gms | German Medical Science

Objectives: Wnt1 plays a crucial role in mediating the skeletal response to mechanical load, but its mechanisms, especially concerning the Wnt1 mutation associated with osteogenesis imperfecta type XV (OI-XV), remain elusive. We investigated the impact of the Wnt1^G177C loss-of-function mutation during mechanical loading and elucidated the signaling cascades governing Wnt1-mediated bone formation.

Methods: In a cyclic axial compression experiment on 12-week-old female Wnt1^+/+ and Wnt1^G177C mice, loading was applied to the right ulna at 2 Hz. Wnt1^+/+ mice received 2.0 N, and Wnt1^G177C mice received 0.5 N, generating a 2000 microstrain surface strain in both strains (Fig. 1A). Loading was performed under anaesthesia for three consecutive days, each lasting one minute, with the left ulna as non-loaded control. Mice were euthanized on day 16 and 4 for microcomputed tomography (µCT) and RNAseq analysis, respectively. RNAseq analysis was performed on bone samples lacking bone marrow. Mechanical stimulation experiments on primary osteoblasts from 12-week-old female Wnt1^+/+ and Wnt1^G177C mice involved laminar fluid flow (LFF) with a shear stress of 10 dynes/cm² for 1h. RNA samples were collected immediately (0h) after LFF.

Results and conclusion: µCT analysis revealed that ulna loading caused a substantial increase of cortical thickness in Wnt1^+/+ control mice, whereas this effect was attenuated in Wnt1^G177C mutant mice (Figure 1B–C [Fig. 1]). Exploring pathways influenced by the Wnt1^G177C mutation during mechanical loading through RNAseq analysis, we identified 1,704 differentially regulated genes (DEGs) (Figure 1D [Fig. 1]). Metascape analysis revealed several modulated pathways, including skeletal system development, and extracellular matrix organization. Comparison with RNAseq on primary osteoblasts, where Wnt1 was deleted using siRNA and underwent LFF, highlighted 16 common genes, with nuclear receptor subfamily 4, group A, member 2 (Nr4a2) consistently dependent on both Wnt1^G177C mutation and stimulation (Figure 1E [Fig. 1]). Depleting NR4A2 in human mesenchymal stem cells limited their osteogenic capacity, thus confirming its positive role in promoting osteoblast differentiation. Our study demonstrated that the Wnt1^G177C mutation impairs mechanically induced bone formation through modulation of Nr4a2 expression.

Our research highlights the significant impact of the Wnt1^G177C mutation on impairing the bone's response to mechanical loading, suggesting that physical therapy to strengthen bone mass in OI-XV patients may have limited success. These findings suggest potential therapeutic interventions targeting these pathways to enhance bone formation in response to mechanical stimuli.