Formononetin Attenuates Osteoclastogenesis via Suppressing the

Publication Date (Web): November 14, 2014. Copyright © 2014 The American Chemical Society and American Society of Pharmacognosy. *(Sang Hoon Lee) ...
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Formononetin Attenuates Osteoclastogenesis via Suppressing the RANKL-Induced Activation of NF-κB, c‑Fos, and Nuclear Factor of Activated T‑Cells Cytoplasmic 1 Signaling Pathway Jeong-Eun Huh,† Wong In Lee,‡ Jung Won Kang,‡ Dongwoo Nam,‡ Do-Young Choi,‡ Dong-Suk Park,§ Sang Hoon Lee,*,‡ and Jae-Dong Lee*,‡ †

Oriental Medicine Research Center for Bone and Joint Disease, East-West Bone & Joint Research Institute, Kyung Hee University, 149, Sangil-dong, Gangdong-gu, Seoul, Korea ‡ Department of Acupuncture and Moxibustion, College of Oriental Medicine, Kyung Hee University, 1, Hoegi-dong, Dongdaemun-gu, Seoul, Korea § Department of Acupuncture and Moxibustion, Kyung Hee University Hospital at Kangdong, 149, Sangil-dong, Gangdong-gu, Seoul, Korea ABSTRACT: Formononetin (1), a plant-derived phytoestrogen, possesses bone protective properties. To address the potential therapeutic efficacy and mechanism of action of 1, we investigated its antiosteoclastogenic activity and its effect on nuclear factor-kappaB ligand (RANKL)-induced bone-marrow-derived macrophages (BMMs). Compound 1 markedly inhibited RANKL-induced osteoclast differentiation in the absence of cytotoxicity, by regulating the expression of osteoprotegerin (OPG) and RANKL in BMMs and in cocultured osteoblasts. Compound 1 significantly inhibited RANKLinduced tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T cell expressed and secreted (RANTES), and macrophage inflammatory protein-1α (MIP-1α) in a concentration-dependent manner. These effects were accompanied by a decrease in RANKLinduced activation of the NF-κB p65 subunit, degradation of inhibitor κBα (IκBα), induction of NF-κB, and phosphorylation of AKT, extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK). NF-κB siRNA suppressed AKT, ERK, JNK, and p38 MAPK phosphorylation. Furthermore, 1 significantly suppressed cFos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), key transcription factors during osteoclastogenesis. SP600125, a specific inhibitor of JNK, reduced RANKL-induced expression of phospho-c-Jun, c-Fos, and NFATc1 and inhibited osteoclast formation. These results suggested that 1 acted as an antiresorption agent by blocking osteoclast activation.

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(RANTES) are potently induced by RANKL, and both MCP-1 and RANTES stimulated the formation of TRAP+, multinucleated cells and increased bone resorption activity in human osteoclasts, suggesting a role for chemokine signaling in osteoclast physiology.9,10 CCL9/macrophage inflammatory protein (MIP-1γ) stimulates cytoplasmic motility and cell spreading in osteoclasts.11,12 Increased osteoclast activity leads to bone loss diseases, such as osteoporosis.9,13 Therefore, the regulation of osteoclastogenesis plays a key role in bone homeostasis. In osteoclast precursor cells, the binding of RANKL to its receptor RANK induces the activation of multiple intracellular signaling pathways, including NF-κB, phosphatidyl-inositol-3kinase (PI3K)/Akt, and mitogen-activated protein kinases (MAPKs), such as extracellular-signal regulated kinase (ERK),

