Department of Obstetrics and Gynecology, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
Endocrinology. 2010 Jan;151(1):394-405. doi: 10.1210/en.2009-0721. Epub 2009 Nov 24.
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 17beta-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.
我们使用两种细胞系，即 Ishikawa 和 EM-E6/E7/hTERT 细胞，研究了克罗米酚（CC）在子宫内膜中的抗雌激素作用的分子机制。CC 或 ICI182780 抑制了 17β-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 ERα在单个活细胞中直接观察配体-雌激素受体（ER）α相互作用。虽然 E2 在 5 分钟内将 GFP-ERα的核定位改变为点状分布，但 CC 或 ICI182780 与 E2 相比，GFP-ERα的移动速度较慢且移动较少。CC 或 ICI182780 的预处理部分阻止了 E2 诱导的 GFP-ERα的核重分布。光漂白后的荧光恢复显示，用 E2 处理的 GFP-ERα的流动性比用 CC 或 ICI182780 处理的流动性更快。由于共激活因子向 ER 的募集对于 ER 依赖性转录至关重要，因此我们研究了 ERα与类固醇受体共激活因子-1（SRC-1）之间的相互作用。用 E2 显著增加了 ERα与 SRC-1 之间的复合物形成，但在存在 CC 或 ICI182780 时通过共免疫沉淀阻止了该复合物形成。此外，根据免疫荧光测定，在存在 CC 或 ICI182780 的情况下，E2 诱导的 GFP-ERα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 降低 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。总的来说，这些结果表明，CC 可能通过抑制 SRC-1 向 ERα的募集来抑制 E2 诱导的子宫内膜上皮细胞增殖和 ERE 反式激活。
