©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ad4BP/SF-1 Regulates Cyclic AMP-induced Transcription from the Proximal Promoter (PII) of the Human Aromatase P450 (CYP19) Gene in the Ovary (*)

M. Dodson Michael (1)(§), Michael W. Kilgore (1)(¶), Ken-ichirou Morohashi (2), Evan R. Simpson (1)(**)

From the (1) Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Departments of Obstetrics/Gynecology and Biochemistry, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9051 and the (2) Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Higashi-ku, Fukuoka 812, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Aromatase P450, which is responsible for the metabolism of C steroids to estrogens, is expressed in the pre-ovulatory follicles and corpora lutea of ovulatory women by means of a promoter proximal to the start of translation (PII). To understand how this transcription is controlled by cAMP, we constructed chimeric constructs containing deletion mutations of the proximal promoter 5`-flanking DNA fused to the rabbit -globin reporter gene. Assay of reporter gene transcription in transfected bovine granulosa and luteal cells revealed that cAMP-stimulated transcription was lost upon deletion from -278 to -100 base pairs, indicating the presence of a functional cAMP-responsive element in this region; however, no classical cAMP-responsive element was found. Mutation of an AGGTCA motif located at -130 base pairs revealed that this element is crucial for cAMP-stimulated reporter gene transcription. When a single copy of this element was placed upstream of a heterologous promoter, it could act as a weak cAMP-response element. Supershift electrophoretic mobility shift assay and UV cross-linking established that Ad4BP/SF-1 binds to this hexameric element. Ad4BP/SF-1 mRNA and protein levels and DNA binding activity are increased in forskolin-treated luteal cells. We conclude that cAMP-stimulated transcription of human aromatase P450 in the ovary is due, at least in part, to increased levels and DNA binding activity of Ad4BP/SF-1.


INTRODUCTION

Aromatase P450 (P450arom, the product of the CYP19 gene) (1, 2, 3, 4, 5) catalyzes the aromatization of the A ring of C steroids with concomitant removal of the C angular methyl group to form C estrogens (6-10). Expression of P450arom in most vertebrate species is limited to the brain and the gonads; however, in humans and some non-human primates, P450arom mRNA has been detected in placenta, adipose, and a variety of fetal tissues (11) . Although the physiological significance of estrogen biosynthesis in the placenta and adipose of humans is unclear at this time, the role of ovarian estrogens has been well documented (12) . In the human ovary, granulosa cells of the pre-ovulatory follicle synthesize increasing amounts of estrogen, primarily estradiol-17, in response to follicle-stimulating hormone. Follicle-stimulating hormone binding to its cell surface receptor causes an elevation of intracellular cyclic AMP (cAMP) which in turn stimulates expression of P450arom. After the gonadotropin surge and ovulation, estrogen levels undergo a transient decline, but increase during the luteal phase due to a marked induction of P450arom mRNA (12).

Previous studies have demonstrated that aromatase activity in human ovarian granulosa-lutein cells is subject to multifactorial regulation and that changes in activity are correlated to changes in the levels of P450arom mRNA (13, 14, 15) . In order to define the molecular basis of the tissue-specific and hormonal regulation of human P450arom gene expression, we have previously isolated and characterized the gene encoding this enzyme. The human CYP19 gene spans at least 75 kilobases and is comprised of 9 coding exons (exons II-X) which encode the entire P450arom protein (16, 17, 18) . Additionally, several untranslated initial exons have been described, all of which have been shown to be spliced into a common 3` splice site located 38 bp() upstream of the start of translation (11). We believe that the use of alternative promoters located upstream of these initial exons may provide multiple modes of transcriptional regulation for P450arom expression. Specifically, the majority of placental transcripts contain a distal exon (exon I.1) located at least 35 kilobases upstream from exon II (16, 19, 20) , whereas in adipose tissue and adipose stromal cells in culture, three different initial exons (exons I.4, I.3, and II) have been detected (21) . In the human corpus luteum, the start site of P450arom transcription was mapped by S1 nuclease protection assay to lie 25 bp downstream of a putative TATA box (hereafter known as promoter II or PII), which is located approximately 140 bp upstream of the start of translation in exon II (19). These results have been confirmed by sequencing clones from a 5`-RACE (rapid Amplification of cDNA Ends) cDNA library prepared from corpus luteum RNA. In addition, exon-specific Northern analysis demonstrated that the majority of P450arom transcripts in human pre-ovulatory follicles contained sequence specific for promoter II, as did human granulosa-lutein cells in culture (22) .

