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. 2017 Jul;14(1):417-423.
doi: 10.3892/etm.2017.4491. Epub 2017 May 22.

(-)-Epigallocatechin gallate but not chlorogenic acid upregulates osteoprotegerin synthesis through regulation of bone morphogenetic protein-4 in osteoblasts

Kazuhiko Fujita  1   2 , Takanobu Otsuka  1 , Naohiro Yamamoto  1   2 , Shingo Kainuma  1   2 , Reou Ohguchi  1   2 , Tetsu Kawabata  1   2 , Go Sakai  1   2 , Gen Kuroyanagi  1   2 , Rie Matsushima-Nishiwaki  2 , Osamu Kozawa  2 , Haruhiko Tokuda  2   3
Affiliations

(-)-Epigallocatechin gallate but not chlorogenic acid upregulates osteoprotegerin synthesis through regulation of bone morphogenetic protein-4 in osteoblasts

Kazuhiko Fujita et al. Exp Ther Med. 2017 Jul.

Abstract

Chlorogenic acid (CGA) is a primary phenolic component of coffee and (-)-epigallocatechin gallate (EGCG) is a primary flavonoid component of green tea, both of which have been documented to possess beneficial health properties. A previous study by the present authors demonstrated that p38 mitogen-activated protein kinase (MAPK) may be associated with osteoprotegerin synthesis stimulated by bone morphogenetic protein-4 (BMP-4) in osteoblast-like MC3T3-E1 cells. In the present study, the effects of CGA and EGCG on BMP-4-stimulated osteoprotegerin synthesis in MC3T3-E1 cells were investigated. It was observed that CGA had no effect on osteoprotegerin release stimulated by BMP-4, whereas EGCG significantly enhanced BMP-4-stimulated osteoprotegerin release (P=0.003). Levels of osteoprotegerin mRNA expression induced by BMP-4 were also significantly increased by EGCG (P=0.03). By contrast, EGCG had no significant effect on phosphorylation of Smad1 or p38 MAPK induced by BMP-4. In addition, EGCG had little effect on BMP-induced phosphorylation of p70 S6 kinase; however rapamycin, as an inhibitor of p70 S6 kinase, significantly suppressed osteoprotegerin release (P=0.007). These data suggest that EGCG but not CGA may upregulate the synthesis of osteoprotegerin induced by BMP-4 in osteoblasts.

Keywords: (−)-epigallocatechin gallate; bone morphogenic protein-4; chlorogenic acid; osteoblast; osteoprotegerin.

