The role of calcium in the activation of estrogen receptor-alpha.

Environmental estrogen mimics, including metalloestrogens that can activate estrogen receptor-alpha (ERα), may contribute to breast cancer risk. However, the underlying mechanisms through which these molecular mimics activate the ERα are generally poorly understood. With concern to this important question, we investigated whether intracellular calcium may mediate the cross-talk between signaling pathways that activate ERα and the ligand-binding domain of ERα. MCF-7 cells treated with EGF, ATP, extracellular calcium, or caffeine to increase intracellular calcium triggered a rapid recruitment of ERα to estrogen-responsive promoters and stimulated expression of estrogen-responsive genes including pS2, complement C3, and progesterone receptor. Induction was blocked by an antiestrogen but also by the chelation of intracellular calcium. Treatment with extracellular calcium also increased the growth of MCF-7 cells through an ER-dependent mechanism. We found that EGF and extracellular calcium activated the C-terminus of ERα and the activation was blocked by the antiestrogen. Mechanistic investigations identified four potential sites on the solvent-accessible surface of the ERα ligand-binding domain as important for calcium activation of the receptor. Taken together, our results suggest that calcium mediates the cross-talk between ERα-activating signaling pathways and the ligand-binding domain of ERα providing a potential explanation for the ability of certain environmental metalloestrogens to activate the receptor.

Activation of estrogen receptor-alpha by the anion nitrite.

In this study, the ability of nitrite and nitrate to mimic the effects of estradiol on growth and gene expression was measured in the human breast cancer cell line MCF-7. Similar to estradiol, treatment of MCF-7 cells with either 1 mumol/L nitrite or 1 mumol/L nitrate resulted in approximately 4-fold increase in cell growth and 2.3-fold to 3-fold increase in progesterone receptor (PgR), pS2, and cathepsin D mRNAs that were blocked by the antiestrogen ICI 182,780. The anions also recruited estrogen receptor-alpha (ERalpha) to the pS2 promoter and activated exogenously expressed ERalpha when tested in transient cotransfection assays. To determine whether nitrite or nitrate was the active anion, diphenyleneiodonium was used to inhibit oxidation/reduction reactions in the cell. The ability of diphenyleneiodonium to block the effects of nitrate, but not nitrite, on the induction of PgR mRNA and the activation of exogenously expressed ERalpha suggests that nitrite is the active anion. Concentrations of nitrite, as low as 100 nmol/L, induced a significant increase in PgR mRNA, suggesting that physiologically and environmentally relevant doses of the anion activate ERalpha. Nitrite activated the chimeric receptor Gal-ER containing the DNA-binding domain of GAL-4 and the ligand-binding domain of ERalpha and blocked the binding of estradiol to the receptor, suggesting that the anion activates ERalpha through the ligand-binding domain. Mutational analysis identified the amino acids Cys381, His516, Lys520, Lys529, Asn532, and His547 as important for nitrite activation of the receptor.