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Molecular Endocrinology 13 (10): 1672-1685
Copyright © 1999 by The Endocrine Society

The Estrogen Receptor Enhances AP-1 Activity by Two Distinct Mechanisms with Different Requirements for Receptor Transactivation Functions

Paul Webb, Phuong Nguyen, Cathleen Valentine, Gabriela N. Lopez, Grace R. Kwok, Eileen McInerney, Benita S. Katzenellenbogen, Eva Enmark, Jan-Åke Gustafsson, Stefan Nilsson and Peter J. Kushner

Metabolic Research Unit (P.W., P.N., C.V., G.N.L., G.R.K., P.J.K.) University of California School of Medicine San Francisco, California 94143
Department of Molecular and Integrative Physiology (E.M., B.S.K.) University of Illinois Urbana, Illinois 61801
KaroBio AB (S.N.) Novum Huddinge, Sweden S-14157
Department of Medical Nutrition and Biosciences (E.E., J.-A.G.) Karolinska Institute Huddinge, Sweden S-14186


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptors (ERs {alpha} and ß) enhance transcription in response to estrogens by binding to estrogen response elements (EREs) within target genes and utilizing transactivation functions (AF-1 and AF-2) to recruit p160 coactivator proteins. The ERs also enhance transcription in response to estrogens and antiestrogens by modulating the activity of the AP-1 protein complex. Here, we examine the role of AF-1 and AF-2 in ER action at AP-1 sites. Estrogen responses at AP-1 sites require the integrity of the ER{alpha} AF-1 and AF-2 activation surfaces and the complementary surfaces on the p160 coactivator GRIP1 (glucocorticoid receptor interacting protein 1), the NID/AF-1 region, and NR boxes. Thus, estrogen-liganded ER{alpha} utilizes the same protein-protein contacts to transactivate at EREs and AP-1 sites. In contrast, antiestrogen responses are strongly inhibited by ER{alpha} AF-1 and weakly inhibited by AF-2. Indeed, ER{alpha} truncations that lack AF-1 enhance AP-1 activity in the presence of antiestrogens, but not estrogens. This phenotype resembles ERß, which naturally lacks constitutive AF-1 activity. We conclude that the ERs enhance AP-1 responsive transcription by distinct mechanisms with different requirements for ER transactivation functions. We suggest that estrogen-liganded ER enhances AP-1 activity via interactions with p160s and speculate that antiestrogen-liganded ER enhances AP-1 activity via interactions with corepressors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen signal transduction is mediated by two related proteins, the estrogen receptors {alpha} (ER{alpha}) and ß (ERß) (1, 2, 3). Both receptors are conditional transcription factors that belong to the nuclear receptor superfamily (4, 5, 6) and are comprised of separable N-terminal (AB), DNA-binding (DBD), and ligand-binding (LBD) domains. The ERs activate gene expression in two ways. In the best understood mode of action, or classical pathway, the ERs bind to specific estrogen response elements (EREs) within target genes and recruit a p160/p300 coactivator complex to the promoter (7, 8). This coactivator complex enhances gene expression by remodeling chromatin and, perhaps, by contacting the basal transcription machinery. In a second mode of action, the ERs utilize unspecified protein-protein interactions to enhance the activity of heterologous transcription factors. For example, both ERs enhance the transcription of genes that contain AP-1 sites, the cognate binding site for the Jun/Fos complex (9, 10, 11, 12, 13, 14). The ERs also enhance the transcription of genes that contain binding sites for other transcription factors (see Refs. 15, 16, 17, 18, 19, 20 and references therein).

While the precise mechanism of ER action at AP-1 sites is unknown, our previous studies have indicated that it shows some striking differences from ER action at EREs (12, 13). First, antiestrogens, such as the breast cancer drug tamoxifen, act as potent agonists of ER action at AP-1 sites, even though they usually block ER action at classical EREs. This is particularly evident in the case of ERß, which potently enhances AP-1-dependent transcription in the presence of antiestrogens, but not estrogen. Second, estrogen action at AP-1 sites does not require the ER{alpha}-DBD and hence does not require specific ERE recognition (9, 12). ER action at AP-1 does require both the site and AP-1 proteins (12). Third, the overall strength of ER action at AP-1 sites often bears little relationship to the strength of ER action at an ERE. For example, ERß is a more potent activator of AP-1-dependent transcription than ER{alpha} (13), but is a weaker activator of classical estrogen response than ER{alpha} (21, 22, 23).

Perhaps more surprisingly, our initial studies of the mechanism of ER action at AP-1 sites also suggested that estrogen and antiestrogen effects are products of completely distinct pathways. We (12) and others (10, 14) found that estradiol effects predominate in several breast cancer cell lines, but we also found that tamoxifen effects predominate in other cell lines, including those of uterine and liver origin. Furthermore, estradiol and tamoxifen action at AP-1 sites showed distinct structure-function requirements. Estradiol activation requires the ER{alpha}-LBD, whereas tamoxifen activation requires the ER{alpha} AB-DBD region. Finally, we found that the estradiol-liganded ER{alpha} targeted the VP16 transactivation function to AP-1 sites in mammalian two-hybrid assays, but the tamoxifen-liganded ER{alpha} did not. Later, we also showed that ERß does not enhance AP-1 activity in the presence of estrogens, but does enhance AP-1 activity in the presence of antiestrogens (13). Together, these results suggest that the ERs participate in distinct sets of protein-protein interactions that lead to stimulation of AP-1 activity, one promoted by estrogens, the other promoted by antiestrogens.

It is well established that ER action at classical EREs is mediated by transactivation functions (4, 5, 6, 24, 25). ER{alpha} contains two separate transactivation functions, AF-1 and AF-2, which synergize strongly to give the overall level of estrogen response (26, 27). While ERß does contain a growth factor-inducible AF-1 (28), its activity stems largely from AF-2 (22, 29). AF-2 of both receptors consists of a small hydrophobic patch on the surface of the estrogen-liganded LBD (30, 31, 32), which binds strongly to specific LXXLL motifs (nuclear receptor boxes) that are found throughout the central region of the p160s (33, 34, 35, 36, 37). ER{alpha} AF-1 consists of a long region of the AB domain that binds the C terminus of glucocorticoid receptor-interacting protein 1 (GRIP1) (NID/AF-1) and other p160s (29). ERß AF-1 also binds weakly to SRC-1, but only when AF-1 is phosphorylated (38). Antiestrogens, including tamoxifen, raloxifene, and ICI 182,780 (ICI), block formation of AF-2, thereby reducing coactivator complex recruitment (31, 32, 39), and restrict ER{alpha} AF-1 activity by promoting association with corepressors (40, 41, 42, 43, 44, 45). Tamoxifen, however, does allow some ER{alpha} AF-1 activity and, consequently, elicits weak agonist effects at classical EREs (24).

Because ER transactivation functions play a central role in classical estrogen response, we ask here whether they might also play a role in ER action at AP-1 sites. We find that the estrogen/AP-1 pathway involves ER transactivation functions, but the antiestrogen/AP-1 pathway does not. Instead, the antiestrogen/AP-1 pathway is strongly inhibited by ER{alpha} AF-1 and weakly inhibited by AF-2 of both receptors. These results reinforce the notion that estrogen and antiestrogen effects at AP-1 sites are mediated by distinct mechanisms and may suggest identities for putative estrogen and antiestrogen pathway targets.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Estrogen/AP-1 Pathway Requires Only the LBD and AF-2
To understand how the ERs enhance AP-1 activity, we examined the role of ER transactivation functions in estrogen and antiestrogen action at AP-1 sites. To perform these experiments, we asked whether transiently transfected wild-type ERs, or ER derivatives bearing specific mutations in AF-1 or AF-2, would elicit estrogen or antiestrogen responses at an AP-1-responsive reporter in HeLa cells.

