Increased Osteoblast GαS Promotes Ossification by Suppressing Cartilage and Enhancing Callus Mineralization During Fracture Repair in Mice

Gα_S, the stimulatory G protein α-subunit that raises intracellular cAMP levels by activating adenylyl cyclase, plays a vital role in bone development, maintenance, and remodeling. Previously, using transgenic mice overexpressing Gα_S in osteoblasts (G_S-Tg), we demonstrated the influence of osteoblast Gα_S level on osteogenesis, bone turnover, and skeletal responses to hyperparathyroidism. To further investigate whether alterations in Gα_S levels affect endochondral bone repair, a postnatal bone regenerative process that recapitulates embryonic bone development, we performed stabilized tibial osteotomy in male G_S-Tg mice at 8 weeks of age and examined the progression of fracture healing by micro-CT, histomorphometry, and gene expression analysis over a 4-week period. Bone fractures from G_S-Tg mice exhibited diminished cartilage formation at the time of peak soft callus formation at 1 week post-fracture followed by significantly enhanced callus mineralization and new bone formation at 2 weeks post-fracture. The opposing effects on chondrogenesis and osteogenesis were validated by downregulation of chondrogenic markers and upregulation of osteogenic markers. Histomorphometric analysis at times of increased bone formation (2 and 3 weeks post-fracture) revealed excess fibroblast-like cells on newly formed woven bone surfaces and elevated osteocyte density in G_S-Tg fractures. Coincident with enhanced callus mineralization and bone formation, G_S-Tg mice showed elevated active β-catenin and Wntless proteins in osteoblasts at 2 weeks post-fracture, further substantiated by increased mRNA encoding various canonical Wnts and Wnt target genes, suggesting elevated osteoblastic Wnt secretion and Wnt/β-catenin signaling. The G_S-Tg bony callus at 4 weeks post-fracture exhibited greater mineral density and decreased polar moment of inertia, resulting in improved material stiffness. These findings highlight that elevated Gα_S levels increase Wnt signaling, conferring an increased osteogenic differentiation potential at the expense of chondrogenic differentiation, resulting in improved mechanical integrity. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.