Biophysical Inhibition of Osteoclast Formation

描述（由申请人提供）：承重期间产生的信号 运动与骨骼质量的保持有关。一个生物物理学 在正常加载过程中产生的因素是细胞应变或变形。我们有 显示了动态机械应变对小鼠骨髓体外应用 培养物强烈抑制破骨细胞生成。应变的目标是骨骼 基质细胞，其通过减少优势分子的表达来响应 控制破骨细胞形成的RANK配体（RANKL）。信号发送机制 在基质细胞应变期间激活，减弱RANKL表达 是本书的主题。我们有证据表明，至少有两个细胞内 在应变过程中激活信号：ERK 1/2 MAPK-激酶（ERK 1/2 MAPK）和一氧化氮合酶（NO）。 氧化物（NO）。抑制ERK 1/2 MAPK可防止RANKL的菌株抑制 提示MAPK的激活是应变效应所必需的。进一步工作 表明ERK 1/2 MAPK的组成性激活导致NO和 一氧化氮供体降低RANKL表达表明，增加NO 可能是菌株和ERK 1/2 MAPK的远端效应器。因此，我们的假设是 机械应变通过激活 ERK 1/2 MAPK，随后上调NO，导致RANKL降低 表情我们将研究这些机械诱导的信号转导 机械敏感的鼠基质细胞中的通路， 体外破骨细胞生成。在具体目标1中，我们将研究 在RANKL下游抑制中应变诱导ERK 1/2 MAPK活化 表情重组腺病毒对RANKL表达的影响 显性负性ERK 1/2 MAPK，或显性负性MEK 1激酶（ 阻断ERK 1/2 MAPK活化）将在对照和应变基质中进行探索 细胞然后，我们将确定上游效应在MAPK途径使用 显性阴性小GTP结合蛋白（ras，raf， Rho和CDC 42）以阻断应变效应。具体目标2 研究NO对RANKL表达调控。我们将调查是否有 增加一氧化氮合酶亚型。然后我们将确定是否应变 NO的诱导需要ERK 1/2 MAPK的激活，以及MAPK是否 最后，在具体目标3中，我们将描述 957 bp RANKL启动子中的机械响应顺式元件驱动 由非复制型逆转录病毒递送的荧光素酶。的缺失映射 RANKL启动子将使我们能够定位菌株所需的序列， 转录抑制。MAPK对RANKL启动子的直接调控 并且将考虑否。随着这些实验的完成， 了解机械应变调节RANKL的转导途径 表达，从而形成破骨细胞。

DESCRIPTION (provided by applicant): Signals generated during weight bearing exercise are involved in preservation of skeletal mass. One of the biophysical factors generated during normal loading is cell strain, or deformation. We have shown that in vitro application of dynamic mechanical strain to murine marrow cultures robustly inhibits osteoclastogenesis. The target of strain is the bone stromal cell, which responds by decreasing expression of the dominant molecule controlling osteoclast formation, RANK ligand (RANKL). The signaling mechanism activated during strain of the stromal cell which attenuates RANKL expression is the subject of this work. We have evidence that at least 2 intracellular signals are activated during strain: ERK1/2 MAP-kinase (ERK1/2MAPK) and nitric oxide (NO). Inhibition of ERK1/2MAPK prevents strain inhibition of RANKL suggesting that activation of MAPK is necessary for strain effect. Further work showing that constitutive activation of ERK1/2MAPK causes increases in NO and that nitric oxide donors decrease RANKL expression suggests that increased NO may be a distal effector of both strain and ERK1/2MAPK. Thus, our hypothesis is that mechanical strain decreases osteoclast recruitment via activation of ERKl/2MAPK, with subsequent upregulation of NO, resulting in decreased RANKL expression. We will study these mechanically induced signal transduction pathways in mechanically sensitive murine stromal cells which support osteoclastogenesis in vitro. In Specific Aim 1 we will investigate the role of strain-induced activation of ERK1/2MAPK in downstream inhibition of RANKL expression. The effect on RANKL expression of adenovirus encoding dominant-negative ERK1/2MAPK, or the dominant-negative MEK1 kinase (which blocks ERK1/2MAPK activation) will be explored in control and strained stromal cells. We will then identify the upstream effectors in the MAPK pathway using adenoviral delivery of dominant-negative small GTP-binding proteins (ras, raf, rho and cdc42) to block the strain effect. In Specific Aim 2 we will investigate NO regulation of RANKL expression. We will probe whether strain increases nitric oxide synthase isoforms. We will then ascertain whether strain induction of NO requires activation of ERK1/2MAPK, as well as whether the MAPK effect on RANKL requires NO. Finally, in Specific Aim 3, we will delineate the mechanically responsive cis-elements in the 957 bp RANKL promoter driving luciferase delivered by a non-replicating retrovirus. Deletion mapping of the RANKL promoter will allow us to localize the sequences necessary for strain inhibition of transcription. Direct regulation of the RANKL promoter by MAPK and NO will be considered. With completion of these experiments we should understand the transduction pathways by which mechanical strain regulates RANKL expression and thereby osteoclast formation.