Genistein and zinc synergistically enhance gene expression and mineralization in osteoblastic MC3T3-E1 cells

Genistein, a phytoestrogen, and zinc, an essential trace element, have an anabolic effect on bone components. We investigated whether the combination of genistein and zinc has additive and synergistic effects in osteoblastic cells in vitro. Osteoblastic MC3T3-E1 cells with subconfluency were cultured for 48 h in medium containing either vehicle, genistein (10(-6) or 10(-5) M), zinc sulfate (10(-5) or 10(-4) M) or genistein (10(-6) or 10(-5) M) plus zinc sulfate (10(-5) or 10(-4) M). The protein content in the osteoblastic cells was significantly increased in the presence of genistein (10(-5) M) or zinc (10(-4) M). This increase was significantly enhanced with a culture of genistein (10(-5) M) plus zinc (10(-4) M). The expression of mRNAs of alpha 1(I) collagen and osteocalcin, which are bone matrix protein, in osteoblastic cells was demonstrated using reverse transcription-polymerase chain reaction (RT-PCR) analysis with a specific primer. This expression was not significantly increased with genistein (10(-6) or 10(-5) M) or zinc (10(-5) or 10(-4) M). The combination of genistein (10(-6) or 10(-5) M) and zinc (10(-5) or 10(-4) M) caused a significant increase in alpha 1(I) collagen and osteocalcin mRNA expression in osteoblastic cells. Alkaline phosphatase mRNA expression in osteoblastic cells was significantly enhanced with a culture of genistein (10(-5) M) plus zinc (10(-4) M). The effect of genistein (10(-5) M) plus zinc (10(-4) M) in enhancing the expression of alpha 1(I) collagen, alkaline phosphatase, or osteocalcin mRNAs in osteoblastic cells was completely prevented with a culture of cycloheximide (10(-7) M), an inhibitor of protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosylbenimidazole (DRB) (10(-6) M), an inhibitor of transcriptional activity. Moreover, a culture with genistein (10(-5) M) or zinc (10(-4) M) for 14 or 21 days caused a significant increase in mineralization. This increase was markedly enhanced with a culture of the combination of genistein (10(-5) M) and zinc (10(-4) M). This study demonstrates that the combination of genistein and zinc can synergistically enhance gene expression and mineralization in osteoblastic cells.