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[Cancer Research 63, 2820-2828, June 1, 2003]
© 2003 American Association for Cancer Research

Experimental Therapeutics

Modulation of the Folate Receptor {alpha} Gene by the Estrogen Receptor

Mechanism and Implications in Tumor Targeting1 ,2

Karen M. M. Kelley, Brian G. Rowan and Manohar Ratnam3

Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614-5804


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The folate receptor (FR) type {alpha} is a promising target for diagnostic imaging agents and therapeutic intervention in major subtypes of gynecological malignancies; however, the receptor levels in the tumors are variable and are generally relatively low in estrogen receptor (ER)-positive tumors. Here we report that the FR-{alpha} gene promoter is repressed in the presence of 17ß-estradiol and derepressed by the antiestrogens tamoxifen and ICI 182,780 in a promoter-specific and ER-{alpha}-dependent manner in carcinoma cell lines including HeLa (cervical carcinoma), BG-1 (ovarian carcinoma), and IGROV-1 (ovarian carcinoma). The ligand and ER dose response of the FR-{alpha} promoter and its time course paralleled those of a classical estrogen response element-mediated effect. Antiestrogens produced an ER-dependent increase of up to 36-fold in the expression of the endogenous FR-{alpha} gene. Deletion analysis and FR-{alpha}/SV40 promoter chimeras showed that the ER effect is mediated exclusively within the G/C-rich region in the TATA-less P4 promoter of FR-{alpha}; electrophoretic mobility shift analysis demonstrated interaction of ER at only one of three G/C-rich elements. ER-ß only modestly affected FR-{alpha} promoter activity but did not diminish the ER-{alpha}-mediated effects. The ER corepressor, SMRT, enhanced the repression by 17ß-estradiol/ER, but ER coactivators, including SRC family members, did not appreciably impact the ER ligand response. The results suggest that in ER+ tumors, FR-{alpha} expression is directly and actively suppressed and predict that a brief treatment with antiestrogens will boost FR-{alpha} expression by passive derepression, enhancing the efficacy of FR-targeted diagnostic and therapeutic applications. They also reveal novel aspects of gene repression by ER.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human FR4 gene family encodes homologous glycopolypeptides termed FR-{alpha} (1 , 2) , FR-ß (3) , and FR-{gamma} (4) . FR-{alpha} and FR-ß are attached to the cell surface by a glycosylphosphatidyl inositol membrane anchor (1 , 5 , 6) , whereas the polymorphic FR-{gamma} is constitutively secreted (7) . Even though the three FR isoforms share ~70% amino acid sequence identity and bind folic acid with a high affinity (KD < 10-9 M) and a 1:1 stoichiometry (8) , each exhibits a distinct and narrow tissue/tumor specificity. Among normal tissues, FR-{alpha} is expressed in epithelial cells of the placenta, breast, lung, kidney proximal tubules, choroid plexus, ovary, fallopian tubes, uterus, endocervix, and salivary glands (9, 10, 11, 12) ; FR-ß is expressed in placenta and mature myelomonocytic cells (3 , 13 , 14) ; FR-{gamma} is expressed by lymphoid cells (4) . Among malignant tissues, FR-{alpha} is consistently expressed in nonmucinous adenocarcinomas of the ovary, uterine and cervical adenocarcinomas, testicular choriocarcinomas, and certain brain tumors and expressed less frequently in breast, colon, and renal carcinomas (9, 10, 11, 12 , 15, 16, 17, 18) .

The {alpha} isoform of FR is the more widely expressed and the best understood in terms of its physiological function (recently reviewed in Ref. 19 ). FR-{alpha} has gained considerable prominence in recent years as a potential target for therapeutic intervention in cancer by virtue of (a) its limited expression in normal tissues, where it is largely restricted to luminal surfaces, not directly accessible to the bloodstream, and (b) the ability of the recycling receptor to bind and internalize (anti)folate compounds and folate conjugates. A wide variety of FR-{alpha}-targeted therapies have shown promise in preclinical and clinical tests (20, 21, 22, 23, 24, 25, 26, 27, 28) . The experimental therapies include folate-cytotoxic conjugates, folate-coated liposomal drug delivery systems, cytotoxic antifolates that require FR-{alpha} for cellular uptake, bifunctional antibodies that produce a host immune response against FR-{alpha}-rich tumors, and peptide and DNA vaccines against FR-{alpha}. In addition, FR-{alpha} has been determined to be a useful target for folate radiopharmaceuticals in diagnostic tumor imaging (reviewed in Ref. 20 ).

Diagnostic/therapeutic targeting of FR-{alpha} has worked extremely well in vivo in xenograft models using human tumor cell lines expressing uniform and high levels of FR-{alpha}, but a major limitation in extending the success to patients is the variability of FR-{alpha} expression levels in a given type of tumor as well as heterogeneity in its expression within a tumor (13) . Therefore, any means of specifically increasing the expression of FR-{alpha} in tumors is of immense potential benefit in enhancing the efficacy of FR-{alpha}-targeted therapies as well as the sensitivity of FR-{alpha}-targeted diagnostic imaging. We have recently demonstrated enhanced efficacy in therapeutic targeting of a related FR (type ß) in acute myelogenous leukemia cells via nuclear receptors for retinoids (29 , 30) . In searching for ways to similarly modulate FR-{alpha}, we were encouraged by the finding of a negative correlation between FR-{alpha} expression levels and ER expression in primary breast cancers (31) , suggesting that FR-{alpha} may be regulated through this nuclear receptor.

