Sulforaphene attenuates multinucleation of pre-osteoclasts by suppressing expression of cell–cell fusion-associated genes DC-STAMP, OC-STAMP, and Atp6v0d2

Abstract

We assessed the effect of sulforaphene (SFE) on osteoclast differentiation. SFE significantly decreased the number of RANKL-induced tartrate-resistant acid phosphatase-positive cells and suppressed pre-osteoclast multinucleation. Furthermore, SFE downregulated mRNA expression of DC-STAMP, OC-STAMP, and Atp6v0d2, which encode cell–cell fusion molecules. Our data suggest that SFE attenuates pre-osteoclast multinucleation via suppression of cell–cell fusion.

Key words:

• sulforaphene
• isothiocyanate
• osteoclast cell fusion
• dendritic cell-specific transmembrane protein (DC-STAMP)
• v-ATPase V0 subunit d2(Atp6v0d2)

Bone quantity is controlled by the balance between bone resorption by osteoclasts and bone formation by osteoblasts.¹⁾ Inhibition of osteoclast differentiation is effective in suppressing osteoporosis. Receptor activator of nuclear factor kappa-B ligand (RANKL) is an essential factor for osteoclast differentiation and is expressed in osteoblasts. RANKL specifically binds to its receptor, RANK, and activates the c-Fos pathway.²⁾ Additionally, this signal results in the activation of nuclear factor of activated T cells c1 (NFATc1), a master regulator of osteoclast differentiation, which controls the expression of the genes encoding tartrate-resistant acid phosphatase (TRAP) and cathepsin K (Ctsk).³⁾ Importantly, osteoclast cell fusion is essential for pre-osteoclast multinucleation. Osteoclast cell fusion through cell–cell fusion molecules, such as dendritic cell-specific transmembrane protein (DC-STAMP), osteoclast stimulatory transmembrane protein (OC-STAMP), and v-ATPase V0 subunit d2 (Atp6v0d2), is one of the main factors involved in the regulation of bone resorption.^4–6) Sulforaphene (SFE) (Fig. 1(A)), a sulfur-containing compound, is derived from Japanese radish, a kind of cruciferous vegetable, and induces apoptosis in several cancer cell lines.^7,8) Recently, we demonstrated that sulforaphane inhibits osteoclast differentiation by suppressing the cell–cell fusion molecules, DC-STAMP and OC-STAMP. ⁹⁾ However, the molecular function of SFE, an isothiocyanate (ITC) with a double bond, in inhibiting osteoclast differentiation has not been documented. In the present study, we investigated the effects of SFE on pre-osteoclast multinucleation and cell fusion in vitro.

Fig. 1. Effects of sulforaphene (SFE) on osteoclast formation in RAW 264.7 cells.

Notes: (A) Chemical structure of SFE. (B) Cell viability of SFE treated RAW 264.7 cells. RAW 264.7 cells (2 × 10³ cells/well) were cultured in a 96-well plate and then treated with various concentrations of SFE for four days in α-MEM containing 10% FBS. Cell viability was assessed using the CCK-8 assay. Cell viability is expressed as a percentage of the values obtained for SFE-untreated cells. (C) RAW 264.7 cells (1 × 10⁴ cells/well) were seeded in a 24-well plate for 24 h. Then, the cells were cultured with various concentrations of SFE (0–1 μM) in the presence of sRANKL (50 ng/mL) for four days. After incubation, the cells were fixed and stained for TRAP, a marker enzyme for osteoclast differentiation. TRAP-positive multinuclear cells (≥3 nuclei) were counted. The effect of SFE on osteoclast differentiation was expressed as rate of multinucleated osteoclast formation, with SFE-untreated cells set at 100%. (D) The number of TRAP-positive multinucleated cells containing fewer than three nuclei, (E) 3–9 nuclei, and (F) 10 or more nuclei. The data are representative of three independent experiments and are expressed as means ± SE. Means with different letters are significantly different (p < 0.05).

SFE was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). RAW 264.7 murine macrophages/monocytes were purchased from American Type Culture Collection (Manassas, VA, USA). In the culture system, RAW 264.7 cells were cultured in α-minimal essential medium (α-MEM) (phenol red-free) (Gibco BRL/Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco BRL/Invitrogen) at 37 °C under 5% CO₂. Total RNA was isolated from RAW 264.7 cells by using the Sepasol-RNA I Super G (Nacalai Tesque, Tokyo, Japan). cDNA was synthesized from total RNA using the PrimeScript RT Reagent Kit (Takara Biotechnology, Otsu, Japan). Real-time PCR was performed using the ABI StepOnePlus System (Applied Biosystems, Foster City, CA, USA) with THUNDERBIRD qPCR Mix (Toyobo, Osaka, Japan). Sequences of primers used have been reported previously.⁹⁾ The following primer sets were used: Atp6v0d2, 5′-GACCCTGTGGCACTTTTTGTATTC-3′ (forward), and 5′-GCTTGCATTTGGGGAATCTATC-3′ (reverse). All reactions were normalized to the housekeeping gene β-actin. Results are presented as means ± S.E. Multiple comparisons were performed using Tukey’s test, after one-way analysis of variance (ANOVA). p < 0.05 was considered statistically significant.