Although it is known that human P450arom gene expression in granulosa-lutein cells is induced by cAMP, the molecular mechanism regulating this induction is not understood. Classically, transcriptional activation in response to increased intracellular cAMP has been found to be regulated by the cAMP-responsive element (CRE) which binds trans-acting factors of the CRE-binding protein (CREB)/activating transcription factor family. The function of the members of this family as components of the cAMP/protein kinase A signal transduction pathway has been extensively characterized (23) . However, no consensus CRE has been found in 945 bp of DNA upstream from the PII start of transcription. On the other hand, a single hexameric element (AGGTCA), which is identical to an estrogen or retinoic acid receptor half-site, is located approximately 125 bp upstream of the start site of transcription (Fig. 1). Recently, this element has been identified in the promoters of all steroidogenic P450 genes (24, 25) as well as in the promoters of the müllerian-inhibiting substance (26) , glycoprotein hormone- subunit (27) , and oxytocin (28) genes. Several orphan members of the steroid receptor superfamily of genes have been characterized to bind this element as monomers, including NGFI-B/nur77 and Ad4BP/SF-1 (29) . Two groups have previously provided evidence for a functional SF-1 site in the rat P450arom promoter (30, 31) . In this study, we have set out to define the molecular basis for cAMP induction of human P450arom gene expression in the ovary.


Figure 1: Sequence of the P450arom promoter II 5`-flanking DNA. Sequences with similarity to the consensus sequences for known transcriptional regulators are boxed, as is the putative TATA box. Nucleotides that diverge from the consensus sequences are indicated with an asterisk. Oligonucleotides used for the PCR generation of 5` deletion mutations are indicated with arrows. Non-annealing restriction sites used for subsequent subcloning steps are shown. The sequence of a double-stranded oligonucleotide used in EMSA and UV cross-linking experiments is shown as bold underline. Bases are numbered such that +1 represents the start of transcription using promoter II. This sequence was previously published (16, 19) and is assigned GenBank/EMBL data bank accession number J05105.




EXPERIMENTAL PROCEDURES

Restriction endonucleases were purchased from either Boehringer Mannheim or Promega and used according to the directions supplied by the manufacturer. Forskolin was purchased from Calbiochem. Radiolabeled nucleoside triphosphates (3000 Ci/mmol) were purchased from Amersham Corp. All other reagents were from Sigma unless otherwise stated.

Reporter Gene Constructs

The OVEC vector (32) , which contains the rabbit -globin gene as reporter, was generously provided by Dr. W. Shaffner. Reporter gene constructs were prepared by creating 5` deletion mutations of the human P450arom PII using PCR and single-stranded oligonucleotides synthesized with sequence complementary to the PII sequence of interest (Fig. 1) plus non-annealing ends for the restriction sites SalI (5` primer) and PstI (3` primer). Thus the -globin promoter is replaced with the PII promoter sequence. Inserts were sequenced using the Sequenase version 1.0 (U. S. Biochemical Corp.) to ensure fidelity of the amplified sequences and were subcloned into the SacI and PstI sites of the OVEC vector. Site-directed mutatgenesis of the hexameric element (AGGTCA AatTCA) was performed using the pALTER mutagenesis system (Promega). This double-stranded oligonucleotide was ligated into the SacI and SalI sites of the OVEC vector, thus allowing the endogenous -globin promoter to drive expression (1Hex construct).

Cell Culture and Transfection

Bovine ovaries were obtained at a local slaughterhouse. Follicles and corpora lutea were dissected from the ovaries, and granulosa and luteal cells were prepared as described previously (33, 34) . Cells were cultured in McCoy's 5A (Life Technologies, Inc.) supplemented as described previously (34) and allowed to grow to confluence (5-6 days) before transfection. JEG-3 choriocarcinoma cells were obtained from ATCC and cultured in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% fetal calf serum.

Transient transfection of bovine granulosa and luteal cells was carried out by electroporation as described (35) using 100 µg of the reporter construct and 2 µg of the OVEC reference plasmid. JEG-3 cells were transfected by the calcium phosphate method (36) using 20 µg of the reporter construct and 2 µg of the OVEC reference plasmid. After an overnight recovery in serum-containing medium, cells were changed to serum-free media either without or with 25 µM forskolin for 9 h.

