BMSC were isolated according to a previously published protocol with some modifications (Krebsbach et al., 1999; Zhang et al., 2009). Briefly, BMSC were isolated from bone marrow, which was aspirated from 8-week old BALB/c mice. All procedures involving mice were carried out in compliance with the relevant laws and institutional guidelines, and approved by the Ethics Committee at Dalian Medical University. BMSC were collected using gradient centrifugation of mesenchymal stem cell-specific gradient solutions (Tianjin Haoyang Biological Manufacture, China). A layer of the bone marrow cell fraction in phosphate-buffered saline (PBS) was placed on top of the gradient solution and centrifuged at 340 g for 20 min. The cell fraction was collected and washed with PBS after centrifugation. The cell samples were resuspended in Minimum Essential Medium Alpha Medium (α-MEM, Gibco, Paisley, UK), supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 μg/ml streptomycin, and maintained at 37°C with 5% CO₂ in a humidified atmosphere. On day 3, the cell suspension was replaced with fresh complete medium. BMSC were further separated from hematopoietic cells by their differential adhesion to tissue culture plastic and prolonged proliferation potential. After 6–7 days in culture, 90% confluence was reached. These cell samples were used in the experiment.

BMSC were cultured with osteogenic medium (OS) (100 nM dexamethasone, 1 mM β-glycerophosphate, and 5 μM L-ascorbic acid 2-phosphate) + 2 μg/ml wedelolactone. Wedelolactone was provided by Key Laboratory of Separation Science for Analytical Chemistry at Dalian Institute of Chemical Physics, Chinese Academy of Sciences (Dalian, China). The purity was >98%. These cells were incubated for 12 days at 37°C with 5% CO₂ in a humidified incubator. Every 3 days the medium was changed and collected. The 3-, 6-, 9-, and 12-day incubation fractions containing wedelolactone-treated or -untreated conditioned media were harvested for use.

Mouse pre-osteoclastic RAW264.7 cells were purchased from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% FCS, 0.03% L-glutamine (Gibco), penicillin (100 U/ml), and streptomycin (100 μg/ml) and maintained at 37°C with 5% CO₂ in a humidified atmosphere.

For differentiation, cells were plated in DMEM or conditioned medium supplemented with 30 ng/ml recombinant RANKL. For drug assays, wedelolactone was added to the culture medium at different concentrations. Cells were incubated for 4 days at 37°C with 5% CO₂ in a humidified incubator and fed daily with RANKL-supplemented medium.

RAW264.7 cells were plated at a density of 1 × 10⁴ cells per well of 96-well plates. After overnight incubation, RANKL, wedelolactone, or conditioned media was added to the plates. Following incubation for 6 days, cell growth was measured by 3-(4, 5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) with a plate reader (Tecan, Switzerland) as previously described (Liu et al., 2014). The percentage of proliferation was calculated with the formula:

RAW264.7 cells were fixed with 60% citrate buffered acetone for 30 s. The fixed cells were then washed with water 3× and were further incubated with 100 μl phosphatase substrate solution containing 10 mM pNPP and 10 mM sodium tartrate in 50 mM citrate buffer (PH 4.6) at 37°C for 1 h. After incubation, the enzyme reaction mixture was transferred to another plate, and the reaction was stopped with 100 μl of 0.1 N NaOH. Absorbance at 405 nm was measured using an ELISA reader (Tecan, Switzerland).

For staining of tartrate-resistant acid phosphatase (TRAP), cells were fixed with 60% citrate buffered acetone for 30 s. The cells were then stained for TRAP with 0.1 M acetate solution (PH 5.0) containing 6.76 mM sodium tartrate, 0.12 mg/ml naphthol AS-MX phosphate, and 0.07 mg/ml of fast Garnet GBC solution as described in the manufacturer’s instruction (Sigma, St. Louis, MO, USA). Photomicrographs were obtained using an Olympus microscope at 200× magnification.

For in vitro osteoblast differentiation, mouse BMSC were isolated from 8-week old BALB/c mice according to a previously published protocol, and were cultured with α-MEM with 10% FBS. After 5 days, cells were reseeded (1 × 10⁴ per cm²) and cultured with OS. Culture medium was changed every third day. After 9 days, alkaline phosphatase (ALP) staining (Sigma) and activity measurements were performed by using an ALP staining kit according to manufactory’s instruction.

