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Molecular Endocrinology 16 (8): 1828-1839
Copyright © 2002 by The Endocrine Society

Estrogen Receptor Inducibility of the Human Na+/H+ Exchanger Regulatory Factor/Ezrin-Radixin-Moesin Binding Protein 50 (NHE-RF/EBP50) Gene Involving Multiple Half-Estrogen Response Elements

Tracy R. Ediger1, Seong-Eun Park1 and Benita S. Katzenellenbogen

Departments of Cell and Structural Biology (T.R.E., B.S.K.) and Molecular and Integrative Physiology (S.-E.P., B.S.K.), University of Illinois and College of Medicine, Urbana, Illinois 61801

Address all correspondence and requests for reprints to: Dr. Benita S. Katzenellenbogen, Department of Molecular and Integrative Physiology, University of Illinois, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801-3704. E-mail: katzenel{at}life.uiuc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Na+/H+ exchanger regulatory factor (NHE-RF; also known as ezrin-radixin-moesin binding protein 50) is a primary response gene under estrogen receptor (ER) control that may provide a link between estrogen action and the regulation of cytoskeletal and cell-signaling pathways. These studies were undertaken to define the human NHE-RF genomic regions and regulatory sequences mediating its robust estrogen responsiveness. Screening of a human genomic library yielded NHE-RF clones comprising the full gene, including the 5'-regulatory region and first exon, which were found to contain a large number (13) of consensus half-estrogen response elements (EREs), but to lack palindromic full EREs. Transfection-transactivation assays with wild-type and mutant ERs and reporter gene constructs linked to progressive deletions, or containing mutations, of the 5'-flanking region including a portion of exon I, and electrophoretic mobility and competitive gel shift assays were performed. These demonstrated direct ER interaction with the multiple half-ERE sites and the importance of the one proximal half-ERE and the multiple upstream half-EREs for eliciting the robust transcription activation of the NHE-RF gene by the estrogen-ER complex. Our findings highlight a paradigm for gene regulation via numerous half-ERE sites that expands the range of modes by which DNA recognition sites mediate the actions of this nuclear receptor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGENS MEDIATE DIVERSE physiological processes such as proliferation and differentiation of reproductive tissues and play important roles in cardiovascular, bone, and brain tissue, through the hormone-activated estrogen receptor (ER). The ER, a member of the large nuclear receptor superfamily, controls these processes, in large measure, via the regulation of gene expression and cross-talk with critical signal transduction pathways (1, 2, 3, 4, 5, 6). The ER appears to be a versatile transcription regulator, utilizing several mechanisms to activate or repress gene expression. These are known to involve both direct receptor interaction with specific DNA sequences in the vicinity of hormone-regulated genes, termed estrogen response elements (EREs), followed by the recruitment of coregulator complexes and activation or repression of estrogen-responsive gene expression, as well as the indirect modulation of transcription, without directly contacting the DNA, through interactions of the estrogen-ER complex with other transcription factors such as stimulatory protein 1 (Sp1) and, in a ligand-dependent manner, stabilizing the binding of these factors to their respective DNA promoter elements to enhance transcription (7, 8, 9, 10).

We showed recently that the Na+/H+ exchanger-regulatory factor [NHE-RF; also known as ezrin-radixin-moesin binding protein 50 (EBP50)] is a primary response gene to estrogen and is under the control of the ER in human breast cancer cells (11). NHE-RF was first described as interacting with and inhibiting the Na+/H+ exchanger type 3 (12) and, since that time, has been shown to play a wide variety of roles in cell signaling (13), promoting the scaffolding of some receptors and signaling molecules such as phospholipase C (14) and interacting with a number of additional channel/ion exchanger proteins, including the Na+/Pi cotransporter, H+ATPase, and the cystic fibrosis transmembrane conductance regulator, a cAMP-regulated Cl- channel (15, 16, 17). NHE-RF contains two PDZ domains, which are the protein-protein interaction domains through which it can localize these and other proteins to the plasma membrane.

In addition, NHE-RF has been found to interact with the ezrin-radixin-moesin family of cytoskeletal linking proteins whereby it can connect the actin cytoskeleton to the previously mentioned channel proteins that contain specific PDZ-interacting domains at their carboxy terminus (18). By acting as a scaffolding protein, NHE-RF has been postulated to alter cell architecture and localize PDZ-binding plasma membrane proteins to specific microdomains. Interestingly, estrogens have been shown to increase the number and density of microvilli and to alter the cytoarchitecture of ER-containing breast cancer cells in a manner that may involve estrogen up-regulation of NHE-RF (19). NHE-RF also binds to and influences the activation of various cell membrane receptors, including the G protein-coupled ß2-adrenergic receptor, the receptor tyrosine kinase platelet-derived growth factor receptor, and the nonreceptor tyrosine kinase-interacting protein Yap65, as well as phospholipase C isotypes ß1 and ß2 (20, 21, 22, 23). Thus, NHE-RF may play an integral role in cell signaling. By estrogen stimulation of NHE-RF gene expression, multiple cellular processes might be modulated, including changes in pH and ion flow, changes in cell cytoarchitecture/ultrastructure, and multiple signal transduction cascades.

Stemmer-Rachamimov et al. (24) demonstrated that ER status and NHE-RF expression correlated closely in human breast carcinoma specimens, and they found that NHE-RF is highly expressed in the estrogen-dominated, proliferative phase endometrium, but only weakly in the endometrium during the secretory phase. Thus, estrogenic regulation of NHE-RF may be important for many functions and in multiple estrogen target tissues.

