VDR dependent and independent effects of 1,25-dihydroxyvitamin D 3 on nitric oxide production by osteoblasts

Abstract

1,25-Dihydroxyvitamin D 3 (1,25(OH) 2D 3) 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) 2D 3 affects NO production by osteoblasts in the presence or absence of mechanical stimulation. We hypothesised that 1,25(OH) 2D 3 stimulates NO production via nuclear actions of the vitamin D receptor (VDR), which requires hours of incubation with 1,25(OH) 2D 3 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) 2D 3 (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) 2D 3. NO production and NO synthase (NOS) expression were quantified. 10 −11 M 1,25(OH) 2D 3 for 24 h, but not 30 min, stimulated NO production by MC3T3-E1 osteoblasts (eightfold). 1,25(OH) 2D 3 for 24 h increased inducible-NOS gene-expression (twofold), suggesting that 1,25(OH) 2D 3 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) 2D 3 for 24 h, or during PFF only. Our results suggest that 1,25(OH) 2D 3 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) 2D 3 may affect mechanical loading-induced NO production independent of genomic VDR action, since 1,25(OH) 2D 3 diminished PFF-induced NO production in VDR −/− bone cells. In conclusion, 1,25(OH) 2D 3 and mechanical loading interact at the level of mechanotransduction, whereby 1,25(OH) 2D 3 seems to act independently of VDR genomic mechanism.