RSpo1调控Wnt通路在小鼠炎性关节炎骨侵蚀中的作用及分子机制

Our previous study funded by Nantional Natural Science Foundation of China (NSFC) suggested that reduced Wnt/β-catenin signaling is involved in the onset and/or progression of bone erosion in rheumatoid arthritis (RA). This could lead to potential new avenues of treatment of bone erosion by enhancing Wnt signaling pathway. One of the R-spondins, RSpo1, a family of 4 proteins unrelated to other Wnt ligands that may act as Wnt agonists has recently been demonstrated. However, its action and mechanism is not well known. The present study will be undertaken to investigate: (1) RSpo1 potentiates Wnt signaling from gene expression and molecule protein; (2) RSpo1 induces osteoblast differentiation and blocks osteoclastogenesis in an OPG- and osteoblast-dependent manner; (3) RSpo1 promotes Wnt signaling and enhances osteoblast activity in murine with β-catenin/TCF promoter; (4) RSpo1 protects against inflammatory joint destruction by up-regulating osteoblast activation and supressing the osteoclast function in murine RA model, which maintain homeostasis of bone metabolism in local joint. The study will reveal the capacity of the RSpo family of proteins to block Dkk activity and enhance Wnt signaling activity, which will render this group of proteins both excellent tools for studying the mechanisms of the Wnt signaling pathway and promising candidates for a therapeutic agent on bone erosion in RA.

我们刚结题的国家自然科学基金课题研究提示：Wnt/β-catenin信号通路活性受抑是类风湿关节炎（RA）骨侵蚀的始动因素，增强该通路有望治疗RA关节破坏。新近发现脊椎蛋白1（RSpo1）可能是Wnt激活剂，但作用和机制不明。本研究拟开展：①从基因和蛋白层面观察RSpo1对内源性Wnt通路的活化作用，深入研究它与Wnt抑制剂的关系。②探讨RSpo1协同和增强Wnt活性对原始成骨（OB）和破骨（OC）细胞分化、成熟及相关功能的影响。③在携带β-catenin/TCF启动子应答的小鼠中，验证Rspo1强化内源性Wnt活性，促进新骨形成的生理功能。④在小鼠RA模型中，确定RSpo1诱导关节β-catenin表达，通过上调OB成骨和抑制OC破骨，从而维持RA关节局部骨代谢稳态。本研究不仅阐明RSpo1功能和机制，为今后研究Wnt激活途径提供有力武器，而且为治疗RA骨侵蚀增添新理论和新手段。

RA关节侵蚀机制一直是风湿免疫性疾病研究热点，有研究结果提示：Wnt/β-catenin信号通路活性受抑是类风湿关节炎（RA）关节破坏的始动因素，增强该通路有望防治RA骨侵蚀。新近发现脊椎蛋白1（RSpo1）可能是Wnt激活剂，但作用和机制不明。本研究通过双荧光素酶报告基因检测系统，利用T细胞因子(TCF)报告质粒检测Wnt/β-catenin信号通路活性。采用蛋白印迹法检测β-catenin蛋白累积情况。通过磷酸酶检测试剂盒检测AKP活性，ELISA法检测OPG的表达，从基因和蛋白层面观察RSpo1对内源性Wnt通路的活化作用，深入研究它与Wnt抑制剂的关系，以及RSpo1协同和增强Wnt活性对原始成骨（OB）细胞分化、成熟及相关功能的影响。本研究观察到RSpo1单独处理上调AKP活力（2.8±0.08 vs 1.7±0.21，T=3.014，P <0.05）及OPG表达能力（1.29±0.13 vs 1.0±0.21，T=3.348，P <0.05）较弱，而 RSpo1及 Wnt3A协同处理可显著增强AKP活力（81.3±5.08 vs 1.7±0.21，T=31.31，P <0.01）并上调OPG表达（5.26±0.60 vs 1.0±0.21，T=6.99，P <0.01）。RSpo1 协同 Wnt3A激活TCF活性并诱导β-catenin蛋白的积累（3.28±0.18 vs 1.0±0.21，T=10.94，P <0.05）。但是，单独RSpo1对TCF的活性及β-catenin的积累（1.25±0.08 vs 1.0±0.21，T=2.225，P＞0.05）无影响。此外，RSpo1可减少DKK1对hFOB1．19细胞碱性磷酸酶活力的抑制作用，并且对OPG的增强作用大于DKK1对其的抑制作用。本研究证实了RSpo1通过抑制DKK1，参与Wnt/β-catenin信号通路，促进成骨细胞分化成熟，分泌骨保护素。这方面研究不仅阐明RSpo1功能和机制，为今后研究Wnt激活途径提供有力武器，而且为防治RA关节侵蚀增添新理论和新手段。