Growth Factors Change Nuclear Distribution of Estrogen Receptor- via Mitogen-Activated Protein Kinase or Phosphatidylinositol 3-Kinase Casca
http://www.100md.com
《内分泌学杂志》
Department of Obstetrics and Gynecology (T.T, M.O., J.K., M.D., T.O., M.S., A.M.-A., B.D., H.I., K.T., H.K.) and Division of Nursing (C.O.), Yamagata University, School of Medicine, Yamagata 990-9585, Japan
Abstract
In the present study, to examine the dynamic changes in the localization of nuclear estrogen receptor (ER) induced by growth factors, we used time-lapse confocal microscopy to directly visualized ER fused with green fluorescent protein (GFP-ER) in single living cells treated with epidermal growth factor (EGF) or IGF-I. We observed that 17-estradiol (E2) changed the normally diffuse distribution of GFP-ER throughout the nucleoplasm to a hyperspeckled distribution within 10 min. Both EGF and IGF-I also changed the nuclear distribution of GFP-ER, similarly to E2 treatment. However, the time courses of the nuclear redistribution of GFP-ER induced by EGF or IGF-I were different from that induced by E2 treatment. In the EGF-treated cells, the GFP-ER nuclear redistribution was observed at 30 min and reached a maximum at 60 min, whereas in the IGF-I-treated cells, the GFP-ER nuclear redistribution was observed at 60 min and reached a maximum at 90 min. The EGF-induced redistribution of GFP-ER was blocked by pretreatment with a MAPK cascade inhibitor, PD98059, whereas the IGF-I-induced redistribution of GFP-ER was blocked by pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002. Analysis using an activation function-2 domain deletion mutant of GFP-ER showed that the change in the distribution of GFP-ER 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 ER by EGF and IGF-I, respectively, and that the activation function-2 domain of ER may be needed for the nuclear redistribution of ER.
Introduction
ESTROGEN PLAYS IMPORTANT physiological roles in the female and male reproductive systems (1, 2), nonreproductive systems (3, 4), and the initiation and proliferation of reproductive cancer cells (5). Most of these diverse effects of estrogen are mediated by binding to and activation of estrogen receptors (ERs) (6, 7, 8). ERs are members of the nuclear receptor superfamily and are ligand-activated transcription factors that regulate the expression of target genes (7, 9, 10). There are two subtypes of ERs, ER and ER, which are synthesized from different genes and are functionally distinct (11, 12, 13). The members of this superfamily display a modular structure with six distinct functional regions termed A–F, which include domains for DNA binding, ligand binding, and transcriptional activation (14, 15, 16). ERs have two inducible transcriptional activation functions (AFs): activation of the C-terminal AF-2 domain, located in the ligand-binding domain, is dependent on agonist binding (14), whereas the amino terminal AF-1 domain can be activated independently of agonists (17). The AF-1 and AF-2 domains activate ERs independently or synergistically, depending on the promoter of the target gene and the cell type (18). On ligand binding, ERs bind to estrogen-responsive elements, which results in activation of the specific ER target genes. Simultaneously, ligand binding to ER alters the ER conformation and thereby affects the interaction of ER with cofactors (7, 19).
Growth factors, such as epidermal growth factor (EGF) and IGF-I, that bind to tyrosine kinase-linked receptors are also important in controlling ER activity in a ligand-independent manner (18, 20). Binding of EGF or IGF-I to its receptor results in activation of the MAPKs, ERK1 and ERK2, which phosphorylate ER at serine 118 in the N-terminal domain and potentiate AF-1 activity (21, 22, 23). Moreover, EGF or IGF-I treatment causes rapid phosphorylation and activation of Akt, which activates ER by phosphorylation of serine 167 in the AF-1 domain in a breast cancer cell line (24, 25). ER phosphorylation induced by both EGF and IGF-I facilitates the association of the coactivators and basal transcriptional factors, which leads to ER-mediated transcriptional activation (26, 27). Moreover, MAPK pathways mediate the phosphorylation and recruitment of steroid receptor coactivator (SRC)-1 (28, 29).
Several recent studies have shown that 17-estradiol (E2) changes the intranuclear distribution of ER fused with green fluorescent protein (GFP-ER) in living cells (30, 31, 32, 33, 34, 35, 36, 37, 38). In the absence of E2, GFP-ER is diffusely distributed throughout the nucleoplasm but is excluded from the nucleolar region. Upon addition of E2, a dramatic redistribution of GFP-ER from a diffuse to a punctate pattern occurs rapidly (10–20 min) within the nucleus (30, 32). Furthermore, the redistribution of GFP-ER correlates with nuclear matrix association and recruitment of ER coactivators, such as SRC-1 (32) or glucocorticoid receptor interacting protein-1 (33). These coactivators have histone acetylase activity (39). Therefore, it is thought that the ligand-dependent redistribution of ER is involved in the transcriptional activation (32, 33, 34, 38).
Whether intranuclear ER redistribution is induced by growth factors, such as EGF or IGF-I, is not yet clear. In the present study, we directly visualized the changes in the nuclear ER distribution using GFP-ER in cells treated with EGF or IGF-I. We found that both EGF and IGF-I induced GFP-ER nuclear redistribution with pattern similar to that induced by E2 but with a different time course than that of E2. Furthermore, we examined whether GFP-ER nuclear redistribution induced by EGF or IGF-I occurred via MAPK or phosphatidylinositol (PI) 3 kinase cascades.
Materials and Methods
Unless otherwise specified, all chemicals were from Sigma Chemical Co. (St. Louis, MO).
Cell culture and transfection
Cos-7 cells were obtained from Cell Resource Center for Biomedical Research Institute of Development, Aging, and Cancer Tohoku University. MCF-7 human breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA). These cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate in the presence of 5% CO2 at 37 C. MCF-7 cells were cultured in DMEM supplemented with 10% charcoal-treated serum for at least 4 d and then subjected to experiments. For all experiments, phenol red-free medium was used. Transient transfection of the cells was performed with LipofectAMINE Plus (Life Technologies, Gaithersburg, MD), according to the manufacturer’s protocol.
