Anabolic effect of β-cryptoxanthin in osteoblastic MC3T3-E1 cells is enhanced with 17β-estradiol, genistein, or zinc sulfate in vitro: the unique effect with zinc on Runx2 and α1(I) collagen mRNA expressions

Abstract

Whether the anabolic effect of β-cryptoxanthin (CRP), a kind of carotenoid, on osteoblastic MC3T3-E1 cells are modulated in the presence of various hormones or nutrient factors were investigated. Cells were cultured for 72 h in a minimum essential medium containing 10% fetal bovine serum (FBS), and the cells with subconfluency were changed to a medium containing either vehicle or CRP (10⁻⁸–10⁻⁶ M) in the presence or absence of various factors without FBS. Cells were cultured for 72 h. Protein content or alkaline phosphatase activity in osteoblastic cells were significantly increased after culture with CRP (10⁻⁷ or 10⁻⁶ M), 1,25-dihydroxyvitamin D₃ (VD₃; 10⁻⁹ or 10⁻⁸ M), 17β-estradiol (E₂; 10⁻⁹ M), genistein (10⁻⁷ or 10⁻⁶ M), or menaquinone-7 (MK-7; 10⁻⁷ or 10⁻⁶ M). The effect of CRP (10⁻⁶ M) in increasing protein content in the cells was significantly enhanced in the presence of E₂ (10⁻⁹ M) or genistein (10⁻⁶ M). Gene expression in osteoblastic cells was determined using reverse transcription-polymerase chain reaction (RT-PCR). Culture with CRP (10⁻⁷ or 10⁻⁶ M) caused a significant increase in the expression of Runx2 and alkaline phosphatase mRNAs in the cells. Runx2 mRNA expression was significantly increased after culture with E₂ (10⁻⁹ M) or MK-7 (10⁻⁷ or 10⁻⁶ M), but not VD₃ (10⁻⁹ or 10⁻⁸ M) or genistein (10⁻⁷ or 10⁻⁶ M). Alkaline phosphatase mRNA expression was significantly increased after culture with VD₃ (10⁻⁹ or 10⁻⁸ M), genistein (10⁻⁷ or 10⁻⁶ M), or MK-7 (10⁻⁷ or 10⁻⁶ M), but not E₂ (10⁻¹⁰ or 10⁻⁹ M). The effect of CRP (10⁻⁷ or 10⁻⁶ M) in increasing Runx2 or alkaline phosphatase mRNA expressions in the cells was not enhanced in the presence of VD₃, E₂, genistein, or MK-7. Culture with zinc sulfate (zinc; 10⁻⁵ M) caused a significant increase in protein content or alkaline phosphatase activity in osteoblastic cells. The effect of CRP (10⁻⁷ M) in increasing protein content or alkaline phosphatase activity in the cells was not significantly enhanced in the presence of zinc (10⁻⁵ M). Culture with zinc (10⁻⁵ M) caused a significant increase in α1(I) collagen mRNA expression, while it did not have a significant effect on Runx2 or osteocalcin mRNA expressions in the cells. The effect of CRP (10⁻⁷ M) in increasing Runx2 or α1(I) collagen mRNA expressions was significantly enhanced in the presence of zinc (10⁻⁶ or 10⁻⁵ M). Such an effect was not seen in the presence of cycloheximide (10⁻⁷ M), an inhibitor of protein synthesis, or 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB; 10⁻⁶ M), an inhibitor of transcriptional activity. This study demonstrates that the stimulatory effect of CRP on protein content in osteoblastic cells was additively enhanced with E₂ or genistein, and that the stimulatory effect of CRP on Runx2 or α1(I) collagen mRNA expressions was enhanced in the presence of zinc. Thus, the anabolic effect of CRP in osteoblastic MC3T3-E1 cells was modulated with a specific factor.