25-Hydroxyvitamin D3 induces osteogenic differentiation of human mesenchymal stem cells

Abstract

25-Hydroxyvitamin D₃ [25(OH)D₃] has recently been found to be an active hormone. Its biological actions are demonstrated in various cell types. 25(OH)D₃ deficiency results in failure in bone formation and skeletal deformation. Here, we investigated the effect of 25(OH)D₃ on osteogenic differentiation of human mesenchymal stem cells (hMSCs). We also studied the effect of 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], a metabolite of 25(OH)D₃. One of the vitamin D responsive genes, 25(OH)D₃-24-hydroxylase (cytochrome P450 family 24 subfamily A member 1) mRNA expression is up-regulated by 25(OH)D₃ at 250-500 nM and by 1α,25-(OH)₂D₃ at 1-10 nM. 25(OH)D₃ and 1α,25-(OH)₂D₃ at a time-dependent manner alter cell morphology towards osteoblast-associated characteristics. The osteogenic markers, alkaline phosphatase, secreted phosphoprotein 1 (osteopontin), and bone gamma-carboxyglutamate protein (osteocalcin) are increased by 25(OH)D₃ and 1α,25-(OH)₂D₃ in a dose-dependent manner. Finally, mineralisation is significantly increased by 25(OH)D₃ but not by 1α,25-(OH)₂D₃. Moreover, we found that hMSCs express very low level of 25(OH)D₃-1α-hydroxylase (cytochrome P450 family 27 subfamily B member 1), and there is no detectable 1α,25-(OH)₂D₃ product. Taken together, our findings provide evidence that 25(OH)D₃ at 250-500 nM can induce osteogenic differentiation and that 25(OH)D₃ has great potential for cell-based bone tissue engineering.

Figure 1. Induction of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) by 25(OH)D₃ and 1α,25-(OH)₂D₃ in hMSCs.

Real-time quantitative PCR was used to determine the induction of CYP24A1 mRNA in response to 25(OH)D₃ (a) and 1α,25-(OH)₂D₃ (b) at the indicated concentrations in the hMSCs during three weeks of treatment. Relative mRNA expression was normalised to the control gene ribosomal protein lateral stalk subunit P0 (RPLP0) and fold inductions were calculated in reference to solvent control (0.1% ethanol), which is set as 1. Results are expressed as means ± SD (n = 3, ***P < 0.001).

Figure 2. Cytochrome P450 family 27 subfamily B member 1 (CYP27B1) mRNA expression.

Real-time quantitative PCR was used to determine the expression of CYP27B1 mRNA in the hMSCs. Relative mRNA expression was normalised to the control gene ribosomal protein lateral stalk subunit P0 (RPLP0). Results are expressed as means ± SD (n = 3, ***P < 0.001).

Figure 3. Morphological changes of hMSCs during the treatments with 25(OH)D₃ or 1α,25-(OH)₂D₃.

(a) hMSCs at day 0; (b) 0.1% ethanol-treated hMSCs at day 21; (c) 500 nM 25(OH)D₃-treated hMSCs at day 21; (d) 10 nM 1α,25-(OH)₂D₃-treated hMSCs at day 21. Scale bars: 60 μm.

Figure 4. Regulation of ALPL activity by 25(OH)D₃ and 1α,25-(OH)₂D₃ in hMSCs.

Alkaline phosphatase (ALPL) activity was measured according to the procedure of ELF97 in the hMSCs during two weeks of treatments with 25(OH)D₃ (a) or 1α,25-(OH)₂D₃ (b) at the indicated concentrations. Results are expressed as means ± SD (n = 9, ***P < 0.001).

Figure 5. Gene regulation by 25(OH)D₃ and 1α,25-(OH)₂D₃ during the osteogenic differentiation of hMSCs.

Real-time quantitative PCR was used to determine the induction of secreted phosphoprotein 1 (SPP1) (a,b), bone gamma-carboxyglutamate protein (BGLAP) (c,d), and runt related transcription factor 2 (RUNX2) (e,f) mRNA in response to the indicated concentrations of 25(OH)D₃ or 1α,25-(OH)₂D₃ in the hMSCs during three weeks of treatments. Relative mRNA expression was normalised to the control gene ribosomal protein lateral stalk subunit P0 (RPLP0) and fold inductions were calculated in reference to vehicle control, which is set as 1. Results are expressed as means ± SD (n = 3, *P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 6. Regulation by 25(OH)D₃ and 1α,25-(OH)₂D₃ on bone gamma-carboxyglutamate protein secretion and intracellular expression in hMSCs.

(a,b) Bone gamma-carboxyglutamate protein secretion in culture media of hMSCs was measured according to the procedure of the Gla-type osteocalcin EIA kit during four weeks of treatments with 25(OH)D₃ or 1α,25-(OH)₂D₃ at the indicated concentrations. Results are expressed as means ± SD. For 25(OH)D₃ n = 3 and for 1α,25-(OH)₂D₃ n = 2. (c–e) Intracellular bone gamma-carboxyglutamate protein staining was performed in 0.1% ethanol-treated hMSCs (c), 500 nM 25(OH)D₃-treated hMSCs (d), and 1 nM 1α,25-(OH)₂D₃-treated hMSCs (e). Scale bars: 50 μm in c and d, 20 μm in e. Controls in which the primary antibody was replaced with non-immunised mouse IgG show no positive staining (data not shown).

Figure 7. Mineralisation of the extracellular matrix of hMSCs.

Mineralisation was determined by measuring the extracellular deposition of calcium in response to the treatment with 25(OH)D₃ or 1α,25-(OH)₂D₃ at indicated concentrations during four weeks. Results are expressed as means ± SD (n = 2, *P < 0.05 and ***P < 0.001).