RNA Isolation and S1 Nuclease Protection Assays

Total RNA was isolated from transfected cells by the procedure of Chomczynski and Sacchi (37) . -Globin transcripts were detected and quantified using a P-labeled, single-stranded rabbit -globin-specific oligonucleotide (93-mer) (35) . Experiments were performed on three separate occasions using different cell preparations, and representative results are shown.

Electrophoretic Mobility Shift Assays

Nuclear extracts were prepared from cultured cells by the method of Dignam et al. (38). Protein concentrations were determined by a modified Bradford assay (Bio-Rad). A double-stranded oligonucleotide corresponding to the hexameric motif flanked by 6 bp of the P450arom-PII sequence on each side (Fig. 1, bold underline) was labeled by Klenow labeling using [-P]dCTP and used as probe. For the competition assays, the unlabeled double-stranded oligonucleotide was added simultaneously with the labeled probe at an approximate 500-fold molar excess. When supershift assays were performed, 1 µl of antiserum was added after the 10 min room temperature incubation, and all samples for that assay were incubated on ice for an additional 30 min.

UV Cross-linking

UV cross-linking was performed as described by Chodosh et al.(39) with the following modifications. A uniformly P-labeled probe spanning -138/-97 bp of P450arom-PII was generated by PCR using 50 µCi each of [-P]dCTP and [-P]dGTP. The binding reaction containing 10 cpm of probe and 25 µg of nuclear protein. Non-radiolabeled competitor oligonucleotide (-138/-119 bp) was added at an approximately 100-fold molar excess. The reaction tube opening was covered with plastic wrap and was exposed to short wave (254 nm) ultraviolet radiation at a distance of 3.5 cm from the source for 1 h at 4 °C. Unprotected DNA probe was digested as described (39) .

Western Blotting

Nuclear protein (25 µg) was separated by 10% SDS-PAGE (reducing conditions) and transferred to nitrocellulose (Bio-Rad) with an electrophoretic tank transfer system. Proteins were detected using a polyclonal Ad4BP antiserum and the ECL detection system (Amersham).

Northern Blotting

Primary cultures of bovine luteal cells were treated in serum-free media either without or with 25 µM forskolin for 9 h. Total RNA was extracted as described above. Poly(A) RNA was then prepared using Poly(A)-Quik push columns (Stratagene). Poly(A) RNA (8 µg) was subjected to Northern analysis as described previously (36) . Ad4BP transcripts were detected by hybridization to a 907-nucleotide probe that was generated from the Ad4BP cDNA by asymmetric PCR using oligonucleotides Ad4#1 (5`-GATGAGCAGGTTGTTTCGGGGC-3`) and Ad4#2 (5`-GGTCCACCAGCTGGGCCACTG-3`). Hybridization of a P-end-labeled oligonucleotide specific for human glyceraldehyde-3-phosphate dehydrogenase (5`-TCTAGACGGCAGGTCAGGTCCACC-3`) to the membrane served as a control for equal loading. Hybridization signals were quantified by means of a PhosphorImager using ImageQuant software (Molecular Dynamics).


RESULTS

Expression of P450arom-PII/OVEC Vectors in Primary Bovine Granulosa and Luteal Cells

In order to determine the genomic elements involved in the regulation of the human CYP19 gene, a series of nested 5` deletion mutations (-946/-17, -693/-17, -278/-17, and -100/-17 bp) was created by PCR. The promoter fragments were ligated upstream of the rabbit -globin reporter gene in the OVEC vector. These reporter constructs have been designed such that the P450arom-PII TATA element is present and the -globin minimal promoter is deleted. Since we have been unable to find satisfactory conditions for the reproducible transfection of primary cultures of human granulosa-lutein cells, we have used primary cultures of bovine granulosa and luteal cells for these transfection experiments. Although the endogenous CYP19 gene is not expressed in the bovine corpus luteum, we have found that human P450arom-PII reporter gene constructs are expressed at equivalent levels in primary bovine luteal cells (Fig. 2) and in primary bovine granulosa cells (data not shown). Although this is a curious observation, we believe that the trans-acting factors for human P450arom-PII expression are present in the bovine ovarian cells; the endogenous bovine CYP19 gene may either lack the necessary cis-acting elements or may be regulated by changes in chromatin structure that prevent luteal phase expression.


