Sophorae Flos extract inhibits RANKL-induced osteoclast differentiation by suppressing the NF-κB/NFATc1 pathway in mouse bone marrow cells

Background Sophorae Flos (SF) is a composite of flowers and buds of Styphnolobium japonicum (L.) Schott and has been used in traditional Korean and Chinese medicine for the treatment of hemostasis and inflammation. Previous studies reported that SF possesses anti-obesity properties, as well as anti-allergic, anti-proliferative, and anti-inflammatory activities. However, the effect of SF in bone resorption has not been studies. In this study, we examined the potential of SF extract (SFE) to inhibit receptor activator of NF-κB ligand (RANKL) -induced osteoclast differentiation in cultured mouse-derived bone marrow macrophages (BMMs). Methods BMMs, that act as osteoclast precursors, were cultured with M-CSF (50 ng/ml) and RANKL (100 ng/ml) for 4 days to generate osteoclasts. Osteoclast differentiation was measured by tartrate-resistant acidic phosphatase (TRAP) staining and the TRAP solution assay. Osteoclast differentiation marker genes were analyzed by the quantitative real-time polymerase chain reaction analysis. RANKLs signaling pathways were confirmed through western blotting. Results SFE significantly decreased osteoclast differentiation in a dose-dependent manner. SFE inhibited RANKL-induced osteoclastogenesis by suppressing NF-κB activation. By contrast, SFE did not affect phospholipase C gamma 2 or subsequent cAMP response element binding activation. SFE inhibited the RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1). Conclusions SFE attenuated the RANKL-mediated induction of NF-κB through inhibition of IκBα phosphorylation, which contributed to inhibiting of RANKL-induced osteoclast differentiation through downregulation of NFATc1.


Background
Osteoclasts differentiate from monocyte/macrophage lineage hematopoietic precursor cells at various stages including proliferation, migration, fusion, and activation [1]. Osteoclasts are specialized as the only bone-resorbing cell type and increased numbers are implicated in the development of bone loss-accompanied diseases such as osteoporosis, periodontitis, rheumatoid arthritis, osteosarcoma, and bone cancer metastases [2][3][4]. Pharmaceutical inhibition of osteoclast differentiation is a current therapeutic approach for the treatment of bone loss-related diseases.
Previous studies indicate that pro-inflammatory cytokines, including IL-17, TNF-α, IL-1, IL-4, and IFN-γ, that are induced during T-cell-mediated immune responses, directly control the expression of RANKL on osteoblasts and that inflammation affects bone metabolism [1,19,20]. Although S. japonicum (L.) Schott has anti-inflammatory activity, effect on bone metabolism has been studie infrequently. The exceptions are studies showing that dichloromethane extracts of Sophora japonica L. stimulate osteoblast differentiation in mesenchymal stem cells [21]. In addition, recent studies show that such extracts prevent bone loss, partly by inhibiting osteoclastic activity [21,22]. However, the potential anti-osteoclast differentiation mechanisms of SF have not been defined clearly.
In our study, we confirmed the inhibitory effects of SF extract (SFE) on RANKL-mediated osteoclast differentiation, provided molecular mechanisms for its activity, and suggested possibilities for the use of SF as a traditional medicine against bone disorders, such as osteoporosis, RA, and periodontitis.

Experimental animals
BALB/c mice (Orient Bio, SeungNam, Korea) were used for all experiments, including osteoclast generation. All mouse studies were performed using protocols approved by the Animal Care and Use Committee of Wonkwang University.

Reagents
Recombinant murine sRANK Ligand and M-CSF were purchased from PeproTech (Rocky Hill, NJ, USA). Fetal bovine serum, α-minimal essential medium, and supplements were obtained from Gibco (Rockford, IL, USA).

Preparation of SFEs
Flower buds of S. japonicum (L.) Schott were purchased from Kwangmyungdang Medicinal Herbs (Ulsan, Korea) and authenticated by Prof. G.S. Lee. SF was extracted from 50 g of S. japonicum flower buds using the reflux method with ethanol. The extract was evaporated and then freezedried. The yield of the final extract was 2.56% (w/w).

