title: VDR dependent and independent effects of 1,25-dihydroxyvitamin D3 on nitric oxide production by osteoblasts.
abstract: 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(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)(2)D(3) affects NO production by osteoblasts in the presence or absence of mechanical stimulation. We hypothesised that 1,25(OH)(2)D(3) stimulates NO production via nuclear actions of the vitamin D receptor (VDR), which requires hours of incubation with 1,25(OH)(2)D(3) to occur. MC3T3-E1 osteoblasts and long-bone osteoblasts of adult wildtype and VDR(-/-) mice were pre-incubated for 24h with or without 1,25(OH)(2)D(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)(2)D(3). NO production and NO synthase (NOS) expression were quantified. 10(-11)M 1,25(OH)(2)D(3) for 24h, but not 30 min, stimulated NO production by MC3T3-E1 osteoblasts (eightfold). 1,25(OH)(2)D(3) for 24h increased inducible-NOS gene-expression (twofold), suggesting that 1,25(OH)(2)D(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)(2)D(3) for 24h, or during PFF only. Our results suggest that 1,25(OH)(2)D(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)(2)D(3) may affect mechanical loading-induced NO production independent of genomic VDR action, since 1,25(OH)(2)D(3) diminished PFF-induced NO production in VDR(-/-) bone cells. In conclusion, 1,25(OH)(2)D(3) and mechanical loading interact at the level of mechanotransduction, whereby 1,25(OH)(2)D(3) seems to act independently of VDR genomic mechanism.
authors.affiliation: Department of Oral Cell Biology, ACTA-University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands. h.willems@acta.nl
authors.forename: Hubertine M E
authors.initials: HM
authors.lastname: Willems
authors.forename: Ellen G H M
authors.initials: EG
authors.lastname: van den Heuvel
authors.forename: Geert
authors.initials: G
authors.lastname: Carmeliet
authors.forename: Anne
authors.initials: A
authors.lastname: Schaafsma
authors.forename: Jenneke
authors.initials: J
authors.lastname: Klein-Nulend
authors.forename: Astrid D
authors.initials: AD
authors.lastname: Bakker
mesh_terms: D000818:Animals; D001842:Bone and Bones / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D015870:Gene Expression / Q000187:drug effects; D008297:Male; D040542:Mechanotransduction, Cellular; D051379:Mice; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D061251:Primary Cell Culture; D011673:Pulsatile Flow; D012333:RNA, Messenger / Q000096:biosynthesis; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D012333:RNA, Messenger; D018167:Receptors, Calcitriol; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse