Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways
Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways
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DOI：
10.1016/j.steroids.2003.12.003
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发表时间：
2004-03-01
期刊：
影响因子：
2.7
通讯作者：
Watson, CS
中科院分区：
文献类型：
--
作者：
Bulayeva, NN;Gametchu, B;Watson, CS
最近研究表明，雌二醇（E（2））和其他类固醇可启动质膜的各种细胞内信号级联反应，包括刺激促分裂原活化蛋白激酶（MAPK），特别是细胞外调节激酶（ERK）。在这项研究中，我们证明了E（2）在GH 3/B6/F10垂体瘤细胞系中激活ERK的能力，最初选择该细胞系是因为其膜雌激素受体α（mER α）的表达增强。我们将E2与其细胞不可渗透的类似物（E（2）与过氧化物酶结合，E（2）-P）以及合成雌激素己烯雌酚（DES）进行了比较。时间依赖性ERK活化用一种新的基于固定细胞的免疫测定法定量，该免疫测定法开发用于有效测定多种化合物对多个参数的活化。E（2）和DES均产生双峰反应，但酶磷酸化（激活）和失活的时间过程明显不同; E（2）-P诱导双相ERK激活。E（2）也以浓度依赖性方式磷酸化ERK，有两个最适浓度（10（-14）和10（-8）M）。采用抑制剂来确定通路（ER、EGFR、膜组织、PI 3激酶、Src激酶、Ca（2+））的参与和通路激活的时间;所有这些都影响ERK激活，最早为3-6分钟，表明同时激活，而不是顺序激活。因此，E（2）和其他雌激素化合物可以通过非基因组途径产生快速ERK磷酸化，使用一种以上的信号产生途径。(C)2004年爱思唯尔公司All rights reserved.
Estradiol (E(2)) and other steroids have recently been shown to initiate various intracellular signaling cascades from the plasma membrane, including those stimulating mitogen-activated protein kinases (MAPKs), and particularly extracellular-regulated kinases (ERKs). In this study we demonstrated the ability of E(2) to activate ERKs in the GH3/B6/F 10 pituitary tumor cell line, originally selected for its enhanced expression of membrane estrogen receptor-alpha (mERalpha). We compared E2 to its cell-impermeable analog (E(2) conjugated to peroxidase, E(2)-P), and to the synthetic estrogen diethylstilbestrol (DES). Time-dependent ERK activation was quantified with a novel fixed cell-based immunoassay developed to efficiently determine activation by multiple compounds over multiple parameters. Both E(2) and DES produced bimodal responses, but with distinctly different time courses of enzyme phosphorylation (activation) and inactivation; E(2)-P induced a monophasic ERK activation. E(2) also phosphorylated ERKs in concentration-dependent manner with two concentration optima (10(-14) and 10(-8) M). Inhibitors were employed to determine pathway (ER, EGFR, membrane organization, PI3 kinase, Src kinase, Ca(2+)) involvement and timing of pathway activations; all affected ERK activation as early as 3-6 min, suggesting simultaneous, not sequential, activation. Therefore, E(2) and other estrogenic compounds can produce rapid ERK phosphorylations via nongenomic pathways, using more than one pathway for signal generation. (C) 2004 Elsevier Inc. All rights reserved.