Table 1.
Primer sequence.
Figure 1.
Effects of 1,25(OH)2D3 and 25(OH)D3 on primary human osteoblast proliferation.
Osteoblasts were cultured in the presence of 0, 1, 10 or 100 nM 1,25(OH)2D3 (A) and 0, 100, 200 or 400 nM 25(OH)D3 (B) and the proliferation was quantified at day 3 and 6. Results (mean ± SEM) are expressed as treatment versus control ratios (time-point 0 was set at 1.0) using cells from 4 (A) or 7 (B) different donors. Results were analysed using Friedman test followed by Dunn’s post hoc test for each timepoint (*p<0.05, **p<0.01, ***p<0.001).
Figure 2.
Effects of 1,25(OH)2D3 and 25(OH)D3 on ALP activity, P1NP and osteocalcin secretion by primary human osteoblast.
Osteoblasts were cultured in the presence of 0 or 100 nM 1,25(OH)2D3 and 0 or 400 nM 25(OH)D3 and ALP activity (A and B respectively), P1NP (C and D respectively) and osteocalcin secretion (E and F respectively) were measured at day 3, 7, 10 and 14 of the differentiation. Results are expressed as mean ± SEM using cells from 5 different donors. Results were analyzed using Wilcoxon signed rank test for each timepoint (*p<0.05, **p<0.01, ***p<0.001).
Figure 3.
Effects of 1,25(OH)2D3 and 25(OH)D3 on mRNA levels of genes involved in primary human osteoblast differentiation.
Osteoblasts were cultured in the presence of 0, 1, 10 and 100 nM 1,25(OH)2D3 or 0, 100, 200 or 400 nM 25(OH)D3 for 10 days in osteogenic medium and mRNA levels of ALP (A), COL1α1 (B), osteocalcin (C) and osteopontin (D) were determined. Results (mean ± SEM) are expressed as treatment versus control ratios (control was set at 1.0; dashed line) using cells from 5 different donors. Results were analysed using Friedman test followed by Dunn’s post hoc test (*p<0.05, **p<0.01, ***p<0.001).
Figure 4.
Effects of 1,25(OH)2D3 and 25(OH)D3 on VDR, CYP27B1 and CYP24 mRNA levels in primary human osteoblast.
Osteoblasts were cultured in the presence of 0, 1, 10 and 100 nM 1,25(OH)2D3 or 0, 100, 200 and 400 nM 25(OH)D3 for 24 hours and mRNA levels of VDR (A), CYP27B1 (B) and CYP24 (C) were determined. Results (mean ± SEM) are expressed as treatment versus control ratios (control was set at 1.0; dashed line) using cells from 5 or 6 different donors. Results were analysed using Friedman test followed by Dunn’s post hoc test (*p<0.05, **p<0.01, ***p<0.001).
Figure 5.
Synthesis of 1,25(OH)2D3 and 24R,25(OH)2D3 by primary human osteoblasts.
Osteoblasts were cultured in the presence of 0, 100, 200, 400 and 1.000 nM 25(OH)D3 for 24 hours and 25(OH)D3 (A) 1,25(OH)2D3 (B) and 24R,25(OH)2D3 (C) levels were measured in non-conditioned and conditioned culture medium. Results are expressed as mean ± SEM using cells from 3 different donors.
Figure 6.
Effects of 25(OH)D3 on mRNA levels of genes involved in primary human osteoblast differentiation after CYP27B1 silencing.
CYP27B1-silenced and control cells were incubated in the presence of 0 or 400 nM 25(OH)D3 for 3 days. CYP27B1 knock down was determined before 25(OH)D3 treatment (A). After 72 hours incubation with 25(OH)D3, we examined levels of 1,25(OH)2D3 (B), 24R,25(OH)2D3 (C) and 25(OH)D3 (D), and mRNA levels of CYP27B1 (E), CYP24 (F), VDR (G), ALP (H), osteocalcin (I) and osteopontin (J) in CYP27B1-silenced and control cells. Results are expressed as mean ± SEM using cells from 5 different donors. Results were analysed using Friedman test followed by Dunn’s post hoc test (*p<0.05, **p<0.01, ***p<0.001).
Figure 7.
Effects of 24R,25(OH)2D3 on mRNA levels of genes involved in osteoblast differentiation.
Osteoblasts were cultured in the presence of 0, 100, 200 or 400 nM 24R,25(OH)2D3 and mRNA levels of COL1α1 (A), ALP (B), osteocalcin (C) and osteopontin (D) were determined after 72 hours. Results (mean ± SEM) are expressed as treatment versus control ratios (control was set at 1.0; dashed line) using cells from 4 different donors. Results were analysed using Friedman test followed by Dunn’s post hoc test (*p<0.05, **p<0.01, ***p<0.001).
Figure 8.
Effects of 24R,25(OH)2D3 on VDR, CYP27B1 and CYP24 mRNA levels in primary human osteoblasts.
Osteoblasts were cultured in the presence of 0, 100, 200 or 400 nM 24R,25(OH)2D3 and mRNA levels of VDR (A), CYP27B1 (B) and CYP24 (C) were determined after 72 hours. Results (mean ± SEM) are expressed as treatment versus control ratios (control was set at 1.0; dashed line) using cells from 4 different donors. Results were analysed using Friedman test followed by Dunn’s post hoc test (*p<0.05, **p<0.01, ***p<0.001).