Full Activation of Estrogen Receptor α Activation Function-1 Induces Proliferation of Breast Cancer Cells
2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês
10.1074/jbc.m301031200
ISSN1083-351X
AutoresTetsuo Fujita, Yôko Kobayashi, Osamu Wada, Yukiyo Tateishi, Lina Kitada, Yasuji Yamamoto, Hisashige Takashima, Akiko Murayama, Tetsu Yano, Tadashi Baba, Shigeaki Kato, Yoh-ichi Kawabe, Junn Yanagisawa,
Tópico(s)Inflammatory mediators and NSAID effects
ResumoThe effects of estrogen and anti-estrogen are mediated through the estrogen receptors (ER) α and β, which function as ligand-induced transcriptional factors. Recently, one of the phthalate esters, n-butylbenzyl phthalate (BBP), has been shown to induce estrogen receptor-mediated responses. By using the truncated types of ER mutants, we revealed that activation function-1 (AF-1) activity was necessary for the BBP-dependent transactivation function of ERα. AF-1 is also known to be responsible for the partial agonistic activity of tamoxifen. Whereas tamoxifen exhibits an anti-estrogenic effect on proliferation of the MCF-7 breast cancer cell line, BBP showed an estrogenic effect on MCF-7 to stimulate proliferation. In vivo and in vitro binding assays revealed that whereas 4-hydroxytamoxifen (OHT) induced binding of ERα to both an AF-1 coactivator complex (p68/p72 and p300) and corepressor complexes (N-CoR/SMRT), BBP selectively enhanced the binding to the AF-1 coactivators. We also showed that the transcriptional activity of OHT-bound ERα was modulated by the ratio between the AF-1 coactivator and corepressor complexes. Expression of a dominant-negative type of N-CoR inhibited the interaction between OHT-bound ERα and N-CoR/SMRT and enhanced the transcriptional activity of OHT-bound ERα. Furthermore, the cell growth of MCF-7 stably expressing the dominant-negative type of N-CoR was enhanced by the addition of OHT. These results indicated that fully activated AF-1 induces the stimulation of breast cancer growth and that the ratio between AF-1 coactivators and corepressors plays a key role to prevent proliferation of tumor by tamoxifen. The effects of estrogen and anti-estrogen are mediated through the estrogen receptors (ER) α and β, which function as ligand-induced transcriptional factors. Recently, one of the phthalate esters, n-butylbenzyl phthalate (BBP), has been shown to induce estrogen receptor-mediated responses. By using the truncated types of ER mutants, we revealed that activation function-1 (AF-1) activity was necessary for the BBP-dependent transactivation function of ERα. AF-1 is also known to be responsible for the partial agonistic activity of tamoxifen. Whereas tamoxifen exhibits an anti-estrogenic effect on proliferation of the MCF-7 breast cancer cell line, BBP showed an estrogenic effect on MCF-7 to stimulate proliferation. In vivo and in vitro binding assays revealed that whereas 4-hydroxytamoxifen (OHT) induced binding of ERα to both an AF-1 coactivator complex (p68/p72 and p300) and corepressor complexes (N-CoR/SMRT), BBP selectively enhanced the binding to the AF-1 coactivators. We also showed that the transcriptional activity of OHT-bound ERα was modulated by the ratio between the AF-1 coactivator and corepressor complexes. Expression of a dominant-negative type of N-CoR inhibited the interaction between OHT-bound ERα and N-CoR/SMRT and enhanced the transcriptional activity of OHT-bound ERα. Furthermore, the cell growth of MCF-7 stably expressing the dominant-negative type of N-CoR was enhanced by the addition of OHT. These results indicated that fully activated AF-1 induces the stimulation of breast cancer growth and that the ratio between AF-1 coactivators and corepressors plays a key role to prevent proliferation of tumor by tamoxifen. The effects