Constructs
GFP-ER and GFP-ER ligand binding domain deletion mutant (GFP-mtER) expression vectors were kind gifts from Dr. Michael A. Mancini (Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX) (32). The estrogen response element (ERE)-containing reporter gene, ptk-ERE-luc, was a kind gift from Dr. J. Larry Jameson (Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, IL) (40).
Western blot analysis
MCF-7 cells plated on 35-mm dishes were transfected with the GFP-ER construct and then cultured overnight. They were then washed twice with PBS and lysed in ice-cold buffer composed of 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium orthovanadate, 100 mM NaF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride (41). The lysates were centrifuged at 12,000 x g at 4 C for 15 min, and the protein concentrations of the supernatants were determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blocking was done in 10% BSA in 1x Tris-buffered saline. Western blot analyses were performed with an anti-ER polyclonal antibody (1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) or an anti-GFP polyclonal antibody (1:1000 dilution, Santa Cruz Biotechnology). Immunoreacted bands in the immunoblots were visualized with horseradish peroxidase-coupled goat antirabbit IgG secondary antibody (1:2000 dilution, Santa Cruz Biotechnology) by using the enhanced chemiluminescence Western blotting system.
Luciferase assays
Approximately 2 x 105 Cos-7 cells and MCF-7 cells were plated per well of a 6-well tissue culture plate 24 h before transfection. Cos-7 cells were cotransfected with 0.5 μg ptk-ERE-luc, 0.5 μg GFP or 0.5 μg GFP-ER or GFP-mtER expression vector, and 0·1 μg pRLCMV (ToYo Ink, Tokyo, Japan) as an internal control. MCF-7 cells were transfected with 1 μg ptk-ERE-luc and 0·1 μg pRLCMV. After transfection, these cells were plated for 3 h in 10% fetal bovine serum-containing DMEM to allow cell attachment, and then the medium was replaced with serum-free DMEM. After 24 h of serum starvation, Cos-7 cells were treated with 10–8 M E2 or vehicle (ethanol) and MCF-7 cells were treated with 10–8 M E2, vehicle (ethanol), 100 ng/ml EGF, or 100 ng/ml IGF-I. In some experiments, MCF-7 cells were pretreated with 20 μM PD98059 (Cell Signaling Technology Inc., Beverly, MA) or 20 μM LY294002 (Calbiochem, La Jolla, CA) and then treated with 100 ng/ml EGF or 100 ng/ml IGF-I. After 24 h, the cells were harvested and luciferase assays were performed with the Picagene dual-sea pansy luminescence kit (ToYo Ink). Firefly-luciferase activity and sea pansy-luciferase activity were measured using a luminometer (Lumat LB9507; EG&G, Berthold, Bad Wildbad, Germany). The firefly-luciferase activity was then normalized relative to the sea pansy-luciferase activity to determine the transfection efficiency.
Live microscopy
Live microscopy was performed on cells transfected with GFP-ER or GFP-mtER. The living cell image acquisition was performed at 37 C. After addition of 10–8 M E2, vehicle (ethanol), 100 ng/ml EGF, or 100 ng/ml IGF-I, fluorescence images of the transfected cells were acquired with a confocal microscope (TCS4D, Leica, Heidelberg, Germany). In some experiments, cells were pretreated with 20 μM PD98059 or 20 μM LY294002 for 30 min and then treated with 100 ng/ml EGF or 100 ng/ml IGF-I. Cell images were obtained through a x63 objective lens by excitation with the 488-nm line from a krypton-argon laser, and the emission was viewed through a 506- to 538-nm band pass filter. Fluorescent images were acquired every 10 min throughout a period of 120 min. For nuclear staining, Hoechst 33342 was used.
Statistical analysis
All data are presented as mean ± SEM. Two-way comparisons were made with Student’s t test; multiple group comparisons were made by one-way ANOVA followed by Scheffé’s F test using StatView software (Abacus Concepts, Berkeley, CA). Significant differences were defined as those with P < 0.05.
Results
Characterization of GFP-ER and GFP-mtER
To study the ligand-dependent or independent changes in the nuclear distribution of ER in living cells, we used vectors expressing chimeras in which GFP was fused to ER or an ER deletion mutant (GFP-mtER) lacking the ligand binding domain of ER (32). Extracts obtained from MCF-7 cells or MCF-7 cells transfected with GFP-ER were analyzed by Western blotting with anti-ER (Fig. 1A, upper panel) or anti-GFP (Fig. 1A, lower panel) antibody. MCF-7 cells transfected with GFP-ER expressed both ER (66 kDa) and GFP-ER chimera protein (94 kDa). In MCF-7 cells transfected with GFP-ER, the fluorescence of GFP-ER was observed only in the nucleus (Fig. 1B). To verify the functional properties of the GFP-ER or GFP-mtER, these constructs were cotransfected with an ER-responsive reporter plasmid, ptk-ERE-luc, into Cos-7 cells, which lack endogenous ER. Treatment with 10–8 M E2 for 24 h significantly induced the transcriptional activation of the ER-responsive reporter gene in Cos-7 cells transfected with GFP-ER but not Cos-7 cells transfected with GFP-mtER (Fig. 1C).
E2, EGF, and IGF-I induce GFP-ER nuclear redistribution
We next examined the effects of E2, EGF, and IGF-I on GFP-ER distribution by using time-lapse confocal microscopy. Under the basal conditions, GFP-ER showed a diffuse distribution throughout the nucleoplasm but was excluded from the nucleoli (Figs. 2–4, images at 0 min). Treatment with 10–8 M E2 changed the localization of GFP-ER to a punctate or hyperspeckled distribution (Fig. 2B). This nuclear redistribution of GFP-ER induced by E2 treatment appeared within 10 min and was sustained thereafter (Fig. 2B). Similar nuclear redistribution of GFP-ER was also induced by treatment with EGF (100 ng/ml) (Fig. 3B) or IGF-I (100 ng/ml) (Fig. 4B). However, the time courses of the GFP-ER nuclear redistribution induced by EGF or IGF-I treatment were different from that induced by E2 treatment. EGF-induced nuclear redistribution of GFP-ER was observed at 30 min and reached a maximum at 60 min (Fig. 3B), whereas IGF-I-induced nuclear redistribution of GFP-ER was observed at 60 min and reached a maximum at 90 min (Fig. 4B). To determine whether the ligand binding domain of ER was necessary for the nuclear redistribution of GFP-ER, GFP-mtER was used for transfection (32). As seen in Fig. 2C, treatment with E2 had no effect on the GFP-mtER nuclear distribution. Treatment with EGF (Fig. 3C) or IGF-I (Fig. 4C) also had no effect of the GFP-mtER nuclear distribution. These data suggest that E2, EGF, and IGF-I induce nuclear redistribution of GFP-ER, and the ligand binding domain of ER is needed for the redistribution.
