ORGANIZATION OF MECHANICAL SIGNALS VIA MEMBRANE SCAFFOLD

描述（由申请人提供）：加载过程中产生的生物物理因子通过调节 RANKL 和 eNOS 两个分子抑制破骨细胞生成。该反应需要激活 ERK1/2，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，低强度菌株会激活 Ras 的单一亚型，即 H-Ras；进一步我们发现 H-Ras 的 RNA 沉默可以阻止菌株调节的基因表达。选择性 Ras 激活为机械传感器的性质提供了线索：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“信号传导中心”内。因此，对于 H-Ras 来说，膜充当了 MARK 信号传导事件受到调节的平台。我们的数据进一步表明，脂筏的破坏可以防止 H-Ras 的菌株激活。对完整组织膜的要求导致我们假设信号分子的膜组织是 RANKL 和 eNOS 表达的机械调节所必需的，并且组织膜可以充当机械传感器。我们建议重点关注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反应中对 H-Ras 的需求，从而导致 RANKL 和 eNOS 的远端变化。 SA#2 检查 H-Ras 与组织膜的关联，包括机械转导中对 Caveolin-1 的需求。这些目标利用受到底物应变攻击的 1 度基质鼠细胞，并测量特定 Ras 激活、RANKL 和 eNOS 反应，以及通过 siRNA 沉默关键分子。 SA#3 询问膜组织是否与对小鼠体内施加负载相关。我们将对 wt C57/B6 的胫骨以及 H-Ras null 和 Caveolin-1 null 小鼠施加负载。加载后将分析基因（RANKL、eNOS、Ras 同工型、caveolin）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塑的应变调节所涉及的机制，而且还将为通用机械传感器生成一个新的范例，该传感器通过扰动有组织的质膜将机械信息转换为细胞内信号。

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.