In vitro osteoclast differentiation
Collected from mice tibiae and femur, BMMs were cultured with M-CSF (30 ng/ml). After 3 days, attached BMMs were used as osteoclast precursor. To form osteoclasts, BMMs were treated with M-CSF (50 ng/ml) and RANKL (100 ng/ml) and cultured for 4 days [23]. For TRAP staining, cells were fixed with 10% formalin and stained. Total TRAP activity was measured at an absorbance of 405 nm using p-nitrophenyl phosphate (Sigma Aldrich, St. Louis, MO, USA) as a substrate.

Real-time quantitative polymerase chain reaction (qRT-PCR)
Total RNA was isolated from cells using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). One microgram of total RNA was synthesized to first strand cDNA using a PrimeScript™ RT reagent kit (TaKaRa Bio, Shiga, Japan). qRT-PCR was performed using the SYBR Green and the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). All results were normalized to the housekeeping gene glyceraldehyde 3phosphate dehydrogenase (Gapdh). PCR primers used were: mouse Acp5

Statistical analysis
Results were analyzed using Student's two-tailed t-test. Data are presented as means ± standard deviation (SD). P-values less than 0.05 were considered significant.

Cytotoxic effects of SFE
To investigate the cytotoxicity of SFE on BMMs (osteoclast precursor), it treated with several concentrations of SFE (0, 25, 50, 100, and 200 μg/ml) for 1 day. Concentrations of SFE, up to 100 μg/ml, did not cause any significant change in cell viability (Fig. 1a). BMMs were also treated with 100 μg/ml SFE and cell viability was measured daily for 4 days. There were no significant differences in viability between control and SFE at any day (Fig. 1b).

SFE treatment suppressed RANKL-mediated osteoclastogenesis in a dose-dependent manner
To investigate the effects of SFE on osteoclast differentiation, RANKL-stimulated BMMs were treated with SFE at the indicated concentrations for 4 days (Fig. 2a). After incubation, osteoclast differentiation and formation were measured by TRAP staining and the total TRAP activity assay. TRAP + multimuclear cells present in each well were identified by the presence of more than three nuclei and a cell size larger than 100 μm in diameter, and were counted. Total TRAP activity was measured in fused and non-fused cells. SFE treatment markedly inhibited RANKL-induced osteoclast formation from BMMs in a dose-dependent manner (Fig. 2a, b). Total TRAP activity of osteoclasts was reduced significantly by SFE in a concentration dependent manner (Fig. 2c). These results suggest that SFE is able to repress osteoclast differentiation.

Abrogation of differentiation-related gene expression by SFE
Acp5 (TRAP), Oscar, Ctsk, Tm7sf4 (dendritic cell-specific transmembrane protein, DC-STAMP), and Atp6v0d2 are well-known as osteoclast differentiation-related marker genes crucial for cell motility, fusion, and bone resorptive activities. To evaluate the inhibitory effect of SFE on RANKL-induced osteoclast differentiation, the expression of these marker genes and a major transcription factor Nfatc1, was measured during RANKL-induced osteoclast differentiation. SFE dramatically inhibited the expression of all tested osteoclast differentiationa b Fig. 1 Effects of Sophorae Flos extract (SFE) on bone marrow marcrophages. a BMMs were cultured with various concentrations of SFE for 1 day. b BMMs were cultured with or without (control) 100 μg/ml SFE for 4 days. Cell viability was measured as described in methods. Data from three independent experiments are expressed as relative proliferation (% of control) ± SD related marker genes, as well as Nfatc1 (Fig. 3). These results suggest that SFE affects the differentiationmediating signal pathway from an early-stage of osteoclastogenesis.