Effect of MAPK inhibitor or PI3 kinase inhibitor on GFP-ER nuclear redistribution induced by EGF or IGF-I
We next examined the mechanism of the nuclear redistribution of GFP-ER induced by E2, EGF, and IGF-I. Because both EGF and IGF-I affect the cells via a MAPK or PI3 kinase-Akt cascade (21, 22, 23, 24, 25), the role of the MAPK or PI3 kinase cascade in the nuclear redistribution of GFP-ER was examined. Pretreatment with either 20 μM PD98059, a MAPK cascade inhibitor, or LY294002, a PI3 kinase inhibitor, had no effect on the E2-induced nuclear distribution of GFP-ER (Fig. 5). Although pretreatment with dimethylsulfoxide (vehicle) or 20 μM LY294002 had no effect on the EGF-induced nuclear redistribution of GFP-ER (Fig. 6, top and lower panels), pretreatment with 20 μM PD98059 prevented the EGF-induced nuclear redistribution of GFP-ER (Fig. 6, middle panel). On the other hand, pretreatment with dimethylsulfoxide (vehicle) or 20 μM PD98059 had no effect on the IGF-I-induced nuclear redistribution of GFP-ER (Fig. 7, top and middle panels). Pretreatment with LY294002 prevented the IGF-I-induced nuclear redistribution of GFP-ER (Fig. 7, lower panel). These results suggest that EGF and IGF-I induced the redistribution of GFP-ER via the MAPK and the PI3 kinase cascades, respectively.
Transcriptional activation of ER by EGF and IGF-I
We examined the effects of EGF and IGF-I on the transcriptional activation of ER. The MCF-7 cells were transfected with an ER-responsive reporter plasmid. Whereas E2 caused about an 8-fold increase in the luciferase activity, both EGF (100 ng/ml) and IGF-I (100 ng/ml) caused about a 3-fold increase in the luciferase activity (Fig. 8). Pretreatment with PD98059 but not LY294002 significantly inhibited the EGF-induced transcriptional activation of the ER-responsive reporter. Similarly, pretreatment with LY294002 but not PD98059 significantly inhibited the IGF-I-induced transcriptional activation of ER-responsive reporter.
Discussion
In the present study, we showed that EGF or IGF-I induced the nuclear redistribution of GFP-ER. Whereas the morphological pattern of the nuclear redistribution induced by EGF or IGF-I was very similar to that induced by E2, the time courses of the nuclear redistribution were quite different. We also showed that the nuclear redistribution of GFP-ER induced by EGF or IGF-I was dependent on the MAPK or PI3 kinase cascades, respectively.
Recently a direct visualization technique using GFP in single living cells revealed a change in the intranuclear localization of ER in the presence of E2. E2 changes the diffuse distribution of GFP-ER in the nucleus to a punctate distribution within 10–20 min (30, 31, 32, 33, 34, 35, 36, 37, 38). We also observed that a similar nuclear redistribution of GFP-ER occurred within 10 min after E2 treatment (Fig. 2). It is thought that the ligand-dependent nuclear redistribution of ER is involved in the transcriptional activity (32, 33, 34, 38) because the ligand-dependent nuclear redistribution of ER correlates with nuclear matrix association and recruitment of ER coactivators, such as SRC-1 (32) and glucocorticoid receptor interacting protein-1 (33), which have histone acetylase activity (39). However, Stenoien et al. (32) and Matsuda et al. (38) reported that the ligand-dependent nuclear redistribution of fluorescent protein tagged-ER did not result in complete association of the redistributed protein with phosphorylated RNA polymerase II, Brg-1 or AcH4, which contribute to the transcriptional process (34, 38). These results suggest that the redistribution of ER is not completely correlated with the transcriptional process in the presence of E2. Therefore, the physiological meaning of the ligand-dependent nuclear redistribution of ER remains unclear.
We also observed that EGF or IGF-I induced nuclear redistribution of GFP-ER that was morphologically similar to that induced by E2 (Figs. 3 and 4). However, this redistribution of GFP-ER induced by EGF or IGF-I had different time courses from that induced by E2 (Figs. 3 and 4). Because E2 is a small and lipid soluble molecule, E2 penetrates into the cell and binds to ER easily. E2 binding to ER results in dimerization, phosphorylation of ER, and subsequent binding of the E2-ER complex to ERE to induce initiation of the transcriptional process, including the recruitment of coactivators of ER (42). In fact, Denton et al. reported that E2 phosphorylates ER within 2 min in MCF-7 cells (43). Moreover, Llopis et al. (44) demonstrated that the E2-ER complex interacted with SRC-1 within few minutes by using fluorescence resonance energy transfer analysis. These evidences are in accord with the rapid (within 10 min) nuclear redistribution of GFP-ER by E2. On the other hand, binding of EGF or IGF-I to its receptor expressed in the plasma membrane results in activation of MAPK or PI3 kinase cascade (45). In fact, Kato et al. (21) demonstrated that ER was phosphorylated by MAPK on Ser118 within 15 min after the addition of EGF or IGF-I to cells in vivo and in vitro. Moreover, PI3 kinase, which is activated by IGF-I and other growth factors, phosphorylates ER within 1 h after its addition, and then active Akt phosphorylates Ser167 of ER (25). These evidences are consistent with the time course of nuclear redistribution of ER by EGF or IGF-I. However, the physiological meaning of the different time course of nuclear redistribution of GFP-ER is unknown. It was reported that both E2 and EGF or IGF-I synergistically stimulates the cell proliferation from ER-positive breast cancer cell lines through ER activation (46). Thus, the different time course of ER activation might be involved in the synergistic stimulation of cell proliferation.