In the classical model of ER action, an ER agonist induces a conformational change in the receptor and, upon the receptor binding to an ERE on a target gene, allows its transactivation domain to interact with the transcription initiation complex. The FR-{alpha} gene lacks an intact ERE. However, it is now well established that ER can activate a gene either through a partial ERE or without directly binding to DNA. Such actions by ER have been shown to occur through association with other DNA binding proteins (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42) . ER may also indirectly regulate genes through the products of its immediate target genes. Another important consideration in the action of ER on a gene is the available complement of ER coregulators (coactivators and corepressors; Refs. 43, 44, 45, 46 ) and ER isoforms ({alpha} and ß) that are known to be determinants of the variable promoter and cell type context of ER action. There are relatively few examples of target genes that are repressed by ER or the estrogen-ER complex (37 , 42 , 47) .

The FR-{alpha} gene comprises 7 exons and 6 introns spanning approximately 7.7 kb in length and is located on chromosome 11q13 (48 , 49) . Multiple FR-{alpha} transcripts are initiated from two distinct promoters in the gene termed P1 and P4, located in exon 1 and exon 4, respectively (50) . There appears to be some tissue specificity in promoter usage, but the P4 promoter-driven transcript is more efficiently translated than the P1 promoter transcripts. The P4 promoter in the FR-{alpha} gene is TATA-less, with a cluster of three G/C-rich, noncanonical Sp1 binding elements, each of which contributes to basal promoter activity (51 , 52) . The P1 promoter has not yet been adequately characterized; however, it has been shown to generate multiple alternately spliced transcripts containing variable lengths of 5'-untranslated region (50) .

The present study was undertaken with the goal of investigating the possibility of modulating FR-{alpha} gene expression by ER ligands and understanding such a possible regulation in sufficient mechanistic detail to enable a general prediction of the response of the FR-{alpha} gene to ER ligands in human gynecological tumors. Both the FR-{alpha} and the ER genes are frequently inactivated or selected against during immortalization and long-term culture of cell lines. As a result, even though major subtypes of ovarian and uterine cancers are known to express both FR-{alpha} and ER, established cell lines in which both of these genes are functional appear to be scarce. Our approach, therefore, was to express FR-{alpha} promoter constructs and ER by transient transfection in model cell lines expressing endogenous ER and FR, respectively. Because restoring normal levels of wild-type ER expression in established cell lines by stable transfection leads to cell growth inhibition and/or cell death, investigators generally use transient transfection systems for studies of its transcriptional effects. The HeLa cell line is a widely accepted model for such studies of ER. The inherent difficulty of generating stable recombinant cell lines expressing wild-type ER may also be overcome by stably transfecting a less active mutant form of ER (53) . Such recombinant cells were used in this study to examine regulation of the endogenous FR-{alpha} gene by ER.

Here we report transcriptional repression of FR-{alpha} by ER or estrogen/ER and derepression by antiestrogens and elucidate the nature of this ER-mediated regulation including the target site of ER action and the effects of ER coregulators and alternate ER isoforms. The results open a new avenue for the design of future strategies in FR-targeted diagnostics and therapies and also expose novel mechanistic aspects of gene repression by ER.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents.
LipofectAMINE, Opti-MEM I reduced serum medium, MEM, and DMEM were purchased from Life Technologies, Inc. FBS was purchased from Irvine Scientific. FuGENE 6 was from Roche Diagnostics. TAM and E2 were purchased from Sigma. ICI 182,780 was a kind gift from Astra Zeneca. [3H]Folic acid was purchased from Moravek with a specific radioactivity of 20.2 Ci/mmol. T4 polynucleotide kinase and HeLa cell nuclear extracts were purchased from Promega. [{gamma}-32P]ATP (6000 Ci/mmol) was purchased from Amersham Pharmacia. Recombinant ER-{alpha} was purchased from Calbiochem. Normal IgG and affinity-purified IgG fractions of antihuman Sp1, antihuman Sp3, and antihuman ER were purchased from Santa Cruz Biotechnology.

DNA Constructs.
Construct design used either natural restriction sites or restriction sites created by the PCR using Vent DNA polymerase (New England Biolabs) and custom oligonucleotides from Life Technologies, Inc. In cases where a natural restriction site could not be found for use, complementary primers containing an appropriate restriction site or point mutation were used in conjunction with upstream and downstream primers containing restriction sites. Alternatively, mutagenic oligonucleotides were used as end primers to amplify the desired fragment. The PCR products were first digested at both ends with the appropriate restriction enzymes and cloned into the PGL3-basic plasmid (Promega) or subcloned into the FR-{alpha}-promoter construct (-3394 nt to +33 nt, corresponding to the transcription initiation site at +1 nt) inserted into the PGL3 basic plasmid at MluI and XhoI sites of the polylinker. The FR-{alpha}/SV40(GC)6-Luc construct was generated using the upstream primer 5'-GTCAGCATATGTAGTCCCGCCC-3' containing a synthetic restriction site (NdeI) and the downstream primer 5'-AAACTTAAGCAGCGATGGGGC-3' containing a synthetic restriction site (AflIII) corresponding to regions in the SV40 promoter of the pGL3-control plasmid (Promega). The FR-{alpha}/SV40-Inr construct was generated using the upstream primer 5'-ATTCTCCGCGGCATCGCTGAC-3' containing a synthetic restriction site (SacII) corresponding to a region in the SV40 promoter and the downstream primer 5'-CACTGCATACGACGATTCTGTG-3' corresponding to a region in the luciferase gene of the pGL3-control plasmid. The downstream restriction site (NarI) used in the subcloning occurred naturally in the plasmid. The recombinant plasmids were amplified in XL1Blue and purified using the Qiagen plasmid kit (Qiagen) or by CsCl gradient centrifugation followed by phenol chloroform extraction and ethanol precipitation. The entire cloned sequence was verified using the Beckman CEQ 2000 automated sequencer. The PCR reaction for sequencing was carried using the dye terminator cycle sequencing kit from Beckman.

