Recombinant human IL-1β was purchased from R&D System (Minneapolis, MN, USA). Atorvastatin was obtained from Axon MedChem (Groningen, the Netherland). Human soluble RANKL and the macrophage-colony stimulating factor (M-CSF) were obtained from PeproTech (Rocky Hill, NJ, USA). Monoclonal antibodies (Abs) against extracellular signal-regulated kinase (ERK) 1/2, phosphate (p)-ERK, c-Jun N-terminal kinases (JNK), p-JNK, p38, p-p38, NF-κB (p65), inhibitor of NF-κBα (IκBα), and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Abs against c-Fos, NFATc1, and tartrate-resistant acid phosphatase (TRAP) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The goat anti-rabbit immunoglobulin G (IgG, ENZO Life Sciences, Farmingdale, NY, USA) and rabbit anti-goat horseradish peroxidase (HRP)-conjugate were purchased from Bethyl Laboratories (Montgomery, TX, USA).

Bone marrow cells (BMCs) were collected from the tibias and femurs with α-minimum essential media (α-MEM, WelGENE, Gyeongsan, Korea) containing antibiotics (Sigma-Aldrich, St. Louis, MO, USA) of 5-week-old male Institute for Cancer Research (ICR) mice. The animal experiments were approved by the Institutional Animal Care and Use Committee of Chonbuk National University (CNU), and they were conducted in accordance with the guidelines of CNU. The BMCs were cultured in α-MEM containing 10% fetal bovine serum (FBS), antibiotics, and M-CSF (10 ng/mL), and then they were cultured for 1 day. Nonadherent cells were plated on 100mm petri dishes and were then cultured for 3 days in the presence of M-CSF (30 ng/mL). Adherent cells were used as bone marrow-derived macrophages (BMMs). The BMMs were cultured for 3 days with M-CSF (30 ng/mL) and RANKL (100 ng/mL) to induce pre-fusion osteoclasts. To differentiate osteoclasts from the BMMs, they were cultured for 4 days with M-CSF (30 ng/mL) and RANKL (50 ng/mL) in the presence or absence of IL-1β (10 ng/mL) or atorvastatin (0.5 μM).

Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommended protocol. The complement (c)-DNA was synthesized using a Maxime RT Premix Kit (iNtRON Biotechnology, Seongnam, Korea), and then cDNA was amplified using a Maxime polymerase chain reaction (PCR) Premix Kit (iNtRON Biotechnology). The primers used in the PCR were as follows: NFATc1 forward, 5′-tgtgcaagccaaattccctg-3′, reverse, 5′-atacacccccagaccgcatc-3′; c-Fos forward, 5′-atcggaggagggagctgaca-3′, reverse, 5′-ggaaccggacaggtccacat-3′; TRAP forward, 5′-gtgctggctggaaaccatga-3′, reverse, 5′-cccactcagcacatagccca-3′; E-cadherin forward, 5′-aaatgatgtggctcccaccc-3′, reverse, 5′-catctcccatggtgccacac-3′; cathepsin K forward, 5′-cagagtgggaaggcagggtc-3′, reverse, 5′-gctttctcgttccccacagg-3′; osteoclast-associated receptor (OSCAR) forward, 5′-ctttttctttctggccgcgt-3′, reverse, 5′-catggtggctcacacccatc-3′; dendritic cell-specific transmembrane protein (DC-STAMP) forward, 5′-gcaaggaacccaaggagtcg-3′, reverse, 5′-cagttggcccagaaagaggg-3′; c-Src forward, 5′-ctggttccacggcaagatca-3′, reverse, 5′-ttcatgttcagtgcccagcc-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5′-tcatcatctccgccccttct-3′, reverse, 5′-agcttcccgttcagctctgg-3′. PCR products were electrophoresed on 1% agarose gel stained with ethidium bromide. Densitometric analysis was performed using FusionCapt Advance software (Vilber Lourmat, Eberhardzell, Germany).

