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Endocrinology Vol. 139, No. 4 1684-1691
Copyright © 1998 by The Endocrine Society

ARTICLES

Characterization of a Region Upstream of Exon I.1 of the Human CYP19 (Aromatase) Gene That Mediates Regulation by Retinoids in Human Choriocarcinoma Cells

Tiejun Sun, Ying Zhao, David J. Mangelsdorf and Evan R. Simpson

Cecil H. and Ida Green Center for Reproductive Biology Sciences (T.S., Y.Z., E.R.S.), Dallas, Texas 75235-9051; and the Departments of Obstetrics/Gynecology, Biochemistry (T.S., Y.Z., E.R.S.), and The Howard Hughes Medical Institute and Department of Pharmacology (D.J.M.), The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9038

Address all correspondence and requests for reprints to: Tiejun Sun, M.D., Ph.D., Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9038.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The biosynthesis of estrogens is catalyzed by aromatase P450 (P450arom), the product of the CYP19 gene. The tissue-specific expression of the CYP19 gene is regulated by means of tissue-specific promoters through the use of alternative splicing mechanisms. Thus, transcripts containing various 5'-untranslated termini are present in ovary, brain, adipose stromal cells, and placenta. Sequence corresponding to untranslated exon I.1 is present uniquely in 5'-termini of transcripts expressed in human placenta and choriocarcinoma cells, as a consequence of expression driven by a distal promoter, I.1. The goal of the present study was the identification of regulatory elements in this promoter region. Various deletion mutations of the upstream flanking region of exon I.1 were constructed using the PCR or restriction enzyme digestion. The genomic fragments were fused upstream of the luciferase reporter gene. These constructs were transfected into human choriocarcinoma (JEG3) cells. The longest construct employed, -924/+10 bp, expressed the highest luciferase reporter gene activity. The -64/+10 bp and -125/+10 bp constructs showed no reporter gene expression. Transfection of the -201/+10 bp construct resulted in reporter gene expression, but at a lower level than that of the -924/+10 bp construct, and this expression was induced by serum as well as by LG69 and TTNPB, ligands specific for RXR and RAR respectively, as well as by vitamin D. These results parallel the actions of the ligands on aromatase activity. Mutation or deletion of an imperfect palindromic sequence (AGGTCATGCCCC) located at -183 to -172 bp upstream of the transcriptional start site of exon I.1 resulted in loss of basal- and retinoid-induced reporter gene expression. Gel retardation analysis using nuclear extracts of JEG3 cells treated with retinoids and the imperfect palindromic sequence as probe, showed that proteins present in the nuclear extracts bound to this sequence in a specific fashion. The binding activities were elevated by incubation of the cells with LG69 and TTNPB, ligands specific for RXR and RAR respectively. Binding of nuclear proteins to the palindromic sequence was displaced either by anti-RXR{alpha} serum or by anti-VDR serum, suggesting the formation of a heterodimer of RXR{alpha} and VDR. These results suggest that the imperfect palindromic sequence upstream of exon I.1 plays an important but novel role in the regulated expression of the CYP19 gene in choriocarcinoma cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE BIOSYNTHESIS of estrogens from C19 steroids is catalyzed by aromatase P450 (1, 2, 3, 4) [P450arom; the product of the CYP19 gene (5)], which catalyzes the aromatization of the A ring of C19 steroids to form the phenolic A ring characteristic of estrogens, with concomitant loss of the C19 angular methyl group as formic acid. P450arom is located in the endoplasmic reticulum of estrogen-producing cells and uses the flavoprotein, NADPH-P450 reductase, a ubiquitous enzyme in most cell types, to provide reducing equivalents from NADPH.