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Figures

Figure 1.
Figure 1.
Effect of CGA on BMP-4-stimulated OPG release in MC3T3-E1 cells. The cultured cells were pretreated with the indicated doses of CGA for 60 min, then stimulated with 30 ng/ml BMP-4 or vehicle (●) or vehicle (○) for 48 h. Levels of OPG in the culture medium were determined by ELISA. Data are presented as the mean ± standard error of the mean of three replicate experiments from three independent cell preparations. CGA, chlorogenic acid, BMP-4, bone morphogenic protein-4; OPG, osteoprotegrin; N.S., no significant difference.
Figure 2.
Figure 2.
Effect of EGCG on BMP-4-stimulated OPG release in MC3T3-E1 cells. (A) Cultured cells were pretreated with 30 µM EGCG (●, ○) or vehicle (▲, ∆) for 60 min, then stimulated with 30 ng/ml BMP-4 (●, ▲) or vehicle (○, ∆) for the indicated time periods. (B) Cultured cells were pretreated with the indicated doses of EGCG for 60 min, then stimulated with 30 ng/ml BMP-4 (●) or vehicle (○) for 48 h. Levels of osteoprotegerin in the culture medium were determined by ELISA. Data are presented as the mean ± standard error of the mean of three replicate experiments from three independent cell preparations. *P<0.05 vs. control cells and **P<0.05 vs. cells stimulated by BMP-4 and pretreated with vehicle. EGCG, (−)-epigallocatechin gallate; BMP-4, bone morphogenic protein-4; OPG, osteoprotegrin.
Figure 3.
Figure 3.
Effect of EGCG on BMP-4-induced expression of OPG mRNA in MC3T3-E1 cells. Cultured cells were pretreated with 10 µM EGCG or vehicle for 60 min, then stimulated with 30 ng/ml BMP-4 or vehicle for 6 h. Total RNA was isolated and quantified using reverse transcription-quantitative polymerase chain reaction. Data are presented as the mean ± standard error of the mean of three replicate experiments from three independent cell preparations *P<0.05 vs. control cells and **P<0.05 vs. cells stimulated by BMP-4 and pretreated with vehicle. EGCG, (−)-epigallocatechin gallate; BMP-4, bone morphogenic protein-4; OPG, osteoprotegrin.
Figure 4.
Figure 4.
Effect of EGCG on BMP-4-induced phosphorylation of Smad1 in MC3T3-E1 cells. Cultured cells were pretreated with the indicated doses of EGCG for 60 min, then stimulated with 30 ng/ml BMP-4 or vehicle for 45 min. Cell extracts were then subjected to SDS-PAGE and subsequent western blot analysis using primary antibodies against phospho-Smad1 and GAPDH. The bar chart shows the levels of BMP-4-induced phosphorylation determined by laser-based densitometric analysis. Data are presented as the mean ± standard error of the mean of three replicate experiments from three independent cell preparations. *P<0.05 vs. control cells. N.S., no significant difference; EGCG, (−)-epigallocatechin gallate; BMP-4, bone morphogenic protein-4; phospho, phosphorylated.
Figure 5.
Figure 5.
Effect of EGCG on BMP-4-induced phosphorylation of p38 MAPK in MC3T3-E1 cells. Cultured cells were pretreated with various doses of EGCG for 60 min, then stimulated with 30 ng/ml BMP-4 or vehicle for 90 min. Cell extracts were then subjected to SDS-PAGE and subsequent western blot analysis using antibodies against phospho-p38 MAPK and p38 MAPK. The bar chart shows the levels of BMP-4-induced phosphorylation determined by laser-based densitometric analysis. Data are presented as the mean ± standard error of the mean of three replicate experiments from three independent cell preparations. *P<0.05 vs. control cells treated with vehicle alone. N.S., no significant difference; EGCG, (−)-epigallocatechin gallate; BMP-4, bone morphogenic protein-4; p38 MAPK, p38 mitogen-activated protein kinase; phospho, phosphorylated.
Figure 6.
Figure 6.
Effect of rapamycin on BMP-4-stimulated OPG release in MC3T3-E1 cells. Cultured cells were pretreated with the indicated doses of rapamycin for 60 min, then stimulated with 30 ng/ml BMP-4 (●) or vehicle (○) for 48 h. Levels of OPG in the culture medium were determined by ELISA. Data are presented as the mean ± standard error of the mean of three replicate experiments from three independent cell preparations. *P<0.05 vs. BMP-4 alone. BMP-4, bone morphogenic protein-4; OPG, osteoprotegrin.
Figure 7.
Figure 7.
Effect of EGCG on BMP-4-induced phosphorylation of p70 S6 kinase in MC3T3-E1 cells. Cultured cells were pretreated with the indicated doses of EGCG for 60 min, then stimulated with 30 ng/ml BMP-4 or vehicle for 120 min. Cell extracts were then subjected to SDS-PAGE and subsequent western blot analysis using antibodies against phospho-p70 S6 kinase and p70 S6 kinase. The bar chart shows the levels of BMP-4-induced phosphorylation determined by laser-based densitometric analysis. Data are presented as the mean ± standard error of the mean of three replicate experiments from three independent cell preparations. *P<0.05 vs. control cells treated with vehicle alone. N.S., no significant difference; EGCG, (−)-epigallocatechin gallate; BMP-4, bone morphogenic protein-4; phospho, phosphorylated.
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