steoclasts are large multinucleated giant cells that are derived from cell-to-cell contact, fusion, and differentiation of monocyte/macrophage lineage cells.1−4 Osteoclasts are generated from bone-marrow-derived macrophages (BMMs) by macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL), which provides tartrate-resistant acid phosphatase (TRAP) activity.5−7 Several inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin (IL)-1β and IL-6, contribute to osteoclastogenesis by modulating the induction of RANKL, osteoprotegerin (OPG), and M-CSF.7,8 In addition, some chemokines are associated with the generation and fusion of osteoclasts.9 RANKL induces the expression of CC chemokines, such as chemokine (C−C motif) ligands (CCL)2, CCL3, CCL4, CCL5, and CCL9, and CXC chemokines.9−12 Previous reports demonstrated that CCL2/ monocytes chemoattractant protein-1 (MCP-1) and CCL5/ regulated on activation normal T cell expressed and secreted © 2014 American Chemical Society and American Society of Pharmacognosy

Received: June 7, 2014 Published: November 14, 2014 2423

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positive multinucleated cells (by 1.3-fold to 21.5-fold) in a concentration-dependent manner (Figure 1A). Near-complete inhibition of osteoclastogenesis was observed in the presence of 5 μM 1 (Figure 1A). If 1 was cytotoxic to BMMs, osteoclast formation would be decreased irrespective of the specific effect of this compound on osteoclast differentiation. To exclude this possibility, we performed cell viability assays after 3 days in culture. No cytotoxicity was observed in the presence of up to 100 μM 1 for 72 h (Figure 1B). We also investigated whether the antiosteoclastogenic activity of 1 was direct, targeting osteoclast precursor cells, or indirect, targeting the supporting osteoblasts. In a coculture system with BMMs and osteoblasts, 1 significantly inhibited the expression of RANKL in a concentration-dependent manner (from 1.5-fold to 6.5-fold), and 1 increased the level of OPG by 1.2- to 2.9-fold in osteoclastogenic-factor-induced osteoblasts (Figure 1C). These data indicated that 1 affected the ability of osteoblasts to support antiosteoclastogenesis, resulting in reduced osteoclast formation. Formononetin (1) Inhibited RANKL-Induced Inflammatory Cytokines and Chemokines. The production of proinflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6) and chemokines (e.g., MCP-1, RANTES, and MIP-1α) stimulates osteoclastogenesis in vivo and in culture systems.8,9,37 Therefore, we investigated the effect of 1 on the expression of inflammatory cytokines and chemokines. Although the levels of TNF-α, IL-1β, IL-6, MCP-1, RANTES, and MIP-1α were almost undetectable in unstimulated cell culture medium, we observed significant increases in response to RANKL and MCSF stimulation (TNF-α by 3.2-fold, IL-1β by 7.8-fold, IL-6 by 16-fold, MCP-1 by 6.9-fold, RANTES by 2.8-fold, and MIP-1α by 43-fold) (Figure 2A−F). Compound 1 (1−10 μM) significantly reduced RANKL induction of TNF-α, IL-1β, IL6, MCP-1, RANTES, and MIP-1α in a dose-dependent manner (by 27.1−63.6% for TNF-α, 25.1−87.5% for IL-1β, 16.7−9.2% for IL-6, 27.4−76.4% for MCP-1, 7.1−42.9% for RANTES, and 23.1−48.7% for MIP-1α) (Figure 2A−F). The typical inflammatory cytokines involved in alveolar bone absorption have been reported to be TNF-α, IL-1, and IL-6.8,37 Of theses, IL-1 and TNF-α have the strongest involvement in alveolar bone resorption, because monocytes and macrophages produce these two cytokines, promoting the signaling cascades involved in osteoclast formation and activation.37 MCP-1, RANTES, and MIP-1α also stimulated the formation of TRAP+ multinucleated cells and increased bone resorption activity in osteoclasts, suggesting a role for chemokine signaling in osteoclasts.9,12,38 Our data identified significant reduction of TNF-α, IL-1β, IL-6, MCP-1, RANTES, and MIP-1α levels by 1, indicating that 1 may attenuate osteoclastogenesis by suppressing RANKL-induced inflammatory cytokines and chemokines that are induced during osteoclast differentiation. Previous studies have shown that 1 had a positive effect on bone remodeling by inhibiting inflammatory mediators and enhancing osteogenesis in osteoblasts of subchondral bone35 and fracture healing.36 Collectively, These results indicate that 1 possesses anti-inflammatory action in osteolysis and may have therapeutic applications in osteoclast-related diseases. Formononetin (1) Inhibits Phosphorylation of NF-κB and Degradation of IκBα during RANKL-Mediated Osteoclastogenesis. The activation of NF-κB is critical for RANKL-induced osteoclastogenesis.14,15,18 NF-κB is inactive in the cytosol because it is bound to IκBα and becomes active once IκBα has been phosphorylated and subsequently

