The SpA was purchased from Sino Biological (Beijing, China); fetal bovine serum (FBS) was purchased from Gibco; Thermo Fisher Scientific Inc. (Waltham, MA, USA); penicillin-streptomycin solution and high-glucose Dulbecco's modified Eagle's medium (DMEM) were purchased from HyClone; GE Healthcare (Chicago, IL, USA). Soluble RANKL was obtained from R&D Systems Inc. (Minneapolis, MN, USA); JSH-23 was from Selleck Chemicals (Houston, TX, USA); acid phosphatase leukocyte kit (TRAP, 387A) and methyl thiazolyl tetrazolium (MTT) were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). A GeneJET RNA purification kit was purchased from Thermo Fisher Scientific Inc.; A PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time) and SYBR Premix Ex Taq™ II (Tli RNase H Plus) were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Oligonucleotide primer sets were synthesized by Augct DNA-Syn Biotechnology Co., Ltd. (Beijing, China). Antibodies against ERK1/2 (cat. no. 9926), p38 (cat. no. 9926), JNK (cat. no. 9926), NF-κB p65 (cat. no. 8242), inhibitor of κB-α (IκB-α; cat. no. 4812), protein kinase (Akt; cat. no. 4691), GAPDH (cat. no. 8884) and NFATc1 (cat. no. 8032), or the phosphorylated form of p38 (cat. no. 9910), ERK1/2 (cat. no. 9910), JNK (cat. no. 9910), Akt (cat. no. 4060), and NF-κB p65 (cat. no. 3033) were purchased from Cell Signaling Technology, Inc. (Danvers, MA USA); Immobilon Western Chemiluminescent horseradish peroxidase (HRP) Substrate and polyvinylidene difluoride (PVDF) membranes were purchased from Merck KGaA. A mouse tumor necrosis factor (TNF)-α SimpleStep ELISA^® kit (cat. no. ab208348), an interleukin-1 (IL)-1α mouse in vitro SimpleStep ELISA™ kit (cat. no. ab199076), and an IL-6 mouse ELISA kit (cat. no. ab46100) were purchased from Abcam (Cambridge, UK).

The Raw264.7 mouse monocytes/macrophage cell line (TIB-71; American Type Culture Collection, Manassas, VA, USA) was used as osteoclast precursors, which can differentiate into osteoclast-like cells in the presence of RANKL (28). The cells were grown in high-glucose DMEM, supplemented with 10% FBS and 1% penicillin-streptomycin solution, in a humidified atmosphere of 95% air and 5% CO₂ at 37°C; media were changed every 3 days.

Raw264.7 cells were plated in 96-well plates at a density of 1×10⁴ cells/well with 10 replicates in each group and cultured for 24 h. Cells were induced with SpA (concentrations of 50, 100, 200, 400, 800 and 1,600 ng/ml) and equal volume of phosphate buffered saline (PBS) alone, as control for 1, 3 and 5 days; media and stimuli were changed every 3 days. After culturing for the above indicated days, 10 µl MTT solution was added to the cells and cells were incubated at 37°C for 4 h away from light. Subsequently, media were removed, and 100 µl dimethyl sulfoxide was added to the plates and cells were agitated at room temperature for 10 min. Subsequently, the optical density (OD) values of every well were detected with a Model 680 Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 570 nm.

To differentiate into osteoclasts, Raw264.7 cells were seeded in 96-well plates at a density of 1×10⁴ cells/well with 5 duplicates in each group and cultured at 37°C for 24 h. Subsequently cells were stimulated with SpA (concentrations of 50, 100, 200, 400 and 800 ng/ml), SpA + RANKL (50 ng/ml), SpA+JSH-23 (20 µM), RANKL (100 ng/ml) or equal volumes of PBS (control) at 37°C for 5 days; media and stimuli were replaced every 3 days. Following this, cells were fixed and stained using the TRAP staining kit according to the manufacturer's protocol. All cells in every well were counted, and TRAP-positive multi-nucleated (≥3 nuclei) cells were counted as osteoclast-like cells.

Raw264.7 cells were seeded in 24-well Corning^® Osteo Assay Surface Multiple Well Plates (Corning Inc., Corning, NY, USA) at a density of 5×10⁴ cells/well with 4 duplicates in each group and cultured at 37°C for 24 h. Subsequently, cells were stimulated with SpA (concentrations of 50, 100, 200, 400 and 800 ng/ml), SpA+ RANKL (50 ng/ml), SpA+JSH-23 (20 µM), RANKL (100 ng/ml) and equal volumes of PBS (control) for 5 days; media and stimuli were changed every 3 days. After 5 days, media and cells were removed, and the areas of resorption pits were imaged by an inverted microscope and analyzed with Scion image software version 4.0.3.2 (Scion Corp., Frederick, MD, USA).

