Molecular Mechanism of the Inhibition of Estradiol-Induced Endometrial Epithelial Cell Proliferation by Clomiphene Citrate
Molecular Mechanism of the Inhibition of Estradiol-Induced Endometrial Epithelial Cell Proliferation by Clomiphene Citrate
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DOI：
10.1210/en.2009-0721
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发表时间：
2010-01-01
期刊：
影响因子：
4.8
通讯作者：
Kurachi, Hirohisa
中科院分区：
文献类型：
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作者：
Amita, Mitsuyoshi;Takahashi, Toshifumi;Kurachi, Hirohisa
我们利用石川细胞系和EM-E6/E7/hTERT细胞系研究了枸橼酸氯米芬（CC）在子宫内膜中的抗雌激素作用的分子机制。CC或ICI182780抑制17 β -雌二醇（E2）诱导的子宫内膜细胞增殖和雌激素反应元件（ERE）基因的转录激活。我们使用绿色荧光蛋白（GFP）标记的ER α在单个活细胞中直接可视化配体与雌激素受体(ER) α的相互作用。E2在5分钟内改变GFP-ER α的核定位为点状分布，而CC或ICI182780使GFP-ER α的动员比E2更慢、更少。CC或ICI182780预处理部分阻止e2诱导的GFP-ER α核重分布。光漂白后的荧光恢复显示，E2处理的GFP-ER α迁移率比CC或ICI182780处理的更快。由于向内质网募集共激活因子对于内质网依赖性转录至关重要，我们研究了内质网α和类固醇受体共激活因子-1 （SRC-1）之间的相互作用。E2显著增加了ER α和SRC-1之间复合物的形成，但CC或ICI182780存在时，通过共免疫沉淀阻止了复合物的形成。此外，根据免疫荧光分析，在CC或ICI182780存在的情况下，e2诱导的GFP-ER α和SRC-1的共定位被阻止。我们还观察到，使用SRC-1的小干扰RNA减少SRC-1导致e2诱导的细胞增殖和ERE基因的转录激活受到抑制。综上所述，这些结果表明CC可能通过抑制SRC-1向ER α的募集来抑制e2诱导的子宫内膜上皮细胞增殖和ERE反激活。（内分泌学15:394-405,2010）
We examined the molecular mechanisms of the antiestrogenic effects of clomiphene citrate (CC) in the endometrium using two types of cell lines, Ishikawa and EM-E6/E7/hTERT cells. CC or ICI182780 inhibited 17 beta-estradiol (E2)-induced endometrial cell proliferation and transcriptional activation of the estrogen response element (ERE) gene. We directly visualized the ligand-estrogen receptor (ER)alpha interaction using green fluorescent protein (GFP)-tagged ER alpha in a single living cell. Whereas E2 changed the nuclear localization of GFP-ER alpha to a punctate distribution within 5 min, CC or ICI182780 changed the slower and less mobilization of GFP-ER alpha compared with E2. Pretreatment with CC or ICI182780 partly prevented the E2-induced nuclear redistribution of GFP-ER alpha. Fluorescence recovery after photobleaching revealed that GFP-ER alpha mobility treated with E2 was more rapid than that treated by CC or ICI182780. As coactivator recruitment to the ER is essential for ER-dependent transcription, we examined the interaction between ER alpha and steroid receptor coactivator-1 (SRC-1). The complex formation between ER alpha and SRC-1 was significantly increased by E2 but was prevented in the presence of CC or ICI182780 by coimmunoprecipitation. Moreover, the E2-induced colocalization of GFP-ER alpha and SRC-1 was prevented in the presence of CC or ICI182780 according to an immunofluorescence assay. We also observed that the reduction of SRC-1 using small interfering RNA for SRC-1 resulted in the inhibition of E2-induced cell proliferation and transcriptional activation of the ERE gene. Collectively, these results suggest that CC may inhibit E2-induced endometrial epithelial cell proliferation and ERE transactivation by inhibiting the recruitment of SRC-1 to ER alpha. (Endocrinology 151: 394-405, 2010)