In view of the potential importance of NHE-RF in the actions of estrogens, and because of its rapid up-regulation by this hormone, we have investigated in this manuscript, the molecular mechanism of estrogen receptor action at the human NHE-RF gene. We have isolated and characterized the human NHE-RF gene and its 5'-flanking region and have identified proximal and distal portions that are responsible for conferring estrogen inducibility. The human NHE-RF gene lacks a consensus or imperfect palindromic full ERE in the upstream 5'-flanking region, but instead contains 13 half-palindromic ERE motifs. Our transfection, gel mobility shift, and mutation and deletion data described herein document an interaction between ER and the half-palindromic EREs, and detail an intriguing mode of regulation involving the proximal and numerous distal half-EREs in mediating the robust estrogen up-regulation of NHE-RF gene expression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of the Human NHE-RF 5'-Flanking Region and Identification of the Transcription Start Site of the Gene
A human genomic DNA library was screened using 483 bp of the 5'-end of the human NHE-RF cDNA and 897 bp of the 3'-end of the human NHE-RF cDNA as probes (Fig. 1Go). Screening the genomic DNA library with each probe resulted in the isolation of several positive clones that together encompassed the full gene and contained 3.5 kb of the 5'-region of the NHE-RF gene.



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  		Figure 1. Isolation and Organization of the Human NHE-RF Gene and Its 5'-Flanking Region

A, A human genomic library was screened with both the 5'- and 3'-probes indicated under the NHE-RF cDNA. Positive clones were further analyzed through restriction digestion and sequencing to determine the intron and exon organization and 5'-flanking region of the NHE-RF gene. Sequencing primers used are indicated under the NHE-RF cDNA. B, A schematic of the NHE-RF gene, showing exons as solid rectangles (numbered I–VI) with intron sizes being indicated above the gaps. The locations of junctions within the NHE-RF cDNA and protein are indicated. The 5'-flanking region is depicted with an open, unfilled horizontal bar. The locations of restriction sites and half-EREs in the 3.5-kb 5'-flanking region are noted in the expanded region diagram. The asterisk denotes the translation start site.

 
As shown in Fig. 1AGo, several site-specific sequencing primers were designed from the cDNA for use in characterizing the NHE-RF gene. Using DNA sequencing, we were able to identify exon-intron junctions as those sequences that diverged from the cDNA sequence. As shown in Fig. 1BGo, the NHE-RF gene consists of six exons and five introns spanning approximately 26.8 kb, and our clones included 3.5 kb of the putative promoter region in the 5'-flanking region of the NHE-RF gene, the sequence of which is shown in Fig. 2AGo. Each exon-intron boundary sequence matched well with the known consensus sequence for 5'- and 3'-splice donor/acceptor sites, and our intron/exon boundary findings were identical with those reported recently from the Human Genome Project sequencing of chromosome 17.



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  		Figure 2. The 5'-Flanking Region and Part of Exon I of the Human NHE-RF Gene, and Identification of the Transcriptional Start Site

A, The nucleotide sequence of the 3-kb 5'-flanking region of the NHE-RF gene is shown (lowercase letters), along with exon I up to the SalI restriction site (bp 489, uppercase letters). The transcriptional start site is marked with an asterisk, and the translation start site (ATG) is indicated with a box and arrow. The corresponding amino acids are indicated in bold capital letters below the nucleotide sequence. Restriction sites mentioned in the text are indicated at the correct location within the genomic DNA, and the half-ERE and Sp1 sequences are marked in bold and underlined with the corresponding names indicated at the right. Eight of the half-ERE motifs and the proximal half-ERE motif mentioned in the text are also labeled beneath the appropriate site. B, RPA used to identify the transcriptional start site. An RPA probe, as described in Materials and Methods, was designed to encompass the transcriptional start site. RNase was used to digest those portions of the probe that did not bind to complementary RNA, and thus the fragment in the gel corresponds to the length of hybridized RNA, which is equal to the length of the transcribed fragment. The base pair markers to the side are numbered with reference to the translation start site, and thus the transcriptional start site is found to be 198 bp upstream of the translation start site (arrow), which corresponds to the thymidine indicated by the asterisk in Fig. 2AGo. RNA for use in the RPA was isolated from MCF-7 cells, and from MDA-MB-231 cells containing a stably integrated ER. It is evident that NHE-RF is more abundant in the MCF-7 cell line, but both cells have identical bands confirming the same transcriptional start site. The yeast RNA control does not show a band at this location.

 
A fragment from the NHE-RF promoter region, extending from bp -115 to + 48 was labeled and used as a probe in a ribonuclease protection assay (RPA) to determine the transcriptional start site. We found this start site, in the RNA from both breast cancer cell lines, to be located 198 bp upstream of the translation start site (Fig. 2BGo).

Analysis of the Regulatory Elements of the NHE-RF Gene
Sequencing analysis of 3.5 kb of the 5'-flanking and exon I region (Fig. 2AGo) revealed that this region, assumed to contain the putative promoter, did not contain a TATA box or a downstream promoter element (DPE motif) sometimes present in TATA-less promoters (25, 26), nor a CAAT box. In addition, this region lacked any consensus or imperfect full estrogen response elements (EREs) but instead contained a very large number (13) of consensus half-EREs, as well as several Sp1 binding sites (Fig. 2AGo).

To determine whether this region did indeed confer estrogen inducibility to the NHE-RF gene, the entire Bam/Sal 3.5-kb fragment was subcloned into the pTZ-thymidine kinase (TK)-chloramphenicol acetyltransferase (CAT) reporter vector (27) and transiently transfected along with wild-type ER{alpha} into MDA-MB-231 cells, an ER-negative breast cancer cell line. The BS 3.5 fragment (entry 1 of Fig. 3Go) showed an approximately 29-fold increase in its transactivation with estradiol (E2) treatment relative to the no-estrogen control (set at 100% E2 induction). To define the most E2-responsive regions within the putative promoter of the NHE-RF gene, deletion constructs were made with various restriction enzymes, as described in Materials and Methods, and used for reporter gene assays (see Fig. 3Go).



