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Endocrinology, doi:10.1210/en.2003-1043
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Endocrinology Vol. 145, No. 1 149-160
Copyright © 2004 by The Endocrine Society

Estrogen Receptor ß Isoforms Exhibit Differences in Ligand-Activated Transcriptional Activity in an Estrogen Response Element Sequence-Dependent Manner

Timothy L. Ramsey, Kelly E. Risinger, Sarah C. Jernigan, Kathleen A. Mattingly and Carolyn M. Klinge

Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, Kentucky 40292

Address all correspondence and requests for reprints to: Carolyn Klinge, Department of Biochemistry, Room 603, 319 Abraham Flexner Way, Louisville, Kentucky 40202. E-mail: carolyn.klinge{at}louisville.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen receptor ß (ERß) has been reported to have lower estradiol (E2)-induced transcriptional activity than human (h)ER{alpha} from estrogen response element (ERE)-driven reporters in transiently transfected cells. Conflicting data for activities of full-length and short hERß [hERß1, 530 amino acids (aa); and hERß1s, 477aa] have been reported. To test the hypothesis that hERß1 has higher transcriptional activity than hERß1s, we compared E2, 2,3-bis(4-hydroxyphenyl)propionitrile (a selective ERß agonist), and resveratrol-induced transcription by hERß1, hERß1s, and rat (r) ERß with hER{alpha} on different EREs in transiently transfected CHO-K1 and HEC-1A cells. Our results demonstrate for the first time that hERß1 has similar E2-induced activity to hER{alpha} and greater activity than rERß or hERß1s on a consensus palindromic ERE, either as a single or double copy; a minimal ERE; and the nonpalindromic pS2 ERE. 2,3-Bis(4-hydroxyphenyl)propionitrile showed greater efficacy with hERß1 and hERß1s than for rERß or hER{alpha}. We found that transcriptional differences between the ERß isoforms and ER{alpha} depend on the ERE sequence, confirming that the DNA sequence bound by ER is an allosteric effector of ER action. For the minimal 13-bp ERE and the pS2 ERE, the increase in transcriptional activity with hERß1 correlated with increased binding affinity. Coactivators steroid receptor coactivator-1 and cAMP response element binding protein-binding protein synergistically activated hER{alpha} and ERß transcription and showed reduced efficacy with rERß and hERß1s, suggesting a role for the N terminus of ERß1 in coactivator interaction. Collectively, these data indicate that the cellular expression of ERß isoforms may differentially impact ERE-regulated target gene expression in a ligand-dependent manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS PLAY A critical role in the regulation of many physiological processes in both females and males, including growth, reproduction, mammary gland differentiation, central nervous system development and homeostasis, and maintenance of bone density (1). Additionally, estrogen plays a role in breast and prostate cancer progression (2, 3). The regulation of gene transcription by estrogen is mediated by estrogen receptors (ER) {alpha} and ß. ER{alpha} and ERß share highest homology within the DNA binding domain (DBD) and diverge in N and C termini (4). Differences in the binding affinity of 17-ß-estradiol (E2) and other ligands, e.g. phytoestrogens, to ER{alpha} and ERß have been reported (5). The ligand binding domain (LBD) encompasses the ligand-dependent activation function (AF)-2, which comprises a shallow hydrophobic groove that is formed by residues between helices H3, H4, H5, and H12 with which the LXXLL receptor interaction domain of coactivators interacts (6). The ER{alpha} N terminus contains AF-1 (7). Truncation of the N terminus of ER{alpha} but not that of ERß decreased E2-induced transcription, indicating a lack of AF-1 in ERß (8). One possible mechanism accounting for differences between the N termini functions of ER{alpha} and ERß is that TATA box-binding protein interacts with the N terminus of ER{alpha}, but not ERß, in vitro (9).

Upon E2 binding, ER undergoes a conformational change that enhances DNA binding and activates AF-2 (6). ER binds directly to specific estrogen response elements (EREs) or interacts with other DNA-bound transcription factors, e.g. Sp1 (10) or activator protein (AP)-1 (11). Both processes result in recruitment of coactivators and components of the RNA polymerase II transcription initiation complex that enhance target gene transcription (12). Binding to different ERE sequences alters the conformation of ER{alpha}, allowing interaction with coactivators in a cell-type and DNA context-dependent manner (13, 14, 15, 16, 17).

Since the initial cloning of ERß, several different isoforms of human (h)ERß have been identified (18). There are at least six isoforms of hERß (19, 20). Moore et al. (19) proposed a numbering sequence for the various ERß isoforms. Three ERß isoforms are numbered ERß1–3, based on their amino acid composition [ERß1 has 530 amino acids (aa), ERß2 has 495 aa, and ERß3 has 513 aa]. ERß1 corresponds to the full-length transcript and has a molecular mass of 58 kDa; ERß1s is generated by an internal ribosome entry site within the hERß coding sequence and thus lacks the first 54 aa of hERß1, corresponding to a size of 53–54 kDa (21). ERß4 and ERß5 have extensive N-terminal deletions resulting in expression only in a portion of the LBD (19). The longest identified human ERß (hERß548) contains 548 aa and had greater transcriptional activity than hERß1 in transfected HEK-293 cells (22). However, it is noteworthy that none of the other ERß genes in GenBank contain that A nucleotide (23). Moreover, a recent study did not detect any alleles corresponding to hERß548 in a total of 324 samples from Africans, Caucasians, or Asians, or in the human testis cDNA used in the original study (24). The possibility that these ERß variants play distinct roles in mediating estrogen action has not been examined.

