Osteocyte Control of Bone Remodeling

 描述（由申请人提供）：本项目的总体目标是了解骨细胞（嵌入骨基质的细胞）如何在生理和病理条件下控制破骨细胞的骨吸收过程。破骨细胞的形成、功能和存活依赖于NF κ B B配体（RANKL）的细胞因子受体激活剂，其作用被可溶性诱饵受体骨保护素（OPG）抑制。最近的研究表明，骨细胞是参与破骨细胞形成的RANKL的重要来源。破骨细胞形成对骨细胞RANKL的需求提出了一个问题，即当这种细胞因子由包埋在骨基质中的细胞产生时，该细胞因子如何到达破骨细胞祖细胞。RANKL最初是作为一种整合的膜蛋白产生的，然后可以被蛋白酶切割以产生可溶性形式，两者都能够促进骨吸收。目前尚不清楚骨细胞来源的RANKL促进的破骨细胞形成是否涉及膜结合形式、可溶性形式或两者兼而有之。皮质孔隙是一种随年龄增长和糖皮质激素过量而增加的骨丢失类型，一些研究表明，前者与皮质骨中RANKL表达增加有关。然而，尚不清楚骨细胞是否是RANKL的来源，以及这种增加是否涉及与衰老或糖皮质激素过量相关的皮质孔隙的产生。一些研究表明OPG也由骨细胞产生，并且OPG产生的这些变化可能有助于病理性骨吸收。然而，OPG是由许多细胞类型产生的，目前还不清楚哪些细胞提供的OPG对控制骨重建很重要。基于这些观察结果，我们提出了一个中心假设，即骨细胞通过多种机制控制破骨细胞的形成，包括产生可溶性RANKL和OPG，这有助于生理性和病理性骨吸收。为了解决这一假设，在目标1中，我们将确定骨细胞是否通过产生可溶性或膜结合RANKL来控制破骨细胞的形成。将产生表达抗蛋白水解切割的RANKL蛋白形式的小鼠，并在生理和病理条件下对其骨骼进行结构和组织学分析。在目标2中，我们将确定骨细胞源性RANKL是否有助于由老化或糖皮质激素过量引起的皮质多孔性。将在骨细胞中缺乏RANKL基因的20月龄小鼠和对照同窝小鼠中测量骨量和结构，包括皮质孔隙率以及破骨细胞数量。还将在相同基因型的成年小鼠中检查糖皮质激素过量对皮质孔隙率的影响。最后，在目标3中，我们将确定骨细胞是否是OPG的重要来源。使用Dmp 1-Cre和Sost-Cre转基因小鼠从骨细胞中删除OPG基因的条件等位基因，并将骨骼表型与OPG种系缺失的小鼠进行比较。

 DESCRIPTION (provided by applicant): The overall goal of this project is to understand how osteocytes, cells embedded in the bone matrix, control the process of bone resorption by osteoclasts under physiological and pathological conditions. Osteoclast formation, function, and survival depend on the cytokine receptor activator of NFB ligand (RANKL), whose actions are inhibited by the soluble decoy receptor osteoprotegerin (OPG). Recent studies have shown that osteocytes are an important source of the RANKL involved in osteoclast formation. The requirement of osteocyte RANKL for osteoclast formation raises the question of how this cytokine reaches osteoclast progenitors when produced by cells embedded in the bone matrix. RANKL is initially produced as an integral- membrane protein, which can then be cleaved by proteases to yield a soluble form, both of which are able to promote bone resorption. It remains unclear whether the osteoclast formation promoted by osteocyte-derived RANKL involves the membrane-bound form, the soluble form, or both. Cortical porosity is a type of bone loss that increases with aging and glucocorticoid excess, and some studies suggest that the former is associated with increased expression of RANKL in cortical bone. However, it is unknown whether osteocytes are the source of this RANKL and whether this increase is involved in the generation of cortical porosity associated with aging or glucocorticoid excess. Some studies suggest that OPG is also produced by osteocytes and those changes in OPG production may contribute to pathological bone resorption. However, OPG is produced by many cell types and it is unclear which cells provide the OPG important for control of bone remodeling. Based on these observations, we propose the central hypothesis that osteocytes control osteoclast formation via multiple mechanisms including production of soluble RANKL and OPG and this contributes to physiological as well as pathological bone resorption. To address this hypothesis, in Aim 1 we will determine whether osteocytes control osteoclast formation via production of soluble or membrane-bound RANKL. Mice expressing a form of the RANKL protein that is resistant to proteolytic cleavage will be generated and their skeletons analyzed structurally and histologically under physiological and pathological conditions. In Aim 2, we will determine whether osteocyte-derived RANKL contributes to the cortical porosity caused by aging or glucocorticoid excess. Bone mass and structure, including cortical porosity, as well as osteoclast number, will be measured in 20-month-old mice lacking the RANKL gene in osteocytes and control littermates. The impact of glucocorticoid excess on cortical porosity will also be examined in adult mice of the same genotypes. Lastly, in Aim 3 we will determine whether osteocytes are an important source of OPG. A conditional allele of the OPG gene will be deleted from osteocytes using Dmp1-Cre and Sost-Cre transgenic mice and the skeletal phenotype compared to that of mice with germline deletion of OPG.