Estrogen (17β-estradiol) and genistein, a phytoestrogen, are both endowed with anabolic activities on bone in vivo and stimulate osteoblastic differentiation and mineralization in vitro. However, the mechanisms by which these agents promote osteoblastic differentiation and bone anabolic responses are multifactorial and only partly understood. Recently, the NF-κB signal transduction pathway was implicated as a negative regulator of osteoblastic differentiation and suppression of this pathway leads to osteoblastic differentiation and mineralization in vitro. To examine whether estrogen and/or genistein regulate osteoblast differentiation by modulating the NF-κB pathway, we examined the effect of 17β-estradiol and genistein on basal and TNFα-stimulated NF-κB activity in the preosteoblastic cell line MC3T3. MC3T3 cells were transiently transfected with an NF-κB responsive luciferase reporter and cultured for 24 h with either vehicle, or physiological doses of 17β-estradiol (10−9 to 10−7 M), or genistein (10−6 to 10−5 M). Our data reveal that while 17β-estradiol had no effect on basal NF-κB activity in MC3T3 cells, it significantly antagonized NF-κB activity induced by TNFα (1 or 10 ng/ml). By contrast, genistein (10−6 or 10−5 M) significantly increased NF-κB activity, and showed no antagonistic effects on TNFα-induced NF-κB promoter activity. These studies suggest that the estrogenic compounds, 17β-estradiol and genistein, mediate very different actions on osteoblastic cells. While 17β-estradiol may stimulate bone anabolism, in part, by antagonizing TNFα-induced NF-κB activation, genistein not only fails to prevent cytokine-induced NF-κB activation, but directly promotes NF-κB activation in MC3T3 cells. These data suggest important mechanistic differences in the mechanisms by which 17β-estradiol and genistein promote osteoblast differentiation.

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