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  		Figure 3. Identification of Estrogen-Responsive Regions Within the Human NHE-RF 5'-Flanking Region

Schematic representation of the NHE-RF-CAT reporter constructs used for transient transfection into MDA-MB-231 human breast cancer cells along with an ER{alpha} expression vector. After transfection, the cells were treated with control ethanol vehicle or 10-8 M E2. The activity of each construct was analyzed by CAT assay and normalized for internal reference ß-galactosidase as described in Materials and Methods. Each result represents the mean ± SEM of at least three separate experiments and is reported as % E2 induction with the full-length BS 3.5-kb region set at 100%, and representing a 29-fold increase in activity. The hatched rectangular boxes denote the half-EREs, and the solid ovals denote Sp1 sites. The constructs are labeled according to the 5'- and 3'-restriction sites from the human NHE-RF upstream regulatory region and the approximate length of the construct (in kilobases). The solid horizontal line extending somewhat 5' from the Sal site in constructs 1–5 and 11 indicates the transcribed region at the start of the 5'-region analyzed in this study. The restriction enzymes used included B, BamHI; E, EcoRI; N, NcoI; X, XhoI; Sm, SmaI; and S, SalI. The numbered constructs are as follows: 1, BS 3.5; 2, ES 2.7; 3, XS 1.3; 4, SmS 0.5; 5, SmS{Delta}TK 0.5; 6, SmSm 0.4; 7, BSm 2.5; 8, BX 2.0; 9, BE 0.8; 10, BN 1.3; and 11, NS 2.2.

 
Transfection of the ES 2.7-kb 5'-deletion fragment (entry 2, Fig. 3Go) demonstrated a large percentage induction (~62%) relative to the full-length BS 3.5 fragment (set at 100% E2 induction), indicating that this region contains much of the E2 responsiveness seen in the full-length promoter. This is consistent with the observation that the deleted portion, containing the two most distal half-EREs, is responsible for only a minimal amount of estrogen responsiveness (BE 0.8 fragment, entry 9 of Fig. 3Go).

When ES 2.7 is further reduced to the 3'-most SmS 0.5 fragment (entry 4, Fig. 3Go), which contains the most proximal half-ERE and continues into the coding region, transfection assays reveal that approximately 60% of the estrogen inducibility is retained, indicating this region alone plays a very important role in the estrogen induction of the NHE-RF gene. A further 3'-deletion of the NHE-RF coding region in this construct at +25 to give a 0.3-kb Sma/Nar fragment, showed induction identical with the SmS 0.5-kb construct, and thus the SmS 0.5 construct was used for subsequent experiments. In addition, when the SmS 0.5 construct is evaluated after the removal of the TK promoter (entry 5, Fig. 3Go), estrogen inducibility persists, suggesting that this region contains the endogenous promoter. Constructs lacking both the SmS 0.5 and TK promoter showed little activity of any sort and no estrogen inducibility (data not shown).

Surprisingly, the inducibility of the XS 1.3 fragment (entry 3, Fig. 3Go) was significantly lower than might have been expected when compared with the ES 2.7 and SmS 0.5 constructs (entries 2 and 4). This suggests the presence of a silencing element within the XS 1.3-kb region that is eliminated when this sequence is truncated to the SmS 0.5. Furthermore, it suggests that when the eight half-EREs more distal to the start site are present, this silencing effect is overcome. The SmSm 0.4 fragment (entry 6), which contains four Sp1 sites, does not appear to contribute to the estrogen-induced transcriptional activity.

To define more clearly the role of the distal half-EREs, several additional deletion mutants were generated. Transfection results demonstrate that both the BSm 2.5 and BX 2.0 constructs (entries 7 and 8, Fig. 3Go), which contain 12 or 10 half-EREs, respectively, show a similar magnitude of estrogen responsiveness (~40% of maximal induction), suggesting that the half-EREs are functional. To determine which elements were essential, the half-EREs were further divided into the BN 1.3 and NS 2.2 regions (entries 10 and 11). Each of these fragments exhibited a similar level of inducibility (42% and 39%, respectively). Taken together, the findings suggest that the multiple half-ERE sites in the upstream region, and the one proximal half-ERE site near the translation start site, are involved in the transactivation of the NHE-RF gene by the estrogen-occupied ER. All of the E2 stimulations shown in Fig. 4Go could be fully repressed by cotreatment with the pure antiestrogen ICI182,780 (data not shown), supporting the fact that these transactivations were indeed ER mediated.



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  		Figure 4. Assessment of the Role of DNA Binding in E2-ER Stimulation of NHE-RF

MDA-MB-231 cells were transfected with the reporter constructs BS 3.5 and SmS 0.5 along with either the wild-type ER or the ER-DBDmut 1 or -mut2 and were treated with control ethanol vehicle or 10-8 M E2. CAT assays and ß-galactosidase assays were performed as described. Values are the mean ± SEM from three separate experiments.

 
Direct Interaction Between ER and DNA Is Required for Estrogen Inducibility
To further understand the mechanism through which the ER activates NHE-RF transcription, we used two DNA-binding domain (DBD) mutants. The first (DBDmut1) contains a triple point mutation in the DBD (amino acids E203, G204, and A207 changed to G203, S204, and V207), which is known to greatly reduce the affinity of ER for consensus ERE DNA (28, 29), and in the second (DBDmut2), most of the DBD is deleted (amino acids 185–251 removed) (30). The latter mutant has previously been shown to eliminate estrogen responsiveness when mediated through the classical ERE direct DNA binding pathway, but not when functioning through an alternate pathway, such as the Sp1/ER interaction, in which ER works through tethering and not by direct contact with the DNA (10). Figure 4Go shows that both full-length (BS 3.5) and the SmaI/SalI (SmS 0.5) fragments demonstrated strong E2 inducibility with the wild-type ER{alpha}, and that stimulation of both constructs was reduced to approximately 30% with DNAmut1 and to the basal, control level with DNAmut2. These results indicate that the predominant regulation of NHE-RF by ER involves interaction with DNA-regulatory sites, suggesting a role for the multiple half-ERE sites in this regulatory region.

