Loss of ERE binding activity by estrogen receptor-α alters basal and estrogen-stimulated bone-related gene expression by osteoblastic cells

Estrogen receptor (ER)-alpha can signal either via estrogen response element (ERE)-mediated pathways or via alternate pathways involving protein-protein or membrane signaling. We previously demonstrated that, as compared to wild type (WT) controls, mice expressing a mutant ER-alpha lacking the ability to bind EREs (non-classical estrogen receptor knock-in (NERKI)) display significant impairments in the skeletal response to estrogen. To elucidate the mechanism(s) underlying these in vivo deficits, we generated U2OS cells stably expressing either WT ER-alpha or the NERKI receptor. Compared to cells transfected with the control vector, stable expression of ER-alpha, even in the absence of E2, resulted in an increase in mRNA levels for alkaline phosphatase (AP, by 400%, P< 0.01) and a decrease in mRNA levels for insulin growth factor-I (IGF-I) (by 65%, P< 0.001), with no effects on collagen I (col I) or osteocalcin (OCN) mRNA levels. By contrast, stable expression of the NERKI receptor resulted in the suppression of mRNA levels for AP, col I, OCN, and IGF-I (by 62, 89, 60, and 70%, P< 0.001). While E2 increased mRNA levels of AP, OCN, col I, and IGF-I in ER-alpha cells, E2 effects in the NERKI cells on AP and OCN mRNA levels were attenuated, with a trend for E2 to inhibit col I mRNA levels. In addition, E2 had no effects on IGF-I mRNA levels in NERKI cells. Collectively, these findings indicate that ERE signaling plays a significant role in mediating effects of estrogen on osteoblastic differentiation markers and on IGF-I mRNA levels.