c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38 MAPK).14,15 These signaling cascades lead to the induction and activation of osteoclastogenic transcription factors such as activator protein-1 (AP-1), c-Fos, and nuclear factor of activated T cells 1 (NFATc1).14−18 When cells are stimulated by RANKL, the IκB kinase (IKK) complex phosphorylates IκB, which is then targeted for degradation by the proteasome, leading to the activation of NF-κB signaling.18 NF-κB subunits are released from IκB in the cytosol and translocate to the nucleus, where they enhance the transcription of target genes. NFATc1 and c-Fos are master transcription factors regulating the terminal differentiation of osteoclasts.18−20 In addition, cFos and NFATc1 transcription factors play critical roles in osteoclast differentiation and bone destruction.21 Mice deficient in c-Fos develop osteopetrosis due to their failure to commit to the osteoclast lineage.22 Ectopic expression of NFATc1 in cFos-deficient cells is sufficient to restore the transcription of osteoclastogenesis-associated genes, leading to osteoclast formation and bone resorption.23

Formononetin (1) is a major constituent of the root of Astragalus membranaceus, a plant that is known to contain phytoestrogens.24,25 Phytoestrogens have attracted the attention of the general public and the medical communities due to their potentially beneficial role in the prevention and treatment of vascular disease, bone metabolism disorders, breast cancer, and menopausal symptoms.26−29 Specifically, the beneficial effects of phytoestrogens in the prevention of bone resorption and augmentation of bone density are active areas of investigation.25,30,31 Phytoestrogens have been shown to reduce bone loss and bone resorption in experimental models and in clinical trials dealing with osteoporosis.31−34 Our recent research demonstrated that 1 displayed a positive biphasic effect on normal and osteoarthritic osteoblasts by modifying their biological synthetic capacities,35 and it promoted fracture healing in the early stage of fracture repair.36 However, the precise molecular effects of 1 on osteoclastogenesis are largely unknown. In this study, the antiosteoclastogenic activities of 1 and its mechanism of action were investigated in RANKL-induced BMMs. The effects on osteoclast formation, the inflammatory cytokines and chemokines, and the expression of OPG/ RANKL in RANKL-induced BMMs cocultured with osteoblasts were examined. To clarify the molecular effects of 1, we examined the activation of AKT, ERK, JNK, p38 MAPK, and NF-κB, as well as osteoclastogenic transcription factors such as c-Fos and NFATc1, in RANKL-stimulated BMMs.

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RESULTS AND DISCUSSION Formononetin (1) Inhibited Osteoclastogenesis in Mouse BMMs. The effect of 1 was examined during mouse BMM differentiation into osteoclasts, in response to a mixture of RANKL and M-CSF. The formation of osteoclast-like cells was monitored by observing the formation of giant multinucleated cells and by measuring the activity of TRAP, an osteoclast marker enzyme. Compound 1 (concentration range 1−10 μM) significantly inhibited the formation of TRAP2424