Much evidence indicates that growth factors transcriptionally activate ER (21, 22). Both EGF and IGF-I activate E2-regulated genes, such as progesterone receptor and pS2, through ER in MCF-7 cells (24). We also confirmed that both EGF and IGF-I activated an ER-responsive gene (Fig. 8). Moreover, EGF induces phosphorylation of the AF-1 domain of ER or ER via MAPK and recruits coactivators, such as p68 RNA helicase and SRC-1 (29, 47). Taken together, these facts suggest that the nuclear redistribution of ER by EGF or IGF-I may be involved in the transcriptional process. However, further studies will be required to elucidate the physiological role of the growth factor-induced nuclear redistribution of ER.
To examine which domain of ER is essential for the nuclear redistribution induced by EGF or IGF-I, we used an AF-2 deletion mutant that was transcriptionally inactive (Fig. 1). Stenoien et al. (32) reported that the nuclear distribution of the AF-2 deletion mutant of GFP-ER expressed in transfected cells did not change in the presence of E2; that observation was consistent with our results (Fig. 2). Furthermore, we tested the effect of deletion of the AF-2 domain of ER on the nuclear redistribution of ER in the presence of EGF or IGF-I. We observed that the AF-2 deletion mutant of GFP-ER was not redistributed in the presence of EGF (Fig. 3C) or IGF-I (Fig. 4C). These results suggested that at least the AF-2 domain of ER is necessary for the nuclear redistribution of ER by EGF or IGF-I. These results leave open the question of whether other domain(s) of ER are necessary for the nuclear redistribution of ER. Although Matsuda et al. (38) determined that the AF-1 domain (amino acids 81–140) was essential for the ligand-dependent nuclear redistribution of ER, there is no direct evidence that AF-1 domain is involved in the nuclear redistribution of ER induced by EGF or IGF-I. Kato et al. (21) and Bunone et al. (22) demonstrated that EGF or IGF-I induced Ser118 phosphorylation in the AF-1 domain. Joel et al. (48) reported that EGF also induced Ser167 phosphorylation in the AF-1 domain through activation of the 90-kDa ribosomal S6 kinase, which was phosphorylated by MAPK. Moreover, Martin et al. (24) demonstrated that both EGF and IGF-I activated the PI3 kinase/Akt pathway in MCF-7 cells and that constitutively active Akt activates ER, whose phosphorylation sites are Ser104, 106, 118, and/or Ser167 of the AF-1 domain. These evidences suggest that EGF or IGF-I can phosphorylate at least Ser118 or Ser167 of the AF-1 domain. Further studies will be required to determine whether the AF-1 domain is also necessary for the nuclear redistribution of ER induced by EGF or IGF-I.
In addition to ER activation by the ligand-dependent transcriptional pathway, the so-called classical or genomic pathway, a growing body of evidence suggests that E2 exerts rapid cellular effects that are mediated by MAPK or PI3 kinase/Akt cascades (3, 49, 50). This alternative pathway of E2 action is known as a nongenomic pathway (1, 3). It is believed that the E2-dependent cytoplasmic signaling pathway is mediated through binding to membrane ER (50). For example, E2 rapidly activates endothelial nitric oxide synthase activity in endothelial cells, which is mediated by the PI3 kinase/Akt cascade independent of transcription (51, 52). Moreover, the E2-dependent cytoplasmic signaling pathway also involves ER transactivation (25, 53, 54). In the present study, neither PD98059, a MAPK inhibitor, nor LY294002, a PI3 kinase inhibitor, prevented E2-induced nuclear redistribution of GFP-ER (Fig. 5). This result suggests that E2 binding to ER is more critical for the E2-induced nuclear redistribution of GFP-ER than the E2-activated cytoplasmic signaling via the MAPK or PI3 kinase cascade. We also observed that EGF-induced nuclear redistribution of GFP-ER was prevented by pretreatment with PD98059 but not by LY294002 (Fig. 6). Moreover, IGF-I-induced nuclear redistribution of GFP-ER was prevented by pretreatment with LY294002 but not with PD98059 (Fig. 7).
These results suggest that EGF and IGF-I-induced nuclear redistribution of GFP-ER is dependent on the MAPK and the PI3 kinase cascade, respectively. What evidences that support the results of induction of these specific cascades are present In fact, the regulation of MAPK or PI3 kinase activated by growth factors is involved in the control of cell growth in breast cancer cells (55, 56). Overexpression of EGF receptor or IGF-I receptor is associated with poor clinical outcome in breast cancer patients (57, 58). The antiestrogen tamoxifen is widely used as a first-line endocrine agent for the treatment of ER-positive breast cancer, with approximately 50% of patients benefiting from this therapy, almost all responsive tumors eventually relapse due to the development of tamoxifen resistance (59). Much evidence indicates that activation of growth factor signaling cascades, including those downstream of EGF receptor or IGF-I receptor can induce tamoxifen resistance (25, 60, 61, 62), which presents a major clinical problem. Recently, Chu et al. (63) reported that the ErbB1/ErbB2, members of EGF receptor, inhibitor in combination with tamoxifen prevents both cell proliferation and ER-dependent gene expression in tamoxifen-resistant ER-positive breast cancer cell lines. Thus, these cascades may be involved in a mechanism of tamoxifen resistance in breast cancer (64).
In this study, we demonstrated for the first time that EGF and IGF-I induced the nuclear redistribution of ER in living cells and that the redistribution was mediated through the MAPK and PI3 kinase pathways, respectively. Although the precise mechanism of this phenomenon is still unclear, this study may provide new clues for studying the molecular mechanism of the activation of ER by growth factors.
Footnotes
This work was supported by Research Grants JSPS KAKEN 15591726 (to T.T.), JSPS KAKEN 14370523, and JSPS Grant-in-Aid for Exploratory Research 16659444 (to H. K.), and the Center of Excellence 21 Program (03COE105) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Abbreviations: AF, Activation function; E2, 17-estradiol; EGF, epidermal growth factor; ER, estrogen receptor; ERE, estrogen response element; GFP, green fluorescent protein; GFP-ER, ER fused with GFP; GFP-mtER, GFP-ER ligand binding domain deletion mutant; PI, phosphatidylinositol; SRC, steroid receptor coactivator.
