CGA and EGCG were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). BMP-4 and a mouse osteoprotegerin enzyme-linked immunosorbent assay (ELISA) kit (cat. no. MOP00) were obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Rapamycin was obtained from Merck KGaA. Antibodies against phosphorylated Smad1 (cat. no. 13820), p38 MAPK (cat. no. 9212), phosphorylated (phospho)-p38 MAPK (cat. no. 4511), p70 S6 kinase (cat. no. 9202) and phospho-p70 S6 kinase (cat. no. 9205S) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against GAPDH (cat. no. sc-25778) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Secondary antibodies, peroxidase-labeled goat anti-rabbit immunoglobulin G (cat. no. 074-1506) were purchased from Seracare (Milford, MA, USA). An enhanced chemiluminescence (ECL) western blotting detection system was obtained from GE Healthcare Life Sciences (Chalfont, UK). EGCG and rapamycin were individually diluted in dimethyl sulfoxide (100 mM) and CGA was diluted in ethanol (100 mM). Both DMSO and ethanol were used at a maximum concentration of 0.1%, which did not affect the assay for osteoprotegerin or western blot analysis.

Cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria (20) were maintained as described previously (21). Briefly, cells were seeded into 35 mm diameter dishes (5×10⁴ cells/dish) for ELISA and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) experiments or 90 mm diameter dishes (2×10⁵ cells/dish) for Western blotting and cultured in α-minimum essential medium (α-MEM; cat. no. M8042, Sigma-Aldrich; Merck KGaA) containing 10% fetal bovine serum (FBS; cat. no. 12483-020; Gibco; Thermo Fisher Scientific Inc. Waltham, MA, USA) at 37°C in a humidified atmosphere of 5% CO2/95% air. After 5 days, the medium was replenished with α-MEM containing 0.3% FBS at 37°C for 48 h. Cells were then used in the following experiments.

Cultured MC3T3-E1 cells were pretreated with 1, 3, 10, 30 or 50 µM of CGA or 1, 3 or 10 µM of EGCG at 37°C for 60 min, then stimulated with 30 ng/ml BMP-4 or PBS supplemented with 0.01% bovine serum albumin (Sigma-Aldrich; Merck KGaA) containing 0.1% ethanol (as a vehicle) in 1 ml α-MEM containing 0.3% FBS at 37°C for 0, 12, 24, 36 or 48 h. The conditioned medium was collected following the incubation periods and the concentration of osteoprotegerin was measured using the mouse osteoprotegerin ELISA kit, according to the manufacturer's protocol.

Cultured MC3T3-E1 cells were pretreated with 10 µM EGCG or vehicle at 37°C for 60 min, then stimulated with 70 ng/ml BMP-4 or vehicle in 1 ml of α-MEM containing 0.3% FBS at 37°C for 6 h. Total RNA was isolated by TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and was reverse transcribed into complementary DNA using an Omniscript Reverse Transcriptase kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's protocol. qPCR was performed using a LightCycler Capillary system and Fast Start DNA Master SYBR-Green I kit (Roche Diagnostics, Basel, Switzerland). The forward and reverse primers for mouse osteoprotegerin mRNA were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). The sequences were as follows: Forward 5′-CAATGGCTGGCTTGGTTTCATAG-3′ and reverse 5′-CTGAACCAGACATGACAGCTGGA-3′. The forward and reverse primers for mouse GAPDH mRNA were synthesized based on the report of Simpson et al (22) and obtained from Sigma-Aldrich (Merck KGaA). The sequences were as follows: Forward 5′-AACGACCCCTTCATTGAC-3′ and reverse 5′-TCCACGACATACTCAGCAC-3′. The reaction mixtures were incubated at 95°C for 10 min, followed by 40 cycles at 60°C for 5 sec and 72°C for 7 sec. The amplified products were determined by a melting curve analysis (23) according to the system protocol. The data were analyzed by second derivative maximum method using LyghtCycler3 Data Analysis (Version 3.5.28; Roche Diagnostics). Levels of osteoprotegerin mRNA were normalized to those of GAPDH mRNA.

Cultured MC3T3-E1 cells were pretreated with 10, 20 or 30 µM of EGCG at 37°C for 60 min, then stimulated with 30 ng/ml BMP-4 or vehicle, PBS supplemented with 0.01% bovine serum albumin containing 0.1% ethanol, in 1 ml of α-MEM containing 0.3% FBS at 37°C for 45, 90 or 120 min according to our previous reports (19,24–26). Cells were then washed twice with phosphate-buffered saline and lysed, homogenized and sonicated with 20 short 1 sec bursts using a TOMY Ultrasonic Disruptor UD-211 (TOMY Digital Biology, Co., Ltd., Tokyo, Japan) in a lysis buffer containing 62.5 mM Tris/HCl (pH 6.8) 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol and 10% glycerol. SDS-PAGE was performed according to the Laemmli method (27) on 10% polyacrylamide gels. The proteins were fractionated and transferred onto Immuno-Blot polyvinyl difluoride (PVDF) membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked with 5% fat-free dry milk in Tris-buffered saline-Tween-20 [TBS-T; 20 mM Tris-HCl (pH 7.6) 137 mM NaCl and 0.1% Tween-20) at room temperature for 2 h prior to incubation with primary antibodies. Western blot analysis was performed as described previously (28) using antibodies against phospho-Smad1, GAPDH, phospho-p70 S6 kinase, p70 S6 kinase, phospho-p38 MAPK and p38 MAPK as primary antibodies and peroxidase-labeled goat anti-rabbit immunoglobulin G as secondary antibodies. The primary and secondary antibodies were diluted to 1:1,000 with 5% fat-free dry milk in TBS-T. Peroxidase activity on the PVDF membranes was detected on an X-ray film using the ECL western blotting detection system.

