Role of Notch Signaling in Osteocytes

描述(申请人提供)：本实验室以往的研究表明，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可导致继发性骨量减少，进而抑制成骨细胞的生成。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，Notch提高了矿物质的沉积率，结构保持了骨小梁的外观。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，而且Notch在骨细胞中的作用是独特的，与成骨细胞中观察到的不同。Noch诱导骨保护素，抑制Wnt拮抗剂skerostin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丢失，我们假设Notch可以保护骨骼免受卸载的有害影响。这项研究的目的是了解Notch在骨细胞中的功能，并确定涉及的机制。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，以及在骨细胞中Notch1和Notch2条件失活的模型。本研究的主要目的是：1)利用诱导表达模型，在成骨细胞中通过靶向Notch1和Notch2的条件性缺失，在Dmp1启动子控制下进行Notch的转基因表达，以确定Notch在成人骨骼骨细胞中的功能。错误表达Notch的小鼠的骨骼表型将与野生型小鼠进行比较，并通过接触射线照相、密度计量学、显微CT扫描和组织形态计量学确定。此外，我们还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。Notch的作用机制将被建立，我们将确定Notch规范信号通路是否与Notch在骨细胞中的作用有关，以及机械转导在体内对规范Notch信号的作用。Notch对骨细胞中硬化素和护骨素表达的影响，它们对观察到的表型的贡献，以及转录和转录后调控水平将在体内和体外模型中进行检验；以及3)确定Notch是否像防止肌肉瘫痪那样保护骨骼免受卸载的有害影响。

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.