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Abstract
In the present study, to examine the dynamic changes in the localization of nuclear estrogen receptor (ER) induced by growth factors, we used time-lapse confocal microscopy to directly visualized ER fused with green fluorescent protein (GFP-ER) in single living cells treated with epidermal growth factor (EGF) or IGF-I. We observed that 17-estradiol (E2) changed the normally diffuse distribution of GFP-ER throughout the nucleoplasm to a hyperspeckled distribution within 10 min. Both EGF and IGF-I also changed the nuclear distribution of GFP-ER, similarly to E2 treatment. However, the time courses of the nuclear redistribution of GFP-ER induced by EGF or IGF-I were different from that induced by E2 treatment. In the EGF-treated cells, the GFP-ER nuclear redistribution was observed at 30 min and reached a maximum at 60 min, whereas in the IGF-I-treated cells, the GFP-ER nuclear redistribution was observed at 60 min and reached a maximum at 90 min. The EGF-induced redistribution of GFP-ER was blocked by pretreatment with a MAPK cascade inhibitor, PD98059, whereas the IGF-I-induced redistribution of GFP-ER was blocked by pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002. Analysis using an activation function-2 domain deletion mutant of GFP-ER showed that the change in the distribution of GFP-ER 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 ER by EGF and IGF-I, respectively, and that the activation function-2 domain of ER may be needed for the nuclear redistribution of ER.
Introduction
ESTROGEN PLAYS IMPORTANT physiological roles in the female and male reproductive systems (1, 2), nonreproductive systems (3, 4), and the initiation and proliferation of reproductive cancer cells (5). Most of these diverse effects of estrogen are mediated by binding to and activation of estrogen receptors (ERs) (6, 7, 8). ERs are members of the nuclear receptor superfamily and are ligand-activated transcription factors that regulate the expression of target genes (7, 9, 10). There are two subtypes of ERs, ER and ER, which are synthesized from different genes and are functionally distinct (11, 12, 13). The members of this superfamily display a modular structure with six distinct functional regions termed A–F, which include domains for DNA binding, ligand binding, and transcriptional activation (14, 15, 16). ERs have two inducible transcriptional activation functions (AFs): activation of the C-terminal AF-2 domain, located in the ligand-binding domain, is dependent on agonist binding (14), whereas the amino terminal AF-1 domain can be activated independently of agonists (17). The AF-1 and AF-2 domains activate ERs independently or synergistically, depending on the promoter of the target gene and the cell type (18). On ligand binding, ERs bind to estrogen-responsive elements, which results in activation of the specific ER target genes. Simultaneously, ligand binding to ER alters the ER conformation and thereby affects the interaction of ER with cofactors (7, 19).
Growth factors, such as epidermal growth factor (EGF) and IGF-I, that bind to tyrosine kinase-linked receptors are also important in controlling ER activity in a ligand-independent manner (18, 20). Binding of EGF or IGF-I to its receptor results in activation of the MAPKs, ERK1 and ERK2, which phosphorylate ER at serine 118 in the N-terminal domain and potentiate AF-1 activity (21, 22, 23). Moreover, EGF or IGF-I treatment causes rapid phosphorylation and activation of Akt, which activates ER by phosphorylation of serine 167 in the AF-1 domain in a breast cancer cell line (24, 25). ER phosphorylation induced by both EGF and IGF-I facilitates the association of the coactivators and basal transcriptional factors, which leads to ER-mediated transcriptional activation (26, 27). Moreover, MAPK pathways mediate the phosphorylation and recruitment of steroid receptor coactivator (SRC)-1 (28, 29).
Several recent studies have shown that 17-estradiol (E2) changes the intranuclear distribution of ER fused with green fluorescent protein (GFP-ER) in living cells (30, 31, 32, 33, 34, 35, 36, 37, 38). In the absence of E2, GFP-ER is diffusely distributed throughout the nucleoplasm but is excluded from the nucleolar region. Upon addition of E2, a dramatic redistribution of GFP-ER from a diffuse to a punctate pattern occurs rapidly (10–20 min) within the nucleus (30, 32). Furthermore, the redistribution of GFP-ER correlates with nuclear matrix association and recruitment of ER coactivators, such as SRC-1 (32) or glucocorticoid receptor interacting protein-1 (33). These coactivators have histone acetylase activity (39). Therefore, it is thought that the ligand-dependent redistribution of ER is involved in the transcriptional activation (32, 33, 34, 38).
Whether intranuclear ER redistribution is induced by growth factors, such as EGF or IGF-I, is not yet clear. In the present study, we directly visualized the changes in the nuclear ER distribution using GFP-ER in cells treated with EGF or IGF-I. We found that both EGF and IGF-I induced GFP-ER nuclear redistribution with pattern similar to that induced by E2 but with a different time course than that of E2. Furthermore, we examined whether GFP-ER nuclear redistribution induced by EGF or IGF-I occurred via MAPK or phosphatidylinositol (PI) 3 kinase cascades.
Materials and Methods
Unless otherwise specified, all chemicals were from Sigma Chemical Co. (St. Louis, MO).
Cell culture and transfection
Cos-7 cells were obtained from Cell Resource Center for Biomedical Research Institute of Development, Aging, and Cancer Tohoku University. MCF-7 human breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA). These cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate in the presence of 5% CO2 at 37 C. MCF-7 cells were cultured in DMEM supplemented with 10% charcoal-treated serum for at least 4 d and then subjected to experiments. For all experiments, phenol red-free medium was used. Transient transfection of the cells was performed with LipofectAMINE Plus (Life Technologies, Gaithersburg, MD), according to the manufacturer’s protocol.
Constructs
GFP-ER and GFP-ER ligand binding domain deletion mutant (GFP-mtER) expression vectors were kind gifts from Dr. Michael A. Mancini (Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX) (32). The estrogen response element (ERE)-containing reporter gene, ptk-ERE-luc, was a kind gift from Dr. J. Larry Jameson (Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, IL) (40).
