The R-spondin family of proteins are Wnt agonists, and the complete embryonic disruption of Rspo2 results in skeletal developmental defects that recapitulate the phenotype observed with Lrp5/6 deficiency. Previous work has shown that R-spondin-2 (Rspo2, RSPO2) is both highly expressed in Wnt-stimulated pre-osteoblasts and its overexpression induces osteoblast differentiation in the same cells, supporting its putative role as a positive autocrine regulator of osteoblastogenesis. However, the role of Rspo2 in regulating osteoblastogenesis and bone formation in postnatal bone has not been explored. Here we show that limb-bud progenitor cells from Rspo2 knockout mice undergo reduced mineralization during osteoblastogenesis in vitro and have a corresponding alteration in their osteogenic gene expression profile. We also generated the first Rspo2 conditional knockout (Rspo2^floxed) mouse and disrupted Rspo2 expression in osteoblast-lineage cells by crossing to the Osteocalcin-Cre mouse line (Ocn-Cre + Rspo2^f/f). Ocn-Cre + Rspo2^f/f male and female mice at 1, 3, and 6 months were examined. Ocn-Cre + Rspo2^f/f mice are decreased in overall body size compared to their control littermates and have decreased bone mass. Histomorphometric analysis of 1-month-old mice revealed a similar number of osteoblasts and mineralizing surface per bone surface with a simultaneous decrease in mineral apposition and bone formation rates. Consistent with this observation, serum osteocalcin in 3-month-old Ocn-Cre + Rspo2^f/f was reduced, and bone marrow-mesenchymal stem cells from Ocn-Cre + Rspo2^f/f mice undergo less mineralization in vitro. Finally, gene expression analysis and immunohistochemistry of mature bone shows reduced beta-catenin signaling in Ocn-Cre + Rspo2^f/f. Overall, RSPO2 reduces osteoblastogenesis and mineralization, leading to reduced bone mass.

R-spondin蛋白家族是Wnt激动剂，Rspo2的完全胚胎破坏导致骨骼发育缺陷，从而概括了Lrp5 / 6缺乏症所观察到的表型。先前的工作表明，R-spondin-2（Rspo2，RSPO2）在Wnt刺激的成骨细胞中都高度表达，并且其过表达诱导同一细胞中的成骨细胞分化，从而支持其作为成骨细胞生成的正向自分泌调节剂的假定作用。但是，Rspo2在调节产后骨骼中成骨细胞生成和骨形成中的作用尚未探索。在这里，我们显示了来自Rspo2的肢体芽祖细胞基因敲除小鼠体外成骨细胞过程中矿物质减少，成骨基因表达谱发生相应变化。我们还通过与Osteocalcin -Cre小鼠品系（Ocn-Cre + Rspo2 ^{f / f}）杂交，产生了第一只Rspo2条件性基因敲除（Rspo2 ^flxed）小鼠，并破坏了成骨细胞系细胞中Rspo2的表达。在1、3和6个月时检查Ocn-Cre + Rspo2 ^{f / f}雄性和雌性小鼠。Ocn-Cre + Rspo2 ^{f / f}与对照同窝仔相比，小鼠的整体体积减小，并且骨骼质量降低。1个月大小鼠的组织形态分析显示，成骨细胞的数量和每个骨表面的矿化表面数量相似，同时矿物质沉积和骨形成速率降低。与该观察结果一致，3个月大的Ocn-Cre + Rspo2 ^{f / f的}血清骨钙素减少，并且Ocn-Cre + Rspo2 ^{f / f}小鼠的骨髓间充质干细胞在体外的矿化较少。最后，成熟骨骼的基因表达分析和免疫组化显示Ocn-Cre + Rspo2 ^{f / f中的}β-catenin信号减少。总体而言，RSPO2减少了成骨细胞的生成和矿化，从而导致骨量减少。