Ablation of Gsα signaling in osteoclast progenitor cells adversely affects skeletal bone maintenance

Abstract

Gsα, the alpha stimulatory subunit of heterotrimeric G proteins that activates downstream signaling through the adenylyl cyclase and cAMP/PKA pathway, plays an important role in bone development and remodeling. The role of Gsα in mesenchymal stem cell (MSC) differentiation to osteoblasts has been demonstrated in several mouse models of Gsα inactivation. Previously, using mice with heterozygous germline deletion of Gsα (Gnas^+/p-), we identified a novel additional role for Gsα in bone remodeling, and showed the importance of Gnas in maintaining bone quality by regulating osteoclast differentiation and function. In this study, we show that postnatal deletion of Gsα (CreERT2;Gnas^fl/fl) leads to reduction in trabecular bone quality parameters and increased trabecular osteoclast numbers. Furthermore, mice with deletion of Gsα specifically in cells of the macrophage/osteoclast lineage (LysM-Cre;Gnas^fl/fl) showed reduced trabecular bone quality and increased trabecular osteoclasts, but to a reduced extent compared to the CreERT2;Gnas^fl/fl global knockout. This demonstrates that while Gsα has a cell autonomous role in osteclasts in regulating bone quality, Gsα expression in other cell types additionally contribute. In both of these mouse models, cortical bone was more subtly affected than trabecular bone. Our results support that Gsα is required postnatally to maintain trabecular bone quality and that Gsα function to maintain trabecular bone is regulated in part through a specific activity in osteoclasts.

Keywords: Bone remodeling; GNAS; Gsα; LysM; Osteoclast; Trabecular bone.

Figure 1. Postnatal homozygous deletion of Gnas/Gsα negatively affects trabecular bone quality

(A) Reduced Gsα mRNA expression in bone samples from CreERT2;Gnas^fl/fl mice compared to control (Cre-negative) Gnas^fl/fl samples was confirmed by RT-PCR (n = 5 per genotype). (B) Representative μCT scans of control and CreERT2;Gnas^fl/fl mice at 6 weeks after tamoxifen injections (n = 5 per genotype) showing cross-section of trabecular bone from distal femurs. (C–F) Measured trabecular bone parameters show reduced bone volume fraction (BV/TV), trabecular number (Tb.N/mm), and trabecular thickness (Tb.Th) and increased trabecular spacing (Tb.Sp, mm) in CreERT2;Gnas^fl/fl. *p < 0.05.

Figure 2. Mice with postnatal homozygous deletion of Gnas/Gsα show increased trabecular osteoclast numbers

(A) TRAP staining of femurs at 4 weeks after tamoxifen injection shows multi-nucleated osteoclasts along trabecular bone. (B) Quantification of trabecular osteoclast numbers (Oc.N/mm) at 4 weeks after tamoxifen injection in control and CreERT2;Gnas^fl/fl mice (n= 4 per genotype). *p < 0.05

Figure 3. Macrophage-specific Gnas/Gsα deletion reduced trabecular bone quality less severely than global deletion of Gnas/Gsα

(A) Gsα mRNA expression in bone marrow macrophages (osteoclast lineage cells) isolated from LysMCre;Gnas^fl/fl mice is reduced relative to controls (Cre-negative; n= 4 per genotype). (B) Representative μCT scans of 12-week-old control and LysMCre;Gnas^fl/fl mice showing cross-sections of trabecular bone from distal femurs. (C–F) Measured trabecular bone parameters show reduced bone volume fraction (BV/TV), trabecular number (Tb.N, and trabecular thickness (Tb.Th) and increased trabecular spacing (Tb.Sp) in LysMCre;Gnas^fl/fl (n= 5 per genotype). **p< 0.01; *p < 0.05; #p≤0.1