Suppression of NFATc1 expression by SFE-mediated NF-κB inactivation
Osteoclast differentiation is regulated by the recruitment of multiple downstream signaling molecules, including PLCγ2, p38, JNK, and ERK, as well as the transcription factors NF-κB, NFATc1, and c-fos, in response to RANKL and its binding to RANK [25]. We confirmed the effects of SFE on RANKL-induced early signaling pathways. First, we confirmed the activation of mitogen-activated protein kinases (MAPKs) and NF-κB by western blotting (Fig. 4). SFE showed had no effect on MAPK activation. By contrast, IκBα phosphorylation, proportional to the NF-κB pathway activity, was diminished in SFE-treated compared with control cells (Fig. 4a). Next, we investigated the expression levels of NFATc1 and c-fos proteins. In SFE-treated cells, the expression of c-fos protein was unchanged during RANKLinduced osteoclast differentiation (Fig. 4b). However, the expression of NFATc1 protein was inhibited dramatically by SFE (Fig. 4c). Concomitantly, we found that SFE failed to inhibit the activation of PLCγ2 and CREB during RANKL-induced osteoclast differentiation (Fig. 5a, b). These results suggest that SFE affects the regulation of RANKL-induced osteoclast differentiation through the NF-κB pathway.

Discussion
SF has been used commonly in traditional medicine because of its various hemostatic, anti-inflammatory, and anti-oxidative effects [26]. Additionally, recent studies indicated that S. japonicum (L.) Schott extracts showed preventive effects against bone loss, partly by inhibiting osteoclastic activity [19,20]. However, the inhibitory potential and molecular mechanisms of SF on RANKLinduced osteoclast differentiation have not been elucidated. Here, we demonstrated that the inhibitory effect of SFE was elicited through a reduction of NFATc1 expression during the differentiation of osteoclasts, cells responsible for bone destruction and associated with inflammation-related bone diseases. BMMs are precursor cells that differentiate into osteoclasts in response to RANKL, which is expressed in osteoblasts, osteocytes, and T cells, and a critical factor in osteoclastogenesis [27].
NF-κB is transcription factor and an inducible dimeric protein consisting of two subunits, p65 and a b c Fig. 2 Effects of Sophorae Flos extract (SFE) on osteoclast differentiation. Bone marrow macrophages were cultured with various concentrations of SFE and treated with RANKL (100 μg/ml) and M-CSF (50 μg/ml) for 4 days. a Osteoclasts stained for tartrate-resistant acidic phosphatase (TRAP). b TRAP + multinuclear cells (MNCs) with more than three nuclei were considered to be mature osteoclasts. c Total TRAP activity from TRAP + mono-, di-, and multinuclear cells was quantified as described in methods. Data from three independent experiments are expressed as mean ± SD. *P < 0.05, # P < 0.01 versus control (0 μg/ml SFE) p50 [29]. In unstimulated cells, NF-κB is located in the cytoplasm in a dormant form complexed with its inhibitory factor, IκB. Various inducers can dissociate this complex, presumably by phosphorylating IκB, resulting in the release of NF-κB. NF-κB then translocates into the nucleus, where it binds to specific DNA sites to regulate gene transcription. In several studies, NF-κB was shown to play a key role in osteoclastogenesis [30,31], and its suppression affected NFATc1 expression. Takatsuna et al. have shown that the NF-κB inhibitor, (-)-dehydroxymethylepoxyquinomicin regulates RANKL-induced osteoclastogenesis through downregulation of NFATc1 [32]. In the current study, we found that IκBα phosphorylation was inhibited by SFE, whereas the induction of MAPK was unaffected (Fig. 4).
RANKL/RANK binding activates PLCγ2 and induces calcium ion signaling, followed by CREB phosphorylation [23,25,37]. CREB is crucial factor for the RANKLstimulated induction of NFATc1 and c-Fos in osteoclast precursors [37]. Nevertheless, in our study, SFE failed to inhibit PLCγ2 and CREB activation, which are essential signaling molecules for osteoclast differentiation, through the repression of c-Fos and NFATc1 expression. These results indicate that SFE is an effective inhibitory agent of osteoclast differentiation through its control of NF-κB induction following RANKL/RANK binding.

Conclusions
Our results demonstrate that SFE reduces the RANKLmediated induction of the NF-κB pathway by inhibiting of IκBα phosphorylation. This effect, in turn, contributes to the downregulation of NFATc1 and inhibition of RANKL-induced osteoclast differentiation. These findings reveal SFE as an effective traditional therapeutic medicine for the treatment of inflammatory bone diseases, such as osteoporosis, rheumatoid arthritis, and periodontitis.