ERN was purchased from Cayman Chemical (Ann Arbor, MI, USA). 1α,25(OH)2D3 was obtained from Sigma-Aldrich (St. Louis, MO, USA). Osteoblast-inducer reagent was purchased from Takara Bio Inc. (Shiga, Japan). Soluble RANKL (sRANKL) was purchased from R&D Systems (Minneapolis, MN, USA). α-minimal essential medium (α-MEM) (phenol red-free) was obtained from Gibco BRL/Invitrogen (Carlsbad, CA, USA).Fetal bovine serum (FBS) was obtained from Biowest (Nuaillé, France). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan).
BMCs were obtained from the femur and tibia of 8week-old male ddY mice. Male ddY mice were purchased from Japan SLC Co. (Hamamatsu, Japan). The mice were fed AIN-93G diet and given distilled water freely for three days as the acclimatization period. The mice were euthanized with an intraperitoneal injection of anesthesia (medetomidine hydrochloride 0.3 mg / kg + midazolam 4 mg / kg + butorphanol tartrate 5 mg / kg) followed by cervical dislocation. BMCs were isolated from femora and tibias, and were collected by centrifugation at 6,000rpm for 20 sec in 2.0 mL microcentrifuge tubes, followed by α-MEM. In the experiment, BMCs from two mice were mixed and used as the BMC samples. BMC samples were randomly divided into two groups, a control group and an ERN treated group. The animal protocols and procedures used in this study were approved by the Tokyo University of Agriculture Animal Use Committee, and mice were maintained in accordance with the guidelines of the University for the care and use of laboratory animals. Marrow cells were flushed from bones, and cells were cultured in α-MEM (phenol red-free) supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco BRL/Invitrogen) at 37°C in a humidified 5% CO2 atmosphere. RAW264.7 cells, mouse macrophage/monocytes, were obtained from the American Type Culture Collection (Manassas, VA, USA) and were cultured in α-MEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco BRL/Invitrogen) at 37°C in a humidified 5% CO2 atmosphere.
To evaluate the effect of ERN on the cell viability of BMCs, cytotoxicity assays were performed using the CCK-8. Briefly, BMCs (1 × 105 cells/well) were cultured in 96-well plates. Then, treated with the presence or absence of ERN (0.01–5 µM) for 6 days in α-MEM containing 10% FBS. The effect of ERN on cell viability was calculated as percent cell viability, with ERN-untreated cells set at 100%.
Osteoclast differentiation assay
To form multinucleated osteoclasts, BMCs were differentiated into osteoclasts using 1α,-25(OH)2D3. BMCs (1 × 106 cells/well) were treated with 10− 8 M of 1α,-25(OH)2D3 to induce differentiation in the presence of ERN at a concentration of 0–1 µM in a 96-well plate for 6 days. After 6 days of incubation, the cells were fixed in 10% formaldehyde and then stained for TRAP, a marker enzyme of differentiated osteoclasts. TRAP-positive cells with ≥ 3 nuclei were scored as differentiated osteoclasts. The effect of ERN on osteoclast differentiation was calculated as the osteoclast formation rate, with ERN-untreated control cells set at 100%.
Real-time PCR analysis
BMCs (1×107 cells/well) were seeded in a 24-well plate, treated with 10− 8 M of 1α,-25(OH)2D3 and various concentrations of ERN (0.1 and 1 µM) for 6 days. Total RNA was isolated from BMCs using Sepasol-RNA I Super G (Nacalai Tesque, Tokyo, Japan). Then, Single-stranded cDNA was synthesized from total RNA using reverse transcriptase (Takara Bio Inc.). Real-time PCR was performed using the THUNDERBIRD qPCR Mix (Toyobo, Osaka, Japan) and results were analyzed using the ABI StepOnePlus System (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed using the following primers: c-Fos, 5′-GAGTGATGCCGAAGGGATAA-3′ (forward) and 5′-GAGAAGCATTCCGGTCAGAG-3′ (reverse); NFATc1, 5′-GCTTCACCCATTTGCTCCAG-3′ (forward) and 5′-ATGGTGTGGAAATACGGTTGGTC-3′ (reverse); TRAP, 5′-ACTTCCCCAGCCCTTACTAC-3′ (forward) and 5′-TCAGCACATAGCCCACACCG-3′ (reverse); Ctsk, 5′-CCAGTGGGAGCTATGGAAGA-3′ (forward) and 5′-CTCCAGGTTATGGGCAGAGA-3′ (reverse); DC-STAMP, 5′-TCCTCCATGAACAAACAGTTCCA-3′ (forward) and 5′-AGACGTGGTTTAGGAATGCAGCTC-3′ (reverse); OC-STAMP, 5′-TGTCCTACAGTGCAGCCAAC-3′ (forward) and 5′-TCTCCTGAGTGATCGTGTGC-3′ (reverse); β-Actin, 5′-TGTCCACCTTCCAGCAGATGT-3′ (forward) and 5′-AGCTCAGTAACAGTCCGCCTAGA-3′ (reverse). All reactions were normalized to the housekeeping gene β-actin (ACTB).
BMCs (1 × 106 cells/well) were seeded in a 96-well plate for 24 h. Cells were then cultured with various concentrations of ERN (0–1 µM) in the presence of osteoblast-inducer reagents (ascorbic acid, β-glycerophosphate, and hydrocortisone) for 15 days. After incubation, the cells were fixed and stained with 1% alizarin red. For quantitative analysis, cells were destained with ethylpyridinium chloride and transferred to a 96-well plate to measure optical absorbance at 570 nm using a microplate reader. The effect of ERN on osteoblast differentiation is expressed as the degree of mineralization, with ERN-untreated cells set at 100%.
Results were presented as means ± SE of measurements performed on 3–6 cultures in each experimental or control group (there was no exclusion for any experimental unit.). All experiments were independently analyzed at least three times to confirm the results. For statistical significance, multiple comparisons were performed using Tukey’s test, after one-way analysis of variance (ANOVA). Statistical significance was set at P < 0.05.