Most studies have reported that ERß has lower E2-induced transcriptional activity than ER{alpha} ERE-reporters in a variety of transiently transfected cells (25, 26, 27, 28). However, there appears to be a cell-type dependence for the lower activity of ERß. Cells in which AF-1 is required and predominates over AF-2 activity, e.g. HepG2 (29), show lower activity of ERß than ER{alpha}, whereas ER{alpha} and ERß exhibit similar activity in cells where AF-2 predominates, e.g. HEK-293 (27). Recently, baculovirus-expressed hERß1 was reported to have approximately 40–50% lower transcriptional activity than ER{alpha} on a reconstituted chromatin template in vitro (23). A chimeric ER{alpha}/ß protein containing the N terminus of ER{alpha} linked to the DBD, LBD, and F domain of ERß1 showed activity approximately 68% of ER{alpha} activity, indicating that the N terminus of ER{alpha} has an activity necessary for transcription on a chromatin template in vitro (23). Clearly, the nature of the assay affects the conclusions, and further experiments will be needed to examine the function of the N terminus of ERß.

To examine the molecular function of the N terminus of hERß, a region having a role in AF-1–AF-2 interaction in ER{alpha} (7), we tested the hypothesis that ERß1 has higher transcriptional activity than ERß1s. We compared the activity of hERß1 with that of hERß1s and rat (r) ERß. ERß1 was reported to have twice the E2-induced activity of ERß1s from a luciferase reporter containing two tandem copies of the Xenopus vitellogenin (vit) A2-ERE luciferase reporter in transiently transfected HepG2 cells (30). Because hER{alpha} exhibits greater AF-1 than AF-2 activity in HepG2 cells (29), these data imply an N-terminal function in the ERß1 isoform that is missing in hERß1s. However, another study showed that ERß1 and ERß1s had similar E2-induced activity from a reporter containing two tandem copies of the vit A2-ERE in transiently transfected HepG2 cells (8). Therefore, the transcriptional activity of ERß1 and ERß1s require further study. The activity of both ERß1 and ERß1s was only 40% that of ER{alpha} in HEK-293 and only 10% of the activity of hER{alpha} in HepG2 cells (8). However, both ERß1 and ERß1s showed 75% of the activity of hER{alpha} in HeLa cells (8) in which AF-2 has greater activity than AF-1 (29). Deletion of the first 93 aa at the N terminus of hERß1 reduced transcriptional activity in HEK293 and HepG2 cells but increased activity in HeLa cells (8). These results imply that the N terminus of hERß1s may target different cell-type specific cofactors than hERß1.

The goal of this study was to compare the transcriptional activities of rERß, hERß1, hERß1s, and hER{alpha} on different ERE sequences in transfected cells in which both AF-1 and AF-2 are active (29). We compared agonist activity of E2 with that of all-trans-resveratrol, a phytoestrogen that has chemopreventive and chemotherapeutic activity (31), and determined the activities of the different ERs on a palindromic ERE as well as on naturally occurring nonpalindromic, sequence variant EREs from estrogen-target genes. Our results collectively indicate that hERß1 (the long, 530 aa form) has greater transcriptional activity than the short form of hERß (hERß1s, 487 aa) or rERß and has transcriptional activity equal to that of hER{alpha} in cells in which both AF-1 and AF-2 are active.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
pCMV-hER{alpha} was provided by Dr. Benita S. Katzenellenbogen (University of Illinois, Urbana, IL) (32). Rat ERß (4), human short ERß (8), and human long ERß (33) expression plasmids pCMV5-rERß, pBK-CMV-hERß1s, and pSG5-hERß1 were provided by Drs. J.-A. Gustafsson, Eckardt Treuter, and Eva Enmark (all from Karolinska Institute, Huddinge, Sweden), respectively.

The sequence of the EREs is listed in Table 1Go. One or two tandem copies of EREc38 (2EREc38) or single copies of the other EREs were cloned into pGL3-pro-luciferase (Promega, Madison, WI) (13, 34). Plasmids were amplified in Escherichia coli strain DH5{alpha} and purified using the Bio-Rad Maxi/Midiprep kit (Bio-Rad Laboratories, Inc., Hercules, CA).


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  		TABLE 1. Sequences of EREs as referred to in the text

 
Transient transfection assay
CHO-K1 cells were purchased from American Type Culture Collection (Manasas, VA) and was maintained in Iscove’s modified Dulbecco’s medium (IMDM, Life Technologies, Inc., Grand Island, NY) supplemented with 10% newborn calf serum (Atlanta Biologicals, Norcross, GA). For transient transfection, cells were plated in 24-well plates at a density of 1.5 x 106 cells per well in IMDM without phenol red (Life Technologies, Inc.) supplemented with 10% charcoal-stripped, heat-inactivated calf serum and pen/strep. Transient transfection was performed using Transfast (Promega) as described (35). Each well received 250 ng of a pGL3-pro-luciferase reporter (Promega) containing various EREs (Table 1Go), 5 ng of a Renilla luciferase reporter (pRL-tk; Promega), and 10 ng of the indicated ER-expressing plasmid(s), depending on the individual experiment. Our results agreed with an earlier report (36) that centrifugation enhanced transfection efficiency. Twenty-four hours after transfection, triplicate wells were treated with ethanol (EtOH; vehicle control), E2 (Sigma Chemical, St. Louis, MO), 4-hydroxytamoxifen (4-OHT) (Research Biochemicals International, Natick, MA), 2,3-bis(4-hydroxyphenyl)propionitrile [DPN, a selective ERß agonist (37); Tocris, Ellisville, MO], ICI 182,780 (Tocris), or resveratrol (generously provided by Pharma Science, Montreal, Canada). Ligands were administered alone or in combination in charcoal-stripped, heat-inactivated calf serum-supplemented IMDM medium. The cells were harvested 30 h post treatment using Passive Lysis buffer (Promega). Luciferase and Renilla luciferase activities were determined in cell extracts using dual luciferase assay (Promega) in an EG&G Berthold Lumat LB 9507 luminometer (Wallac, Inc., Gaithersburg, MD). Firefly luciferase was normalized by Renilla luciferase to correct for transfection efficiency (42, 50, 52). Values detected in the EtOH treatment group were used as a baseline, and fold induction was determined by dividing the averaged triplicates from each treatment by the EtOH value for each transfection condition within that experiment. Statistical analyses were performed using one-way ANOVA followed by Dunn’s multiple comparison or Dunnett’s post hoc test using GraphPad Prism (San Diego, CA).

