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Suppression by estrogen receptor β of AP-1 mediated transactivation through estrogen receptor α
Journal of Steroid Biochemistry & Molecular Biology 78 (2001) 177– 184 www.elsevier.com/locate/jsbmb
Suppression by estrogen receptor b of AP-1 mediated transactivation through estrogen receptor a Satoshi Maruyama, Nariaki Fujimoto *, Kohsuke Asano, Akihiro Ito Department of Cancer Research, Research Institute for Radiation Biology and Medicine (RIRBM), Hiroshima Uni6ersity, 1 -2 -3 Kasumi, Minami-ku, Hiroshima 734 -8553, Japan Accepted 26 March 2001
Abstract The estrogen receptor (ER) is known to mediate gene transcription from AP-1 enhancer elements as well as the well-documented estrogen responsive elements (EREs). Investigations of AP-1 mediated transactivation through ER have been performed with rather complex promoters such as insulin like growth factor 1 (IGF-1) and collagenase promoters. In the present study, we investigated AP-1 mediated transactivation through ERa and ERb with a less complicated reporter consisting of only consensus AP-1 motifs. NIH 3T3 cells were transiently transfected with human ERa and ERb expression plasmids and AP-1-luc and ERE-luc reporters. 17b-Estradiol failed to activate ERb-AP-1 responses while activating ERa-AP-1, ERa-ERE, ERb-ERE mediated transcription. On the other hand, antiestrogens such as tamoxifen enhanced AP-1 mediated transactivation through both ERa and ERb. An ERa positive human breast cancel cell line, MCF-7, also showed the same manner of AP-1 mediated transactivation through ERa. When NIH 3T3 with ERa and MCF-7 were co-transfected with ERb, E2 dependent AP-1 responses decreased in both cell lines depending on the amount of the ERb expression plasmid. These results suggest that ERa and ERb may function in opposition with ERb actually suppressing the function of ERa in AP-1 mediated transactivation. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Estrogen receptor a and b; Differential transcription; Consensus AP-1 motif
1. Introduction The estrogen receptor (ER) is a conditional transcription factor, which belongs to the nuclear receptor superfamily and activates transcription of the specific genes only when a ligand is bound [1 – 3]. An estrogen responsive element (ERE), a DNA motif with palindromic structure, has been identified as a binding site for ER in the promoter region of several estrogen responsive genes such as these for vitellogenin and prolactin [4]. Once ER binds to EREs, it recruits a p160/p300 coactivator complex and enhances gene expression by interactions with the basic transcriptional machinery [5]. However, several lines of evidence have shown that estrogen can also enhance transcription from gene promoters containing the AP-1 motif which * Corresponding author. Tel.: + 81-82-257-5820; fax: +81-82-2567107. E-mail address: [email protected] (N. Fujimoto).
is a binding site for c-fos/c-jun protein and also known to be the TPA responsive element. It has been reported that AP-1 sites in the promoter region are involved in E2 dependent transcription of collagenase, insulin-like growth factor 1 (IGF-1) and ovalbumin genes [6–9]. Recently a novel type of ER was cloned and designated as ERb [10,11]. Differences in structure and tissue distribution between ERa and ERb suggest different biological roles of the two receptor subtypes. Transfection experiments with ERE containing reporters have indicated, however, that there is no difference in E2 dependent transactivation through the two types of receptor, even through some ligands such as genistein and coumestrol have a higher affinity for ERb [12,13]. When the E2 dependent response of the collagenase promoter which contains AP-1 motifs was examined, it was observed only with ERa but not with ERb. Interestingly partial estrogen antagonists or AF-1 agonists such as tamoxifen, which have known to induce ERa-AP-1 responses, also did activate ERb-AP-1 medi-
0960-0760/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 0 7 6 0 ( 0 1 ) 0 0 0 8 3 - 8
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ated transcription. Although these findings suggested a biological importance of AP-1 mediated E2 dependent transactivation especially for understanding functional differences between the two types of ER, very limited numbers of models have been examined and all of them involve rather complex promoters. In the present study, we therefore reconstructed and examined a model for E2 dependent AP-1 mediated transactivation through ERs using a reporter containing only consensus AP-1 motifs.
2. Materials and methods
2.1. Hormones 17b-Estradiol (E2) and OH-tamoxifen (OH-TAM) were purchased from Sigma Chemicals, St. Louis and dissolved in ethanol to give stock solutions. ICI182,782 (ICI) was generously provided by Zeneca KK (Hyogo, Japan).
2.2. Cell culture The NIH 3T3 cell line was obtained from the Japanese Collection of Research Bioresources (JCRB) and maintained in DME (Sigma Chemicals) containing penicillin and streptomycin with 10% calf serum (CS, Life Technologies, Rockville, MD, USA). For hormone treatments, the medium was changed to phenol red free DME (Sigma Chemicals) containing the same antibiotics along with dextran-charcoal treated CS for a week. The MCF-7 cell line was maintained in MEM (Sigma Chemicals) containing penicillin and streptomycin with 5% fetal bovine serum (FBS, Life Technologies). The medium was changed to phenol red free MEM (Sigma Chemicals) containing the antibiotics and dextran-charcoal treated FBS for a week.