Raw264.7 cells were seeded in 6-well plates at a density of 1×10⁵ cells/well and cultured for 24 h. Subsequently, cells were stimulated with SpA (concentrations of 50, 100, 200, 400 and 800 ng/ml), SpA+ RANKL (50 ng/ml), RANKL (100 ng/ml) or equal volumes of PBS (control) for 5 days; media and stimuli were changed every 3 days. After 5 days, cells were harvested and total RNA was isolated with a GeneJET RNA purification kit according to the manufacturer's protocol, and the concentration of total RNA was measured with a micro spectrophotometer (Thermo Fisher Scientific Inc., NanoDrop 2000). Total RNA of 500 ng was used to synthesize cDNA by reverse transcription using PrimeScript™ RT reagent kit with gDNA Eraser. PCR was performed in a CFX96 Real-time system (Bio-Rad Laboratories, Inc.) with a SYBR^® Premix Ex Taq™. Reactions were initiated by incubation at 94°C for 5 min, and PCR [94°C for 30 sec, 60°C (58°C for GAPDH) for 34 sec, 72°C for 30 sec] was performed for 40 cycles; all reactions were performed in triplicate. Relative quantities of the tested genes were normalized to GAPDH, and the normalized data were expressed using the comparative 2^−ΔΔCq method (29). Primers used in this study are presented in Table I.

Raw264.7 cells were cultured in 25 cm² cell culture flasks; when cells reached 80% confluency, the culture medium was replaced, and cells were cultured at 37°C for another 1 h in the cell incubator. Following that, cells were stimulated with SpA (400 ng/ml) for 0, 5, 10, 20, 40 and 60 min, and for NFATc1 measurement, Raw264.7 cells were treated with SpA (400 ng/ml) or JSH-23+ SpA (400 ng/ml) for 0, 1, 2 and 3 days. At the indicated time point, cells were harvested and lysed using high efficiency tissue/cell lysis solution (Beijing Solarbio Science and Technology Co, Ltd., Beijing, China), and were followed by centrifugation at 4°C and 13,800 × g for 15 min, proteins in the supernatant were collected. Subsequently, protein concentrations were measured by Bicinchoninic Acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China), and protein samples were mixed with the sample buffer and boiled at 95°C for 5 min. Following that, protein samples (20 µg) were loaded onto 8–12% polyacrylamide gels, transferred to PVDF membranes and subsequently blocked with 5% skimmed milk or bovine serum albumin (Beijing Solarbio Science and Technology Co, Ltd., Beijing, China)-TBS (0.05% Tween-20) for 1 h. Following that, membranes were probed with specific antibodies against total ERK1/2 (1:1,000), p38 (1:1,000), JNK (1:1,000), NF-κB p65 (1:1,000), IκB-α (1:500), Akt (1:1,000), GAPDH (1:2,000) and NFATc1 (1:1,000), or the phosphorylated form of p38 (1:1,000), ERK1/2 (1:1,000), JNK (1:1,000), Akt (1:1,000) and NF-κB p65 (1:1,000), and were incubated at 4°C for 12 h. Detection was carried out using a HRP-linked rabbit IgG antibody (cat. no. 7074; 1:1,000; Cell Signaling Technology, Inc. Danvers, MA USA) at room temperature for 1 h, followed by an enhanced chemiluminescence western blotting detection reagent at room temperature for 1 min. Densitometry of the blots were performed using the Bio-Rad Universal Hood 2 Electrophoresis Imaging Cabinet (Bio-Rad Laboratories, Inc.), and were analyzed by Bio-Rad Image Lab Software version 5.2.1 (Bio-Rad Laboratories, Inc.).

Each experiment was performed in triplicate and similar results were obtained. Data are expressed as the mean ± standard deviation. SPSS 19.0 software (IBM Corp., Armonk, NY, USA) was used to perform the statistical analysis, and the statistical significance was determined by one-way analysis of variance followed by the Least Significant Difference/Student-Newman-Keuls test. P<0.05 was considered to indicate a statistically significant difference.

The cytotoxic effect of SpA on the Raw264.7cells was measured by MTT assay. As depicted in Fig. 1, SpA promoted cell proliferation on the first day under the concentration of 100–400 ng/ml, but this effect disappeared on days 3 and 5. Overall, SpA had no cytotoxicity to Raw264.7 cells below the concentration of 800 ng/ml. However, when the concentration was raised to 1,600 ng/ml, the cell proliferation activity was significantly inhibited. Since SpA did not show any toxic effect on cell viability up to 800 ng/ml, concentrations of 50–800 ng/ml were used in the following experiments.

