Characterization of the Regulatory Regions of the Human Aromatase (P450arom) Gene Involved in Placenta-Specific Expression
Amrita Kamat,
Joseph L. Alcorn,
Cheryl Kunczt and
Carole R. Mendelson
Departments of Biochemistry (A.K., J.L.A., C.K., C.R.M.)
and Obstetrics-Gynecology (C.R.M.), The Cecil H. and Ida Green
Center for Reproductive Biology Sciences University of Texas
Southwestern Medical Center at Dallas Dallas, Texas 75235
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ABSTRACT
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Aromatase P450 (P450arom), a product of the CYP19
gene, catalyzes the conversion of C19-steroids
to estrogens. Human P450arom is expressed in placental
syncytiotrophoblast, ovarian granulosa cells, and adipose stromal cells
by use of tissue-specific promoters that are located 5' of unique
untranslated first exons. Mononuclear cytotrophoblasts isolated from
midterm human placenta spontaneously fuse in culture to form
multinucleated syncytiotrophoblast. These morphological changes are
associated with a marked induction of P450arom gene expression. The
majority of P450arom transcripts in placental syncytiotrophoblast
contain sequences encoded by exon I.1, which lies more than 35 kb
upstream of the translation initiation site in exon II. To functionally
map genomic sequences required for placenta-specific P450arom
expression, fusion genes containing various amounts of DNA flanking the
5'-end of placenta-specific exon I.1 linked to the human GH (hGH) gene,
as reporter, were introduced into primary cultures of human trophoblast
cells and other cell types. Since the trophoblast cells manifest high
levels of aromatase P450 expression, we believe that this provides a
physiologically relevant system for characterizing the regulatory
regions of this gene. Expression of the fusion genes increased as a
function of time in culture in concert with syncytiotrophoblast
differentiation and induction of aromatase activity and of P450arom
gene expression. P450arom-hGH fusion genes containing 923 and 501 bp of
exon I.1 5'-flanking DNA were expressed at comparable levels; these
levels were more than 3-fold greater than those of fusion genes
containing 2400 bp of exon I.1 5'-flanking DNA, suggesting the presence
of an upstream silencer element(s). Expression of these fusion genes
was undetectable in cell lines that do not express aromatase or that
express aromatase utilizing a nonplacental P450arom promoter. By
contrast, P450arom I.1-hGH fusion genes containing 246, 201, or 125 bp
of exon I.1 5'-flanking sequence were expressed both in trophoblast
cells and in other cell lines. These findings demonstrate that 501 bp
of exon I.1 5'-flanking DNA contain response elements required for
trophoblast-specific expression of P450arom. These results also suggest
the presence of regulatory elements between -501 bp and -246 bp of
exon I.1 5'-flanking sequence that bind inhibitory transcription
factors expressed in nontrophoblast cells. Deletion and site-directed
mutagenesis experiments further suggest that cis-acting
elements, including a GC box and two hexameric sequences present within
246 bp of sequence flanking the 5'-end of exon I.1, contribute to the
high levels of P450arom promoter activity in primary cultures of
placental cells. By competitive and supershift electrophoretic mobility
shift assays, it was observed that the ubiquitously expressed
transcription factor Sp1 comprises one of the proteins binding to the
GC box in the 5'-flanking sequence of P450arom exon I.1.
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INTRODUCTION
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Aromatase, an enzyme complex of the endoplasmic reticulum,
catalyzes the conversion of C19-steroids to estrogens.
Aromatase is comprised of two polypeptides, the ubiquitous
flavoprotein, NADPH-cytochrome P450 reductase, and a unique form of
cytochrome P450, P450arom (a product of the CYP19 gene), which is
present exclusively in estrogen-producing cells (1, 2, 3). In humans,
aromatase is expressed in various tissues including the
syncytiotrophoblast layer of the placenta (4, 5), gonads (6), brain (7, 8), and adipose tissue (8, 9), as well as in different fetal tissues,
including skin, intestine, and liver (10, 11, 12). The human placenta has a
remarkable capacity to aromatize C19-steroids secreted by
the fetal adrenals; after the ninth week of gestation, the placenta
provides the primary source of circulating estrogens (13).
