The Role of Transcription Factor NFAT in Osteoblast Differentiation

描述(由申请人提供)：Zayzafoon实验室的长期目标是开发新的治疗方法，以建立和保留人类的骨量。这项拟议工作的近期目标是明确确定钙调神经磷酸酶(CN)和活化T细胞核因子(NFAT)信号通路在调节骨量中的作用。骨骼是一种高度动态的结构，通过一个称为重塑的过程不断更新。CN和NFAT是破骨细胞分化和骨吸收的关键促进剂。相反，CN和NFAT在成骨细胞分化和骨形成中的作用尚不清楚。我们发现，低浓度环孢素A(CsA)对CN的药理抑制作用可增加体外成骨细胞分化和体内骨量。这种成骨细胞分化的增加与AP-1家族成员Fra-2表达的类似增加有关。为了确定CN是否直接影响成骨细胞的分化，我们利用Cre介导的重组酶技术创建了成骨细胞特有的钙调神经磷酸酶调节亚单位(Cnb1)缺失的小鼠。我们的结果表明，当CN/NFAT信号通路被限制在成骨细胞中时，通过直接增加成骨细胞的分化和间接减少破骨细胞的生成，导致骨量增加。因此，我们假设CN/NFAT信号通路通过调节AP-1转录因子家族的表达和激活来负向调节成骨细胞分化和骨量。本研究的具体目的是：1.确定成骨细胞中CN/NFAT信号的中断对骨表型的影响。2.研究CN/NFAT在成骨细胞生长和分化中的作用。3.确定CN/NFAT调节骨量的分子机制。以前的文献将CN/NFAT通路描述为骨形成的正向调节因子。然而，来自成骨细胞特异性敲除这一途径的小鼠的数据表明相反-CN/NFAT是骨形成的负面调节因子。我们致力于最终解决这个问题，因为使用CN/NFAT抑制剂来合成增加骨量具有治疗潜力。我们的初步数据提供了强有力的证据，证明CN/NFAT是骨量的负调节因子。这些目标的完成将使我们能够明确地确定CN/NFAT信号在成骨细胞分化和骨量中的作用。我们的工作的影响可能是深远的，因为它将提供更好的了解CN/NFAT调节成骨细胞分化的机制，并将对开发针对CN/NFAT通路的新的合成代谢药物来治疗骨质疏松和骨丢失具有重要意义。公共卫生相关性：我们将明确确定成骨细胞中CN/NFAT信号在调节骨形成和骨量中的作用。我们的工作的影响可能是深远的，因为它将对开发针对CN/NFAT途径的治疗骨质疏松症和骨丢失的新的合成代谢药物具有重要意义。

DESCRIPTION (provided by applicant): The long-term goal of the Zayzafoon laboratory is to develop novel therapeutic approaches for building and retaining bone mass in humans. The immediate goal of this proposed work is to unequivocally determine the role of the Calcineurin (Cn) and Nuclear Factor of Activated T Cells (NFAT) signaling pathway in regulating bone mass. Bone is a highly dynamic structure that is constantly renewing through a process called remodeling. Cn and NFAT are key promoters of osteoclast differentiation and bone resorption. In contrast, the role of Cn and NFAT in osteoblast differentiation and bone formation is not well defined. We discovered that the pharmacologic inhibition of Cn by low concentrations of Cyclosporin A (CsA) increased osteoblast differentiation in vitro and bone mass in vivo. This increase in osteoblast differentiation was associated with a similar increase in the expression of Fra-2, an AP-1 family member. To determine whether Cn directly impacts osteoblast differentiation, we utilized Cre-mediated recombinase technology to create mice lacking the calcineurin regulatory subunit (Cnb1) specifically in osteoblasts. Our results demonstrate that a block in the Cn/NFAT signaling pathway, when restricted to osteoblasts, leads to an increase in bone mass by directly increasing osteoblast differentiation and indirectly decreasing osteoclastogenesis. Therefore, we hypothesize that the Cn/NFAT signaling pathway negatively regulates osteoblast differentiation and bone mass by regulating the expression and activation of AP-1 family of transcription factors. The Specific Aims of this study are: 1. Determine how the disruption of CN/NFAT signaling in osteoblasts affects bone phenotype. 2. Characterize the roles of Cn/NFAT in osteoblasts growth and differentiation. 3. Determine the molecular mechanisms by which Cn/NFAT regulates bone mass. Previous publications characterize the Cn/NFAT pathway as a positive regulator of bone formation. However, the data from mice with osteoblast-specific knockout of this pathway suggest the opposite - that Cn/NFAT is a negative regulator of bone formation. We are dedicated to definitively resolving this issue because of the therapeutic potential of using inhibitors of Cn/NFAT to anabolically increase bone mass. Our preliminary data provide strong evidence that Cn/NFAT is a negative regulator of bone mass. Completion of these aims will enable us to unequivocally determine the roles of Cn/NFAT signaling in osteoblast differentiation and bone mass. The impact of our work may be far reaching, because it will provide better understanding of the mechanisms by which Cn/NFAT regulates osteoblast differentiation and will have important implications in the development of new anabolic drugs targeting Cn/NFAT pathway to treat osteoporosis and bone loss. PUBLIC HEALTH RELEVANCE: We will unequivocally determine the roles of Cn/NFAT signaling in osteoblasts in regulating bone formation and bone mass. The impact of our work may be far reaching, because it will have important implications in the development of new anabolic drugs targeting Cn/NFAT pathway to treat osteoporosis and bone loss.