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Figure 1. Effects of formononetin (1) on osteoclastogenesis. (A) BMMs were untreated (a) or treated with RANKL (100 ng/mL) (b) and increasing concentrations of 1 (c−e) for 7 days. The cells were then fixed, subjected to TRAP staining, and observed under a light microscope. The bar graph shows the number of TRAP-positive multinucleated osteoclasts. (B) BMMs were cultured with RANKL (100 ng/mL) and the indicated concentration of 1 for 72 h. Cell viability was determined using WST-8 assays, as described in the Experimental Section. (C) Expression of RANKL and OPG in cocultures of mouse calvariae osteoblasts and BMMs. Cells were stimulated with RANKL (100 ng/mL) for 1 h and treated with 1 for 48 h. The amounts of RANKL and OPG were determined by ELISA. Values represent the mean ± SEM of two independent experiments, and significance was determine using Student’s t tests. ns means nonsignificant. ###p < 0.001 compared with control. *p < 0.05 and ***p < 0.001 compared with vehicle. 2425

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Figure 2. Effect of formononetin (1) on RANKL-induced inflammatory cytokines and chemokines in BMMs. Cells were stimulated with RANKL (100 ng/mL) for 1 h and treated with 1 for 48 h. The levels of TNF-α, IL-1β, IL-6, MCP-1, RANTES, and MIP-1α α (panels A−F, respectively) were determined by ELISA. Values represent the mean ± SEM of two independent experiments, and significance was determined using Student’s t tests. ###p < 0.001 compared with control. *p < 0.05 and ***p < 0.001 compared with vehicle.

examined the phosphorylation of AKT, ERK, JNK, and p38 MAPKs by Western blot. This study found that phospho-AKT, phospho-ERK, phospho-JNK, and phospho-p38 MAPK were suppressed by 1 in a concentration-dependent manner (Figure 3C). These results indicated that 1 inhibited RANKL-induced activation of AKT and MAPKs in osteoclasts. We further investigated the molecular mechanisms by which 1 inhibited RANKL-induced phosphorylation of AKT, ERK, JNK, and p38 MAPK using specific siRNAs (Figure 3D). Interestingly, NF-κB siRNA effectively blocked the phosphorylation of AKT, ERK, JNK, and p38 MAPK. In addition, cotreatment with NF-κB siRNA and 1 (5 μM) completely inhibited the phosphorylation of AKT, ERK, JNK, and p38 MAPK (Figure 3D). AKT and ERK play a functional role in both osteoclast differentiation and survival.39,40 AKT could induce osteoclast differentiation through regulating the NFATc1 signaling cascade.39 Inhibition of RANKL-stimulated phosphorylation of AKT has been reported to block bone resorption by mature osteoclasts.41 ERK is known to induce c-Fos, promoting osteoclastogenesis, while a specific inhibitor of ERK (PD98059) inhibited

degraded. Thus, we examined whether 1 inhibited IκB degradation and NF-κB activation in RANKL-stimulated BMMs. Compound 1 significantly suppressed RANKL-induced phosphorylation of the NF-κB p65 subunit and degradation of IκBα in BMMs (Figure 3A). Furthermore, the inhibitory effects of 1 on NF-κB activation were confirmed in NF-κB-transfected BMMs. The transcriptional activity of NF-κB increased by 3.5fold following treatment with RANKL, and this activation was inhibited by 1 by 1.3-fold to 2.9-fold in a concentrationdependent manner (Figure 3B). Thus, 1 inhibits degradation of IκBα, resulting in reduced levels of NF-κB transactivation. These results suggest that down-regulation of NF-κB signaling is one of the mechanisms involved in the antiosteoclastogenic activity of 1. Formononetin (1) Inhibited Activation of AKT and MAPKs during RANKL-Mediated Osteoclastogenesis. Besides the NF-κB signaling pathway, activation of AKT, ERK, JNK, and the p38 MAPK pathways plays a pivotal role in osteoclastogenesis. To evaluate the effect of 1 on AKT and MAPKs following the stimulation of BMMs with RANKL, we 2426