Cell Culture and Transfection.
HeLa I-1 cells were kindly provided by Dr. S. T. Rosen. BG-1 cells were provided by Dr. Randolf Ruch. HeLa (American Type Culture Collection) and HeLa I-1 cells were routinely cultured in phenol red-free MEM supplemented with FBS (10%), penicillin (100 units/ml), streptomycin (100 mg/ml), and L-glutamine (2 mM). BG-1 and IGROV1 (American Type Culture Collection) cells were routinely cultured in DMEM and supplemented as described above. Treated or transfected cells were grown in phenol-red free media supplemented with charcoal-stripped FBS (5% v/v), penicillin (100 units/ml), streptomycin (100 mg/ml), L-glutamine (2 mM), insulin (2 µg/ml), and transferrin (40 µg/ml), unless otherwise noted. In addition, treated HeLa I-1 cells were grown in culture with 50 µg/ml G418. E2, TAM, or ICI 182,780 was used where indicated at the concentrations specified. Transfections with the cDNA constructs were carried out in 6-well plates (Corning) using LipofectAMINE (Life Technologies, Inc.) or FuGENE 6 (Roche Diagnostics), according to the manufacturer’s suggested protocol. Uniformity of transfection was routinely monitored through cotransfections with the ß-galactosidase expression plasmid, pSV-ß-gal (Promega). ß-Galactosidase activity was measured colorimetrically using the assay system available from Promega.

Luciferase Assay.
Forty-eight h after transfection, the cells were washed once with PBS [10 mM sodium phosphate (pH 7.5), 150 mM NaCl] and harvested in 400 µl of reporter lysis buffer provided with the luciferase assay system (Promega). The samples were centrifuged at 12,000 x g for 2 min at room temperature. The supernatant was assayed for luciferase activity in a luminometer (Lumat LB9501; Berthold) using the luciferase substrate from Promega.

EMSA.
EMSA-grade HeLa cell nuclear extract was purchased from Promega. Equimolar quantities of complementary oligonucleotides corresponding to specific regions in the FR-{alpha} P4 promoter, a consensus Sp1 sequence probe (5'-ATTCGATCGGGGCGGGGCGAG-3'; Promega) or an ERE sequence probe (5'-GTCAGGTCACAGTGACCTGA-3'; Invitrogen) were denatured in TE buffer [100 mM Tris-Cl, 10 mM EDTA (pH 7.5)] at 100°C for 5 min and annealed by cooling to room temperature in a thermal cycler at a rate of -1°C/min. The oligonucleotides were labeled using [{gamma}32P]ATP and T4 polynucleotide kinase (Promega). Ten µg of nuclear extract were incubated with 32P-labeled probes (40,000 cpm) in 10 µl of binding solution [25 mM HEPES buffer (pH 8.0) containing 50 mM KCl, 0.5 mM MgCl2, 0.5 mM DTT, 2 mg of poly(dI-dC)-poly(dI-dC), and 10% glycerol]. Recombinant ER was added to particular samples in the amounts indicated. The samples were incubated at room temperature for 15 min. After addition of the appropriate antibody in certain reaction tubes, the samples were incubated at room temperature for an additional 30 min. The reaction mixture was then run on a 4% polyacrylamide gel at 275 V for 45 min. The resulting gel was subjected to either autoradiography on X-ray film or phosphorimaging on a phosphor screen (Molecular Dynamics) using a Storm 840 scanner (Molecular Dynamics) and ImageQuant version 1.1 imaging software (Molecular Dynamics).

Western Blots.
The samples were electrophoresed on a SDS-polyacrylamide gel and transferred electrophoretically to nitrocellulose. The blots were probed with a rabbit antibody to FR-{alpha}, and the receptor was visualized using a chemiluminescence kit (Amersham) following the protocol provided by the vendor. The densitometric scans of the blots were analyzed using the ImageQuant software (Molecular Dynamics).

[3H]Folic Acid Binding Assay.
Whole cells in 6-well tissue culture dishes were first washed with ice-cold PBS. Endogenously bound folate was removed by washing with 1 ml of ice cold acid buffer [10 mM sodium acetate (pH 3.5), 150 mM NaCl]. The cells were washed again with PBS and then incubated in a 1-ml solution of PBS containing 30 pmol of [3H]folic acid (Moravek) for 60 min at 4°C. The cells were then washed twice with PBS. Ice-cold acid buffer was used to remove bound [3H]folic acid, and the radioactivity was measured by liquid scintillation counting. In duplicate wells, cells were preincubated with an excess (3 nmol) of unlabeled folic acid to determine the extent of nonspecific binding of [3H]folic acid.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
FR-{alpha} Promoter Usage in Model Cell Lines.
The full promoter region of the FR-{alpha} gene contains two basal promoters (P1 and P4) with distinct transcription initiation sites, but the predominant mRNA species detected in malignant cells by RNase protection assay is the P4 promoter-driven transcript. Because we used transient transfections to study the FR-{alpha} promoter activity in the following sections, it was of importance to first test whether this promoter preference of the endogenous FR-{alpha} gene is reflected in the promoter-luciferase reporter constructs during transient transfection of the model cell lines used (i.e., HeLa cervical carcinoma, IGROV-1 ovarian carcinoma, and BG-1 ovarian carcinoma). As shown in Table 1Citation , the FR-{alpha}-P1-promoter-luciferase activity was much lower than that of P4-luciferase, and the FR-{alpha} promoter activity was greatly diminished by deleting the Sp1 elements in the P4 promoter, indicating that the promoter preference of the chromosomal gene is retained in the model cell lines even outside the chromosomal context.