2.3. Plasmid construction A reporter plasmid, pAP1-luc, which contains six tandem copies of the AP-1 enhancer. 5%-(TGACTAA)6, connected to a TATA box located upstream from the luciferase gene [15], and its control reporter lacking AP-1 motifs, pTA-luc, were purchased from Clontech (Palo Alto, CA, USA). The (ERE)3-SV40-luc plasmid, which contains 5%-CGGTCACAGTGACCAGTGGTCACGTGA-CCAGTC GGTCACAGTGACCC was a gift from Dr M. Kudoh, Yamanouchi Pharmaceutical Co., Tsukuba, Japan [16]. Human ERa and hERb, gifts from Dr S. Kato at Tokyo University, Tokyo), were inserted into the EcoRI site of the pSG5 expression vector, to give pSG5-hERa and pSG5-hERb, respectively. PRL-CMV (Promega, Madison, WI, USA) was used as the internal control.
2.4. Transient transfection with expression and reporter plasmids NIH 3T3 cells were plated at 1× 105/well in 12 well plates (Nalge Nunc International, Rochester, NY, USA). After 24 h, cells in each well were co-transfected with total 2.0 mg of plasmid DNA with TransFast transfection reagent containing a synthetic cationic lipid (Promega) following the supplied protocol. The weight ratio of TransFast reagent to DNA was 1:1. After 24 h incubation with hormones, cells were harvested with 100 ml of cell lysis buffer (Promega) and 10 ml of each lysate was mixed with firefly luciferase substrate followed by mixing with renilla luciferase substrate to determine luciferase activities (Dual-luciferase reporter assay system, Promega). Luminescence was measured with a Micro-beta scintillation counter (Amersham Pharmacia Biotech, Uppsala, Sweden). Firefly luciferase activity was normalized to renilla luciferase activity from pRL-CMV.
2.5. Immunoblot detection of c-fos and c-jun For immunoblot analysis, cells were lysed in NP40 lysis buffer (10 mM NaMo, 5 mM EDTA, 300 mM NaCl, 50 mM Tris–HCl, pH 8.0, 0.5% NP40, 0.2% Sarcosyl) with 0.2 mM PMSF. Aliquots (100 mg) of lysate protein were applied for 10% SDS-polyacrylamide gel electrophoresis. The bands were then transferred to P-bound nitrocellulose filters (Amersham Pharmacia Biotech, Uppsala, Sweden) with an electric transblot apparatus and immunostained with an ECL kit (Amersham Pharmacia Biotech) according to the company’s recommended procedure. Polyclonal anti-cfos and anti-c-jun antibodies were purchased from Santa Cruz Biotechnology, CA, USA for this purpose. The second antibody, peroxidase labeled anti-rabbit IgG, was from MBL, Nagoya, Japan. Developed bands were scanned with an image scanner and intensities were quantified using Scion Image software (Scion Corp., Frederick, MD, USA).
2.6. Re6erse transcription-polymerase chain reaction (RT-PCR) detection of ERs This was performed as described previously [17]. Briefly, total RNAs from cells were prepared by a modified acid guanidium thiocyanate-phenol–chloroform extraction method and treated with RQ1 DNase (Promega). One microgram of total RNA was reversetranscribed with 100 U of MMLV-RT (Life Technologies) and 1.25 pmol of oligo-dT primers. Amplification was carried out with Ex-Taq DNA polymerase (Takara, Tokyo, Japan) in the presence of various amounts of competitor DNAs.
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2.7. Statistical analysis Statistical comparisons were made using the Student’s t-test.
3. Results
3.1. ER dependence of AP1 -luc transacti6ation by E2 E2 responsive AP1-luc transcription was activated only when the ER expression plasmid, pSG5-hERa was co-transfected in NIH 3T3. The maximum response of 2.8-fold induction was found at 10 − 9 M of E2 (Fig. 1A). With the same cell line, induction of the (ERE)3SV40-luc response by E2 was also low, approximately 3.7 times the control level (Fig. 1B). Fig. 1C shows that E2 dependent AP-1 responses increase according to the amount of pSG5-hERa co-transfected.
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3.2. Effects of E2 on c-fos and c-jun protein le6els in NIH 3T3 cells Fig. 2 indicates time dependent changes in c-fos and c-jun proteins levels in NIH 3T3 cells transfected with either ERa or ERb and treated with E2. Immunoblot analysis showed that amounts of both proteins to be unaltered after hormone treatment. More importantly there was no difference in c-fos/c-jun levels between the cells transfected with ERa and ERb.
3.3. AP-1 mediated transcription in NIH 3T3 cells co-transfected with ERh and ERi E2 exerted effects on both ERE and AP-1 mediated transcription when cells were co-transfected with pSG5hERa. However, OH-TAM activated only AP-1 mediated transcription (Fig. 3A, B). The ICI compound also activated AP-1 response while suppressing the ERE dependent response to below the control level. When
Fig. 1. Dependency of transactivation of pAP1-luc on ER. (A) E2 dependent transcription activity of AP-1 with and without ER in NIH 3T3 cells. NIH 3T3 cells were transiently transfected with pAP1-luc along with an expression plasmid for hERa, then treated with E2 at the concentrations indicated, and harvested 24 h later. The responses are expressed as fold changes in the activity relative to the control level (mean 9 S.E.M.). (B) E2 dependent transactivation through ERE for comparison. The plasmid, p(ERE)3-SV40-luc, was used instead of pAP1-luc. (C) Effects of amounts of ER expression plasmid on AP-1 transcription activity. The responses are expressed as fold changes based on the activity of the cells without ER transfection or E2 treatment. * and ** indicated values are significantly different from control values at PB 0.05 and PB 0.01, respectively.