SpA is an important virulence factor of S. aureus, the effect of SpA on osteoclast differentiation was explored. Raw264.7cells were grown in 96-well plates and stimulated with various concentrations of SpA in the absence or presence of RANKL for 5 days. SpA significantly promoted the formation of osteoclast-like cells in a dose-dependent manner in the absence of RANKL, but the number of osteoclast-like cells formed was lower than that of RANKL -induced (Fig. 2A and B). As reported previously, Raw264.7 cells stimulated by RANKL alone could differentiate into mature osteoclasts (28), however, Staphylococcal lipoteichoic acid, another virulence factor of S. aureus, inhibits osteoclast differentiation in the presence of RANKL (30). Therefore, the induction effect of SpA on osteoclast differentiation in the presence of RANKL was also examined (Fig. 2A). However, SpA did not inhibit the formation of osteoclast-like cells in the presence of RANKL, and the number of osteoclast-like cells was higher than that without RANKL under the same concentration (Fig. 2B), which indicated that RANKL enhanced the promotion effect of SpA on osteoclast differentiation. In addition, when treated with JSH-23, an inhibitor of NF-κB activation, the formation of osteoclast-like cells induced by SpA was inhibited (Fig. 2).

Since SpA promoted the formation of osteoclast-like cells derived from Raw264.7 cells, and bone resorption is the feature that identifies mature osteoclasts (31), the bone resorption activity of osteoclast-like cells induced by SpA from Raw264.7 cells was evaluated next. In the bone resorption assay, Raw264.7 cells were grown in COAS plates and stimulated with various concentrations of SpA and RANKL for 5 days. As illustrated in Fig. 3, SpA induced the formation of resorption pits in a dose-dependent manner in the absence or presence of RANKL (Fig. 3A and B), and the area of resorption pits with RANKL were higher than that without RANKL under the same SpA concentration (200, 400 and 800 ng/ml; Fig. 3B). However, when treated with JSH-23, the formation of resorption pits induced by SpA was decreased (Fig. 3). Collectively, these results suggested that SpA induced the formation of osteoclasts with bone resorption activity, and this effect was inhibitedby JSH-23.

Osteoclasts originated from hematopoietic cells of the monocyte/macrophage family (17), and Raw264.7 cells have been demonstrated to differentiate into mature osteoclasts treated with RANKL alone (17,32). mRNA expression levels of osteoclast-specific genes, such as TRAP, MMP-9, cathepsin K, CTR and Atp6v0d2 were measured. As demonstrated in Fig. 4, SpA increased the mRNA expression levels of TRAP, MMP-9, cathepsin K, CTR and Atp6v0d2 in a dose-dependent manner, and these expression levels were higher in the presence of RANKL rather than when treated with SpA alone. However, when JSH-23 was added, the mRNA expression levels of osteoclast-specific genes induced by SpA were inhibited (Fig. 4).

It was reported that, three mitogen-activated protein kinase (MAPK; including p38, ERK and JNK) (33), NF-κB (21,34) and PI-3K/Akt (23,35) signaling pathways have been involved in osteoclast differentiation. To elucidate the signaling pathways by which SpA promoted osteoclast differentiation, the protein expression of NFATc1 and the above-mentioned signaling pathways was measured in Raw264.7 cells stimulated by SpA. As illustrated in Fig. 5, the degradation of IκBα occurred 10 min after SpA treatment (Fig. 5A and B), while the phosphorylation of NF-κB increased markedly at 5 and 10 min (Fig. 5A and C). Furthermore, proteins in the signaling pathways of Akt and MAPKs showed no obvious change when treated with SpA (Fig. 5A). Because of this, the culture medium was supplemented with JSH-23, which is an inhibitor of NF-κB (36,37). Subsequently, Raw264.7 cells were treated with SpA and the formation of osteoclast-like cells and resorption pits was decreased; the expression levels of osteoclast-specific genes were inhibited too, which indicated that NF-κB signaling pathway was involved in the SpA-induced osteoclast differentiation (Fig. 5A and C).

NFATc1 is an important transcription factor in osteoclast differentiation; the induction of NFATc1 is a hallmark in the cell terminal determination of osteoclasts (38,39); and its presence in osteoclast precursors could promote their differentiation into mature osteoclasts in the absence of RANKL (39). Thus, the expression of NFATc1 in Raw264.7 cells treated with SpA for 1, 2 and 3 days was examined, and the results demonstrated that SpA significantly promoted the expression of NFATc1 on days 2 and 3 (Fig. 5D and E), but JSH-23 inhibited the expression of NFATc1 induced by SpA. These findings suggested that SpA may induce osteoclast differentiation through NF-κB activation, which subsequently activates NFATc1 determining osteoclast differentiation.