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  		Table 1 FR-{alpha} promoter usage in model cell lines

 
ER-mediated Repression of the FR-{alpha} Promoter and Derepression by Antiestrogens.
Cotransfection of HeLa and IGROV-1 cells with a plasmid expressing ER-{alpha} and the FR-{alpha} promoter-luciferase reporter plasmid caused a 71% and 61% reduction in the promoter activity, respectively (Fig. 1A)Citation ; the promoter activities were either partially or completely restored to the uninhibited values in the presence of pharmacological concentrations of either of the antiestrogens TAM and ICI 182,780 (Fig. 1A)Citation . To ensure that the endogenous ER expressed in malignant cells can modulate the FR-{alpha} promoter, BG-1 ovarian carcinoma cells, which express endogenous ER, were transfected with FR-{alpha} promoter-luciferase. As expected, treatment with ICI 182,780 increased the promoter activity in BG-1 cells (Fig. 1B)Citation . It may be noted that in Fig. 1BCitation , the promoter activity in the presence of antiestrogens exceeded the value in the absence of E2. As seen below, this may be ascribed to the repressive effect of ER even in the absence of E2.



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  		Fig. 1. Promoter-specific repression of FR-{alpha} transcription by ER in model cell lines. A, HeLa and IGROV-1 cells (106) were cotransfected with the FR-{alpha} promoter-luciferase construct (400 ng) and either an ER expression plasmid (25 ng) or the plasmid vector alone (25 ng) in the presence of 1 nM E2. Antiestrogens were introduced at the time of transfection. TAM and ICI 182,780 did not alter FR-{alpha} promoter activity in the absence of ER (data not shown). B, the FR-{alpha} promoter-luciferase construct (400 ng) was transfected into BG-1 cells (106) in the absence or in the presence of E2 (1 nM), and antiestrogens were introduced at the time of transfection. C, HeLa cells (106) were transfected with the TK-promoter-luciferase (200 ng) or RSV-promoter-luciferase (200 ng) constructs and cotransfected with either ER plasmid (25 ng) or the plasmid vector (25 ng). Antiestrogens were introduced at the time of transfection.

 
The ER and ER ligand response of the FR-{alpha} promoter was clearly promoter specific because other promoters (RSV and TK promoters) did not respond appreciably to ER in either the presence or absence of antiestrogens under the cell culture and transfection conditions use in this study (Fig. 1C)Citation .

ER Ligand Dose Response of the FR-{alpha} Promoter.
E2 activates ERE-driven promoters at a subnanomolar concentration; the estrogen antagonist TAM, which has approximately 100-fold lower affinity for ER compared with E2, generally counteracts the estrogen effect at a higher relative concentration, whereas the pure antiestrogen, ICI 182,780, effectively counteracts the E2 effect at the lower concentration. Such a ligand dose response is a hallmark of ER-mediated transcriptional effects. Contrary to the ERE-mediated effect on a control ERE-driven promoter (ERE2E1b; Fig. 2ACitation ), the FR-{alpha} promoter was repressed by E2, and the repression was counteracted by TAM and ICI 182,780 (Fig. 2BCitation ; please note in Fig. 2Citation that the Y axis in A indicates promoter activation, whereas the Y axis in B indicates promoter repression). The dose responses of the ligand effects on the FR-{alpha} promoter, however, occurred within concentration ranges comparable with those required to regulate the ERE2E1b promoter. It may also be noted that the ER effects on both the ERE2E1b promoter and the FR-{alpha} promoter occurred (albeit suboptimally) even in media containing charcoal-stripped FBS, suggesting that the unliganded receptor may also repress the FR-{alpha} promoter and that this effect is enhanced by E2.



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  		Fig. 2. Estrogen and antiestrogen dose response of a control ERE-driven promoter and the FR-{alpha} promoter in HeLa cells. Please note that the Y axis in A indicates promoter activation, whereas the Y axis in B indicates promoter repression. The cells (106) were transfected with either ERE2E1b-luciferase (100 ng; A) or FR-{alpha} promoter-luciferase (100 ng; B) and cotransfected with the ER expression plasmid (50 ng) where indicated. Cells that were not cotransfected with the ER plasmid were cotransfected instead with the plasmid vector alone (50 ng). E2, TAM, or ICI 182,780 was included from the time of transfection.

 
ER Dose Response for Repression of the FR-{alpha} Promoter.
The level of ER in various tumors is variable, but in general, all ER+ tumors have adequate ER to mediate the classical physiological effects of estrogen. In view of the potential clinical benefit of modulating the FR-{alpha} gene with antiestrogens, it was important to compare the ER dose dependence for modulating the FR-{alpha} promoter with that of a classical ERE-dependent gene. Table 2Citation shows the degree of activation of an ERE-dependent promoter luciferase or repression of FR-{alpha} promoter-luciferase in response to cotransfection with different amounts of the expression plasmid for ER in the presence of E2. The similarity in the ER plasmid dose response of the two promoters supports the view that ER repression of the FR-{alpha} promoter occurs at ER levels comparable with those required for activation of a classical estrogen-responsive gene.


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  		Table 2 ER dose response of the FR-{alpha} promoter

 
Time Course and Reversibility of Antiestrogen Effects on the FR-{alpha} Promoter.
When antiestrogens (TAM and ICI 182,780) were introduced 24 h after transfection with ER and either the ERE2E1b-luciferase (Fig. 3A)Citation or the FR-{alpha} promoter-luciferase (Fig. 3B)Citation , the repression or activation, respectively, of the promoters began within hours and progressively increased. As seen in Fig. 4Citation , even when antiestrogens were introduced 24 h after cotransfection of cells with FR-{alpha} promoter-luciferase and ER, there was a doubling of the reporter activity at the end of 48 h after transfection. A brief 6-h treatment with ICI 182,780 was adequate to produce full derepression of the FR-{alpha} promoter that lasted for at least 18 h after withdrawing the compound (Fig. 4)Citation . In contrast, the derepression of the FR-{alpha} promoter by TAM was partially lost after withdrawing the compound. This result is entirely consistent with the known modes of ER-mediated actions of TAM and ICI 182,780 in vivo; TAM acts by reversibly modulating ER, whereas the net result of the interaction of ICI 182,780 with ER is by down-regulation of the receptor.