In the human, in contrast to most vertebrate species in which estrogen biosynthesis is limited to the gonads and brain, aromatase is expressed in a number of tissues throughout the body including adipose stromal cells (6), syncytiotrophoblasts of the placenta (7), and various sites in the brain (8, 9), in addition to the granulosa and luteal cells of ovary (10, 11) and Leydig cells of the testis (12). In addition, a number of fetal tissues express aromatase, especially liver, but also intestine, skin, and brain (13, 14, 15). In the human, tissue-specific expression is regulated, in part, by the use of tissue-specific promoters as a consequence of alternative-splicing mechanisms (16, 17, 18). Thus expression in gonads uses the proximal promoter II (P II) (16, 19, 20), whereas expression in placenta employs a distal promoter I. 1 (P I.1) (16, 18). On the other hand, expression in adipose tissue depends on both promoter II and another distal promoter, P I.4 (21, 22).

The placenta is the primary site of estrogen synthesis in pregnant women, and 16{alpha}-hydroxydehydroisoandrosterone sulfate synthesized by the combined actions of the fetal adrenal and liver is the major precursor for placental estrogen biosynthesis. Human aromatase messenger RNA expressed in placenta has a unique 5'-untranslated exon, exon I.1, due to the use of the distal promoter I.1. In addition to placenta, messenger RNA containing exon I.1-specific sequence is also present in human choriocarcinoma cells of the JEG3 line. This cell line expresses aromatase activity endogenously, and the expression can be induced by a number of stimulants, suggesting that the JEG3 cell line is a suitable model to study the transcriptional regulation of aromatase expression. Several investigators have studied aromatase gene expression in human choriocarcinoma cells. Thus, Toda et al. (23) characterized a distal element between -2141 and -2115 bp upstream of exon I.1, which binds NF-IL6, a member of the CCAAT/enhancer-binding protein family. Yamada et al. (24) identified two elements within -300 bp upstream of exon I.1, which recognize the same transacting factor that binds to the trophoblast-specific element previously located in the enhancer region of the human glycoprotein hormone {alpha}-subunit gene.

The nuclear hormone receptors constitute a large superfamily of structurally related transcription factors that regulate the expression of genes responsive to steroids, thyroid hormone, 1,25 D3, retinoids, peroxisome proliferators, and fatty acids (25). These ligand-modulated receptors regulate gene expression by binding to specific hormone response elements present in the promoter regions of hormone-responsive genes. The subfamily of nuclear hormone receptors that includes, among others, retinoid receptors [retinoid acid receptor (RAR) and retinoid X receptor (RXR)], thyroid hormone receptors (TRs), 1,25 D3 receptor (VDR), and peroxisome proliferator activated receptors (PPARs), binds to response elements that consist of one or more conserved DNA elements related to the core half-site hexameric sequence, AGGTCA. High affinity binding of many receptors within this subfamily to cognate response elements is manifest through heterodimerization with common auxiliary cofactors, one of which has been identified as the 9-cis-retinoic acid receptor RXR. The RXRs ({alpha}, ß, and {gamma}), along with the RARs (, {alpha}, ß, and {gamma}) mediate the cellular response to retinoids and their metabolites, and as such play diverse and essential roles in development and differentiation. RXR has been shown to form heterodimers with RAR, VDR, TR, and PPAR to generate high affinity DNA binding complexes specific for different response elements (26).

In the present study, we have sought to characterize regulatory elements associated with promoter I.1 of the CYP19 gene and to determine the role of these elements in the expression of the CYP19 gene in human placenta. To this end, the 5'-flanking region upstream of exon I.1 was characterized for its ability to regulate transcriptional activity, by means of transient transfection of chimeric CYP19/luciferase gene constructs into JEG3 cells. We found that the sequence -201 bp upstream of the transcriptional start site of exon I.1 is sufficient for basal expression and induction by retinoic acid and 1,25 D3. Moreover, endogenous aromatase activity as well as reporter gene activity of JEG3 cells were increased by metabolites of retinoic acid when added to the culture medium. This region of promoter I.1 contains a core sequence, 5'-AGGTCATGCCCC-3', which is essential for basal and stimulated activity, and which is an imperfect palindrome with two mismatches from the perfect sequence AGGTCATGACCT (TREpal). This signal transduction pathway appears to play an important role in the expression of the CYP19 gene in human choriocarcinoma cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human JEG-3 choriocarcinoma cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The Firefly Luciferase report system kit was purchased from Analytical Luminescence Laboratory. The internal standard report system kit was purchased from Tropix, Inc. (Bedford, MA). The protein certificate analysis kit (TNT T7 Coupled Reticulocyte Lysate System) was purchased from Promega Corp. (Pittsburgh, PA). Polyclonal antiserum against human RXR{alpha} was a kind gift (from Dr. Ronald M. Evans, The Salk Institute). Polyclonal antiserum against the human 1,25 D3 receptor was obtained from Dr. Joseph Zerwekh of this institution. Polyclonal antiserum against Ad4BP/SF1 was a kind gift from Dr. K. Morohashi (National Institute for Basic Biology, Okazaki, Japan). Polyclonal antiserum against human RAR{alpha}1, monoclonal antiserum against human RXRß, and monoclonal antiserum against human thyroid hormone receptor (TR{alpha}1) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Specific ligands [LG69 for RXR, a generous gift from Ligand Pharmaceuticals, Inc. (San Diego, CA); and TTNPB for RAR (27)] were obtained as described. Thyroid hormone (T3) and 1,25 D3 were obtained from Sigma Chemical Co. (St. Louis, MO).

