An Activator Protein 1-Like Motif Mediates 17ß-Estradiol Repression of Gonadotropin-Releasing Hormone Receptor Promoter via an Estrogen Receptor {alpha}-Dependent Mechanism in Ovarian and Breast Cancer Cells

Chi Keung Cheng, Billy K. C. Chow and Peter C. K. Leung

Department of Obstetrics and Gynecology, University of British Columbia (C.K.C., P.C.K.L.), Vancouver, Canada V6H 3V5; and Department of Zoology, University of Hong Kong (B.K.C.C.), Hong Kong, China

Address requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, 2H30-4490 Oak Street, British Columbia Women’s Hospital, Vancouver, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although it is recognized that estrogen is one of the most important regulators of GnRH receptor (GnRHR) gene expression, the mechanism underlying the regulation at the transcriptional level is unknown. In the present study, we demonstrated that 17ß-estradiol (E2) repressed human GnRHR promoter via an activator protein 1-like motif and estrogen receptor-{alpha}, of which the DNA-binding domain and the ligand-binding domain were indispensable for the repression. Interestingly, the same cis-acting motif was also found to be important for both the basal activity and phorbol 12-myristate 13-acetate responsiveness of the GnRHR promoter. EMSAs indicated that multiple transcription factors including c-Jun and c-Fos bound to the activator protein 1-like site and that their DNA binding activity was not significantly affected by E2 treatment. In addition, we demonstrated that the E2 repression could be antagonized by phorbol 12-myristate 13-acetate, which stimulated c-Jun phosphorylation on serine 63, a process that is a prerequisite for recruitment of the transcriptional coactivator cAMP response element binding protein (CREB)-binding protein (CBP). Concomitantly, we found that overexpression of CBP could reverse the suppression in a dose-dependent manner. Taken together, our data indicate that E2-activated estrogen receptor-{alpha} represses human GnRHR gene transcription via an indirect mechanism involving CBP and possibly other transcriptional regulators.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IN ADDITION TO its pivotal role in stimulating gonadotropin secretion, GnRH has been shown to function as an autocrine and/or paracrine regulator in certain extrapituitary tissues (1, 2, 3, 4, 5). One of the well established autocrine actions of GnRH is to suppress the proliferation of hormone-sensitive tumors derived from various reproductive tissues such as the ovary, endometrium, and prostate (6). Because the growth of these cancer cells can be significantly inhibited by nanomolar concentrations of GnRH analogs, it is believed that the antiproliferative action of the hormone is mediated via the high-affinity GnRH receptor (GnRHR), as supported by the fact that the nucleotide sequence of the receptor in these tissues is identical to that in the pituitary (7). Modulation of the mitogenic signal transduction pathways has been implicated to be one of the major mechanisms by which GnRH exerts its growth-inhibitory effect in these tumors (6). For instance, it has been demonstrated that the GnRH agonist leuprolide mediates its antiproliferative effect in ovarian adenocarcinoma Caov-3 cells via a sustained stimulation of the ERK signaling cascade, leading to dephosphorylation of the retinoblastoma protein (8), an event that is known to prevent cell cycle progression from G1 to S phase. Alternatively, GnRH agonists have been shown to be capable of antagonizing growth factor-induced mitogenic signaling in certain ovarian and endometrial cancer cell lines, thereby suppressing their proliferation (9). Other mechanisms in mediating the antiproliferative actions of GnRH or its analogs in cancer cells include inhibition of phosphatidylinositol kinase activity (10) and down-regulation of telomerase reverse transcriptase and vascular permeability factor expression (11, 12).

Estrogen has been implicated in the pathogenesis and positive growth regulation of carcinomas arising from the ovary, breast, and uterus (13, 14, 15, 16, 17, 18). Previous studies from our laboratory have shown that E2 can inhibit GnRHR mRNA expression in a dose- and time-dependent manner in ovarian carcinoma OVCAR-3 cells via a receptor-mediated mechanism (19). A similar down-regulatory effect of E2 on the GnRHR mRNA level has also been observed in primary-cultured granulosa-luteal cells (20). Accordingly, pretreatment or cotreatment with E2 has been found to antagonize the antiproliferative effect of GnRH in OVCAR-3 cells (19), suggesting the existence of a functional interaction between the GnRH/GnRHR and E2/estrogen receptor (ER) systems such that the balance of antiproliferative and proliferative signals from these pathways may play an essential role in regulating tumor cell growth. However, the molecular mechanism underlying this E2-mediated inhibition of the GnRHR gene expression has not been clearly addressed to date.

The human GnRHR 5'-flanking region is characterized by the presence of multiple promoter elements that direct tissue-specific expression of the gene (21, 22, 23). In addition, a number of hormone response cis-acting motifs have been identified in the flanking region (24, 25). Recently, we have demonstrated that a putative progesterone response element located between nucleotide (nt) -535 and -521 is responsible for mediating the differential activity of progesterone receptor-A and -B isoforms in transcriptional regulation of the human GnRHR gene (26). On the contrary, a search on the 5'-regulatory region of the mammalian GnRHR genes has revealed no consensus estrogen response element (ERE) (25, 27, 28), which is a palindrome of A/GGGTCA motifs separated by 3 bp. Until now, transcriptional regulation of GnRHR genes by estrogen has been a far-from-understood issue. Earlier studies on the proximal 1.9-kb 5'-flanking region of the mouse GnRHR gene have failed to demonstrate any E2 regulation in vivo (29). Intriguingly, it was later shown that E2 responsiveness of the ovine GnRHR promoter was undetectable in vitro but was only revealed in transgenic mice (30), suggesting that the in vivo model may represent one of the few viable avenues for defining the mechanism mediating E2 regulation of GnRHR gene expression. Nevertheless, it remains unclear whether this phenomenon is also applicable to other mammalian GnRHR promoters. In the present study, we demonstrated for the first time that E2 could repress human GnRHR gene transcription via an AP-1-like motif located in the proximal promoter in the ovarian cancer OVCAR-3 and breast cancer MCF-7 cells. Also, we showed that the repression was mediated by ER{alpha} and involved the transcriptional coactivator cAMP response element binding protein (CREB)-binding protein (CBP).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E2 Repression of the GnRHR Promoter Requires ER{alpha} But Not ERß
To investigate whether E2 down-regulates GnRHR gene expression at the transcriptional level, the human GnRHR promoter-luciferase construct p(-1671/+1)-Luc was transiently cotransfected with expression plasmids encoding ER{alpha}, ERß, ER{alpha} and ERß, or progesterone receptor-B into OVCAR-3 cells, followed by stimulation with 100 nM E2 for 24 h. As shown in Fig. 1AGo, E2 treatment had no apparent effect on the GnRHR promoter when either the empty pCMV5 or pSG5-PR-B expression vector was introduced into the cells. However, a 46% reduction (P < 0.001 vs. control) of promoter activity was observed when ER{alpha} was forcibly expressed in the cancer cells. In contrast, overexpression of ERß produced an insignificant reduction of promoter activity, suggesting the involvement of ER{alpha}, but not ERß, in mediating the repression. Because it has been demonstrated that ERß can serve as a transdominant repressor of ER{alpha} transcriptional activity (31), we cotransfected both ER subtypes simultaneously into OVCAR-3 cells to investigate any functional interference of ER{alpha} activity by ERß or vice versa. Our present data revealed that an elevated expression of ERß did not significantly alter the overall sensitivity of the ER{alpha}-overexpressing cells to E2 (Fig. 1AGo) although it has been reported that coexpression of ER{alpha} and ERß results in the preferential formation of receptor heterodimers instead of homodimers that may possess unique transcriptional activities (31, 32, 33). The differential transrepressing activity observed for ER{alpha} and ERß is unlikely due to different transfection/translation efficiency of the ER expression plasmids, as Western blot analysis revealed similar levels of the two ER subtypes after cotransfection (data not shown). The specificity of the E2 action was demonstrated by the observation that repression of the GnRHR promoter could be attenuated by the ER antagonist tamoxifen (Fig. 1BGo), which by itself had no apparent effect on the promoter activity.



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Fig. 1. ER{alpha}, But Not ERß, Mediates E2 Repression of the GnRHR Promoter

A, The human GnRHR promoter-luciferase construct p(-1671/+1)-Luc was transiently transfected with expression plasmids encoding various steroid hormone receptors into OVCAR-3 cells by LIPOFECTAMINE reagent. The RSV-lacZ vector was also cotransfected to normalize the transfection efficiency. The cells were treated 24 h after transfection with 100 nM E2 under serum- and phenol red-free conditions for an additional 24 h before harvest. Lane 1, 1 µg of empty pCMV5; lane 2, 1 µg pCMV5-ER{alpha}; lane 3, 1 µg pCMV5-ERß; lane 4, 0.5 µg each of pCMV5-ER{alpha} and pCMV5-ERß; lane 5, 1 µg each of pCMV5-ER{alpha} and pCMV5-ERß; lane 6, 1 µg pSG5-PR-B. B, The ER antagonist tamoxifen attenuates E2-dependent repression of the GnRHR promoter. The construct p(-1671/+1)-Luc was cotransfected with pCMV5-ER{alpha} and RSV-lacZ into OVCAR-3 cells. The cells were treated 24 h after transfection with 100 nM E2, 100 nM tamoxifen (T), or 100 nM E2 and 100 nM T for 24 h. C, Upper level, Western blot analysis of the endogenous ER{alpha} and ERß proteins in OVCAR-3 (O) and MCF-7 (M) cells. Lower panel, Quantitative comparison of the ER{alpha} protein level in the ovarian and breast cancer cell lines by densitometry. D, The construct p(-1671/+1)-Luc was transiently cotransfected with RSV-lacZ into OVCAR-3 and MCF-7 cells. Twenty-four hours after transfection, the cells were treated with 100 nM E2 for 24 h. E, As a control experiment, the reporter plasmid ERE2-tk109-Luc containing two copies of ERE was cotransfected with 1 µg of either pCMV5-ER{alpha} or pCMV5-ERß into OVCAR-3 cells. The cells were treated 24 h after transfection with 100 nM E2 for 48 h. The relative promoter activity is represented as the percentage of the respective control group (vehicle-treated), of which the activity is set as 100% after being normalized by ß-galactosidase activity. Values in all panels represent the mean ± SEM of three independent experiments each performed in triplicate. a, P < 0.001 vs. control; b, P < 0.05 vs. control; c, P < 0.001 vs. E2; d, P < 0.05 vs. OVCAR-3 cells.