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both E2 and OH-TAM activated the AP-1 mediated response in a dose dependent manner, while the ERE response was evoked only with E2 (Fig. 5). ICI did not stimulate any response with either AP-1 or ERE reporters. When ERb was co-transfected in MCF-7, E2 dependent transcription of pAP-1-luc was suppressed, depending on the amount of the ERb expression plasmid (Fig. 6).
4. Discussion
Fig. 2. Time dependent changes in c-fos and c-jun protein levels in NIH 3T3 cells after E2 treatment. NIH 3T3 cells were transfected with hERa and hERb and treated with E2 at 10 − 9 M. After the time periods as indicated, cells were harvested and whole cell lysates were separated with SDS-PAGE and transferred to nitrocellulose filters. C-fos and c-jun were detected with ECL reagents and quantified with an image analyzer. The amounts are expressed as fold changes relative to the control level.
cells were transfected with ERb E2 did not initiate AP-1 dependent transactivation, while the ERE response was activated to the same level as with ERa (Fig. 3C, D). OH-TAM caused AP-1 mediated transcription with ERb but did not initiate ERE transactivation. With ERb, interestingly, ICI failed to activate either AP-1 or ERE dependent transactivation. Responses to E2 were further examined with cells transfected with both ERa and ERb at different ratios. In Fig. 4A, AP-1 mediated responses decreased as the transfected amount of ERb increased. On the other hand, with the (ERE)3-SV40-luc reporter, the response to E2 was independent of the ERa/ERb ratio (Fig. 4B). OH-TAM activated AP-1 response at any ERa/b ratio but did not induce ERE mediated transcription (Fig. 4C, D).
3.4. AP-1 mediated transcription in MCF-7 cells AP-1 responses were examined in a human breast cancer cell line, MCF-7, in comparison with ERE responses. RT-PCR detection for ERa and ERb showed that MCF-7 cells in the present study preferentially expressed ERa. The expressed mRNA levels for ERa and ERb were 2.29 0.19 fg/mg total RNA and 0.0289 0.004 fg/mg total RNA, respectively. In this cell line,
In the present study, for the first time, we reconstructed a model system for AP-1 mediated E2 dependent transcription with a simple AP-1 reporter containing only consensus AP-1 motifs. An E2 dependent response with the AP-1 reporter in NIH/3T3 cells was only found with ERa but not with ERb, while tamoxifen enhanced AP-1 mediated transcription through both ERa and ERb. The results are consistent with data for the collagenase promoter containing AP-1 motifs previously described [14]. There was a discrepancy between our findings and those for the collagenase promoter, however, in response to ICI. No response was noted in our model with ERb but high induction was found with the collagenase promoter through both ERa and ERb. This ICI induction may be due to the interaction at the other transcriptional motifs in the complex collagenase promoter or, simply, due to the difference between cell lines used. It is well known that ER is a conditional transcription factor which belongs to the nuclear receptor superfamily. When ligands bind to ER, the conformation is changed so that interaction with ERE motifs of DNA is facilitated. The liganded receptor at the ERE recruits several transcriptional cofactors including p160/p300 complexes to initiate transcription of specific genes. A few genes such as collagenase and IGF-1 are known to be regulated by estrogen (E2) without containing any ERE motifs in their gene promoters and it has been found that this E2 inducible gene regulation is mediated through the AP-1 motif via interactions of ER and AP-1 [6 –8]. In general, however, AP-1 and nuclear receptor mediated activities are known to interfere with each other, possibly because of interaction with overlapping binding sites [18]. In 1997, a novel type of ER was cloned and designated as ERb. Besides structural differences, the tissue distribution of this new receptor subtype is very different from that of ERa. In rats, high expression of ERb was found in the prostate, ovary and testis, contrasting with the predominant expression of ERa in the uterus and the mammary gland, which may suggest different biological roles [10,12]. Transfection experiments with ERE containing reporters indicated, however, that there is no difference in E2 dependent transactivation
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through the two types of receptor, even through it was found that certain ligands such as genistein and coumestrol have higher affinity for ERb. Differences in biological roles between two receptors may be associated with differential interactions among ER, ERE and transcriptional cofactors. On the other hand, a difference was very evident when E2 dependent responses in the collagenase promoter containing AP-1 motifs were examined [14]. In this model, it was found that E2 could enhance the transcription only with ERa but not with ERb. Interestingly partial estrogen antagonists or AF-1 agonists such as tamoxifen and nafoxidin, which are known to induce collagenase promoter responses through ERa, also did activate the transcription through ERb. The present study with the consensus AP-1 reporter confirmed previous findings and sug-
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gested the AP-1 pathway functions generally in E2 responsive transactivation. We further examined the AP-1 responses through ERs in MCF-7 cells which expressed preferentially ERa and which should provide a biologically more relevant model. In the cell line transfected with the AP-1 reporter, E2 was able to induce AP-1 mediated transactivation through endogenously expressed ERa. In the collagenase promoter model, a pure antiestrogen, ICI, also was reported to enhance the response. With our reporter, this could only be confirmed in NIH 3T3 cells transfected with ERa, which may indicate the specificity of the collagenase reporter or may be due to variations of cell lines. Further investigations are needed. Recently, several lines of evidence have indicated that ERb acts as a modulator of ERa or may have a
Fig. 3. Transactivation of pAP1-luc in NIH 3T3 cells transfected with ERa and ERb by E2, tamoxifen and ICI. Transcription activity of AP-1 with ERa (A) and ERb (C) in NIH 3T3 cells. The ERE response was examined with ERa (B) and ERb (D) for comparison. NIH 3T3 cells were transiently transfected with pAP1-luc or p(ERE)3-SV40-luc along with expression plasmids for hERa or hERb, then treated with E2, OH-tamoxifen (OH-TAM) and ICI182,782 (ICI) at the concentrations indicated, and harvested 24 h later. The responses are expressed as fold changes relative to the control activity (Mean 9 S.E.M.). * and ** indicate values that are significantly different from control values at P B0.05 and P B 0.01, respectively.
