Activation of the G Protein-Coupled Estrogen Receptor Elicits Store Calcium Release and Phosphorylation of the Mu-Opioid Receptors in the Human Neuroblastoma SH-SY5Y Cells.

Xiaowei Ding,Ping Luo,Xueyin Shi,Po Gao,Li Dong,Guohua Zhang,Ting Gao,Youqiang Meng,Weifang Rong,Yi Zheng

doi:10.3389/fnins.2019.01351

Abstract

Estrogens exert extensive influences on the nervous system besides their well-known roles in regulation of reproduction and metabolism. Estrogens act via the nuclear receptor ERα and ERβ to regulate gene transcription (classical genomic effects). In addition, estrogens are also known to cause rapid non-genomic effects on neuronal functions including inducing fast changes in cytosolic calcium level and rapidly desensitizing the μ type opioid receptor (MOR). The receptors responsible for the rapid actions of estrogens remain uncertain, but recent evidence points to the G protein-coupled estrogen receptor (GPER), which has been shown to be expressed widely in the nervous system. In the current study, we test the hypothesis that activation of GPER may mediate rapid calcium signaling, which may promote phosphorylation of MOR through the calcium-dependent protein kinases in neuronal cells. By qPCR and immunocytochemistry, we found that the human neuroblastoma SH-SY5Y cells endogenously express GPER and MOR. Activation of GPER by 17β-estradiol (E2) and G-1 (GPER selective agonist) evoked a rapid calcium rise in a concentration-dependent manner, which was due to store release rather than calcium entry. The GPER antagonist G15, the PLC inhibitor U73122 and the IP3 receptor inhibitor 2-APB each virtually abolished the calcium responses to E2 or G-1. Activation of GPER stimulated translocation of PKC isoforms (α and ε) to the plasma membrane, which led to MOR phosphorylation. Additionally, E2 and G-1 stimulated c-Fos expression in SH-SY5Y cells in a PLC/IP3-dependent manner. In conclusion, the present study has revealed a novel GPER-mediated estrogenic signaling in neuroblastoma cells in which activation of GPER is followed by rapid calcium mobilization, PKC activation and MOR phosphorylation. GPER-mediated rapid calcium signal may also be transmitted to the nucleus to impact on gene transcription. Such signaling cascade may play important roles in the regulation of opioid signaling in the brain.

Highlights

Estrogens exert an extraordinarily wide spectrum of actions in the human body
G protein-coupled estrogen receptor (GPER) mRNA appeared to be more abundant in SH-SY5Y cells than in N2A and N2AMT cells (Figure 1A)
Whilst it is clear that two nuclear receptors, ERα and ERβ, mediate the slow genomic effects, the receptor(s) responsible for the rapid non-genomic effects in the nervous system remain uncertain

Summary

Introduction

Estrogens exert an extraordinarily wide spectrum of actions in the human body. Besides the well-known roles in regulation of reproduction and metabolism, estrogens exert multifaceted influences on neuronal development and neuronal functions (Jensen and Desombre, 1973). Estrogens are known to act by interacting with two nuclear receptors, ERα and ERβ, which function as ligand-activated transcription factors to regulate gene transcription (Jensen and Desombre, 1973; Kuiper et al, 1996; Mosselman et al, 1996; Paech et al, 1997) In addition to this slow genomic mode of actions which typically develop with latencies ranging from an hour to several days, estrogens directly alter neuronal electrical activity in various brain regions within seconds to minutes, which may underlie the fast effects of estrogens on brain functions such as female reproductive behavior, memory and cognition, neuroprotection and pain (Woolley, 1999; Kelly and Ronnekleiv, 2009; Ogawa et al, 2018). A number of candidate receptors have been proposed, including the classical ERα that may alternatively be bound to the plasma membrane, several ERα variants (ERα-52, ERα-46, and ERα-36), membrane-associated ER-X, the Gαqcoupled mERs and more recently the G protein-coupled estrogen receptor (GPER, known as GPR30) (Rainville et al, 2015)

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