 
The functional importance of ER{alpha}, but not ERß, in mediating the repressive effect of E2 on the GnRHR promoter was further demonstrated by using the MCF-7 breast adenocarcinoma cell line, which was found to express a significantly higher level of ER{alpha} protein than OVCAR-3 cells (Fig. 1CGo). In stark contrast, the expression level of ERß in MCF-7 cells was virtually undetectable (Fig. 1CGo), and similar observations have also been reported elsewhere (34). Treatment of MCF-7 cells with 100 nM E2 for 24 h resulted in a 26% reduction (P < 0.001 vs. control) of promoter activity, whereas the same treatment exerted no apparent effect in OVCAR-3 cells in the absence of ER{alpha} overexpression (Fig. 1DGo). These findings thus implicate that a threshold level of ER{alpha} may be required to confer negative E2 regulation of the GnRHR promoter. In support of this speculation, we found that the repressive effect of E2 on the GnRHR promoter in OVCAR-3 cells was ER{alpha} dose dependent and that the optimal repression was achieved when 1 µg pCMV5-ER{alpha} was cotransfected (data not shown). This amount was then used in all subsequent experiments.

To rule out the possibility that E2 nonspecifically suppressed the GnRHR promoter, we cotransfected the reporter plasmid ERE2-tk109-Luc with either pCMV5-ER{alpha} or pCMV5-ERß into OVCAR-3 cells and then treated the cells with or without the estrogen. The reporter plasmid ERE2-tk109-Luc contains two copies of the vitellogenin gene ERE upstream of a 109-bp fragment of the thymidine kinase promoter and has been described previously (35). The activity of the heterologous promoter was found to increase by about 2.1-fold and 1.5-fold in response to E2 treatment when ER{alpha} and ERß were overexpressed in the cells, respectively (Fig. 1EGo). These results thus indicate that E2 selectively represses the GnRHR promoter via an ER{alpha}-dependent mechanism.

At low E2 concentrations (10-13 and 10-11 M), no significant decrease in the transcriptional activity of the GnRHR promoter could be observed in the ER{alpha}-overexpressing OVCAR-3 cells (Fig. 2AGo). However, a dose-dependent repression of promoter activity was detected when the cells were treated with an increasing concentration of the steroid hormone (10-9 to 10-7 M) (Fig. 2AGo). Repression of the GnRHR promoter by E2 was also time dependent. Short-term E2 treatment (4 h and 8 h) had no apparent effect on the GnRHR promoter (Fig. 2BGo). A significant inhibition of promoter activity was only evident after 12 h, and repression was found to increase with time, with a maximal of 80% reduction being observed after 48 h of treatment (Fig. 2BGo).



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Fig. 2. Dose- and Time-Dependent Effects of E2 on the GnRHR Promoter Activity in ER{alpha}-Overexpressing OVCAR-3 Cells

The construct p(-1671/+1)-Luc was transiently cotransfected with pCMV5-ER{alpha} and RSV-lacZ into the cancer cells. The cells were treated 24 h after transfection with different concentrations of E2 for 24 h (A) or with 100 nM E2 for different time periods (B). The relative promoter activity is represented as the percentage of the control group (vehicle treated), of which the activity is set as 100% after being normalized by ß-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate. a, P < 0.001 vs. control; b, P < 0.05 vs. control.

 
Domains of ER{alpha} Required for Repression of the GnRHR Promoter
Human ER{alpha} is a 595-amino acid protein that has been studied extensively by mutational analysis (36, 37, 38). It has been demonstrated that the transcriptional activity of ER{alpha} is mediated by two transactivation functions (AFs) located in the amino terminus (AF-1) and carboxyl terminus (AF-2). Although both AFs work in a synergistic manner in most circumstances, they can also function independently in a cell- and promoter-specific manner (39, 40). To define the ER{alpha} domains required for repression of the GnRHR promoter, expression vectors encoding a series of 5'- and 3'-deletion mutants of the steroid hormone receptor were constructed (Fig. 3AGo) and cotransfected with p(-1671/+1)-Luc into OVCAR-3 cells. Cotransfection of the wild-type receptor (ER{alpha}-wt) caused a 50% reduction of promoter activity in response to E2 (Fig. 3BGo). Deletion of the ligand-binding domain (LBD) created a mutant (ER{alpha}-mut1) that was not able to repress the GnRHR promoter (Fig. 3BGo). In contrast, an ER{alpha} mutant with a deletion of the amino-terminal AF-1 (ER{alpha}-mut2) still mediated E2 inhibition of the GnRHR gene transcription (Fig. 3BGo). Further deletion of the amino terminus up to amino acid 262 (ER{alpha}-mut3) or internal deletion of the DNA-binding domain (DBD) (ER{alpha}-mut4) generated mutants that lose the ability to suppress the GnRHR promoter (Fig. 3BGo). Collectively, these data indicate that both the DBD and LBD are required for the E2-mediated repression of the GnRHR gene transcription.



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Fig. 3. ER{alpha} Domains Required for GnRHR Promoter Repression Overlap Those for Classical Transactivation at ERE

A, Structural domains of the wild-type and mutant human ER{alpha}. Numbers represent the amino acid (aa) positions. B, The construct p(-1671/+1)-Luc was transiently cotransfected with the wild-type or mutant ER{alpha} expression plasmid into OVCAR-3 cells. The cells were treated 24 h after transfection with 100 nM E2 for 24 h. C, For studies of the classical mode of ER transactivation, the reporter plasmid ERE2-tk109-Luc was cotransfected with the wild-type or mutant ER{alpha} expression plasmid. The cells were treated 24 h after transfection with 100 nM E2 for 48 h. The relative promoter activity is represented as the percentage of the respective control group (vehicle treated), of which the activity is set as 100% after being normalized by ß-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate. a, P < 0.001 vs. control; b, P < 0.05 vs. control.

 
In parallel, we also examined the ability of these ER{alpha} mutants to activate the reporter plasmid ERE2-tk109-Luc. As shown in Fig. 3CGo, the AF-1 was dispensable for the activation (ER{alpha}-mut2), whereas both the DBD and LBD were essential for transactivation at ERE since deletion of either of these regions nearly abrogated transactivation of the reporter gene (Fig. 3CGo). Thus, these findings suggest that the same ER{alpha} domains are required for mediating its classical mode of transactivation and transrepression of the GnRHR promoter.

An AP-1-Like Motif at Nucleotide (nt) -130/-124 Mediates Both Basal Activity and E2 Repression of the GnRHR Promoter
Progressive deletion analysis was performed between nt -1671 and +1 to locate the sequence(s) that confers negative E2 regulation of the GnRHR promoter. Deletion of the distal region from nt -1671 to -1018 did not abolish the E2 responsiveness of the GnRHR promoter (Fig. 4Go), indicating that the core E2-response element(s) resides in the proximal 1-kb region. This observation was supported by the finding that removal of this proximal region [i.e. the construct p(-1700/-1018)-Luc] could completely abrogate the E2 response (Fig. 4Go). To further delineate the location of the negative E2-response region(s), a more detailed 5'-deletion mapping was performed. As shown in Fig. 4Go, although the basal GnRHR promoter activity was found to decrease when the sequence between nt -1018 and -266 was removed, no significant change in the E2 responsiveness could be detected. However, further deletion to nt -117 totally abolished the basal activity as well as the E2 sensitivity of the GnRHR promoter, suggesting that the core E2-response element(s) lies in the 150-bp region between nt -266 and -117.



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Fig. 4. Localization of the E2-Response Region to nt -266/-117 of the GnRHR 5'-Flanking Region

A panel of 5'- and 3'-deletion mutants of the GnRHR promoter was cotransfected with pCMV5-ER{alpha} and RSV-lacZ into OVCAR-3 cells by LIPOFECTAMINE reagent. The cells were treated 24 h after transfection with 100 nM E2 or vehicle (control) for 24 h. The relative promoter activity is represented as the fold induction when compared with the promoterless pGL2-Basic vector. Values represent the mean ± SEM of three independent experiments each performed in triplicate. a, P < 0.001 vs. control.

 
Sequence analysis revealed a number of putative transcription factor binding sites including an ERE-like element in the 150-bp E2-response region (Fig. 5Go). These elements have been demonstrated to mediate diverse transcriptional responses triggered by the ERs (41, 42, 43, 44, 45, 46, 47, 48, 49). Site-directed mutagenesis indicated that alteration of an AP-1-like motif [AP-1-like-mut (a), from 5'-TGACATA-3' to 5'-TAGGCCT-3'] resulted in an almost complete loss of the E2 suppression in OVCAR-3 cells (Fig. 5Go). Interestingly, this mutation also abolished the basal promoter activity, indicating that the motif may concomitantly involve basal transcription of the GnRHR gene. In contrast, mutations (or deletion) of other regulatory elements neither significantly affected the basal activity nor impaired the E2 responsiveness of the GnRHR promoter (Fig. 5Go). To further examine the role of the AP-1-like motif in mediating suppression, we generated a mutant construct in which the AP-1-like motif was changed to a consensus AP-1 sequence [AP-1-like-mut (b), from 5'-TGACATA-3' to 5'-TGACTCA-3']. As shown in Fig. 5Go, this nucleotide substitution had no apparent effect on the sensitivity of the GnRHR promoter to E2 but, rather, it strongly stimulated the basal activity by 2.3-fold. Therefore, it is apparent that a consensus AP-1 sequence and the AP-1-like motif possess opposing effects on the GnRHR promoter.