The Distal Half-EREs Need a Proximal Promoter Element to Function to Full Potential
To delineate the roles of the distal half-EREs and the one proximal half-ERE within the transcribed NHE-RF gene, and to more carefully localize the estrogen responsiveness of the proximal SmS 0.5 fragment, two sets of NHE-RF mutants were generated. In the first, the proximal half-ERE TGACC motif was mutated to TGCTC for the full-length BS 3.5 (entry 2, Fig. 5Go) and the SmS 0.5 fragment (entry 4, Fig. 5Go) in the pTZ-TK-CAT reporter, or in this same reporter without the thymidine kinase promoter element (entry 6, Fig. 5Go) to study the endogenous NHE-RF promoter activity. This double-point mutation has previously been shown to eliminate E2 inducibility (31). Mutation of the proximal half-ERE reduced the estrogen induction by 70% for the full-length promoter (entry 2 vs. entry 1), and by 40–50% for both SmS 0.5 mutants (entry 4 vs. 3 and entry 6 vs. 5). This suggests that the proximal half-ERE is indeed necessary for full estrogen-induced transcription.



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  		Figure 5. The Proximal Half-ERE Motif Is Necessary for Full E2 Induction of the Human NHE-RF Gene

Schematic representation of the NHE-RF-CAT reporter constructs used for transient transfection into MDA-MB-231 cells along with an ER{alpha} expression vector. The bold X over the half-ERE indicates that it contained a double-point mutation that eliminates ERE activity. The constructs used included 1, BS 3.5; 2, BS 3.5 proxmut; 3, SmS 0.5; 4, SmS 0.5 proxmut; 5, SmS{Delta}TK; 6, SmS{Delta}TK proxmut; and 7, EX 1.3. Construct 8 is the EX 1.3 fragment adjacent to an inserted oligonucleotide containing the proximal half-ERE motif. Each value represents the mean ± SEM of at least three separate experiments and is reported as percent E2 induction with the full-length BS 3.5 region set at 100%.

 
In the second experiment, an oligonucleotide encompassing the proximal half-ERE was cloned downstream of the NHE-RF fragment containing the more distal eight half-EREs to generate a promoter consisting of the regions most strongly induced by estrogen (Fig. 5Go, entry 8). Of note (entry 8 vs. 7), addition of the proximal half-ERE oligonucleotide reconstituted full activity observed with the full-length BS 3.5-kb region (entry 1), suggesting that these two regions work together to elicit the robust estrogen inducibility of the NHE-RF gene.

In Vitro Binding of ER to the Half-Palindromic ERE Sequences
Transfection experiments revealed that the eight half-EREs in the far upstream region and the proximal half-ERE could contribute to activate estrogen-regulated transcription of NHE-RF. To determine whether the ER in fact binds to the half-ERE sites, a gel mobility shift assay was performed with synthetic oligonucleotides containing each of the half-palindromic ERE sequences and neighboring flanking sequences (Fig. 6Go). [See Figs. 2AGo and 5Go, entry 1, for motif numbering and position.] Gel shifts with the motif 5&6 half-EREs showed strong intensity, similar to that of the consensus palindromic ERE, whereas the motif 1 half-ERE showed somewhat less ER binding than motif 5&6. The other motifs, 2, 3, 4, 7, 8 and the proximal half-ERE, showed an interaction with ER, although much weaker.



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  		Figure 6. ER Interaction with the Multiple Half-Palindromic EREs of the 5'-Flanking Region of the Human NHE-RF Gene

Gel mobility shift assays were conducted with probes containing either one half-ERE (motif 1, 2, 3, 4, 7, 8 and proximal half-ERE, denoted P) or two half-EREs (motif 5&6) and the adjacent sequence as described in Materials and Methods, with the position of these half-EREs illustrated in Figs. 2AGo and 5Go. 32P-labeled probes were incubated with Baculovirus-expressed and purified E2-occupied ER. The position of the ER-half-ERE complexes is indicated with an arrowhead at the left. Lanes 1 and 2 contain the consensus palindromic ERE without or with ER, and lanes 3–10 contain the proximal half-ERE (P, lane 3) followed by the distal half-ERE motifs (lanes 4–10) as indicated.

 
A competitive gel mobility shift assay using nonlabeled synthetic oligonucleotides revealed that ER binds to the half-ERE motifs with differing affinities (Fig. 7Go, A and B). When compared with the affinity of ER binding to the consensus palindromic ERE, the binding affinities to motif 5&6 and motif 1 were about 3-fold and 40-fold lower, respectively. Therefore, these observations raise the possibility that binding of the E2-occupied ER to multiple half-EREs may be needed to elicit the full transcription activation of the human NHE-RF gene in response to the E2-ER complex.



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  		Figure 7. Competitive Gel Mobility Shift Assay to Determine Relative Binding Affinities of ER to the Half-Palindromic ERE Motifs

A, The binding of purified E2-occupied ER to a 32P-labeled consensus palindromic ERE oligomer was competed with increasing amounts of radioinert consensus palindromic ERE (panel 1), the half-ERE motif 5&6 (panel 2), or motif 1 (panel 3) in a competitive gel mobility shift assay. The position of ER-half-ERE complexes is marked with an arrowhead. (B) The assays were quantified by phosphorimager analysis and the results were graphed. The graph is plotted as amount of cold competitor oligonucleotide (picomoles) vs. percent receptor DNA binding. Values are the mean ± range from two separate experiments.

