Elsevier

Neuroscience

Volume 238, 15 May 2013, Pages 345-360
Neuroscience

The key involvement of estrogen receptor β and G-protein-coupled receptor 30 in the neuroprotective action of daidzein

https://doi.org/10.1016/j.neuroscience.2013.02.005Get rights and content

Abstract

Phytoestrogens have received considerable attention because they provide an array of beneficial effects, such as neuroprotection. To better understand the molecular and functional link between phytoestrogens and classical as well as membrane estrogen receptors (ERs), we investigated the effect of daidzein on the glutamate-mediated apoptotic pathway. Our study demonstrated that daidzein (0.1–10 μM) inhibited the pro-apoptotic and neurotoxic effects caused by glutamate treatment. Hippocampal, neocortical and cerebellar tissues responded to the inhibitory action of daidzein on glutamate-activated caspase-3 and lactate dehydrogenase (LDH) release in a similar manner. Biochemical data were supported at the cellular level by Hoechst 33342 and calcein AM staining. The sensitivity of neuronal cells to daidzein-mediated protection was most prominent in hippocampal cultures at an early stage of development 7th day in vitro. A selective estrogen receptor β (ERβ) antagonist, 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5,-a]pyrimidin-3-yl]phenol (PHTPP), and a selective G-protein-coupled receptor 30 (GPR30) antagonist, 3aS,4R,9bR)-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline (G15), reversed the daidzein-mediated inhibition of glutamate-induced loss of membrane mitochondrial potential, caspase-3 activity, and LDH release. A selective ERα antagonist, methyl-piperidino-pyrazole (MPP), did not influence any anti-apoptotic effect of daidzein. However, a high-affinity estrogen receptor antagonist, 7α,17β-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI) 182,780, and a selective GPR30 agonist, (±)-1-[(3aR,4S,9bS)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone (G1), intensified the protective action of daidzein against glutamate-induced loss of membrane mitochondrial potential and LDH release. In siRNA ERβ- and siRNA GPR30-transfected cells, daidzein did not inhibit the glutamate-induced effects. Twenty-four hour exposure to glutamate did not affect the cellular distribution of ERβ and GPR30, but caused greater than 100% increase in the levels of the receptors. Co-treatment with daidzein decreased the level of ERβ without significant changing of the GPR30 protein level.

Here, we elucidated neuroprotective effects of daidzein at low micromolar concentrations and demonstrated that the phytoestrogens may exert their effects through novel extranuclear GPR30 and the classical transcriptionally acting ERβ. These studies uncover key roles of the ERβ and GPR30 intracellular signaling pathways in mediating the anti-apoptotic action of daidzein and may provide insight into new strategies to treat or prevent neural degeneration.

Highlights

► This study has demonstrated a key role of ERβ and GPR30 in the neuroprotective action of daidzein. ► Daidzein inhibited glutamate-induced apoptosis and neurotoxicity. ► Daidzein inhibited glutamate-stimulated protein expression of ERβ, without a significant change of GPR30. ► Involvement of ERβ and GPR30 in daidzein neuroprotection was verified with selective ligands and siRNAs. ► Biochemical data were supported by ERβ- and GPR30-specific immunofluorescence and confocal microscopy.

Introduction

Phytoestrogens are present in numerous dietary supplements, and their global consumption is rapidly increasing. Phytoestrogens have received considerable attention because they provide an array of beneficial effects, including preventive or therapeutic actions in carcinogenesis, atherosclerosis, and osteoporosis. Phytoestrogens are also thought to reduce menopausal symptoms. The soy-derived isoflavone daidzein inhibits the cell growth of many cancer cell lines, such as HeLa, MCF-7, or human thyroid carcinoma cells, by inducing the mitochondrial apoptotic pathway (Jin et al., 2010, Zhang et al., 2010, Reiter et al., 2011, Xiao et al., 2011, Somjen et al., 2012). There is also a line of evidence pointing to neuroprotective actions of daidzein against excitotoxicity, β-amyloid peptide1–42, stroke-like injury, and endoplasmic reticulum stress-mediated neuronal degeneration (Occhiuto et al., 2008, Schreihofer and Redmond, 2009, Zhao et al., 2009). Recent studies have demonstrated that daidzein induces axonal outgrowth in hippocampal neurons and improves their viability and proliferation via the BDNF-Trk pathway (Wang et al., 2008, Pan et al., 2012). Daidzein has also been found to inhibit glycogen synthase kinase-3β during homocysteine-induced degeneration of SH-SY5Y cells, and daidzein reduces cytochrome c release and reactive oxygen species formation during low potassium-induced apoptosis of cerebellar granule cells (Atlante et al., 2010, Park et al., 2010). Despite these data, the mechanisms by which daidzein imparts apoptotic processes in neuronal cells are poorly understood and require further investigation.