Human P450arom is encoded by a single-copy gene that spans more than 75
kb in the human genome (14, 15, 16). The protein-coding sequence is
contained within nine exons (exons IIX); the translation initiation
and termination codons are present in exons II and X, respectively. The
5'-untranslated regions of P450arom mRNA transcripts in human ovary,
testis, adipose stromal cells, and placenta are encoded by alternative
first exons that are spliced onto a common site that lies
39 bp
upstream of the start of translation in exon II (17, 18). The
ovary-specific first exon lies proximal to exon II, whereas the major
first exon encoding the 5'-untranslated region of P450arom in placenta
(exon I.1) lies more than 35 kb upstream of exon II. The genomic clones
containing exons I.1 and II do not overlap, so the exact distance
between them is not known (16). The major human adipose-specific first
exon (exon I.4) lies
20 kb upstream of exon II, although alternative
downstream first exons (exons I.3 and II) are used in human adipose
stromal cells treated with cAMP and phorbol esters (19, 20).
In rodents, P450arom is not expressed in placenta; rather, P450arom
expression is restricted to gonads and the brain. As is the case in
humans, gonadal expression of P450arom appears to be under control of
promoter II. Therefore, it appears that the ovary-specific promoter
(PII) is the primordial promoter, whereas the placenta-specific
promoter (PI.1) was recruited much later in phylogeny with the
evolution of the primates and the hemochorial type of placenta
expressing high levels of aromatase.
The rapid growth of human placenta during the first trimester of
pregnancy is driven by the replication of mononuclear cytotrophoblast
cells, which sit on a basement membrane. When these cells mature, they
stop dividing and fuse to form the syncytiotrophoblast layer, which has
numerous secretory and transport/processing functions (21).
Syncytiotrophoblast differentiation entails the generation of a cascade
of regulatory signals that result in expression of genes encoding
different polypeptide hormones and steroid- metabolizing enzymes.
However, the molecular events that promote and maintain
syncytiotrophoblast differentiation and culminate in expression of
various genes, including P450arom, are not clearly
understood.
Cell transfection studies to functionally define the regulatory regions
of the human P450arom gene required for trophoblast-specific expression
have thus far been confined to use of choriocarcinoma cell lines (14, 22, 23, 24), which are undifferentiated cytotrophoblasts that express
relatively low levels of aromatase activity (21). In the present
investigation, we sought to develop a more relevant model system for
defining critical regulatory regions of the human P450arom gene
involved in placenta-specific expression by introduction of reporter
gene constructs containing various amounts of DNA flanking the 5'-end
of P450arom exon I.1 into human cytotrophoblasts in primary monolayer
culture. These trophoblast cells, which were isolated from midterm
human placenta, differentiate into syncytiotrophoblast, which manifests
high levels of aromatase activity and P450arom gene expression. The
results of these studies indicated the presence of putative enhancer
elements between -501 bp and -42 bp that are necessary for
trophoblast-specific expression of aromatase. Our findings also suggest
the presence of negative regulatory elements between -501 and -246 bp
that prevent P450arom expression in nonplacental cells.