Preparation of baculovirus-expressed recombinant ER
An N-terminal Flag (aa sequence = DYKDDDDK) was added to the N terminus of the long form (530 aa) of recombinant human ERß1 that was PCR-amplified from pSG5-ERß, generously provided by Dr. Eva Enmark (33), and cloned into pBAC4 x 1 (Novagen, Madison, WI). After restriction digestion screening using BamHI, several clones were sequenced, and one correct clone of pBAC4 x 1-Flag-ERß was used for cotransfection of Sf21 cells with BacVector-3000 viral DNA (Novagen). Recombinant virus plaques were cloned by repeated plaque purification in Sf21 cells. Cloned virus was screened by [3H]E2 binding and adsorption to hydroxyapatite (HAP assay) (38) and by Western blotting using ERß antibody CWK-F12 (39) (provided by Dr. Benita S. Katzenellenbogen), and anti-Flag M2 antibody (Eastman Kodak, Rochester, NY) (data not shown). Nuclear extracts containing recombinant hER{alpha}, rERß, and Flag-tagged hERß1 were prepared from baculovirus-infected IPLB-Sf21AE insect cells as described (35, 40, 41). All ER concentrations, including those assayed in whole cell extracts of CHO cells transfected with the expression vectors for rERß, hERß1s, and hERß1, were determined by specific [3H]E2 binding by HAP assay (38) and refer to dimeric ER, i.e. with two molecules of bound ligand.

EMSA
Protein-DNA binding was measured by EMSA as previously reported (14). For determination of ER-ERE binding affinity, identical molar amounts of baculovirus-expressed rERß or hERß1, based on HAP assay results, were incubated with 2–32 fmol [32P]-labeled EREc38 (sequence in Table 1Go). Binding reactions included 40 mM Tris-HCl (pH 7.5), 10% glycerol, 0.75 µg/µl BSA, and 0.02 µg/µl polydeoxyinosinic deoxycytidylic acid (Midland Certified Reagents, Midland, TX), 111 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonylfluoride. An ERß-specific antibody (Y19; Santa Cruz Biotechnology, Santa Cruz, CA) was included in selected reactions, as indicated in the Fig. 3Go legend. Dried EMSA gels were analyzed using a Packard Instruments (Meriden, CT) InstantImager and associated software, Packard Imager for Windows version 2.04 (42). Kd values were determined as reported (35).



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  		FIG. 3. Recombinant hERß1 binds EREc38 with high affinity. A fixed concentration of hERß1 (50 fmol) was incubated with 2–32 fmol [32P]EREc38, lanes 1–9, as described in Materials and Methods. Lane 10 included 10.7 fmol [32P]EREc38, hERß, and 1 µl of the ERß-specific antibody Y-19. The closed arrow indicates the specific ERß1-EREc38 band, and -SS indicates the supershifted complex formed between the ERß antibody Y-19 and the ERß-EREc38 complex. This autoradiograph is representative of four independent EMSA experiments showing similar results.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Short and long hERß, rERß, and hER{alpha} show different E2-induced transcriptional activity
First, we examined rERß, hERß1, and hERß1s activity on two tandem copies of EREc38, a consensus ERE sequence that includes the commonly used Xenopus vit A2 ERE palindrome (43). This allows comparison with other reports that used two or three tandem, head-to-tail copies of the vit A2 ERE, called 2ERE (14, 34, 43, 44, 45, 46) or 3ERE (7, 47, 48, 49, 50, 51, 52). E2 induced a concentration-dependent activation of rERß, hERß1, and ERß1s from 2ERE in transiently transfected CHO-K1 cells (Fig. 1Go). Notably, the E2-induced activity of hERß1 was significantly higher than that of rERß or hERß1s (Table 2Go). Both 4-OHT and ICI 182,780 suppressed ERß transcription below basal. 4-OHT and ICI 182,780 inhibited the E2-induced activity of each of the three ERß isoforms (Fig. 1Go and data not shown). This indicates that the induction of reporter activity in response to E2 is mediated by E2-ERß binding and not a receptor-independent pathway.