Western blot analysis
MCF-7 cells plated on 35-mm dishes were transfected with the GFP-ER construct and then cultured overnight. They were then washed twice with PBS and lysed in ice-cold buffer composed of 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium orthovanadate, 100 mM NaF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride (41). The lysates were centrifuged at 12,000 x g at 4 C for 15 min, and the protein concentrations of the supernatants were determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blocking was done in 10% BSA in 1x Tris-buffered saline. Western blot analyses were performed with an anti-ER polyclonal antibody (1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) or an anti-GFP polyclonal antibody (1:1000 dilution, Santa Cruz Biotechnology). Immunoreacted bands in the immunoblots were visualized with horseradish peroxidase-coupled goat antirabbit IgG secondary antibody (1:2000 dilution, Santa Cruz Biotechnology) by using the enhanced chemiluminescence Western blotting system.
Luciferase assays
Approximately 2 x 105 Cos-7 cells and MCF-7 cells were plated per well of a 6-well tissue culture plate 24 h before transfection. Cos-7 cells were cotransfected with 0.5 μg ptk-ERE-luc, 0.5 μg GFP or 0.5 μg GFP-ER or GFP-mtER expression vector, and 0·1 μg pRLCMV (ToYo Ink, Tokyo, Japan) as an internal control. MCF-7 cells were transfected with 1 μg ptk-ERE-luc and 0·1 μg pRLCMV. After transfection, these cells were plated for 3 h in 10% fetal bovine serum-containing DMEM to allow cell attachment, and then the medium was replaced with serum-free DMEM. After 24 h of serum starvation, Cos-7 cells were treated with 10–8 M E2 or vehicle (ethanol) and MCF-7 cells were treated with 10–8 M E2, vehicle (ethanol), 100 ng/ml EGF, or 100 ng/ml IGF-I. In some experiments, MCF-7 cells were pretreated with 20 μM PD98059 (Cell Signaling Technology Inc., Beverly, MA) or 20 μM LY294002 (Calbiochem, La Jolla, CA) and then treated with 100 ng/ml EGF or 100 ng/ml IGF-I. After 24 h, the cells were harvested and luciferase assays were performed with the Picagene dual-sea pansy luminescence kit (ToYo Ink). Firefly-luciferase activity and sea pansy-luciferase activity were measured using a luminometer (Lumat LB9507; EG&G, Berthold, Bad Wildbad, Germany). The firefly-luciferase activity was then normalized relative to the sea pansy-luciferase activity to determine the transfection efficiency.
Live microscopy
Live microscopy was performed on cells transfected with GFP-ER or GFP-mtER. The living cell image acquisition was performed at 37 C. After addition of 10–8 M E2, vehicle (ethanol), 100 ng/ml EGF, or 100 ng/ml IGF-I, fluorescence images of the transfected cells were acquired with a confocal microscope (TCS4D, Leica, Heidelberg, Germany). In some experiments, cells were pretreated with 20 μM PD98059 or 20 μM LY294002 for 30 min and then treated with 100 ng/ml EGF or 100 ng/ml IGF-I. Cell images were obtained through a x63 objective lens by excitation with the 488-nm line from a krypton-argon laser, and the emission was viewed through a 506- to 538-nm band pass filter. Fluorescent images were acquired every 10 min throughout a period of 120 min. For nuclear staining, Hoechst 33342 was used.
Statistical analysis
All data are presented as mean ± SEM. Two-way comparisons were made with Student’s t test; multiple group comparisons were made by one-way ANOVA followed by Scheffé’s F test using StatView software (Abacus Concepts, Berkeley, CA). Significant differences were defined as those with P < 0.05.
Results
Characterization of GFP-ER and GFP-mtER
To study the ligand-dependent or independent changes in the nuclear distribution of ER in living cells, we used vectors expressing chimeras in which GFP was fused to ER or an ER deletion mutant (GFP-mtER) lacking the ligand binding domain of ER (32). Extracts obtained from MCF-7 cells or MCF-7 cells transfected with GFP-ER were analyzed by Western blotting with anti-ER (Fig. 1A, upper panel) or anti-GFP (Fig. 1A, lower panel) antibody. MCF-7 cells transfected with GFP-ER expressed both ER (66 kDa) and GFP-ER chimera protein (94 kDa). In MCF-7 cells transfected with GFP-ER, the fluorescence of GFP-ER was observed only in the nucleus (Fig. 1B). To verify the functional properties of the GFP-ER or GFP-mtER, these constructs were cotransfected with an ER-responsive reporter plasmid, ptk-ERE-luc, into Cos-7 cells, which lack endogenous ER. Treatment with 10–8 M E2 for 24 h significantly induced the transcriptional activation of the ER-responsive reporter gene in Cos-7 cells transfected with GFP-ER but not Cos-7 cells transfected with GFP-mtER (Fig. 1C).
E2, EGF, and IGF-I induce GFP-ER nuclear redistribution
We next examined the effects of E2, EGF, and IGF-I on GFP-ER distribution by using time-lapse confocal microscopy. Under the basal conditions, GFP-ER showed a diffuse distribution throughout the nucleoplasm but was excluded from the nucleoli (Figs. 2–4, images at 0 min). Treatment with 10–8 M E2 changed the localization of GFP-ER to a punctate or hyperspeckled distribution (Fig. 2B). This nuclear redistribution of GFP-ER induced by E2 treatment appeared within 10 min and was sustained thereafter (Fig. 2B). Similar nuclear redistribution of GFP-ER was also induced by treatment with EGF (100 ng/ml) (Fig. 3B) or IGF-I (100 ng/ml) (Fig. 4B). However, the time courses of the GFP-ER nuclear redistribution induced by EGF or IGF-I treatment were different from that induced by E2 treatment. EGF-induced nuclear redistribution of GFP-ER was observed at 30 min and reached a maximum at 60 min (Fig. 3B), whereas IGF-I-induced nuclear redistribution of GFP-ER was observed at 60 min and reached a maximum at 90 min (Fig. 4B). To determine whether the ligand binding domain of ER was necessary for the nuclear redistribution of GFP-ER, GFP-mtER was used for transfection (32). As seen in Fig. 2C, treatment with E2 had no effect on the GFP-mtER nuclear distribution. Treatment with EGF (Fig. 3C) or IGF-I (Fig. 4C) also had no effect of the GFP-mtER nuclear distribution. These data suggest that E2, EGF, and IGF-I induce nuclear redistribution of GFP-ER, and the ligand binding domain of ER is needed for the redistribution.
