Role of NFAT in the Skeleton

 描述(申请人提供)：我们实验室的研究表明，骨骼细胞合成激活的T细胞核因子(NFAT)c1至c4。NFAT在成骨细胞分化和功能中的抑制作用。这在体内得到了证实，在I型α-1胶原启动子(COL3.6-NFATC2)控制下的成骨细胞中表达CANFATC2的转基因细胞具有骨量减少和骨形成障碍。在小鼠模型中进行的NFATC2整体失活的研究很难解释，因为它们具有非特异性的炎症反应和各种细胞因子的上调。为了了解NFATC2在成骨细胞中的功能，我们获得了条件性NFATC1loxP/loxP，并创建了条件性NFATC2loxP/LoxP小鼠，将我们置于一个独特的位置来建立其功能，单独和在NFATc1失活的背景下。与COL3.6-NFATC2转基因细胞的骨质疏松表型一致，Osterix表达细胞中NFATc1或NFATC2的失活导致松质骨体积增加。在独立的研究中，我们发现Notch选择性地诱导成骨细胞中NFATC2的表达，并通过转录后机制发挥作用。在Notch2功能获得模型中，Notch2Q2319X突变小鼠由于骨吸收增加和缺乏代偿性骨形成反应而表现出骨量减少。在这个模型中，NFATC2在成骨细胞中被诱导，并可能解释了两个重塑事件的解偶联。该项目的目标是了解NFATC2在成骨细胞系细胞中的功能。为此，我们将研究在成骨细胞系细胞中错误表达NFATC2的小鼠和细胞模型。我们的具体目标是：1)建立NFATC2在成骨细胞中的功能。为此，将利用显微计算机断层扫描和骨组织形态计量学分析，在成骨细胞系的细胞中建立NFATC2条件缺失突变体的骨骼表型，单独和与NFATc1结合。此外，我们还将检测骨骼的生物力学特性，并研究其影响机制；2)通过Notch在成骨细胞中建立NFATC2的转录后调控。这一目标将确定Notch对NFATC2表达的调控，并确定负责NFATC2转录后调控的RNA序列、结合蛋白和microRNAs；以及3)确定NFATC2在成骨细胞中对Notch作用的贡献，并确定Notch诱导的NFATC2是否导致Notch2Q2319X突变体未能表现出促进骨吸收的骨形成反应。为此，将在NFATC2失活的背景下检查Notch2Q2319X的表型。这些研究将阐明NFATC2在成骨细胞系细胞中的功能，并确定骨骼整合不同信号来调节骨重建的新机制。

 DESCRIPTION (provided by applicant): Investigations from our laboratory revealed that skeletal cells synthesize Nuclear factor of activated T- cells (NFAT)c1 through c4. Characterization of cells expressing constitutively active (ca) Nfatc1 or Nfatc2 indicates an inhibitory role of Nfat in osteoblastic differentiation and function. This was confirmed in vivo, ad transgenics expressing caNfatc2 in osteoblasts under the control of the type I α1 collagen promoter (Col3.6- Nfatc2) are osteopenic and have impaired bone formation. Studies in mouse models of global Nfatc2 inactivation are difficult to interpret because they have non-specific inflammatory responses and upregulation of various cytokines. To understand the function of Nfatc2 in osteoblasts, we obtained conditional Nfatc1loxP/loxP and created conditional Nfatc2loxP/loxP mice placing us in a unique position to establish its function, singly and in the context of the Nfatc1 inactivation. In accordance with the osteopenic phenotype of the Col3.6-Nfatc2 transgenics, inactivation of Nfatc1 or Nfatc2 in Osterix expressing cells resulted in an increase in cancellous bone volume. In independent studies, we discovered that Notch induces Nfatc2 expression in osteoblasts selectively and acts by post-transcriptional mechanisms. In a Notch2 gain-of-function model, Notch2Q2319X mutant mice exhibited osteopenia due to increased bone resorption and lack of a compensatory bone forming response. In this model, Nfatc2 is induced in osteoblasts and may account for the uncoupling of the two remodeling events. The goal of this project is to understand the function of Nfatc2 in cells of the osteoblastc lineage. For this purpose, we will study mouse and cellular models misexpressing Nfatc2 in cells of the osteoblastic lineage. Our specific aims are: 1) To establish the function of Nfatc2 in osteoblasts. To this end, the skeletal phenotype of conditional deletion mutants of Nfatc2, singly and in combination with Nfatc1, will be established in cells of the osteoblastic lineage using microcomputed tomography and bone histomorphometric analysis. In addition, we will examine the biomechanical properties of bone and study mechanisms responsible for the effects; 2) To establish the post-transcriptional control of Nfatc2 by Notch in osteoblasts. This Aim will define the control of Nfatc2 expression by Notch and identify RNA sequences, binding proteins and microRNAs responsible for Nfatc2 post-transcriptional regulation; and 3) To establish the contribution of Nfatc2 to Notch actions in osteoblasts and determine whether the Nfatc2 induction by Notch is responsible for the failure of Notch2Q2319X mutants to manifest a bone forming response to enhance bone resorption. To this end, the Notch2Q2319X phenotype will be examined in the context of the Nfatc2 inactivation. These investigations should clarify the function of Nfatc2 in cells of the osteoblastic lineage, and identify novel mechanisms by which the skeleton integrates distinct signals to regulate bone remodeling.