of estrogens are mediated primarily via estrogen receptor α and β (ERα and -β), 1The abbreviations used are: ER, estrogen receptor; AF, activation function; BBP, n-butylbenzyl phthalate; N-CoR, nuclear receptor core-pressor; SMRT, silencing mediator for retinoid and thyroid hormone receptor; LBD, ligand binding domain; ID, interaction domain; OHT, 4-hydroxytamoxifen; ICI, ICI182,780; TSA, trichostatin A; GST, glutathione S-transferase; TK, thymidine kinase; ERE, estrogen-responsive element; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; BrdUrd, 5-bromouridine 5′-triphosphate; E2, 17β-estradiol; SRC-1, steroid receptor coactivator-1; TRAP, TR-associated proteins; TRRAP, transformation/transcription domain-associated protein.1The abbreviations used are: ER, estrogen receptor; AF, activation function; BBP, n-butylbenzyl phthalate; N-CoR, nuclear receptor core-pressor; SMRT, silencing mediator for retinoid and thyroid hormone receptor; LBD, ligand binding domain; ID, interaction domain; OHT, 4-hydroxytamoxifen; ICI, ICI182,780; TSA, trichostatin A; GST, glutathione S-transferase; TK, thymidine kinase; ERE, estrogen-responsive element; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; BrdUrd, 5-bromouridine 5′-triphosphate; E2, 17β-estradiol; SRC-1, steroid receptor coactivator-1; TRAP, TR-associated proteins; TRRAP, transformation/transcription domain-associated protein. which are members of the nuclear hormone receptor superfamily. 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Suzawa M. Kawabata M. Miyazono K. Yanagisawa J. Kato S. J. Biol. Chem. 1999; 274: 12971-12974Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Nuclear receptor interaction region in TRRAP was inserted into pVP16 vector to generate VP-TRRAP (32Yanagisawa J. Kitagawa H. Yanagida M. Wada O. Ogawa S. Nakagomi M. Oishi H. Yamamoto Y. Nagasawa H. McMahon S.B. Cole M.D. Tora L. Takahashi N. Kato S. Mol. Cell. 2002; 9: 553-562Abstract Full Text PDF PubMed Scopus (145) Google Scholar). C-terminal fragments of N-CoR and SMRT (including the NR interaction domains ID1 and ID2) were inserted into the pVP16 vector (Clontech) to generate VP-N-CoR, VP-SMRT, and pGEX-2T vector to GST-ID1-2 of N-CoR and SMRT and pcDNA3 vector (Invitrogen) for FLAG-N-CoR ID1-2. ERα mutation in amino acid replacement D351Y was introduced into full-length ERα and GAL-DEF plasmid by PCR-based point mutagenesis (Stratagene). Transfection, Luciferase Assay, Mammalian Two-hybrid Assay, and Repression Assay—293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Two days before transfection, medium was changed to phenol red-free DMEM containing 5% charcoal-stripped FBS. Transfection was performed with Lipofectin reagent (Invitrogen) according to the manufacturer's protocol. For luciferase assays, 250 ng of ERE3-tk-luc plasmid was cotransfected with 25 ng of ER expression vector (HEG0/ERGβ0) or mutants. For mammalian two-hybrid assays, 250 ng of ERE3-tk-luc or 17m5-luc vector was cotransfected with 250 ng of HEG0, GAL-DEF, or GAL-DEF(D351Y) in combination with 250 ng of indicated VP16-conjugated constructs and/or p300, p68, N-CoR, or N-CoR ID1-2 plasmids. For repression analysis, 1 μg of MH100-tk-luc vector was cotransfected with 250 ng of GAL-DEF. As a reference plasmid to normalize for transfection efficiency, either 5 ng of pRL-CMV vector (Promega) or 125 ng of pRSVβGAL vector was cotransfected in all experiments. Six hours after transfection, culture medium was replaced with fresh medium containing 0.2% FBS. At this time, either E2 (10 nm), OHT (100 nm), ICI182,780 (100 nm), ethanolic vehicle, or 5 ng/ml trichostatin A (TSA) was added, and cells were incubated for an additional 24 h. Preparation of cell extracts and luciferase assays were performed following the manufacturer's