A novel signal transduction pathway of estrogen in neuron

神经元中雌激素信号转导的新途径

基本信息

批准号：
11694328
负责人：
KITO Shozo
金额：
$ 4.86万
依托单位：
Hyogo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11694328/
关键词：
hippocampus H19-7 cells IGF-1 estrogen tamoxifen ICI182,780 cAMP Ca ion 不死化エストロゲン ICI182、780

项目摘要

Experiments were done with use of the H19-7 cell, the immortalized rat fetal hippocampal neural cell line which was established by transduction of a temperature-sensitive SV 40 large T antigen. The H19-7 cell was differentiated into neurons expressing neuronal markers at the nonpermissive temperature (39℃) in defined medium by several agents, including basic fibroblast growth factor (bFGF).It was observed by RNase Protection assay that 10^<-9>M βestradiol increased IGF-1 mRNA expression in the differentiated H19-7 neuronal cell with was inhibited by simultaneous addition of tamoxifen, a partial antagonist of estrogen.On the other hand, the effects of βestradiol on nuclear estrogen receptorα (ER α) mRNA expression in the differentiated H19-7 cell was observed by RNase Protection Assay. As the result, administration of 10^<-9>M βestradiol significantly increased expression of ER αmRNA showing estrogen-induced ER up-regulation that was inhibited by ICI182,780, but not by tamoxifen.We conf … More irmed that βestradiol caused an increase of immediate and transient intracellular Ca ion concentration in the differentiated H19-7 neuronal cell suggesting existence of the membranous estrogen receptor. It has been advocated that the membrane receptor of estrogen is ligand-dependently conjugated with G-protein. It is assumed that thus increased cyclic AMP causes an increase of intracellular Ca ion concentration through the cyclic AMP-gated channel. Accordingly, we tried to observe the time course of intracellular protein kinase A (PKA) activity in the living differentiated H19-7 neuronal cell by the method of Kudo et al in which DR2 was used as fluorescent PKA indicator. We confirmed that estrogen induced an immediate increase of intracellular PKA activity in H19-7 neuronal cells. Moreover, we noticed that estrogen caused increases of AP-1, CREB binding activities and increases of GAP43 mRNA expression in the H19-7 neuronal cells by means of gel shift assay and real time PCR-based RNA analysis, respectively together with induction of IGF-1 mRNA expression. These estrogen-induced responses in H19-7 neuronal cells including expressions of IGF-1, GAP43 mRNAs and increases of CREB, AP-1 binding activities were all inhibited by simultaneous administration of ICI182,780. Less

使用 H19-7 细胞进行实验，该细胞是通过转导温度敏感的 SV 40 大 T 抗原建立的永生化大鼠胎儿海马神经细胞系。H19-7 细胞分化为表达神经元标记的神经元。在规定的培养基中使用多种试剂，包括碱性成纤维细胞生长因子（bFGF），在不允许的温度（39℃）下进行。通过RNase保护测定观察到10^<-9>M β雌二醇增加分化的H19-7神经元细胞中IGF-1 mRNA的表达，同时添加雌激素部分拮抗剂他莫昔芬可抑制β雌二醇对分化的H19-7神经元细胞中IGF-1 mRNA表达的影响。通过RNase保护测定观察分化的H19-7细胞。结果，给予10^-9Mβ雌二醇显着增加ERαmRNA的表达，显示雌激素诱导。 ER 上调被 ICI182,780 抑制，但不被他莫昔芬抑制。我们确信 β 雌二醇导致分化的 H19-7 神经元细胞中立即和短暂的细胞内 Ca 离子浓度增加，表明膜雌激素受体的存在有人认为雌激素的膜受体与 G 蛋白配体依赖性结合，因此认为环 AMP 的增加会导致细胞内 Ca 的增加。因此，我们尝试通过 Kudo 等人的方法观察活体分化的 H19-7 神经元细胞内蛋白激酶 A (PKA) 活性的时间浓度过程，其中 DR2 用作我们证实雌激素诱导 H19-7 神经元细胞内 PKA 活性立即增加，此外，我们注意到雌激素导致 AP-1、CREB 结合活性增加和 GAP43 mRNA 增加。分别通过凝胶位移和基于实时 PCR 的 RNA 测定，以及 IGF-1 mRNA 表达的诱导，研究 H19-7 神经元细胞中的这些雌激素诱导的反应，包括 IGF-1 的表达。、GAP43 mRNA 和 CREB、AP-1 结合活性的增加均被 ICI182,780 的同时施用所抑制。