The absorbance of enzyme immunosorbent assay samples was measured at 450 nm with an EL 340 Bio Kinetic Reader (BioTek Instruments, Inc., Winooski, VT, USA). Densitometric analysis of western blotting was performed using a scanner and Image J 1.47 software (National Institutes of Health, Bethesda, MD, USA). Levels of phosphorylated protein were calculated as the background-subtracted signal intensity of each phosphorylation signal normalized to the respective total protein signal, and plotted as a fold increase relative to that of control cells treated with vehicle reagent.

Data were analyzed using one-way analysis of variance followed by a Bonferroni method for multiple comparisons between pairs, using Microsoft Office Excel 2013 for Windows (Microsoft Corporation, Redmond, WA, USA) and P<0.05 was considered to indicate a statistically significant difference. All data are presented as the mean ± standard error of the mean of three replicate experiments from three independent cell preparations.

In a previous study by the present authors (19), it was demonstrated that BMP-4 stimulates osteoprotegerin synthesis in osteoblast-like MC3T3-E1 cells. The present study initially investigated the effect of CGA on BMP-4-stimulated osteoprotegerin release in MC3T3-E1 cells. It was observed that CGA (≤50 nM) + BMP-4 had no significant effect on BMP-4-stimulated osteoprotegerin release in comparison to the value of vehicle + BMP-4 (Fig. 1).

The effect of EGCG on BMP-4-stimulated osteoprotegerin release was also investigated in MC3T3-E1 cells. In contrast to CGA, it was observed that 30 µM of EGCG + BMP-4 (Fig. 2A) significantly enhanced BMP-4-stimulated osteoprotegerin release in a time-dependent manner compared with the value of vehicle + BMP-4 (P<0.005; Fig. 2A). In addition, the stimulatory effects of EGCG + BMP-4 (Fig. 2B) on osteoprotegerin release were dose-dependent between 1 and 10 µM in comparison to the value of vehicle + BMP-4 (P<0.003; Fig. 2B).

To determine whether the stimulatory effects of EGCG on BMP-4-stimulated osteoprotegerin release was mediated by transcriptional events in osteoblast-like MC3T3-E1 cells, the effect of EGCG on BMP-4-induced expression of osteoprotegerin mRNA was subsequently evaluated. It was observed that EGCG (10 µM) + BMP-4 significantly enhanced BMP-4-induced osteoprotegerin mRNA expression in comparison to the value of vehicle + BMP-4 (P=0.03; Fig. 3).

The Smad1/5/8 pathway is an intracellular signal transduction system that principally mediates the effects of BMPs (15). To determine whether the stimulatory effects of EGCG on osteoprotegerin synthesis were dependent on the activation of Smad1/5/8 in MC3T3-E1 cells, the present study evaluated the effect of EGCG on BMP-4-induced phosphorylation of Smad1. It was observed that vehicle alone or EGCG + vehicle did not stimulate the phosphorylation of Smad1, whereas vehicle + BMP-4 did. It was observed that EGCG (10–30 µM) + BMP-4 had no effect on BMP-4-induced phosphorylation of Smad1 in comparison to the value of vehicle + BMP-4 (Fig. 4).

In previous studies by our group (19,24), BMP-4 induced the activation of p38 MAPK in osteoblast-like MC3T3-E1 cells, and p38 MAPK was associated with the BMP-4-stimulated osteoprotegerin synthesis in these cells. Therefore, to determine whether the effect of EGCG on BMP-4-stimulated osteoprotegerin synthesis was associated with activation of p38 MAPK in MC3T3-E1 cells, the present study evaluated the effect of EGCG on BMP-4-induced phosphorylation of p38 MAPK. It was observed that EGCG (10–30 µM) + BMP-4 had no significant effect on BMP-4-induced phosphorylation of p38 MAPK in comparison to the value of vehicle + BMP-4 (Fig. 5).

BMP-4 has been demonstrated to induce the synthesis of vascular endothelial growth factor through p70 S6 kinase in osteoblast-like MC3T3-E1 cells (25). Therefore, the present study also evaluated whether p70 S6 kinase serves a role in BMP-4-stimulated osteoprotegerin synthesis in MC3T3-E1 cells. It was observed that rapamycin, as an inhibitor of mammalian target of rapamycin (mTOR) that activates p70 S6 kinase (29), significantly suppressed BMP-4-stimulated osteoprotegerin release in a dose-dependent manner between 1 and 50 ng/ml in comparison to the value of vehicle + BMP-4 (P<0.007; Fig. 6).

To determine whether the effect of EGCG on BMP-4-stimulated osteoprotegerin synthesis was due to the activation of p70 S6 kinase in MC3T3-E1 cells, the present study evaluated the effect of EGCG on BMP-4-induced phosphorylation of p70 S6 kinase. However, it was observed that EGCG (10–30 µM) + BMP-4 had no significant effect on BMP-4-induced phosphorylation of p70 S6 kinase in comparison to the value of vehicle + BMP-4 (Fig. 7).