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  		FIG. 1. hERß1 has higher E2-induced transcriptional activity compared with rERß or hERß1s. A, CHO-K1 cells were cotransfected with rERß (open bars), hERß1s (solid bars), or hERß1 (hatched bars) plus pGL3-2EREc38-luciferase reporter and pRL-CMV as described in Materials and Methods. Twenty-four hours after transfection, the cells were treated with EtOH, the indicated concentration of E2 for 30 h. Cell extracts were prepared and assayed as described in Materials and Methods. Data are displayed as luciferase activity divided by the RL-luc activity in each well and normalized by EtOH control (which is set to 1). Within each experiment, each treatment was performed in triplicate. The data shown are the mean ± SEM from at least eight separate experiments. Asterisks indicate ERß1 values that are statistically different from rERß or hERß1s values, P < 0.05. B, Western blot of identical amounts (100 µg protein) of whole cell extracts from CHO-K1 cells transfected with 10 ng of the expression vectors for rERß, hERß1s, or hERß1. The blot was stripped and probed for ß-actin. The films were quantitated by densitometric scanning and the amount of ERß on the Western normalized by ß-actin. Data are the value (in pixels) of the ERß divided by the value (in pixels) of ß-actin in the same lane and normalized to the rERß expression level, which was set to 1. The bar graph shows the mean ± SD of two separate experiments.

 

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  		TABLE 2. E2-induced transcriptional activity from different EREs

 
To confirm equivalent ERß expression, extracts from CHO cells transfected with equal amounts (10 ng) of the expression vectors for rERß, hERß1s, or hERß1 were separated by SDS-PAGE and analyzed for ERß expression by Western blot (Fig. 1BGo). When the data were normalized by ß-actin, the levels of ERß were equivalent. These extracts also showed equal specific [3H]E2 binding by HAP assay with 0.49, 0.54, and 0.59 nM ER dimer concentration in 50 µg protein. Together these data confirm that differences in transcriptional activity between the ERß isoforms are not the result of differences in expression in the CHO cells.

Next, we compared the activity of hER{alpha} with rERß, hERß1, or hERß2 on a single ERE or on two tandem EREs (Fig. 2Go). Higher activity was induced by hER{alpha} and hERß1 vs. hERß1s or rERß, and these differences were statistically significant (P < 0.05; Fig. 2Go, A and B). Similar to the results in CHO-K1 (Fig. 2AGo), hER{alpha} and hERß1 showed significantly (P < 0.05) higher activity than either rERß or hERß1s on a single ERE in transiently transfected HEC-1A human endometrial cancer cells (Fig. 2CGo). These results indicate that the observed higher activity of hERß1 than hERß1s and rERß is not unique to CHO-K1 cells. In both cell lines, the E2-induced transcriptional activity of hER{alpha} and the three ERß isoforms was inhibited by 4-OHT. We conclude that hERß1 has higher transcriptional activity than hERß1s or rERß. Furthermore, the E2-induced activity of hERß1 is indistinguishable from hER{alpha} on one or two tandem copies of the vit A2 ERE in both CHO-K1 and HEC-1A cell lines.



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  		FIG. 2. hER{alpha} and hERß1 have similar E2-induced transcriptional activity that is higher than that of rERß or hERß1s. CHO-K1 (A and B) or HEC-1A (C) cells were cotransfected with hER{alpha}, rERß, hERß1s, or hERß1, as indicated by the different fills, pGL3-pro-EREc38 (A and C) or pGL3-2EREc38-luciferase reporter and pRL-CMV (B) and treated as indicated and described in Materials and Methods and Fig. 1Go. The data shown are the mean ± SEM from at least eight separate experiments. a, E2 values that are statistically different from the EtOH control value, P < 0.05. b, E2 values that are statistically different from the ER{alpha} value, P < 0.05.

 
rERß and hERß1 bind EREc38 with high affinity
Because ER binding affinity is correlated with transcriptional response from ERE-driven reporters in transiently transfected cells (35, 53), one logical hypothesis to account for the similar transcriptional activities of hER{alpha} and hERß1 is that hERß1 binds EREc38 with higher affinity than rERß and comparable to that of hER{alpha}. Human ER{alpha} and rERß bind a single copy of EREc38 with affinities of 0.11 and 0.64 nM, respectively (35). Figure 3Go is representative of six EMSAs directly measuring the affinity of E2-hERß1 binding to [32P]EREc38, which was calculated to have a Kd = 0.9 nM (Table 3Go). This is not significantly different from the Kd for rERß-EREc38 interaction, but is significantly lower than the binding affinity of hER{alpha} for EREc38. Therefore, we conclude that the higher transcriptional activity of hERß1 vs. rERß or hERß1s is not accounted for by higher binding affinity for the ERE as measured in vitro.


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  		TABLE 3. Affinity of ER-ERE binding

 
ERß inhibits ER{alpha} transcriptional activity
ER{alpha} and ERß are coexpressed in some tissues, e.g. mammary gland (54), and heterodimerize in vitro and in transfected cells (26). We compared the abilities of rERß and hERß1 to affect ER{alpha} activity in transiently transfected CHO-K1 cells (Fig. 4Go). The shape of the E2 dose-response curves is in agreement with the reported affinity of E2 binding to hER{alpha}, rERß, and hERß1 (5, 33, 48). As anticipated based on previous reports showing that ERß was a transdominant repressor of hER{alpha} in HepG2 or HeLa (27) or COS-1 (55) cells, rERß inhibited E2-activated hER{alpha} activity. In contrast, when hERß1 and hER{alpha} were mixed, the E2-induced transcriptional activity was similar to that for each alone.



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  		FIG. 4. Transcriptional activity of hER{alpha}, rERß, and hERß1 on ERE-regulated gene expression. CHO-K1 cells were cotransfected with 10 ng of hER{alpha}, 10 ng of rERß, or 5 ng of both hER{alpha} and rERß (A) or 10 ng of hER{alpha}, 10 ng hERß1, or 5 ng of both hER{alpha} and hERß1 (B), as indicated by the different symbols, pGL3-pro-2EREc38 luciferase reporter, and pRL-CMV and treated as indicated and described in Materials and Methods and Fig. 1Go. The data shown are the mean ± SEM from three separate experiments.

