Skeletal effects of estrogen are mediated by opposing actions of classical and nonclassical estrogen receptor pathways

ERα acts either through classical (ERE-mediated) or nonclassical (non-ERE) pathways. The generation of mice carrying a mutation that eliminates classical ERα signaling presents a unique opportunity to study the relative roles of these pathways in bone. This study defines the skeletal phenotype and responses to ovariectomy and estrogen replacement in these mice. Introduction: Estrogen receptor α (ERα) can act either through classical estrogen response elements (EREs) or through non-ERE (nonclassical) pathways. To unravel these in bone, we crossed mice heterozygous for a knock-in mutation abolishing ERE binding (nonclassical ERα knock-in [NERKI]) with heterozygote ERα knockout mice and studied the resulting female ERα^+/+, ERα^+/NERKI, and ERα ^-/NERKI mice. The only ERα present in ERα ^-/NERKI mice is incapable of activating EREs but can signal through nonclassical pathways, whereas ERα^+/NERKI mice may have a less drastic alteration in the balance between classical and nonclassical estrogen signaling pathways. Materials and Methods: BMD was measured using DXA and pQCT at 3 months of age (n = 46-48/genotype). The mice were randomly assigned to sham surgery, ovariectomy, ovariectomy + estradiol (0.25 μg/day), or ovariectomy + estradiol (1.0 μg/day; n = 10-12/group) and restudied 60 days later. Results and Conclusions: At 3 months of age, both the ERα ^+/NERKI and ERα^-/NERKI mice had deficits in cortical, but not in trabecular, bone. Remarkably, changes in cortical bone after ovariectomy and estrogen replacement in ERα^-/NERKI mice were the opposite of those in ERα^+/+ mice. Relative to sham mice, ovariectomized ERα^-/NERKI mice gained more bone (not less, as in ERα^+/+ mice), and estrogen suppressed this increase (whereas augmenting it in ERα^+/+ mice). Estrogen also had opposite effects on bone formation and resorption parameters on endocortical surfaces in ERα^-/NERK1 versus ERα^+/+ mice. Collectively, these data show that alteration of the balance between classical and nonclassical ERα signaling pathways leads to deficits in cortical bone and also represent the first demonstration, in any tissue, that complete loss of classical ERE signaling can lead to paradoxical responses to estrogen. Our findings strongly support the hypothesis that there exists a balance between classical and nonclassical ERα signaling pathways, which, when altered, can result in a markedly aberrant response to estrogen.