Regulation of Osteoclastogenesis by Calcium

我们将研究钙对破骨细胞生成的调节，重点是最近发现的一种 钙释放激活钙通道通路。激活破骨细胞的吸收是最终的 病理性骨破坏的共同途径，钙信号在RANKL刺激中起关键作用 破骨细胞生成。在破骨细胞前体中，钙信号激活转录调节因子 NFATc1，它驱动破骨细胞特异性蛋白的表达。NFATc1的其他影响包括 在破骨细胞前体融合中的作用，目前还不清楚。然而，时空上的 调控分化的钙离子通量的模式在很大程度上是未知的。 我们发现钙释放激活的钙通道Orai1是一种细胞外介质 体外破骨细胞前体细胞内钙离子内流。在体外的人类破骨细胞分化中，我们发现 Orai1的缺失或抑制阻碍了成熟的多核破骨细胞的形成，并极大地 减少骨矿物质的吸收。对Orai1-/-小鼠的研究在体内也显示了类似的变化， 与骨骼异常相关，证实了Orai1在骨调节中的重要性。 我们假设Orai1通过以下方式介导调节破骨细胞形成的关键钙信号 控制前体细胞的融合。 在目标1中，我们将在体内确定Orai1在破骨细胞前体中的功能。我们将生成一个 小鼠允许组织选择性地从破骨细胞前体中删除Orai1。这将避免出现以下缺陷 完全Orai1缺乏症，影响多种细胞类型，导致早期死亡。我们会比较一下 一种新的组织选择性Orai1基因敲除小鼠对全局Orai1基因的骨骼效应 基因敲除，以区分Orai1对破骨细胞形成和骨骼的直接和间接影响 监管。我们将分析选择性破骨细胞前体Orai1缺乏症对骨骼的影响 并鉴定破骨细胞前体Orai1缺失引起的分化异常。 在目标2中，我们将确定Orai1钙信号调节破骨细胞的机制 前体基因转录调节破骨细胞的形成，特别是细胞融合。这项工作将 包括确定破骨细胞分化信号响应Orai1依赖的钙离子流量的研究 通过测量与破骨细胞分化和活性有关的野生型钙振荡 Orai1缺陷细胞。通路分析将进一步将NFATc1调节的基因表达定义为 钙激活在Orai1缺乏和WT细胞中的作用，我们将鉴定和分析融合- 依赖于Orai1的相关基因。 这些研究将确定Orai1调节破骨细胞生成的机制，即 将指导开发控制病理性破骨细胞活动的新策略。

We will study regulation of osteoclastogenesis by calcium, focusing on a recently discovered calcium-release activated calcium channel pathway. Activation of osteoclast resorption is the final common pathway in pathologic bone destruction, and Ca2+ signaling is critical to RANKL-stimulated osteoclastogenesis. In osteoclast precursors, Ca2+ signals activate the transcriptional regulator NFATc1, which drives expression of osteoclast-specific proteins. Other effects of NFATc1 include a role, now poorly defined, in fusion of osteoclast precursors. However, the the spatial and temporal patterns of Ca2+ fluxes regulating differentiation were largely unknown. We identified the calcium release activated calcium channel Orai1 as a mediator of extracellular Ca2+ influx in osteoclast precursors in vitro. In human osteoclast differentiation in vitro, we showed that absence or inhibition of Orai1 blocked the formation of mature multinucleated osteoclasts and greatly reduced bone mineral resorption. Studies of Orai1-/- mice showed similar changes in vivo, with associated with skeletal abnormalities, confirming the importance of Orai1 in bone regulation. We hypothesize that Orai1 mediates critical Ca2+ signals that regulate osteoclast formation by controlling the fusion of precursor cells. In Aim 1 we will define in vivo the function of Orai1 in osteoclast precursors. We will generate a mouse allowing tissue-selective deletion of Orai1 from osteoclast precursors. This will avoid pitfalls of complete Orai1 deficiency, which affects multiple cell types, leading to early mortality. We will compare the skeletal effects of a novel tissue-selective Orai1 knockout mouse to those of the global Orai1 knockout, to distinguish direct from indirect effects of Orai1 on osteoclastogenesis and skeletal regulation. We will analyze the skeletal effects of selective Orai1 deficiency in osteoclast precursors in vivo and identify abnormalities of differentiation caused by loss of Orai1 from osteoclast precursors. In Aim 2 we will determine the mechanisms by which Orai1 calcium signals modulate osteoclast precursor gene transcription to regulate osteoclast formation, particularly cell fusion. This work will include studies defining Orai1-dependent Ca2+ fluxes in response to osteoclast differentiation signals via measurement of Ca2+ oscillations with respect to osteoclast differentiation and activity in wild type and Orai1 deficient cells. Pathway analysis will, further, define NFATc1-regulated gene expression as a function of Ca2+ activation in Orai1-deficient and WT cells, and we will identify and analyze fusion- related genes dependent on Orai1. These studies will define mechanisms through which Orai1 regulates osteoclastogenesis, which will guide the development of new strategies to control pathologic osteoclastic activity.