We previously showed that estrogen action at AP-1 sites could be obtained in the absence of the ER{alpha} N-terminal domain and DBD, suggesting that the LBD is necessary for the estrogen/AP-1 pathway (12). We therefore asked whether the LBD was also sufficient to obtain an estrogen response at AP-1 sites. Figure 1Go shows that the ER{alpha}-LBD gave weak constitutive activation of the AP-1 responsive reporter gene relative to empty expression vector. Addition of estradiol elicited a further 10-fold increase in the activity of the AP-1-responsive promoter. Tamoxifen (shown) and raloxifene and ICI (not shown) failed to enhance AP-1 activity in the presence of the isolated LBD. Thus, the ER{alpha}-LBD is sufficient to obtain a potent estrogen response at AP-1 sites.



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  		Figure 1. The Estrogen Pathway Requires only the ER{alpha}-LBD and AF-2

Results of a HeLa cell transfection in which the effect of ligands upon the activity of a transiently transfected AP-1-responsive reporter is monitored in the presence of empty expression vector or expression vectors for the isolated ER{alpha}-LBD (amino acids 282–595) or similar LBDs bearing mutations in AF-2. The mutant LBDs contained a double hydrophobic mutation in AF-2 (LBDmAF-2; M543A,L544A) or a complete truncation of helix 12 and F domain sequences downstream of amino acid 537 (LBD{Delta}AF-2). The individual bars in each graph represent luciferase activity in extracts of cells treated with ethanolic vehicle (open bars), 5 µM tamoxifen (gray bars) or 10 nM estradiol (black bars). The bars show average luciferase activities calculated from triplicate wells in a representative transfection.

 
We then asked whether the LBD-mediated estrogen response required AF-2. ER{alpha}-LBDs bearing either a specific mutation in AF-2 (LBDmAF-2; M543A,L544A) or a complete truncation of helix 12 (LBD{Delta}AF-2, truncated at position 537) gave weak constitutive activation at the AP-1 site, but failed to yield further estradiol activation. We conclude that AF-2 plays a key role in the estrogen/AP-1 pathway. We also note that there are strong parallels between the behavior of the ER{alpha}-LBD at AP-1 sites and the behavior of the glucocorticoid receptor LBD at the herpes simplex virus thymidine kinase promoter (46), raising the possibility that AF-2-dependent modulation of heterologous transactivation factor activity could be a common feature of nuclear receptor action.

Estrogen Responses at AP-1 Sites Require AF-1, but Tamoxifen Responses Are Independent of AF-1
We next turned our attention to AF-1, which synergises with AF-2 in estrogen response at classical EREs and also mediates weak antiestrogen agonist effects at classical EREs. In Hela cells, full ER{alpha} AF-1 activity requires a long region of the AB domain (amino acids 41–129, Fig. 2AGo) (29, 47, 48, 49, 50). The two flanking regions (iAF-1A, amino acids 1–41, iAF-1B, amino acids 129–178) weakly inhibit AF-1 activity at EREs (29).



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  		Figure 2. AF-1 Suppresses Tamoxifen Effects at AP-1 Sites

A, The human ER{alpha} N-terminal AB domain (amino acids 1–178). Amino acids 1–41 weakly inhibit AF-1 activity and are designated iAF-1A. The extent of sequences required for full AF-1 activity in HeLa cells (amino acids 41–129) is marked in gray. A second weak inhibitory function (amino acids 129–178) is designated iAF-1B. B, Behavior of deletions that eliminate different portions of the ER{alpha} N-terminal (AB) domain at classical EREs. The experiment was carried out using the EREII-LUC reporter gene shown at top. Included in each transfection were an empty SV40 expression vector, SG5 (none), or SV40 expression vectors for wild-type ER{alpha}, or ER{alpha} truncations lacking the first 101 (N101), 109 (N109), 117 (N117) amino acids of the AB domain, the entire AB domain ({Delta}AB), or containing an internal deletion of iAF-1B sequences ({Delta}129–178). A schematic of these deletions is shown at the lefthand side. The effect of ER ligands upon the activity of the transiently transfected EREII-LUC reporter gene is shown in the accompanying graph. Individual points show average luciferase activities, calculated from triplicate wells in a representative transfection and expressed relative to the activity of the promoter in the absence of ligand and ER{alpha} expression vector that was assigned a value of 1. C, The behavior of the same ER deletions at an AP-1-responsive reporter gene, coll73-LUC. The individual points show luciferase activities, calculated similarly to those of panel B.

 
In accordance with our previously published results, ER{alpha} N-terminal deletions lacking AF-1 activity (N101, N109, N117, {Delta}AB) elicited reduced estrogen response and no tamoxifen response at a classical ERE-responsive reporter (EREII-LUC; Fig. 2BGo). Moreover, an ER{alpha} internal deletion that retains AF-1, but lacks iAF-1B ({Delta}129–178), elicited normal activity in the presence of both ligands and also showed a modest enhancement of ligand-independent activity.

At an AP-1-responsive reporter (coll73-LUC), the ER{alpha} truncations showed a similar activity profile in the presence of estradiol (Fig. 2CGo). Estradiol action was abolished by certain N-terminal deletions that abolish AF-1 activity (N117, {Delta}AB) but was unaffected by deletion of iAF-1B (ER{Delta}129–178). In the presence of tamoxifen, however, the same deletions showed a sharply contrasting phenotype. Two truncations that abolish AF-1 activity (N101, N109) showed accentuated tamoxifen response. This result indicates that tamoxifen responses at AP-1 sites are independent of AF-1 activity per se and may actually be suppressed by AF-1. Longer N-terminal deletions (N117 or {Delta}AB) and the iAF-1B deletion ({Delta}129–178) each failed to elicit tamoxifen responses from the AP-1-responsive reporter. Thus, in the context of full-length ER{alpha}, estrogen responses at AP-1 sites require AF-1, but tamoxifen responses are independent of AF-1 and require iAF-1B.

Serine 118 Is Dispensable For Tamoxifen Responses at AP-1 Sites
At classical EREs, full ER{alpha} AF-1 activity requires a serine residue (S118) that forms part of a mitogen-activated protein kinase consensus recognition sequence (PXXSP) and is a target for phosphorylation (51, 52, 53, 54, 55, 56). At AP-1 sites, ER{alpha}-dependent tamoxifen effects require an unspecified function whose N-terminal boundary lies between amino acids 109 and 117 of the AB domain (Fig. 2Go). We therefore asked whether S118 played any role in ER{alpha} action at AP-1 sites. We changed S118 to either alanine (A), arginine (R), which has a bulky charged side chain and should disrupt S118-dependent protein-protein contacts, or glutamic acid (E), which mimics an active phosphorylated serine residue (55), and asked whether any of these mutants affected tamoxifen response at EREs or AP-1 sites.

