Fig. 6
Raising the intracellular level of calcium by ionomycin rescued ALPL deficiency-induced age-related osteoporosis. a MicroCT analysis showed that alpl+/− mice (n = 12) had significantly decreased bone mineral density (BMD), Bone volume relative to tissue volume (BV/TV), and trabecular number (Tb.N) in trabecular bone (TB) of the distal femur compared the levels in WT littermates and alpl+/− mice treated with ionomycin at a dose of 1 mg/kg/day for 28 days (n = 10). b H&E staining showed a decreased TB volume (yellow circled area) in the distal femurs of alpl+/− mice compared with the WT group and alpl+/− mice treated with ionomycin. Scale bar, 1 mm. c Representative histological images of distal femurs showed a significantly increased number of adipocytes in alpl+/− mouse bone marrow, as assessed by oil red O staining. Scale bar, 25 μm. d The calcein double labeling assay showed a significantly decreased bone formation rate in alpl+/− mice compared with that of WT and alpl+/− mice treated with ionomycin. Scale bar, 25 μm. e The intracellular level of Ca2+ in alpl+/− BMSCs was decreased compared with that in WT BMSCs, and ionomycin treatment elevated the intracellular level of Ca2+ in alpl+/− BMSCs. f Alizarin red staining showed that alpl+/− BMSCs cultured under osteoinductive conditions had a decreased capacity to form mineralized nodules compared with that of WT BMSCs. Ionomycin treatment of alpl+/− BMSCs showed an elevated capacity to form mineralized nodules. g Western blot analysis showed that alpl+/− BMSCs had decreased expression of osteogenic-related proteins RUNX2 and Sp7 compared with WT BMSCs and ionomycin treatment of alpl+/− BMSCs. β-actin was used as a protein loading control. alpl+/− BMSCs showed an increased number of oil red O-positive cells when cultured under adipogenic conditions (h), and western blotting showed that there was an upregulation of the adipogenic-related proteins PPARγ2 and LPL (i) compared with levels in WT BMSCs and alpl+/− BMSCs treated with ionomycin. The representative results from three independent experiments are shown. Error bars represent the s.d. from the mean values. *P < 0.05; **P < 0.01; ***P < 0.001