For Western blot analysis, cells were lysed using lysis buffer containing 10 mM Tris/HCl (PH 7.5), 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, 1 mM Na₃CO₄, 1 mM PMSF, and 0.1 mM aprotinin. The cells were scraped from the plates and centrifuged at 16,060 g for 30 min at 4°C. For nuclear protein extraction, the pellet from the 700 g centrifugation was washed by buffer A, resuspended in buffer B containing 20 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 420 mM NaCl, 0.2 mM EDTA, 10 mM NaF, 2 mM Na₃VO₄, 1 mM pyrophosphoric acid, and Complete TM protease inhibitors (Cell Signaling, USA) and incubated on ice for 5 min. The protein concentration in the cell lysates was determined using the Bradford protein assay. Western blot analysis was performed as previously described using the several antibodies (Bennett et al., 2005). Sema3A, plexin A1, and NRP1 antibodies were obtained from ABcam, Cambridge, UK. PLCγ2, phospho-PLCγ2, β-cateinin, and DAP12 antibodies were from Cell Signaling, USA.

For immunoprecipitation assay, cells were harvested and lysed in radioimmuno- precipitation assay (RIPA) buffer. Cell lysates were then centrifuged at 12,000 rpm for 10 min at 4°C (Hayashi et al., 2012). PlexinA1 or IgG antibodies were added into the supernatant and incubated at 4°C overnight with rotation. Protein A or G agarose beads (Thermo, Rockford, IL, USA) were then added and incubated with rotation for 3 h at 4°C. After centrifugation, proteins were subjected to Western blotting.

All work was carried out in a designated PCR-clean area. RNA was extracted from cells using Trizol reagent (Gibco-BRL, Rockville, MD, USA) and isolated as specified by the manufacturer. The RNA was DNAse-treated (DNase I-RNase-Free, Ambion) to remove any contaminating DNA; 200 ng of total RNA was reverse-transcribed with oligodT primers using the High Capacity cDNA RT Kit (Takara, Japan) in a 20-μl cDNA reaction, as specified by the manufacturer. For quantitative PCR, the template cDNA was added to a 20 μl reaction with SYBR GREEN PCR Master Mix (Applied Biosystems) and 0.2 μM of primer. The amplification was carried out using an ABI Prism 7000 for 40 cycles under the following conditions: (1) an initial denaturation of 95°C for 10 min plus 40 cycles of 95°C for 15 s and (2) 60°C for 1 min. The -fold changes were calculated relative to β-actin (NM_007393.5) using the ΔΔCt method for Sema3A (NM_001243073.1) mRNA analysis. The following primer sets consisted of mouse β-actin: forward, 5′-GTACGCCAACACAGTGCTG-3′; reverse, 5′-CGTCATACTCCTGCTTGCTG-3′ and mouse Sema3A: forward, 5′-AGTGTCCGTACGATCCCAAG-3′; reverse, 5′-GAACAGTGACCACCGTCATC-3′.

Differences between experimental groups were evaluated by one-way analysis of variance (ANOVA) using SPSS 17.0 software. Differences with a P-value < 0.05 were considered statistically significant. All experimental data are presented as the mean ± SEM with values from more than three experiments.

To determine the influence of wedelolactone-treated conditioned media from BMSC on osteoclastic differentiation, TRAP activity of osteoclastic RAW264.7 cells was examined after treatment with wedelolactone or conditioned media. As shown in Figure 1, when RAW264.7 cells were exposed to RANKL for 4 days, TRAP activity was significantly increased. After addition of different conditioned media fractions, which were treated with 2 μg/ml wedelolactone for 3, 6, 9, and 12 days, TRAP activity decreased but to different levels. The decreased amount of TRAP activity was most potent with the addition of the 9-day conditioned medium fraction (Figure 1). The effects of wedelolactone and 9-day conditioned medium on RAW264.7 cell viability was evaluated.

The 9-day conditioned medium fraction or wedelolactone at 2 μg/ml did not influence cell viability in the MTT assay (Figure 2A). However, TRAP staining confirmed that the RANKL-induced increased number of TRAP-stained positive RAW264.7 cells was significantly reduced by incubation with 2 μg/ml wedelolactone. In contrast, treatment with the 9-day conditioned medium fraction led to a more significant decrease in the number of TRAP-stained positive RAW264.7 cells (Figure 2B).

