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
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
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G 蛋白偶联雌激素受体的激活引起人神经母细胞瘤 SH-SY5Y 细胞中钙的释放和 Mu-阿片受体的磷酸化
DOI：
10.3389/fnins.2019.01351
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发表时间：
2019-12-17
影响因子：
4.3
通讯作者：
Rong, Weifang
中科院分区：
文献类型：
--
作者：
Ding, Xiaowei;Gao, Ting;Rong, Weifang
雌激素除了在生殖和新陈代谢调节方面发挥众所周知的作用外，还对神经系统产生广泛的影响。雌激素通过核受体ERα和ERβ调节基因转录(经典基因组效应)。此外，雌激素还可以引起神经元功能的快速非基因组效应，包括诱导胞浆钙水平的快速变化和迅速脱敏mu型阿片受体(MOR)。负责雌激素快速作用的受体尚不清楚，但最近的证据表明G蛋白偶联雌激素受体(GPER)已被证明在神经系统中广泛表达。在目前的研究中，我们验证了一种假说，即GPER的激活可能介导了快速的钙信号转导，这可能通过神经细胞中的钙依赖蛋白激酶促进MOR的磷酸化。通过定量聚合酶链式反应和免疫细胞化学方法，我们发现人神经母细胞瘤SH-SY5Y细胞内源性表达GPER和MOR。17β-雌二醇(E_2)和G-1(GPER选择性激动剂)激活GPER可引起钙离子浓度依赖地迅速升高，这是由于钙离子释放而不是钙离子内流所致。GPER拮抗剂G15、PLC抑制剂U73122和IP3受体拮抗剂2-APB几乎都取消了对E2或G-1的钙反应。GPER的激活刺激PKC亚型(α和epsilon)移位到质膜，从而导致MOR磷酸化。此外，E2和G-1以PLC/IP3依赖的方式刺激SH-SY5Y细胞c-Fos的表达。综上所述，本研究揭示了神经母细胞瘤细胞中一种新的GPER介导的雌激素信号，其中GPER激活后伴随着快速的钙动员、PKC激活和MOR磷酸化。GPER介导的快速钙信号也可能被传递到细胞核，影响基因转录。这种信号级联可能在脑内阿片类药物信号的调节中发挥重要作用。
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 ER alpha and ER beta 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 mu 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 17 beta-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 (alpha and epsilon) 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.