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Fig. 5. An AP-1-Like Site at nt -130/-124 Mediates Both Basal Activity and E2 Responsiveness of the GnRHR Promoter

A diagrammatic representation of the mutant promoter constructs is shown on the left side of the figure. Nucleotide positions of various potential transcription factor binding sites in the core E2-response region are indicated. Each motif was mutated by introducing a restriction enzyme digestion site into the core binding sequence. The AP-1-like-mut (b) was prepared by mutating the wild-type sequence into a consensus AP-1 site (5'-TGACTCA-3'). Mutations are marked with black crosses. Wild-type [p(-266/+1)-Luc and p(-214/+1)-Luc] or the mutant constructs were transiently cotransfected with pCMV5-ER{alpha} and RSV-lacZ into OVCAR-3 cells. The cells were treated 24 h after transfection with 100 nM E2 or vehicle (control) for 24 h. The relative promoter activity is represented as the fold induction when compared with the promoterless pGL2-Basic vector. Values represent the mean ± SEM of three independent experiments each performed in triplicate. a, P < 0.001 vs. control. DRE, Dioxin-response element.

 
Multiple Transcription Factors Including c-Jun and c-Fos Form Complexes with the AP-1-Like Motif
To examine whether E2 represses the GnRHR promoter via altering DNA binding activity of transcription factors at the AP-1-like site, we performed EMSAs using nuclear extracts prepared from E2- or vehicle-treated OVCAR-3 cells transfected with pCMV5-ER{alpha}. Three DNA-protein complexes (A, B, and C) were formed with the AP-1-like motif, and we found that the intensity of these complexes was not significantly altered after E2 treatment (Fig. 6AGo). Formation of these complexes could be inhibited by the unlabeled probe or a consensus AP-1 motif but not by other nonspecific sequences [nuclear factor-{kappa}B (cNF-{kappa}B) and cAP-2], suggesting specific interactions of AP-1 transcription factors with the AP-1-like site (Fig. 6AGo). The specificity of these complexes was further confirmed by the observation that mutation of the AP-1-like site could completely abolish complex formation (Fig. 6AGo). Antibody supershift assays showed that c-Jun and c-Fos were present in complex C but not in the two upper complexes (Fig. 6BGo). The identity of complex A and B remains to be determined as antibody targeted against the CREB family transcription factors (CREB-1, activating transcription factor 1, and cAMP response modulator 1), which also recognize AP-1 motif, did not affect complex formation (Fig. 6BGo). Despite the finding that the DBD of ER{alpha} was required for repression of the GnRHR promoter, we found that addition of anti-ER{alpha} (or anti-ERß, data not shown) antibody also had no effect on complex formation (Fig. 6BGo). Moreover, although in vitro translated ER{alpha} proteins could interact with a consensus ERE sequence in the presence or absence of E2, they did not bind to the AP-1-like motif (Fig. 6CGo). These results thus suggest that ER{alpha} represses the GnRHR gene transcription via a mechanism that does not rely on direct binding to the GnRHR promoter.



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Fig. 6. Binding of the AP-1 Transcription Factors c-Jun and c-Fos, But Not ER{alpha}, to the AP-1-Like Motif

A, Five micrograms of nuclear extract (NE) from vehicle-treated or E2-treated ER{alpha}-overexpressing OVCAR-3 cells were incubated with 50 fmol of the wild-type (wt) or mutant (mut) AP-1-like probe in the presence of the unlabeled sequence, a consensus AP-1 (cAP-1), NF-{kappa} B (cNF-{kappa} B), or AP-2 (cAP-2) oligonucleotide. B, Five micrograms of NE from vehicle-treated cells were preincubated with 3 µg of anti-ER{alpha}, anti-c-Jun, anti-c-Fos, anti-CREB, or anti-GATA-4 antibody for 30 min at room temperature before the addition of the wild-type probe. Similar binding results were obtained from E2-treated cells (data not shown). C, In vitro translated (IVT) human ER{alpha} proteins (2.5 µl) were incubated with the wild-type AP-1-like probe or three copies of ERE in the presence or absence of 100 nM E2.

 
Phorbol 12-Myristate 13-Acetate (PMA) Antagonizes E2-Dependent Repression of the GnRHR Promoter
An essential feature of AP-1 binding sites is their ability to confer phorbol ester responsiveness to the promoter. To examine whether the AP-1-like motif at nt -130/-124 behaves as a classical AP-1 element, we transiently transfected the GnRHR promoter-luciferase construct p(-266/+1)-Luc into OVCAR-3 cells and treated the cells with various concentrations of PMA. A dose-dependent stimulation of promoter activity was observed, with a maximal 1.8-fold increase being detected at 10 nM PMA treatment (Fig. 7AGo). Higher doses (100 nM and 1 µM) reduced the PMA responsiveness of the promoter (Fig. 7AGo). In contrast, under the conditions that have been shown to produce the optimal forskolin response of the GnRHR promoter in gonadotropes and placental cells (50, 51), the same treatment had an insignificant stimulatory effect on the activity of the construct in OVCAR-3 cells (Fig. 7AGo). Importantly, mutation of the AP-1-like motif significantly attenuated the PMA sensitivity of the GnRHR promoter in the cancer cells (Fig. 7BGo), indicating that it functions as a typical PMA-response element.



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Fig. 7. Functional Antagonism Between the ER and AP-1 Signaling Pathways in Regulating the GnRHR Gene Transcription

A, The human GnRHR promoter construct p(-266/+1)-Luc was transiently cotransfected with pCMV5-ER{alpha} and RSV-lacZ into OVCAR-3 cells. The cells were treated 24 h after transfection with various concentrations of PMA for 24 h or forskolin for 6 h. B, The construct p(-266/+1)-Luc or AP-1-like-mut (a) was cotransfected with pCMV5-ER{alpha} and RSV-lacZ into OVCAR-3 cells, and the transfected cells were treated with 100 nM PMA for 24 h. C, The construct p(-266/+1)-Luc was cotransfected with pCMV5-ER{alpha} and RSV-lacZ into OVCAR-3 cells, and the transfected cells were treated with 100 nM E2, 100 nM PMA, or 100 nM E2 and 100 nM PMA for 24 h. The relative promoter activity is represented as the percentage of the control group (DMSO treated), of which the activity is set as 100% after being normalized by ß-galactosidase activity. Values in all panels represent the mean ± SEM of three independent experiments each performed in triplicate. a, P < 0.001 vs. control; b, P < 0.05 vs. control. EtOH, Ethanol. D, Western blot analysis of the phosphorylation (P) and total expression (T) levels of c-Jun and c-Fos in OVCAR-3 cells after transient transfection and pharmacological treatment as described in panel C. E, Transcription factor binding to the AP-1-like site is not affected by PMA or cotreatment with PMA and E2. Nuclear extract (NE) 5 µg, from ER{alpha}-overexpressing OVCAR-3 cells (treated for 24 h with DMSO, 100 nM PMA, or 100 nM PMA and 100 nM E2) was incubated with 50 fmol of the radiolabeled AP-1-like probe for 15 min at room temperature before being analyzed by a 6% native gel.

 
Because it is apparent that both the negative E2 and positive PMA responses of the GnRHR promoter converge at the single AP-1-like motif, we sought to investigate whether PMA stimulation could antagonize the repressive effect of E2 on the GnRHR promoter. As shown in Fig. 7CGo, cotreatment of OVCAR-3 cells with PMA completely abolished E2 repression of the GnRHR promoter, suggesting the existence of a functional antagonism between the ER and AP-1 signaling pathways in regulating GnRHR gene transcription. To elucidate the mechanism by which this antagonism occurs, we examined the phosphorylation and expression levels of c-Jun and c-Fos in OVCAR-3 cells after the transfection and pharmacological treatment as described in Fig. 7CGo. Treatment with E2 alone had no apparent effect on AP-1 protein expression and c-Jun phosphorylation, as revealed by Western blot analysis (Fig. 7DGo). Similarly, stimulation with PMA or PMA in the presence of E2 did not affect AP-1 expression significantly (Fig. 7DGo). Interestingly, we found a strong stimulation of c-Jun phosphorylation on serine 63 after 24 h of PMA treatment; this change was unaffected by cotreatment with E2 (Fig. 7DGo). To examine the functional consequence of this enhanced c-Jun phosphorylation in terms of AP-1 binding to the AP-1-like motif, we performed EMSAs using nuclear extracts prepared from ER{alpha}-transfected OVCAR-3 cells treated with PMA or PMA in the presence of E2. Consistent with an earlier finding from Baker et al. (52), we found that the enhanced phosphorylation did not increase the binding activity of the c-Jun/c-Fos heterodimer to the AP-1-like site (Fig. 7EGo), suggesting that the ER-AP-1 antagonism occurs independently of the AP-1 DNA binding activity.