protocol (Promega). β-Galactosidase activity was measured to control the efficiency for each transfection. Individual transfections, each consisting of triplicate wells, were repeated at least three times. For establishing MCF-7 stable transfectant of N-CoR ID1-2, Lipofectin reagent was used for introduce pcDNA-ID1-2, and transfectants were selected by 500 μg/ml G418 (Sigma), and several clones were isolated. GST Pull-down Assay—For GST pull-down assays, bacterially expressed GST fusion proteins or GST bound to glutathione-Sepharose 4B beads (Amersham Biosciences) were incubated on ice with [35S]methionine-labeled proteins expressed by in vitro translation using the TnT-coupled transcription-translation system (Promega). After 1 h of incubation, free proteins were removed by washing the beads 5 times with phosphate-buffered saline containing 10% glycerol and protease inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μm phenylmethylsulfonyl fluoride). Specifically bound proteins were eluted by boiling in SDS sample buffer and analyzed by 6% SDS-PAGE. After electrophoresis, radiolabeled proteins were visualized by autoradiography. Coimmunoprecipitation and Western Blotting—293T cells were transfected with the indicated plasmids, lysed in TNE (10 mm Tris-HCl (pH 7.8), 1% Nonidet P-40, 0.15 m NaCl, 1 mm EDTA, 1 μm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin) buffer, and immunoprecipitated with anti-FLAG M2 monoclonal antibody (Sigma) or anti-ERα (Chemicon). Interacting proteins were separated by 6% SDS-PAGE, transferred onto polyvinylidine difluoride membranes (Millipore), and detected with anti-ERα, anti-p300 (Santa Cruz Biotechnology), anti-FLAG M2, or anti-V5 tag (Invitrogen), and secondary antibodies were conjugated with horseradish peroxidase. For detecting the expression of ERα and N-CoR ID1-2, isolated clones were lysed in TNE, and each lysate was detected by immunoblotting using anti-ER or anti-FLAG and secondary antibodies. Cell Proliferation Analysis—Two days before assay, MDA-MB-231 (ERα-negative) and MCF-7 (ERα-positive) cells were cultured in a 24-well plate in phenol red-free DMEM supplemented with 0.2% charcoal-stripped fetal bovine serum. As experimental medium, either E2 (10 nm), OHT (1 μm), BBP (1 μm), or ethanol vehicle was supplemented. Cells were harvested for the indicated times, and the number of viable cells was counted with hemocytometer. S-phase Entry Analysis—For S-phase entry analysis, NIH3T3 cells were cultured in phenol red-free DMEM supplemented with 5% charcoal-stripped FBS and seeded onto glass coverslips at 60–70% confluence. Transfection with 3 μg of wild-type or mutant ERα expression plasmid was performed by using Perfectin Reagent (Gene Therapy Systems) according to the manufacturer's protocol. Incubation medium was changed after 24 h into phenol red-free DMEM supplemented with 0.2% charcoal-stripped FBS. Cells were left in this medium for 24 h and then cultured with 100 μm 5-bromouridine 5′-triphosphate (BrdUrd) in the presence of either E2 (10 nm), BBP (1 μm), or OHT (1 μm) for an additional 24 h. After incubation, the cells were fixed for immunostaining. BBP Binds Ligand-binding Pocket of ERα and Induces the Transcriptional Activity of AF-1—It has been reported that BBP binds to ERα and enhances the transcriptional activity of ERα. To confirm the binding of BBP to ERs, we performed an in vitro competitive ligand binding assay to investigate the abilities of BBP to compete with E2 for binding to ERα and -β. E2 exhibited an IC50 of ∼1.0 nm to both ERs (Fig. 1B), which is within the range of previously reported IC50 values (40Zacharewski T.R. Meek M.D. Clemons J.H. Wu Z.F. Fielden M.R. Matthews J.B. Toxicol. Sci. 1998; 4
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