Phytoestrogens such as daidzein produce both estrogenic and antiestrogenic effects, depending on the status of endogenous estrogen and the distribution of estrogen receptors. Most of the biological effects of estrogen are mediated by the classical estrogen receptors α and β (ERα and ERβ, respectively), which act as hormone-inducible transcription factors by binding to the estrogen-responsive element (ERE) in the promoter region of target genes (Hall et al., 2001). Recent studies have shown that G-protein-coupled receptor 30 (GPR30), also known as G-protein-coupled ER 1 (GPER) mediates non-genomic estradiol signaling in a variety of tissues, including estrogen-responsive cancer cells (e.g. MCF-7), where it primarily activates the epidermal growth factor receptor (EGFR) transduction pathway (Albanito et al., 2007, Prossnitz and Barton, 2011). GPR30 is a seven-transmembrane G protein-coupled receptor identified as a membrane-bound estrogen receptor in several cell lines and in brain and peripheral tissues (Hazell et al., 2009). GPR30 specifically binds estradiol with high nanomolar affinity, leading to the activation of adenylyl cyclase via GαS protein followed by intracellular calcium mobilization and the accumulation of phosphatidylinositol-3,4,5-triphosphate in the nucleus. GPR30-dependent activation of extracellular-signal-regulated kinase 1/2 (ERK1/2) signaling via the Gβγ pathway has been observed in human breast cancer and liver cell lines (Revankar et al., 2005, Thomas et al., 2005). GPR30 is expressed in many regions of the brain, with particularly high expression in the hypothalamus, hippocampus, cortex and striatum (Brailiou et al., 2007). Recently, it has been indicated that GPR30 mediates the neuroprotective effects of estradiol in murine hippocampal and cortical cells (Gingerich et al., 2010, Liu et al., 2012). Little is known, however, about GPR30 involvement in the neuroprotective and potentially anti-apoptotic actions of phytoestrogens.

Our previous study demonstrated strong neuroprotective effects of genistein at low micromolar concentrations and provided evidence for involvement of the estrogen receptor/GSK-3β intracellular signaling pathway in the anti-apoptotic action of the phytoestrogen (Kajta et al., 2007). Thus, in this study, we aimed to evaluate mechanisms by which daidzein acts on neuronal cells during apoptosis and neurotoxicity. Daidzein is known to bind classical estrogen receptors with lower affinities than genistein (Muthyala et al., 2004), but phytoestrogen binding affinity to extranuclear GPR30 remains unknown. Recent identification of the first GPR30-selective antagonist (G15) and agonist (G1) provided pharmacological tools to differentiate between the functions of the classical ERs and the membrane GPR30 in mediating the estrogen pathway. In addition, a high-affinity estrogen receptor antagonist, ICI 182,780, has been known since recently to possess properties of a GPR30 agonist (Thomas et al., 2005). Therefore, in the present study we evaluated the effects of daidzein on glutamate-induced apoptotic and neurotoxic parameters, such as loss of mitochondrial potential, activation of caspase-3, and lactate dehydrogenase (LDH) release. Biochemical data were complemented with cellular analyses, including Hoechst 33342 and calcein AM staining, to visualize apoptotic DNA-fragmentation and to assess cell survival, respectively. To assess whether the effects of daidzein were age- and tissue-dependent, we studied hippocampal, neocortical, and cerebellar tissues from 7- and 12-day-old cultures. The involvement of classical estrogen receptors was verified using an estrogen receptor antagonist exhibiting high affinity to ERα and ERβ, a selective ERα antagonist, and a selective ERβ antagonist and agonist. The involvement of GPR30 was analyzed using a specific antagonist and agonist. In addition to ligands, siRNAs toward ERβ and GPR30 were employed to confirm a crucial role of the receptors in the neuroprotective action of daidzein. The levels of these two receptors were measured with enzyme-linked immunosorbent assays (ELISAs), and their cellular distributions were demonstrated with confocal microscopy.

Section snippets

Materials

Ac-DEVD-pNA (N-acetyl-asp-glu-val-asp p-nitro-anilide), CaCl2, DMSO (dimethyl sulfoxide), EGTA, glucose, glutamic acid, HEPES, KCl, K-gluconate, MgCl2, NaCl, NaOH, poly-ornithine, sodium ATP, and sodium GTP were purchased from Sigma–Aldrich (St. Louis, MO, USA). Alexa 488-conjugated anti-goat IgG, calcein AM, and Hoechst 33342 were purchased from Molecular Probes (Eugene, OR, USA). The B27 and neurobasal media were obtained from Gibco (Grand Island, NY, USA), and Bradford reagent was from

Effects of daidzein on glutamate-induced caspase-3 activity and LDH release in hippocampal cultures on seven DIV

In hippocampal cultures exposed to 1 mM glutamate on seven DIV, the activity of caspase-3 increased by 47% at 6 h, and it was enhanced by 38% over the control until the end of the experiment (Fig. 1, panel a). LDH release increased with the duration of glutamate treatment. The LDH release was elevated to 186% and 250% of the control values, at 6 and 24 h, respectively (Fig. 1, panel b).

In the presence of daidzein (0.1–10 μM) the activity of caspase-3 was diminished by 18–40% compared to the

Discussion

This study demonstrated that treatment with daidzein at a dose range of 0.1–10 μM inhibited the apoptotic and neurotoxic effects of glutamate treatment. In our previous study (Kajta et al., 2007) we used another phytoestrogen genistein in concentrations of 0.01, 0.1, 1, and 10 μM. The lowest concentration of genistein inhibited the effects of glutamate at 3, 6 and 24 h in hippocampal cultures, but it was not effective in inhibiting all the glutamate-induced effects in neocortical and cerebellar

Acknowledgments

The authors gratefully acknowledge financial support from the Polish Ministry of Education and Science, Grant No. N N401 572138. We also wish to thank professor Elzbieta Pyza for her expert suggestions and kindly provided access to confocal microscope LSM 510 META, Axiovert 200 M, ConfoCor 3 (Carl Zeiss MicroImaging GmbH, Jena Germany) in the Department of Cell Biology and Imaging of Institute of Zoology at the Jagiellonian University in Krakow.

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