Effect of MAPK inhibitor or PI3 kinase inhibitor on GFP-ER nuclear redistribution induced by EGF or IGF-I
We next examined the mechanism of the nuclear redistribution of GFP-ER induced by E2, EGF, and IGF-I. Because both EGF and IGF-I affect the cells via a MAPK or PI3 kinase-Akt cascade (21, 22, 23, 24, 25), the role of the MAPK or PI3 kinase cascade in the nuclear redistribution of GFP-ER was examined. Pretreatment with either 20 μM PD98059, a MAPK cascade inhibitor, or LY294002, a PI3 kinase inhibitor, had no effect on the E2-induced nuclear distribution of GFP-ER (Fig. 5). Although pretreatment with dimethylsulfoxide (vehicle) or 20 μM LY294002 had no effect on the EGF-induced nuclear redistribution of GFP-ER (Fig. 6, top and lower panels), pretreatment with 20 μM PD98059 prevented the EGF-induced nuclear redistribution of GFP-ER (Fig. 6, middle panel). On the other hand, pretreatment with dimethylsulfoxide (vehicle) or 20 μM PD98059 had no effect on the IGF-I-induced nuclear redistribution of GFP-ER (Fig. 7, top and middle panels). Pretreatment with LY294002 prevented the IGF-I-induced nuclear redistribution of GFP-ER (Fig. 7, lower panel). These results suggest that EGF and IGF-I induced the redistribution of GFP-ER via the MAPK and the PI3 kinase cascades, respectively.
Transcriptional activation of ER by EGF and IGF-I
We examined the effects of EGF and IGF-I on the transcriptional activation of ER. The MCF-7 cells were transfected with an ER-responsive reporter plasmid. Whereas E2 caused about an 8-fold increase in the luciferase activity, both EGF (100 ng/ml) and IGF-I (100 ng/ml) caused about a 3-fold increase in the luciferase activity (Fig. 8). Pretreatment with PD98059 but not LY294002 significantly inhibited the EGF-induced transcriptional activation of the ER-responsive reporter. Similarly, pretreatment with LY294002 but not PD98059 significantly inhibited the IGF-I-induced transcriptional activation of ER-responsive reporter.
Discussion
In the present study, we showed that EGF or IGF-I induced the nuclear redistribution of GFP-ER. Whereas the morphological pattern of the nuclear redistribution induced by EGF or IGF-I was very similar to that induced by E2, the time courses of the nuclear redistribution were quite different. We also showed that the nuclear redistribution of GFP-ER induced by EGF or IGF-I was dependent on the MAPK or PI3 kinase cascades, respectively.
Recently a direct visualization technique using GFP in single living cells revealed a change in the intranuclear localization of ER in the presence of E2. E2 changes the diffuse distribution of GFP-ER in the nucleus to a punctate distribution within 10–20 min (30, 31, 32, 33, 34, 35, 36, 37, 38). We also observed that a similar nuclear redistribution of GFP-ER occurred within 10 min after E2 treatment (Fig. 2). It is thought that the ligand-dependent nuclear redistribution of ER is involved in the transcriptional activity (32, 33, 34, 38) because the ligand-dependent nuclear redistribution of ER correlates with nuclear matrix association and recruitment of ER coactivators, such as SRC-1 (32) and glucocorticoid receptor interacting protein-1 (33), which have histone acetylase activity (39). However, Stenoien et al. (32) and Matsuda et al. (38) reported that the ligand-dependent nuclear redistribution of fluorescent protein tagged-ER did not result in complete association of the redistributed protein with phosphorylated RNA polymerase II, Brg-1 or AcH4, which contribute to the transcriptional process (34, 38). These results suggest that the redistribution of ER is not completely correlated with the transcriptional process in the presence of E2. Therefore, the physiological meaning of the ligand-dependent nuclear redistribution of ER remains unclear.
We also observed that EGF or IGF-I induced nuclear redistribution of GFP-ER that was morphologically similar to that induced by E2 (Figs. 3 and 4). However, this redistribution of GFP-ER induced by EGF or IGF-I had different time courses from that induced by E2 (Figs. 3 and 4). Because E2 is a small and lipid soluble molecule, E2 penetrates into the cell and binds to ER easily. E2 binding to ER results in dimerization, phosphorylation of ER, and subsequent binding of the E2-ER complex to ERE to induce initiation of the transcriptional process, including the recruitment of coactivators of ER (42). In fact, Denton et al. reported that E2 phosphorylates ER within 2 min in MCF-7 cells (43). Moreover, Llopis et al. (44) demonstrated that the E2-ER complex interacted with SRC-1 within few minutes by using fluorescence resonance energy transfer analysis. These evidences are in accord with the rapid (within 10 min) nuclear redistribution of GFP-ER by E2. On the other hand, binding of EGF or IGF-I to its receptor expressed in the plasma membrane results in activation of MAPK or PI3 kinase cascade (45). In fact, Kato et al. (21) demonstrated that ER was phosphorylated by MAPK on Ser118 within 15 min after the addition of EGF or IGF-I to cells in vivo and in vitro. Moreover, PI3 kinase, which is activated by IGF-I and other growth factors, phosphorylates ER within 1 h after its addition, and then active Akt phosphorylates Ser167 of ER (25). These evidences are consistent with the time course of nuclear redistribution of ER by EGF or IGF-I. However, the physiological meaning of the different time course of nuclear redistribution of GFP-ER is unknown. It was reported that both E2 and EGF or IGF-I synergistically stimulates the cell proliferation from ER-positive breast cancer cell lines through ER activation (46). Thus, the different time course of ER activation might be involved in the synergistic stimulation of cell proliferation.
