Estrogen Receptor β-Selective Agonists Stimulate Calcium Oscillations in Human and Mouse Embryonic Stem Cell-Derived Neurons

Lili Zhang,Isaac Cohen,Tatjana Zogovic-Kapsalis,Dale C Leitman,Renee A Reijo Pera,Heather A Harris,Synthia H Mellon,Mary Tagliaferri,Marcus D Schonemann,Richard I Weiner,Brigitte E Blackman,Xiaoyu Pan,Huibert D Mansvelder

doi:10.1371/journal.pone.0011791

Abstract

Estrogens are used extensively to treat hot flashes in menopausal women. Some of the beneficial effects of estrogens in hormone therapy on the brain might be due to nongenomic effects in neurons such as the rapid stimulation of calcium oscillations. Most studies have examined the nongenomic effects of estrogen receptors (ER) in primary neurons or brain slices from the rodent brain. However, these cells can not be maintained continuously in culture because neurons are post-mitotic. Neurons derived from embryonic stem cells could be a potential continuous, cell-based model to study nongenomic actions of estrogens in neurons if they are responsive to estrogens after differentiation. In this study ER-subtype specific estrogens were used to examine the role of ERα and ERβ on calcium oscillations in neurons derived from human (hES) and mouse embryonic stem cells. Unlike the undifferentiated hES cells the differentiated cells expressed neuronal markers, ERβ, but not ERα. The non-selective ER agonist 17β-estradiol (E2) rapidly increased [Ca2+]i oscillations and synchronizations within a few minutes. No change in calcium oscillations was observed with the selective ERα agonist 4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT). In contrast, the selective ERβ agonists, 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN), MF101, and 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3 benzoxazol-5-ol (ERB-041; WAY-202041) stimulated calcium oscillations similar to E2. The ERβ agonists also increased calcium oscillations and phosphorylated PKC, AKT and ERK1/2 in neurons derived from mouse ES cells, which was inhibited by nifedipine demonstrating that ERβ activates L-type voltage gated calcium channels to regulate neuronal activity. Our results demonstrate that ERβ signaling regulates nongenomic pathways in neurons derived from ES cells, and suggest that these cells might be useful to study the nongenomic mechanisms of estrogenic compounds.

Highlights

Estrogens are critical for the development of reproductive organs and regulating reproductive function
Using these selective estrogen receptors (ER) subtype agonists, we show that ERb mediates the stimulation of calcium oscillations in neurons derived from mouse and human embryonic stem (ES) cells, which demonstrates that these neurons might be a useful cell based model to study the role of ERs and the nongenomic mechanisms of estrogens
To convert human embryonic stem (hES) cells into human neuronal progenitor cells by embryonic body (EB) formation, the cells were dissociated into cell aggregates with collagenase IV, replated into low attachment 6-well plates and cultured in hNPC medium, which is a 1:1 mixture of the knockout serum replacer (KSR) medium and neurobasal medium supplemented with N2 supplements, 10 mM retinoic acid (RA, Sigma-Aldrich), 500 ng/ml epidermal growth factor (EGF, R&D systems), 2 mM glutamine, 0.1 mM non-essential amino acids (NEAA) and 100 units/ml penicillin and streptomycin

Summary

Introduction

Estrogens are critical for the development of reproductive organs and regulating reproductive function. In GnRH neurons, estradiol (E2) and DPN rapidly increased the firing rate, whereas PPT did not have any effect on neuronal activity [33] These studies indicate that ERb has an important role in mediating nongenomic actions in neurons, which is consistent with the observations that ERb is present in the hippocampus and hypothalamus [34,35]. We examined the role of ERb on calcium oscillations by comparing the non-selective ER agonist, E2, with the ERb-selective agonists, DPN [30], ERB-041 [17,31], and MF101 [19] Using these selective ER subtype agonists, we show that ERb mediates the stimulation of calcium oscillations in neurons derived from mouse and human ES cells, which demonstrates that these neurons might be a useful cell based model to study the role of ERs and the nongenomic mechanisms of estrogens

Materials and Methods

Results

Experiments

Discussion