 
Effect of resveratrol on transcriptional activity of ERß
Resveratrol, trans-3,5,4'-trihydroxystilbene, is a bioflavonoid that is found naturally in grapes and is enriched in red wine (56). We reported that resveratrol is an hER{alpha} and rERß agonist in CHO-K1 cells, as indicated by induction of ERE-driven reporter gene activity (31). Interestingly, resveratrol had greater potency as a ligand for rERß than hER{alpha} (31). Because those experiments used rERß, it was of interest to determine whether resveratrol, like E2, stimulates higher induction of reporter activity from the hERß1 vs. rERß or hERß1s. Resveratrol stimulated ERß-driven transcription in a concentration-dependent manner (Fig. 5Go). hERß1s was more transcriptionally active than rERß or hERß1 at low concentrations of resveratrol (1–100 nM). hERß1 showed highest activity at 25 and 50 µM resveratrol, concentrations that inhibit the proliferation of breast and prostate cancer cells (31, 57). The resveratrol-stimulated activity of the three ERßs was equivalent at 75 and 100 µM, and the activity observed at 100 µM was similar to that detected with 10 nM E2 for rERß and hERß1s. We conclude that resveratrol is an ERß agonist with lower efficacy than E2, which shows a preferential activation of hERß1s at nanomolar concentrations. Only at 25 and 50 µM resveratrol was the activity of hERß1 greater than that of the other two forms of ERß tested. Therefore, resveratrol has a different impact than E2 on ERß transcriptional activity.



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  		FIG. 5. Resveratrol has ERß agonist activity. CHO-K1 cells were cotransfected with rERß, hERß1s, or hERß1, as indicated by the different fills, pGL3-pro-EREc38 luciferase reporter, and pRL-CMV and treated as indicated and described in Materials and Methods and Fig. 1Go. The data shown are the mean ± SEM from at least four separate experiments. Asterisks indicate E2 or resveratrol values that are statistically different from the EtOH control value, P < 0.05.

 
The ERß selective ligand DPN shows lower activity with rERß than with hERß1 or hERß1s
The Katzenellenbogen laboratories developed an ERß-selective ligand, DPN, that shows 70-fold higher affinity for ERß than ER{alpha} and selectively activates ERß-induced transcription from a 4ERE-luciferase reporter in transiently transfected HEC-1 human endometrial cells (37). We compared the transcriptional activity of DPN with rERß, hERß1, hERß1s, and hER{alpha} on 2EREc38 in transiently transfected CHO-K1 cells. In agreement with the previous report (37), DPN selectively stimulated transcription by ERß compared with hER{alpha} (Fig. 6Go). Interestingly, hERß1s and hERß1 showed virtually identical stimulation by DPN and greater responsiveness to DPN than rERß. These data contrast with those detected for E2-induced transcription, where rERß = hERß1s < hERß1. We conclude that DPN elicits a different transcriptional activity with ERß1s than E2.



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  		FIG. 6. Comparison of ERß-selective agonist DPN activity with ERß and ER{alpha}. CHO-K1 cells were cotransfected with hER{alpha}, rERß, hERß1s, or hERß1, indicated by the symbols, plus pGL3-2EREc38 and pRL-CMV, and treated as indicated and described in Materials and Methods and Fig. 1Go. Values are expressed as percentage of the ER{alpha} or ERß response with 10 nM E2. The data shown are the mean ± SEM from at least three separate experiments.

 
Reduction of ERE palindrome length to 13 bp reduces ERß transcriptional activity
Next, we compared the activity of ER{alpha} and the three forms of ERß on EREc13, the minimal ERE (58) featuring a consensus, 13 bp perfectly palindromic ERE (Fig. 7Go). Notably, hER{alpha} and hERß1 showed similar transcriptional activity that was higher than rERß and ERß1s. Cotreatment with 4-OHT inhibited E2-induced activity, indicating the stimulation to be ER-mediated. ANOVA revealed statistically significant (P < 0.05) differences between hER{alpha} and hERß1 vs. rERß, and hERß1s (Table 2Go). This result is similar to the data for EREc38.



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  		FIG. 7. hER{alpha} and hERß1 have higher activity than hERß1s and rERß on the minimal EREc13 palindrome. CHO-K1 cells were cotransfected with hER{alpha}, rERß, hERß1s, or hERß1, indicated by the different fills, plus pGL3-EREc13 luciferase reporter, and pRL-CMV. Cells were treated as indicated and described in Materials and Methods and Fig. 1Go. The data shown are the mean ± SEM from at least three separate experiments. a, E2 values that are statistically different from the EtOH control value, P < 0.05. b, E2 values that are statistically different from the ER{alpha} value, P < 0.05.

 
Because ER-ERE binding affinity is correlated with transcriptional response in transiently transfected cells (15, 35, 42, 53, 59), one hypothesis stemming from these findings is that hERß1 binds EREc13 with higher affinity than rERß or hERß1s. We reported that the affinity of hER{alpha} and rERß binding to EREc13 was 90% lower than binding to EREc38 (EREc13 = 1.08 and 1.72 nM; and EREc38 = 0.11 and 0.64 nM for hER{alpha} and rERß, respectively (35) (Table 3Go). Direct binding EMSA experiments were used to estimate the Kd for hERß1 interaction with EREc13 (data not shown). The affinity of hERß1 binding to EREc13 was calculated to be 0.84 nM (Table 3Go). This value is significantly different from the affinity of rERß binding to EREc13 and is not different from the affinity of hER{alpha}-EREc13 interaction. The increase in hERß1 binding affinity to EREc13 correlates with the higher transcriptional activity (Fig. 7Go).