Figure 3AGo reveals that ER{alpha}-S118E showed enhanced tamoxifen response at the classical ERE relative to wild-type ER{alpha} and to the S118A and S118R mutants. This result is consistent with the notion that S118E mimics the phosphorylated, active state of AF-1 and that S118A and S118R block phosphorylation and do not allow full AF-1 activity. By contrast, each ER{alpha} S118 mutant elicited tamoxifen responses at AP-1 sites that were comparable to those obtained with wild type ER{alpha} (Fig. 3BGo). Similar results (not shown) were also obtained with an S118A mutation in the context of the isolated ER{alpha} AB-DBD region, which enhances gene expression constitutively in a manner that resembles tamoxifen-liganded ER{alpha} (12, 57). Thus, S118 is dispensable for tamoxifen effects at AP-1 sites. The fact that ER-S118E showed enhanced tamoxifen activation at the ERE, but did not affect tamoxifen activation at the AP-1 site, also reinforces the notion that conventional AF-1 activity is not required for tamoxifen activation at AP-1 sites.



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  		Figure 3. Serine 118 Is Dispensable For ER{alpha} Action at AP-1 Sites

A, The effect of three-point mutations in phosphorylation target residue serine 118 on ER{alpha} action at EREs. S118A, Alanine; S118R, arginine; S118E, glutamic acid. The figure represents an average of four experiments. Each point was normalized to the activity of the EREII-LUC reporter gene in the absence of ER{alpha} and ligand. B, Experiment as in Fig. 3AGo, except that the coll73-LUC reporter is used. The figure represents one typical experiment.

 
Removal of ER{alpha} AF-1 Creates an ERß-Like Phenotype
Removal of ER{alpha} AF-1 leads to enhanced tamoxifen responses at AP-1 sites in HeLa cells (Fig. 2Go). This phenotype is similar to ERß (13), which naturally lacks constitutive AF-1 activity (29). We therefore asked whether removal of ER{alpha} AF-1 created other ERß-like phenotypes.

We previously showed that full-length ER{alpha} only enhances AP-1 activity in the presence of tamoxifen and estradiol in HeLa cells and that ERß strongly enhances AP-1 activity in the presence of a range of antiestrogens, but not estrogens, in the same cell type (13). Figure 4Go shows that, as expected, wild-type ER{alpha} enhanced AP-1 activity in the presence of tamoxifen, estradiol, and the synthetic agonist diethylstilbestrol (DES), but not raloxifene or ICI, in HeLa cells. Likewise, ERß enhanced AP-1 activity in the presence of ICI, raloxifene, and tamoxifen, but not estradiol or DES. In parallel, short ER{alpha} N-terminal truncations (N21, N41) showed ligand responses that were similar to wild-type ER{alpha}. However, longer truncations that partially (N87), or completely (N109, {Delta}AB), eliminate AF-1 activity showed enhanced antiestrogen effects, especially with raloxifene and ICI. Thus, ER{alpha} AF-1 suppresses antiestrogen action at AP-1 sites in HeLa cells, and its elimination allows ER{alpha} to behave like ERß in its ligand preferences for action at AP-1.



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  		Figure 4. Deletion of ER{alpha} AF-1 Creates an ERß-Like Phenotype

AF-1 suppresses the capacity of ER{alpha} to enhance AP-1 activity in the presence of antiestrogens in HeLa cells. The effect of various ER ligands upon the activity of a transiently transfected coll73-LUC reporter gene was examined. Also included in the transfection were an empty CMV expression vector (none) or CMV expression vectors for human ER{alpha}, ERß (ERß485), or ER{alpha} truncations lacking different portions of the ER N terminus. The ligands were ICI 182,780 (10 nM); raloxifene (10 nM), tamoxifen (5 µM), estradiol (10 nM), or DES (10 nM).

 
We also previously showed that ER{alpha} only elicits weak estrogen responses at AP-1 sites in breast cells (12), but ERß elicits strong antiestrogen effects (13). Accordingly, wild-type ER{alpha} only enhanced AP-1 activity weakly in the presence of estrogens in MCF-7 cells, and ERß enhanced AP-1 activity in the presence of antiestrogens, but not estrogens (Fig. 5AGo). In parallel, ER{alpha} N-terminal deletions that eliminate AF-1 (N87, N109, {Delta}AB) elicited extremely potent antiestrogen effects. A similar profile was also obtained in MDA-MB-453 breast cells (data not shown). Thus, AF-1 restricts ER{alpha} action in breast cells and, here again, removal of AF-1 allows an ERß-like spectrum of ligand preferences for action at AP-1 sites.



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  		Figure 5. Deletion of AF-1 Changes the Ligand Preference of ER{alpha} Action in Breast Cells

A, Removal of AF-1 Enhances ER{alpha} action in MCF-7 Cells in the presence of antiestrogens. Results of a similar series of transfections, carried out in MCF-7 breast cells, using the same CMV-driven expression vectors for each receptor, and ligand treatments, as shown in Fig. 4Go. The figure represents the average of five separate transfections. To ease comparison of different experiments, luciferase activity obtained in the absence of transfected ER expression vector and ligand was assigned the value of 1. Other luciferase activities were expressed relative to this value. B, Ligand preference of ER{alpha} N-terminal truncations in MCF-7 cells. The activity of different ligands at AP-1 sites was plotted as a function of the ER{alpha} N-terminal deletion endpoint. In each case, basal AP-1 activity obtained in the absence of ER and ligand was subtracted, and the maximal activity that was obtained with any N-terminal deletion mutant in the presence of a particular ligand was set to 100%. Each point represents the average of five transfections.

 
Closer examination revealed some interesting aspects to the behavior of the ER{alpha} N-terminal truncations, which were especially prominent in MCF-7 cells (Fig. 5BGo). Estradiol (and DES, not shown) effects at AP-1 sites were enhanced by deletion of iAF-1A at the extreme ER{alpha} N terminus and were reduced by deletion of AF-1. These sequence requirements are very similar to those seen for estrogen action at EREs (29, 47, 48, 49, 50). Conversely, antiestrogen effects were all enhanced by elimination of AF-1. However, different truncations showed distinct ligand preferences. A truncation that partially eliminates AF-1 activity (N87) showed strongly enhanced tamoxifen effects and weaker raloxifene and ICI effects. Longer deletions (N109, N117, {Delta}AB) showed stronger ICI and raloxifene effects. Furthermore, tamoxifen effects were heavily dependent upon the iAF-1B region, but raloxifene (and ICI, not shown) responses were less dependent upon this region, and, in fact, were largely mediated by the DBD-LBD region. Thus, different antiestrogens utilize different regions of ER{alpha} to enhance AP-1 activity. Despite these differences, the stark separation of estrogen and antiestrogen effects at AP-1 sites underscores our notion that different ER functions contribute to estrogen and antiestrogen effects at AP-1 sites.

Do Longer ERß Isoforms Possess a Constitutive AF-1 Function That Suppresses Antiestrogen Action at AP-1 Sites?
Removal of ER{alpha} AF-1 creates a phenotype that resembles ERß. This result predicts that addition of a constitutive AF-1 activity back to ERß might suppress its ability to exert antiestrogen effects at AP-1 sites. We previously showed that a short form of ERß [ERß485, (1, 58)] lacks a constitutive AF-1 activity (29) and strongly enhances AP-1 activity in the presence of antiestrogens, but not estrogens (13). Because a longer form of ERß (ERß530) has now been identified (59, 60), we asked whether it might contain such an AF-1 activity.