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RESULTS
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Aromatase Expression Is Induced in Primary Cultures of Placental
Trophoblasts from Midgestation Human Placenta in Association with
Syncytiotrophoblast Differentiation
We have modified a primary cell culture system developed for term
placenta by Kliman et al. (25) for isolation and culture of
cytotrophoblast cells from midterm human placental tissue. We have
found that the yield of cytotrophoblast cells per gram of tissue from
midgestational placenta is at least 5-fold greater than that from
term placenta. Upon isolation, the midgestation placental
cytotrophoblasts were found to have low or undetectable levels of
aromatase activity (Fig. 1A
) and P450arom
mRNA (Fig. 1B
). Within 24 h after plating, aromatase activity and
P450arom mRNA were detectable and continued to increase further between
24 and 72 h of incubation (Fig. 1
). The two species of P450arom
mRNA (3.4 kb and 2.9 kb, Fig. 1B
) result from alternative use of
different polyadenylation signals present in exon X of the P450arom
gene (16). After 4 days in culture, aromatase activity was induced to
levels as high as 812 pmol androgen metabolized to estrogen/mg of
protein/min (Fig. 1A
). As previously observed using cytotrophoblasts
from term placenta (25, 26), as a function of time in culture, the
human cytotrophoblast cells migrated toward each other and fused to
form syncytiotrophoblast. Our findings, therefore, indicate that
differentiation of placental cytotrophoblasts to syncytiotrophoblast is
associated with a marked induction of aromatase activity and P450arom
gene expression. In consideration of this finding, we reasoned that
midgestational human placental cells in primary culture should provide
a relevant model system for functional mapping of genomic elements that
mediate tissue-specific expression of the P450arom gene in
syncytiotrophoblast.

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Figure 1. Aromatase Activity (A) and Northern Blot Analysis
of P450arom mRNA (B) in Human Trophoblasts before and after
Differentiation to Syncytiotrophoblast in Culture
A, Freshly isolated cytotrophoblasts were suspended in DMEM containing
2% FBS and plated at a density of 2 x 106 cells per
dish. The aromatase activity (picomoles androgen metabolized to
estrogen per mg protein/min) was assayed over a 4-day period by the
incorporation of tritium from [1ß-3H]androstenedione
into water. The values are the mean ± SEM of data
from triplicate dishes of cells. B, Total RNA (20 µg/lane) was
obtained from freshly isolated cytotrophoblasts and syncytiotrophoblast
after 24, 48, and 72 h in culture and analyzed for P450arom mRNA
by Northern blotting using a full-length 32P-labeled human
P450arom cDNA probe.
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Cultures of replicating trophoblast cell lines derived from human
choriocarcinomas are commonly used for study of placental function and
trophoblast-specific gene expression (18, 22, 27, 28). The human
choriocarcinoma cell line JEG-3, like normal trophoblasts, lacks
17
-hydroxylase/17,20 lyase activity but produces human CG,
progesterone, and estradiol-17ß (21, 27). However, aromatase activity
(Fig. 2A
) of JEG-3 choriocarcinoma cells
was found to be very low (
0.2 pmol of androgen metabolized to
estrogen/mg protein/min) as compared with primary syncytiotrophoblast
(
11 pmol of androgen metabolized to estrogen/mg protein/min);
P450arom mRNA levels of the JEG-3 cells were essentially undetectable
when compared with the trophoblast cells in culture (Fig. 2B
).

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Figure 2. Aromatase Activity ( A) and Northern Blot Analysis
of P450arom mRNA ( B) in Human Trophoblasts and JEG-3 Cells
A, Aromatase activity (picomoles androgen metabolized to estrogen per
mg protein/min) in trophoblast cells in primary culture and in JEG-3
cells was assayed by analysis of the incorporation of tritium from
[1ß-3H]androstenedione into water. Values shown are the
mean ± SEM of aromatase activity of triplicate dishes
of cells on day 4 of culture. B, Total RNA (20 µg) obtained from
freshly isolated cytotrophoblasts, syncytiotrophoblast after 4 days in
culture, and JEG-3 cells at 8090% confluence was analyzed for
P450arom mRNA by Northern blotting using a full-length
32P-labeled human P450arom cDNA as probe.
