VDR dependent and independent effects of 1,25-dihydroxyvitamin D3 on nitric oxide production by osteoblasts

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) strongly mediates bone mass. Mechanical stimulation also affects bone mass, partly via enhancing nitric oxide (NO) production by osteoblasts. We aimed to determine whether 1,25(OH)2D3 affects NO production by osteoblasts in the presence or absence of mechanical stimulation. We hypothesised that 1,25(OH)2D3 stimulates NO production via nuclear actions of the vitamin D receptor (VDR), which requires hours of incubation with 1,25(OH)2D3 to occur.

MC3T3-E1 osteoblasts and long-bone osteoblasts of adult wildtype and VDR−/− mice were pre-incubated for 24 h with or without 1,25(OH)2D3 (10−13-10−9 M), followed by 30 min pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) or static culture with or without 1,25(OH)2D3. NO production and NO synthase (NOS) expression were quantified.

10−11 M 1,25(OH)2D3 for 24 h, but not 30 min, stimulated NO production by MC3T3-E1 osteoblasts (eightfold). 1,25(OH)2D3 for 24 h increased inducible-NOS gene-expression (twofold), suggesting that 1,25(OH)2D3 stimulated NO production via activation of NOS gene transcription. PFF rapidly increased NO production by MC3T3-E1 osteoblasts, wildtype osteoblasts, and VDR−/− osteoblasts. This PFF effect was abolished after incubation with 1,25(OH)2D3 for 24 h, or during PFF only.

Our results suggest that 1,25(OH)2D3 stimulates inducible-NOS expression and NO production by osteoblasts in the absence of mechanical stimulation, likely via genomic VDR action. In contrast, 1,25(OH)2D3 may affect mechanical loading-induced NO production independent of genomic VDR action, since 1,25(OH)2D3 diminished PFF-induced NO production in VDR−/− bone cells. In conclusion, 1,25(OH)2D3 and mechanical loading interact at the level of mechanotransduction, whereby 1,25(OH)2D3 seems to act independently of VDR genomic mechanism.