VDR dependent and independent effects of 1,25-dihydroxyvitamin D 3 on nitric oxide production by osteoblasts Hubertine M.E. Willems a , Ellen G.H.M. van den Heuvel b , Geert Carmeliet c , Anne Schaafsma d , Jenneke Klein-Nulend a,⇑ , Astrid D. Bakker a a Department of Oral Cell Biology, ACTA-University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands b Royal FrieslandCampina Research, Harderwijkerstraat 6, 7418 BA Deventer, The Netherlands c Department of Experimental Medicine-Endocrinology, Katholieke Universiteit Leuven, O&N I Herestraat 49, 3000 Leuven, Belgium d Royal FrieslandCampina Research, Innovation International, P. Stuyvesantweg 1, 8937 AC Leeuwarden, The Netherlands article info Article history: Received 15 July 2011 Received in revised form 30 September 2011 Accepted 31 October 2011 Available online 7 November 2011 Keywords: Osteoblasts Mechanical loading 1,25-Dihydroxyvitamin D 3 Vitamin D receptor VDR À/À mice Nitric oxide synthase 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 deter- mine whether 1,25(OH) 2 D 3 affects NO production by osteoblasts in the presence or absence of mechan- ical 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 À/À mice were pre -incubated for 24 h with or without 1,25(OH) 2 D 3 (10 À13 –10 À9 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 À11 M 1,25(OH) 2 D 3 for 24 h, but not 30 min, stimulated NO production by MC3T3-E1 osteoblasts (eightfold). 1,25(OH) 2 D 3 for 24 h 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 À/À osteoblasts. This PFF effect was abolished after incubation with 1,25(OH) 2 D 3 for 24 h, or during PFF only. Our results suggest that 1,25(OH) 2 D 3 stimulates inducible-NOS expression and NO production by oste- oblasts 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 À/À 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. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction 1,25-Dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) is a steroid hormone, which serves as a signalling molecule in almost every human tissue several target tissues, including bone [1]. The action of 1,25(OH) 2 D 3 on bone is ambiguous. On the one hand, 1,25(OH) 2 D 3 increases bone mineralization, stiffness, and strength directly via stimulation of cal- cium and phosphate supply, mainly by absorption from the gut [2]. On the other hand, 1,25(OH) 2 D 3 has clear direct effects on osteo- blasts and osteoclasts [3]. 1,25(OH) 2 D 3 not only stimulates osteocal- cin and alkaline phosphatase production by osteoblasts [3], but also affects osteopontin expression in osteoblasts [4], and bone resorp- tion in vitro. Considering the biological significance of 1,25(OH) 2 D 3 in the regulation of bone mass, it is surprising how little is known about the mechanism by which it stimulates bone mass, and how the actions of 1,25(OH) 2 D 3 on bone interact with other bone mass- regulating factors, such as mechanical loading. The genomic responses to 1,25(OH) 2 D 3 are mediated by the for- mation of a ligand-receptor complex of 1,25(OH) 2 D 3 with the vita- min D receptor (VDR), a member of the super-family of steroid 0039-128X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2011.10.015 Abbreviations: 1,25(OH) 2 D 3 , 1,25-dihydroxyvitamin D 3 ; 1,25D 3 -MARSS, 1,25 (OH) 2 D 3 -membrane-associated rapid response to steroid; NO, nitric oxide; NOS, nitric oxide synthase; PFF, pulsating fluid flow; RANKL, receptor activator of nuclear factor kappa-B ligand; VDR, vitamin D receptor; VDR À/À , VDR knock out; VDRE, VDR response element; wt, wildtype. ⇑ Corresponding author. Tel.: +31 205980881. E-mail addresses: h.willems@acta.nl (H.M.E. Willems), ellen.vandenheuvel@ frieslandcampina.com (E.G.H.M. van den Heuvel), geert.carmeliet@med.kuleuven.be (G. Carmeliet), anne.schaafsma@frieslandcampina.com (A. Schaafsma), j.kleinnulen d@acta.nl (J. Klein-Nulend), a.bakker@acta.nl (A.D. Bakker). Steroids 77 (2012) 126–131 Contents lists available at SciVerse ScienceDirect Steroids journal homepage: www.elsevier.com/locate/steroids