Cell culture and determination of aromatase activity
Human JEG-3 choriocarcinoma cells were maintained in RPMI media, and aromatase activity was determined by the incorporation of tritium into [3H] water from [1ß-3H] androstenedione as described previously (6).

Deletion mutagenesis
Figure 2Go shows the promoter I.1 deletion constructs that were prepared either by PCR or restriction enzyme digestion. The pGL2-control luciferase reporter vector (pGL2-C), which contains the SV40 promoter and enhancer, served as a positive control, whereas the pGL2-basic luciferase reporter vector (pGL2-B), which is promoterless and enhancerless, served as a negative control. The constructs (-924/+10 bp, -414/+10 bp, -246/+10 bp, -201/+10 bp, -125/+10 bp, -64/+10 bp) were subcloned into the pGL2-basic luciferase reporter vector employing SalI and PstI sites (inserted between the XhoI and HindIII sites of the pGL2-basic luciferase reporter vector). All constructs were sequenced to confirm their authenticity. The pCMV ß-galactosidase (Clontech Laboratories Inc., Palo Alto, CA) (lac Z) vector was cotransfected in all experiments as an internal standard for transfection efficiency.



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  		Figure 2. Schematic diagram of reporter gene constructs used in transient transfection assays. These constructs include the transcriptional start site of promoter I.1 of the CYP19 gene. In these luciferase constructs, deletion mutations of the genomic region flanking the 5'-end of exon I.1 have been fused to the firefly luciferase reporter gene, as described in Materials and Methods.

 
Site-specific mutagenesis
In this procedure, the -201/+10 bp fragment containing the imperfect palindromic sequence (AGGTCATGCCCC) was digested by SalI and PstI and gel-purified to generate the template for mutagenesis. Site-specific mutagenesis was performed as follows (see Fig. 4Go). 1) 5'-AatTATGCCCC-3' (mutation 1); 2) 5' AGGTCAT{alpha}CgCg-3' (mutation 2); 3) 5' -AatTCATaCgCg-3' (mutation 3). Additionally, by mean of the PCR technique, a series of deletion mutations of the -201/+10 bp sequence was constructed as shown in Fig. 5Go: 1) 5'-T CATTCCAGAGGAGGTCATGCCCCATACCCTGGAGGAGGAA-3' (deletion 1); 2) 5'-AGGAGGTCATGCCCCATACCCTGGAGGAAGGAA-3' (deletion 2); 3) 5'-TGCCCCATACCCTGGAGGAAGGAA-3' (deletion 3); 4) 5'-ATACC-CTGGAGGAAGGAA-3' (deletion 4). These oligonucleotide fragments were subcloned into the pCR II vector, sequenced, and then fused upstream of the pGL2-basic luciferase reporter vector and resequenced. The resulting constructs (mutated and deleted) were transfected into JEG3 cells and luciferase reporter gene activity determined. The fidelity of the mutagenesis was verified by dideoxy sequencing using the Sequenase DNA sequencing kit.



