Regulation of Osteoclastogenesis by Calcium

钙对破骨细胞生成的调节

基本信息

批准号：
8735616
负责人：
Harry C. Blair
金额：
$ 32.39万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-16 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8735616
关键词：
Affect Animals Ants Arthritis Binding Bone Resorption Calcium Calcium Channel Calcium Signaling Cell fusion Cells Clinical Complex Development Disease Figs - dietary Gene Expression Genes Genetic Transcription Human ITAM ITPR1 gene In Vitro Knock-out Knockout Mice Life Malignant Neoplasms Measurement Measures Mediating Mediator of activation protein Minerals Modeling Molecular Molecular Conformation Mus Normalcy Osteoclasts Osteoporosis Pathologic Pathway Analysis Pathway interactions Pattern Postmenopausal Osteoporosis Process Proteins Regulation Rheumatoid Arthritis Role Signal Pathway Signal Transduction TNFSF11 gene Tissues Work bone cell type cytokine design extracellular in vivo mortality novel osteoclastogenesis precursor cell public health relevance receptor response skeletal skeletal abnormality tartrate-resistant acid phosphatase

项目摘要

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.

描述（由申请人提供）：我们将研究钙对破骨细胞生成的调节，重点关注最近发现的钙释放激活钙通道途径。破骨细胞吸收的激活是病理性骨破坏的最终共同途径，Ca2+信号传导对于 RANKL 刺激的破骨细胞生成至关重要。在破骨细胞前体中，Ca2+ 信号激活转录调节因子 NFATc1，从而驱动破骨细胞特异性蛋白的表达。 NFATc1 的其他作用包括在破骨细胞前体融合中的作用，但目前尚不清楚。然而，Ca2+通量调节分化的空间和时间模式在很大程度上是未知的。我们在体外鉴定了钙释放激活的钙通道 Orai1 作为破骨细胞前体细胞外 Ca2+ 流入的介质。在人破骨细胞体外分化中，我们发现Orai1的缺失或抑制会阻碍成熟多核破骨细胞的形成，并大大减少骨矿物质吸收。对 Orai1-/- 小鼠的研究显示了类似的体内变化，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 介导关键的 Ca2+ 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 Orai1 在破骨细胞前体中的功能。我们将培育出一种能够从破骨细胞前体中组织选择性删除 Orai1 的小鼠。这将避免 Orai1 完全缺乏的缺陷，这种缺陷会影响多种细胞类型，导致早期死亡。我们将比较新型组织选择性 Orai1 敲除小鼠与整体 Orai1 敲除小鼠的骨骼效应，以区分 Orai1 对破骨细胞生成和骨骼调节的直接和间接影响。我们将分析体内破骨细胞前体中选择性 Orai1 缺陷对骨骼的影响，并识别破骨细胞前体中 Orai1 缺失导致的分化异常。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。这项工作将包括通过测量野生型和 Orai1 缺陷细胞中破骨细胞分化和活性的 Ca2+ 振荡来定义响应破骨细胞分化信号的 Orai1 依赖性 Ca2+ 通量的研究。通路分析将进一步将 NFATc1 调节的基因表达定义为 Orai1 缺陷细胞和 WT 细胞中 Ca2+ 激活的函数，并且我们将鉴定和分析依赖于 Orai1 的融合相关基因。这些研究将明确 Orai1 调节破骨细胞生成的机制，这将指导开发控制病理性破骨细胞活性的新策略。