Dose-dependent estrogen inhibits osteo-adipogenic transdifferentiation of MC3T3-E1 cells likely via modulating canonical Wnt singling pathway.

<p>After 14 days’ osteogenesis, two groups of MC3T3-E1 cells were divided. The first group was cultured in adipogenic cocktail medium accompanied with 10⁻⁷ M of 17beta-estradiol for 14 days and 50 ng/ml DKK-1 was added to block canonical Wnt signaling pathway simultaneously. The second group was cultured in adipogenic cocktail medium accompanied with 10⁻⁷ M of 17beta-estradiol and ICI together for 14 days and 50 ng/ml Wnt-3a was added to activate canonical Wnt signaling pathway simultaneously. O14+A14∶14 days’ adipogenesis after 14 days’ osteogenesis; O14+A14+E₂∶14 days’ adipogenesis accompanied with 10⁻⁷ M of 17beta-estradiol after 14 days’ osteogenesis; O14+A14+E₂+ICI: 14 days’ adipogenesis accompanied with 10⁻⁷ M of 17beta-estradiol and ICI after 14 days’ osteogenesis. O14+A14+E₂+ICI+Wnt-3a: 14 days’ adipogenesis accompanied with 10⁻⁷ M of 17beta-estradiol, ICI and 50 ng/ml Wnt-3a after 14 days’ osteogenesis; O14+A14+E₂+DKK-1∶14 days adipogenesis accompanied with 10⁻⁷ M 17beta-estradiol and 50 ng/ml DKK1 after 14 days’ osteogenesis. A: ALP staining of osteo-adipogenic transdifferentiation of MC3T3-E1 cells; B: Alizarin red staining of osteo-adipogenic transdifferentiation of MC3T3-E1 cells; C: Oil red staining of osteo-adipogenic transdifferentiation of MC3T3-E1 cells; D: ALP activity of osteo-adipogenic transdifferentiation of MC3T3-E1 cells. The control value for ALP activity was 0.687±0.014 unit/mg protein; E: The mineralization of osteo-adipogenic transdifferentiation of MC3T3-E1 cells. The control value for mineralization was 0.795±0.020 OD; F: Quantification of lipid; G: Effect of Wnts and 17beta-estradiol on the osteogenic mRNA expression; H: Effect of Wnts and 17beta-estradiol on the adipogenic mRNA expression; I: Effect of Wnts and 17beta-estradiol on the canonical Wnt signaling mRNA expression. Expression of each target gene was calculated as a relative expression to beta-actin and represented as normalized fold expression. Data are represented as mean±SD of 3 independent experiments. *P<0.05 and **P<0.01.