Sui Xin, Deng Shijian, Liu Mengmeng, Fan Linlin, Wang Yunfei, Xu Huaxing, Sun Yao, Kishen Anil, Zhang Qi
Department of Endodontics, School & Hospital of Stomatology, Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai, China.
Department of Implantology, School & Hospital of Stomatology, Tongji University, Shanghai, Engineering Research Center of Tooth Restoration and Regeneration, Shanghai, China.
Cell Physiol Biochem. 2018;48(5):2091-2102. doi: 10.1159/000492549. Epub 2018 Aug 14.
BACKGROUND/AIMS: Activation of the Wnt/β-catenin signalling pathway has been widely investigated in bone biology and shown to promote bone formation. However, its specific effects on osteoclast differentiation have not been fully elucidated. Our study aimed to identify the role of β-catenin in osteoclastogenesis and bone homeostasis.
In the present study, exon 3 in the β-catenin gene (Ctnnb1) allele encoding phosphorylation target serine/threonine residues was flanked by floxP sequences. We generated mice exhibiting conditional β-catenin activation (Ctsk-Cre;Ctnnb1flox(exon3)/+, designated CA-β-catenin) by crossing Ctnnb1flox(exon3)/flox(exon3) mice with osteoclast-specific Ctsk-Cre mice. Bone growth and bone mass were analysed by micro-computed tomography (micro-CT) and histomorphometry. To further examine osteoclast activity, osteoclasts were induced from bone marrow monocytes (BMMs) isolated from CA-β-catenin and Control mice in vitro. Osteoclast differentiation was detected by tartrate-resistant acid phosphatase (TRAP) staining, immunofluorescence staining and reverse transcription-quantitative PCR (RT-qPCR) analysis.
Growth retardation and low bone mass were observed in CA-β-catenin mice. Compared to controls, CA-β-catenin mice had significantly reduced trabecular bone numbers under growth plates as well as thinner cortical bones. Moreover, increased TRAP-positive osteoclasts were observed on the surfaces of trabecular bones and cortical bones in the CA-β-catenin mice; consistent results were observed in vitro. In the CA-β-catenin group, excessive numbers of osteoclasts were induced from BMMs, accompanied by the increased expression of osteoclast-associated marker genes.
These results indicated that the constitutive activation of β-catenin in osteoclasts promotes osteoclast formation, resulting in bone loss.
背景/目的：Wnt/β-连环蛋白信号通路的激活在骨生物学中已得到广泛研究，并显示可促进骨形成。然而，其对破骨细胞分化的具体影响尚未完全阐明。我们的研究旨在确定β-连环蛋白在破骨细胞生成和骨稳态中的作用。
在本研究中，编码磷酸化靶丝氨酸/苏氨酸残基的β-连环蛋白基因（Ctnnb1）等位基因的外显子3两侧为floxP序列。我们通过将Ctnnb1flox(exon3)/flox(exon3)小鼠与破骨细胞特异性Ctsk-Cre小鼠杂交，培育出表现出条件性β-连环蛋白激活的小鼠（Ctsk-Cre;Ctnnb1flox(exon3)/+，称为CA-β-连环蛋白）。通过微计算机断层扫描（micro-CT）和组织形态计量学分析骨生长和骨量。为了进一步检查破骨细胞活性，体外从CA-β-连环蛋白和对照小鼠分离的骨髓单核细胞（BMMs）中诱导破骨细胞。通过抗酒石酸酸性磷酸酶（TRAP）染色、免疫荧光染色和逆转录定量PCR（RT-qPCR）分析检测破骨细胞分化。
在CA-β-连环蛋白小鼠中观察到生长迟缓和低骨量。与对照组相比，CA-β-连环蛋白小鼠生长板下的小梁骨数量显著减少，皮质骨也更薄。此外，在CA-β-连环蛋白小鼠的小梁骨和皮质骨表面观察到TRAP阳性破骨细胞增加；体外观察到一致的结果。在CA-β-连环蛋白组中，BMMs诱导出过多的破骨细胞，同时破骨细胞相关标记基因的表达增加。
这些结果表明破骨细胞中β-连环蛋白的组成性激活促进破骨细胞形成，导致骨质流失。