 
Assessment of the Effect of Sp1 on E2 Inducibility of NHE-RF Gene Transcription
In addition to the multiple half-ERE sites, the human NHE-RF promoter region also contains several Sp1 sites. Because the ER has been shown to be able to cooperate with Sp1 in promoting transcriptional activation of some genes (10, 32, 33, 34, 35), we investigated the effect of Sp1 and ER in modulating NHE-RF gene transcription. Studies were conducted using Drosophila Schneider insect cells because these cells are deficient in ER and the Sp1 transcription factor, but have been shown to reconstitute the transcriptional activity of estrogen-regulated genes when these factors are introduced via transfection (36, 37). We could not use the mammalian MDA-MB-231 cells for these studies because they contain high levels of Sp1, so much so that we observed no effect of transfected Sp1 on NHE-RF activity in these cells (data not presented), as we observed previously for the regulation of other genes in this human cell line (37).

As shown in Fig. 8Go, E2-occupied ER alone was able to stimulate NHE-RF activity (~4.7-fold) in the absence of Sp1 in these Drosophila cells. An approximately 2-fold stimulation of control NHE-RF activity was observed with added Sp1 alone and, as expected, the activity of Sp1 was not affected by addition of E2. Cotransfection of ER along with Sp1 did not elicit increases in fold E2 induction of NHE-RF activity above that seen with ER alone (5.2-fold vs. 4.7-fold); however, the control CAT activity (without E2) of ER + Sp1, like that of Sp1 alone, was approximately 1.8 times higher than that of ER alone. These data suggest that the mechanism of activation of NHE-RF gene expression by the E2-occupied ER may be largely independent of Sp1.



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  		Figure 8. Examination of Estrogen-Occupied ER and Sp1 Effects on NHE-RF Promoter Activity in Drosophila Cells

Drosophila cells were transfected with human NHE-RF BS 3.5 fragment in pTZ-TK-CAT or with pPac-ER{alpha} or pPac-Sp1, or with both. At 24 h after transfection, cells were treated with 0.1% ethanol vehicle control or with 10-8 M E2 as indicated. After 48 h, cells were harvested and cell extracts were assayed for CAT activity and ß-galactosidase activity. Bars show the mean ± SEM from three separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The findings described in this report provide a molecular mechanism for understanding how the hormone-occupied ER regulates expression of the human NHE-RF gene. Previously, we had demonstrated that NHE-RF mRNA was increased rapidly in response to estrogen via an ER-mediated pathway in two human breast cancer cell lines (11). Another report extended these observations using NHE-RF protein immunostaining to demonstrate that ER status and NHE-RF presence correlated closely in human breast carcinoma specimens, and that NHE-RF was more highly expressed in the endometrium during the estrogen-stimulated portion of the menstrual cycle (24), further supporting estrogen regulation of this gene. However, neither of these earlier reports detailed the molecular mechanism through which estrogen induces this expression.

In this study, we analyzed the molecular basis for the marked hormonal responsiveness of this gene. Although ER-regulated genes often contain palindromic full (13 bp) EREs that differ in 1 or 2 bp from the consensus, there is increasing evidence for diversity in the nature and positioning of EREs. It is of interest that the NHE-RF promoter does not possess any consensus or imperfect palindromic full EREs, although it contains more than 12 consensus half-EREs. Transfection experiments revealed that estrogen responsiveness tracked with two regulatory portions of the NHE-RF gene. The first encompasses one proximal half-ERE near the translation start site, with the second encompassing the multiple half-ERE sites localized upstream from the translation start site. The direct interaction of ER with these half-ERE sites is supported by the fact that mutations in the DBD of ER greatly reduced or eliminated estrogen inducibility of NHE-RF gene transactivation, that ER could be shown to interact directly with half-ERE elements of the 5'-flanking and exon I region, and that mutation or deletion of the sites resulted in a marked loss in hormone responsiveness.

Gel mobility shift assays demonstrated that ER can bind to the half-ERE motifs, although with lower affinity than to a consensus palindromic ERE. The different affinities observed when different half-ERE-ER complexes were compared implied that sequences adjacent to the half-ERE sites affect the stability of the binding of ER to these motifs, as noted previously (38). Of interest, the two half-ERE motif (motif 5&6), which is identical with a consensus full ERE except for the absence of the three-nucleotide spacing element between the half-ERE sequences, was found to bind to ER with significant affinity, only 3-fold less than that of the consensus ERE. Our findings are consistent with observations that ER-DNA complexes are observed on direct repeat half-EREs with different spacings between half-sites and that binding of ERs to direct repeats is more flexible than binding to palindromic response elements (38, 39).

It is of note that the proximal half-ERE near the translational start site was important for maximal estrogen regulation of the human NHE-RF gene and that when it was mutated, induction was greatly reduced. Also, when this half-ERE sequence was added to the more distal fragment containing the eight half-ERE motifs, full estrogen induction was restored. These findings imply the involvement of proximal and distal elements that together mediate estrogen regulation of NHE-RF gene expression.

