G-protein coupled receptor EBI2 regulation of bone mass

描述(申请人提供)：破骨细胞与骨附近的循环单核细胞不同，需要两种关键信号：M-CSF和RANKL。单核细胞和破骨细胞前体如何迁移到骨骼与表达适当信号的细胞相互作用尚不清楚。在接收到这些信号后，破骨细胞前体经历了一个需要细胞-细胞相互作用和多个细胞融合事件的分化过程。由于细胞-细胞相互作用和融合是一个动态过程，它预测破骨细胞前体继续 收到分化信号后要动起来。然而，调控单核细胞/破骨细胞前体运动和向骨表面迁移的机制(S)仍未解决。细胞运动在很大程度上是由G？I蛋白偶联受体(G？iPCR)控制的。我们最近发现破骨细胞前体和破骨细胞强烈表达EBI2(GPR183，G？iPCR)，EBI2配体是由位于破骨细胞形成的骨髓细胞内的细胞产生的。有趣的是，体外培养的EBI2基因缺陷小鼠的破骨细胞大小有缺陷。 EBI2缺乏会导致骨密度增加，并保护小鼠免受卵巢切除引起的骨质疏松的影响。以前的研究表明，在初级T依赖抗体应答过程中，EBI2及其配体引导B细胞向次级淋巴器官的外层和滤泡间壁龛迁移。因此，我们考虑了EBI2促进BMDM和破骨细胞前体运动的可能性，从而增加了细胞-细胞相互作用和细胞-细胞融合的可能性，这是大型多核破骨细胞分化所必需的。在初步实验中，我们发现EBI2缺陷的细胞在体外以较慢的速度移动，并随着时间的推移表现出减少的移位。最后，我们的初步研究表明，成骨细胞分泌大量的EBI2配体，这可能促进表达EBI2的破骨细胞前体定向迁移到骨龛。事实上，EBI2逆转录病毒在破骨细胞前体细胞中的过度表达使这些细胞比对照细胞更接近骨表面。这一建议的一个主要焦点是揭示EBI2调节单核细胞/破骨细胞前体运动和向破骨细胞发生部位迁移的分子机制。我们将使用双光子和细胞成像相结合的方法来表征EBI2(GPR183，一种G？iPCR)在破骨细胞分化、移动和向骨表面迁移中所起的特殊作用。鉴于GPCR是治疗干预的主要靶点，拟议的研究有可能阐明破骨细胞生物学的新方面，这些方面应该在骨骼疾病中产生翻译影响。

DESCRIPTION (provided by applicant): Osteoclasts differentiate from recirculating monocytes in niches adjacent to bone and require two critical signals, M-CSF and RANKL. How monocytes and osteoclast precursors migrate to the bone to interact with cells expressing the appropriate signals is unknown. Upon receiving these signals, osteoclast precursors undergo a differentiation process that requires cell-cell interaction and multiple cell fusion events. As cel-cell interaction and fusion is a dynamic process it predicts that osteoclast precursors continue to be motile after receiving differentiation signals. However, the mechanism(s) regulating monocyte/osteoclast precursor motility and migration to bone surfaces remain unresolved. Cell motility is largely controlled by G¿i-protein coupled receptors (G¿iPCR). We have recently uncovered that osteoclast precursors and osteoclasts robustly express EBI2 (Gpr183, a G¿iPCR) and EBI2 ligand is produced by cells located within osteoclastogenic bone marrow niches. Interestingly, osteoclasts derived from EBI2 -deficient mice in vitro are defective in size and EBI2 deficiency results in increased bone density and protects mice from ovariectomy- induced osteoporosis. Previous studies demonstrated that EBI2 and its ligands direct B cell migration to outer and interfollicular niches in secondary lymphoid organs during primary T-dependent antibody response. Therefore, we considered the possibility that EBI2 promotes BMDM and osteoclast precursor motility thereby increasing the probability of cell-cell interaction and cell-cell fusion essential for differentiation of large multinucleated osteoclasts. In preliminry experiments we found that EBI2-deficient cells moved with slower velocity and exhibited reduced displacement over time in vitro. Finally, our preliminary studies show that osteoblasts secrete substantial amounts of EBI2-ligand, which may promote directional migration of EBI2-expressing osteoclast precursors to bone niches. Indeed, retroviral overexpression of EBI2 in osteoclast precursors preferentially positions these cells in closer proximity to the bone surface than in control cells. A major focus of this proposal is to uncover the molecular mechanisms underlying EBI2 regulation of monocyte/osteoclast precursor motility and migration to sites of osteoclastogenesis. We will use a combination of 2-photon and cellular imaging approaches to characterize the role played specifically by EBI2 (Gpr183, a G¿iPCR) in osteoclast differentiation, motility, and migration to the bone surface. Given that GPCRs are prime targets for therapeutic interventions, the proposed studies have the potential to elucidate new aspects of osteoclast biology that should have a translational impact in bone disease.