS抑制破骨细胞生成。这种反应需要激活ERK 1/2，表明菌株必须启动MARK信号级联中的近端事件。我们对上游分子的研究表明，低量级菌株激活Ras的单一同种型，H-Ras;并且我们进一步表明，H-Ras的RNA沉默阻止菌株调节的基因表达。选择性Ras激活提供了机械转换器性质的线索：Ras亚型在膜内具有特定的空间分布。H-Ras位于与有组织的膜（脂筏或小窝）相关的“信号中心”内。因此，对于H-Ras，膜充当MARK信号传导事件被调节的平台。我们的数据进一步表明，脂筏的破坏阻止了H-Ras的菌株活化。对完整的组织化膜的要求导致我们的假设，即RANKL和eNOS表达的机械调节需要信号分子的膜组织，并且组织化膜可以作为机械换能器。我们建议把重点放在近端的要求转换成细胞内的化学信号的机械信号。在SA#1中，我们将检查导致RANKL和eNOS远端变化的细胞应变反应中对H-Ras的需求。SA#2检查H-Ras与组织化膜的关联，包括机械转导中对小窝蛋白-1的需求。这些目标利用了用底物菌株激发的1度基质鼠细胞，并测量了特异性Ras活化、RANKL和eNOS反应，以及通过siRNA沉默关键分子。SA#3询问膜组织是否与向小鼠体内施加负荷相关。我们将对wt C57/B6的胫骨以及H-Ras缺失和小窝蛋白-1缺失小鼠施加负荷。加载后将分析基因（RANKL、eNOS、Ras亚型、小窝蛋白）的局部表达和骨组织形态计量学。通过这项工作，我们不仅将定义骨重建的应变调节机制，而且还将产生一个新的范例，通过扰动有组织的质膜，将机械信息转化为细胞内信号的一般mechanotransducer。