Regulation of Osteoclastogenesis by Calcium

描述（由申请人提供）：我们将研究钙对破骨细胞生成的调节，重点是最近发现的钙释放激活的钙通道途径。破骨细胞再吸收的激活是病理性骨破坏的最终共同途径，Ca 2+信号传导对RANKL刺激的破骨细胞生成至关重要。在破骨细胞前体中，Ca 2+信号激活转录调节因子NFATc 1，其驱动破骨细胞特异性蛋白的表达。NFATc 1的其他作用包括在破骨细胞前体融合中的作用，现在还不清楚。然而，调控分化的Ca 2+通量的时空格局在很大程度上是未知的。 我们确定了钙释放激活的钙通道Orai 1作为介导细胞外Ca 2+内流的破骨细胞前体在体外。在体外人破骨细胞分化中，我们发现Orai 1的缺乏或抑制阻断了成熟多核破骨细胞的形成，并大大减少了骨矿物质的吸收。对Orai 1-/-小鼠的研究显示了与骨骼异常相关的体内类似变化，证实了Orai 1在骨调节中的重要性。 我们假设Orai 1介导的关键Ca 2+信号，通过控制前体细胞的融合来调节破骨细胞的形成。 在目标1中，我们将在体内定义Orai 1在破骨细胞前体中的功能。我们将产生一个小鼠允许组织选择性删除Orai 1破骨细胞前体。这将避免Orai 1完全缺乏的陷阱，这种缺陷会影响多种细胞类型，导致早期死亡。我们将比较一种新的组织选择性Orai 1基因敲除小鼠的骨骼效应与全球Orai 1基因敲除小鼠的骨骼效应，以区分Orai 1对破骨细胞生成和骨骼调节的直接和间接效应。我们将分析在体内破骨细胞前体中选择性Orai 1缺乏的骨骼效应，并确定由破骨细胞前体中Orai 1缺失引起的分化异常。 在目标2中，我们将确定Orai 1钙信号调节破骨细胞前体基因转录以调节破骨细胞形成，特别是细胞融合的机制。这项工作将包括研究定义Orai 1依赖的Ca 2+通量响应破骨细胞分化信号通过测量Ca 2+振荡相对于破骨细胞的分化和活性在野生型和Orai 1缺陷细胞。通路分析将进一步确定NFATc 1调节的基因表达作为Orai 1缺陷和WT细胞中Ca 2+激活的函数，我们将鉴定和分析依赖于Orai 1的融合相关基因。 这些研究将确定Orai 1调节破骨细胞生成的机制，这将指导控制病理性破骨细胞活性的新策略的开发。

DESCRIPTION (provided by applicant): 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 type, 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.