Fisetin Inhibits Osteoclast Differentiation via Downregulation of p38 and c-Fos-NFATc1 Signaling Pathways

Abstract

The prevention or therapeutic treatment of loss of bone mass is an important means of improving the quality of life for patients with disorders related to osteoclast-mediated bone loss. Fisetin, a flavonoid dietary ingredient found in the smoke tree (Continus coggygria), exhibits various biological activities, but its effect on osteoclast differentiation is unknown. In this study, fisetin dose-dependently inhibited the RANKL-induced osteoclast differentiation with downregulation of the activity or expression of p38, c-Fos, and NFATc1 signaling molecules. The p38/c-Fos/NFATc1-regulated expression of genes required for cell fusion and bone resorption, such as DC-STAMP and cathepsin K, was also inhibited by fisetin. Considering the rescue of fisetin's inhibitory action by NFATc1 over-expression, the cascade of p38-c-Fos-NFATc1 could be strongly involved in the inhibitory effect of fisetin on osteoclast differentiation. Furthermore, fisetin inhibited the bone-resorbing activity of mature osteoclasts. In conclusion, fisetin may be of use in the treatment of osteoclast-related disorders, including osteoporosis.

Figure 1

Fisetin inhibits RANKL-mediated osteoclast differentiation. (a) Structure of fisetin. (b) BMM cells were cultured for 4 days in the presence of RANKL (5 ng/mL) and M-CSF (30 ng/mL) with the vehicle (DMSO) or fisetin. Multinucleated osteoclasts were visualized to red-colored giant cells by TRAP staining. (c) Total TRAP-positive multinucleated osteoclasts (TRAP + MNCs; left graph) and TRAP + MNCs with more than 3 nuclei (N > 3; right graph) were counted. **P < 0.01; ***P < 0.001 (versus “the control”). (d) TRAP activity was measured. ^### P < 0.001 (versus “the negative control”). **P < 0.01; ***P < 0.001 (versus “the positive control”). (e) After pretreatment with the vehicle (DMSO) or fisetin (5 μM) for 1 h, BMMs were treated with RANKL (5 ng/mL) for the indicated number of days, and then mRNA expression levels were analyzed by real-time PCR. *P < 0.05; **P < 0.01; ***P < 0.001 (versus “the vehicle control”). (f) Effect of fisetin on the viability of BMMs was evaluated by CCK-8 assay.

Figure 2

Fisetin inhibits RANKL-induced phosphorylation of p38 and expression of c-Fos and NFATc1. (a) BMMs were pretreated with vehicle or fisetin (5 μM) for 1 h prior to RANKL stimulation (5 ng/mL) at indicated time periods. Then, protein expression levels were evaluated by western blot analysis. Actin was used as the internal control. (b) BMMs were stimulated with RANKL (5 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of fisetin (5 μM) for the indicated times. Then, total RNA was isolated from cells using TRIzol reagent, and mRNA expression levels were evaluated by real-time PCR. *P < 0.05 (versus “the vehicle control”). (c) Effects of fisetin on protein expression levels of c-Fos and NFATc1 were evaluated by western blot analysis. Actin was used as the internal control.

Figure 3

NFATc1 over-expression restores fisetin-mediated inhibition of osteoclast differentiation. (a) BMMs were infected with pMX-IRES-GFP (GFP) or pMX-IRES-CA-NFATc1-GFP (CA-NFATc1-GFP) for 8 h with polybrene (10 μg/mL). Infected BMMs were cultured with M-CSF (30 ng/mL) and RANKL (5 ng) for 4 days in the presence or absence of fisetin (5 μM). After 4 days, cells were fixed and GFP expression was visualized under a fluorescence microscope. (b) BMMs were infected with GFP or CA-NFATc1-GFP and then cells were cultured as described in (a). After 4 days, mature TRAP-positive multinucleated osteoclasts were visualized by TRAP staining. (c) TRAP-positive cells were counted as osteoclasts. (d) TRAP activity was measured at 405 nm.

Figure 4

Fisetin inhibits osteoclastic bone resorption. (a) Effect of fisetin on mRNA expression of cathepsin K was analyzed by real-time PCR as described in Figure 1(e). **P < 0.01; ***P < 0.001 (versus “the vehicle control”). (b) Attached cells on BioCoat Osteologic MultiTest slides were removed and photographed under a light microscope (bottom images). Pit areas were quantified using ImageJ program (upper graph). **P < 0.01; ***P < 0.001 (versus “the control”). (c) Mature osteoclasts were plated on BioCoat slides and treated for 24 h with the indicated concentrations of fisetin. Cells were fixed, permeabilized, and stained with TRAP. TRAP-positive multinucleated cells were counted (upper graph) and photographed under a light microscope (bottom images).