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  		Fig. 3. Time course of response of the control ERE2E1b-luciferase (A) and FR-{alpha} promoter luciferase (B) to antiestrogens in HeLa cells. The cells (106) were transfected with the indicated promoter-luciferase construct (100 ng) and cotransfected with ER expression plasmid (50 ng) in the presence of E2 (1 nM). Twenty-four h after transfection, TAM or ICI 182,780 was introduced, and cells were harvested at different intervals to determine luciferase activity.

 


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  		Fig. 4. Short-term reversibility of antiestrogen effects on the FR-{alpha} promoter. Antiestrogen (TAM or ICI 182,780) was introduced 24 h after cotransfection of HeLa cells (106) with the FR-{alpha} promoter-luciferase construct (100 ng) and the ER expression plasmid (50 ng). In one set of experiments, the antiestrogen was removed after a 6-h treatment. Cells were harvested 48 h after transfection and assayed for luciferase activity.

 
Modulation of Endogenous FR-{alpha} Expression by ER.
Transcriptional effects observed in model promoter-reporter constructs commonly translate into more pronounced effects in the corresponding endogenous genes, in the chromosomal context. The relatively low transcription rate of the FR-{alpha} gene, together with the relatively slow mRNA and protein turnover rates (~24 h) of FR-{alpha} (54) , confounds efforts to directly quantitate the change in endogenous FR-{alpha} expression caused by transfected ER during the short-term (2 or 3 days) transient transfections. Furthermore, only a small fraction of the FR-{alpha}+ cells (<10%) may be transfected in transient transfections. Given the above considerations, even profound transcriptional effects on the endogenous FR-{alpha} gene may be expected to result in very small changes in FR-{alpha} levels against a high background in transient transfection systems. To examine the effect of ER/ER ligands on endogenous FR-{alpha} expression, it is therefore imperative to use a cell culture system in which all of the cells express ER over an extended period. Because restoration of wild-type ER expression would adversely affect the viability of established (FR-{alpha}+) cell lines, we used a stable recombinant HeLa cell line (HeLa I-1; Ref. 53 ) expressing a mutant (V400G) form of ER that has a lower affinity for E2. The mutant ER allows long-term cell survival in culture but may have altered E2 response characteristics due to different E2-induced conformations of the protein compared with wild-type ER. As seen from the Western blot in Fig. 5ACitation , treatment of HeLa-I-1 cells with either TAM or ICI 182,780 resulted in a high level of endogenous FR-{alpha} (up to ~36-fold over the untreated control) over a period of 6 days. Consistent with the anticipated alteration in E2 response of the mutant ER in HeLa-I-1 cells, E2 also produced an increase in FR-{alpha}, notably at supraphysiological concentrations, as expected from the decreased affinity of the mutant ER for E2 (Fig. 5A)Citation . As seen in Fig. 5BCitation , the up-regulated FR-{alpha} was functional in that it was capable of binding [3H]folic acid. The antiestrogen effects occurred at pharmacological concentrations of the ligands. These results underscore a profound effect of ER and ER ligands on the endogenous FR-{alpha} gene expression.



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  		Fig. 5. Effect of ER ligands on the expression of the endogenous FR-{alpha} gene in HeLa I-1 cells. A, the cells were either untreated (DMSO) or treated with E2, ICI 182,780, or TAM at the beginning of day 1 or day 3, and all of the cells were harvested at the end of day 6. The cell lysates were subjected to Western blot analysis as described in "Materials and Methods." The FR-{alpha} band intensities are indicated in relative units, using a value of 1 for the untreated control. The ER ligand treatment did not appreciably alter the viable cell count at the end of the 6-day period. FR-{alpha} expression was not altered by E2 or antiestrogen treatment in the ER- parental HeLa cells (data not shown). B, the cells (106) were either untreated (DMSO) or treated with E2, ICI 182,780, and/or TAM as indicated for 4 days. The amount of functional FR-{alpha} was assayed by measuring [3H]folic acid cell binding to the cell surface.

 
To ensure that ER ligands will modulate FR-{alpha} expression selectively in FR-{alpha}+ and ER+ tissues and that the ligands will not alter the tissue specificity of FR-{alpha} expression, TAM and ICI 182,780 were tested on a variety of ER+/FR{alpha}- cells including the ER+ BG-1 cells discussed above. In no case did we observe induction of endogenous FR-{alpha} expression (data not shown). It appears that ER will only modulate the endogenous FR-{alpha} gene in cells in which the gene is transcriptionally active.

Mapping the ER-responsive Element in the FR-{alpha} Promoter.
A short FR-{alpha} fragment (-173 nt to +33 nt; Fig. 6ACitation ) containing the basal P4 promoter retained the ER ligand response of the FR-{alpha} promoter in HeLa and IGROV-1 cells (data not shown). This fragment lacks a classical ERE. However, the fragment does contain two cis elements known in other genes to mediate an ER ligand response, i.e., an AP-1-like element and a cluster of three G/C-rich (Sp1 binding) elements (Fig. 6A)Citation . Deletion of the AP-1-like element failed to abrogate the ER modulation of the FR-{alpha} promoter (Fig. 6B)Citation , excluding a role for AP-1 in mediating the ER effect. Because deletion of the G/C-rich region would abolish the basal promoter activity of the TATA-less P4 promoter, the entire cluster of Sp1 elements was substituted with a G/C-rich region from the heterologous SV40 early promoter. The resulting chimeric construct retained promoter activity but was activated by E2 in the presence of ER; this activation was counteracted by ICI 182,780, in striking contrast to the ER modulation of the native P4 promoter (Fig. 6C)Citation . These results pinpoint the G/C-rich region in the FR-{alpha} P4 promoter as an essential site for the action of ER ligands. Fig. 6CCitation also shows that, when the TATA box containing initiator region of the SV40 early promoter was substituted in the FR-{alpha} promoter, ER repression as well as derepression by antiestrogens was retained. This result suggests that the promoter specificity of ER response of FR-{alpha} resides entirely in the G/C-rich region of the P4 promoter.