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Expression of P450aromI.1-hGH Fusion Genes in Human Trophoblast
Cells in Primary Monolayer Culture Increases in Association with
Syncytiotrophoblast Differentiation
Sequence analysis of
2.4 kb of 5'-flanking sequence of P450arom
exon I.1 revealed the existence of a number of putative
cis-acting elements including a purine-rich sequence or PU
box (5'-GAGGAA-3') (29) at -164 bp, two hexameric sequences between
-191 bp and -183 bp (5'-ATTCCAGAGGAGGTCATGC-3',
shown in bold); the downstream sequence is similar to the
binding site for an orphan member of the nuclear receptor superfamily,
Ad4BP/SF1 (30, 31), a GC box consensus-binding site for Sp1
(5'-GGCGGG-3') (32) at -233 bp, a reverse Pit-1 site (5'-ATGAATAAA-3')
(33) at -460 bp, a so-called trophoblast-specific element or TSE
(5'-CCAATGGG-3') (34) at -825 bp and human aromatase cytochrome P450
gene transcriptional regulatory elements or hATRE-1
(5'-CTTTCTGGCCTAAGGGTTGGAGAGC-3') and hATRE-2
(5'-CTTTTATGTTGCCCAATACCTGCTCTGCCTCGAG-GGTCACTCTC-3')
previously characterized by Toda and Shizuta (23) at -2238 and -2141
bp, respectively (Fig. 3
). To
functionally define the genomic regions involved in P450arom expression
of promoter I.1 in primary trophoblast cultures, P450aromI.1-hGH fusion
genes were constructed comprised either of 42, 125, 201, 246,
501, 923, or 2400 bp of DNA upstream and 103 bp of sequence downstream
of the transcription initiation site of exon I.1, linked to the hGH
structural gene, as reporter (Fig. 3
). As a control, a P450aromII-hGH
fusion gene containing 952 bp of 5'-flanking DNA and +29 bp of
ovary-specific exon II linked to the hGH structural gene also was
constructed. At this point, we found that the primary cultures of human
placental cells were resistant to conventional methods of DNA
transfection, such as calcium phosphate,
diethylaminoethyl-dextran, lipofection, and electroporation
(our unpublished observations). To circumvent this barrier, the
fusion genes were incorporated into the genome of a
replication-defective human adenovirus (35). The recombinant adenoviral
particles were then used for transfer of the P450arom-hGH fusion genes
into the placental cells in primary monolayer culture by infection. By
this means, the fusion genes were transferred into the cells with high
efficiency and without perturbation of cellular integrity. Fusion gene
expression was analyzed by RIA of hGH secreted into the culture medium,
which was collected at 24-h intervals.

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Figure 3. Schematic Diagram of Exon I.1 of the Human P450arom
Gene and Its 5'-Flanking Region with Putative Regulatory Elements, and
of P450aromI.1-hGH Fusion Genes
The positions of putative regulatory elements (indicated by labeled
small boxes) within 2400 bp of DNA flanking the 5'-end
of P450arom exon I.1 are shown in the top portion of the
figure. These elements include hATRE-1 and hATRE-2, TSE, Pit-1, GC box,
Hex sequences, and PU box (defined in text). Just upstream of exon I.1
at -27 bp from the transcription start site is the TATA box, ATAAA.
The flanking DNA is indicated by the black lines, while
the black boxes indicate exon I.1 sequences. The
arrow indicates the transcription initiation site and
the direction of transcription. The various P450aromI.1-hGH fusion gene
constructs used in this study are shown in the lower
part of the figure; the human P450arom DNA sequences are shown
in black, and the hGH sequences are indicated by the
white boxes. For cell transfection studies, the fusion
genes were incorporated into the genome of a replication-defective
human adenovirus (details of the procedure are described in
Materials and Methods). The trophoblast cells in primary
culture were incubated overnight with the appropriate recombinant
adenoviruses. Media from infected cells were collected at 24-h
intervals and assayed for hGH by RIA. Media were changed daily.
