Effect of 17 β-estradiol on the human osteosarcoma cell line MG-63

Abstract

We present evidence that 17 β-estradiol (17 β-E 2) regulates 1,25(OH) 2D 3-induced alkaline phosphatase synthesis and osteocalcin secretion by the human osteosarcoma cell line MG-63. When cells were pre-treated with 17 β-E 2 for 48 h prior to treatment with 1,25(OH) 2D 3 (50 nM) for another 48 h, alkaline phosphatase activity increased by 40% ( P < 0.025) with 2 nM 17 β-E 2 and plateaued at levels of 20 and 200 nM 17 β-E 2. Under the same experimental conditions, osteocalcin secretion was enhanced by 37% ( P < 0.005) with 2 nM E 2. However, 17 β-E 2 had no effect on basal alkaline phosphatase or on osteocalcin secretion. Moreover, simultaneous addition of 17 β-E 2 and 1,25(OH) 2D 3 to cells did not result in any additional effect over l,25(OH) 2D 3 treatment alone. Tamoxifen (10 nM) inhibited 17 β-E 2-induced activities in l,25(OH) 2D 3-treated cells while not affecting control cells. Dexamethasone pretreat-ment (100 nM, 48 h) also stimulated alkaline phosphatase activity in MG-63 cells. Moreover, dexamethasone pretreatment followed by treatment with 17 β-E 2 and l,25(OH) 2D 3 gave an additive effect for alkaline phosphatase activity. 17 α-Estradiol (17 α-E 2), a less active form of estrogen, failed to modify, at low concentrations, control or l,25(OH) 2D 3-induced alkaline phosphatase synthesis and osteocalcin secretion. In fact, a 100–1000-fold higher concentration of 17 α-E 2 was necessary to reproduce the effects of 17 β-E 2 on osteocalcin secretion. The addition of insulin-like growth factor I (IGF-I) for 24 h (1–50 ng/ml) to MG-63 cells did not modify 1,25(OH) 2D 3-induced osteocalcin release from these cells. However, longer incubations with 50 ng/ml IGF-I did reproduce some of the effects observed with 17 β-E 2. Thus, the effects of 17 β-E 2 are probably not related to IGF-I production in MG-63 cells since under these conditions the addition of IGF-I alone should have produced a response at shorter incubation times and in the presence of lower concentrations of IGF-I. Since 17 β-E 2 pretreatment was necessary to observe any effects on l,25(OH) 2D 3-induced activities, we hypothesized that 17 β-E 2 regulated 1,25(OH) 2D 3 receptors in MG-63 cells. When cells were treated with 100 nM 17 β-E 2 for 48 h, the binding affinity was unchanged: 37.3 ± 1.9 versus 35.1 ± 0.4 pM for cells whether treated or not with l7 β-E 2, respectively. In contrast, a significant increase in binding capacity (B max) was noted (15 ± 3.5%; P < 0.025). These results suggest that the estrogen analogue 17 β-E 2 induces the differentiation of MG-63 cells into a more osteoblastic-like phenotype while 17 α-E 2 is without physiological effect. They also suggest that estrogens may regulate bone remodeling by modulating hormonal-induction of proteins involved in bone mineralization. This effect is indirect since it does not modify basal activities, but involves a regulation of 1,25(OH) 2D 3 receptor levels in these MG-63 cells.