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控RANKL和eNOS两种分子抑制破骨细胞生成。这种反应需要激活ERK1/2，这表明菌株必须在MARK信号级联中启动近端事件。我们对上游分子的研究表明，低强度菌株激活Ras的单一异构体H-Ras；进一步，我们发现H-Ras的RNA沉默可以阻止菌株调节基因的表达。选择性Ras激活为机械换能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位于与有组织膜（脂筏或小泡）相关的“信号中心”内。因此，对于H-Ras来说，膜是MARK信号事件调控的平台。我们的数据进一步表明，脂筏的破坏阻止了H-Ras的菌株激活。对完整的有组织膜的要求导致了我们的假设，信号分子的膜组织是RANKL和eNOS表达的机械调节所必需的，并且有组织的膜可以作为机械换能器。我们建议将重点放在将机械信号转换为细胞内化学信号的近端要求上。在SA#1中，我们将研究导致RANKL和eNOS远端变化的细胞株反应对H-Ras的需求。SA#2研究了H-Ras与有组织膜的关联，包括在机械转导中对小窝蛋白-1的需求。这些目标利用底物菌株挑战的1度间质小鼠细胞，测量特异性Ras激活，RANKL和eNOS反应，以及通过siRNA沉默关键分子。SA#3询问膜组织是否与小鼠体内负荷的施加有关。我们将对wt C57/B6小鼠胫骨以及H-Ras和caveolin-1缺失小鼠施加负荷。加载后将分析基因（RANKL, eNOS， Ras亚型，caveolin）和骨组织形态学的局部表达。通过这项工作，我们不仅将定义骨重塑应变调节的机制，而且还将为通过扰动有组织的质膜将机械信息转换为细胞内信号的通用机械换能器产生新的范例。

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.