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  		Fig. 6. Functional mapping of the ER response element in the FR-{alpha} P4 promoter. A, the nt sequence of the ER-responsive P4 promoter fragment. An AP-1-like element is highlighted, and three G/C-rich (Sp1) elements are shown in bold. The nt are numbered in relation to the transcription initiation site (+1 nt). The translation start site is underlined. B, HeLa cells (106) were transfected with a FR-{alpha} promoter-luciferase construct (400 ng) in which the AP-1-like site (-154 nt to -143 nt) was deleted. The cells were cotransfected with either ER expression plasmid (25 ng) or the plasmid vector alone (25 ng). The transfected cells were grown in media containing E2 (1 nM) and in the absence of antiestrogen or the presence of ICI 182,780 or TAM. The cells were harvested 48 h after transfection for luciferase assays. C, HeLa cells (106) were transfected with FR-{alpha} promoter-luciferase (FR-{alpha}-Luc; 400 ng), FR-{alpha}-Luc in which the G/C-rich region (-47 nt to -18 nt) of the P4 promoter was replaced by the G/C-rich region in the SV40 early promoter [FR-{alpha}/SV40 (GC)6; 400 ng] or with FR-{alpha}-Luc in which the initiator region (-28 nt to +33 nt) was replaced by the TATA box-containing initiator region of the SV40 early promoter (FR-{alpha}/SV40-Inr; 400 ng). ER expression plasmid (25 ng) or the plasmid vector (25 ng) was cotransfected as indicated. The transfected cells were grown in the presence of E2 (1 nM) and in the absence or presence of ICI 182,780 for 48 h before harvesting them for luciferase assays.

 
Identification of an ER-interacting Site in the P4 Promoter of FR-{alpha}.
The association of HeLa cell nuclear proteins and of ER with the P4 promoter was initially examined by EMSA of synthetic 40-bp DNA probes, covering the entire P4 promoter fragment including the G/C-rich and initiator regions; the EMSA probes were designed to have 20-bp overlaps. Only one of the overlapping probes (–89 nt to -50 nt) showed a shift that could be attributed to ER (Fig. 7)Citation . This probe contains one of the three Sp1 elements of the FR-{alpha} promoter together with its flanking sequences. The ER-associated shift was not observed when either a portion of the Sp1 element or the 3'-flanking sequence was deleted (data not shown), indicating a requirement for both the Sp1 binding and the flanking sequences. Antibodies to either Sp1 or ER completely blocked specific and distinct EMSA bands as indicated in Fig. 7ACitation . Antibody to Sp3 (but not an isotype control) also blocked the Sp1 band, presumably due to cross-reactivity with Sp1, as seen in Fig. 7BCitation for a consensus (control) Sp1 element. The quantitative blocking of this band by the Sp1-specific antibody (Fig. 7A)Citation and the different mobilities of binary DNA complexes of Sp1 and Sp3 (Fig. 7B)Citation exclude the possibility that Sp3 may bind to the -89 nt to -50 nt sequence of the P4 promoter. ER, on the other hand, failed to show a shift with the consensus Sp1 element (Fig. 7B)Citation , demonstrating specificity for the FR-{alpha} probe. Further characterization of the EMSA band resulting from interaction of the -89 nt to -50 nt probe with ER is shown in Fig. 7, A and CCitation . A 100-fold excess of an unlabeled probe containing a classical ERE sequence blocked the ER band in Fig. 7ACitation , confirming that the band is due to ER. E2 increased the intensity of the ER band, whereas both ICI 182,780 and TAM abolished it (Fig. 7C)Citation . In contrast, when a control probe containing a classical ERE sequence was used, TAM, but not ICI 182,780, abolished the EMSA band due to ER (Fig. 7D)Citation . The above results demonstrate a direct interaction of ER with a specific G/C-rich region of the FR-{alpha} P4 promoter and show that this interaction is profoundly affected by ER ligands.



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  		Fig. 7. EMSA of the interaction of ER and nuclear proteins in the G/C-rich region of the FR-{alpha} P4 promoter. A HeLa cell nuclear extract (HeLa NE; 10 µg) was used in all cases. 32P-labeled probes corresponding to a FR-{alpha} promoter sequence (-89 nt to -50 nt; A and C), a 21-mer consensus Sp1 probe (B), or a 20-mer consensus ERE probe (D) was used (described in "Materials and Methods"). In A, Lanes 3 and 4, 0.25 and 0.75 µg of ER were used, respectively. In all other reactions containing ER, 0.75 µg of the protein was used. The various EMSA conditions are indicated in the figure, and the assay procedure is described in "Materials and Methods."

 
Effect of ER-ß on FR-{alpha} Promoter Activity.
Because ER+ gynecological tumors are known to frequently coexpress ER-{alpha} and ER-ß, it was of interest to test whether ER-ß may modulate FR-{alpha} similar to ER-{alpha} or may otherwise counteract the effect of ER-{alpha}. As seen in Fig. 8Citation , ER-ß caused a relatively modest repression of the FR-{alpha} promoter, even though its full functionality was evident from its activation of the control ERE2E1b promoter. The modest repression of the FR-{alpha} promoter by ER-ß was reversed by ICI 182,780 (Fig. 8)Citation . Cotransfection of ER-ß and ER-{alpha}, however, did not mitigate the ER-{alpha}-mediated modulation of the FR-{alpha} promoter (Fig. 8)Citation , suggesting that ER-ß will not competitively inhibit the effect of ER-{alpha} on the FR-{alpha} promoter.