Sequence variant EREs show differential transactivation by ER{alpha} and ERß
Many endogenous estrogen-responsive genes contain nonpalindromic EREs (58). One such nonpalindromic ERE is located between -418 and -378 in the promoter, the human pS2 gene (60) (sequence in Table 1Go). Figure 8AGo shows a comparison of the activities of hER{alpha}, rERß, hERß1, or hERß1s on a pS2-ERE-luciferase reporter in transiently transfected CHO-K1 cells. As seen for EREc38 (Figs. 1Go and 2Go), hER{alpha} and hERß1 had significantly higher E2-induced transcriptional activity than rERß or hERß1s (Table 2Go). The E2-induced transcriptional activity was inhibited by 100 nM 4-OHT. This indicates that the induction of reporter activity in response to E2 is mediated by E2- ER{alpha} or ERß binding and not a receptor-independent pathway. Direct binding EMSA experiments were used to estimate the Kd for hERß1 interaction with the pS2 ERE (data not shown). The affinity of hERß1 binding to the pS2 ERE was calculated to be 1.33 nM (Table 3Go). This value is significantly different from the affinity of rERß binding to the pS2 ERE and is not different from the affinity of hER{alpha}-pS2 ERE interaction. The increase in hERß1 binding affinity to the pS2 ERE correlates with the increased transcriptional activity (Fig. 8AGo).



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  		FIG. 8. Alterations in the ERE sequence from natural estrogen target genes impacts the E2-induced activities of hER{alpha}, hERß1, hERß1s, and rERß. CHO-K1 cells were cotransfected with hER{alpha}, rERß, hERß1s, or hERß1, indicated by the different fills, plus A, pGL3-pS2-ERE; B, Fos-900; C, Fos-1211; D, PR1148; or E, PR540 luciferase reporters, and pRL-CMV. Cells were treated as indicated and described in Materials and Methods and Fig. 1Go. The data shown are the mean ± SEM from at least three separate experiments. a, E2 values that are statistically different from the EtOH control value, P < 0.05. b, E2 values that are statistically different from the ER{alpha} value, P < 0.05.

 
The human c-fos gene has two imperfect EREs at positions -1211 and -900 (61, 62). We compared the activity of hER{alpha} and the three forms of ERß on both Fos-900 and Fos-1211 reporter constructs in transiently transfected CHO-K1 cells (Fig. 8Go, B and C). Human ER{alpha} and all three ERß isoforms exhibit similar E2-induced activity on Fos-900, whereas hER{alpha} showed significantly higher activity on Fos-1211 than any of the ERß isoforms (Table 2Go). These data differ from the activities of hER{alpha} and the ERßs on EREc38, EREc13, and pS2, indicating that the ERE sequence directly impacts E2-induced ER transcription in a receptor-specific manner. The progesterone receptor (PR) promoter contains two imperfect EREs at positions +540 and +1148, which lie between the transcription start sites for the A and B forms of PR (63). E2 treatment of CHO-K1 cells transfected with the PR1148 reporter and either hER{alpha} or any of the three ERß isoforms induced reporter activity (Fig. 8DGo) that was inhibited by 4-OHT. All receptors showed similar activity on PR1148 (Table 2Go). In contrast, hERß1 showed significantly greater E2-induced activity on PR540 compared with the other ERß isoforms or hER{alpha} (Fig. 8EGo and Table 2Go). E2-induced reporter activity from PR540 was inhibited by 4-OHT. These results indicate that the ERE sequence impacts E2-induced ER transcriptional activity in an ER subtype- and isoform-specific manner.

Table 2Go gathers the results of the transient transfection assays in tabular form for direct comparison of the E2-induced transcriptional activities of hER{alpha}, rERß, hERß1s, and hERß1. The results summarized in Table 2Go provide strong support for our hypothesis that the different forms of ERß examined are not functionally equivalent, at least under the conditions in which they were tested in CHO-K1 cells.

Impact of coactivators on E2-induced activity of hER{alpha}, hERß1, hERß1s, and rERß
One hypothesis for the observed higher transcriptional activity of hERß1 vs. hERß1s and rERß is that hERß1 has greater interaction with coactivators than hERß1s and rERß. To test this idea, CHO-K1 cells were transfected with the EREc38-luciferase reporter and 250ng of steroid receptor coactivator-1 (SRC-1), ACTR, cAMP response element binding protein-binding protein (CBP), or both SRC-1 and CBP. These amounts were determined to give maximal E2-induced stimulation of hER{alpha}, rERß, hERß1, and hERß1s activity in this cell line (data not shown). Cells transfected with each coregulator were treated with EtOH and 10 nM E2; the luciferase activity detected with E2 was normalized by that for EtOH. Thus, any effect of a coactivator on basal transcription within that experiment was excluded from subsequent analysis. All the coactivators stimulated the basal activity, i.e. ligand-independent activity of each ER, from the ERE reporter but did not affect transcription from the empty pGL3-pro luciferase reporter (data not shown).