Figure 2: PII-OVEC reporter gene construct expression in bovine luteal cells in primary culture. Bovine luteal cells were transfected with OVEC vectors containing -945, -694, -278, -278, or -100 bp of PII 5`-flanking sequence. The -278 construct contains an inactivating mutation of the core hexameric element (AGGTCA AatTCA). An SV40-OVEC construct (+) and the reporter plasmid with no insert (OVEC) were included as controls. Cells were treated for 9 h as indicated. C, control; F, forskolin, 2.5 10M. RNA was prepared, and each sample (15 µg) was analyzed by S1 nuclease protection assay (see ``Experimental Procedures''). Location of the undigested probe (93mer), correctly initiated transcripts (75mer), and the reference transcript (ref) are indicated.



Expression of the human P450arom-PII/OVEC constructs in both luteal (Fig. 2) and granulosa (data not shown) cells revealed low basal transcriptional activity from the -945/-17-bp construct. The level of -globin transcripts driven by the -945/-17-bp construct was markedly induced by forskolin treatment. Deletion of the promoter to -278 bp did not drastically affect the level of basal or forskolin-stimulated transcription, even though two CRE-like sequences were deleted. However, we observed that cAMP-responsive transcription was lost upon deletion from -278 to -100 bp, indicating that a cAMP-responsive element was present in this region. Data base analysis of this 178-bp region identified a hexameric AGGTCA sequence reported by Lala et al.(25) and by Morohashi et al.(24) to be present in the promoters of all steroidogenic P450 genes. When the hexameric element was mutated to AatTCA in the -278/-17 bp construct, transcriptional activation in response to forskolin was markedly diminished (Fig. 2). These results directly show that the hexameric element is necessary for cAMP-induced P450arom-PII transcription in the ovary.

P450arom-PII/OVEC Constructs Are Expressed in a Tissue-specific Manner

The P450arom-PII/OVEC constructs were transfected into JEG-3 human choriocarcinoma cells to determine if these constructs would be expressed with the same tissue specificity as the endogenous gene. JEG-3 cells express the CYP19 gene at high levels from the placental-specific promoter, promoter I.1. Whereas a -926/-10-bp P450arom-PI.1/OVEC construct is expressed at high levels in JEG-3 cells (data not shown), no detectable transcription could be seen from P450arom-PII/OVEC constructs (Fig. 3). Transfection of A549 human lung adenocarcinoma cells, which do not express CYP19, with P450arom-PII constructs also showed no detectable transcription (data not shown). These data are consistent with the observations of endogenous gene expression, in that the P450arom-PII constructs are expressed in a tissue-specific manner.


Figure 3: PII-OVEC reporter gene constructs are not expressed in JEG-3 cells. JEG-3 human choriocarcinoma cells were transfected with PII-OVEC reporter vectors as indicated. An SV40-OVEC construct (+) and the reporter plasmid with no insert (-) were included as controls. Cells were treated for 9 h. C, control; F, forskolin, 2.5 10M. Other conditions were as described in the legend to Fig. 2.



One Copy of the Hexameric Motif Supports cAMP-induced Transcription in Ovarian Cells

Since we had determined that the hexameric element was crucial for cAMP-stimulated transcription from P450arom-PII, we designed a reporter construct (1Hex) to test the functionality of this element as a cAMP-responsive element. This construct, which contained only one copy of the hexameric element positioned immediately 5` to the minimal -globin promoter, was transfected into primary cultures of luteal cells followed by treatment with or without forskolin. S1 nuclease analysis revealed that the hexameric element alone could act as a functional cAMP-responsive element, although the level of cAMP-induction (17-fold over control) was not as great as seen with the -278/-17-bp P450arom-PII/OVEC construct (45-fold over control) (Fig. 4). Nevertheless, this was a significant effect, since with the promoterless OVEC plasmid, no trace of transcription has ever been detected, either in the absence or presence of forskolin. This is the first demonstration to our knowledge that such an element can act alone as a functional CRE. These data are also indicative of an enhancer element that is located in the -278/-17-bp construct which acts to enhance the level of cAMP-induction from the hexameric element.


