Investigating the in vivo effects of osteoblast-specific overexpression of Gαs and Gαq/11 on bone formation and remodeling during fracture repair

ABSTRACT

G proteins mediate a multitude of physiological processes by transducing extracellular signals from G protein-coupled receptors to various intracellular signaling cascades. Of particular importance in bone are GαS that raises cAMP levels by activating adenylyl cyclase and Gαq/11 that increases intracellular calcium levels via activation of phospholipase C. Using transgenic mice that overexpress either GαS (Gs-Tg) or Gαq/11 (G11-Tg) in osteoblast lineage cells, previous work in our lab has demonstrated that alterations in GαS and Gαq/11 levels in the skeleton can have profound effects on bone mass, microarchitecture, mineral density, and quality. It remains unclear, however, how variation in GαS and Gαq/11 levels affects bone regeneration and repair. To test our hypothesis that increasing Gαs and Gα11 in osteoblasts leads to differential fracture healing responses, we performed stabilized tibial osteotomy in male Gs-Tg and G11-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.

Gs-Tg mouse fractures exhibited diminished cartilage formation, increased callus mineralization, increased bone formation, and improved material stiffness. Histological analyses also revealed excess fibroblasts along the newly formed woven bone surfaces and elevated osteocyte density at the times of increased bone formation and elevated osteoclastic bone resorption in the bone remodeling stage. Coincident with enhanced osteogenesis, elevated osteoblastic Wnt secretion and Wnt/-catenin signaling was evidenced by quantitative reverse transcription PCR and immunofluorescence staining implicating increased Wnt signaling activation as a mechanism by which increased GαS levels promote osteogenesis and induce fibrosis during bone repair in Gs-Tg mice. In contrast, normal endochondral healing progressed in G11-Tg mice. However, their reduced osteogenic differentiation capacity led to diminished formation of highly mineralized bone and poor structural integrity of healing bone. The lack of changes in osteoclast parameters and osteoclast marker expression suggested that the G11-Tg fracture repair phenotype is driven by osteogenic abnormalities. Overall, our results confirm our hypothesis that GαS and Gαq/11 differentially regulate bone formation and remodeling during fracture repair and highlight that osteoblast GαS and Gαq/11 levels play a significant role in determining the pattern, rate, and extent of fracture healing.