Molecular mechanism of the inhibition of estradiol-induced endometrial epithelial cell proliferation by clomiphene citrate.
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.
Amita
Mitsuyoshi
M
Department of Obstetrics and Gynecology, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
Takahashi
Toshifumi
T
Tsutsumi
Seiji
S
Ohta
Tsuyoshi
T
Takata
Keiko
K
Henmi
Noriko
N
Hara
Shuichiro
S
Igarashi
Hideki
H
Takahashi
Kazuhiro
K
Kurachi
Hirohisa
H
D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D002996:Clomiphene / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects; D004717:Endometrium / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D004958:Estradiol / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000037:antagonists & inhibitors / Q000378:metabolism; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D056920:Nuclear Receptor Coactivator 1 / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology; D020218:Response Elements / Q000187:drug effects / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics; D014018:Tissue Distribution; D015533:Transcriptional Activation / Q000187:drug effects
C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D002996:Clomiphene; D004958:Estradiol; D056920:Nuclear Receptor Coactivator 1