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Fig. 4. Transactivation of pAP1-luc by E2 in NIH 3T3 cells transfected with ERa and ERb at different ratios. NIH 3T3 cells were transiently transfected with pAP1-luc along with expression plasmids for hERa and hERb, then treated with E2 at 10 − 9 M (A) or OH-TAM at 10 − 7M (C), and harvested 24 h later. (ERE)3-SV40-luc was transfected for comparison (B, D). The responses are expressed as fold changes relative to each control activity (Mean 9S.E.M.). * and ** indicate values are significantly different from the responses with ERa alone at P B0.05 and PB 0.01, respectively.
suppressive role against its proliferative functions [19– 21]. Our results are in favor of the concept that ERa and ERb can act in opposition, since ERa – AP-1 responses were suppressed by co-transfection of ERb in NIH 3T3 cells. No synergistic or dominant-negative effects of ERb were indicated. Importantly AP-1 mediated responses through endogenous ERa in MCF-7 cells were also interfered with transfection of ERb in a dose dependent manner. The mechanism of ER action at AP-1 sites is not well understood. Earlier studies with a chicken IGF-1 promoter which contains the AP-1 motif showed that E2 enhances the binding of c-fos/c-jun to the AP-1 motif [8]. However, our gel mobility shift assay using six tandemly repeated AP-1 motifs did not show any increase in the shifted band by E2 (data not shown). It is possible that both E2 and tamoxifen induce c-fos/c-jun proteins in general, but that was also not the case in our model system as shown in Fig. 2. The major structural differences between ERa and ERb are in the
N-terminus A/B domain, which is shorter in ERb. It is well known that ERa contains two different activation function domains, AF-1 in the N-terminus A/B domain and AF-2 in the ligand binding domain. Partial estrogen antagonists suppress the latter but activate AF-1 to induce some agonistic effects at ERE sites [22]. Previous investigations with the collagenase promoter have suggested that, with AP-1 mediated transactivation, the A/B domain of ERb has a suppressive function on E2 dependent stimulation [23]. Besides AP-1 mediated actions, several other mechanisms have been reported for E2 dependent transactivation through ER without ERE interaction. Alu DNA repeats may function as ER dependent transcription enhancers [24]. The NF-Y-Sp-1 complex is known to be essential for E2F1 gene regulation by E2 [25] and the Sp-1 promoter element alone is also able to activate transcription depending on liganded ER [26,27]. These findings suggest that E2 dependent transactivation not interacting with ERE accounts for a significant part of
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Fig. 5. Transactivation of pAP1-luc by E2 in MCF-7 cells. (A) MCF-7 cells were transiently transfected with pAP1-luc, then treated with E2, OH-tamoxifen (OH-TAM), ICI182,782 (ICI), and harvested 24 h later. (B) (ERE)3-SV40-luc was transfected for comparison. The ratio of endogenous ERa and ERb in this cell line was 62:1 in terms of the mRNA levels. The responses are expressed as fold changes relative to the control activity (Mean 9 S.E.M.). * and ** indicate values that are significantly different from control values at PB0.05 and PB 0.01, respectively.
E2 dependent gene regulation. Interestingly, substantial transcriptional differences between two ER subtypes (a/b) have been found for AP-1 and Sp-1 mediated systems, rather than for classical ERE mediated model, although more recent studies indicated the clear differential transcription through two receptor subtypes at the complex rat promoter of arginine-vasopressin containing EREs as well as AP-1 sites [28]. Moreover, in the present study, we showed that suppressive effects of ERb co-existing with ERa were also seen only with AP-1 mediated transactivation. Estrogen responses in vivo should consist of both classical ERE and non-ERE mediated responses. Both types of ER may act similarly
in the former and the balance of the two subtypes may be more important for transactivation in the latter case.
Acknowledgements We thank Ms K. Hashimoto for her expert technical assistance, Dr J. Kanno for providing ICI182,782, and Dr M.A. Moore for reading the manuscript and suggesting English clarification and JCRB for providing NIH 3T3 cells. This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare, Japan.
References
Fig. 6. Effects of co-transfection of ERb on pAP1-luc transactivation by E2 in MCF-7. MCF-7 cells were transiently transfected with pAP1-luc and various amounts of a hERb expression plasmid, then treated with E2 at 10 − 9 M, and harvested 24 h later. The responses are expressed as fold changes relative to the control activity (Mean 9 S.E.M.). * and ** indicate values that are significantly different from the control values at P B 0.05 and PB 0.01, respectively.