Our experiments do not eliminate the possibility that some of the estrogen regulation of the NHE-RF gene might be due to protein-protein interactions between ER and other transcription factors. DNAmut2 fully eliminated estrogen regulation of NHE-RF. However, because this mutant ER is missing most of the DBD, some of the reduction of ER activity could be the result of reduced protein interactions with this domain. We tried to address this point by using DNAmut1, in which the strength of DNA interaction is reduced by a triple-point mutation that changes ER recognition of response elements from an ERE to a glucocorticoid response element (28). This mutant gave NHE-RF stimulation only 30% that of wild-type ER. Hence, it is possible that up to 30% of ER activity at this gene could arise from protein-protein interactions. If tethering via other transcription factors occurs, our studies using Sp1-deficient Drosophila cells suggest that Sp1 protein and Sp1 sites in NHE-RF are not involved, although the limited involvement of other transcription-regulatory factors cannot be ruled out. However, these interactions would have to be restricted to the ER DBD, because no activity is observed when most of the DBD is eliminated (DBDmut2). Also, it is possible that the residual activity of DBDmut1 arises from a low affinity that it may have for the multiple, widely spaced half-EREs in the NHE-RF gene.

Although half-ERE sites have been noted in other estrogen-regulated genes, these gene promoters often contain consensus or nonconsensus palindromic full EREs as well. For example, the rat PRL gene contains an imperfect full ERE and four half-EREs close to one another, all at 1.5–1.8 kb upstream from the promoter. Mutation of either the imperfect full ERE or one of the half-sites resulted in complete loss of estrogen inducibility, implying the importance of both of these elements and most notably that the half-EREs cannot function alone to confer estrogen inducibility (40). The ovalbumin gene, by contrast, contains four widely spaced upstream half-EREs and a proximal half-ERE close to the TATA box, and studies by Chambon and co-workers (41) have shown that the four upstream half-EREs function synergistically with the proximal half-ERE.

Hence, although there are some similarities in the function of half-ERE sites in the ovalbumin, PRL, and NHE-RF promoters, there are also notable differences. In particular, NHE-RF contains many more half-EREs (13) than the other two gene promoters, and in NHE-RF, as opposed to the situation observed for ovalbumin, the upstream half-EREs showed substantial estrogen-regulated activity, even in the absence of the proximal half-ERE. In both genes, however, the proximal site was either very important (NHE-RF) or essential [ovalbumin (41)] for achieving maximum estrogen inducibility. Thus, there appears to be an emerging paradigm for estrogen regulation of some genes that can involve only ERE half-sites. The number of ERE half-sites in these regulated genes appears to be variable [prothymosin {alpha} 2 (37), ovalbumin 5, and NHE-RF 13], but they are capable of supporting a high level of estrogen inducibility.

Estrogens are known to stimulate growth and maintenance of microvilli in several estrogen-sensitive tissues, such as mammary tissue, pituitary gland, gall bladder, uterus, and male reproductive efferent duct (19, 42, 43, 44). Of interest, immunocytochemical analysis has shown that NHE-RF is localized to the apical membrane of epithelial cells and is particularly abundant in cells rich in microvilli, where it directly binds to specific microvillar proteins (45), and in cells specialized for ion transport or absorption (24). Thus far, the means by which estrogen induces these microvilli is unknown; however, it is appealing to consider that the increase in NHE-RF after treatment with estrogen might allow for an increase in the plasma membrane protein-cytoskeleton interaction and thereby alter the cell ultrastructure. In addition, changes in the cytoskeleton have also been shown to alter gene expression. Thus, estrogen, by stimulation of the synthesis of NHE-RF, may influence gene expression through this more indirect mechanism as well.

The marked NHE-RF up-regulation by estrogen, and the fact that NHE-RF can interact with several receptor systems [ß2-adrenergic receptor, platelet derived growth factor receptor, Yap65, and others (20, 21, 22, 23)], suggest that NHE-RF could act as a possible link in cross-talk between ER and other signaling pathways such as the cAMP/protein kinase A (46, 47, 48) and epidermal growth factor pathways (49) that are known to be involved in estrogen signaling. It is also significant that the NHE-RF gene is located on chromosome 17q25.1, a site showing frequent genetic alterations in human breast tumors (50, 51, 52, 53). By characterizing the human NHE-RF gene and its upstream regulatory region, we have identified multiple half-EREs that cooperate in promoting robust ER-regulated hormonal stimulation of this gene. It will be of interest to determine which of the multiple cell signaling and cell cytoarchitectural and organizational functions served by NHE-RF are most prominently affected by NHE-RF up-regulation by estrogen in the diverse target tissues in which estrogens act.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Chemicals and Materials
Cell culture media were purchased from Life Technologies, Inc. (Gaithersburg, MD). Calf serum was obtained from HyClone Laboratories, Inc. (Logan, UT). E2 was purchased from Sigma (St. Louis, MO). The antiestrogen ICI182,780 was kindly provided by Alan Wakeling and Zeneca Pharmaceuticals (Macclesfield, UK).

Isolation of the NHE-RF Gene and 5'-Flanking Region
A human genomic library (CLONTECH Laboratories, Inc., Palo Alto, CA) was plated at 30,000 plaque-forming units/plate onto a K802 host strain lawn. The plaques were transferred and fixed to HybondN nylon filters (Amersham Pharmacia Biotech, Arlington Heights, IL). For hybridization, the filters were wet with 6x sodium chloride/sodium citrate (SSC) and then washed with 50 mM Tris (pH 8), 1 M NaCl, 1 mM EDTA, 0.1% sodium dodecyl sulfate (SDS) for 2 h in a 65 C shaking water bath. The filters were then prehybridized in 6x SSC, 5x Denhardt’s solution, 10 mM EDTA, 0.1% SDS, 200 µg/ml denatured salmon sperm DNA overnight with shaking at 65 C. Radiolabeled probes, prepared using the Rediprime DNA labeling system from Amersham Pharmacia Biotech, were added and hybridization continued for more than 18 h at 65 C. Filters were washed in 2x SSC, 0.1% SDS at 65 C in a shaking water bath twice for 1 h, and then washed two times in 0.2% SDS, 0.1% SDS under similar conditions. Filters were air dried and exposed to film with intensifying screens for more than 18 h at -80 C. Positive plaques were isolated, the phage was eluted, and secondary and tertiary screenings were completed as above.

