Role of Notch Signaling in Osteocytes

描述（由申请人提供）：我们实验室的先前研究表明，Notch在细胞分化中起关键作用，其在未成熟成骨细胞中的表达导致继发于成骨细胞生成抑制作用的骨质减少。Notch的作用是细胞环境依赖性的，并且与在成骨细胞中观察到的作用相反，当Notch在牙本质基质蛋白1（Dmp 1）启动子的控制下在骨细胞中表达时，其引起小梁和皮质骨体积的显著增加。在松质骨中，由于破骨细胞数量减少，Notch抑制骨吸收。在皮质骨中，Notch增强了矿物质沉积速率，结构保持了小梁外观。我们的观察导致了这样的假设，即Notch在骨细胞功能的调节中起着核心作用，并且Notch在骨细胞中的作用是独特的，与在成骨细胞中观察到的作用不同。Notch诱导骨保护素并抑制Wnt拮抗剂硬化素的表达，从而增强Wnt靶基因的表达。在最初的实验中，我们发现Notch在骨细胞中的表达可以防止肌肉麻痹后的骨丢失，我们假设Notch可以保护骨免受卸载的不利影响。这项研究的目的是了解Notch在骨细胞中的功能，并确定相关机制。为此，我们将研究在骨细胞环境中表达Notch的小鼠和细胞模型，以及骨细胞中Notch 1和Notch 2的条件性失活模型。我们的具体目标是：1）通过使用诱导型表达模型在Dmp 1启动子的控制下转基因表达Notch和通过骨细胞中靶向的Notch 1和Notch 2条件性缺失来确定Notch在成人骨骼中骨细胞中的功能。将错表达Notch的小鼠的骨骼表型与野生型小鼠的骨骼表型进行比较，并通过接触放射照相术、光密度测定法、显微CT扫描和组织形态测定法进行测定。此外，我们将分析骨的生化和生物力学特性; 2）确定Notch在骨细胞中的作用机制。将建立负责Notch效应的机制，并且我们将确定Notch经典信号传导途径是否负责Notch在骨细胞中的效应以及机械转导对体内经典Notch信号传导的作用。将在体内和体外模型中检查Notch对骨细胞中硬化蛋白和骨保护素表达的影响、它们对观察到的表型的贡献以及转录和转录后调节的水平;以及3）确定Notch是否保护骨骼免受卸载的有害影响，就像它免受肌肉麻痹的有害影响一样。

DESCRIPTION (provided by applicant): Previous investigations from our laboratory revealed that Notch plays a critical role in cell differentiation, and its expression in immature osteoblast causes osteopenia secondary to an inhibitory effect on osteoblastogenesis. The effect of Notch is cell context-dependent, and in contrast to the effects observed in osteoblasts, when Notch is expressed in osteocytes under the control of the Dentin matrix protein 1 (Dmp1) promoter it causes a pronounced increase in trabecular and cortical bone volume. In cancellous bone, Notch inhibits bone resorption due to a decrease in the number of osteoclasts. In cortical bone Notch enhances mineral apposition rate and the structure maintains a trabecular appearance. Our observations led to the hypothesis that Notch plays a central role in the regulation of osteocyte function, and Notch effects in osteocytes are unique and distinct from those observed in osteoblasts. Notch induces osteoprotegerin and inhibits the expression of the Wnt antagonist sclerostin enhancing the expression of Wnt target genes. In initial experiments, we discovered that expression of Notch in osteocytes prevents the bone loss that follows muscle paralysis, and we postulate that Notch protects bone from the detrimental effects of unloading. The aim of the proposed research is to understand the function of Notch in osteocytes and define the mechanisms involved. For this purpose, we will study mouse and cellular models expressing Notch in the osteocyte environment, and models of conditional inactivation of Notch1 and Notch2 in osteocytes. Our specific aims are: 1) To determine the function of Notch in osteocytes in the adult skeleton by transgenic expression of Notch under the control of the Dmp1 promoter using inducible models of expression and by targeted Notch1 and Notch2 conditional deletion in osteocytes. The skeletal phenotype of mice misexpressing Notch will be compared to that of wild type mice and determined by contact radiography, densitometry, micro CT scanning and histomorphometry. In addition, we will analyze the biochemical and biomechanical properties of bone; 2) To determine the mechanism of action of Notch in osteocytes. Mechanisms responsible for the effects of Notch will be established, and we will determine whether the Notch canonical signaling pathway is responsible for the effects of Notch in osteocytes and the role of mechanotransduction on canonical Notch signaling in vivo. The impact of Notch on the expression of sclerostin and osteoprotegerin in osteocytes, their contribution to the phenotype observed and levels of transcriptional and post-transcriptional regulation will be examined in in vivo and in vitro models; and 3) To establish whether Notch protects the skeleton from the detrimental effects of unloading, as it does from those of muscle paralysis.