We examined the cytotoxic effect of SFE on RAW 264.7 cells, osteoclast precursor cells, using the Cell Counting Kit-8 (CCK-8; DOJINDO, Kumamoto, Japan) assay. Low-dose SFE had no cytotoxicity, but decreased cell viability at a concentration of 5 μM (Fig. 1(B)). These results indicate that the maximum concentration used in our subsequent experiments (1 μM) had no cytotoxic effects against RAW 264.7 cells. Next, to examine the effects of SFE on pre-osteoclast multinucleation, the cells were incubated with SFE in the presence of soluble RANKL (sRANKL; R&D Systems, Minneapolis, MN, USA). Compared to that reported for sRANKL-induced cells, SFE decreased the rate of multinucleated osteoclast formation (Fig. 1(C)). Interestingly, the osteoclasts with 10 nuclei or more decreased in number, whereas the cells with fewer than 3 nuclei increased drastically (Fig. 1(D–F)). Furthermore, we analyzed the effect of SFE on the mRNA expression levels of osteoclast-associated genes, such as c-Fos, NFATc1, TRAP, and Ctsk using real-time PCR. Compared to that in sRANKL-induced cells, SFE dose dependently decreased mRNA expression levels of TRAP and Ctsk, whereas the expression of c-Fos did not change (Fig. 2(A–D)). NFATc1 mRNA expression was downregulated by SFE at 0.l μM, but there was no dose dependency. Additionally, the effects of SFE on cell viability and pre-osteoclast multinucleation of bone marrow cells, which are closer to in vivo conditions, were similar to those observed with RAW 264.7 cells (Supplemental Fig. 1; see Biosci. Biotechnol. Biochem Website). This result indicates that SFE inhibits osteoclast differentiation by suppressing RANKL/NFATc1 signal, but there might be other reasons.

Fig. 2. Effects of SFE on expression of osteoclast-differentiation associated genes.

Notes: RAW 264.7 cells were treated with indicated doses of SFE for four days in the presence of sRANKL (50 ng/mL). mRNA expression levels of (A) c-Fos, (B) NFATc1, (C) TRAP, and (D) Ctsk were analyzed by real-time PCR and the results were normalized to the expression of β-actin. The data are representative of three independent experiments and are expressed as means ± SE. Means with different letters are significantly different (p < 0.05).

Monomer osteoclasts promote cell–cell fusion by increasing the expression levels of DC-STAMP or OC-STAMP, which are major factors for activating osteoclasts and inducing bone resorption.^10,11) Additionally, according to a previous study, Atp6v0d2-deficient mice exhibit impaired osteoclast cell fusion.⁶⁾ Thus, these reports indicate that DC-STAMP, OC-STAMP, and Atp6v0d2 play an important role in osteoclast cell fusion. We also found that SFE inhibited pre-osteoclast multinucleation and decreased the number of multinuclear cells (10 or more nuclei) as shown in Fig. 1(D–F). Therefore, we revealed that SFE affects osteoclast cell fusion. As shown in Fig. 3(A–C), SFE dose dependently decreased the mRNA expression levels of DC-STAMP, OC-STAMP, and Atp6v0d2 in sRANKL-induced multinucleated osteoclasts. Importantly, Kim et al. reported that NFATc1 induces the expression of DC-STAMP and Atp6v0d2 by binding to their promoter regions.¹²⁾ These data indicate that NFATc1/DC-STAMP and Atp6v0d2 signals play a key role in the pre-osteoclast multinucleation process. In addition, OC-STAMP is induced by sRANKL treatment, and it promotes the formation of multinucleated osteoclasts.¹³⁾

Fig. 3. Effects of SFE on expression of osteoclast cell fusion molecules.

Notes: RAW 264.7 cells were treated with sRANKL (50 ng/mL) for four days in the absence or presence of SFE at various concentrations. mRNA expression levels of (A) DC-STAMP, (B) OC-STAMP, and (C) Atp6v0d2 were analyzed by real-time PCR and the results were normalized to the expression of β-actin. The data are representative of three independent experiments and are expressed as means ± SE. Means with different letters are significantly different (p < 0.05).

In conclusion, we demonstrated that SFE plays a novel role in inhibiting osteoclast cell fusion. In addition, SFE decreased the number of multinucleated osteoclasts by suppressing RANKL/NFATc1 signal. Particularly, this study was the first to demonstrate that SFE strongly inhibited multinucleation of mononuclear pre-osteoclasts by downregulating not only DC-STAMP and OC-STAMP, but also Atp6v0d2. Further studies of the mechanisms by which SFE improves bone resorption and osteoporosis in vivo are still required.

Author contributions

T. T., H. I, N. T., and M. U. designed the experiments. T. T. and H. I. performed the experiments. R. K-T. participated in cell culture. T. T., H. I., N. T., R. K-T., and M. U. analyzed the data. T. T., H. I., and M. U. wrote the manuscript. N. T. contributed to the development of the manuscript. All authors read and approved the final manuscript.

Supplemental materials

The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2017.1281729.

Funding

This work was supported by Grant-in-Aid for Scientific Research (A) [No. 15H01767] from the Japan Society for the Promotion of Science (JSPS).

Disclosure statement

No potential conflict of interest was reported by the authors.

Notes

Abbreviations: SFE,sulforaphene; ITC, isothiocyanate; RANKL, Receptor activator of nuclear factor kappaB ligand; NFATc1, nuclear factor of activated T cells c1; TRAP, tartrate-resistant acid phosphatase; Ctsk, cathepsin K; DC-STAMP, dendritic-cell specific transmembrane protein; OC-STAMP, osteoclast stimulatory transmembrane protein; Atp6v0d2, v-ATPase V0 subunit d2; α-MEM, α-minimal essential medium; CCK-8,cell counting kit-8; BMCs, bone marrow cells.