A novel signal transduction pathway of estrogen in neuron

实验使用H19-7细胞进行，H19-7细胞是通过转导温度敏感SV 40大T抗原建立的永生化大鼠胎海马神经细胞系。用碱性成纤维细胞生长因子（bFGF）等诱导剂在非允许温度（39℃）下诱导H19-7细胞分化为表达神经元标志物的神经元细胞，RNase保护实验观察到10 μ <-9>M β-雌二醇促进分化的H19-7细胞IGF-1 mRNA的表达，同时加入三苯氧胺可抑制其表达，另一方面，用RNA酶保护实验观察β雌二醇对分化后的H19-7细胞核雌激素受体α（ER α）mRNA表达的影响。结果表明，给予10 μ <-9>M β雌二醇显著增加ER αmRNA的表达，表明雌激素诱导的ER上调可被ICI 182，780抑制，但不被他莫昔芬抑制。 ...更多信息 结果表明，β-雌二醇可引起分化H19-7神经细胞内即刻和瞬时钙离子浓度升高，提示细胞膜上存在雌激素受体。有人认为雌激素的膜受体与G蛋白是配体依赖性结合的。据推测，这样增加的环AMP通过环AMP门控通道引起细胞内Ca离子浓度的增加。因此，我们尝试用Kudo等的方法，以DR 2作为PKA荧光指示剂，观察活的分化的H19-7神经元细胞内蛋白激酶A（PKA）活性的时间过程。我们证实，雌激素诱导的H19-7神经元细胞内PKA活性的立即增加。凝胶迁移实验和基于真实的时间PCR的RNA分析发现，雌激素可诱导H19-7神经细胞AP-1、CREB结合活性和GAP 43 mRNA表达增加，并诱导IGF-1 mRNA表达。同时给予ICI 182，780可抑制雌激素诱导的H19-7神经元细胞IGF-1、GAP-43 mRNA的表达和CREB、AP-1结合活性的增加。少

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