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  		Figure 4. Role of the imperfect palindromic sequence in the transient expression of -201/+10 bp/Luc fusion gene constructs. Fusion gene constructs containing the wild-type -201/+10 bp sequence, as well sequences in which the imperfect palindrome was mutated (A), were linked to the pGL2-B luciferase reporter vector, and transfected into JEG3 cells in culture. Cells were treated for 24 h with LG69 (1 µM) or TTNPB (1 µM). Cells were then harvested, and lysates were prepared for assay of luciferase activity (B). Other details were described in Materials and Methods.

 


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  		Figure 5. Role of the imperfect palindromic sequence in the transient expression of the -201/+10 bp/Luc fusion gene construct. Fusion gene constructs containing the wild type -201/+10 bp sequence, as well as several 5' deletions of this sequence (A), were linked to the luciferase vector and transfected into JEG3 cells in culture. Cells were treated for 24 h with LG69 (1 µM), TTNPB (1 µM), or T3 (100 µM), or LG69+T3. Cells were then harvested, and lysates were prepared for luciferase assay (B). Other details are as described in Materials and Methods.

 
Transient transfection and determination of luciferase reporter gene activity
Human JEG3 choriocarcinoma cells were maintained in MEM containing 10% FCS. Transfection by means of calcium phosphate copre-cipitation was carried out with minor modification as described (28). When JEG3 cells in culture were approaching confluence, the cells were transfected with 2 µg of cesium chloride-prepared plasmid DNA containing the requisite construct, together with 0.5 µg of pCMVß (lacZ) plasmid as an internal standard to assess the transfection efficiency, and the transfected cells were incubated at 37 C for 4 h. Fifteen percent glycerol shock was carried out for 1 min. The cells were allowed to recover overnight in 10% FCS-containing media for 24 h and then incubated with or without stimulatory factors in the absence of FCS. The transfected cells were maintained for 24 h and then harvested for the purpose of luciferase and ß-galactosidase determination. The luciferase assay was performed with minor modifications as described. Medium was removed and the cells were washed with 1 x PBS, harvested, and lysed in lysis buffer. Luciferase activity was expressed as relative light units, and the relative luciferase activity was calculated by dividing the total luciferase activity per dish by the total ß-galactosidase activity per dish. Each set of experiments was repeated at least three times and results shown are the average obtained (SEM for the three transfections).

Preparation of nuclear extracts and electrophoretic mobility shift assay
Nuclear extracts from JEG3 cells in culture were prepared according to the method of Dignam et al. (29) with slight modification. After the cells were swollen in 2 ml of hypotonic buffer, they were homogenized with 12 strokes of a Kontes all glass Dounce homogenizer (B type pestle). The homogenate was centrifuged at 2000 rpm for 2 min. The pellet was then suspended in 500 µl of chilled buffer containing 20 mM HEPES, pH 7.4, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 20% glycerol. After centrifuging at 60000 rpm for 5 min, the supernatant was taken and stored at -70 C until use. Double-stranded oligonucleotides were Klenow-labeled using [{alpha}-32P] dCTP and then were incubated (5000 cpm) with nuclear extracts (6 µg) on ice for 10 min. For the competition assay, the unlabeled oligonucleotide used as competitor was added simultaneously with the labeled fragment at 100-fold excess. The resulting DNA-protein complexes were analyzed by electrophoresis using a 8% polyacrylamide gel with 0.5 x Tris borate-EDTA as running buffer. The gel was vacuum-dried and exposed to XR film at -70 C for 24 h. The sequence employed as probe corresponded to the native imperfect palindromic CYP19 sequence: 5'-AGGTCATGCCCCATACCCTGGA-3' (i.e. wild-type -183/-162 bp).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LG69, TTNPB and 1,25-dihydroxycholecalciferol stimulate aromatase activity in cultured JEG 3 cells
Previous studies in our laboratory showed that increasing concentration of serum can induce aromatase activity in JEG 3 cells. To determine which factors in serum induce aromatase expression in JEG3 cells and localize which area residing in promoter I.1 is responsible for the induction, several compounds were tested in the first instance for their ability to stimulate endogenous aromatase activity (Fig. 1Go). Synthetic ligands that bind to RXR and RAR specifically, namely LG69 and TTNPB, respectively (27), were employed, because all-trans and 9-cis retinoic acids can be metabolized. LG69 stimulated aromatase activity over 3-fold, TTNPB stimulated aromatase activity over 2.5-fold, and 1,25 D3 stimulated aromatase activity about 2.8-fold following exposure for 24 h of JEG 3 cells. The actions of LG69 and 1,25 D3, LG69 and TTNPB, and TTNPB and 1,25 D3 appeared to be additive. Half-maximum stimulation by LG69 and TTNPB was achieved at concentrations of 5 x 10-8 M (data not shown). On the other hand, dexamethasone, T3, Bt2 cAMP and PPAR{gamma}-specific ligand BRL49653 (30) had little or no effect on aromatase gene expression.



