Takahashi Toshifumi, Ohmichi Masahide, Kawagoe Jun, Ohshima Chika, Doshida Masakazu, Ohta Tsuyoshi, Saitoh Maki, Mori-Abe Akiko, Du Botao, Igarashi Hideki, Takahashi Kazuhiro, Kurachi Hirohisa
Department of Obstetrics and Gynecology, Yamagata University, School of Medicine, 2-2-2 Iidanishi, Yamagata 990-9585, Japan.
Endocrinology. 2005 Sep;146(9):4082-9. doi: 10.1210/en.2005-0302. Epub 2005 Jun 9.
In the present study, to examine the dynamic changes in the localization of nuclear estrogen receptor (ER)alpha induced by growth factors, we used time-lapse confocal microscopy to directly visualized ERalpha fused with green fluorescent protein (GFP-ERalpha) in single living cells treated with epidermal growth factor (EGF) or IGF-I. We observed that 17beta-estradiol (E2) changed the normally diffuse distribution of GFP-ERalpha throughout the nucleoplasm to a hyperspeckled distribution within 10 min. Both EGF and IGF-I also changed the nuclear distribution of GFP-ERalpha, similarly to E2 treatment. However, the time courses of the nuclear redistribution of GFP-ERalpha induced by EGF or IGF-I were different from that induced by E2 treatment. In the EGF-treated cells, the GFP-ERalpha nuclear redistribution was observed at 30 min and reached a maximum at 60 min, whereas in the IGF-I-treated cells, the GFP-ERalpha nuclear redistribution was observed at 60 min and reached a maximum at 90 min. The EGF-induced redistribution of GFP-ERalpha was blocked by pretreatment with a MAPK cascade inhibitor, PD98059, whereas the IGF-I-induced redistribution of GFP-ERalpha was blocked by pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002. Analysis using an activation function-2 domain deletion mutant of GFP-ERalpha showed that the change in the distribution of GFP-ERalpha was not induced by E2, EGF, or IGF-I treatment. These data suggest that MAPK and phosphatidylinositol 3-kinase cascades are involved in the nuclear redistribution of ERalpha by EGF and IGF-I, respectively, and that the activation function-2 domain of ERalpha may be needed for the nuclear redistribution of ERalpha.
在本研究中，为了检测生长因子诱导的细胞核雌激素受体（ER）α定位的动态变化，我们使用延时共聚焦显微镜直接观察在用表皮生长因子（EGF）或胰岛素样生长因子-I（IGF-I）处理的单个活细胞中与绿色荧光蛋白融合的ERα（GFP-ERα）。我们观察到，17β-雌二醇（E2）在10分钟内将GFP-ERα在整个核质中的正常弥散分布改变为超斑点分布。EGF和IGF-I也改变了GFP-ERα的核分布，类似于E2处理。然而，EGF或IGF-I诱导的GFP-ERα核重新分布的时间进程与E2处理诱导的不同。在EGF处理的细胞中，30分钟时观察到GFP-ERα核重新分布，60分钟时达到最大值，而在IGF-I处理的细胞中，60分钟时观察到GFP-ERα核重新分布，90分钟时达到最大值。EGF诱导的GFP-ERα重新分布被丝裂原活化蛋白激酶（MAPK）级联抑制剂PD98059预处理所阻断，而IGF-I诱导的GFP-ERα重新分布被磷脂酰肌醇3-激酶抑制剂LY294002预处理所阻断。使用GFP-ERα的激活功能-2结构域缺失突变体进行分析表明，E2、EGF或IGF-I处理未诱导GFP-ERα分布的变化。这些数据表明，MAPK和磷脂酰肌醇3-激酶级联分别参与EGF和IGF-I诱导的ERα核重新分布，并且ERα的激活功能-2结构域可能是ERα核重新分布所必需的。