Figure 6AGo shows that estrogen-liganded ERß530 gave comparable levels of transcriptional activity to ERß485 at classical EREs. Similar results were also obtained with both forms of estradiol-liganded ERß in the presence of overexpressed coactivators, GRIP1 and p300 (+CoAcs). Overexpression of GRIP1 and p300 also enhanced the activity of both forms of ERß in the absence of ligand, a phenomenon that stems from p160 interaction with the weak inducible ERß AF-1 function (38), but did not enhance ERß activity in the presence of tamoxifen (shown) or raloxifene and ICI (not shown). In parallel, ER{alpha} did elicit extremely potent AF-1-dependent tamoxifen responses in the presence of GRIP1 and p300 (lower panel, note expanded scale). Thus, ERß530, like ERß485, lacks a constitutive AF-1 activity.



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  		Figure 6. ERß Isoforms Lack AF-1 Activity and Enhance AP-1 Activity in the Presence of Antiestrogens

A, Overexpression of GRIP1 and p300 fail to elicit tamoxifen responses at classical EREs in Hela cells in the presence of either form of ERß. Effects of a combination of GRIP1 and p300 (CoAcs) on EREII-LUC expression in the presence of SV40-driven expression vectors for human ERß or ER({alpha}). Note the expanded scale for the transfection containing ER({alpha}). B, Autoradiogram of an SDS-PAGE gel showing labeled GRIP1 protein retained by various GST-fusion proteins. The lanes represent 10% input GRIP1, GRIP1 protein retained by GST-beads, GST-ERß AB domain fusion, GST-ER{alpha} AB domain, GST-ER{alpha} LBD, and GST-ER{alpha} LBD in the presence of estrogen. C, Effect of ERß530 and ERß485 upon AP-1-responsive transcription in HeLa cells. A range of ligands was used as in Fig. 4Go.

 
We also previously showed that the ER{alpha} AB domain contains a constitutive binding site for GRIP1, but the AB domain of ERß485 does not (29). We therefore generated a glutathione-S-transferase (GST) fusion protein containing the entire AB domain of the long form of ERß (amino acids 1–144) and asked whether it would bind GRIP1. Figure 6BGo shows that the ER{alpha} AB domain bound GRIP1 strongly and the ERß AB domain did not. Thus, ERß530 lacks a constitutive binding site for GRIP1.

Finally, we asked whether the longer form of ERß would enhance AP-1-responsive transcription. Figure 6CGo shows that ERß530 actually enhanced AP-1 activity more potently than ERß485 in the presence of antiestrogens. Neither form of receptor enhanced AP-1 activity in the presence of estradiol or DES. Thus, human ERß530 does not possess a constitutive AF-1 activity that suppresses antiestrogen action at AP-1 sites. It remains to be seen whether even longer forms, such as the 549 amino acid rodent ERß (38), might contain such an activity.

AF-2 Is Dispensable For Antiestrogen Action at AP-1 Sites
Next, we examined the role of AF-2 in antiestrogen action at AP-1 sites. AF-2 consists of a hydrophobic cleft, made up of a cluster of residues from LBD helices 3, 5, and 12, that forms upon the surface of the holo-ER (30, 31, 32). To probe the requirement for AF-2 in ER action at AP-1 sites, we used a mutation (K>A) in a helix 3 lysine residue (61), which forms key hydrogen bonds with the GRIP1 nuclear receptor box (32).

We first examined how the AF-2 mutant would affect ER{alpha} action at AP-1 sites in HeLa cells. Figure 7AGo shows that the ER{alpha}K362A mutant gave reduced estradiol response and normal tamoxifen response at AP-1 sites. The equivalent ERß AF-2 mutant (K269A) gave ICI, raloxifene, and tamoxifen responses that were slightly larger than those obtained with the wild-type ERß (Fig. 7BGo). Likewise, the ER{alpha} K362A mutation gave modestly enhanced raloxifene and ICI responses in the context of the isolated DBD-LBD region at AP-1 sites (not shown). Control transfections revealed that each AF-2 mutant receptor was inactive at an ERE-responsive reporter (not shown). Thus, AF-2 is either dispensable or weakly inhibitory for antiestrogen action at AP-1 sites.



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  		Figure 7. Mutation of AF-2 Does Not Affect Antiestrogen Action at AP-1 Sites

A, Effect of tamoxifen or estradiol upon AP-1-responsive transcription in the presence of either full-length ER{alpha}, or the AF-2 mutant (K362A). The data are shown as fold increase of luciferase activity in the presence of ligand over no hormone. B, Effects of different ligands upon AP-1-responsive transcription in HeLa cells in the presence of full-length ERß or the ERß AF-2 mutant (K269A).

 
The Estrogen/AP-1 Pathway Requires ER{alpha} Recognition Sites within the p160 Coactivator GRIP1
Estradiol effects at AP-1 sites require ER{alpha} transactivation functions. Because the actions of the ER transactivation functions at classical EREs are mediated by p160s, we next asked whether the actions of the ER transactivation functions at AP-1 sites were also mediated by p160s. Figure 8Go shows that overexpression of the p160 GRIP1 enhanced basal AP-1 activity and ER action at the AP-1 site. By contrast, overexpression of a GRIP1 derivative with mutations in NR boxes II and III (NR box mutant), which is unable to bind AF-2, enhanced basal AP-1 activity but showed weakened estradiol and DES effects. Overexpression of another GRIP1 derivative with a truncation of NID/AF-1 region ({Delta}1121C), which is unable to bind AF-1, completely failed to potentiate estradiol and DES effects at the AP-1 site. Thus, estrogen action at AP-1 sites requires the integrity of ER{alpha} binding regions within the GRIP1 molecule. It therefore appears that estrogen action at AP-1 sites, like estrogen action at EREs, is mediated by contacts with p160s (see Discussion).



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  		Figure 8. Estrogen Effects at AP-1 Sites Require ER{alpha} Recognition Sites within GRIP1

The effect of ligands upon the coll73-LUC reporter gene was examined in the presence of an ER{alpha}V400 expression vector and SV40-driven expression vectors for wild-type GRIP1, a GRIP1 mutant that cannot bind ER{alpha} AF-2 (NR Box mutant) or a GRIP1 truncation that cannot bind ER{alpha} AF-1 ({Delta}1121C). Schematics of each GRIP1 molecule are shown at the lefthand side of the diagram. The positions of the NR boxes are marked with black bands; the position of the AF-1 binding region (NID/AF-1) is marked with a gray box. Shown at the righthand side is a typical experiment in which the effect of different ER ligands upon the coll73-LUC reporter gene was evaluated in the presence of ER{alpha} and the GRIP1 expression vectors. Ligand treatments were the same as described in Fig. 4Go.

