Negative Regulation of RANKL-induced Osteoclastic Differentiation in RAW264.7 Cells by Estrogen and Phytoestrogens

Abstract

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␣ but not ER␤. In contrast to RANKL, which decreased ER␣ expression and induced osteoclast differentiation, 10 nM estradiol, 3 M genistein, or 3 M daidzein all increased ER␣ 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␣ expression by more than ϳ50%. 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 IB degradation and NF-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.Estrogen is a key regulator of skeletal mass. Most estrogen effects are mediated by estrogen receptors (ERs) 1 ␣ and ␤, which are ligand-dependent transcriptional regulators. ER␣, the classical sex-related receptor, has a wide tissue distribution and is found in osteoclasts and osteoclast precursors as well as osteoblasts; ER␤ is primarily found in epithelial and mesenchymal tissues, including the mesenchymal stem cell-derived bone-forming cells, osteoblasts. There are also estrogen-binding proteins that are not transcription factors, and some estrogenic effects are mediated by membrane receptors linked to calcium (1, 2). The effects of estrogens on skeletal cells are complex, and the mechanism(s) of action are controversial (reviewed in Ref.3). However, transgenic and knock-out mice with varying ER␣ and ER␤ expression established that estrogen effects on bone involve ER␣ and ER␤, which modulate signaling pathways involving Erk and nitric oxide and perform direct transcriptional activity (4). Estrogen responses in mesenchymal stem cell-derived bone-forming cells, osteoblasts, are extensively studied. The effects of estrogens on osteoblasts include regulation of synthesis of the osteoclast differentiation factor RANKL relative to its inhibitor osteoprotegerin (5, 6), which may secondarily regulate osteoclast formation and activity.In contrast, primary estrogen effects on osteoclast differentiation and function are largely uncharacterized. ER␣ is present in osteoclasts (7), whereas ER␤ is...

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