Sema3A is reported to be produced from osteoblasts (Hayashi et al., 2012). To examine whether wedelolactone stimulated the production of Sema3A from BMSC, qRT-PCR assay was first performed. It was observed that the Sema3A mRNA expression level was upregulated after wedelolactone treatment of BMSC for 9 days (Figure 3). We added different concentrations of Sema3A antibody to BMSC, and then the activity of ALP, a marker enzyme for osteoblast differentiation, was determined. The increased number of ALP staining-positive BMSC by wedelolactone was reduced by addition of 0.5 or 1 μg/ml Sema3A antibody (Figure 4A). Morphological changes further confirmed that 1 μg/ml Sema3A antibody blocked the increase in the number of ALP-staining positive BMSC (Figure 4B). In addition, although 1 μg/ml Sema3A antibody did not affect the reduction in wedelolactone-induced TRAP activity in osteoclastic RAW264.7 cells, the decrease in TRAP activity caused by exposure to the 9-day conditioned medium fraction was reversed by Sema3A antibody (Figure 4C). These results suggest that wedelolactone-stimulated Sema3A production contributed to the enhancement of ALP activity in BMSC and inhibition of TRAP activity in RAW264.7 cells.

The activity of Sema3A on osteoblasts is mediated by its binding to the cell surface receptor consisting of Nrp1 and plexinA1. To determine the mechanism by which wedelolactone stimulated the production of Sema3A and subsequently enhanced osteoblastogenesis, the binding of NRP1, plexin-A1, and DAP12 was examined after treatment of BMSC with wedelolactone. Wedelolactone treatment (2 μg/ml) enhanced the binding of NRP1 and plexin-A1, while inhibition of Sema3A by its antibody reduced the formation amount of NRP1-plexin-A1 complex. The formation of the Sema3A-plexin-A1 complex was increased by wedelolactone, but the Sema3A antibody had no effect on the increased amount of the wedelolactone-induced Sema3A-plexin-A1 complex (Figure 5A).

The Sema3A signaling pathway is known to induce activation of the Wnt/β-catenin signaling pathway, which is crucial for osteoblast differentiation (Hens et al., 2005; Pederson et al., 2009). Therefore, we focused on nuclear translocation of β-catenin, a transcription factor involved in osteoblast differentiation. When BMSC were treated with wedelolactone, an increase in nuclear accumulation of β-catenin was observed. However, addition of the Sema3A antibody reversed the enhanced wedelolactone-induced nuclear translocation (Figure 5B). These results suggest that wedelolactone promoted the formation of a Sema3A-NRP1-plexin-A1 complex and subsequently activated β-catenin, which resulted in BMSC differentiation toward osteoblasts.

In contrast to osteoblasts, the activity of Sema3A on osteoclasts is mediated through inhibition of RANKL-induced formation of the plexinA1-DAP12 complex but without affecting the plexinA1-Nrp1 complex. To examine the impact of wedelolactone on the activation of the Sema3A signaling pathway in osteoclasts, the expression of plexinA1 associated with Nrp1 or DAP12 was determined. As shown in Figure 6A, addition of RANKL to RAW264.7 cells induced the formation of the plexinA1-DAP12 complex by sequestering plexinA1 from Nrp1, which was consistent with that of previously reported results (Hayashi et al., 2012). The RANKL-induced increase in the amount of DAP12 associated with plexinA1 was downregulated by conditioned media, while the decrease in the amount of Nrp1 associated with PlexinA1 was upregulated by wedelolactone. Furthermore, incubation with the 9-day conditioned medium fraction enhanced the effect of wedelolactone on the plexinA1-NRP1 complex formation, indicating that wedelolactone combined with Sema3A activated the plexinA1-NRP1 pathway. Phospholipase C (PLC)γ2 acts downstream of the plexinA1/NRP1/DAP12 pathway, which participates in osteoclastogenesis. To determine whether PLCγ2 activation was involved in wedelolactone-reduced osteoclastogenesis, phosphorylation of PLCγ2 was monitored. RANKL-induced phosphorylation of PLCγ2 was inhibited by wedelolactone treatment. Addition of the 9-day conditioned medium fraction resulted in a more significant decrease in phosphorylation of PLCγ2 expression (Figure 6B), indicating that wedelolactone activated the plexin A1//NRP1/DAP12 pathway and subsequently inhibited PLCγ2 activation, thus leading to reduced osteoclastogenesis.