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  		Fig. 8. Effect of ER-{alpha} versus ER-ß on FR-{alpha} promoter activity. HeLa cells (106) were transfected with the FR-{alpha} promoter-luciferase construct (200 ng) and cotransfected individually or in combination with expression plasmids (25 ng) for ER-{alpha} or ER-ß or with the plasmid vector. The cells were treated from the time of transfection with E2 (1 nM) ± ICI 182,780.

 
Effect of ER Coregulators on ER Modulation of the FR-{alpha} Promoter.
Because the nature of the physiological and pharmacological effects of ER in a given tissue or cell type may be determined by the complement of coactivators and corepressors expressed in those cells, we examined the effects of a variety of known ER coregulators on ER-mediated regulation of the FR-{alpha} promoter. From Table 3Citation , it is evident that whereas coactivators of the major SRC family (SRC-1, SRC-1E, TIF-2, and RAC3) as well as Creb binding protein enhanced ER activation of the control ERE2E1b promoter to a variable degree, none of the coactivators appreciably altered either ER-mediated repression of the FR-{alpha} promoter or its activation by TAM. In contrast, the classical corepressor, SMRT, increased ER repression of the FR-{alpha} promoter (Table 3)Citation .


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  		Table 3 Effect of coregulators on ER modulation of the FR-{alpha} promoter

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates that both the endogenous FR-{alpha} gene and the plasmid FR-{alpha} promoter-reporter constructs are modulated by ER/ER ligands. The magnitude of the ER-ligand-induced synthesis of FR-{alpha} was considerable in the chromosomal context of the endogenous FR-{alpha} gene. Antiestrogens, however, did not induce FR-{alpha} expression in a variety of ER+/FR-{alpha}- cell lines, indicating that they will not alter the tissue specificity of FR-{alpha} expression. ER ligands did not alter FR-{alpha} expression when the gene was driven by a constitutive (RSV) promoter (data not shown), excluding the possibility of posttranscriptional effects of ER on FR-{alpha} expression.

The experiments in this study were designed to address key mechanistic issues that pertain to the predictability of an in vivo tumor response of FR-{alpha} up-regulation by antiestrogens. The close parallel in the ER and ER ligand dose response of repression of the FR-{alpha} promoter and the classical activation of the ERE2E1b promoter support the view that the ER ligands will modulate FR-{alpha} in tumors at physiological/pharmacological levels of ER, estrogen, and antiestrogens. In both promoters, transcriptional modulation by ER was observed even when the culture media were devoid of estrogenic molecules, and the serum in the culture was previously treated with dextran-coated charcoal to deplete estrogen, although the addition of E2 did further increase the transcriptional effects of ER. Because ER is known to activate promoters in a ligand-independent (phosphorylation state-dependent) manner (46 , 55) , the above observation suggests that unliganded ER is capable of repressing the FR-{alpha} promoter and that optimal repression is obtained in the presence of subnanomolar (physiological) levels of estrogen. The short-term reversibility of the effect of TAM but not ICI 182,780 on both the ERE2E1b and FR-{alpha} promoters also supports a fundamental similarity between the two promoter responses in terms of the mode of action of the antiestrogens on ER in vivo, i.e., reversible alteration of the conformation of ER by TAM (56) and down-regulation of ER by ICI 182,780 (57, 58, 59) . The time course of the antiestrogen response of the FR-{alpha} promoter was relatively rapid, consistent with the view that the FR-{alpha} gene is a direct target of ER action (discussed below).

Extensive investigations of ER have not revealed any DNA sequence element to which ER can bind directly besides the consensus ERE and its variants or ERE half-sites (60) . However, in recent years, estrogen-ER complexes have been found to activate promoters through AP-1 or AP-1-like elements, a nuclear factor {kappa}B response element, G/C-rich (Sp1 binding) elements, or other elements (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42) . Repression of promoters by E2/ER is relatively less frequent and not as well characterized and may occur by direct interaction of ER with transcription factors such as nuclear factor {kappa}B, Sp3, or GATA-1 (37 , 42 , 47) . The ER repression of the FR-{alpha} promoter was mapped to the proximal region of the P4 promoter, which lacks an ERE but contains an AP-1-like element and a cluster of G/C-rich elements. Mutational analysis excluded a role for the AP-1-like element but identified the G/C-rich region as the site of ER action. A growing number of genes are activated by E2 via an ER-Sp1 complex [at G/C-rich elements (reviewed in Ref. 40 )]. There is a single reported example of promoter repression by E2/ER at a G/C-rich element, i.e., the vascular endothelial growth factor gene (42) . In that example, the ER repression appears to be mediated by an ER-Sp3 complex, but a role for the remainder of the promoter context was not ruled out. The present studies of chimeras of the TATA-less FR-{alpha} promoter and the TATA box-dependent SV40 early promoter suggest that the entire promoter specificity of E2/ER repression of the FR-{alpha} promoter resides within the G/C-rich region of the FR-{alpha} gene. In the chimeric promoter in which the G/C-rich region of the FR-{alpha} promoter was replaced by the known Sp1 binding sequence of the SV40 promoter, E2/ER activated the promoter, consistent with the well known action of the ER-Sp1 complex. Thus, the FR-{alpha} promoter specificity for repression by ER must be determined by the specific sequence of the G/C-rich region of the P4 promoter. From EMSA, it appears that ER directly interacts with only one of the three Sp1 binding sites in the P4 promoter. The specificity for this site includes both the Sp1 binding element and its 3'-flanking sequence. In contrast to the vascular endothelial growth factor gene promoter, Sp1 but not Sp3 bound at this site. The formation of this ER complex occurred in the absence of E2 but increased in the presence of E2. This observation is consistent with the functional data on repression of the FR-{alpha} promoter by unliganded ER and its further repression when E2 is present. Furthermore, both TAM and ICI 182,780 prevented formation of the complex, offering a mechanistic explanation for the antagonistic effects of the ligands on ER repression of the FR-{alpha} gene. It may also be noted that in contrast to the FR-{alpha} promoter element, ICI 182,780 did not prevent the binding of ER to the ERE sequence in vitro, indicating a difference in the modes of interaction of ER with the two elements. Indeed the unique manner in which ER associates with the FR-{alpha} promoter may enable it to recruit transcriptional corepressors rather than coactivators (discussed below).

