From the
Aromatase P450, which is responsible for the metabolism of
C
Aromatase P450 (P450arom, the product of the CYP19 gene)
(1, 2, 3, 4, 5) catalyzes the aromatization of the A ring of C
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
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-
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 (
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.
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
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
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
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).
(
)
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) .
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.
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 (1
Hex 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.
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).
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
10
M. 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 10
M. 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
10
M.
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
10
M 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
10
M 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
10
M 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.
-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 1
Hex 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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.