Overexpression of the Transcriptional Coactivator CBP Attenuates E2-Dependent Repression of the GnRHR Promoter
So how does ER{alpha} suppress the GnRHR gene transcription in the cancer cells? It has been proposed that nuclear receptor-AP-1 antagonism can occur through direct competition for limiting transcriptional coactivators that are commonly required to induce gene expression (53, 54, 55, 56). A potential candidate of these cofactors is CBP, which has been reported to interact with both nuclear receptors and AP-1 transcription factors (56). It could be imagined that under the condition of coactivation, AP-1 and ligand-activated ER would compete for a limiting amount of CBP, leading to suppression of AP-1 transcriptional activity. To examine whether this mechanism is involved in repression of the GnRHR promoter, we cotransfected the construct p(-1671/+1)-Luc with an increasing amount of a CBP expression plasmid into OVCAR-3 cells and then examined the degree of repression after 24 h of E2 treatment. As shown in Fig. 8Go, overexpression of CBP resulted in a dose-dependent reduction of the E2-induced repression of the GnRHR promoter such that a significant decrease (51 to 28% repression) was observed when 1.5 µg pRc/RSV-CBP-HA were cotransfected, and the decrease was even more evident (to 12% repression) when 2 µg of the expression plasmid were used. These findings thus implicate that CBP is involved in transcriptional repression of the GnRHR gene by ligand-activated ER{alpha}.



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Fig. 8. Overexpression of the Transcriptional Coactivator CBP Attenuates the E2-Dependent Repression of the GnRHR Promoter

The construct p(-1671/+1)-Luc was cotransfected with pCMV5-ER{alpha} and RSV-lacZ in the presence of an increasing amount of pRc/RSV or pRc/RSV-CBP-HA into OVCAR-3 cells. Twenty four hours after transfection, the cells were treated with 100 nM E2 for 24 h. Data are expressed as the percentage of repression relative to each respective control group (vehicle treated). Values represent the mean ± SEM of three independent experiments each performed in triplicate. a, P < 0.001 vs. no CBP overexpression.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
There is convincing evidence that GnRH functions as an important autocrine and/or paracrine factor in some extrapituitary tissues (1, 2, 3, 4, 5), in addition to its essential role in stimulating gonadotropin secretion. A well known example of these extrapituitary functions is to serve as a negative growth regulator in a number of tumors derived from the reproductive tracts (6). Since its discovery some 30 yr ago, many GnRH analogs have been developed and studied extensively (57). Clinically, some of these synthetic analogs have been used as an effective treatment for a variety of reproductive endocrinopathies such as prostate cancer (58). Estrogen, on the other hand, has been implicated as a positive regulator of carcinogenesis in various reproductive organs (13, 14, 15, 16, 17, 18). Although previous studies from our laboratory have shown that E2 down-regulates the GnRHR mRNA level in the human ovary and antagonizes the growth-inhibitory effect of GnRH in ovarian cancer cells via an ER-mediated process (19, 20), the mechanism underlying these negative E2 actions is obscure. In this report, we demonstrated for the first time that an AP-1-like motif was responsible for mediating E2 repression of the GnRHR promoter via an ER{alpha}-dependent mechanism. Also, we showed that this repression was apparently independent of direct ER binding to the promoter and involved the transcriptional coactivator CBP, which is recruited by both nuclear receptors and AP-1 proteins for activating gene expression.

In the present study, we found that cotransfection of a wild-type ER{alpha} expression plasmid into OVCAR-3 cells was necessary to restore the estrogen response. This phenomenon is probably due to down-regulation of the receptor expression in cultures as the ER gene is known to locate in an unstable chromosomal region (41). This scenario would be advantageous as we could definitely dissect the role of the ER subtype in mediating the E2 effect in the cancer cells. Significantly, the data obtained from MCF-7 cells (without ER{alpha} overexpression) (Fig. 1DGo) suggest that estrogen repression of the GnRHR promoter does occur at a physiological level of ER{alpha} protein. On the other hand, we do not have a clear explanation for the modest transactivation response observed for ERE2-tk109-Luc in OVCAR-3 cells (Fig. 1EGo), but we speculate that the cancer cells may lack certain transcription factors, which can interact with the vitellogenin gene ERE in addition to the ER (59) and may be essential for full expression of the estrogen response. Despite the fact that ER{alpha} and ERß share a high degree of homology in their DBDs and LBDs so that they recognize the same consensus ERE and have similar affinities to estrogens, they do respond differentially to E2 in controlling gene transcription in various cellular systems (60, 61, 62). Furthermore, gene-knockout studies have also revealed that ER{alpha} possesses biological functions distinct from those of ERß, as evidenced by the different phenotypes of the {alpha} ER-knockout and ß ER-knockout mice (63, 64). Our data showed that ER{alpha}, but not ERß, was able to repress the GnRHR promoter in OVCAR-3 and MCF-7 cells, providing an indication that ER{alpha} may play a predominant role in controlling cell proliferation in ovarian and breast cancers, where both receptor subtypes are expressed (65, 66, 67, 68). In fact, these observations are supported by previous findings, which demonstrated an up-regulation of ER{alpha} relative to ERß gene expression during ovarian and mammary carcinogenesis (69, 70). Because repression of the GnRHR promoter is ER{alpha} dose dependent, it is conceivable that E2 may exert stronger inhibitory effects on GnRHR gene expression as tumorigenesis proceeds to later stages.

The human ER belongs to the nuclear receptor superfamily of ligand-inducible transcription factors (71), the members of which include the receptors for steroids, thyroid hormone, retinoic acid, and vitamin D and orphan receptors for which no ligands have been identified. In the classical mode of ER action, the receptor binds to ERE as homodimers (36) or heterodimers (72, 73, 74) in estrogen-responsive promoters and then recruits an array of transcriptional cofactors by means of which the nuclear receptor interferes with other transcription factors including components of the general transcription factor machinery (75). In addition, some of the cofactors possess chromatin-remodeling activities or yet recruit additional proteins to the nuclear receptor-cofactor complex to mediate transcription regulation of the target genes (75). However, it has also been well reported that EREs are absent in many genes that are regulated by ERs. Multiple lines of evidence indicate that this nonclassical mode of ER action occurs in the absence of direct ER binding to DNA and is achieved via protein-protein interactions with other transcription factors that bind to their response sequences. For instance, it has been demonstrated that physical interaction of ER{alpha} with NF-{kappa}B and CCAAT/enhancer binding protein transcription factors can lead to transcriptional repression of the NF-{kappa}B- and CCAAT/enhancer binding protein-dependent IL-6 promoter (41, 42). Also, repression of erythropoiesis by estrogen has been found to involve estrogen-dependent inhibition of the transcriptional activity of GATA-1 by direct interaction with ER{alpha} (47). Furthermore, repression of the IGF-I receptor gene transcription has been shown to be mediated by ER{alpha} inhibition of Sp1 binding to the target promoter (62).

Our finding that mutation of the AP-1-like site reduced the promoter activity to the basal level does not oppose its involvement in mediating E2 suppression. Rather, it implies that the estrogen exerts its repressive effect by interfering with the basal transcription of the GnRHR gene. The fact that the AP-1-like site is quite different from the consensus sequence may contribute to their opposing effects on the GnRHR promoter (Fig. 5Go). Other genes that are also repressed by E2 via AP-1 sites include the human hepatic lipase (48), murine lipoprotein lipase (49), ovine FSHß (76), as well as human choline acetyltransferase (77) genes. It should be noted that ER{alpha} has been shown to physically interact with promoter-bound c-Jun to mediate the nonclassical pathway and that the DBD of the receptor is dispensable for its activity at AP-1 sites (35, 78, 79, 80). Accordingly, our present data revealed that E2 repressed the GnRHR promoter in the absence of direct DNA binding. Also, our data suggested that ER{alpha} was unlikely to be tethered to the promoter via interaction with c-Jun as we had repeatedly failed to interfere c-Jun binding with anti-ER{alpha} antibody. In addition, our data also ruled out the possibility that E2 repressed the GnRHR promoter via altering AP-1 binding activity, as reported in some instances (48, 49). Furthermore, we found that the DBD together with the LBD, which are necessary for gene activation at classical ERE, were required for transrepression of the GnRHR promoter. The LBD harbors the ligand-dependent AF-2, which mediates activation of gene transcription by recruiting a large coactivator complex composed of p160, CBP/p300, and P/CAF (p300 and CBP-associated factor) (81, 82, 83). Because tamoxifen has been shown to prevent the formation of an active AF-2 surface (84), the observation that the antiestrogen could antagonize E2-mediated suppression of the GnRHR promoter suggests that the AF-2 surface is involved in the repression process.

Our present findings suggest that c-Jun phosphorylation is involved in regulating the GnRHR gene transcription via a pathway independent of its DNA binding activity. There are several lines of evidence indicating that this posttranslational modification may affect c-Jun interaction with other protein factors such as CBP (54, 85, 86). The transcriptional coactivator CBP has been proposed as an integrator of multiple signal transduction pathways by virtue of its ability to enhance transcriptional activation by CREB, AP-1, and nuclear receptors (53, 56, 87). Recruitment of CBP by c-Jun requires phosphorylation of the AP-1 protein on serines 63 and 73 by the c-Jun amino-terminal kinase (88, 89). In contrast to the finding that ligand-activated nuclear receptors, including those for glucocorticoids, retinoic acid, and T3, could antagonize AP-1 activity by inhibiting c-Jun phosphorylation (90), our current data found that E2-bound ER{alpha} did not reduce the phosphorylation induced by PMA. It is speculated that the ER{alpha}-AP-1 antagonism observed in the GnRHR gene transcription may occur via competition for a limiting amount of CBP that is shared by both classes of the transcription factors for activating gene expression. Nonetheless, it is possible that other transcriptional cofactor(s) may participate in this antagonism, as it has been reported that steroid receptor coactivator 1 and CAPER are also coactivators for ER{alpha} and AP-1 proteins (91, 92). In addition, it is not yet known whether other CBP-recruiting factors can repress the GnRHR promoter equally. Furthermore, one should note that as the E2 effect could only be observed after 12 h of the estrogen treatment (Fig. 2BGo), the repression may be indirect and involve activation of certain early response genes, the products of which can interfere with AP-1 induction. For instance, it has been demonstrated that the expression of early growth response factor-1 (Egr-1) gene can be rapidly and strongly induced by E2 via ERK (93). Because early growth response factor-1 is also capable of physically and functionally interacting with CBP to activate gene transcription (94), it is possible that the early response protein, instead of the ligand-activated ER, competes with AP-1 for the limiting CBP. Nevertheless, as cofactor sharing appears to be the major mechanism mediating E2 suppression of the GnRHR gene transcription, our data strengthen the notion that ER{alpha} and ERß have different preferences and affinities in cofactor recruitment (95, 96) that may account for their differential repressive activities on the GnRHR promoter.