Figure 4: The hexameric element acts as a weak cAMP-responsive element. Primary bovine luteal cells were transfected with the -278/-17-bp P450arom-PII/OVEC reporter construct and 1Hex, which contains a single copy of the hexameric element upstream of the -globin minimal promoter. An SV40-OVEC construct (+) was included as control. Cells were treated for 9 h as indicated. C, control; F, forskolin, 2.5 10M. Other conditions were as described in the legend to Fig. 2.



Ad4BP/SF-1 Forms a Specific DNA-Protein Complex with the Hexameric Element in P450arom-PII

Nuclear extract prepared from primary cultures of bovine luteal cells and a radiolabeled hexameric element were used for electrophoretic mobility shift assay (EMSA) to determine if these cells contained a protein(s) capable of binding the hexameric element in vitro. One DNA-protein complex (complex A, Fig. 5) was observed that was competed with an approximately 500-fold molar excess of non-radiolabeled competitor oligonucleotide. Identical results were observed using bovine granulosa cell nuclear extract (data not shown).


Figure 5: EMSA shows luteal cell nuclear protein binding to the hexameric element. Bovine luteal cell nuclear protein (10 µg) was incubated with a P-labeled probe (-138/-119 bp) in the absence (+bLC NE) or presence (+cold) of 500-fold molar excess of non-radiolabeled homologous competitor. The crescendo triangle represents increasing volumes (0.5-2 µl) of polyclonal Ad4BP antiserum added to the binding reaction. DNA-protein complexes were separated from free probe by electrophoresis and were visualized by autoradiography. fp, free probe. The position of the specific complex is indicated as A.



Since the hexameric element is present as a half-site rather than as a direct or inverted repeat, we sought after proteins which could bind this element as a monomer such as NGFI-B/nur77 and Ad4BP/SF-1. We performed ultraviolet (UV) cross-linking analysis to determine the molecular weight of the protein bound to the hexameric element (Fig. 6). A probe spanning -138/-97 bp of P450arom-PII was incubated with 25 µg of bovine luteal cell nuclear extract in the absence or presence of UV irradiation. In the absence of UV exposure, no binding was observed. With UV exposure, several proteins showed binding; however, only the band at 50 kDa was competed when a 100-fold molar excess of a non-radiolabeled, double-stranded oligonucleotide corresponding to -138/-119 bp was added to the binding reaction. These data suggested that NGFI-B/nur77 is not present in this complex, since NGFI-B/nur77 migrates as a broad band greater than 70 kDa on SDS-PAGE analysis. This result lead us to believe that Ad4BP/SF-1, a 53-kDa protein, was present in the DNA-protein complex. To address this hypothesis, we conducted supershift EMSA using increasing amounts of a polyclonal rabbit antiserum directed against bovine Ad4BP (Fig. 5). Although a supershift was not observed, the formation of complex A was significantly diminished in a dose-dependent manner. In order to check the cross-reactivity of this action, the ability of the antiserum to displace proteins present in nuclear extracts which bound to a consensus GC box (Sp1 binding site) was examined. No displacement was observed under these conditions, indicative of the specificity of the antiserum toward Ad4BP/SF-1 (data not shown). Together, the results from UV cross-linking analysis and antibody displacement EMSA provide strong evidence that the protein bound to the hexameric element is indeed Ad4BP/SF-1.


Figure 6: UV cross-linking analysis of proteins bound to the hexameric element. Bovine luteal cell nuclear protein (25 µg) was incubated with 10 cpm of a uniformly labeled probe spanning -138/-97 bp in the absence or presence (+cold) of a 100-fold molar excess of non-radiolabeled competitor (-138/-119 bp). Reactions were exposed to UV light (+UV) or not (-UV). Unprotected DNA was digested, and proteins were separated by 11% SDS-PAGE. Products were visualized by autoradiography. fp, free probe. The position of molecular weight markers is shown to the left. The specifically competed complex is indicated with an arrow.



cAMP-induced Transcription by the Hexameric Element Is Due in Part to Increased Levels of Ad4BP/SF-1

To begin to understand the mechanism whereby the hexameric element and Ad4BP/SF-1 could directly confer cAMP-responsive gene transcription, we performed gel shift analysis using nuclear extracts isolated from bovine luteal cells cultured in serum-free medium in the absence or presence of 25 µM forskolin and a radiolabeled hexameric element as probe. A representative experiment is shown in Fig. 7A. Nuclear extracts isolated from untreated cells showed a basal level of binding activity that could be competed by a 500-fold molar excess of non-radiolabeled probe. This binding activity was increased approximately 4-fold when nuclear extracts from forskolin-treated bovine luteal cells was used. The forskolin-induced binding activity could be directly correlated to a forskolin induction of the level of Ad4BP/SF-1 protein present in these nuclear extracts as determined by Western blot analysis employing a polyclonal Ad4BP antiserum (Fig. 7B).