For the initial screen, the 897-bp fragment from the 3'-NHE-RF cDNA was used as a probe and produced eight different purified plaques; however, none contained the 5'-upstream region for which we were searching. Thus, we repeated the screen using the 5'-483-bp NHE-RF fragment, and seven plaques were isolated, at least two of which contained the 5'-region. Sequencing of the DNA employed a fluorescent sequencing method using the BigDye DNA Sequencing Kit (PE Applied Biosystems, Foster City, CA).

Cell Culture and Transient Cell Transfections
Transfections were done in ER-negative MDA-MB-231 human breast cancer cells that were maintained as previously described (11). The cells were plated in 24-well plates and transfected when approximately 80% confluent. Transfections were performed using 2 µg of the pTZ-NHE-RF-CAT construct, 0.4 µg of the internal reference ß-galactosidase reporter plasmid pCMVß, 0.2 µg ER expression vector, and carrier plasmid to 5 µg total DNA. A premix containing 5 µg/well lipofectin, 16 µg/well transferrin, and 29 µl/well Hank’s balanced salt solution was mixed with DNA in Hank’s balanced salt solution (100 µl/well) for 15 min at room temperature. The cells were washed with serum-free medium, and then the liquid was aspirated and replaced with 300 µl serum-free media/well. One hundred fifty microliters of the DNA/lipofectin/transferrin mixture (100 µl DNA and 50 µl lipofectin/transferrin mixture) were added to each well. After incubation for 8 h at 37 C in a 5% CO2 incubator, the cells were washed one time with medium containing 5% charcoal dextran-treated calf serum and then replaced with 1 ml medium plus serum. Cells were treated with the indicated ligand or 0.1% ethanol control and incubated for 24 h at 37 C with 5% CO2. After this, cells were scraped into 300 µl Tris/NaCl/EDTA solution and lysates were prepared by freeze/thawing three times. ß-Galactosidase assay to standardize transfection efficiency and CAT assays were performed as previously described (54, 55).

Drosophila Schneider SL2 insect cells were cultured with Schneider’s Drosophila medium (Life Technologies, Inc.) containing 10% fetal calf serum and 100 µg/ml of streptomycin, 100 IU/ml of penicillin G, and 0.025 µg/µl of gentamycin. Cells were transfected in 24-well plates using FuGene 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the user manual provided. NHE-RF BS 3.5-pTZ-TK-CAT (1.0 µg) was cotransfected with 0.2 µg of pPac-ER{alpha} or with 0.1 µg of pPac-Sp1, or both. pCMVß (1.5 µg) was used as a ß-galactosidase internal control. At 24 h after transfection, cells were washed and treated with 0.1% ethanol vehicle control or with 10-8M E2 for 48 h. Cells were then harvested and cell extracts were assayed for CAT activity and ß-galactosidase activity.

Gel EMSAs and Competition Assays
The oligonucleotides for the EMSAs and for the competition assays were synthesized as follows:

Motif 1: 5'-GATCACATGTATCCCTTTGACCCCAAGCTCTGCCT-3'; Motif 2: 5'-GATCTCTGCCTCCTCCCTGACCACCCATGCCCTTT-3'; Motif 3: 5'-GATCTTAGGTGGTGGGAGGTCACCCATTTCCGAGT-3'; Motif 4: 5'-GATCATGTGTCCTCCTTGGTCACATATCTCCCAAA-3'; Motif 5&6: 5'-GATCGGAAACATGAGGTCATGACCTGCAGGCATCT-3'; Motif 7: 5'-GATCTCTGTCCTCAGATGGTCAGCTCCCCGCTCAA-3'; Motif 8: 5'-GATCTCTCCCGCCTCTGTGACCAGCCTCTCTTTGGC-3'; Proximal: 5'-GATCCAAGTCGCGCCGCTGACCCGTCGCAGGGCGAG-3'.

The synthesized single-stranded oligomers were annealed to their complements. The resultant double-stranded oligomers were gel purified and 32P labeled as described previously (56). Baculovirus expressed and purified ER (200 fmol) was assembled in 1x binding buffer (57). The 32P-labeled response element DNA was added to the reaction mixtures and then incubated for 10 min at 25 C. E2 (10 nM) was included in all binding reactions containing ER. Samples were loaded onto an 8% polyacrylamide gel, run at 300 V in 1x Tris/acetate/EDTA running buffer, and visualized by autoradiography. For the competition assay, a radiolabeled consensus palindromic ERE was incubated with different concentrations of the nonlabeled oligonucleotides. Free and ER-bound DNA were separated on 8% nondenaturing acrylamide gel, and signal intensity was visualized and quantified by phosphorimager analysis.