In addition to the c-Jun/c-Fos heterodimer, two unidentified complexes (complex A and B) were formed with the AP-1-like motif (Fig. 6Go). We found that the formation of these complexes was also unaffected by antibodies specific to other AP-1 proteins such as JunB, JunD, and FosB (data not shown). Indeed, a similar situation has also been observed in the lipoprotein lipase promoter, in which an AP-1-like sequence mediating E2 repression is bound by an unknown AP-1-related protein (49). Whether these complexes are derived from other existing or unknown members of AP-1 proteins remains to be elucidated. Also, the failure to detect ER{alpha} binding to the GnRHR promoter may be due to inappropriate experimental conditions used, such as dilution of transcription factors in the nuclear extracts and the use of nonphysiological buffers. Importantly, the identification of these complexes suggests that additional transcriptional regulators, which may be more important than CBP, are involved in mediating the E2 effect. Further studies are warranted to elucidate the complex cross-talk between the ER and AP-1 signaling pathways in transcriptional regulation of the GnRHR gene.

It should be noted that pituitary GnRHR gene expression is also tightly regulated by estrogen. Like the ovary, an increase in GnRHR mRNA level in rat pituitary has been observed after ovariectomy, and replacement therapy of the castrated animals with E2 can markedly decrease GnRHR mRNA expression (97). However, E2 also exerts a positive feedback action, presumably by enhancing hypothalamic GnRH secretion, on pituitary GnRHR gene expression, which is important for sensitizing gonadotropes to GnRH during the preovulatory gonadotropin surge (98). Accordingly, a positive estrogen response on pituitary luciferase expression has been observed for the ovine GnRHR promoter in vivo (30). In contrast, using the gonadotrope-derived {alpha}T3–1 cells, our preliminary data have failed to reveal any E2 regulation of the human promoter even with ER overexpression (data not shown). Whether this discrepancy is due to species differences of the GnRHR promoters or concomitant requirement of the hypothalamic input to mediate the estrogen action on pituitary GnRHR gene transcription requires further investigations.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells and Cell Culture
Human ovarian adenocarcinoma OVCAR-3 and human breast adenocarcinoma MCF-7 cells were obtained from American Type Culture Collection (Manassas, VA). The cancer cells were maintained in DMEM (Invitrogen, Inc., Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT). Cultures were maintained at 37 C in humidified atmosphere of 5% CO2 in air. Culture media were renewed every 3 d. Cells were passaged when they reached about 80% confluence using trypsin-EDTA solution (0.05% trypsin and 0.53 mM EDTA).

Western Blot Analysis
Cells were lysed in a lysis buffer containing 1x PBS (pH 7.4), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10 µg/ml phenylmethylsulfonyl fluoride, 30 µg/ml aprotinin, and 10 µg/ml leupeptin for 15 min on ice. Cell lysates were collected and debris was cleared by centrifugation. Proteins were resolved by 10% SDS-PAGE, and separated proteins were transferred onto Hybond-C nitrocellulose membranes (Amersham Pharmacia Biotech, Morgan, Canada). Membranes were blocked with 5% (wt/vol) nonfat dried milk in Tris-buffered saline containing 20 mM Tris-Cl (pH 8.0), 140 mM NaCl, and 0.05% (vol/vol) Tween 20 for 2 h at room temperature before incubating with primary antibodies specific to ER{alpha}, ERß, phospho-c-Jun (Ser63), c-Jun, and c-Fos overnight at 4 C. All antibodies except anti-phospho-c-Jun (New England Biolabs Ltd., Ontario, Canada) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Immunostained proteins were detected using the ECL Western Blot Analysis System (Amersham Pharmacia Biotech).

Plasmid Construction and Site-Directed Mutagenesis
Human GnRHR promoter-luciferase constructs p(-1671/+1)-Luc, p(-1346/+1)-Luc, p(-1018/+1)-Luc, and p(-1700/-1018)-Luc (the numbering is relative to the ATG initiation codon) were prepared as previously described (23). Other constructs for deletion mapping were generated by PCR of the corresponding regions of the GnRHR 5'-flanking region and subsequent cloning of the amplified fragments into the promoterless pGL2-Basic vector (Promega Corp., Nepean, Canada). PCR reactions were carried out for 30 cycles with denaturation for 30 sec at 94 C, annealing for 1 min at 60 C, extension for 1 min at 72 C, and a final extension for 15 min at 72 C. Restriction sites (KpnI or HindIII) were introduced into the primers for directional cloning.

To identify the cis-acting regulatory element(s) in mediating E2 regulation of the GnRHR promoter, a three-step PCR mutagenesis method described by Chow et al. (99) was employed using site-specific mutagenic primers NF-Y-mut, ERE-like-mut, GATA-mut, AP-1-like-mut (a), and AP-1-like-mut (b) as well as universal primers MPB, MPD, and 1H (Table 1Go) to generate the corresponding mutant constructs. E box mutants were produced by direct PCR amplification with primers E box-1-mut, E box-2-mut, and 1H using p(-266/+1)-Luc as the template. All the amplified fragments were cloned into the KpnI and HindIII sites of the pGL2-Basic vector. Desired mutations were confirmed by restriction mapping and nucleotide sequencing.


View this table:
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Table 1. Nucleotide Sequences of Primers Used in Site-Directed Mutagenesis

 
Full-length human ER{alpha} (pCMV5-ER{alpha}) and ERß (pRST7-ERß) expression plasmids were kindly provided by Dr. B. S. Katzenellenbogen (Department of Molecular and Integrative Physiology, University of Illinois at Urbana Champaign) and Dr. D. P. McDonnell (Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC). The ERß coding region was released by restriction digestion and subcloned into an empty pCMV5 vector to generate pCMV5-ERß. ER{alpha} deletion mutants were prepared by PCR amplification of the corresponding coding regions and subsequent cloning of the amplified fragments into the pCMV5 vector. Initiation codons (ATG) or termination codons (TGA) were introduced into the primers for proper translation. PCR reactions were carried out in the presence of 10% dimethyl sulfoxide (DMSO) for 30 cycles with denaturation for 30 sec at 94 C, annealing for 1 min at 59 C, extension for 1.5 min at 72 C, and a final extension for 15 min at 72 C. The authenticity of the deletion mutants was confirmed by nucleotide sequencing. Human progesterone receptor-B expression plasmid (pSG5-PR-B) was provided by Dr. P. Chambon (Institut National de la Santé et de la Recherche Médicale, Universite Louis Pasteur, Paris, France) and has been described previously (26). The reporter plasmid ERE2-tk109-Luc was provided by Dr. J. L. Jameson (Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, IL). Expression vector carrying the full-length cDNA for CBP (pRc/RSV-CBP-HA) was generously provided by Dr. R. H. Goodman (Oregon Health Sciences University, Eugene, OR). Plasmid DNA for transient transfection was prepared using the QIAGEN Plasmid Midi Kits (QIAGEN, Chatsworth, CA) following the manufacturer’s suggested procedures. The concentration and quality of DNA were determined by measuring absorbance at 260 nm and by agarose gel electrophoresis, respectively.

Transient Transfection and Reporter Gene Assay
Transient transfection was carried out using LIPOFECTAMINE Reagent (Invitrogen, Inc.) following the manufacturer’s suggested procedures. To correct for different transfection efficiencies of various luciferase constructs, the Rous sarcoma virus (RSV)-lacZ plasmid was cotransfected into the cells with the GnRHR promoter-luciferase constructs. Briefly, 2.5 x 105 of cells were grown on six-well tissue culture plates in phenol red-free DMEM supplemented with 10% charcoal-dextran-treated fetal bovine serum (Hyclone Laboratories, Inc.) for 48 h before the day of transfection. One microgram of GnRHR promoter-luciferase constructs, 0.5 µg RSV-lacZ plasmid, and an indicated amount of expression plasmids was cotransfected into the cells under phenol red- and serum-free conditions. After 5 h of transfection, 1 ml of medium containing 20% charcoal-dextran-treated serum was added, and the cells were allowed to recover overnight (18 h). After incubation, the cells were washed twice with phenol red-free DMEM and then treated with various concentrations of E2, tamoxifen, PMA, or forskolin (all from Sigma-Aldrich, St. Louis, MO) for different time periods as indicated before harvest. Ethanol and DMSO were added to the control media in the same final solvent concentration (typically 0.1%). Cellular lysates were collected with 150 µl of Reporter Lysis Buffer (Promega Corp., Madison, WI) and assayed for luciferase activity with the Luciferase Assay System (Promega Corp.). Luminescence was measured using the Lumat LB 9507 luminometer (E.G&G, Berthold, Germany). ß-Galactosidase activity was measured using the ß-Galactosidase Enzyme Assay System (Promega Corp.) and used to normalize the transfection efficiency. Promoter activity was calculated as luciferase activity/ß-galactosidase activity.