 
The behavior of the GRIP1 truncation ({Delta}1121C) requires further comment. First, it failed to enhance basal AP-1 activity, suggesting that the GRIP1 C-terminal region contains unspecified functions that are essential for p160 coactivation at the AP-1 site. Second, it strongly potentiated antiestrogen effects, especially those of tamoxifen. We have previously shown that this GRIP1 truncation acts as a specific dominant negative for ER{alpha} AF-1 (29). The ability of GRIP1{Delta}1121C to enhance antiestrogen effects at AP-1 sites is therefore consistent with our notion that AF-1 suppresses antiestrogen action at AP-1 sites. Accordingly, GRIP1{Delta}1121C failed to enhance antiestrogen effects in the presence of ER{alpha} truncations that lack AF-1 (N109, {Delta}AB, not shown). Thus, our studies with GRIP1 overexpression also provide further support for the notion that the antiestrogen/AP-1 pathway is independent of ER transactivation functions and is strongly suppressed by AF-1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Activation Function-Mediated and Independent Pathways of ER Action at AP-1 Sites
Several lines of evidence indicate that the ER can modulate the activity of heterologous transcription factors via estrogen- and antiestrogen-specific mechanisms. Differences in the cell specificity, structure-function requirements, and behavior in mammalian two-hybrid assays led us to propose that ER{alpha}-dependent estradiol and tamoxifen effects at AP-1 sites are products of distinct mechanisms (12). Later, we showed that ERß does not enhance AP-1-dependent transcription in the presence of estrogens, but does enhance AP-1-dependent transcription in the presence of antiestrogens (13). Other genes have also been described that respond weakly to estrogens, and strongly to antiestrogens, in the presence of either form of ER (15, 17, 62, 63). Here, we have shown that different ER functions contribute to estrogen and antiestrogen effects at AP-1 sites. Estrogen effects only require the isolated ER{alpha}-LBD and its AF-2 function. Tamoxifen effects require the proximal part of the ER{alpha} B region (iAF-1B), along with an intact DBD (12), and raloxifene and ICI effects require the ER{alpha} DBD-LBD region, but not AF-2. Thus, our study reinforces the notion that ER activates AP-1-dependent transcription via estrogen- and antiestrogen-specific mechanisms.

Even though the estrogen/AP-1 pathway does not require the ER-DBD, it does share some features with classical estrogen response. Estrogen response at AP-1 sites requires AF-1 and AF-2 in the context of the full-length ER and AF-2 in the context of the isolated ER-LBD. Furthermore, estrogen effects at AP-1 sites require the integrity of the GRIP1 NR boxes, which bind AF-2, and the NID/AF-1 region, which binds AF-1. By contrast, the antiestrogen/AP-1 pathway is independent of ER transactivation functions and is strongly suppressed by ER{alpha} AF-1. We also suggest that the lack of a constitutive AF-1 in ERß, perhaps coupled with its relatively weak AF-2 (64), accounts for its ability to enhance AP-1 activity in the presence of antiestrogens, but not estrogens. Based on these results, we propose that the estrogen/AP-1 pathway should be renamed the activation function (AF)-mediated/AP-1 pathway and the antiestrogen/AP-1 pathway should be renamed the AF-independent/AP-1 pathway. This new nomenclature reflects important mechanistic features of each pathway and makes no assumptions about ligand preference.

We expect that estrogen enhancement of AP-1 activity will always occur through the AF-mediated pathway and that antiestrogen enhancement of AP-1 activity will largely occur through the AF-independent pathway. However, most of our studies have been conducted in HeLa cells that exhibit low AF-1 activity, and high AF-2 activity. Because tamoxifen is an AF-1 releasing agonist, we are presently exploring the idea that tamoxifen, but not raloxifene and ICI, will be able to work through the AF-mediated/AP-1 pathway in cell types that exhibit high AF-1 activity.

Mutations That Block One Pathway of ER Action at AP-1 Sites Enhance ER Action in the Other Pathway
One recurring observation of our studies has been that mutations that block one pathway of ER action at AP-1 sites enhance the other (12). Accordingly, we showed here that ER{alpha} AF-1 promotes estrogen effects at AP-1 sites but also inhibits antiestrogen effects. We also note that the ER{alpha} DBD-LBD region elicits potent AF-2-independent antiestrogen effects at AP-1 sites, but the LBD alone elicits only strong AF-2-dependent estrogen effects at AP-1 sites. We have also recently identified an ER{alpha}-DBD point mutant that blocks antiestrogen effects at AP-1 sites and, instead, allows ER{alpha} to elicit extremely potent AF-2-dependent estrogen effects at AP-1 sites (R. M. Uht, C. A. Anderson, P. Webb, D. B. Starr, and P. J. Kushner, unpublished data). Together, these results suggest that the ER-DBD promotes antiestrogen effects at AP-1 sites but also inhibits estrogen effects. Thus, disruption of the ER’s ability to participate in one pathway often frees the receptor to participate in the other.

A Model to Explain the Two Pathways of ER Action at AP-1 Sites
What are the molecular interactions that underlie ER action at AP-1 sites? ER/p160 contacts play an important role in the AF-mediated/AP-1 pathway, just as they do in classical estrogen response. Nonetheless, we suspect that there may be significant differences between the role of ER/p160 contacts at AP-1 sites and at EREs. The isolated ER{alpha}-LBD enhances AP-1-responsive transcription in an AF-2-dependent manner, even though it does not bind jun/fos (12). Thus, the ER-LBD cannot enhance AP-1 activity by binding jun/fos and recruiting p160s to the jun/fos complex, as the ERs do at EREs. It is known that AP-1 proteins activate transcription by recruiting a p300-p160 complex to the promoter via direct contacts with p300/CREB-binding protein (34). We therefore propose that the ER transactivation functions may serve to recognize p160s within the Jun/Fos-coactivator complex and to bring the ER to the AP-1-responsive promoter (Fig. 9AGo). The ER, positioned on the complex, would then enhance its activity. We are presently testing this hypothesis by asking whether ER can enhance the activity of isolated coactivator proteins.



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  		Figure 9. Models for the Two Pathways of ER Action at AP-1 Sites

A, The AF-mediated/AP-1 pathway. Estrogen-liganded ER uses its own transactivation functions (AF-1 and AF-2) to recognize the cognate surfaces (NID/AF-1; NR boxes) upon p160s that are present in the jun/fos (J/F)-associated complex. From this location, ER enhances AP-1-dependent transcription. The ER-DBD (striped) is dispensable for this process. B, The AF-independent/AP-1 pathway. Antiestrogen-liganded ER (with AF-2 inactive, as designated by a cross) binds a nuclear receptor corepressor (CoR) and sequesters unspecified repressors (R) from the AP-1 complex. This interaction is mediated by regions of ER that do not interact with coactivators including the ER-DBD (striped). AP-1 activity is thereby enhanced by ERs that do not directly participate in the AP-1 complex.

 
Our studies do not directly address the mechanism of the AF/independent AP-1 pathway. However, any hypothesis to account for antiestrogen effects at AP-1 sites must account for one puzzling observation. Tamoxifen-liganded ER{alpha} and isolated ER{alpha} AB-DBD region both enhance AP-1 activity yet do not target the strong VP16 transactivation function to AP-1 sites in mammalian two-hybrid assays (12). Thus, antiestrogen-liganded ER{alpha} seems to be able to enhance AP-1 activity without directly participating in the AP-1 complex. How is this possible? One attractive idea is that the antiestrogen-liganded ER enhances AP-1 activity by binding, and sequestering, AP-1-associated repressors (Fig. 9BGo). Two pieces of evidence are consistent with this notion. First, nuclear receptor corepressors preferentially associate with receptor/antihormone complexes (43, 65). Second, in some circumstances, the iAF-1B region represses ER{alpha} action at classical EREs, suggesting that it recruits a repressor (29). The same iAF-1B region plays an important role in antiestrogen responses at AP-1 sites (Figs. 2Go, 4Go, and 5Go). Thus, corepressors are possible agents in antiestrogen action at AP-1 sites. We are presently exploring this hypothesis by asking whether ER/N-CoR interactions might play a role in antiestrogen action at AP-1 sites. We stress that N-CoR itself need not be present in the AP-1 complex. Rather, other functional components of the repressor complex, such as histone deacetylases, could be associated with the jun/fos-coactivator complex and be diverted from that location by formation of the antiestrogen-liganded ER/N-CoR complex.

