ORGANIZATION OF MECHANICAL SIGNALS VIA MEMBRANE SCAFFOLD

描述(申请人提供)：加载过程中产生的生物物理因素通过调节RANKL和eNOS两个分子来抑制破骨细胞的形成。这种反应需要ERK1/2的激活，这表明该菌株必须启动Mark信号级联中的近端事件。我们对上游分子的研究表明，低强度的菌株激活了单一的RAS亚型，H-RAS；此外，我们还表明，H-RAS的RNA沉默阻止了菌株调节的基因表达。RAS的选择性激活为了解机械转导的性质提供了线索：RAS亚型在膜内有特定的空间分布。H-RAS位于与有组织膜(脂筏或小窝)相关的“信号中心”内。因此，对于H-RAS，膜作为调节Mark信号事件的平台。我们的数据进一步表明，脂筏的破坏阻止了H-RAS的应变激活。对完整的有组织膜的要求导致了我们的假设，即RANKL和eNOS表达的机械调节需要信号分子的膜组织，并且有组织的膜可能作为机械转导。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远端变化的细胞应变反应中对H-RAS的需求。SA#2检查了H-RAS与有组织的膜的联系，包括机械转导中对小窝蛋白-1的需求。这些目标利用1度基质小鼠细胞挑战底物应变和测量特定的RAS激活，RANKL和eNOS反应，以及通过siRNA沉默关键分子。SA#3询问膜组织是否与在活体内对小鼠施加负荷有关。我们将载荷施加于wt C57/B6小鼠的胫骨，以及H-RAS基因缺失和小窝蛋白-1基因缺失的小鼠。加载后将分析局部基因(RANKL、eNOS、RAS亚型、小窝蛋白)的表达和骨组织形态计量学。通过这项工作，我们不仅将确定骨重建的应变调节机制，而且还将产生一种新的通用力学换能器范例，通过对有组织的质膜的扰动将力学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.