SRC-1 increased E2-induced ER{alpha} activity on EREc38, but SRC-1 had no significant (P < 0.05) effect on rERß, hERß1s, or hERß1 transcriptional activity (Fig. 9Go). In contrast, ACTR had no significant effect on transcriptional activity. CBP significantly (P < 0.05) enhanced the E2-induced activity hER{alpha}, but not hERß1. The combination of SRC-1 and CBP resulted in synergistic activation of hER{alpha} and all forms of ERß with hERß1 showing the highest response of all the ERß isoforms. These data are in agreement with a previous report for mouse ERß (64). These data indicate that SRC-1 shows greater selectivity for E2-ER{alpha} than ERß.



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  		FIG. 9. Coactivators SRC-1 and ACTR show differential impact on ER{alpha} and ERß transcriptional activity in transfected cells. CHO-K1 cells were cotransfected with hER{alpha}, rERß, hERß1s, or hERß1, indicated by the different fills, plus EREc38-luciferase, pRL-CMV, and either 250 ng of pcDNA3 (as a negative control), SRC-1, or ACTR and treated as indicated and described in Materials and Methods and Fig. 1Go. The data shown are the mean ± SEM from at least three separate experiments. Asterisks indicate E2 values that are statistically different from the EtOH control value, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Despite the homology between the DBD and LBD of ER{alpha} and ERß (4, 5), most cell-based assays indicate that ERß has significantly lower transcriptional activity than ER{alpha} (e.g., see Refs.25, 26, 27, 28). However, most previous studies used either the rERß, a 485-aa protein, or hERß1s. Here, we tested the hypothesis that hERß1 has higher transcriptional activity than the shorter forms of rERß and hERß1s using different ERE reporters in transiently transfected CHO-K1 cells. The data presented here demonstrate for the first time that hERß1 has similar activity to hER{alpha} and higher E2-induced transcriptional activity compared with rERß or hERß1s on the palindromic vit A2 ERE, either as a single or double copy, the minimal ERE, i.e. EREc13, and the nonpalindromic pS2 ERE. Previous reports (8, 65) examined activities of ER{alpha} and ERß using reporter genes containing two or three tandem copies of the vit A2 ERE. However, it is well established that most estrogen-responsive genes contain only a single, nonpalindromic ERE or are regulated via the tethering of ER to AP-1 or Sp1 transcription factors bound to their response elements (58). Our study is unique in comparing the activities of hER{alpha} and different ERß isoforms not only on the vit A2 ERE, but also on a minimal 13-bp ERE and the nonpalindromic EREs from the human pS2, Fos, and PR genes.

The greater transcriptional activity of hERß1 vs. rERß can be accounted for by the higher ERE binding affinity of hERß1 for EREc13 and pS2 ERE. These observations support previous studies showing that ER-ERE binding affinity correlates with transcriptional response in transfected cells (reviewed in Ref.58). On the other hand, ERß binding affinity and transcription were not correlated for the vit A2 ERE, EREc38. A possible mechanism accounting for this difference is that hERß1 interacts more strongly with coactivators, perhaps through interactions between its extended N terminus and the C terminus of ERß (8), compared with hERß2 or rERß. A notable observation in our studies is that ER{alpha} and ERß show different functional interaction with coactivators SRC-1 and CBP in transiently transfected cells. Whereas E2-induced hER{alpha} transcriptional activity was stimulated by SRC-1 or CBP, neither coactivator stimulated E2-induced transcription by any of the three ERß isoforms tested. Cotransfection with both SRC-1 and CBP synergistically activated transcription by hER{alpha} and all ERßs, but rERß and hERß1s showed significantly reduced response compared with hER{alpha} and hERß1. Our data are in agreement with data from a mammalian two-hybrid assay showing greater interaction between E2-ER{alpha} and SRC-1 than between SRC-1 and E2-ERß1s and that immobilized nuclear receptor (NR) boxes from SRC-1 interact with ER{alpha} with higher affinity than ERß1s (66).

To our knowledge, ours is the first report showing that the combination of coactivators SRC-1 and CBP enhances the E2-dependent transcription in an ERß isoform-specific manner, i.e. stimulating hERß1 more strongly than hERß1s or rERß. Thus, estrogen target genes in tissues expressing the long form of hERß1 may be activated by SRC-1 and CBP. Our data are in agreement with a report showing that the coactivator p300, which is closely related to CBP, increased E2-induced N- to C-terminal synergy for both ER{alpha} and ERß (67).

The transcriptional activities of hER{alpha} and the ERß isoforms varied with the ERE sequence. For example, hER{alpha} showed higher activity on the Fos-1211 ERE than any of the ERß isoforms. On the other hand, hERß1 showed higher activity than hER{alpha} or the other ERß isoforms on PR540. We suggest that, like hER{alpha} (14, 16, 17), ERE sequence alters ERß conformation, which in turn impacts ER-coactivator interaction. We have included the native nucleotides surrounding the ERE in each of our natural ERE reporters (see Table 1Go). A search of the pS2, PR540, PR1148, Fos-1211, and Fos-900 EREs using the Transfac database (68) revealed that each oligomer also contains AP-1 response elements (AP-1 RE), which is not surprising given that the consensus AP-1 RE, i.e. 5'-TGAG/CTCA-3', shares nucleotide homology with an ERE. However, there was no correlation between the presence of a particular transcription factor binding site and the transcriptional activity of hER{alpha} or any ERß isoform on that ERE sequence. We suggest that the ERE sequence itself, rather than other regulatory elements, governs E2-induced ER activity.

