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Whether the anabolic effect of beta-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 D(3) (VD(3); 10(-9) or 10(-8) M), 17beta-estradiol (E(2); 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 E(2) (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 E(2) (10(-9) M) or MK-7 (10(-7) or 10(-6) M), but not VD(3) (10(-9) or 10(-8) M) or genistein (10(-7) or 10(-6) M). Alkaline phosphatase mRNA expression was significantly increased after culture with VD(3) (10(-9) or 10(-8) M), genistein (10(-7) or 10(-6) M), or MK-7 (10(-7) or 10(-6) M), but not E(2) (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 VD(3), E(2), 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 alpha1(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 alpha1(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-beta-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 E(2) or genistein, and that the stimulatory effect of CRP on Runx2 or alpha1(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.

The effect of beta-cryptoxanthin, which is greatly present in fruits, has not been clarified so far on bone metabolism. The effect of beta-cryptoxanthin on bone formation and bone resorption was investigated in tissue culture in vitro. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48 h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-8)-10(-5) M beta-cryptoxanthin. The presence of beta-cryptoxanthin (10(-6) or 10(-5) M) caused a significant increase in calcium content, alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal and metaphyseal tissues. These increases were completely prevented in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis. beta-Carotene (10(-6) or 10(-5) M) or xantine (10(-6) or 10(-5) M) had no effect on the diaphyseal and metaphyseal calcium contents. The bone-resorbing factors parathyroid hormone (1-34) (PTH; 10(-7) M) or prostaglandin E2 (PGE2; 10(-5) M) caused a significant decrease in calcium content in the diaphyseal and metaphyseal tissues. The decrease in bone calcium content induced by PTH or PGE2 was completely inhibited by beta-cryptoxanthin (10(-8)-10(-6) M). In addition, beta-cryptoxanthin (10(-8)-10(-6) M) completely inhibited the PTH (10(-7) M)- or PGE, (10(-5) M)-induced increase in medium glucose consumption and lactic acid production by diaphyseal and metaphyseal tissues. The inhibitory effect of beta-cryptoxanthin (10(-7) M) on PTH (10(-7) M)- or PGE2 (10(-5) M)-stimulated decrease in the diaphyseal calcium content was significantly prevented in the presence of 10(-3) M vanadate, an inhibitor of protein tyrosine phosphatase. Vanadate (10(-3) M) did not have a significant effect on calcium content and lactic acid production in control bone tissues. The present study demonstrates that beta-cryptoxanthin has a direct stimulatory effect on bone formation and an inhibitory effect on bone resorption in tissue culture in vitro.