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  		Figure 1. LG69, TTNPB, and 1,25 D3 increase aromatase activity in JEG 3 cells. Confluent JEG 3 cells were maintained in the absence of serum (column 1) or in the presence of serum (10% FCS, column 2). The following ligands were added in the absence of serum: LG69 (1 µM, column 3), LG69 plus 1,25 D3 (column 4), TTNPB (1 µM, column 5), LG69 plus TTNPB (column 6), 1,25 D3 (100 nM, column 7), TTNPB plus 1,25 D3 (column 8), BRL 49653 (100 nM, column 9), dexamethasone (250 nM, column 10), T3 (100 nM, column 11), and Bt2cAMP (100 nM, column 12). The method of determining aromatase activity was described in Materials and Methods. Data of aromatase activities were analyzed by Wilcoxon’s matched-paired signed rank test. When P < 0.05, it was considered significant.

 
Deletion analysis of the 5'-flanking region
To determine the mechanism of this stimulation, as well as to localize sequences of the CYP19 gene responsible for basal expression and induction by retinoids, experiments were carried out to examine the effects of deletion of the flanking region upstream of promoter I.1. A series of fragments of different sizes were generated using PCR and restriction enzyme digestion. The various fragments were fused upstream of the pGL2-basic luciferase reporter vector as shown in Fig. 2Go. These constructs were transfected into JEG3 cells in the presence and absence of 10% FCS, and the results are shown in Fig. 3Go. The construct containing -924/+10 bp expressed the highest luciferase reporter gene activity, which was induced about 2.8-fold by 10% FCS. The construct containing -201/+10 bp, which contains the imperfect palindromic sequence AGGTCATGCCCC, was also capable of basal expression of the luciferase reporter gene. This basal expression was about 48% of that of the -924/+10 bp construct. However, the fold-induction by FCS was about the same as that of the longest construct. On the other hand, the -125/+10 bp and -64/+10 bp constructs exhibited no luciferase reporter gene activity. These results suggest that the response to FCS maps between -201 and -125 in the CYP 19 promoter.



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  		Figure 3. Transient expression of luciferase constructs in JEG3 cells. JEG3 cells were transfected with the various fusion gene constructs as indicated, as well as pGL2-C as a positive control and the negative control pGL2-B. Cells were treated for 24 h in the presence or absence of FCS (10%). The cells were harvested and cell lysates were prepared. Firefly luciferase activities were determined as described. Luciferase values were corrected for transfection efficiency, using the pCMV ß-galactosidase-containing vector as standard. Results are represented as relative luciferase activity as described in Materials and Methods.