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Suppression by estrogen receptor b of AP-1 mediated transactivation through estrogen receptor a Satoshi Maruyama, Nariaki Fujimoto *, Kohsuke Asano, Akihiro Ito Department of Cancer Research, Research Institute for Radiation Biology and Medicine (RIRBM), Hiroshima Uni6ersity, 1 -2 -3 Kasumi, Minami-ku, Hiroshima 734 -8553, Japan Accepted 26 March 2001
Abstract The estrogen receptor (ER) is known to mediate gene transcription from AP-1 enhancer elements as well as the well-documented estrogen responsive elements (EREs). Investigations of AP-1 mediated transactivation through ER have been performed with rather complex promoters such as insulin like growth factor 1 (IGF-1) and collagenase promoters. In the present study, we investigated AP-1 mediated transactivation through ERa and ERb with a less complicated reporter consisting of only consensus AP-1 motifs. NIH 3T3 cells were transiently transfected with human ERa and ERb expression plasmids and AP-1-luc and ERE-luc reporters. 17b-Estradiol failed to activate ERb-AP-1 responses while activating ERa-AP-1, ERa-ERE, ERb-ERE mediated transcription. On the other hand, antiestrogens such as tamoxifen enhanced AP-1 mediated transactivation through both ERa and ERb. An ERa positive human breast cancel cell line, MCF-7, also showed the same manner of AP-1 mediated transactivation through ERa. When NIH 3T3 with ERa and MCF-7 were co-transfected with ERb, E2 dependent AP-1 responses decreased in both cell lines depending on the amount of the ERb expression plasmid. These results suggest that ERa and ERb may function in opposition with ERb actually suppressing the function of ERa in AP-1 mediated transactivation. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Estrogen receptor a and b; Differential transcription; Consensus AP-1 motif
1. Introduction The estrogen receptor (ER) is a conditional transcription factor, which belongs to the nuclear receptor superfamily and activates transcription of the specific genes only when a ligand is bound [1 – 3]. An estrogen responsive element (ERE), a DNA motif with palindromic structure, has been identified as a binding site for ER in the promoter region of several estrogen responsive genes such as these for vitellogenin and prolactin [4]. Once ER binds to EREs, it recruits a p160/p300 coactivator complex and enhances gene expression by interactions with the basic transcriptional machinery [5]. However, several lines of evidence have shown that estrogen can also enhance transcription from gene promoters containing the AP-1 motif which * Corresponding author. Tel.: + 81-82-257-5820; fax: +81-82-2567107. E-mail address: [email protected] (N. Fujimoto).
is a binding site for c-fos/c-jun protein and also known to be the TPA responsive element. It has been reported that AP-1 sites in the promoter region are involved in E2 dependent transcription of collagenase, insulin-like growth factor 1 (IGF-1) and ovalbumin genes [6–9]. Recently a novel type of ER was cloned and designated as ERb [10,11]. Differences in structure and tissue distribution between ERa and ERb suggest different biological roles of the two receptor subtypes. Transfection experiments with ERE containing reporters have indicated, however, that there is no difference in E2 dependent transactivation through the two types of receptor, even through some ligands such as genistein and coumestrol have a higher affinity for ERb [12,13]. When the E2 dependent response of the collagenase promoter which contains AP-1 motifs was examined, it was observed only with ERa but not with ERb. Interestingly partial estrogen antagonists or AF-1 agonists such as tamoxifen, which have known to induce ERa-AP-1 responses, also did activate ERb-AP-1 medi-
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ated transcription. Although these findings suggested a biological importance of AP-1 mediated E2 dependent transactivation especially for understanding functional differences between the two types of ER, very limited numbers of models have been examined and all of them involve rather complex promoters. In the present study, we therefore reconstructed and examined a model for E2 dependent AP-1 mediated transactivation through ERs using a reporter containing only consensus AP-1 motifs.
2. Materials and methods
2.1. Hormones 17b-Estradiol (E2) and OH-tamoxifen (OH-TAM) were purchased from Sigma Chemicals, St. Louis and dissolved in ethanol to give stock solutions. ICI182,782 (ICI) was generously provided by Zeneca KK (Hyogo, Japan).
2.2. Cell culture The NIH 3T3 cell line was obtained from the Japanese Collection of Research Bioresources (JCRB) and maintained in DME (Sigma Chemicals) containing penicillin and streptomycin with 10% calf serum (CS, Life Technologies, Rockville, MD, USA). For hormone treatments, the medium was changed to phenol red free DME (Sigma Chemicals) containing the same antibiotics along with dextran-charcoal treated CS for a week. The MCF-7 cell line was maintained in MEM (Sigma Chemicals) containing penicillin and streptomycin with 5% fetal bovine serum (FBS, Life Technologies). The medium was changed to phenol red free MEM (Sigma Chemicals) containing the antibiotics and dextran-charcoal treated FBS for a week.