EMSA
Oligonucleotides containing the putative AP-1-like motif (5'-CAAAATTTGACATACGTCTAA-3', nt -130/-124) and the corresponding mutant sequence (5'-CAAAATTTAGGCCTCGTCTAA-3') were annealed and end-labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase. Underlined sequences represent mutated nucleotides. Radiolabeled probes were purified by Microspin G-25 columns (Amersham Pharmacia Biotech). Consensus AP-1, NF-{kappa}B, and AP-2 oligonucleotides were purchased from Santa Cruz Biotechnology, Inc. Preparation of nuclear extracts from OVCAR-3 cells was performed essentially as previously described (23). Briefly, the cells were grown on 100-mm tissue culture dishes to about 70% confluence in phenol red-free DMEM supplemented with 10% charcoal-dextran-treated fetal bovine serum. Afterward, the cells were transiently transfected with 12 µg pCMV5-ER{alpha} by LIPOFECTAMINE Reagent according to the manufacturer’s suggested protocol. After transfection, the cells were treated with 100 nM E2, 100 nM PMA, or 100 nM E2 and 100 nM PMA for 24 h before harvest. Ethanol and DMSO were added to the control media in the same final solvent concentration (0.1%). Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). In vitro translated human ER{alpha} proteins were generated by the TNT Coupled Reticulocyte Lysate System (Promega Corp.). EMSAs were carried out in 20-µl reactions containing 20 mM HEPES (pH 7.5), 50 mM NaCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 1 µg of poly (dI:dC), 5 µg of nuclear proteins (or 2.5 µl of in vitro translated product), and 50 fmol of radiolabeled probes (30,000 cpm). For the competitive assays, competitor oligonucleotides were added simultaneously with the radiolabeled probe. For the supershift assays, nuclear extracts were preincubated with 3 µg anti-ER{alpha}, anti-c-Fos, anti-c-Jun, anti-CREB, or anti-GATA-4 for 30 min at room temperature before the addition of the radiolabeled probe. Binding reactions were incubated at room temperature for 15 min and then separated by 6% polyacrylamide gels containing 0.5x TBE (0.09 M Tris-borate and 2 mM EDTA, pH 8.0) at constant 200 V and at 4 C. After electrophoresis, gels were dried and exposed to Kodak X-OMAT AR films (Eastman Kodak Co., Rochester, NY) at -70 C.

Data Analysis
For transient transfection assays, data were shown as the mean ± SEM of triplicate assays in three independent experiments. For Western blot and EMSAs, all studies were repeated three times, and consistent results were obtained between experiments. Data were analyzed by one-way ANOVA, followed by Tukey’s multiple comparison tests using the computer software PRISM (GraphPad Software, Inc., San Diego, CA). Data were considered significantly different from each other at P < 0.05.


    ACKNOWLEDGMENTS
 
We thank Dr. B. S. Katzenellenbogen, Dr. D. P. McDonnell, Dr. P. Chambon, Dr. J. L. Jameson, and Dr. R. H. Goodman for providing various plasmid constructs used in this study. Also, we would like to express special thanks to K. Y. Kim and Dr. C. M. Yeung for their assistance during the course of this study.


    FOOTNOTES
 
This work was supported by grants from the Canadian Institutes of Health Research. C.K.C. is a recipient of the British Columbia Research Institute of Children’s and Women’s Health Studentship Award. P.C.K.L. is a Distinguished Scholar of the Michael Smith Foundation for Health Research.

Abbreviations: AF, Transactivation function; AP-1, activator protein 1; CBP, CREB-binding protein; CREB, cAMP response element binding protein; DBD, DNA-binding domain; DMSO, dimethyl sulfoxide; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen-response element; ERE-mut, GnRHR, GnRH receptor; LBD, ligand-binding domain; NF-{kappa}B, nuclear factor {kappa}B; nt, nucleotide; PMA, phorbol 12-myristate 13-acetate; RSV, Rous sarcoma virus.