 
The imperfect palindromic sequence upstream of promoter I.1 is required for retinoid induction
To determine whether the -201/+10 bp portion of the promoter including the AGGTCATGCCCC palindromic sequence plays a role in retinoid regulation of promoter I.1-driven transcription, -201/+10 bp-pGL2-basic-luciferase reporter genes were constructed in which this sequence was mutated as shown in Fig. 4aGo. Additionally, by means of PCR, a series of deletion mutations of the -201/+10 bp sequence was constructed as shown in Fig. 5aGo. The mutated and deleted luciferase reporter constructs were transfected into JEG3 cells that were incubated in the presence or absence of LG69 and TTNPB. As can be seen in Figs. 4Go and 5Go, mutation or deletion of each half-site of the imperfect palindromic sequence dramatically decreased the luciferase reporter gene activity. Both mutation or deletion of the imperfect palindromic sequence resulted in not only loss of basal luciferase reporter gene expression but also expression stimulated by the RXR or RAR ligands. The RXR- and RAR-specific ligands, LG69 and TTNPB, stimulated expression of the -201/+10 bp-containing construct. However, T3 inhibited expression stimulated by LG69 (Fig. 5Go).

1,25 D3 and LG69 can additively increase aromatase gene expression in JEG 3 cells
Because the experiment of Fig. 1Go showed that 1,25 D3 and LG69 can additively increase aromatase activity, -201/+10-luc constructs were transfected into JEG 3 cells to examine whether these compounds can induce reporter gene expression additively. As shown in Fig. 6Go, luciferase reporter gene activity was increased by addition of LG69, TTNPB and 1,25 D3. When added together, by LG69 plus 1,25 D3, LG69 plus TTNPB and TTNPB plus 1,25 D3, acted in a more-or-less additive fashion, paralleling the results primarily shown for aromatase activity. (Fig. 1Go).



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  		Figure 6. LG69, TTNPB and 1,25 D3 increase luciferase reporter gene activity in JEG3 cells. Fusion gene constructs containing the wild type -201/luc were transfected into JEG 3 cells. Cells were treated without serum (bar 10) and with serum (10% FCS, bar 9). The following ligands were added in the absense of serum: LG69 (1 µM, bar 3), TTNPB (1 µM, bar 4), 1,25 D3 (100 nM, bar 5), LG69 plus 1,25 D3 (bar 6), LG69 plus TTNPB (bar 7), and TTNPB plus 1,25 D3 (bar 8). Bars 1and 2 represent pGL-2 positive control and pGL-2 basic negative control respectively. Other details are described in Materials and Methods.

 
Nuclear proteins bound to the imperfect palindromic sequence are a heterodimer formed by RXR{alpha} and VDR
Gel mobility and antibody supershift experiments were employed to further characterize the nuclear binding proteins, employing nuclear extracts from JEG3 cells incubated for 24 h with 10% FCS or LG69 (1 x 10-7 M). As shown in Fig. 7Go, when anti-RXR{alpha} and anti-VDR sera were employed separately, each of these antisera could displace the band, which was indicative of binding of nuclear protein to the probe, 5'-AGGTCATGCCCCATACCTGGA-3'. On the other hand, antisera against RAR{alpha}, RXRß, TR{alpha}1 and Ad4BP/SF1 were ineffective, suggesting that the nuclear proteins that bound to the probe were RXR{alpha} and VDR. To further test whether the nuclear proteins bound to this imperfect palindromic sequence are a heterodimer of RXR{alpha} and VDR, gel mobility-shift assays were performed using in vitro synthesized receptor proteins and 32P-labeled -183/-162 sequence as a probe (Fig. 8Go). VDR, RXR{alpha} and RAR{alpha} did not bind to the -183/-162 sequence individually and the mixture of RXR{alpha} and RAR{alpha} or RAR{alpha} and VDR did not bind either, but only when RXR{alpha} and VDR were mixed together was a strong protein-DNA complex formed (lane 7), and this binding could be displaced by either anti-RXR{alpha} or anti-VDR sera, but not by anti-RXRß serum, further suggesting that the proteins binding to the imperfect palindromic sequence are a heterodimer formed by RXR{alpha} and VDR.



