Role of Notch Signaling in Osteocytes

描述（由申请人提供）：我们实验室之前的研究表明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成骨细胞减少，继发于成骨细胞形成的抑制作用。Notch的作用依赖于细胞环境，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 （Dmp1）启动子的控制下在骨细胞中表达时，它会导致骨小梁和皮质骨体积的显著增加。在松质骨中，Notch由于破骨细胞数量的减少而抑制骨吸收。在皮质骨中，切迹提高了矿物质的附着率，结构保持了小梁的外观。我们的观察结果导致Notch在骨细胞功能调节中起核心作用的假设，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，抑制Wnt拮抗剂硬化蛋白的表达，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瘫痪后的骨质流失，我们假设Notch可以保护骨骼免受卸载的有害影响。本研究的目的是了解Notch在骨细胞中的功能，并确定其机制。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Notch1和Notch2的条件失活模型。我们的具体目标是：1)在Dmp1启动子的控制下，通过诱导表达模型和骨细胞中靶向Notch1和Notch2条件缺失，通过Notch转基因表达来确定成人骨骼中Notch在骨细胞中的功能。将Notch错表达小鼠的骨骼表型与野生型小鼠进行比较，并通过接触x线摄影、密度测定、显微CT扫描和组织形态学测定来确定。此外，我们将分析骨骼的生化和生物力学特性；2)确定Notch在骨细胞中的作用机制。Notch的作用机制将被建立，我们将确定Notch经典信号通路是否负责Notch在骨细胞中的作用，以及体内机械转导对经典Notch信号的作用。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和转录和转录后调节水平的贡献将在体内和体外模型中进行研究；3)确定Notch是否保护骨骼免受卸载的有害影响，就像它对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.