Role of FoxOs in Skeletal Homeostasis

描述（由申请人提供）：Wnt/2-连环蛋白信号传导对于成骨细胞生成和骨形成是必不可少的。在导致本申请的研究中，阐明了Wnt/2-连环蛋白拮抗作用的新机制，其中FoxO转录因子的激活将有限的2-连环蛋白库从Wnt诱导的TCF/Lef转录转移到FoxO介导的转录。骨中的这种拮抗级联反应是由活性氧（ROS）启动的，代表了氧化应激的介体和防御机制。FoxOs通过调节参与细胞周期、DNA修复和寿命的基因的表达来介导氧化应激反应。还确定了组蛋白脱乙酰酶Sirt 1参与ROS对Wnt/2-连环蛋白的抑制;以及2-连环蛋白对促进成骨细胞在应激条件下存活的FoxO靶基因表达的共激活。此外，获得的证据表明，糖皮质激素或TNF 1对骨的作用至少部分是由ROS诱导的FoxO激活介导的。上述观察结果形成了以下假设的基础，即氧化应激对FoxO转录因子的激活代表了Wnt/2-连环蛋白拮抗作用的先前未被认识到的细胞自主机制，其通过将2-连环蛋白从TCF-介导的转录转移到FoxO-介导的转录而导致衰老、糖皮质激素过量和炎性细胞因子对骨的不利影响。为了检验这一假设，将研究ROS诱导的FoxO翻译后修饰对成骨细胞中FoxO与β-连环蛋白结合的作用，以及ROS是否导致β-连环蛋白募集到FoxO靶基因的启动子（目的1）。此外，将进行体外研究以确定FoxO活化在成骨细胞寿命和分化中的结果（目的2）。最后，在成骨细胞前体及其后代（成骨细胞，骨细胞）中过表达FoxO 3a的小鼠;以及条件性缺失三种主要FoxO亚型FoxO 1，3a和4的小鼠将用于检查FoxO在骨骼稳态中的作用（目的3）。这项工作应该推进知识如何老化，糖皮质激素过量或炎性细胞因子减少骨量。此外，它应该提供一个更好的了解如何优化这种情况的治疗。 公共卫生相关性：拟议的研究旨在确定衰老、糖皮质激素过量或炎性细胞因子导致骨丢失的方式。这将通过研究控制骨形成细胞的蛋白质功能的变化来实现。对控制骨形成的机制的进一步了解将为开发维持或增加骨量和强度的疗法提供重要信息，从而降低骨质疏松性骨折的风险

DESCRIPTION (provided by applicant): Wnt/2-catenin signaling is indispensable for osteoblastogenesis and bone formation. In studies leading to this application, a novel mechanism of Wnt/2-catenin antagonism, whereby activation of FoxO transcription factors diverts the limited pool of 2-catenin from Wnt induced TCF/Lef- transcription to FoxO-mediated transcription was elucidated. This antagonistic cascade in bone is initiated by reactive oxygen species (ROS) and represents both a mediator of, and a defense mechanism against, oxidative stress. FoxOs mediate oxidative stress responses by regulating the expression of genes involved in cell cycle, DNA repair, and lifespan. The involvement of the histone deacetylase Sirt1 in the inhibition of Wnt/2-catenin by ROS; and co- activation by 2-catenin of the expression of FoxO-target genes that promote osteoblast survival under stress conditions was also established. Moreover, evidence was obtained that the actions of glucocorticoids or TNF1 on bone are mediated, at least in part, by ROS-induced FoxO activation. The above observations form the foundation of the hypothesis that activation of FoxO transcription factors by oxidative stress represents a previously unappreciated cell-autonomous mechanism of Wnt/2-catenin antagonism which contributes to the adverse effects of aging, glucocorticoid excess and inflammatory cytokines on bone, by diverting 2-catenin from TCF- to FoxO-mediated transcription. To test this hypothesis, the role of ROS-induced FoxO post- translational modifications on the binding of FoxOs to 2-catenin, in osteoblastic cells, and whether ROS lead to recruitment of 2-catenin to the promoter of FoxO-target genes will be investigated (Aim 1). In addition, in vitro studies will be done to establish the consequences of FoxO activation in osteoblast lifespan and differentiation (Aim 2). Finally, mice overexpressing FoxO3a in osteoblast precursors and their progeny (osteoblasts, osteocytes); and mice in which the three main FoxO isoforms FoxO1, 3a, and 4 are conditionally deleted will be used to examine the role of FoxOs in skeletal homeostasis (Aim 3). This work should advance knowledge of how aging, glucocorticoid excess or inflammatory cytokines decrease bone mass. Furthermore, it should provide a better understanding of how to optimize the treatment of this condition. PUBLIC HEALTH RELEVANCE: The proposed studies seek to identify the means by which aging, glucocorticoid excess or inflammatory cytokines cause bone loss. This will be accomplished by studying changes in the function of proteins that control bone-forming cells. Increased understanding of the mechanisms that control bone formation will provide important information for the development of therapies to maintain or increase bone mass and strength, thereby reducing the risk of osteoporotic fractures