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  		Figure 7. Gel mobility analysis of proteins binding to the imperfect palindrome sequence. Nuclear extracts prepared from JEG 3 cells in the absence of serum (lane 2) or in the presence of serum (10% FCS, lane 3), and LG69 (1 µM, lane 4) were incubated with radiolabeled -183/-162 bp fragment and the reaction mixtures were subjected to PAGE in an 8% gel. For competition, a 100-fold molar excess of nonradiolabeled -183/-162 (lane 5) and mutated -183/-162 (5'-AatTCATaCgCgATACCCTGG-3' lane 6) oligonucleotides was added to the incubation mixture. To determine which nuclear receptor is the component of the protein binding to the probe, anti-RXR{alpha} antibody (lane 7), anti VDR serum (lane 8), anti-RAR{alpha}1 antibody (lane 9), anti-RXRß antibody (lane 10), and anti Ad4/SF-1 serum (lane 11) were incubated with radiolabeled DNA probe in the presence of nuclear extracts. Lane 1: free probe without nuclear extracts. Other details are described in Materials and Methods. The arrow indicates the position of the radiolabeled band.

 


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  		Figure 8. RXR{alpha} and VDR form a heterodimer binding to the imperfect palindromic sequence. Proteins were generated by in vitro transcription-translation employing various templates as indicated. Nuclear extracts from JEG 3 cells treated with LG69 were incubated with radiolabeled -183/-162 fragment as probe (lane 2). Lane 3, 100-fold molar excess of nonradiolabeled probe was added to the reaction mixture. In vitro transcription-translation generated VDR (lane 4), RXR{alpha} (lane 5), RAR{alpha} (lane 6), VDR plus RXR{alpha} (lane 7), RXR{alpha} plus RAR{alpha} (lane 8), RAR{alpha} plus VDR (lane 9) were incubated with the radiolabeled probe as indicated above and subjected to electrophoresis. To determine whether RXR{alpha} and VDR heterodimer bound to the imperfect palindrome sequence, anti-RXR{alpha} serum (lane 10), anti-VDR serum (lane 11) and anti-RXRß serum (lane 12) were incubated with radiolabeled probe in the presence of the in vitro transcription-translation-generated proteins. Lane 1: free probe.

 
The sequence bound by a heterodimer of RXR{alpha} and VDR is a palindromic element
It has been shown that the heterodimer formed between RXR and other nuclear receptors can bind and activate through DNA response elements that are repeats of the core sequence AGGTCA spaced by 0–5 nucleotides. To further confirm that the RXR{alpha}/VDR heterodimer binds the palindromic sequence, gel mobility-shift analysis was performed using nuclear extracts from JEG cells treated with LG69 and 32P-labeled -183/-162 sequence as a probe. Degenerate hexad sequences of AGGTCA spaced by 0–5 nucleotides and the TRE palidromic (TREpal) sequence were used as a competitor. As revealed in Fig. 9Go, only the DR0 or TRE palindromic sequence could displace the DNA-protein complex band, suggesting that the imperfect palindromic sequence upstream exon I.1 is a functional element and the sequence bound by RXR{alpha}/VDR heterodimer is an imperfect palindromic sequence.



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  		Figure 9. RXR{alpha}/VDR heterodimer binds to a DR0 response element or palindromic sequence. Competition mobility gel shift assay was performed as indicated above in Fig. 7Go except that competitor oligonucleotides were perfect tandem repeats (DR0-DR5) of the sequence AGGTCAnAGGTCA, where n = 0–5 nucleotides. In addition, the palindromic TRE element was also used as a competitor. Nuclear extract from JEG 3 cells treated with LG69 was incubated with radiolabeled probe (-183/-162 fragment, lane 2), 100-fold molar excess of nonradiolabeled probe was added to the reaction mixture as a competitor (lane 3). In lane 4, a mutated nonradiolabeled DNA probe was incubated with nuclear extracts from JEG 3 cells treated with LG69. Nonradiolabeled, direct repeat oligonucleotides; DR0 (lane 5), DR1 (lane 6), DR2 (lane 7), DR3 (lane 8), DR4 (lane 9), DR5 (lane 10) and TRE pal (lane 11) were added to reaction as indicated above in 100-fold molar excess.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we report that aromatase activity of JEG 3 choriocarcinoma cells is stimulated by ligands of the RXR and RAR retinoid receptors, as well as by vitamin D, and have characterized an element associated with promoter I.1 of the CYP19 gene that is responsible for the induction of aromatase expression by these retinoids. By creating a series of deletion chimeric constructs, we have found that a construct containing -924/+10 bp of flanking sequence exhibited highest luciferase reporter gene expression when transfected into choriocarcinoma cells. The construct containing -201/+10 bp was sufficient for both basal expression and expression induced either by serum or retinoid receptor ligands, whereas the -125/+10 bp and -64/+10 bp constructs were inactive. So our present study focused on the region between -201 and -125 bp upstream of exon I.1. A sequence (AGGTCATGCCCC) resides at -183/-172 bp, which differs by 2 bp from a consensus DR0 palindrome (AGGTCATGACCT) (TRE pal). Expression of fusion gene constructs (-201/+10 bp) containing this sequence is induced markedly in the presence of LG69 and TTNPB, which are specific ligands of the RXR and RAR receptors, respectively, 1,25 D3 also increased expression of these constructs in human JEG3 cells. Both mutation or deletion of this palindromic sequence resulted in loss of basal expression and responsiveness to retinoid ligands. The action of these ligands to stimulate reporter gene expression mirrors that of the ligands to stimulate endogenous aromatase activity, whereas T3 inhibited expression induced by these ligands.

