A novel signal transduction pathway of estrogen in neuron

用H19-7细胞进行实验，H19-7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海马神经细胞系。在不允许的温度(39℃)条件下，用碱性成纤维细胞生长因子(BFGFs)等多种因素诱导H19-7细胞分化为表达神经元标志物的神经元。用核糖核酸酶保护实验观察10β雌二醇促进分化的H19-7神经元细胞IGF1mRNA的表达，同时加入雌激素的部分拮抗剂他莫昔芬，观察β雌二醇对分化的H19-7细胞雌激素受体α(ERα)基因表达的影响。结果表明，10β雌二醇显著增加雌激素诱导的ERα的表达，这种上调可被ICI182,780抑制，但不受他莫昔芬的抑制。我们认为…更重要的是，β雌二醇引起分化的H19-7神经细胞内即刻和短暂的细胞内钙离子浓度增加，这表明膜雌激素受体的存在。有观点认为，雌激素的膜受体与G蛋白是配体依赖的偶联。由此推测，环磷酸腺苷的增加通过环磷酸腺苷门控通道引起细胞内钙离子浓度的增加。因此，我们试图用Kudo等人的方法，以DR2作为PKA的荧光指示剂，观察活的分化的H19-7神经元细胞内蛋白激酶A(PKA)活性的时间进程。我们证实雌激素可立即诱导H19-7神经细胞内PKA活性增加。此外，通过凝胶漂移实验和实时定量聚合酶链式反应(Real-Time-PCR)RNA分析，我们发现雌激素诱导H19-7神经细胞AP-1、CREB结合活性增加，GAP43 mRNA表达增加，同时诱导IGF-1mRNA表达。ICI182、780可抑制雌激素诱导的H19-7神经细胞IGF-1、GAP43mRNAs表达及CREB、AP-1结合活性的升高。较少

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