Sun Weiwei, Shi Yu, Lee Wen-Chih, Lee Seung-Yon, Long Fanxin
Department of Orthopaedic Surgery, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Anatomy, Histology and Embryology, Nanjing Medical University, Nanjing, China.
Department of Orthopaedic Surgery, Washington University School of Medicine, St. Louis, MO 63110, USA.
Bone. 2016 Apr;85:1-8. doi: 10.1016/j.bone.2016.01.013. Epub 2016 Jan 15.
Wnt signaling has emerged as a major target pathway for the development of novel bone anabolic therapies. Neutralizing antibodies against the secreted Wnt antagonist sclerostin (Scl-Ab) increase bone mass in both animal models and humans. Because we have previously shown that Rictor-dependent mTORC2 activity contributes to Wnt signaling, we test here whether Rictor is required for Scl-Ab to promote bone anabolism. Mice with Rictor deleted in the early embryonic limb mesenchyme (Prx1-Cre;Rictor(f/f), hereafter RiCKO) were subjected to Scl-Ab treatment for 5weeks starting at 4months of age. In vivo micro-computed tomography (μCT) analyses before the treatment showed that the RiCKO mice displayed normal trabecular, but less cortical bone mass than the littermate controls. After 5weeks of treatment, Scl-Ab dose-dependently increased trabecular and cortical bone mass in both control and RiCKO mice, but the increase was significantly blunted in the latter. Dynamic histomorphometry revealed that the RiCKO mice formed less bone than the control in response to Scl-Ab. In addition, the RiCKO mice possessed fewer osteoclasts than normal under the basal condition and exhibited lesser suppression in osteoclast number by Scl-Ab. Consistent with the fewer osteoclasts in vivo, bone marrow stromal cells (BMSC) from the RiCKO mice expressed less Rankl but normal levels of Opg or M-CSF, and were less effective than the control cells in supporting osteoclastogenesis in vitro. The reliance of Rankl on Rictor appeared to be independent of Wnt-β-catenin or Wnt-mTORC2 signaling as Wnt3a had no effect on Rankl expression by BMSC from either control or RICKO mice. Overall, Rictor in the limb mesenchymal lineage is required for the normal response to the anti-sclerostin therapy in both bone formation and resorption.
Wnt信号通路已成为新型骨合成代谢疗法开发的主要靶点途径。针对分泌型Wnt拮抗剂硬化蛋白的中和抗体（Scl-Ab）在动物模型和人类中均可增加骨量。由于我们之前已表明，依赖Rictor的mTORC2活性有助于Wnt信号传导，因此我们在此测试Rictor对于Scl-Ab促进骨合成代谢是否是必需的。在胚胎早期肢体间充质中缺失Rictor的小鼠（Prx1-Cre;Rictor(f/f)，以下简称RiCKO）在4月龄时开始接受Scl-Ab治疗5周。治疗前的体内微型计算机断层扫描（μCT）分析表明，RiCKO小鼠的小梁骨正常，但皮质骨量比同窝对照小鼠少。治疗5周后，Scl-Ab剂量依赖性地增加了对照小鼠和RiCKO小鼠的小梁骨和皮质骨量，但后者的增加明显减弱。动态组织形态计量学显示，RiCKO小鼠对Scl-Ab的反应形成的骨比对照小鼠少。此外，RiCKO小鼠在基础条件下的破骨细胞比正常小鼠少，并且Scl-Ab对破骨细胞数量的抑制作用较小。与体内破骨细胞较少一致，RiCKO小鼠的骨髓基质细胞（BMSC）表达的Rankl较少，但Opg或M-CSF水平正常，并且在体外支持破骨细胞生成方面比对照细胞效果差。Rankl对Rictor的依赖性似乎独立于Wnt-β-连环蛋白或Wnt-mTORC2信号传导，因为Wnt3a对对照小鼠或RiCKO小鼠的BMSC的Rankl表达均无影响。总体而言，肢体间充质谱系中的Rictor是骨形成和骨吸收对抗硬化蛋白治疗正常反应所必需的。