Received for publication June 6, 2003. Accepted for publication August 22, 2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Peng C, Fan NC, Ligier M, Vaananen J, Leung PC 1994 Expression and regulation of gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acids in human granulosa-luteal cells. Endocrinology 135:1704–1746
  2. Minaretzis D, Jakubowski M, Mortola JF, Pavlou SN 1995 Gonadotropin-releasing hormone receptor gene expression in human ovary and granulosa-lutein cells. J Clin Endocrinol Metab 80:430–434[Abstract]
  3. Lin LS, Roberts VJ, Yen SS 1995 Expression of human gonadotropin-releasing hormone receptor gene in the placenta and its functional relationship to human chorionic gonadotropin secretion. J Clin Endocrinol Metab 80:580–585[Abstract]
  4. Kang SK, Tai CJ, Nathwani PS, Choi KC, Leung PC 2001 Stimulation of mitogen-activated protein kinase by gonadotropin-releasing hormone in human granulosa-luteal cells. Endocrinology 142:671–679[Abstract/Free Full Text]
  5. Bussenot I, Azoulay-Barjonet C, Parinaud J 1993 Modulation of the steroidogenesis of cultured human granulosa-lutein cells by gonadotropin-releasing hormone analogs. J Clin Endocrinol Metab 76:1376–1379[Abstract]
  6. Emons G, Muller V, Ortmann O, Schulz KD 1998 Effects of LHRH-analogues on mitogenic signal transduction in cancer cells. J Steroid Biochem Mol Biol 65:199–206[CrossRef][Medline]
  7. Kakar SS, Grizzle WE, Neill JD 1994 The nucleotide sequences of human GnRH receptors in breast and ovarian tumors are identical with that found in pituitary. Mol Cell Endocrinol 106:145–149[CrossRef][Medline]
  8. Kimura A, Ohmichi M, Kurachi H, Ikegami H, Hayakawa J, Tasaka K, Kanda Y, Nishio Y, Jikihara H, Matsuura N, Murata Y 1999 Role of mitogen-activated protein kinase/extracellular signal-regulated kinase cascade in gonadotropin-releasing hormone-induced growth inhibition of a human ovarian cancer cell line. Cancer Res 59:5133–5142[Abstract/Free Full Text]
  9. Emons G, Muller V, Ortmann O, Grossmann G, Trautner U, von Stuckrad B, Schulz KD, Schally AV 1996 Luteinizing hormone-releasing hormone agonist triptorelin antagonizes signal transduction and mitogenic activity of epidermal growth factor in human ovarian and endometrial cancer cell lines. Int J Oncol 9:1129–1137
  10. Takagi H, Imai A, Furui T, Horibe S, Fuseya T, Tamaya T 1995 Evidence for tight coupling of gonadotropin-releasing hormone receptors to phosphatidylinositol kinase in plasma membrane from ovarian carcinomas. Gynecol Oncol 58:110–115[CrossRef][Medline]
  11. Nagai N, Oshita T, Mukai K, Shiroyama Y, Shigemasa K, Ohama K 2002 GnRH agonist inhibits human telomerase reverse transcriptase mRNA expression in endometrial cancer cells. Int J Mol Med 10:593–597[Medline]
  12. Olson TA, Mohanraj D, Ramakrisknan S 1995 The selective inhibition of vascular permeability factor (VPF) expression in ovarian carcinoma cell lines by a gonadotropin-releasing hormone (GnRH) agonist. Int J Oncol 6:905–910
  13. Hoover R, Gray LA, Fraumeni JF 1977 Stilboestrol (diethylstilbestrol) and the risk of ovarian cancer. Lancet 2:533–534[CrossRef][Medline]
  14. Chien C, Wang F, Hamilton TC 1994 Transcriptional activation of c-myc proto-oncogene by estrogen in human ovarian cancer cells. Mol Cell Endocrinol 99:11–19[CrossRef][Medline]
  15. Galtier-Dercure F, Capony F, Maudelonde T 1992 Estradiol stimulates cell growth and secretion of procathepsin D and a 120-kilodalton protein in the human ovarian cancer cell line BG-1. J Clin Endocrinol Metab 75:1497–1502[Abstract]
  16. Langon SP, Hirst GI, Miller EP, Hawkins RA, Tesdale AI, Smyth JF, Miller WR 1994 The regulation of growth and protein expression by estrogen in vitro: a study of 8 human ovarian carcinoma cell lines. J Steroid Biochem Mol Biol 50:131–135[CrossRef][Medline]
  17. Clemons M, Goss P 2001 Estrogen and the risk of breast cancer. N Engl J Med 344:276–285[Free Full Text]
  18. Persson I 2000 Estrogens in the causation of breast, endometrial and ovarian cancers—evidence and hypotheses from epidemiological findings. J Steroid Biochem Mol Biol 74:357–364[CrossRef][Medline]
  19. Kang SK, Choi KC, Tai CJ, Auersperg N, Leung PC 2001 Estradiol regulates gonadotropin-releasing hormone (GnRH) and its receptor gene expression and antagonizes the growth inhibitory effects of GnRH in human ovarian surface epithelial and ovarian cancer cells. Endocrinology 142:580–588[Abstract/Free Full Text]
  20. Nathwani PS, Kang SK, Cheng KW, Choi KC, Leung PC 2000 Regulation of gonadotropin-releasing hormone and its receptor gene expression by 17ß-estradiol in cultured human granulosa-luteal cells. Endocrinology 141:1754–1763[Abstract/Free Full Text]
  21. Ngan ES, Cheng PK, Leung PC, Chow BK 1999 Steroidogenic factor-1 interacts with a gonadotrope-specific element within the first exon of the human gonadotropin-releasing hormone receptor gene to mediate gonadotrope-specific expression. Endocrinology 140:2452–2462[Abstract/Free Full Text]
  22. Cheng KW, Chow BK, Leung PC 2001 Functional mapping of a placenta-specific upstream promoter for human gonadotropin-releasing hormone receptor gene. Endocrinology 142:1506–1516[Abstract/Free Full Text]
  23. Cheng CK, Yeung CM, Chow BK, Leung PC 2002 Characterization of a new upstream gonadotropin-releasing hormone receptor promoter in human ovarian granulosa-luteal cells. Mol Endocrinol 16:1552–1564[Abstract/Free Full Text]
  24. Kakar SS 1997 Molecular structure of the human gonadotropin-releasing hormone receptor gene. Eur J Endocrinol 137:183–192[Medline]
  25. Fan NC, Peng C, Krisinger J, Leung PC 1995 The human gonadotropin-releasing hormone receptor gene: complete structure including multiple promoters, transcription initiation sites, and polyadenylation signals. Mol Cell Endocrinol 107:R1–R8
  26. Cheng KW, Cheng CK, Leung PC 2001 Differential role of PR-A and -B isoforms in transcription regulation of human GnRH receptor gene. Mol Endocrinol 15:2078–2092[Abstract/Free Full Text]
  27. Campion CE, Turzillo AM, Clay CM 1996 The gene encoding the ovine gonadotropin-releasing hormone (GnRH) receptor: cloning and initial characterization. Gene 170:277–280[CrossRef][Medline]
  28. Albarracin CT, Kaiser UB, Chin WW 1994 Isolation and characterization of the 5'-flanking region of the mouse gonadotropin-releasing hormone receptor gene. Endocrinology 135:2300–2306[Abstract]
  29. McCue JM, Quirk CC, Nelson SE, Bowen RA, Clay CM 1997 Expression of a murine gonadotropin-releasing hormone receptor-luciferase fusion gene in transgenic mice is diminished by immunoneutralization of gonadotropin-releasing hormone. Endocrinology 138:3154–3160[Abstract/Free Full Text]
  30. Duval DL, Farris AR, Quirk CC, Nett TM, Hamernik DL, Clay CM 2000 Responsiveness of the ovine gonadotropin-releasing hormone receptor gene to estradiol and gonadotropin-releasing hormone is not detectable in vitro but is revealed in transgenic mice. Endocrinology 141:1001–1010[Abstract/Free Full Text]
  31. Hall JM, McDonnell DP 1999 The estrogen receptor ß-isoform (ERß) of the human estrogen receptor modulates ER{alpha} transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140:5566–5578[Abstract/Free Full Text]
  32. Cowley SM, Hoarse S, Mosselman S, Parker MG 1997 Estrogen receptors {alpha} and ß form heterodimers on DNA. J Biol Chem 272:19858–19862[Abstract/Free Full Text]
  33. Pettersson K, Grandien K, Kuiper GG, Gustafsson JA 1997 Mouse estrogen receptor ß forms estrogen response element-binding heterodimers with estrogen receptor {alpha}. Mol Endocrinol 11:1486–1496[Abstract/Free Full Text]
  34. Shanmugam M, Krett NL, Maizels ET, Cutler RE, Peters CA, Smith LM, O’Brien ML, Park-Sarge OK, Rosen ST, Hunzicker-Dunn M 1999 Regulation of protein kinase C{delta} by estrogen in the MCF-7 human breast cancer cell line. Mol Cell Endocrinol 148:109–118[CrossRef][Medline]
  35. Jakacka M, Ito M, Weiss J, Chien PY, Gehm BD, Jameson JL 2001 Estrogen receptor binding to DNA is not required for its activity through the nonclassical AP1 pathway. J Biol Chem 276:13615–13621[Abstract/Free Full Text]
  36. Kumar V, Chambon P 1988 The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell 55:145–156[Medline]
  37. Mader S, Kumar V, de Verneuil H, Chambon P 1989 Three amino acids of the oestrogen receptor are essential to its ability to distinguish an oestrogen from a glucocorticoid-responsive element. Nature 338:271–274[CrossRef][Medline]
  38. Tora L, White J, Brou C, Tasset D, Webster N, Scheer E, Chambon P 1989 The human estrogen receptor has two independent nonacidic transcriptional activation functions. Cell 59:477–487[Medline]
  39. Berry M, Metzger D, Chambon P 1990 Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen. EMBO J 9:2811–2818[Abstract]
  40. Tzuckerman MT, Esty A, Santiso-Mere D, Danielian P, Parker MG, Stein RB, Pike J W, McDonnell DP 1994 Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions. Mol Endocrinol 8:21–30[Abstract]
  41. Stein B, Yang MX 1995 Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-{kappa}B and C/EBPß. Mol Cell Biol 15:4971–4979[Abstract]
  42. Ray P, Ghosh SK, Zhang DH, Ray A 1997 Repression of interleukin-6 gene expression by 17ß-estradiol: inhibition of the DNA-binding activity of the transcription factors NF-IL6 and NF-{kappa}B by the estrogen receptor. FEBS Lett 409:79–85[CrossRef][Medline]
  43. Xing W, Archer TK 1998 Upstream stimulatory factors mediate estrogen receptor activation of the cathepsin D promoter. Mol Endocrinol 12:1310–1321[Abstract/Free Full Text]
  44. Farsetti A, Narducci M, Moretti F, Nanni S, Mantovani R, Sacchi A, Pontecorvi A 2001 Inhibition of ER{alpha}-mediated trans-activation of human coagulation factor XII gene by heteromeric transcription factor NF-Y. Endocrinology 142:3380–3388[Abstract/Free Full Text]
  45. Wang W, Dong L, Saville B, Safe S 1999 Transcriptional activation of E2F1 gene expression by 17ß-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions. Mol Endocrinol 13:1373–1387[Abstract/Free Full Text]
  46. Holth LT, Sun JM, Coutts AS, Murphy LC, Davie JR 1997 Estrogen receptor diminishes DNA-binding activities of chicken GATA-1 and CACCC-binding proteins. DNA Cell Biol 16:1477–1482[Medline]
  47. Blobel GA, Sieff CA, Orkin SH 1995 Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor. Mol Cell Biol 15:3147–3153[Abstract]
  48. Jones DR, Schmidt RJ, Pickard RT, Foxworthy PS, Eacho PI 2002 Estrogen receptor-mediated repression of human hepatic lipase gene transcription. J Lipid Res 43:383–391[Abstract/Free Full Text]