We also note that our model suggests an explanation for the apparent mutual exclusivity of the two pathways. The estrogen- and antiestrogen-liganded ERs must be present in completely distinct subnuclear locations, either at or away from the promoter, to enhance AP-1 activity. Furthermore, nuclear receptors are unable to interact with coactivators and corepressors simultaneously (7, 65). Disruption of ER/coactivator or ER/corepressor interactions would free ER to participate more strongly in the other type of interaction.

Differences between Tamoxifen and Raloxifene Action at AP-1 Sites
There are several differences between the behavior of tamoxifen and raloxifene at AP-1 sites. Tamoxifen-liganded ER{alpha} enhances AP-1 activity in HeLa cells, but raloxifene-liganded ER{alpha} does not (13). More subtly, ER{alpha} AF-1 partially suppresses tamoxifen effects at AP-1 sites, but completely suppresses raloxifene action at AP-1 sites, and different regions of AF-1 suppress tamoxifen and raloxifene effects (Fig. 5BGo). Furthermore, tamoxifen effects at AP-1 sites are heavily dependent upon iAF-1B, and raloxifene effects are less dependent upon this region, and, in fact, are largely mediated by the DBD-LBD region. On the basis of our model, we speculate that the tamoxifen-liganded ER{alpha} relies on iAF-1B to bind corepressors, and work through the AF-independent pathway at AP-1 sites, and that raloxifene-liganded ER{alpha} relies on the DBD-LBD region to achieve the same effect.

Tamoxifen and raloxifene also show different properties at classical EREs. Antiestrogens all permit ER{alpha} to bind DNA (66, 67), yet tamoxifen allows high AF-1 activity in some contexts (24), and ICI and raloxifene do not (67, 68). Thus, antiestrogens exert distinct effects upon ER{alpha} LBD/AB domain cross-talk in diverse gene regulation assays. Perhaps understanding the way that different ligands influence ER/corepressor interactions will clarify both the ligand specificity of ER action at AP-1 sites and release of AF-1 activity at EREs.

Do AF-Mediated and AF-Independent Antiestrogen Effects Occur in Vivo?
Antiestrogens block estrogen response by blocking AF-2 activity. They also possess the capacity to enhance gene expression at EREs and AP-1 sites, but with completely opposite requirements for AF-1. At EREs, tamoxifen releases AF-1 activity, which can result in quite substantial cell and promoter-specific tamoxifen effects (57). However, raloxifene releases considerably less AF-1 activity and ICI none (67). At AP-1 sites, as we have shown here, tamoxifen, raloxifene, and ICI enhance AP-1 activity via an activation function-independent mechanism.

Because each type of antiestrogen effect shows characteristic ligand preferences, it may be possible to guess which mechanisms are active in vivo. In breast, antiestrogens block estrogen action and show few agonist effects (69, 70). Thus, AF-2 may play an important role in breast physiology. In uterus, tamoxifen acts as an ER agonist, but raloxifene does not. Thus, ER{alpha} AF-1 may play an important role in uterine physiology. We stress, however, that ER{alpha} AF-1 lacks strong activity at simple EREs in a tamoxifen-responsive uterine cell line (12), suggesting that any putative uterine AF-1 activity must be of a type that is only observed at more complex promoters, perhaps containing spaced ERE half-sites (71), and binding sites for heterologous transcription factors. Finally, in bone and cardiovascular system, tamoxifen and raloxifene both act as ER agonists. Even ICI, which has the additional effect of promoting ER protein degradation in vivo (72), shows some agonist activity in bone (73). Thus, these effects are candidates for an AF-independent mechanism.

We recognize that ER action in vivo will likely prove to be much more complex than ER action at our simple transiently transfected reporter genes. As a first step to understanding the contributions of AF-1-mediated and AF-1-independent antiestrogen effects in vivo, it will be interesting to determine whether both types of effect are active upon chromosomally integrated target genes or, even better, upon native ER target genes in their normal chromosomal context. Should both types of mechanism indeed prove to be active in vivo, an informed strategy to block or promote specific antiestrogen effects will require an understanding of the contributions of each mechanism to regulation of key ER target genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mammalian Reporter Genes and Expression Vectors
Coll73-LUC and ERE-II-LUC have been previously described (12, 13). The ER{alpha} expression vectors have been previously described but, for ease of comparison, we have given them a consistent nomenclature. The previous names and sources of each construct are as follows: SG5-ER = HEG0; SG5-ERV400 = HE0, n101 = HE302; n117 = HE303; {Delta}129–178 = HE316 (49), CMV-ER; n21 = n21; n41 = E41; n87 = A87; n109 = M109; ER{Delta}AB (50). For some experiments the effects of ER-N109 were compared with SV40-driven ER expression vectors. For these experiments, the ER N109 cDNA was moved out of the CMV expression vector as an EcoRI/BamHI fragment and recloned into pSG5. The coactivator expression vectors (GRIP1, GRIP1 NR box II and III mutant, and GRIP1 {Delta}1121C) have each been previously described (29, 36).

The ER{alpha}-LBD expression vector SG5-LBD and its mutant derivatives were constructed from pKCR2-HE14 (74), which encodes human ER-LBD amino acids 282–595. First, the LBD-coding sequence was moved into pSG5 as an EcoRI fragment. A V400 mutation, which was present in the original human cDNA clone (75), was corrected by incorporating wild-type human sequences from the full-length pSG5-ER{alpha} as a HindIII/BamHI fragment. The resulting ER{alpha}-LBD expression vector was subjected to point mutagenesis with a PCR-based method designed to incorporate oligonucleotides into the LBD cDNA (Stratagene). The mutations converted methionine 543 and leucine 544 to alanine residues (LBDmAF-2) or introduced a stop codon after tyrosine 537 (LBD{Delta}AF-2). ERs bearing specific amino acid substitutions at serine 118 and at lysine 362 (ER{alpha}) or lysine 269 (ERß) were derived by similar methods. The nature of each mutant ER was confirmed by sequence analysis.

ERß530 was generated from a CMV vector containing a full-length cDNA clone (S. Nilsson, unpublished). An EcoRI fragment spanning the 5'-end of the longer cDNA was obtained and substituted into our existing ERß485 expression vector (13). The orientation of the insert was confirmed by restriction analysis and sequencing. The GST-ERß AB domain fusion protein was generated by amplification of the sequences homologous to the AB domain (amino acids 1–144) by PCR and subcloning the resulting fragment into the BamHI/EcoRI sites of pGEX-5X-3.

