pone-0013305-g001: RA inhibits differentiation of osteoclast progenitors.TRAP activity in medium in RAW264.7 cells incubated with RANKL (RL, 100 ng/ml) with various concentrations of RA (A), or in the presence or absence of 4 nM of RA or 8 nM of RAR pan-antagonist (AGN) (B, C) on bone slices or plastic for 7 days was measured as described in materials and methods. Human CD14+ blood monocytes were incubated with M-CSF (M, 25 ng/ml), RANKL (RL, 25 ng/ml) and various concentrations of RA (D), or in the presence or absence of 4 nM RA or 8 nM AGN on bone slices for 14 days or on plastic for 10 days (E–G). Release of TRAP activity in the culture medium was determined using an adapted Sigma protocol (A, B, D, E). The TRAP staining was carried out using the Acid Phosphatase Leukocyte (TRAP) kit (C, F). CTX was determined by CrossLaps ELISA kit (G). Each data point represents the average ± SD of triplicate wells. Similar results were obtained in more than three independent experiments. NS means non-significant difference. A, D, compared with RL group. * P<0.05, *** P<0.001.

Mentions: Secreted TRAP activity, a marker of the osteoclast resorption capacity [37], in the medium of murine RAW264.7 cells was significantly increased 3 to 5 times compared to cells not receiving RANKL on plastic (Figure 1A, B). TRAP staining of these cells revealed an abundance of osteoclasts with more than 3 nuclei both on plastic and bone slices (Figure 1C, expanded figure in Figure S2). However, no functional osteoclasts were formed, measured as CTX fragment generation, when the RAW264.7 cells were cultured with RANKL on bone slices (data not shown). RA inhibited the RANKL-induced osteoclast differentiation when it was added to the RAW 264.7 cell cultures on plastic (Figure 1A-C). RA at a concentration as low as 0.4 nM was sufficient to inhibit TRAP release in RAW 264.7 cells (Figure 1A). No multiple-nuclear TRAP positive cells were found in the presence of 4 nM RA (Figure 1C). The RAR pan-antagonist (AGN) partially reversed the inhibition of osteoclastic TRAP release and multi-nuclear TRAP-positive osteoclast formation by RA (Figure 1B, C). To test the effect of RA on human cells we used a standard, validated culture system for human osteoclastogenesis involving culturing peripheral blood purified CD14+ cells with M-CSF and RANKL for 10–14 days [35]. In these cultures TRAP-release was increased 42- and 13-fold compared with the cells not receiving RANKL seeded on plastic or bone slices, respectively (Figure 1E). Importantly, these cells were functional osteoclasts as the bone resorption activity was highly induced by RANKL, measured as CTX fragment generation, when cells were cultured on bone slices (Figure 1G). RA inhibited the RANKL-induced osteoclast differentiation when it was added to human CD14+ cells both on plastic and bone slices (Figure 1D–F). The inhibition can even be observed at a concentration as low as 0.04 nM of RA on human CD14+ cells (Figure 1D and Figure S1). RA at 4 nM in the human CD14+ cell culture in the presence of M-CSF and RANKL almost completely abolished the formation of multi-nuclear TRAP-positive cells together with a near total elimination of TRAP- and CTX-release (Figure 1D–G, Figure 1F was expanded in Figure S2). Osteoclast formation, TRAP- and CTX-release were largely restored in the presence of the RAR-pan-antagonist (Figure 1E–G). These results were consistent irrespective of the carrier for the cell culture, i.e. plastic or bone.

Hu L, Lind T, Sundqvist A, Jacobson A, Melhus H - PLoS ONE (2010)

Bottom Line: Also, RA demonstrated differential effects depending on the material supporting the cell culture.RA did not affect TRAP activity in the culture supernatant in the bone slice culture system, but inhibited the release of TRAP activity if cells were cultured on plastic.In conclusion, our results suggest that retinoic acid increases proliferation of human osteoclast progenitors and that it inhibits RANK-stimulated osteoclast differentiation by suppressing RANK.

Affiliation: Department of Medical Sciences, Uppsala University, Uppsala, Sweden. Lijuan.Hu@medsci.uu.se

Abstract: It has been shown that high vitamin A intake is associated with bone fragility and fractures in both animals and humans. However, the mechanism by which vitamin A affects bones is unclear. In the present study, the direct effects of retinoic acid (RA) on human and murine osteoclastogenesis were evaluated using cultured peripheral blood CD14(+) monocytes and RAW264.7 cells. Both the activity of the osteoclast marker tartrate resistant acid phosphatase (TRAP) in culture supernatant and the expression of the genes involved in osteoclast differentiation together with bone resorption were measured. To our knowledge, this is the first time that the effects of RA on human osteoclast progenitors and mature osteoclasts have been studied in vitro. RA stimulated proliferation of osteoclast progenitors both from humans and mice. In contrast, RA inhibited differentiation of the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis of human and murine osteoclast progenitors via retinoic acid receptors (RARs). We also show that the mRNA levels of receptor activator of nuclear factor κB (RANK), the key initiating factor and osteoclast associated receptor for RANKL, were potently suppressed by RA in osteoclast progenitors. More importantly, RA abolished the RANK protein in osteoclast progenitors. This inhibition could be partially reversed by a RAR pan-antagonist. Furthermore, RA treatment suppressed the expression of the transcription factor nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and increased the expression of interferon regulatory factor-8 (IRF-8) in osteoclast progenitors via RARs. Also, RA demonstrated differential effects depending on the material supporting the cell culture. RA did not affect TRAP activity in the culture supernatant in the bone slice culture system, but inhibited the release of TRAP activity if cells were cultured on plastic. In conclusion, our results suggest that retinoic acid increases proliferation of human osteoclast progenitors and that it inhibits RANK-stimulated osteoclast differentiation by suppressing RANK.