2.3. Plasmid construction A reporter plasmid, pAP1-luc, which contains six tandem copies of the AP-1 enhancer. 5%-(TGACTAA)6, connected to a TATA box located upstream from the luciferase gene [15], and its control reporter lacking AP-1 motifs, pTA-luc, were purchased from Clontech (Palo Alto, CA, USA). The (ERE)3-SV40-luc plasmid, which contains 5%-CGGTCACAGTGACCAGTGGTCACGTGA-CCAGTC GGTCACAGTGACCC was a gift from Dr M. Kudoh, Yamanouchi Pharmaceutical Co., Tsukuba, Japan [16]. Human ERa and hERb, gifts from Dr S. Kato at Tokyo University, Tokyo), were inserted into the EcoRI site of the pSG5 expression vector, to give pSG5-hERa and pSG5-hERb, respectively. PRL-CMV (Promega, Madison, WI, USA) was used as the internal control.
2.4. Transient transfection with expression and reporter plasmids NIH 3T3 cells were plated at 1× 105/well in 12 well plates (Nalge Nunc International, Rochester, NY, USA). After 24 h, cells in each well were co-transfected with total 2.0 mg of plasmid DNA with TransFast transfection reagent containing a synthetic cationic lipid (Promega) following the supplied protocol. The weight ratio of TransFast reagent to DNA was 1:1. After 24 h incubation with hormones, cells were harvested with 100 ml of cell lysis buffer (Promega) and 10 ml of each lysate was mixed with firefly luciferase substrate followed by mixing with renilla luciferase substrate to determine luciferase activities (Dual-luciferase reporter assay system, Promega). Luminescence was measured with a Micro-beta scintillation counter (Amersham Pharmacia Biotech, Uppsala, Sweden). Firefly luciferase activity was normalized to renilla luciferase activity from pRL-CMV.
2.5. Immunoblot detection of c-fos and c-jun For immunoblot analysis, cells were lysed in NP40 lysis buffer (10 mM NaMo, 5 mM EDTA, 300 mM NaCl, 50 mM Tris–HCl, pH 8.0, 0.5% NP40, 0.2% Sarcosyl) with 0.2 mM PMSF. Aliquots (100 mg) of lysate protein were applied for 10% SDS-polyacrylamide gel electrophoresis. The bands were then transferred to P-bound nitrocellulose filters (Amersham Pharmacia Biotech, Uppsala, Sweden) with an electric transblot apparatus and immunostained with an ECL kit (Amersham Pharmacia Biotech) according to the company’s recommended procedure. Polyclonal anti-cfos and anti-c-jun antibodies were purchased from Santa Cruz Biotechnology, CA, USA for this purpose. The second antibody, peroxidase labeled anti-rabbit IgG, was from MBL, Nagoya, Japan. Developed bands were scanned with an image scanner and intensities were quantified using Scion Image software (Scion Corp., Frederick, MD, USA).
2.6. Re6erse transcription-polymerase chain reaction (RT-PCR) detection of ERs This was performed as described previously [17]. Briefly, total RNAs from cells were prepared by a modified acid guanidium thiocyanate-phenol–chloroform extraction method and treated with RQ1 DNase (Promega). One microgram of total RNA was reversetranscribed with 100 U of MMLV-RT (Life Technologies) and 1.25 pmol of oligo-dT primers. Amplification was carried out with Ex-Taq DNA polymerase (Takara, Tokyo, Japan) in the presence of various amounts of competitor DNAs.
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2.7. Statistical analysis Statistical comparisons were made using the Student’s t-test.
3. Results
3.1. ER dependence of AP1 -luc transacti6ation by E2 E2 responsive AP1-luc transcription was activated only when the ER expression plasmid, pSG5-hERa was co-transfected in NIH 3T3. The maximum response of 2.8-fold induction was found at 10 − 9 M of E2 (Fig. 1A). With the same cell line, induction of the (ERE)3SV40-luc response by E2 was also low, approximately 3.7 times the control level (Fig. 1B). Fig. 1C shows that E2 dependent AP-1 responses increase according to the amount of pSG5-hERa co-transfected.
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3.2. Effects of E2 on c-fos and c-jun protein le6els in NIH 3T3 cells Fig. 2 indicates time dependent changes in c-fos and c-jun proteins levels in NIH 3T3 cells transfected with either ERa or ERb and treated with E2. Immunoblot analysis showed that amounts of both proteins to be unaltered after hormone treatment. More importantly there was no difference in c-fos/c-jun levels between the cells transfected with ERa and ERb.
3.3. AP-1 mediated transcription in NIH 3T3 cells co-transfected with ERh and ERi E2 exerted effects on both ERE and AP-1 mediated transcription when cells were co-transfected with pSG5hERa. However, OH-TAM activated only AP-1 mediated transcription (Fig. 3A, B). The ICI compound also activated AP-1 response while suppressing the ERE dependent response to below the control level. When
Fig. 1. Dependency of transactivation of pAP1-luc on ER. (A) E2 dependent transcription activity of AP-1 with and without ER in NIH 3T3 cells. NIH 3T3 cells were transiently transfected with pAP1-luc along with an expression plasmid for hERa, then treated with E2 at the concentrations indicated, and harvested 24 h later. The responses are expressed as fold changes in the activity relative to the control level (mean 9 S.E.M.). (B) E2 dependent transactivation through ERE for comparison. The plasmid, p(ERE)3-SV40-luc, was used instead of pAP1-luc. (C) Effects of amounts of ER expression plasmid on AP-1 transcription activity. The responses are expressed as fold changes based on the activity of the cells without ER transfection or E2 treatment. * and ** indicated values are significantly different from control values at PB 0.05 and PB 0.01, respectively.