Cell Culture and Transfection
HeLa cells were maintained and transfected as previously described (12), except that 2 µg of luciferase reporter were employed. Also included in each transfection was 1 µg of pJ3 ß-galactosidase control. All cells were grown and transfected in phenol red-free medium. Cell lysates were prepared 36–40 h after transfection, and luciferase and ß-galactosidase assays were performed using the standard methods described in the reference above. Where indicated, luciferase activities were corrected for variations in transfection efficiency using ß-galactosidase activity.

Protein Binding Assays
Fusions of GST to various human ER{alpha} domains were prepared as described (76). Bacteria expressing the fusion proteins were resuspended in buffer IPAB-80 (20 mM HEPES, 80 mM KCl, 6 mM MgCl2, 10% glycerol, 1 mM dithiothreitol, 1 mM ATP, 0.2 mM phenylmethylsulfonyl fluoride, and protease inhibitors; pH 7.9) and sonicated mildly, and the debris was pelleted at 12,000 rpm for 1 h in an ss34 rotor. The supernatant was incubated for 2 h with 500 µl of glutathione Sepharose 4B beads that had been previously washed with 5 vol of PBS 0.2% Triton X-100 and equilibrated with 5 vol of IPAB 80. GST-fusion proteins beads were then washed with 5 vol of PBS 0.05% Nonidet P-40 and resuspended in 1 ml of IPAB-80 for storage at 4 C until use. All procedures were performed at 4 C.

Assays of GST-ER fusions were carried out in a volume of 100 µl that contained 40 µl of bead suspension (volume equivalent to 10 µl of compact beads) and 1 µl of 35S in vitro translated GRIP1 in IPAB-80 2.5% nonfat milk and incubated for 1.5 h at 4 C. Beads were washed five to six times with IPAB-80 containing 0.05% NP-40. Input-labeled proteins, proteins bound to GST, and the ER fusion beads were subjected to SDS-PAGE in 10% acrylamide and then to autoradiography.


   ACKNOWLEDGMENTS
 
We would like to thank Drs. P. Chambon, D. Metzger (Strasbourg, France), and D. Leitmann (San Francisco, CA) for gifts of ER expression vectors; Carol Anderson for technical assistance and helpful discussions; and Dr. R. Uht for critical reading of the manuscript.


   FOOTNOTES
 
Address requests for reprints to: Dr. Peter Kushner/Dr. Paul Webb, Metabolic Research Unit, University of California San Francisco School of Medicine, MRU 1119 HSW, San Francisco, California 94122-0540.

This work was supported by the State of California Breast Cancer Research Program Grant 1KB-0188 to P.W.; by the US Army Breast Cancer Research Program and by NIH Grants CA-80210 and DK-51083 to P.J.K.; by NIH Grant CA-18119 to B.S.K.; and by grants from the Swedish Cancer Fund to J.-A.G.

Peter Kushner is a consultant and director with significant financial holdings in KaroBio AB, a Swedish pharmaceutical development company with interests in nuclear receptors.