Figure 7: Ad4BP/SF-1 is increased in forskolin-treated primary bovine luteal cells. A, EMSA using nuclear protein isolated from cultured bovine luteal cells treated in the absence (C) or presence (F) of 2.5 10M forskolin. Nuclear proteins (10 µg) were incubated with a P-labeled hexameric element probe (-138/-119 bp) in the absence (-) or presence (+) of a 500-fold molar excess of non-radiolabeled homologous competitor. B, Western blot analysis of Ad4BP from bovine luteal cells nuclear protein (25 µg) from cells treated in the absence (C) or presence (F) of 2.5 10M forskolin. The position of molecular weight markers is shown on the left. C, Northern analysis of Ad4BP mRNA from bovine luteal cells treated in the absence (C) or presence (F) of 2.5 10M forskolin. Poly(A) RNA (8 µg) from each condition was separated on a 1.25% formaldehyde-agarose gel. Following transfer to Hybond-N+ (Amersham), transcripts were hybridized to a P-labeled, 907-nucleotide probe corresponding to a partial Ad4BP cDNA. The membrane was stripped and reprobed with P-labeled oligonucleotide specific for human glyceraldehyde-3-phosphate dehydrogenase as a control for loading (data not shown).



To determine if the increase in Ad4BP/SF-1 protein could be related to an increase in its transcription, we analyzed the steady-state levels of Ad4BP/SF-1 transcripts from bovine luteal cells by means of Northern analysis employing a 907-nucleotide Ad4BP probe generated by asymmetric PCR. Ad4BP/SF-1 mRNA was increased in forskolin-treated cells approximately 2.5-fold over control. These data suggest a mechanism of cAMP-induced transcription, whereby cAMP may directly affect the transcription of the Ad4BP/SF-1 gene. Increased levels of Ad4BP/SF-1 protein follow the rise in mRNA, which ultimately leads to a higher occupancy of hexameric element binding sites.


DISCUSSION

Aromatase expression in the human ovary is cell-specific and is regulated in a time- and hormone-dependent manner via the proximal promoter PII. Follicle-stimulating hormone and activators of the protein kinase A pathway stimulate aromatase activity at the level of increased gene transcription, yet there are no known consensus CRE sequences in the PII region. This study demonstrates that a single copy of a hexameric element (AGGTCA) is necessary, but not entirely sufficient, for conferring cAMP-dependent transcriptional regulation on PII of the human P450arom gene.

The PII-OVEC reporter construct containing 278 bp of PII 5`-flanking DNA is sufficient to confer cAMP-responsive transcription to the -globin reporter gene; however, none of the PII-OVEC reporter constructs are expressed in other non-ovarian steroidogenic (JEG-3) and non-steroidogenic (A549) cells. These data indicate that the PII-OVEC constructs mimic the tissue-specific pattern of endogenous CYP19 gene expression. Located in this minimal cAMP-responsive region of PII at -132 bp is an element (AGGTCA) which shows identity to the half-site for binding of members of the steroid receptor superfamily such as estrogen or retinoic acid receptors. When this element was mutated to AatTCA, cAMP-responsive reporter gene transcription was lost. This data led us to conclude that this element is necessary for cAMP-responsive transcription of from PII. In primary luteal cells transfected with a construct containing a single copy of the hexameric element immediately upstream of the minimal -globin promoter in the OVEC vector, we observed a weak stimulation of transcription by forskolin; thus, the hexameric element alone is sufficient to act as a weak CRE in luteal cells transfected with the OVEC vector. We have noted, however, that the level of cAMP induction by the 1Hex construct is not as great as that seen with the -278/-17-bp construct, indicating that other elements and factors are necessary to allow full responsiveness to cAMP. Recent reports identified a CRE-like sequence in the rat P450arom promoter at -159 bp (40) . A similar sequence, present in PII at -211 bp (Fig. 1), may act together with the hexameric element to provide a more robust cAMP-dependent stimulation of transcription.