Depending on the target gene and tissue, a variety of ER ligands, collectively known as SERMs, may act as agonists or antagonists of the transcriptional effects of estrogen by reversibly modulating the conformation of the receptor (56) . Contrary to SERMs (e.g., TAM), pure antiestrogens such as ICI 182,780 completely attenuate both ligand-dependent and ligand-independent functions of ER by multiple mechanisms in vivo. ICI 182,780 is known to impair ER dimerization, increase ER degradation, and interfere with nuclear localization of the receptor (57, 58, 59) . Consistent with the known actions of the antiestrogens on ER in vivo, the derepression of the FR-{alpha} promoter by TAM but not ICI 182,780 was reversible in the short term. The ability of estrogen to activate a promoter and for a SERM to either promote or antagonize this activation is known to be determined by specific coregulator(s) (coactivators and corepressors) recruited by ER, which in turn is governed by the tissue-specific complement of the ER coregulators (46 , 56 , 60 , 61) . In E2/ER-mediated gene repression, however, there is little information available on a potential role of coregulators, even though mutational analysis has provided indirect evidence to suggest a role for coregulators in gene repression by an ER-AP-1 complex (35) . None of the ER coactivators tested, including the major SRC family proteins, altered ER repression of the FR-{alpha} promoter or its derepression by TAM; on the other hand, the ER corepressor, SMRT, increased the repression. These observations support the view that FR-{alpha} gene repression by ER is an active process that involves recruitment of corepressors but that the increase in FR-{alpha} promoter activity by antiestrogens may simply represent a passive process of derepression in which the antiestrogens disable ER. The results clearly indicate that the variable coregulator complement of a target tumor should not be a significant concern in extending the findings of antiestrogen modulation in cell lines to tumor tissues in vivo.

Most studies of estrogen action have focused on ER-{alpha} because ER-ß was discovered more recently (61) . The two ER types show differential expression as well as coexpression in various normal tissues, but notably, they have been found to be coexpressed in epithelial cells in ovarian cancers (62) . ER-{alpha} and ER-ß may respond differently to ER ligands in a cell-dependent and target gene context-dependent manner (32 , 39) , and they may even form functional heterodimers; ER-ß can also inhibit ER-{alpha} transcriptional activity (63, 64, 65) . It has been demonstrated that both ER-{alpha} and ER-ß form similar complexes with Sp1, binding to its COOH-terminal region (66) . However, in a model promoter, activation by the ER-Sp1 complex required the AF-1 domain of ER-{alpha}; ER-ß was unable to activate the promoter due to a nonfunctional AF-1 domain (66) . The same study reported that when ER-{alpha} and ER-ß were coexpressed, ER-ß inhibited promoter activation by ER-{alpha}-Sp1. The above considerations raised the concern that in tumor cells expressing both ER-{alpha} and ER-ß, the latter may counteract ER-{alpha}-dependent regulation of the FR-{alpha} gene. However, ER-ß did mediate repression of the FR-{alpha} promoter, albeit to a modest extent, and the promoter activity was restored by antiestrogens. More importantly, ER-ß did not decrease the ER-{alpha}-mediated repression of the FR-{alpha} promoter. This observation underscores potential differences in the ability of ER-{alpha} versus ER-ß to associate with specific non-ERE elements.

Based on the foregoing results, it may be expected that FR-{alpha} expression in ER-{alpha}+ tumors will be repressed by ER in the presence of physiological levels of ER and estrogen and that pharmacological doses of antiestrogens will specifically and substantially increase the receptor levels independent of both ER-ß expression and the cellular coregulator complement. The predicted short-term treatment with ER ligands for the specific purpose of temporarily elevating FR-{alpha} levels should be an entirely acceptable therapeutic strategy because current clinical trials of SERMS as chemopreventatives (67) involve protracted periods of treatment. The present study establishes an experimental groundwork for optimal exploitation of FR-{alpha} modulation with ER ligands for imaging and treatment in major types of gynecological cancers and also points to novel mechanistic aspects of gene repression by ER.


   ACKNOWLEDGMENTS
 
We thank Drs. Carolyn Smith, Nancy Weigel, and Stephen Safe for several plasmid constructs. ICI 182,780 was a kind gift from AstraZeneca.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by National Cancer Institute Grants CA 80183 and CA 70873 (to M. R.) K. M. M. K. was supported by Institutional Pre-Doctoral NRSA Grant CA 79450. Back

2 The findings reported here are a part of a pending patent application. Back

3 To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Biology, Medical College of Ohio, 3035 Arlington Avenue, Toledo, OH 43614-5804. Phone: (419) 383-3862; Fax: (419) 383-6228; E-mail: mratnam{at}mco.edu Back

4 The abbreviations used are: FR, folate receptor; ER, estrogen receptor; E2, 17ß-estradiol; ERE, estrogen response element; EMSA, electrophoretic gel mobility shift assay; FBS, fetal bovine serum; nt, nucleotide(s); TAM, tamoxifen; RSV, Rous sarcoma virus; TK, thymidine kinase; AP-1, activator protein 1; SERM, selective ER modulator. Back

Received 9/12/02. Accepted 3/31/03.


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 DISCUSSION
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