Gel mobility shift analyses employing the radiolabeled probe (5'-AGGTCATGCCCCATACCCTGGA-3') demonstrated that nuclear proteins prepared from human choriocarcinoma cells treated with LG69 bound to this palindromic sequence to give a single band, and the binding activity was higher than that of nuclear proteins from cells without treatment. The binding activities were displaced by anti-RXR{alpha} serum and anti-VDR serum, but not by anti-RAR{alpha}, anti-RXRß, or anti-TR{alpha}1 sera, suggesting that at least one of the protein complexes capable of binding to the probe contained RXR{alpha} and VDR. In addition, a mixture of in vitro transcribed and translated RXR{alpha} and VDR was able to bind to the palindromic sequence. Taken together, these results suggest the existence in JEG 3 cells of a unique interaction between VDR and RXR{alpha} that permits them to heterodimerize on a TRE-like palindromic response element and be responsive to both vitamin D and RXR-selective ligands. RXR/VDR heterodimers have previously been shown to bind optimally to DR3 response elements, and in this context respond only to VDR, but not RXR, ligands. Thus, the finding that the RXR/VDR heterodimer can bind to the CYP19 palindromic sequence and respond to both receptors ligands is surprising and suggests a novel mechanism of interaction exists for this receptor combination on this response element. Furthermore, RXR ligand activation through this element is inhibited by thyroid hormone and stimulated by RAR-selective ligands, suggesting that the element may function as both a negative TRE and a positive RARE. These latter results imply that receptor subtypes other than TR{alpha}1 and RAR{alpha} must be involved because in our experiments anti-RAR{alpha} and anti-TR{alpha}1 sera failed to detect the binding of these specific receptors to the CYP19 palindromic sequence. Ongoing analysis of the other protein components interacting at this response element should lead to an understanding of the observed differential hormonal response of the CYP19 promoter. This analysis may reveal tissue and receptor specific coactivators and/or corepressors, which are known to modify receptor function (31).

Although further study is needed to determine why and how so many endocrine signals converge to regulate aromatase expression in JEG3 cells, the involvement of at least one of these signals (i.e. retinoids) may be predicted from recent studies. Recently, it was reported that levels of RXR and RAR receptor expression increase during the process of cytotrophoblast differentiation into syncytiotrophoblasts in the placenta (32). This change in retinoid receptor levels is coincident with the increase in aromatase expression, suggesting that retinoids may indeed play an important role in the developmental regulation of aromatase gene expression in the placenta, as well as fetal development and differentiation in general.


   Acknowledgments
 
This work was supported, in part, by United States Public Health Service Grant DK-31206. The authors gratefully acknowledge the gift of anti-VDR serum from Dr. Joseph Zerwekh, synthetic retinoid ligands from Dr. Richard Heyman (Ligand Pharmaceuticals, Inc.) and anti-RXR antibody from Drs Jackie Dyckard and Ronald M. Evans (Salk Institute).

Received September 30, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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