Department of Orthopedic Surgery, Brigham and Women's Hospital, Boston, MA 02115, USA.
Department of Orthopedic Surgery, Brigham and Women's Hospital, Boston, MA 02115, USA; Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.
Bone. 2022 Feb;155:116267. doi: 10.1016/j.bone.2021.116267. Epub 2021 Nov 29.
Leucine-rich repeat containing G-protein-coupled receptor 6 (LGR6) is a marker of osteoprogenitor cells and is dynamically expressed during in vitro osteodifferentation of mouse and human mesenchymal stem cells (MSCs). While the Lgr6 genomic locus has been associated with osteoporosis in human cohorts, the precise molecular function of LGR6 in osteogenesis and maintenance of bone mass are not yet known. In this study, we performed in vitro Lgr6 knockdown and overexpression experiments in murine osteoblastic cells and find decreased Lgr6 levels results in reduced osteoblast proliferation, differentiation, and mineralization. Consistent with these data, overexpression of Lgr6 in these cells leads to significantly increased proliferation and osteodifferentiation. To determine whether these findings are recapitulated in vivo, we performed microCT and ex vivo osteodifferentiation analyses using our newly generated CRISPR-Cas9 mediated Lgr6 mouse knockout allele (Lgr6-KO). We find that ex vivo osteodifferentiation of Lgr6-KO primary MSCs is significantly reduced, and 8 week-old Lgr6-KO mice have less trabecular bone mass as compared to Lgr6 wildtype controls, indicating that Lgr6 is necessary for normal osteogenesis and bone mass. Towards mechanism, we analyzed in vitro signaling in the context of two LGR6 ligands, RSPO2 and MaR1. We find that RSPO2 stimulates LGR6-mediated WNT/β-catenin signaling whereas MaR1 stimulates LGR6-mediated cAMP activity, suggesting two ligand-dependent functions for LGR6 receptor signaling during osteogenesis. Collectively, this study reveals that Lgr6 is necessary for wildtype levels of proliferation and differentiation of osteoblasts, and achieving normal bone mass.
富含亮氨酸重复的 G 蛋白偶联受体 6（LGR6）是成骨前体细胞的标志物，在体外小鼠和人间充质干细胞（MSCs）的成骨分化过程中动态表达。虽然 Lgr6 基因组座与人骨质疏松队列有关，但 LGR6 在成骨和维持骨量中的精确分子功能尚不清楚。在这项研究中，我们在小鼠成骨细胞中进行了体外 Lgr6 敲低和过表达实验，发现 Lgr6 水平降低导致成骨细胞增殖、分化和矿化减少。与这些数据一致的是，这些细胞中 Lgr6 的过表达导致增殖和成骨分化显著增加。为了确定这些发现是否在体内得到重现，我们使用我们新生成的 CRISPR-Cas9 介导的 Lgr6 小鼠敲除等位基因（Lgr6-KO）进行了 microCT 和体外成骨分化分析。我们发现 Lgr6-KO 原代 MSC 的体外成骨分化显著减少，并且 8 周龄的 Lgr6-KO 小鼠的小梁骨量比 Lgr6 野生型对照少，表明 Lgr6 对于正常成骨和骨量是必要的。在机制方面，我们分析了两种 LGR6 配体 RSPO2 和 MaR1 背景下的体外信号。我们发现 RSPO2 刺激 LGR6 介导的 WNT/β-catenin 信号，而 MaR1 刺激 LGR6 介导的 cAMP 活性，表明 LGR6 受体信号在成骨过程中有两个配体依赖的功能。总之，这项研究表明 Lgr6 对于成骨细胞的增殖和分化以及达到正常骨量是必需的。
