Synergistic effect of 1α,25-dihydroxyvitamin D3 and 17β-estradiol on osteoblast differentiation of pediatric MSCs.

Vitamin D is essential for mineral homeostasis and contributes to bone metabolism by stimulating osteoblast differentiation of marrow stromal cells (MSCs). In this study, we used MSCs from pre-pubertal girls and boys to test the hypothesis that 1α,25(OH) 2 D and 17β-estradiol have synergistic effects on these MSCs, and what mechanism is involved. With IRB approval, we isolated MSCs from discarded excess iliac marrow graft from children undergoing alveolar cleft repair. Plasma was available from 8 female (9.3 ± 0.2 years) and 8 male (9.6 ± 0.1 years) subjects for hormone assays [25(OH)D, total testosterone, 17β-estradiol, estrone, DHEA-S, Growth Hormone, IGF-I]. RT-PCR was used for gene expression. Alkaline phosphatase (ALP) activity was used to measure osteoblast differentiation at day 7; alizarin red was used to measure matrix mineralization at day 21. All subjects were pre-pubertal based on their hormone levels. Serum 25(OH)D levels ranged from 13.1 to 26.4 ng/mL, with 75% below 20 ng/mL. Constitutive gene expression of VDR and ERα, β varied from subject to subject with no association with sex or serum chemistries. In osteoblastogenic medium, 1α,25(OH) 2 D 3 (10 nM) increased ALP activity by 36% (p < 0.05) in MSCs; 10 nM of E2 was not stimulatory but the combination of 1α,25(OH) 2 D 3 and E2 increased ALP 151% (p < 0.05 vs. control) and by 84.5% (p < 0.05 vs. 1α,25(OH) 2 D3 alone). The combination of 1α,25(OH) 2 D 3 and E2 significantly increased mineralization 11-fold, compared with either agent alone. Twenty-four hour treatment with 1α,25(OH) 2 D 3 (10 nM) or E2 (10 nM) upregulated each other’s receptor by as much as 5.8-fold for ERα and 2.9-fold for the VDR. In summary, 1α,25(OH) 2 D3 stimulated osteoblast differentiation and matrix mineralization with MSCs from pre-pubertal subjects, with a synergistic effect of E2, mediated by upregulated receptor levels, at least in part. These studies add new information about the regulation of human osteoblast differentiation, effects of 1α,25(OH) 2 D 3 and E2 on MSCs, and the importance of vitamin D for skeletal health. [ABSTRACT FROM AUTHOR]