Cell viability was determined using a CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium (CCK-8) was reduced by dehydrogenases in cells to yield an orange-colored product (formazan) [17]. The amount of formazan dye generated by dehydrogenases in the cells was directly proportional to the number of living cells. The BMMs were plated on 96-well plates with α-MEM containing 10% FBS and antibiotics with M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 3 days. The cells were washed with complete α-MEM media and were then incubated for 24 hours with IL-1β (10 ng/mL) or atorvastatin (0.5 μM) in the presence of M-CSF (30 ng/mL) and RANKL (50 ng/mL). CCK-8 (10 μL/well) was added to each well of the plates, and the cells were incubated for 3 hours. The absorbance was measured at 450 nm using a microplate reader.

Cultured cells were lysed in the lysis buffer (50 mM Tris-HCl, 150 mM NACl, 5 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, and 1% deoxycholate) containing protease inhibitors. The protein concentration was determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Samples containing 30 μg of protein were boiled in sodium dodecyl sulfate (SDS) sample buffer at 95ºC for 5 minutes, separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. Primary Abs (10 μg/mL) against NFATc1, c-Fos, TRAP, ERK 1/2, p-ERK, JNK, p-JNK, p38, p-p38 NF-κB (p65), IκBα, and β-actin were incubated overnight at 4ºC. Then the secondary HRP-conjugated Ab, which was goat anti-rabbit IgG or rabbit anti-goat IgG, was washed three times for 30 minutes. Reactive proteins were detected by enhanced chemiluminescence (Amersham Life Sciences, Arlington, IL, USA) using Fusion FX7 (Vilber Lourmat).

The BMMs were seeded at a density of 1.5 × 10⁴ cells/well in a 96-well plate in triplicate for 3 days. The cells were cultured for 4 days with IL-1β (10 ng/mL) or atorvastatin (0.5 μM) in the presence of M-CSF (30 ng/mL) and RANKL (50 ng/mL). After 4 days, the cells were fixed with 3.7% formalin and permeabilized in 0.1% Triton X-100 for 10 minutes. The cells were stained with TRAP stain solution using a leukocyte acid phosphatase kit (Sigma-Aldrich) according to the manufacturer’s instructions. TRAP positive cells containing three or more nuclei were counted as multinuclear osteoclasts. Photographs were taken under a light microscope (Carl Zeiss, Thornwood, NY, USA).

The formation of osteoclasts was evaluated by the resorption pit area as follows. The BMMs (2 × 10⁴ cells/well) were seeded in a 96-well plate containing a dentine slice 5 mm in diameter (Immunodiagnostic Systems, Gaithersburg, MD, USA) with IL-1β (10 ng/mL) or atorvastatin (0.5 μM) in the presence of M-CSF (30 ng/mL) and RANKL (50 ng/mL) for 3 weeks. Cells on the dentine slices were rubbed with a cotton bud and stained with hematoxylin (Sigma-Aldrich) or TRAP. Quantification of the area of resorption was conducted using the IMT i-solution software (Samwoo Scientific Co., Seoul, Korea).

All data are expressed as a mean ± standard deviation of triplicate measurements. The mean values of each group were compared using Student t test or analysis of variance as appropriate, and p < 0.05 was considered statistically significance. Statistical analyses were performed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA).

To determine the effects of atorvastatin on RANKL-mediated or IL-1β-stimulated osteoclastogenesis, we initially examined the formation of TRAP (+) multinuclear cells as an indicator of osteoclast differentiation, which satisfied most of the morphological criteria of osteoclasts. IL-β significantly increased the formation of TRAP (+) cells compared to the absence of this cytokine (48.2 ± 4.3 vs. 149.4 ± 7.5, respectively; p < 0.05). Atorvastatin significantly decreased RANKL-mediated or IL-1β-stimulated formation of TRAP (+) cells compared to the absence of atorvastatin (34.3 ± 4.5 vs. 47.3 ± 5.2, respectively; p < 0.05) (Fig. 1A).