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Figure 3. Formononetin (1) inhibited RANKL-induced activation of NF-κB, AKT, and MAPK signaling pathways. (A) Effect of 1 on RANKLinduced phosphorylation of NF-κB/p65 and IκBα degradation. BMMs were prestimulated with RANKL (100 ng/mL) for 1 h and subsequently treated with 1 for 4 h. Cell lysates were prepared for Western blotting with antibodies as indicated. The Western blots displayed are representative of two different experiments. (B) NF-κB-dependent transcriptional activity. Cells were prestimulated with RANKL (100 ng/mL) for 1 h and cotransfected with NFκB-SEAP-NPT plasmid (vehicle) or vector (control). Twenty-four hours after transfection, cells were treated with 1 for 12 h. Cell supernatants were collected and assayed for fluorescence activity as described in the Experimental Section. Values represent the mean ± SEM of two independent experiments, and significance was determined using Student’s t tests. ###p < 0.001 compared with the control. ***p < 0.001 compared with the vehicle. Cells were stimulated with RANKL (100 ng/L) for 1 h and treated with 1 for 12 h. Whole cell lysates were subjected to Western blotting with the indicated antibodies. (D) NF-κB deficiency regulates the phosphorylation of the AKT and MAPKs pathways. Cells were transfected with control siRNA or NF-κB/p65 siRNA and treated with 1 for 12 h. Whole cell lysates were collected for Western blotting with the indicated antibodies. These experiments were performed at least twice.

osteoclast formation, but not resorption activity.40,42,43 The other two MAPK family members, JNK and p38, are involved only in osteoclastogenesis.44−46 p38 MAPK is important in the early stages of osteoclast generation, while dominant-negative JNK prevented RANKL-induced osteoclastogenesis.44,46 In our study, 1 suppressed the phosphorylation of AKT, ERK, JNK, and p38 MAPK. These results suggest that AKT and MAPKs play pivotal roles in the antiosteoclastogenic effects of 1 in RANKL-stimulated BMMs and, therefore, represent molecular therapeutic targets for bone disease. Formononetin (1) Suppressed the Induction of NFATc1 and c-Fos by RANKL. Activated AKT and MAPKs can translocate to the nucleus and regulate various transcription factors, including c-Fos and NFATc1.14,15,39,40 To determine whether 1 influenced c-Fos and NFATc1 by inhibiting AKT, MAPKs, and NF-κB signaling cascades, we examined the expression of c-Fos and NFATc1 during RANKL-induced osteoclastogenesis. When BMMs were cultured in differentiation medium containing RANKL, the expression of cFos and NFATc1 increased markedly, as indicated by

quantitative real-time polymerase chain reaction (qRT-PCR) analysis (Figure 4A). Compound 1 decreased the extent of cFos and NFATc1 induction in response to RANKL (Figure 4A). Next, we determined the c-Fos and NFATc1 protein levels in cells treated with RANKL and 1. RANKL strongly increased the levels of c-Fos and NFATc1 at 6−12 and 24 h, respectively (Figure 4B), whereas 1 attenuated this RANKL-dependent increase in c-Fos and NFATc1 levels in a concentrationdependent manner (Figure 4C). These results suggested that 1 downregulated NF-κB and IκBα by suppressing RANKLinduced c-Fos and NFATc1 expression during RANKLstimulated differentiation of osteoclasts. Treatment with SP600125, a specific inhibitor of JNK, effectively blocked induction of phospho-c-Jun, c-Fos, and NFATc1 and also impaired RANKL-induced osteoclast formation (Figure 4D and E). RANKL signaling induced binding of c-Fos to the NFATc1 promoter, which in turn induced the expression of NFATc1.15,46,47 c-Fos is a major component of the AP-1 transcription factor complex, which induces members of the Jun family.15 Phosphorylation of c-Jun, in particular, plays an 2427

