Estrogen regulation of human osteoblastic cell proliferation and differentiation

Estrogen (E₂) has been shown to prevent bone loss among postmenopausal women. The molecular mechanism(s) by which this is accomplished is not clear. The discovery of E₂ receptor (ER) in osteoblasts and osteoclasts has implicated these cells as direct targets for E₂. Previous studies on the effects of E₂ on osteoblastic cells in vitro or in organ culture present conflicting results, possibly due to heterogeneity in cell types, stage of differentiation, ER levels, and/or species differences. The effects of E₂ on gene expression during various stages of human osteoblast cell differentiation has not been investigated extensively. In this study we employed a newly developed human fetal osteoblastic cell line (hFOB/ER9) that contains high levels of ER to examine the effects of E₂ on osteoblast proliferation and differentiation. The basal levels and E₂ effects on the expression of various extracellular matrix proteins were also characterized throughout different stages of differentiation. These stages include a proliferative/relatively undifferentiated stage (day 6), a matrix maturation stage (days 10-14), and a mineralization/calcified nodule stage (day 18). During the stage of rapid cell proliferation, E₂ treatment of hFOB/ER9 cells resulted in a dose-dependent decrease in [³H]thymidine incorporation to a maximum of 72% compared to the vehicle control value. Treatment of hFOB/ER9 cells with 10^-9 M E₂ for 48 h resulted in an increase in alkaline phosphatase (AP) activity throughout cell differentiation. The magnitude of AP induction varied from ~200-500%. In contrast, E₂ decreased osteocalcin protein levels to a minimum of 54% compared to the vehicle control value. The steady state messenger RNA levels for AP increased and osteocalcin decreased after E₂ treatment, similar to the responses observed at the protein level. At all stages, there was little or no effect of E₂ on type I collagen protein levels or osteonectin steady state messenger RNA levels. The E₂ responses on hFOB/ER9 cell matrix protein expression and cell proliferation were mediated through the ER, as cultures cotreated with a 100-fold molar excess of a type II anti-E₂ (ICI 182,780) abrogated these effects. These results support the hypothesis that E₂ does have an effect on osteoblastic differentiation by decreasing hFOB/ER9 cell proliferation and differentially regulating extracellular matrix expression.