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KM Wiren, A Chapman Evans, and XW Zhang

Significant levels of estrogen and androgens circulate in men and women, and both play an important role in bone metabolism. While it is well established that either estrogen or androgen replacement therapy is effective at ameliorating bone loss associated with hypogonadism, recent evidence nevertheless suggests that estrogen and androgens have distinct molecular actions on the skeleton. In this study, we have employed normal rat calvarial osteoblast cultures to characterize relative expression profiles of estrogen (ERalpha and ERbeta) and androgen receptors (AR) during osteoblast differentiation. Normal osteoblast cultures can proceed through in vitro differentiation with distinct stages of proliferation, matrix maturation and mineralization in the appropriate differentiation medium containing ascorbic acid. Expression profiles of AR, ERalpha and ERbeta in primary cultures during osteoblast differentiation were characterized both by semi-quantitative relative RT-PCR and by Western analysis. In cultures induced to differentiate by growth in the presence of ascorbic acid, the expression profile for each receptor was unique during the course of differentiation. ERalpha levels were elevated during matrix maturation and then declined during mineralization. ERbeta expression was relatively constant throughout differentiation, exhibiting more constitutive expression. In contrast, AR levels were lowest during proliferation, and then increased throughout differentiation with highest levels in the most mature mineralizing cultures. Since steroid hormone action is generally mediated by specific cognate receptors, these results suggest that androgen actions may target cells during the mineralization stage of osteoblast differentiation, while estrogen action through either receptor isoform is more likely to affect osteoblasts earlier during matrix maturation. Interestingly, sex steroid receptor expression profiles did not exhibit the same patterns of regulation if osteoblast cultures were grown without ascorbic acid in medium that did not support extracellular matrix deposition. Thus, sex steroids may distinctly influence skeletal health by differential modulation of function during osteoblast differentiation.