R-spondin 2/LGR5增强wnt/β-catenin信号通路促进骨质疏松后骨重建的机制

The Wnt/β-catenin signalling pathway plays an important role in regulation processes of osteogenic differentiation. R-spondin 2 unrelated to other Wnt ligands that act as Wnt agonists, are present in bone tissues. Recent studies have shown that R-spondin 2 to promoted osteoblast maturation and mineralization through the Wnt signalling pathway. In our previous study, we have confirmed that Wnt pathway play the important roles in the pathogenesis of osteoporosis, and R-spondin 2 as well as its receptor LGR5 expression was down-regulated in cancellous bone of postmenopausal osteoporosis patients. Therefore, we hypothesize that R-spondin 2/ LGR5 has a major impact on the function of osteoblast for bone modeling through the Wnt signalling pathway. The purpose of this study was to investigate the potential role of R-spondin 2/LGR5 signaling in osteoblasts and the regulatory mechanism involved Wnt pathway. In this study, we will investigate the impacts of exogenous R-spondin 2 on the function of osteoblast for the bone remodeling in vivo and in vitro, and observe whether blockading LGR5 can antagonize the effectiveness by LGR5 gene knockout mice. Then, we will further determine the effect of R-spondin 2 in bone mass and vascularization in ovariotomized R-spondin 2 transgenic mice, and whether Wnt pathway antagonist (Dkk-1) can antagonize the effectiveness. This study lay emphasis on the impacts of R-spondin 2/LGR5 on osteoporosis through Wnt/β-catenin signalling pathway, providing the theoretical and experimental evidence for pathogenesis of osteoporosis, and raising a new idea for the prevention and treatment of osteoporosis.

Wnt/β-catenin通路是维持骨稳态的成骨关键信号。研究表明R-spondin 2可调控Wnt通路，增强成骨细胞矿化作用。但能否通过上调Wnt通路促进骨质疏松后骨重建及其具体机制尚不清楚。我们的预实验结果提示骨质疏松骨组织中R-spondin 2及其受体LGR5表达明显降低。故我们提出假说：R-spondin 2结合LGR5增强wnt通路促进骨质疏松后骨重建。为验证这一假说，我们将通过人体骨组织、原代BMSCs和成骨细胞、去卵巢小鼠模型、R-spondin 2转基因小鼠和LGR5基因敲除小鼠，采用PCR、Western blot、ELISA、RNA干扰、组织学染色等手段，从分子、细胞、组织以及动物体内多层次探讨R-spondin 2/LGR5调控经典Wnt通路在骨重建中的重要作用及可能机制。本项目的研究有助于深入了解Wnt通路调控成骨分化的上游信号传导，为骨质疏松的防治提供新思路。

R-spondin 2可调控Wnt通路，增强成骨细胞矿化作用。但能否通过上调Wnt通路促进骨质疏松后骨重建及其机制尚不清楚。本课题采用重组R-spondin 2蛋白因子处理MC3T3-E1细胞，ALP活性明显升高，矿化结节的数量和面积明显增加，同时成骨细胞标志基因OPG、Osterix和Runx2表达明显升高，但是RANKL基因表达未受影响，RANKL/OPG比值降低。当加入Dkk-1时，R-spondin 2促进活性β-catenin蛋白表达的作用受到抑制；加入Wnt3a时，活性β-catenin蛋白表达进一步增强，同时成骨相关基因表达、矿化结节形成、ALP活性和Wnt信号活性进一步明显增强。干扰MC3T3-E1细胞的LGR4基因功能， R-spondin 2失去对矿化结节形成、ALP活性以及成骨细胞标志基因表达的积极诱导作用，同时R-spondin 2失去了对Wnt信号通路相关蛋白磷酸化-Lrp5和活性β-catenin蛋白表达的促进作用，并且失去对ZNRF3蛋白表达的抑制作用。R-spondin 2刺激共培养体系，培养液中OPG蛋白浓度明显升高，而RANKL蛋白浓度无明显变化，RANKL/OPG比值降低，破骨细胞标志基因Trap和Cd47表达降低，破骨细胞分化和成熟受到抑制，破骨细胞数量明显减少；加入RANKL蛋白因子，破骨细胞数量明显增加，Trap和Cd47表达明显升高，R-spondin 2对破骨细胞分化和成熟的抑制作用被中和。当LGR4-/-MC3T3-E1细胞/破骨细胞共培养时，破骨细胞数量明显增加，Trap和Cd47表达明显升高，R-spondin 2对破骨细胞分化和成熟的抑制作用消失。R-spondin 2治疗卵巢切除小鼠8周后，骨小梁周围破骨细胞数量和面积明显减少，MAR和BFR明显增加，骨微结构明显改善。同时，R-spondin 2促使正常小鼠的骨合成代谢增加，胫骨近端破骨细胞数量和面积减少，骨微结构参数BV/TV、Tb.N和Tb.Th明显增加，Tb.Sp明显降低。结果表明R-spondin 2结合LGR4，通过活化经典Wnt信号通路，促进成骨细胞分化和成熟，诱导骨形成。R-spondin2抑制破骨细胞形成需要LGR4基因功能正常表达的成骨细胞存在。外源性R-spondin 2能够促进骨形成，降低骨量丢失，改善卵巢切除小鼠胫骨近端骨微细结构。