RPA
The following PCR primers were used to amplify 163 bp of the XhoI/SalI fragment from the 5'-NHE-RF genomic DNA. RPA1 (5'-ACACTTCCACTTCCTGAGC-3') and RPA2 (5'-GTGTCCCAGGATCC-3'); note that in RPA2, the underlined T was a point mutation that introduced a novel BamHI restriction site into the genomic DNA. After amplification, the DNA was digested with PstI and BamHI and subcloned into pBluescript II SK. To create a probe, this plasmid was first PCR amplified with T3 and T7 promoter primers (Promega Corp., Madison, WI), and this product was purified using the Qiaex kit (QIAGEN, Chatsworth, CA) following the manufacturer’s instructions. The purified product was labeled with 32P-dCTP using the MaxiscriptT3 (Ambion, Inc., Austin, TX) and polyacrylamide gel purified following manufacturer’s protocol. The RPA was performed using the RPAII kit from Ambion, Inc. Briefly, 20 µg of total RNA from MCF-7cells, MDA-MB-231 cells, or two yeast control RNAs were mixed with 1.6 x 105 cpm radiolabeled probe, and the RNA was precipitated with 0.5M NH4OAc and 2.5 vol EtOH. Pellets were resuspended in 10 µl hybridization buffer, denatured for 4 min at 95 C, and then incubated overnight at 45 C to allow hybridization of the probe to its complement in the sample RNA. Next, 150 µl of ribonuclease (RNase) digestion buffer with 1:80 dilution of RNaseA/RNase T1 Mix were added to each sample, except the second yeast control, to which the RNase digestion buffer alone was added. The samples were incubated for 30 min at 37 C, and then 225 µl of RNase inactivation/precipitation III solution were added along with 150 µl EtOH. These were transferred to a -80 C freezer for 20 min and then microcentrifuged for 20 min to precipitate the RNA fragment. The liquid was aspirated and pellets dried for several minutes, after which they were resuspended in 6 µl loading dye and loaded onto a polyacrylamide sequencing gel. The size of the protected fragment was determined using size standards consisting of 35S-labeled DNA sequence run on either side of the RPA samples. The fragment size corresponded to the distance in base pairs from the transcriptional start site to the end of the novel BamHI sequence (GGATCC) at +53 bp (Fig. 2Go).

Plasmid Constructs
The BS 3.5-kb fragment (-2985 to +469) from the human NHE-RF upstream region was directionally cloned into the BamHI and SalI sites of pTZ-TK-CAT (27) according to standard subcloning protocol. The NHE-RF BS 3.5 was further divided into the following fragments, blunted with Klenow, and subcloned into the SmaI site of the pTZ-TK-CAT vector: ES 2.7 (-2202 to +469), XS 1.3 (-764 to +469), SmS 0.5 (-55 to +469), BSm 2.5 (-2985 to -466), BX 2.0 (-2985 to -938), BE 0.8 (-2985 to -2202), BN 1.3 (-2985 to -1656), NS 2.2 (-1656 to +469), SmSm 0.4 (-466 to -55), EX 1.4 (-2202 to -938). The SmS 0.5 minus TK was created by cloning the -55 to +469 fragment into the pTZ-CAT vector from which the TK promoter region had been released with BglII and BamHI restriction digestion and the compatible ends religated. EX+proximal half-ERE (-2202 to -938 and +134 to +205) was created by designing an oligo containing bp +134 to +205 with BamHI 5'- and SalI 3'-compatible ends, and inserting this into the BamHI and SalI sites of the EX 1.4 construct. The mutations in the proximal ERE were introduced into BS 3.5, SmS 0.5, and SmS 0.5–TK constructs with the QuikChange Site-Directed Mutagenesis Kit from Stratagene (La Jolla, CA) following manufacturer’s instructions using the forward primer 5'-CCAAGTCGCGCCGCTGCTCCGTCGCAGGGCGAGATGAGC-3' and the reverse primer 5'-GCTCATCTCGCCCTGCGACGGAGCAGCGGCGCGACTTGG-3'. BSm 2.5, SmSm 0.4, and SmS 0.5 were also cloned into pBluescriptII SK (Stratagene) for use in sequence analysis. The ER DBD mutants 1 and 2 have been described previously (28, 30). In DBDmut1, the DBD contains three changed amino acids, E203G, G204S, and A207V (28, 29). In DBDmut2, amino acids 185–251 have been deleted (30).


   ACKNOWLEDGMENTS
 
We thank Dr. Ann Nardulli, University of Illinois, Urbana, for providing purified Baculovirus-expressed ER, and Jennifer Schultz for guidance in the gel shift assays.


   FOOTNOTES
 
This work was supported in part by NIH Grant CA-18119 and a grant from The Breast Cancer Research Foundation.

1 T.R.E. and S.-E.P. contributed equally to this work and should be considered equal first authors. Back

Abbreviations: CAT, Chloramphenicol acetyltransferase; DBD, DNA-binding domain; DBDmut, DBD mutant; E2, estradiol; EBP50, ezrin-radixin-moesin binding protein 50; ER, estrogen receptor; ERE, estrogen-response element; NHE-RF, sodium/hydrogen exchanger regulatory factor; RNase, ribonuclease; RPA, RNase protection assay; SSC, sodium chloride/sodium citrate; SDS, sodium dodecyl sulfate; Sp1, stimulatory protein 1; TK, thymidine kinase.

Received for publication October 23, 2001. Accepted for publication March 29, 2002.


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Mol. Endocrinol., September 1, 2006; 20(9): 2020 - 2035.
[Abstract] [Full Text] [PDF]

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T. Simoncini, C. Scorticati, P. Mannella, A. Fadiel, M. S. Giretti, X.-D. Fu, C. Baldacci, S. Garibaldi, A. Caruso, L. Fornari, et al.
Estrogen Receptor {alpha} Interacts with G{alpha}13 to Drive Actin Remodeling and Endothelial Cell Migration via the RhoA/Rho Kinase/Moesin Pathway
Mol. Endocrinol., August 1, 2006; 20(8): 1756 - 1771.
[Abstract] [Full Text] [PDF]

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 J. Biol. Chem.Home page
F. Stossi, V. S. Likhite, J. A. Katzenellenbogen, and B. S. Katzenellenbogen
Estrogen-occupied Estrogen Receptor Represses Cyclin G2 Gene Expression and Recruits a Repressor Complex at the Cyclin G2 Promoter
J. Biol. Chem., June 16, 2006; 281(24): 16272 - 16278.
[Abstract] [Full Text]