The effect of AG490 on the crosstalk between osteoblasts and osteoclasts in vitro

Abstract

Objective To establish a co-culture system of MC3T3-E1 cells and RAW 264.7 cells in vitro, and then explore the effect of AG490 on osteoclastogenesis in this system. Methods The mineralized MC3T3-E1 cells and RAW 264.7 cells were co-cultured after the addition of different concentrations of AG490 (1 μmol/L, 5 μmol/L, 10 μmol/L) . The cell proliferation was analyzed by CCK-8 assay, ALP activity was analyzed by a simultaneous-coupling azo dye method, and osteoblast-related genes OPG, COL1, ALP, OCN were analyzed by RT-PCR in mineralized MC3T3-E1 cells. TRAP and hematoxylin staining were performed to observe the osteoclastogenesis, and Western blot was adopted to analyze the RANK expression in osteoclasts. Data were evaluated by One-Way ANOVA and Two-Way ANOVA. Results AG490 inhibited the ALP activity and the expression of osteoblast-related genes OPG, COL1, ALP, OCN in mineralized MC3T3-E1 cells (FOPG= 30.120, POPG, AG490 1 μmol/L= 0.021, POPG, AG490 5 μmol/L= 0.221, POPG, AG490 10 μmol/L= 0.003; FALP= 67.352, PALP, AG490 1 μmol/L= 0.034, PALP, AG490 5 μmol/L= 0.001, PALP, AG490 10 μmol/L= 0.156; FCOL1= 28.158, PCOL1, AG490 1 μmol/L= 0.054, PCOL1, AG490 5 μmol/L= 0.002, PCOL1, AG490 10 μmol/L= 0.4; FOCN= 125.375, POCN, AG490 1 μmol/L<0.001, POCN, AG490 5 μmol/L<0.001, POCN, AG490 10 μmol/L<0.001) . Compared with RAW264.7 cells alone group (control group) , the indirect co-culture of MC3T3-E1 cells and RAW264.7 cells successfully promoted osteoclast formation, which could be significantly inhibited by AG490 (F= 85.391, Pco-culture-control<0.001, Pco-culture+AG490-co-culture= 0.035) , meanwhile the addition of AG490 down-regulated the RANK in the osteoclasts in protein level (FRANK= 376.25, PRANK, AG490 1 μmol/L= 0.468, PRANK, AG490 5 μmol/L=0.003, PRANK, AG490 10 μmol/L<0.001) . Conclusions AG490 can inhibit osteoblastic differentiation in MC3T3-E1 cells, and might play an important role in osteoclast formation inhibition in co-culture of MC3T3-E1 cells and RAW 264.7 cells by blockade of OPG/RANKL/RANK signal axis. Key words: Culture techniques; Receptor activator of nuclear factor-kappa B; AG490; Osteoblastic differentiation; Osteoclastic differentiation