Estrogens attenuate oxidative stress and the differentiation and apoptosis of osteoblasts by DNA-binding-independent actions of the ERα

Estrogens diminish oxidative stress in bone and bone marrow, attenuate the generation of osteoblasts, and decrease the prevalence of mature osteoblast apoptosis. We have searched for the molecular mechanism of these effects using as tools a mouse model bearing an estrogen receptor α (ERα) knock-in mutation that prevents binding to DNA (ERα^NERKI/-) and several osteoblast progenitor cell models expressing the wild-type ERα or the ERα^NERKI/-. We report that the ability of estrogens to diminish the generation of reactive oxygen species, stimulate the activity of glutathione reductase, and decrease the phosphorylation of p66^shc, as well as osteoblastogenesis and osteoblast number and apoptosis, were fully preserved in ERα^NERKI/- mice, indicating that the DNA-binding function of the ERα is dispensable for all these effects. Consistent with the attenuation of osteoblastogenesis in this animal model, 17β-estradiol attenuated bone morphogenetic protein 2 (BMP-2)-induced gene transcription and osteoblast commitment and differentiation in murine and human osteoblastic cell lines. Moreover, 17β-estradiol attenuated BMP-2-induced differentiation of primary cultures of calvaria- or bone marrow-derived osteoblastic cells from ERα^NERKI/- mice as effectively as in cells from wild-type littermates. The inhibitory effect of the hormone on BMP-2 signaling resulted from an ERα-mediated activation of ERKs and the phosphorylation of Smad1 at the linker region of the protein, which leads to proteasomal degradation. These results illustrate that the effects of estrogens on oxidative stress and the birth and death of osteoblasts do not require the binding of ERα to DNA response elements, but instead they result from the activation of cytoplasmic kinases.