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both E2 and OH-TAM activated the AP-1 mediated response in a dose dependent manner, while the ERE response was evoked only with E2 (Fig. 5). ICI did not stimulate any response with either AP-1 or ERE reporters. When ERb was co-transfected in MCF-7, E2 dependent transcription of pAP-1-luc was suppressed, depending on the amount of the ERb expression plasmid (Fig. 6).
4. Discussion
Fig. 2. Time dependent changes in c-fos and c-jun protein levels in NIH 3T3 cells after E2 treatment. NIH 3T3 cells were transfected with hERa and hERb and treated with E2 at 10 − 9 M. After the time periods as indicated, cells were harvested and whole cell lysates were separated with SDS-PAGE and transferred to nitrocellulose filters. C-fos and c-jun were detected with ECL reagents and quantified with an image analyzer. The amounts are expressed as fold changes relative to the control level.
cells were transfected with ERb E2 did not initiate AP-1 dependent transactivation, while the ERE response was activated to the same level as with ERa (Fig. 3C, D). OH-TAM caused AP-1 mediated transcription with ERb but did not initiate ERE transactivation. With ERb, interestingly, ICI failed to activate either AP-1 or ERE dependent transactivation. Responses to E2 were further examined with cells transfected with both ERa and ERb at different ratios. In Fig. 4A, AP-1 mediated responses decreased as the transfected amount of ERb increased. On the other hand, with the (ERE)3-SV40-luc reporter, the response to E2 was independent of the ERa/ERb ratio (Fig. 4B). OH-TAM activated AP-1 response at any ERa/b ratio but did not induce ERE mediated transcription (Fig. 4C, D).
3.4. AP-1 mediated transcription in MCF-7 cells AP-1 responses were examined in a human breast cancer cell line, MCF-7, in comparison with ERE responses. RT-PCR detection for ERa and ERb showed that MCF-7 cells in the present study preferentially expressed ERa. The expressed mRNA levels for ERa and ERb were 2.29 0.19 fg/mg total RNA and 0.0289 0.004 fg/mg total RNA, respectively. In this cell line,
In the present study, for the first time, we reconstructed a model system for AP-1 mediated E2 dependent transcription with a simple AP-1 reporter containing only consensus AP-1 motifs. An E2 dependent response with the AP-1 reporter in NIH/3T3 cells was only found with ERa but not with ERb, while tamoxifen enhanced AP-1 mediated transcription through both ERa and ERb. The results are consistent with data for the collagenase promoter containing AP-1 motifs previously described [14]. There was a discrepancy between our findings and those for the collagenase promoter, however, in response to ICI. No response was noted in our model with ERb but high induction was found with the collagenase promoter through both ERa and ERb. This ICI induction may be due to the interaction at the other transcriptional motifs in the complex collagenase promoter or, simply, due to the difference between cell lines used. It is well known that ER is a conditional transcription factor which belongs to the nuclear receptor superfamily. When ligands bind to ER, the conformation is changed so that interaction with ERE motifs of DNA is facilitated. The liganded receptor at the ERE recruits several transcriptional cofactors including p160/p300 complexes to initiate transcription of specific genes. A few genes such as collagenase and IGF-1 are known to be regulated by estrogen (E2) without containing any ERE motifs in their gene promoters and it has been found that this E2 inducible gene regulation is mediated through the AP-1 motif via interactions of ER and AP-1 [6 –8]. In general, however, AP-1 and nuclear receptor mediated activities are known to interfere with each other, possibly because of interaction with overlapping binding sites [18]. In 1997, a novel type of ER was cloned and designated as ERb. Besides structural differences, the tissue distribution of this new receptor subtype is very different from that of ERa. In rats, high expression of ERb was found in the prostate, ovary and testis, contrasting with the predominant expression of ERa in the uterus and the mammary gland, which may suggest different biological roles [10,12]. Transfection experiments with ERE containing reporters indicated, however, that there is no difference in E2 dependent transactivation
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through the two types of receptor, even through it was found that certain ligands such as genistein and coumestrol have higher affinity for ERb. Differences in biological roles between two receptors may be associated with differential interactions among ER, ERE and transcriptional cofactors. On the other hand, a difference was very evident when E2 dependent responses in the collagenase promoter containing AP-1 motifs were examined [14]. In this model, it was found that E2 could enhance the transcription only with ERa but not with ERb. Interestingly partial estrogen antagonists or AF-1 agonists such as tamoxifen and nafoxidin, which are known to induce collagenase promoter responses through ERa, also did activate the transcription through ERb. The present study with the consensus AP-1 reporter confirmed previous findings and sug-
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gested the AP-1 pathway functions generally in E2 responsive transactivation. We further examined the AP-1 responses through ERs in MCF-7 cells which expressed preferentially ERa and which should provide a biologically more relevant model. In the cell line transfected with the AP-1 reporter, E2 was able to induce AP-1 mediated transactivation through endogenously expressed ERa. In the collagenase promoter model, a pure antiestrogen, ICI, also was reported to enhance the response. With our reporter, this could only be confirmed in NIH 3T3 cells transfected with ERa, which may indicate the specificity of the collagenase reporter or may be due to variations of cell lines. Further investigations are needed. Recently, several lines of evidence have indicated that ERb acts as a modulator of ERa or may have a
Fig. 3. Transactivation of pAP1-luc in NIH 3T3 cells transfected with ERa and ERb by E2, tamoxifen and ICI. Transcription activity of AP-1 with ERa (A) and ERb (C) in NIH 3T3 cells. The ERE response was examined with ERa (B) and ERb (D) for comparison. NIH 3T3 cells were transiently transfected with pAP1-luc or p(ERE)3-SV40-luc along with expression plasmids for hERa or hERb, then treated with E2, OH-tamoxifen (OH-TAM) and ICI182,782 (ICI) at the concentrations indicated, and harvested 24 h later. The responses are expressed as fold changes relative to the control activity (Mean 9 S.E.M.). * and ** indicate values that are significantly different from control values at P B0.05 and P B 0.01, respectively.
