Journal of Steroid Biochemistry & Molecular Biology 121 (2010) 277–280 Contents lists available at ScienceDirect Journal of Steroid Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb The metabolism of 25-(OH)vitamin D 3 by osteoclasts and their precursors regulates the differentiation of osteoclasts  M. Kogawa a , P.H. Anderson b , D.M. Findlay a,b , H.A. Morris b , G.J. Atkins a,b,∗ a Bone Cell Biology Group, Discipline of Orthopaedics and Trauma, University of Adelaide, Adelaide, South Australia 5000, Australia b Hanson Institute, Adelaide, South Australia 5000, Australia article info Article history: Received 19 October 2009 Accepted 13 March 2010 Keywords: CYP27B1 25-Hydroxyvitamin D3 Osteoclast Metabolism Autocrine Paracrine abstract Current evidence suggests that levels of 25-(OH)vitamin D 3 (25D), rather than 1,25-(OH) 2 vitamin D 3 (1,25D), directly affect bone mineralization and that the skeleton is a site of extra-renal synthesis of 1,25D. Since cells of the monocyte lineage can also metabolise 25D, it is possible that osteoclasts participate in local production of, and the response to, 1,25D. In this study, we investigated the effects of vitamin D metabolism on osteoclastogenesis using both the murine RAW 264.7 cell line and the human peripheral blood mononuclear cell (PBMC) models. PBMC-derived osteoclasts expressed cytoplasmic cyp27b1 and nuclear vdr proteins. PBMC expressed CYP27B1 mRNA, levels of which increased during RANKL induced differentiation into osteoclasts in both cell types. While 1,25D elicited a robust CYP24 transcriptional response in PBMC, the response to 25D was approximately 100-fold less at the concentrations used. Using media devoid of pre-existing vitamin D metabolites, we found that 25D was metabolised by RAW 264.7 cells to 1,25D and resulted in significant elevation in the numbers of TRAP-positive, multinucleated osteoclasts when present in the cultures for the first 3–5 days. These results suggest that vitamin D metabolism by osteoclast lineage cells is an important regulator of osteoclast formation. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Increasing evidence suggests that vitamin D plays a direct role in regulating the activity of bone cells, as recently reviewed [1].A current view is that 1,25D maintains normocalcaemia by directly stimulating bone resorption and increasing the rate and extent of osteoblast-mediated osteoclastogenesis. However, while the pre- cise mechanism of action is not clear, evidence suggests that 1,25D also has direct effects on osteoclast precursors [2–6]. We have recently demonstrated, using a defined serum-free culture sys- tem devoid of contaminating vitamin D metabolites, a direct effect of 1,25D on RANKL-induced osteoclast formation from the mouse osteoclast precursor cell line, RAW 264.7, where 1,25D in the co-presence of RANKL resulted in increased numbers of multin- ucleated TRAP-positive osteoclasts [7]. We [8,9] and others (reviewed in [1]) have demonstrated that osteoblasts are a source of extra-renal synthesis of vitamin D metabolism and convert 25D into functional 1,25D by virtue of their  Special issue selected article from the 14th Vitamin D Workshop held at Brugge, Belgium on October 4–8, 2009. ∗ Corresponding author at: Discipline of Orthopaedics and Trauma, University of Adelaide, North Terrace, Adelaide, South Australia 5000, Australia. Tel.: +61 8 8222 3054; fax: +61 8 8232 3065. E-mail address: gerald.atkins@adelaide.edu.au (G.J. Atkins). expression of CYP27B1. The intriguing possibility exists that osteo- clasts also participate in local production of, as well as response to, 1,25D. Indeed, cells of the monocyte/macrophage lineage are known to express CYP27B1 and convert 25D into 1,25D [10,11]. The RAW 264.7 cell line has been shown to express CYP27B1 mRNA, levels of which increased during their differentiation into osteoclast-like cells [12]. In this study, we further examined the role of vitamin D metabolism during osteoclastogenesis. Our results indicate that the metabolism of 25D into 1,25D by RAW 264.7 osteoclast precur- sors, in a defined, otherwise vitamin D metabolite-free medium, results in enhanced formation of TRAP-positive, multinucleated osteoclasts. We conclude that the metabolism of vitamin D dur- ing osteoclastogenesis maybe an important intrinsic mechanism for controlling the osteoclast formation of these cells. 2. Materials and methods 2.1. Osteoclast formation RAW 264.7 cells were cultured under osteoclast forming condi- tions essentially as described previously [7], with modifications. Briefly, RAW 264.7 cells were seeded into wells of a 96-well plate (1 × 10 4 cells/well) in serum-deprived medium (SDM) [7] and cultured overnight. Media were then replaced with fresh SDM 0960-0760/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2010.03.048