Our results showing similar activities of hER{alpha} and hERß1 on 2EREc38 differ from those of McInerney et al. (49) who reported that ERß1 had approximately 40% of the activity of hER{alpha} on a 2ERE-TATA-luciferase reporter in transfected CHO cells. A possible explanation is the difference between the promoters, i.e. SV-40 in pGL3-pro-luciferase vs. TATA-luciferase in Ref.49 . We note that neither hER{alpha} nor any form of ERß stimulated activity from pGL3-pro-luciferase parental vector lacking EREs in CHO-K1 cells treated with E2, indicating that covert ER binding or tethering sites in the SV-40 promoter do not appear responsible for the activity of ER in our assays.

Our findings of higher activity of hERß1 than hERß1s are different from a study showing indistinguishable activities of hERß1 and hERß1s in HepG2 cells (27). A possible explanation for this disparity is that hERß1 has N-terminal AF-1 activity that is cell-type specific. This hypothesis is based on the greater activity of ER{alpha} AF-1 in HepG2 cells, whereas in CHO-K1 cells AF-1 and AF-2 are equally active (29, 69). Hence, we suggest that the N terminus of hERß1 has greater activity in CHO-K1 than HepG2 cells. Indeed, we have observed lower E2-induced transcriptional activity of hERß1 in HepG2 vs. CHO-K1 cells (Klinge, C. M., and R. A. Prough, unpublished data), a result consistent with this suggestion. Intramolecular interactions between the N and C termini of ER{alpha}, but not ERß, have been characterized (70).

To our knowledge, there is only one report showing that hERß1 has higher transcriptional activity than hERß1s from two tandem vit A2-EREs in transfected HepG2 cells (30). In contrast, another group reported that hERß1 and hERß1s had similar E2-induced activity from two tandem vit A2-EREs in transfected HepG2 and HEK-293 (8). Notably, the activities of both ERß1 and ERß1s were only 40 and 10% of ER{alpha} in HEK-293 and HepG2 cells, respectively (8). Deletion of the first 93 aa at the N terminus of hERß1s reduced hERß transcriptional activity in HEK293 and HepG2 cells, but increased hERß transcriptional activity in HeLa cells (8). Another report stated that hERß1 gave "slightly more robust stimulation by E2" compared with hERß1s (65). However, there was no direct comparison of the transcriptional activities that two ERß isoforms presented. These results imply that the N terminus of hERß1s targets different cell type-specific cofactors, e.g. coactivators and corepressors. AF-1-specific coactivators that stimulate ER{alpha}, but not ERß have been identified, e.g. p68 and p72 (reviewed in Ref.12). The RNA coactivator SRA also interacts with ER{alpha} AF-1 and not ERß (71).

Resveratrol, a naturally occurring phytoestrogen, is a mixed agonist/antagonist with ER{alpha} and an agonist with ERß (25, 31, 72). Resveratrol showed greater efficacy with rERß than with hER{alpha} (31, 72). In contrast to the E2 data, hERß1s was stimulated at lower concentrations of resveratrol compared with rERß or hERß1, but at micromolar resveratrol concentrations, the activities of the three ERßs were identical. These data indicate that E2 and resveratrol differentially impact AF-2 transcriptional activity in the different ERß isoforms.

In contrast to the data for E2, the ERß-selective agonist DPN had similar efficacy with hERß1 and hERß1s. However, DPN had lower agonist activity with rERß. One possible explanation for these data is that DPN may elicit a different conformational change upon binding the LBD of ERß1s compared with E2. Given that the LBDs of hERß1 and hERß1s are identical, the DPN-induced LBD conformation may be transmitted to the N terminus and differentially impact the N terminus of hERß1s compared with E2. As expected on the basis of previous work (37), hER{alpha} showed approximately 70% of agonist activity of DPN but was at least 100-fold less potent.

In conclusion, our data indicate that the E2-induced transcriptional activities of rERß, hERß1, and hERß1s are not functionally equivalent in a CHO cell-based transcription assay. Furthermore, the ERE sequence differentially impacted the E2-induced transcriptional activities of hER{alpha}, hERß1, hERß1s, and rERß. These data add further proof supporting the hypothesis that DNA sequence is an allosteric modulator of ER action. Promoter sequence-dependent alterations in ER activity offer a mechanism by which individual genes are differentially responsive to estrogens in vivo. We conclude that the cellular expression of ERß isoforms may differentially impact ERE-regulated target gene expression in a ligand-dependent manner.


   Acknowledgments
 
We thank Drs. Milan Bagchi, Eva Enmark, Ronald M. Evans, Jan-Ake Gustafsson, Benita Katzenellenbogen, Bert W. O’Malley, Eckhard Treuter, and Michael Stallcup for providing their constructs for use in our experiments. We thank Alejandra P. Clark for technical assistance in preparing CHO cell extracts. We thank Dr. Barbara J. Clark for her insightful comments on this manuscript.


   Footnotes
 
This work was supported by NIH Grant R01 DK 53220 and a University of Louisville School of Medicine Research Grant (to C.M.K.). S.C.J. was supported by NIH Grant T35 GM08561.

Abbreviations: aa, Amino acids; ACTR, activin receptor; AF, activation function; AP, activator protein; CBP, cAMP response element binding protein-binding protein; DBD, DNA binding domain; DPN, 2,3-bis(4-hydroxyphenyl)propionitrile; E2, 17-ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; EtOH, ethanol; h, human; HAP, hydroxyapatite; IMDM, Iscove’s modified Dulbecco’s medium; LBD, ligand binding domain; 4-OHT, 4-hydroxytamoxifen; PR, progesterone receptor; r, rat; vit A2-ERE, Xenopus vitellogenin A2-ERE.

Received August 12, 2003.

Accepted for publication September 9, 2003.


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 Introduction
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 Discussion
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