(-)-Epigallocatechin-3-Gallate Decreases Osteoclastogenesis via Modulation of RANKL and Osteoprotegrin

Abstract

Osteoporosis is the second most common epidemiologic disease in the aging population worldwide. Previous studies have found that frequent tea drinkers have higher bone mineral density and less hip fracture. We previously found that (-)-epigallocatechin gallate (EGCG) (20⁻100 µmol/L) significantly suppressed receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis and pit formation via inhibiting NF-κB transcriptional activity and nuclear transport of NF-κB in RAW 264.7 cells and murine primary bone marrow macrophage cells. The most important regulation in osteoclastogenesis is the receptor activator of nuclear factor-kB/RANKL/osteoprotegrin (RANK/RANKL/OPG) pathway. In this study, we used the coculture of RAW 264.7 cells and the feeder cells, ST2, to evaluate how EGCG regulated the RANK/RANKL/OPG pathway in RAW 264.7 cells and ST2 cells. We found EGCG decreased the RANKL/OPG ratio in both mRNA expression and secretory protein levels and eventually decreased osteoclastogenesis by TRAP (+) stain osteoclasts and TRAP activity at low concentrations-1 and 10 µmol/L-via the RANK/RANKL/OPG pathway. The effective concentration can be easily achieved in daily tea consumption. Taken together, our results implicate that EGCG could be an important nutrient in modulating bone resorption.

Keywords: EGCG; RANK/RANKL/OPG; RAW 264.7 cells; catechin; osteoclast; osteoclastogenesis.

Figure 1

Effects of (−)-epigallocatechin gallate (EGCG) on ST2 cells in MTS. There was no significant change in the MTS assay and cell cycle after EGCG treatment from 1 to 200 µmol/L for 24 and 48 h. With the treatment of EGCG, the viability of ST2 cells was not affected by EGCG at both 1 and 10 µmol/L (both p > 0.05). However, decrease via viability of ST2 cells was noted in EGCG concentrations higher than 20 µmol/L. *: p < 0.05, **: p < 0.01, compared with day 1 0 µM. ##: p < 0.01 Compared with day 2 0 µM.

Figure 2

The mRNA expression of osteoprotegrin (OPG) and receptor activator of nuclear factor-kB ligand (RANKL) genes. (A) The mRNA expression of OPG increased after EGCG treatment for 24 and 48 h at concentrations of both 1 and 10 µmol/L. (B) The mRNA expression of RANKL decreased after EGCG treatment for 24 and 48 h at concentrations of both 1 and 10 µmol/L. However, the difference did not reach statistical significance. (C) The ratio of RANKL/OPG decrease at 10 µmol/L after treatment for 24 (p = 0.06) and 48 h (p = 0.005). ##: p < 0.01, compared with day 2 control.

Figure 3

Secretory OPG and RANKL expression by ELISA. There was no significant change in OPG secretion after EGCG treatment at either 1 or 10 µmol/L (A). Like mRNA expression, secreted RANKL decreased after EGCG treatment for 4 and 7 days at concentrations of both 1 and 10 µmol/L (B). The ratio of RANKL/OPG decreased after EGCG treatment for four (p < 0.05) and 7 days (p < 0.01) at concentrations of both 1 and 10 µmol/L (C). **: p < 0.01 compared with day 4 control. ##: p < 0.01 compared with day 7 control.

Figure 4

EGCG inhibited osteoclast differentiation of RAW 264.7 cells cocultured with ST2 cells. Coculture cells were seeded in 48-well plates containing α-MEM medium plus 10% FBS, 0.1 μmol/L 1,25(OH)₂D₃, and 0.1 μmol/L dexamethasone (DEX). In RAW 264.7 cells, the number of TRAP-positive osteoclasts was reduced by EGCG in a dose-dependent manner (A). After quantification, EGCG effectively suppressed the number of multinucleated TRAP-positive cells by 21% at 1 µmol/L (p < 0.05) and 91% at 10 µmol/L (p < 0.001) (B). EGCG at concentrations of 1 and 10 µmol/L dose-dependently reduced the TRAP activity by 17% (p < 0.05) and 21% (p < 0.01) in RAW 264.7 cells (C).