Characterization of the proteins which bind the hexameric element revealed that both granulosa-lutein and luteal cell nuclear extracts contain protein(s) that specifically bind to the hexameric element. UV cross-linking analysis demonstrated that a nuclear protein of 50 kDa binds to the hexameric element. This size was indicative of the well characterized Ad4BP/SF-1 protein, which has been shown previously to bind DNA as a monomer. Supershift EMSA using a polyclonal antiserum against Ad4BP also demonstrated that Ad4BP/SF-1 could be the hexameric element-binding protein. Taken together with the presence of flanking sequences in the PII hexameric element that are characteristic of Ad4BP/SF-1 binding (CAAGGTCA) (24) , these data indicate that the hexameric element binding protein and Ad4BP/SF-1 are indistinguishable. Characterization of the cAMP regulation of Ad4BP/SF-1 demonstrated that the DNA binding activity of Ad4BP/SF-1 was elevated in forskolin-treated luteal cells. Although Ad4BP/SF-1 mRNA and nuclear protein levels were concomitantly increased, we have not ruled out the possibility that, in addition to these inductive effects, Ad4BP/SF-1 may be post-translationally modified in response to stimulation of the protein kinase A pathway. Recently, Yajima and colleagues (41) have reported that Ad4BP/SF-1 is expressed sporadically in the granulosa and surrounding stromal cells of the human pre-antral follicle preceding the detection of immunoreactive steroidogenic P450s. In dominant antral follicles, Ad4BP/SF-1 was present in both the granulosa and theca interna cells. Ad4BP/SF-1 immunoreactivity was also detected in both the luteinized granulosa and theca cells of the human corpus luteum. Taken together with the results presented in this study, these data are suggestive of other factors that are responsible for restricting aromatase expression to the granulosa cells of the pre-ovulatory follicle. A trans-acting factor(s) that acts to enhance the cAMP responsiveness of the Ad4BP/SF-1 bound to the hexameric element in PII may be expressed in a cell-specific fashion to allow aromatase expression exclusively in the granulosa cells of the pre-ovulatory follicle.


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grant HD13234. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by United States Public Health Service Training Grant 5-T32-HD07190.

Present address: Greenville Hospital System/Clemson University, Cooperative Research and Education Program, Clemson, SC 29364-1912.

**
To whom correspondence should be addressed: Cecil H. and Ida Green Center for Reproductive Biology Sciences, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9051. Tel.: 214-648-3260; Fax: 214-648-8683.

The abbreviations used are: bp, base pair(s); PII, promoter II; CRE, cAMP-responsive element; CREB, CRE-binding protein; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay.


ACKNOWLEDGEMENTS

We gratefully acknowledge the skilled editorial assistance of Melissa Meister. We thank Dr. Gary Means for his input on this project. We also thank Sarita Sharma, Gireesh Sharda, and Carolyn Fisher for providing excellent technical assistance.


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  14. Steinkampf, M. P., Mendelson, C. R., and Simpson, E. R.(1987) Mol. Endocrinol. 1, 465-471 [Abstract]
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  21. Mahendroo, M. S., Mendelson, C. R., and Simpson, E. R.(1993) J. Biol. Chem. 268, 19463-19470 [Abstract/Free Full Text]
  22. Jenkins, C., Michael, D., Mahendroo, M., and Simpson, E.(1993) Mol. Cell. Endocrinol. 97, R1-R6 [Medline] [Order article via Infotrieve]
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  27. Barnhart, K. M., and Mellon, P. L.(1994) Mol. Endocrinol. 8, 878-885 [Abstract]
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  34. Lauber, M. E., Kagawa, N., Waterman, M. R., and Simpson, E. R.(1993) Mol. Cell. Endocrinol. 93, 227-233 [CrossRef][Medline] [Order article via Infotrieve]
  35. Begeot, M., Shetty, U., Kilgore, M. W., Waterman, M. R., and Simpson, E.(1993) J. Biol. Chem. 268, 17317-17325 [Abstract/Free Full Text]
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  40. Fitzpatrick, S. L., and Richards, J. S.(1994) Mol. Endocrinol. 8, 1309-1319 [Abstract]
  41. Takayama, K., Suzuki, T., Sasano, H., Tamura, M., Morohashi, K., Fukaya, T., and Yajima, A.(1994) Abstracts of the Ninth International Congress on Hormonal Steroids, September 24-29, 1994, Dallas, Texas, p. 120

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