Suppression of ligand-dependent estrogen receptor activity by bone-resorbing cytokines in human osteoblasts

Estrogens are important for bone homeostasis and are classified as antiresorptive agents. One of the mechanisms for this effect is the inhibition of cytokine-induced bone resorption, which is mediated in part through an interaction between the estrogen receptor (ER) and nuclear factor (NF)-kappa B in osteoblasts. We present evidence that bone-resorbing cytokines that activate NF-kappa B conversely inhibit ligand-dependent ER activity in the conditionally immortalized human osteoblast cell line, HOB-03-CE6. Treatment of HOB-03-CE6 cells with 17 beta-estradiol (17 beta-E-2) up-regulated reporter gene activity [ERE-thymidine kinase (tk)-luciferase] 3- to 5-fold in a dose-dependent manner (EC50 = 1.0 pM). However, cotreatment of the cells with 17 beta-E-2 and increasing concentrations of either tumor necrosis factor-alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha), or IL-1 beta completely suppressed ERE-tk-luciferase activity in a dose-dependent manner (IC50 = 0.05-5.0 pM). On the other hand, treatment of the cells with growth factors either up-regulated or had no effect on ERF-tk-luciferase expression. Neither TNF alpha, IL-1 alpha, nor IL-1 beta treatment affected basal reporter gene activity in the cells, and the TNF alpha effect was reversed by a neutralizing antibody to the cytokine. TNF alpha treatment also suppressed ligand-dependent ER activity in MCF-7 human breast cancer cells, but not in Chinese hamster ovary cells that overexpressed human ER alpha, even though both cell lines responded to the cytokine as measured by the up-regulation of NF kappa B-tk-luciferase activity. TNF alpha treatment did not affect the steady state levels of either ER alpha or ERP messenger RNA expression by the HOB-03-CE6 cells, nor did it reduce [I-125]17 beta-E-2 binding. Moreover, TNF alpha treatment only weakly inhibited ligand-dependent glucocorticoid receptor activity in the HOB-03-CE6 cells. Bone-resorbing cytokines, which do not signal through the NF-kappa B pathway, did not suppress ERE-tk-luciferase activity in HOB-03-CE6 cells. Treatment of the cells with 17 beta-E-2 partially suppressed the activation of NF-kappa B by TNF alpha, but did not block cytokine-induced IL-6 secretion. Finally, cotreatment of HOB-03-CE6 cells with an antisense oligonucleotide to NF-KB p50 partially reversed the suppression of ERE-tk-luciferase activity by TNF alpha. In summary, these data provide evidence for a potent feedback inhibition of estrogen action in human osteoblasts that is at least partly mediated by the activation of NF-kappa B.