  49. Homma H, Kurachi H, Nishio Y, Takeda T, Yamamoto T, Adachi K, Morishige K, Ohmichi M, Matsuzawa Y, Murata Y 2000 Estrogen suppresses transcription of lipoprotein lipase gene. Existence of a unique estrogen response element on the lipoprotein lipase promoter. J Biol Chem 275:11404–11411[Abstract/Free Full Text]
  50. Cheng KW, Leung PC 2002 Human chorionic gonadotropin-activated cAMP pathway regulates human placental GnRH receptor gene transcription in choriocarcinoma JEG-3 cells. J Clin Endocrinol Metab 87:3291–3299[Abstract/Free Full Text]
  51. Cheng KW, Leung PC 2001 Human gonadotropin-releasing hormone receptor gene transcription: up-regulation by 3',5'-cyclic adenosine monophosphate/protein kinase A pathway. Mol Cell Endocrinol 181:15–26[CrossRef][Medline]
  52. Baker SJ, Kerppola TK, Luk D, Vandenberg MT, Marshak DR, Curran T, Abate C 1992 Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts. Mol Cell Biol 12:4694–4705[Abstract]
  53. Arias J, Alberts AS, Brindle P, Claret FX, Smeal T, Karin M, Feramisco J, Montminy M 1994 Activation of cAMP and mitogen responsive genes relies on a common nuclear factor. Nature 370:226–229[CrossRef][Medline]
  54. Bannister AJ, Oehler T, Wilhelm D, Angel P, Kouzarides T 1995 Stimulation of c-Jun activity by CBP: c-Jun residues Ser63/73 are required for CBP induced stimulation in vivo and CBP binding in vitro. Oncogene 11:2509–2514[Medline]
  55. Chakravarti D, LaMorte VJ, Nelson MC, Nakajima T, Schulman IG, Juguilon H, Montminy M, Evans RM 1996 Role of CBP/P300 in nuclear receptor signaling. Nature 383:99–103[CrossRef][Medline]
  56. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC, Heyman RA, Rose D, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403–414[Medline]
  57. Conn PM, Crowley Jr WJ 1994 Gonadotropin-releasing hormone and its analogs. Annu Rev Med 45:391–405[CrossRef][Medline]
  58. Gommersall LM, Hayne D, Shergill IS, Arya M, Wallace DM 2002 Luteinizing hormone releasing hormone analogues in the treatment of prostate cancer. Expert Opin Pharmacother 3:1685–1692[Medline]
  59. Jost JP, Saluz HP, McEwan I, Feavers IM, Hughes M, Reiber S, Liang HM, Vaccaro M 1990 Tissue specific expression of avian vitellogenin gene is correlated with DNA hypomethylation and in vivo specific protein-DNA interactions. Philos Trans R Soc Lond B Biol Sci 326:231–240[Medline]
  60. Liu MM, Albanese C, Anderson CM, Hilty K, Webb P, Uht RM, Price RH, Pestell RG, Kushner PJ 2002 Opposing action of estrogen receptors {alpha} and ß on cyclin D1 gene expression. J Biol Chem 277:24353–24360[Abstract/Free Full Text]
  61. Shapiro RA, Xu C, Dorsa DM 2000 Differential transcriptional regulation of rat vasopressin gene expression by estrogen receptor {alpha} and ß. Endocrinology 141:4056–4064[Abstract/Free Full Text]
  62. Scheidegger KJ, Cenni B, Picard D, Delafontaine P 2000 Estradiol decreases IGF-1 and IGF-1 receptor expression in rat aortic smooth muscle cells. Mechanisms for its atheroprotective effects. J Biol Chem 275:38921–38928[Abstract/Free Full Text]
  63. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson JA, Smithies O 1998 Generation and reproductive phenotypes of mice lacking estrogen receptor ß. Proc Natl Acad Sci USA 95:15677–15682[Abstract/Free Full Text]
  64. Couse JF, Hewitt SC, Bunch DO, Sar M, Walker VR, Davis BJ, Korach KS 1999 Postnatal sex reversal of the ovaries in mice lacking estrogen receptors {alpha} and ß. Science 286:2328–2331[Abstract/Free Full Text]
  65. Branderberger AW, Tee MK, Jaffe RB 1998 Estrogen receptor {alpha} (ER-{alpha}) and ß (ER-ß) mRNAs in normal ovary, ovarian serous cystadenocarcinoma and ovarian cancer cell lines: down-regulation of ER-ß in neoplastic tissues. J Clin Endocrinol Metab 83:1025–1028[Abstract/Free Full Text]
  66. Lau KM, Mok SC, Ho SM 1999 Expression of human estrogen receptor-{alpha} and -ß, progesterone receptor, and androgen receptor mRNA in normal and malignant ovarian epithelial cells. Proc Natl Acad Sci USA 96:5722–5727[Abstract/Free Full Text]
  67. Shaw JA, Udokang K, Mosquera JM, Chauhan H, Jones JL, Walker RA 198 2002 Oestrogen receptors {alpha} and ß differ in normal human breast and breast carcinomas. J Pathol 198:450–457[CrossRef][Medline]
  68. Dotzlaw H, Leygue E, Watson PH, Murphy LC 1997 Expression of estrogen receptor-ß in human breast tumors. J Clin Endocrinol Metab 82:2371–2374[Abstract/Free Full Text]
  69. Pujol P, Rey JM, Nirde P, Roger P, Gastaldi M, Laffargue F, Rochefort H, Maudelonde T 1998 Differential expression of estrogen receptor-{alpha} and -ß messenger RNAs as a potential marker of ovarian carcinogenesis. Cancer Res 58:5367–5373[Abstract]
  70. Leygue E, Dotzlaw H, Watson PH, Murphy LC 1998 Altered estrogen receptor {alpha} and ß messenger RNA expression during human breast tumorigenesis. Cancer Res 58:3197–3201[Abstract]
  71. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889–895[Medline]
  72. Tremblay GB, Tremblay A, Larbie F, Giguere V 1999 Dominant activity of activation function 1 (AF-1) and differential stoichiometric requirements for AF-1 and -2 in the estrogen receptor {alpha}-ß heterodimeric complex. Mol Cell Biol 19:1919–1927[Abstract/Free Full Text]
  73. Ogawa S, Inoue S, Watanabe T, Hiroi H, Orimo A, Hosoi T, Ouchi Y, Muramatsu M 1998 The complete primary structure of human estrogen receptor ß (hER ß) and its heterodimerization with ER{alpha} in vivo and in vitro. Biochem Biophys Res Commun 243:122–126[CrossRef][Medline]
  74. Pace P, Taylor J, Suntharalingam S, Coombes RC, Ali S 1997 Human estrogen receptor ß binds DNA in a manner similar to and dimerizes with estrogen receptor {alpha}. J Biol Chem 272:25832–25838[Abstract/Free Full Text]
  75. McKenna NJ, Lanz RB, O’Malley BW 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 20:321–344[Abstract/Free Full Text]
  76. Miller CD, Miller WL 1996 Transcriptional repression of the ovine follicle-stimulating hormone-ß gene by 17ß-estradiol. Endocrinology 137:3437–3446[Abstract]
  77. Schmitt M, Bausero P, Simoni P, Queuche D, Greoffroy V, Marschal C, Kempf J, Quirin-Stricker C 1995 Positive and negative effects of nuclear receptors on transcription activation by AP-1 of the human choline acetyltransferase proximal promoter. J Neurosci Res 40:152–164[Medline]
  78. Teyssier C, Belguise K, Galtier F, Chalbos D 2001 Characterization of the physical interaction between estrogen receptor {alpha} and JUN proteins. J Biol Chem 276:36361–36369[Abstract/Free Full Text]
  79. Gaub MP, Bellard M, Scheuer I, Chambon P, Sassone-Corsi P 1990 Activation of the ovalbumin gene by the estrogen receptor involves the fos-jun complex. Cell 63:1267–1276[Medline]
  80. Webb P, Lopez GN, Uht RM, Kushner PJ 1995 Tamoxifen activation of the estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like effects of antiestrogens. Mol Endocrinol 9:443–456[Abstract]
  81. Shibata H, Spencer TE, Onate SA, Jenster G, Tsai SY, Tsai MJ, O’Malley BW 1997 Role of co-activators and co-repressors in the mechanism of steroid/thyroid receptor action. Recent Prog Horm Res 52:141–164[Medline]
  82. Torchia J, Glass C, Rosenfeld MG 1998 Co-activators and co-repressors in the integration of transcriptional responses. Curr Opin Cell Biol 10:373–383[CrossRef][Medline]
  83. Webb P, Nguyen P, Shinsako J, Anderson C, Feng W, Nguyen MP, Chen D, Huang SM, Subramanian S, McKinerney E, Katzenellenbogen BS, Stallcup MR, Kushner PJ 1998 Estrogen receptor activation function 1 works by binding p160 coactivator proteins. Mol Endocrinol 12:1605–1618[Abstract/Free Full Text]
  84. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL 1998 The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927–937[Medline]
  85. Inada H, Mukai J, Matsushima S, Tanaka T 1997 QM is a novel zinc-binding transcription regulatory protein: its binding to c-Jun is regulated by zinc ions and phosphorylation by protein kinase C. Biochem Biophys Res Commun 230:331–334[CrossRef][Medline]
  86. Zeiner M, Gebauer M, Gehring U 1997 Mammalian protein RAP46: an interaction partner and modulator of 70 kDa heat shock proteins. EMBO J 16:5483–5490[Abstract/Free Full Text]
  87. Kwok RP, Lundblad JR, Chrivia JC, Richards JP, Bachinger HP, Brennan RG, Roberts SG, Green MR, Goodman RH 1994 Nuclear protein CBP is a coactivator for the transcription factor CREB. Nature 370:223–226[CrossRef][Medline]
  88. Pulverer BJ, Kyriakis JM, Avurch J, Nikolakaki E, Woodgett JR 1991 Phosphorylation of c-jun mediated by MAP kinases. Nature 353:670–674[CrossRef][Medline]
  89. Smeal T, Binetruy B, Mercola DA, Birrer M, Karin M 1991 Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73. Nature 354:494–496[CrossRef][Medline]
  90. Caelles C, Gonzalez-Sancho JM, Munoz A 1997 Nuclear hormone receptor antagonism with AP-1 by inhibition of the JNK pathway. Genes Dev 11:3351–3364[Abstract/Free Full Text]
  91. Lee SK, Kim HJ, Ma SY, Kim TS, Choi HS, Im SY, Lee JW 1998 Steroid receptor coactivator-1 coactivates activating protein-1-mediated transactivations through interaction with the c-Jun and c-Fos subunits. J Biol Chem 273:16651–16654[Abstract/Free Full Text]
  92. Jung DJ, Na SY, Na DS, Lee JW 2002 Molecular cloning and characterization of CAPER, a novel coactivator of activating protein-1 and estrogen receptors. J Biol Chem 277:1229–1234[Abstract/Free Full Text]
  93. de Jager T, Pelzer T, Muller-Botz S, Imam A, Muck J, Neyses L 2001 Mechanisms of estrogen receptor action in the myocardium. Rapid gene activation via the ERK1/2 pathway and serum response elements. J Biol Chem 276:27873–27880[Abstract/Free Full Text]
  94. Silverman ES, Du J, Williams AJ, Wadgaonkar R, Drazen JM, Collins T 1998 cAMP-response-element-binding-protein-binding protein (CBP) and p300 are transcriptional coactivators of early growth response factor-1 (Egr-1). Biochem J 336:183–189[Medline]
  95. Wong CW, Komm B, Cheskis BJ 2001 Structure-function evaluation of ER {alpha} and ß interplay with SRC family coactivators. ER selective ligands. Biochemistry 40:6756–6765[CrossRef][Medline]
  96. Bramlett KS, Burris TP 2002 Effects of selective estrogen receptor modulators (SERMs) on coactivator nuclear receptor (NR) box binding to estrogen receptors. Mol Genet Metab 76:225–233[CrossRef][Medline]
  97. Kaiser UB, Jakubowiak A, Steinberger A, Chin WW 1993 Regulation of rat pituitary gonadotropin-releasing hormone receptor mRNA levels in vivo and in vitro. Endocrinology 133:931–934[Abstract]
  98. Bauer-Dantoin AC, Weiss P, Jameson JL 1995 Gonadotropin-releasing hormone receptor messenger ribonucleic acid expression in the ovary during the rat estrous cycle. Endocrinology 136:1014–1019[Abstract]
  99. Chow BK, Ting V, Tufaro F, MacGillivray RT 1991 Characterization of a novel liver-specific enhancer in the human prothrombin gene. J Biol Chem 266:18927–18933[Abstract/Free Full Text]