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Figure 4. continued

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Figure 4. Formononetin (1) suppressed RANKL-induced c-Fos and NFATc1 expression. (A) Time-course of the effects of 1 on RANKL-induced cFos and NFATc1 mRNA expression in BMMs. Cells were stimulated with RANKL (100 ng/mL) for 12 h and treated with 1 for the indicated times and concentrations. The c-Fos and NFATc1 mRNA levels were analyzed by real-time PCR. *p < 0.05 and ***p < 0.001 compared with the vehicle (treated with RANKL in cells). (B, C) Effect of 1 on RANKL-induced c-Fos and NFATc1 expression in BMMs. Cells were prestimulated with RANKL (100 ng/mL) for 12 h and treated with 1 for the indicated times and concentrations. Whole cell lysates were subjected to Western blotting with anti-c-Fos and NFATc1 antibodies. β-Actin served as an internal control. (D) SP600125, a specific JNK inhibitor, inhibited RANKL-induced phospho-c-Jun, c-Fos, and NFATc1 expression. Cells were incubated with RANKL (100 ng/mL) for 12 h and treated with SP600125 for 4 h. Whole cell lysates were subjected to Western blotting with antiphospho-c-Jun, anti-c-Jun, anti-c-Fos, and NFATc1 antibodies. β-Actin was used as an internal control. (E) TRAP-positive multinucleated osteoclasts were counted under a light microscope. Values represent the mean ± SEM of two independent experiments, and the significance was determined using Student’s t tests. ###p < 0.001 compared with the control. **p < 0.01 and ***p < 0.001 compared with the vehicle.

essential role in osteoclastogenesis.46,47 The signaling pathways mediated by RANK/TRAF6/JNK/AP-1/c-Fos seem to be critical for osteoclastogenesis, because TRAF6- and c-fosdeficient mice develop osteoporosis by affecting the JNK signaling pathway.48,49 In the present study, inhibition of JNK activation by SP600125 strongly suppressed phospho-c-Jun, cFos, NFATc1, and osteoclast formation in RANKL-induced BMMs. Collectively, our data indicated that c-Fos and NFATc1 were major transcription factors involved in the antiosteoclastogenic activity of 1. In conclusion, 1 suppressed RANKL-stimulated osteoclastogenesis via inhibiting NF-κB, c-Fos, and NFATc1 activation and downregulating AKT, ERK, JNK, and p38 MAPK signaling in osteoclast precursors. These data suggest that 1 represents an attractive lead compound for further development for preventing and treating inflammatory bone-lytic diseases.

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and NFATc1 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Fetal bovine serum (FBS), antibiotics, Dulbecco’s modified Eagle’s medium (DMEM), and Trizol reagent were purchased from Gibco-BRL, which is now part of the Invitrogen Corporation (Carlsbad, CA, USA). Culture of Primary Bone Marrow-Derived Macrophages as Osteoclast Precursors. Bone marrow macrophages were obtained by flushing the tibiae and femurs of 6-week-old ICR mice with αMEM. After the red blood cells were removed using ACK buffer (0.01 mM ethylenediaminetetraacetic acid [EDTA], 0.011 M KHCO, and 0.155 M NH4Cl, pH 7.3), the bone marrow cells were suspended in αMEM containing 10% (v/v) FBS, 100 U/mL penicillin, and 100 μg/ mL streptomycin and incubated for 24 h in the presence of 10 ng/mL M-CSF. Nonadherent cells were transferred and cultured in the presence of 30 ng/mL M-CSF for 3 days. Adherent cells were used as BMMs. To generate osteoclasts, BMMs were cultured in the presence of 30 ng/mL M-CSF and 100 ng/mL RANKL for 4 days. Multinucleated cells staining positive for TRAP and containing three or more nuclei were considered to be osteoclasts. Tartrate-Resistant Acid Phosphatase Staining. BMMs (5 × 105 cells/well) were seeded in 12-well plates, stimulated with 100 ng/ mL RANKL for 1 h and treated with 1 (1, 5, and 10 μM) or SP600125 (1, 10, and 20 μM) in the presence of 30 ng/mL M-CSF. To confirm the generation of osteoclasts, staining was performed using the leukocyte acid phosphatase assay kit (Sigma-Aldrich), according to the manufacturer’s instructions. TRAP-positive multinucleated cells were counted under a light microscope. Cell Viability Assay. BMMs were cultured with RANKL and the indicated concentration of 1 (0, 1, 5, 10, and 100 μM) for 72 h. We performed a WST-8 assay to examine the effect of 1 on the cytotoxicity of BMMs using Cell Counting Kit-8 (Dojindo Molecular Technology, Japan), according to the manufacturer’s instructions.