Much evidence indicates that growth factors transcriptionally activate ER (21, 22). Both EGF and IGF-I activate E2-regulated genes, such as progesterone receptor and pS2, through ER in MCF-7 cells (24). We also confirmed that both EGF and IGF-I activated an ER-responsive gene (Fig. 8). Moreover, EGF induces phosphorylation of the AF-1 domain of ER or ER via MAPK and recruits coactivators, such as p68 RNA helicase and SRC-1 (29, 47). Taken together, these facts suggest that the nuclear redistribution of ER by EGF or IGF-I may be involved in the transcriptional process. However, further studies will be required to elucidate the physiological role of the growth factor-induced nuclear redistribution of ER.
To examine which domain of ER is essential for the nuclear redistribution induced by EGF or IGF-I, we used an AF-2 deletion mutant that was transcriptionally inactive (Fig. 1). Stenoien et al. (32) reported that the nuclear distribution of the AF-2 deletion mutant of GFP-ER expressed in transfected cells did not change in the presence of E2; that observation was consistent with our results (Fig. 2). Furthermore, we tested the effect of deletion of the AF-2 domain of ER on the nuclear redistribution of ER in the presence of EGF or IGF-I. We observed that the AF-2 deletion mutant of GFP-ER was not redistributed in the presence of EGF (Fig. 3C) or IGF-I (Fig. 4C). These results suggested that at least the AF-2 domain of ER is necessary for the nuclear redistribution of ER by EGF or IGF-I. These results leave open the question of whether other domain(s) of ER are necessary for the nuclear redistribution of ER. Although Matsuda et al. (38) determined that the AF-1 domain (amino acids 81–140) was essential for the ligand-dependent nuclear redistribution of ER, there is no direct evidence that AF-1 domain is involved in the nuclear redistribution of ER induced by EGF or IGF-I. Kato et al. (21) and Bunone et al. (22) demonstrated that EGF or IGF-I induced Ser118 phosphorylation in the AF-1 domain. Joel et al. (48) reported that EGF also induced Ser167 phosphorylation in the AF-1 domain through activation of the 90-kDa ribosomal S6 kinase, which was phosphorylated by MAPK. Moreover, Martin et al. (24) demonstrated that both EGF and IGF-I activated the PI3 kinase/Akt pathway in MCF-7 cells and that constitutively active Akt activates ER, whose phosphorylation sites are Ser104, 106, 118, and/or Ser167 of the AF-1 domain. These evidences suggest that EGF or IGF-I can phosphorylate at least Ser118 or Ser167 of the AF-1 domain. Further studies will be required to determine whether the AF-1 domain is also necessary for the nuclear redistribution of ER induced by EGF or IGF-I.
In addition to ER activation by the ligand-dependent transcriptional pathway, the so-called classical or genomic pathway, a growing body of evidence suggests that E2 exerts rapid cellular effects that are mediated by MAPK or PI3 kinase/Akt cascades (3, 49, 50). This alternative pathway of E2 action is known as a nongenomic pathway (1, 3). It is believed that the E2-dependent cytoplasmic signaling pathway is mediated through binding to membrane ER (50). For example, E2 rapidly activates endothelial nitric oxide synthase activity in endothelial cells, which is mediated by the PI3 kinase/Akt cascade independent of transcription (51, 52). Moreover, the E2-dependent cytoplasmic signaling pathway also involves ER transactivation (25, 53, 54). In the present study, neither PD98059, a MAPK inhibitor, nor LY294002, a PI3 kinase inhibitor, prevented E2-induced nuclear redistribution of GFP-ER (Fig. 5). This result suggests that E2 binding to ER is more critical for the E2-induced nuclear redistribution of GFP-ER than the E2-activated cytoplasmic signaling via the MAPK or PI3 kinase cascade. We also observed that EGF-induced nuclear redistribution of GFP-ER was prevented by pretreatment with PD98059 but not by LY294002 (Fig. 6). Moreover, IGF-I-induced nuclear redistribution of GFP-ER was prevented by pretreatment with LY294002 but not with PD98059 (Fig. 7).
These results suggest that EGF and IGF-I-induced nuclear redistribution of GFP-ER is dependent on the MAPK and the PI3 kinase cascade, respectively. What evidences that support the results of induction of these specific cascades are present In fact, the regulation of MAPK or PI3 kinase activated by growth factors is involved in the control of cell growth in breast cancer cells (55, 56). Overexpression of EGF receptor or IGF-I receptor is associated with poor clinical outcome in breast cancer patients (57, 58). The antiestrogen tamoxifen is widely used as a first-line endocrine agent for the treatment of ER-positive breast cancer, with approximately 50% of patients benefiting from this therapy, almost all responsive tumors eventually relapse due to the development of tamoxifen resistance (59). Much evidence indicates that activation of growth factor signaling cascades, including those downstream of EGF receptor or IGF-I receptor can induce tamoxifen resistance (25, 60, 61, 62), which presents a major clinical problem. Recently, Chu et al. (63) reported that the ErbB1/ErbB2, members of EGF receptor, inhibitor in combination with tamoxifen prevents both cell proliferation and ER-dependent gene expression in tamoxifen-resistant ER-positive breast cancer cell lines. Thus, these cascades may be involved in a mechanism of tamoxifen resistance in breast cancer (64).
In this study, we demonstrated for the first time that EGF and IGF-I induced the nuclear redistribution of ER in living cells and that the redistribution was mediated through the MAPK and PI3 kinase pathways, respectively. Although the precise mechanism of this phenomenon is still unclear, this study may provide new clues for studying the molecular mechanism of the activation of ER by growth factors.
Footnotes
This work was supported by Research Grants JSPS KAKEN 15591726 (to T.T.), JSPS KAKEN 14370523, and JSPS Grant-in-Aid for Exploratory Research 16659444 (to H. K.), and the Center of Excellence 21 Program (03COE105) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Abbreviations: AF, Activation function; E2, 17-estradiol; EGF, epidermal growth factor; ER, estrogen receptor; ERE, estrogen response element; GFP, green fluorescent protein; GFP-ER, ER fused with GFP; GFP-mtER, GFP-ER ligand binding domain deletion mutant; PI, phosphatidylinositol; SRC, steroid receptor coactivator.
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