Fig 1.
Effects of 6-week ALF or ELD administration in Cyp27b1+/+ or Cyp27b1–/–mice.
(A) Body weight. *P < 0.05, **P < 0.01 vs. 3-week old mice, Dunnett’s test. (B) Plasma Ca concentration. Significantly different from each 3-week old mice; *P < 0.05, **P < 0.01, ***P < 0.001, Dunnett’s test. (C) Plasma PTH concentration. *P < 0.05 vs. Cyp27b1+/+ mice administered vehicle, Student’s t-test. ##P < 0.01 vs. Cyp27b1–/–mice administered vehicle, Dunnett’s test. (D) ECaC2 mRNA expression level in the duodenum. †P < 0.05 vs. Cyp27b1+/+ mice administered ALF, Student’s t-test. (E) Calbindin-D9k mRNA expression level in the duodenum. ##P < 0.01 vs. Cyp27b1+/+ mice administered vehicle, Student’s t-test. (F) CYP2R1 mRNA expression levels in the liver. (G) CYP27A1 mRNA expression levels in the liver. (H) CYP24A1 mRNA expression level in the kidneys. #P < 0.05, ###P < 0.001 vs. Cyp27b1+/+ or Cyp27b1–/–mice administered vehicle, Dunnett’s test.
Fig 2.
Appearance and soft X-ray image of the femurs of Cyp27b1+/+ or Cyp27b1–/–mice administered ALF or ELD.
(A) Appearance and soft X-ray image of femur. (B) Femur bone length. **P < 0.01 vs. Cyp27b1+/+ mice administered vehicle, Student’s t-test. ##P < 0.01 vs. Cyp27b1–/–mice administered vehicle, Dunnett’s test.
Fig 3.
Bone histology in Cyp27b1+/+ or Cyp27b1–/–mice administered ALF or ELD.
(A) Analysis of bone mineralization by von Kossa staining. Areas stained black indicate calcification sites in which Ca has been deposited. Scale bar: 500 μm. (B) Analysis of cartilage formation by toluidine blue staining. Areas stained purple indicate cartilage cells. Scale bar: 500 μm. (C) Analysis of osteoid formation by Villanueva staining. Areas stained reddish purple indicate osteoid. Scale bar: 500 μm. (D) Analysis of bone formation rate by calcein double labeling. Scale bar: 50 μm.
Fig 4.
Bone mineral density and bone strength of the femurs in Cyp27b1+/+ or Cyp27b1–/–mice administered ALF or ELD.
(A) Total bone mineral density of femoral metaphysis. ##P < 0.01 vs. Cyp27b1+/+ or Cyp27b1–/–mice administered vehicle, Student’s t-test. (B) Cortical bone mineral density of femoral metaphysis. **P < 0.01 vs. Cyp27b1+/+ mice administered vehicle, Student’s t-test. ##P < 0.01 vs. Cyp27b1–/–mice administered vehicle, Dunnett’s test. (C) Trabecular bone density of metaphysis of the femur. #P < 0.05 vs. Cyp27b1+/+ mice administered vehicle, Student’s t-test. †P < 0.05 vs. Cyp27b1+/+ or Cyp27b1–/–mice administered ALF, Student’s t-test. (D) Bone strength in the X-axis direction of the femur. **P < 0.01 vs. Cyp27b1+/+ mice administered vehicle, Student’s t test. #P < 0.05, ##P < 0.01 vs. Cyp27b1–/–mice administered vehicle, Dunnett’s test. (E) Bone strength in the Y-axis direction of the femur. **P < 0.01 vs. Cyp27b1+/+ mice administered vehicle, Student’s t-test. #P < 0.05, ##P < 0.01 vs. Cyp27b1–/–mice administered vehicle, Dunnett’s test. (F) Bone strength of twisted femur. **P < 0.01 vs. Cyp27b1+/+ mice administered vehicle, Student’s t-test. #P < 0.05, ##P < 0.01 vs. Cyp27b1–/–mice administered vehicle, Dunnett’s test.
Fig 5.
Structural analysis of trabecular bone of femur of Cyp27b1+/+ or Cyp27b1–/–mice administered ALF and ELD.
(A) 2D trabecular structure of femur by micro-CT. Scale bar: 500 μm. (B) 3D trabecular structure of femur by micro-CT. Scale bar: 500 μm.
Fig 6.
Effect of vitamin D derivatives on the proliferation and the differentiation of ATDC5 cells.
(A) Proliferation of ATDC5 cells. Vitamin D homologs and vitamin D derivatives inhibited cell proliferation in a dose-dependent manner. (B) Evaluation of chondrocyte differentiation by Alcian blue staining. Vitamin D homologs as well as derivatives were found to inhibit the differentiation of cartilage cells in a dose-dependent manner.
Fig 7.
VDR-dependent transcriptional activity of vitamin D derivatives in ATDC5 cells and analysis of the effect of binding to DBP.
(A) Concentration dependence of the effects of vitamin D derivatives on VDR-dependent transcriptional activity of CYP24A1 in ATDC5 cells. When 1α,25D3 was added, CYP24A1 transcriptional activity increased in a concentration-dependent manner. Although no transcriptional activity was noted upon treatment with ALF and 25D3, ELD markedly induced transcriptional activity at 10−8 M. (B) Effects of vitamin D derivatives on VDR-dependent transcriptional activity of CYP24A1 in the presence or absence of DBP in ATDC5 cells. Although ALF was not affected by DBP, 1α,25D3 increased CYP24A1 transcriptional activity in the presence of DBP. However, ELD and 25D3 showed decreased transcriptional activity in a dose-dependent manner in the presence of DBP. (C, D) Megalin-mediated VDR-dependent transcriptional activity of CYP24A1 mediated by vitamin D derivatives in the presence of DBP in ATDC5 cells. (C) Megalin mRNA expression in si-megalin-transfected ATDC5 cells. In si-megalin transfected ATDC5 cells, megalin mRNA expression was knocked down by approximately 90%. (D) VDR-dependent transcriptional activity of CYP24A1 in megalin-knockdown ATDC5 cells. In megalin-knockdown ATDC5 cells, while 1α,25D3 and ALF had no effect, ELD and 25D3 significantly lowered CYP24A1 transcriptional activity.
Fig 8.
Schematic illustration of ELD action in trabecular bone.