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Fig. 4. Transactivation of pAP1-luc by E2 in NIH 3T3 cells transfected with ERa and ERb at different ratios. NIH 3T3 cells were transiently transfected with pAP1-luc along with expression plasmids for hERa and hERb, then treated with E2 at 10 − 9 M (A) or OH-TAM at 10 − 7M (C), and harvested 24 h later. (ERE)3-SV40-luc was transfected for comparison (B, D). The responses are expressed as fold changes relative to each control activity (Mean 9S.E.M.). * and ** indicate values are significantly different from the responses with ERa alone at P B0.05 and PB 0.01, respectively.
suppressive role against its proliferative functions [19– 21]. Our results are in favor of the concept that ERa and ERb can act in opposition, since ERa – AP-1 responses were suppressed by co-transfection of ERb in NIH 3T3 cells. No synergistic or dominant-negative effects of ERb were indicated. Importantly AP-1 mediated responses through endogenous ERa in MCF-7 cells were also interfered with transfection of ERb in a dose dependent manner. The mechanism of ER action at AP-1 sites is not well understood. Earlier studies with a chicken IGF-1 promoter which contains the AP-1 motif showed that E2 enhances the binding of c-fos/c-jun to the AP-1 motif [8]. However, our gel mobility shift assay using six tandemly repeated AP-1 motifs did not show any increase in the shifted band by E2 (data not shown). It is possible that both E2 and tamoxifen induce c-fos/c-jun proteins in general, but that was also not the case in our model system as shown in Fig. 2. The major structural differences between ERa and ERb are in the
N-terminus A/B domain, which is shorter in ERb. It is well known that ERa contains two different activation function domains, AF-1 in the N-terminus A/B domain and AF-2 in the ligand binding domain. Partial estrogen antagonists suppress the latter but activate AF-1 to induce some agonistic effects at ERE sites [22]. Previous investigations with the collagenase promoter have suggested that, with AP-1 mediated transactivation, the A/B domain of ERb has a suppressive function on E2 dependent stimulation [23]. Besides AP-1 mediated actions, several other mechanisms have been reported for E2 dependent transactivation through ER without ERE interaction. Alu DNA repeats may function as ER dependent transcription enhancers [24]. The NF-Y-Sp-1 complex is known to be essential for E2F1 gene regulation by E2 [25] and the Sp-1 promoter element alone is also able to activate transcription depending on liganded ER [26,27]. These findings suggest that E2 dependent transactivation not interacting with ERE accounts for a significant part of
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Fig. 5. Transactivation of pAP1-luc by E2 in MCF-7 cells. (A) MCF-7 cells were transiently transfected with pAP1-luc, then treated with E2, OH-tamoxifen (OH-TAM), ICI182,782 (ICI), and harvested 24 h later. (B) (ERE)3-SV40-luc was transfected for comparison. The ratio of endogenous ERa and ERb in this cell line was 62:1 in terms of the mRNA levels. The responses are expressed as fold changes relative to the control activity (Mean 9 S.E.M.). * and ** indicate values that are significantly different from control values at PB0.05 and PB 0.01, respectively.
E2 dependent gene regulation. Interestingly, substantial transcriptional differences between two ER subtypes (a/b) have been found for AP-1 and Sp-1 mediated systems, rather than for classical ERE mediated model, although more recent studies indicated the clear differential transcription through two receptor subtypes at the complex rat promoter of arginine-vasopressin containing EREs as well as AP-1 sites [28]. Moreover, in the present study, we showed that suppressive effects of ERb co-existing with ERa were also seen only with AP-1 mediated transactivation. Estrogen responses in vivo should consist of both classical ERE and non-ERE mediated responses. Both types of ER may act similarly
in the former and the balance of the two subtypes may be more important for transactivation in the latter case.
Acknowledgements We thank Ms K. Hashimoto for her expert technical assistance, Dr J. Kanno for providing ICI182,782, and Dr M.A. Moore for reading the manuscript and suggesting English clarification and JCRB for providing NIH 3T3 cells. This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare, Japan.
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Fig. 6. Effects of co-transfection of ERb on pAP1-luc transactivation by E2 in MCF-7. MCF-7 cells were transiently transfected with pAP1-luc and various amounts of a hERb expression plasmid, then treated with E2 at 10 − 9 M, and harvested 24 h later. The responses are expressed as fold changes relative to the control activity (Mean 9 S.E.M.). * and ** indicate values that are significantly different from the control values at P B 0.05 and PB 0.01, respectively.
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