Membrane estrogen receptor-α-mediated nongenomic actions of phytoestrogens in GH3/B6/F10 pituitary tumor cells

To investigate the activation of different nongenomic pathways, and determine the involvement of mERα, we measured prolactin (PRL) release by radio-immunoassay, MAPK activations (ERK1/2 and JNK1/2/3) via a quantitative plate immunoassay, and intracellular [Ca2+] by Fura-2 fluorescence imaging in cells treated with E2 or four different phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol).Coumesterol and daidzein increased PRL release similar to E2 in GH3/B6/F10 cells, while genistein and trans-resveratrol had no effect. All of these compounds except genistein activated ERK1/2 signaling at 1–10 picomolar concentrations; JNK 1/2/3 was activated by all compounds at a 100 nanomolar concentration. All compounds also caused rapid Ca2+ uptake, though in unique dose-dependent Ca2+ response patterns for several aspects of this response. A subclone of GH3 cells expressing low levels of mERα (GH3/B6/D9) did not respond to any phytoestrogen treatments for any of these responses, suggesting that these nongenomic effects were mediated via mERα.Phytoestrogens were much more potent in mediating these nongenomic responses (activation of MAPKs, PRL release, and increased intracellular [Ca2+]) via mERα than was previously reported for genomic responses. The unique non-monotonic dose responses and variant signaling patterns caused by E2 and all tested phytoestrogens suggest that complex and multiple signaling pathways or binding partners could be involved. By activating these different nongenomic signaling pathways, phytoestrogens could have significant physiological consequences for pituitary cell functions.Binding of 17β-estradiol (E2) to estrogen receptors (ERs) elicits important changes in growth, differentiation, maturation, and other functions of target tissues [1-4]. In the pituitary, E2 and its pharmaceutical mimic diethylstilbesterol (DES) have many effects, including proliferation of several pituitary cell types, formation of new blood vessels [5], regulatio