We also measured the resorption area as another indicator of osteoclast formation. IL-1β significantly increased the resorption area compared to the absence of IL-1β (48.3 ± 4.5 vs. 80.8 ± 3.2, respectively; p < 0.05). Atorvastatin also significantly decreased the RANKL-mediated or IL-1β stimulated formation of resorption pits compared to the absence of atorvastatin (40.9 ± 3.5 vs. 53.4 ± 2.5, respectively; p < 0.05) (Fig. 1B).

To evaluate the effects of atorvastatin on the growth properties of the BMCs from the 5-week-old male ICR mice, we measured cell survival with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence or absence of atorvastatin (0.5 μM) or IL-1 (10 ng/mL) for 3 days as previously described. As shown in Fig. 2A, IL-1β significantly increased the survival of osteoclast precursor cells compared to the control without IL-1β (p < 0.05). Atorvastatin significantly inhibited the survival of osteoclast precursor cells compared to IL-1β (p < 0.05). However, there was no difference in the survival of osteoclast precursor cells between the control and cultures with atorvastatin. To determine the dose-dependent effects of atorvastatin on IL-1β-induced survival of osteoclast precursor cells, we added various doses of atorvastatin (0.5, 1, and 5 μM) to the osteoclast precursor cell cultures with IL-1β (10 ng/mL) for 3 days and performed a CCK-8 assay. The inhibitory effects of atorvastatin were significantly enhanced as the concentration of atorvastatin increased (Fig. 2B). These effects were also time dependent (data not shown).

Osteoclast differentiation is regulated by the induction of various genes in response to RANKL and other osteotropic agents, including IL-1β and TNF-α. IL-1β is a very important inflammatory cytokine in RA and causes bone loss by increasing osteoclast formation. Both c-Fos and NFATc1 play essential roles in the differentiation of osteoclast precursors [10]. Therefore, we examined whether atorvastatin regulated the expression of c-Fos, NFATc1 messenger RNAs (m-RNAs), and proteins in response to RANKL or IL-1β. The densitometric value of reverse transcription (RT)-PCR normalized by the GAPDH intensity showed that atorvastatin significantly decreased the RANKL-mediated or IL-1β-stimulated expression of c-Fos and NFATc1 mRNA (Fig. 3A). Consistent with the results of the RT-PCR analyses, expression of c-Fos and NFATc1 protein levels increased in response to RANKL and IL-1β, and the expression of c-Fos and NFATc1 was significantly inhibited by atorvastatin (Fig. 3B). Thus, atorvastatin may inhibit osteoclast differentiation by inhibiting c-Fos and NFATc1 expressions in response to RANKL and IL-1β. Many molecules are implicated in cell-cell fusion, including E-cadherin [18], DC-STAMP [19], and Src family kinases [20]. Therefore, we examined whether atorvastatin regulated the expression of E-cadherin, DC-STAMP, and c-Src. The densitometric value of RT-PCR normalized by the GAPDH intensity showed that atorvastatin significantly decreased RANKL or IL-1β, as well as the expression of E-cadherin, DC-STAMP, and c-Src mRNA (Fig. 4).

To determine the involvement of signal transduction and the mechanisms underlying the effect of atorvastatin on IL-1β-stimulated, RANKL-mediated osteoclast differentiation, we evaluated the activation of MAPKs in the BMCs. The densitometric values on immunoblots normalized according to β-actin intensity showed that IL-1β activated intracellular MAPKs, including ERK, p38, JNK (p < 0.05), and atorvastatin significantly inhibited RANKL-mediated or IL-1β-stimulated activation of intracellular MAPK, including ERK and JNK, except p38 (p < 0.05) (Fig. 5). These results indicated that atorvastatin inhibits RANKL-mediated or IL-1β-stimulated osteoclast differentiation and survival via intracellular MAPKs pathways.