Fig. 6: Knockout of PGE₂ receptor 4 (EP4) in sensory nerves abolishes divalent cation-induced bone formation.

a EP4 expression in DRG neurons stimulated by 10 mM Mg²⁺ with or without the conditioned medium from Mg²⁺-treated macrophages (n = 3). Reconstructed µCT images (b, scale bars = 1 mm), corresponding measurements (c), and representative H&E staining images (d) showing the new bone formation in EP4^wt mice or EP4_Avil^−/− mice (n = 3). Lower images are high-resolution versions (scale bars = 100 µm) of the boxed regions in the upper images (scale bars = 500 µm). e The running-wheel activity of EP4^wt or EP4_Avil^−/− mice at week 4 postoperatively. f The phosphorylation of CREB in the VMH of EP4^wt or EP4_Avil^−/− mice at week 1 postoperatively (n = 3, scale bars = 200 µm). Reconstructed µCT images (g, scale bars = 1 mm), corresponding measurements (h), and representative H&E staining images (i) showing the new bone formation in mice injected with vehicle (Veh) or propranolol (PROP) Lower images (scale bars = 100 µm) are high-resolution versions of the boxed regions in the upper images (scale bars = 500 µm). j The running-wheel activity of mice injected with vehicle or propranolol at week 4 postoperatively. k Schematic diagram showing the findings of this study. The divalent cations triggered the production of PGE₂ from macrophages, which activated the EP4 at the sensory nerve to tune down sympathetic tones via the CREB signaling in the VMH, resulting in increased osteogenesis and decreased osteoclastogenesis in the periosteum. Data are mean ± s.d. *P < 0.05, **P < 0.01 by one-way ANOVA with Tukey’s post hoc test (a, c, e, h, j). Source data are provided as a source data file.