Received for publication February 25, 1999. Revision received June 7, 1999. Accepted for publication June 30, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Kuiper GGJMA, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA 1996 Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930[Abstract/Free Full Text]
  2. Katzenellenbogen BS, Korach KS 1997 Editorial: A new actor in the estrogen receptor drama—enter ER ß. Endocrinology 138:861–862[Free Full Text]
  3. Giguere V, Tremblay A, Tremblay GB 1998 Estrogen receptor ß: re-evaluation of estrogen and antiestrogen signaling. Steroids 63:335–339[CrossRef][Medline]
  4. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  5. Ribeiro RC, Kushner PJ, Baxter JD 1995 The nuclear hormone receptor gene superfamily. Annu Rev Med 46:443–453[CrossRef][Medline]
  6. Katzenellenbogen BS 1996 Estrogen receptors: bioactivities and interactions with cell signaling pathways. Biol Reprod 54:287–293[Abstract]
  7. Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:1167–1177[Abstract]
  8. Glass CK, Rose DW, Rosenfeld MG 1997 Nuclear receptor coactivators. Curr Opin Cell Biol 9:222–232[CrossRef][Medline]
  9. Gaub MP, Bellard M, Scheuer I, Chambon P, Sassone CP 1990 Activation of the ovalbumin gene by the estrogen receptor involves the fos-jun complex. Cell 63:1267–1276[CrossRef][Medline]
  10. Philips A, Chalbos D, Rochefort H 1993 Estradiol increases and anti-estrogens antagonize the growth factor-induced activator protein-1 activity in MCF7 breast cancer cells without affecting c-fos and c-jun synthesis. J Biol Chem 268:14103–14108[Abstract/Free Full Text]
  11. Umayahara Y, Kawamori R, Watada H, Imano E, Iwama N, Morishima T, Yamasaki Y, Kajimoto Y, Kamada T 1994 Estrogen regulation of the insulin-like growth factor I gene transcription involves an AP-1 enhancer. J Biol Chem 269:16433–16442[Abstract/Free Full Text]
  12. 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]
  13. Paech K, Webb P, Kuiper GGJM, Nilsson S, Gustafsson JA, Kushner PJ, Scanlan TS 1997 Differential ligand activation of estrogen receptors ER {alpha} and ER ß at AP1 sites. Science 277:1508–1510[Abstract/Free Full Text]
  14. Philips A, Teyssier C, Galtier F, Rivier-Covas C, Rey JM, Rochefort H, Chalbos D 1998 FRA-1 expression level modulates regulation of activator protein-1 activity by estradiol in breast cancer cells. Mol Endocrinol 12:973–985[Abstract/Free Full Text]
  15. Yang N, Venugopalan M, Hardikar S, Glasebrook A 1996 Identification of an estrogen response element activated by metabolites of 17-ß-estradiol and raloxifene. Science 273:1222–1225[Abstract]
  16. Elgort MG, Zou A, Marschke KB, Allegretto EA 1996 Estrogen and estrogen receptor antagonists stimulate transcription from the human retinoic acid receptor-alpha 1 promoter via a novel sequence. Mol Endocrinol 10:477–487[Abstract]
  17. Montano MM, Jaiswal AK, Katzenellenbogen BS 1998 Transcriptional regulation of the human quinone reductase gene by antiestrogen-liganded estrogen receptor-{alpha} and estrogen receptor-ß. J Biol Chem 273:25443–25449[Abstract/Free Full Text]
  18. Liu HL, Golder-Novoselsky E, Seto MH, Webster L, McClary J, Zajchowski DA 1998 The novel estrogen-responsive B-box protein (EBBP) gene is tamoxifen-regulated in cells expressing an estrogen receptor DNA-binding domain mutant. Mol Endocrinol 12:1733–1748[Abstract/Free Full Text]
  19. Xie W, Duan R, Safe S 1999 Estrogen induces adenosine deaminase gene expression in MCF-7 human breast cancer cells: role of estrogen receptor-Sp1 interactions. Endocrinology 40:219–227
  20. 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]
  21. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors {alpha} and ß. Endocrinology 138:863–870[Abstract/Free Full Text]
  22. McInerney EM, Weis KE, Sun J, Mosselman S, Katzenellenbogen BS 1998 Transcription activation by the human estrogen receptor subtype ß (ERß) studied with ERß and ER{alpha} receptor chimeras. Endocrinology 139:4513–4522[Abstract/Free Full Text]
  23. Tremblay GB, Tremblay A, Labrie F, VG 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]
  24. Gronemeyer H, Benhamou B, Berry M, Bocquel MT, Gofflo D, Garcia T, Lerouge T, Metzger D, Meyer ME, Tora L, Vergezac A, Chambon P 1992 Mechanisms of antihormone action. J Steroid Biochem Mol Biol 41:217–221[CrossRef][Medline]
  25. Parker MG 1993 Steroid and related receptors. Curr Opin Cell Biol 5:499–504[CrossRef][Medline]
  26. 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[CrossRef][Medline]
  27. Lees JA, Fawell SE, Parker MG 1989 Identification of constitutive and steroid-dependent transactivation domains in the mouse oestrogen receptor. J Steroid Biochem 34:33–39[CrossRef][Medline]
  28. Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, Giguere V 1997 Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor ß. Mol Endocrinol 11:353–365[Abstract/Free Full Text]
  29. Webb P, Nguyen P, Shinsako J, Anderson C, Feng W, Nguyen MP, Chen D, Huang S-M, Subramanian S, McInerney 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]
  30. Feng W, Ribeiro RC, Wagner RL, Nguyen H, Apriletti JW, Fletterick RJ, Baxter JD, Kushner PJ, West BL 1998 Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors. Science 280:1747–1749[Abstract/Free Full Text]
  31. Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson JA, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758[CrossRef][Medline]
  32. 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[CrossRef][Medline]
  33. Voegel JJ, Heine MJS, Tini M, Vivat V, Chambon P, Gronemeyer H 1998 The coactivator TIF2 contains three nuclear receptor-binding motifs and mediates transactivation through CBP binding dependent and independent pathways. EMBO J 17:507–519[CrossRef][Medline]
  34. Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK, Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function. Nature 387:677–684[CrossRef][Medline]
  35. Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387:733–736[CrossRef][Medline]
  36. Ding XF, Anderson CM, Ma H, Hong H, Uht RM, Kushner PJ, Stallcup MR 1998 Nuclear receptor-binding sites of coactivators glucocorticoid receptor interacting protein1 (GRIP1) and steroid receptor coactivator 1 (SRC-1): multiple motifs with different binding specificities. Mol Endocrinol 12:302–313[Abstract/Free Full Text]
  37. Kalkhoven E, Valentine JE, Heery DM, Parker MG 1998 Isoforms of steroid receptor co-activator 1 differ in their ability to potentiate transcription by the oestrogen receptor. EMBO J 17:232–243[CrossRef][Medline]
  38. Tremblay A, Tremblay GB, Labrie F, Giguere V 1999 Ligand-independent recruitment of SRC-1 to estrogen receptor ß through phosphorylation of activation function AF-1. Mol Cell 3:513–519[CrossRef][Medline]
  39. Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ, Baxter JD, Fletterick RJ, Yamamoto KR 1998 Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev 12:3343–3356[Abstract/Free Full Text]
  40. Smith CL, Nawaz Z, O’Malley BW 1997 Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen. Mol Endocrinol 11:657–666[Abstract/Free Full Text]
  41. Lavinsky RM, Jepsen K, Heinzel T, Torchia J, Mullen TM, Schiff R, Del-Rio AL, Ricote M, Ngo S, Gemsch J, Hilsenbeck SG, Osborne CK, Glass CK, Rosenfeld MG, Rose DW 1998 Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes. Proc Natl Acad Sci USA 95:2920–2925[Abstract/Free Full Text]
  42. Jackson TA, Richer JK, Bain DL, Takimoto GS, Tung L, Horwitz KB 1997 The partial agonist activity of antagonist-occupied steroid receptors is controlled by a novel hinge domain-binding coactivator L7/SPA and the corepressors N-CoR or SMRT. Mol Endocrinol 11:693–705[Abstract/Free Full Text]
  43. Wagner BL, Norris JD, Knotts TA, Weigel NL, McDonnell DP 1998 The nuclear corepressors N-CoR and SMRT are key regulators of both ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of the human progesterone receptor. Mol Cell Biol 18:1369–1378[Abstract/Free Full Text]
  44. Zhang X, Jeyakumar M, Petukhov S, Bagchi MK 1998 A nuclear receptor corepressor modulates transcriptional activity of antagonist-occupied steroid hormone receptor. Mol Endocrinol 12:513–524[Abstract/Free Full Text]
  45. Laherty CD, Billin AN, Lavinsky RM, Yochum GS, Bush AC, Sun JM, Mullen TM, Davie JR, Rose DW, Glass CK, Rosenfeld MG, Ayer DE, Eisenman RN 1998 SAP30, a component of the mSin3 corepressor complex involved in N-CoR-mediated repression by specific transcription factors. Mol Cell 2:33–42[CrossRef][Medline]
  46. Boruk M, Savory JG, Hache RJ 1998 AF-2-dependent potentiation of CCAAT enhancer binding protein ß-mediated transcriptional activation by glucocorticoid receptor. Mol Endocrinol 12:1749–1763[Abstract/Free Full Text]
  47. Imakado S, Koike S, Kondo S, Sakai M, Muramatsu M 1991 The N-terminal transactivation domain of rat estrogen receptor is localized in a hydrophobic domain of eighty amino acids. J Biochem (Tokyo) 109:684–689[Abstract/Free Full Text]
  48. Danielian PS, White R, Lees JA, Parker MG 1992 Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors. EMBO J 11:1025–1033[Medline]
  49. Metzger D, Ali S, Bornert JM, Chambon P 1995 Characterization of the amino-terminal transcriptional activation function of the human estrogen receptor in animal and yeast cells. J Biol Chem 270:9535–9542[Abstract/Free Full Text]
  50. McInerney EM, Katzenellenbogen BS 1996 Different regions in activation function-1 of the human estrogen receptor required for antiestrogen- and estradiol-dependent transcription activation. J Biol Chem 271:24172–24178[Abstract/Free Full Text]
  51. Ali S, Metzger D, Bornert JM, Chambon P 1993 Modulation of transcriptional activation by ligand-dependent phosphorylation of the human oestrogen receptor A/B region. EMBO J 12:1153–1160[Medline]
  52. Le Goff P, Montano MM, Schodin DJ, Katzenellenbogen BS 1994 Phosphorylation of the human estrogen receptor. Identification of hormone-regulated sites and examination of their influence on transcriptional activity. J Biol Chem 269:4458–4466[Abstract/Free Full Text]
  53. Lahooti H, White R, Danielian PS, Parker MG 1994 Characterization of ligand-dependent phosphorylation of the estrogen receptor. Mol Endocrinol 8:182–188[Abstract]
  54. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270:1491–1494[Abstract/Free Full Text]
  55. Bunone G, Briand PA, Miksicek RJ, Picard D 1996 Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J 15:2174–2183[Medline]
  56. Trowbridge JM, Rogatsky I, Garabedian MJ 1997 Regulation of estrogen receptor transcriptional enhancement by the cyclin A/Cdk2 complex. Proc Natl Acad Sci USA 94:10132–10137[Abstract/Free Full Text]
  57. 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[Medline]
  58. Kuiper GG, Gustafsson JA 1997 The novel estrogen receptor-ß subtype: potential role in the cell- and promoter-specific actions of estrogens and anti-estrogens. FEBS Lett 410:87–90[CrossRef][Medline]
  59. Moore JT, McKee DD, Slentz-Kesler K, Moore LB, Jones SA, Horne EL, Su JL, Kliewer SA, Lehmann JM, Willson TM 1998 Cloning and characterization of human estrogen receptor ß isoforms