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

G alpha(S), the stimulatory G protein alpha-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 G alpha(S) in osteoblasts (G(S)-Tg), we demonstrated the influence of osteoblast G alpha(S )level on osteogenesis, bone turnover, and skeletal responses to hyperparathyroidism. To further investigate whether alterations in G alpha(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 beta-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/beta-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 G alpha(S) levels increase Wnt signaling, conferring an increased osteogenic differentiation potential at the expense of chondrogenic differentiation, resulting in improved mechanical integrity. (c) 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.