Negative regulation of RANKL-induced osteoclastic differentiation in RAW264.7 cells by estrogen and phytoestrogens

We studied estrogen effects on osteoclastic differentiation using RAW264.7, a murine monocytic cell line. Differentiation, in response to RANKL and colony-stimulating factor 1, was evaluated while varying estrogen receptor (ER) stimulation by estradiol or nonsteroidal ER agonists was performed. The RAW264.7 cells were found to express ER alpha but not ER beta. In contrast to RANKL, which decreased ER alpha expression and induced osteoclast differentiation, 10 nM estradiol, 3 mu M genistein, or 3 mu M daidzein all increased ER alpha expression, stimulated cell proliferation, and decreased multinucleation, with the effects of estrogen >= daidzein > genistein. However, no estrogen agonist reduced RANKL stimulation of osteoclast differentiation markers or its down-regulation of ER alpha expression by more than similar to 50%. Genistein is also an Src kinase antagonist in vitro, but it did not decrease Src phosphorylation in RAW264.7 cells relative to other estrogen agonists. However, both phytoestrogens and estrogen inhibited RANKL-induced I kappa B degradation and NF-kappa B nuclear localization with the same relative potency as seen in proliferation and differentiation assays. This study demonstrates, for the first time, the direct effects of estrogen on osteoclast precursor differentiation and shows that, in addition to effecting osteoblasts, estrogen may protect bone by reducing osteoclast production. Genistein, which activates ERs selectively, inhibited osteoclastogenesis less effectively than the nonselective phytoestrogen daidzein, which effectively reproduced effects of estrogen.