EXPERIMENTAL SECTION

General Experimental Procedures. Formononetin (1) (99% pure) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The purity of compound 1 was determined by high-performance liquid chromatography. Recombinant human RANKL, human M-CSF, OPG, and RANKL were measured using specific enzyme-linked immunosorbent assay (ELISA) purchased from R&D Systems (Minneapolis, MN, USA). WST-8 was obtained from the Dojindo Lab (Tokyo, Japan). A real-time PCR system using SYBR Green PCR Master Mix kit was purchased from Roche Diagnostics (Mannheim, Germany). Specific antibodies for phospho-AKT, phospho-ERK, phospho-JNK, phospho-p38, AKT, ERK, JNK, p38, NF-κB/p65, phospho-IκBα, IκBα, and phospho-c-Jun were obtained from Cell Signaling Technology (Danvers, MA, USA), and specific antibodies for c-Fos 2429

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Measurement of RANKL and OPG Expression in the Coculture System. Primary osteoblasts were prepared from the calvariae of 1-day-old mice. BMMs (3 × 105 cells/well) and calvarial osteoblasts (2.5 × 104 cells/well) were seeded in 48-well plates, prestimulated with 100 ng/mL RANKL for 1 h, and treated with 1 (5 μM) in the presence of 30 ng/mL M-CSF for 48 h. The amounts of RANKL protein in the cell lysate and OPG in the cell culture media were determined using RANKL and OPG ELISA (R&D Systems), according to the manufacturer’s instructions. Cytokine and Chemokine Measurement in Bone-MarrowDerived Macrophages. BMMs (3 × 105 cells/well) were seeded in 48-well plates, prestimulated with 100 ng/mL RANKL for 1 h, and treated with 1 (1, 5, and 10 μM) in the presence of 30 ng/mL M-CSF. After 7 days, the levels of TNF-α, IL-1β, IL-6, MCP-1, RANTES, and MIP-1γ in the cell culture media were determined using ELISA (R&D Systems), according to the manufacturer’s instructions. NF-κB Transcriptional Activity. We analyzed NF-κB activity in BMMs transiently transfected with a pNF-κB-SEAP-NPT construct, which encoded four copies of the NF-κB sequence and the SEAP gene as a reporter, using Lipofectamine 2000 (Invitrogen). Briefly, BMMs were seeded in six-well plates (5 × 105 cells/well) and stimulated with RANKL (100 ng/mL). After 1 h, the cells were co-transfected with 300 ng of pNF-κB-SEAP-NPT or the control SEAP-NPT plasmid in the presence of M-CSF and RANKL. After incubation for 24 h, the cells were treated with 1 (0, 1, 5, and 10 μM) in the presence of MCSF and RANKL for 12 h at 37 °C. NF-κB activity was measured using a 96-well plate fluorometer (Molecular Devices, F max) at 449 nm (emission) and 360 nm (excitation) and expressed as percent of control. Western Blotting. Cells were lysed with lysis buffer (Invitrogen). Protein concentrations were measured using the Bio-Rad protein assay with bovine serum albumin as a standard (Bio-Rad Laboratories, Mississauga, ON). Proteins (20 μg/lane) were size-fractionated using 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel (Invitrogen) electrophoresis under reducing conditions and transferred onto Hybond-C nitrocellulose membranes (Amersham Biosciences, NJ, USA). After blocking with 5% skim milk, the membranes were reacted with primary antibodies (1:1000 dilution) against NF-κB/p65, phospho-IκBα, IκBα, phospho-AKT, phospho-ERK, phospho-JNK, phospho-p38, AKT, ERK, JNK, p38, c-Fos, NFATc1, β-actin, and nonimmunized mouse IgG (Sigma-Aldrich Co.). The samples were then incubated with horseradish-peroxidase-labeled anti-goat IgG or anti-mouse IgG, and immunoreactive bands were detected with ECL Western blotting reagents (Amersham Biosciences). siRNA Transfection. BMMs were transiently transfected with siRNAs against NF-κB (Cell Signaling Tech., MA, USA) using Lipofectamine 2000 (Gibco), according to the manufacturer’s protocol. Briefly, NF-κB siRNA (200 nM) was suspended in 100 μL of Lipofectamine solution and mixed with an equal volume of serumfree DMEM. The mixture was added to 90% confluent BMMs cultured in six-well plates. Control siRNA (Dharmacon, Seoul, Korea) was used as a negative control. After 6 h of incubation, the transfected cells were washed twice with phosphate-buffered saline, replenished with fresh medium containing RANKL (100 ng/mL) and M-CSF (30 ng/mL), and incubated in a CO2 incubator for 24 h. The cells were then incubated with or without 1 (5 μM) for 12 h at 37 °C. Whole cell lysates were fractionated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The nitrocellulose membranes were reacted with primary antibodies (1:1000 dilution) against phospho-AKT, phospho-ERK, phosphoJNK, phospho-p38 MAPK, AKT, ERK, JNK, and p38 MAPK. Quantitative RT-PCR. Total RNA was isolated using TRIzol reagent, according to the manufacturer’s protocol. Reverse transcription was performed using M-MLV reverse transcriptase (TaKaRa Biotechnology, Seoul, Korea), according to the manufacturer’s specifications. Briefly, first-strand cDNA was synthesized at 37 °C for 1 h in a 20 μL reaction mixture using 1 μg of isolated mRNA. Quantitative RT-PCR was carried out in a 25 μL volume containing SYBR Green PCR Master Mix. The template source was either 5 ng of cDNA or purified DNA standard. Oligonucleotide primers of c-Fos

(forward, 5′-AACACTGCAACGTCCAGAT-3′; reverse, 5′CTGCAGCACGGTATAGGTGA-3′), NFATc1 (forward, 5′CCGTTGCTTCCAGAAAATAACA-3′; reverse, 5′-TGTGGGATGTGAACTCG GAA-3′), and β-actin (forward, 5′-GCTCTCCAGAACATCACTCCTGCC-3′; reverse, 5′-CGTTGTCATACCAGGAAATGAGCTT-3′) were designed on the basis of published sequences.21 To standardize mRNA levels, we amplified β-actin as an internal control. The cycle threshold (CT) for each sample was averaged and normalized to β-actin. The results were then analyzed using the comparative ΔΔCT method (2(−ΔΔCT)) for relative quantification of gene expression. Statistical Analysis. Data are expressed as the mean ± SEM. Groups were compared using one-way analysis of variance (ANOVA), followed by Duncan’s multiple range tests or Student’s t tests to compare two samples. p values < 0.05 were considered significant.

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AUTHOR INFORMATION

Corresponding Authors

*(Sang Hoon Lee) Tel: +82-2-958-9157. Fax: 82-2-440-6799. E-mail: [email protected]. *(Jae-Dong Lee) Tel: +82-2-958-9208. Fax: 82-2-440-6799. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by a grant from the Oriental Medicine R & D Project, Ministry of Health and Welfare, Republic of Korea (B110047).

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