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Equivalent numbers of cytotrophoblast cells (2 x 106
cells per dish) were infected with the same number of functional
recombinant adenoviral particles in terms of plaque forming units
(1 x 106 pfu); these were limiting with respect to
the number of cells per dish (multiplicity of infection,
0.5). Since
titration of adenoviral particles in 293 cells provides a reliable
index of the number of infectious recombinant viruses, the same number
of trophoblast cells (1 x 106 cells) were infected in
each experiment using each fusion gene construct. In this manner,
highly reproducible results were obtained from one experiment to
another, and the results of a number of independent experiments could
be combined without normalization.
When placental cells were infected with recombinant adenoviruses
containing P450aromI.1-hGH fusion genes carrying -923 bp or -501 bp
of exon I.1 5'-flanking sequence, hGH production was essentially
undetectable on the first day after infection. However, expression of
these fusion genes increased as a function of time in culture in
association with syncytiotrophoblast differentiation and the induction
of P450arom expression and reached maximal levels after 4 days of
incubation (Fig. 4
). On the other hand,
expression of the P450aromI.1-42-hGH fusion gene
construct, which contains the minimal promoter region, remained
essentially undetectable throughout the 5-day culture period (Fig. 4
).
A comparative tritiated water assay of aromatase activity indicated
that infection of placental cells with recombinant adenoviruses did not
affect the aromatase activity levels that increased as a function of
time in culture and were similar to those of uninfected placental cells
in culture (data not shown).

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Figure 4. Expression of P450aromI.1-hGH Fusion Genes in Human
Trophoblasts as a Function of Time in Culture
Freshly isolated cytotrophoblasts in culture were infected
with 1 x 106 recombinant adenoviruses con-
taining P450aromI.1-923-hGH,
P450aromI.1-501-hGH, or P450aromI.1-42-hGH
fusion genes within an hour of plating. After an overnight incubation,
the media were collected and replaced with DMEM containing 2% FBS.
Media from the infected cells were collected over the next 4 days and
replaced with fresh media containing 2% FBS. The collected media were
assayed for hGH by RIA. Shown here are the levels of hGH secreted into
the medium for each day of culture. Values are the mean ±
SEM of data from three independent experiments, each
conducted in triplicate.
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To further delineate the genomic elements that mediate P450arom gene
expression in placenta, trophoblast cells in primary culture were
infected with recombinant adenoviruses containing P450aromI.1-hGH
fusion genes containing either 2400, 923, 501, 246, 201, 125, or 42 bp
of exon I.1 5'-flanking sequence. On the third day after infection,
when most of the cells had fused to form syncytia, fusion genes
containing 923 bp and 501 bp of P450arom exon I.1 5'-flanking sequence
were found to be expressed at comparable levels; these levels of
expression were
2- to 3-fold higher than those of the
P450aromI.1-2400-hGH fusion gene construct (Fig. 5
). By contrast, P450arom-hGH fusion
genes containing only 246 bp of exon I.1 5'-flanking DNA were
expressed at levels
3-fold greater than those of the -923 and
-501 bp fusion gene constructs. These findings suggest the presence of
upstream silencer elements between -2400 bp and -923 bp and also
between -501 bp and -246 bp of exon I.1 5'-flanking sequence.
Expression of a P450aromI.1-201-hGH fusion gene construct
that does not contain the GC box (-233 bp) was
50% lower than the
-246 bp-containing fusion gene. Further deletion of exon I.1
5'-flanking sequence to 125 bp resulted in an 80% reduction of fusion
gene expression when compared with the P450aromI.1-246-hGH
fusion gene construct. The P450aromI.1-125-hGH fusion gene
does not con-tain the Hex sequences between -191 bp and -183 bp or
the PU box (-164 bp). In cells infected with recombinant
adenoviruses containing P450aromI.1-42-hGH fusion gene
construct or the fusion gene containing -952 bp of DNA flanking the
5'-end of ovarian-specific exon II, hGH production was essentially
undetectable. Taken together, these results suggest that as little as
-246 bp of exon I.1 5'-flanking sequence mediates maximal levels of
P450arom gene expression in human syncytiotrophoblast in primary
culture.

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Figure 5. Expression of P450aromI.1-hGH Fusion Genes
Containing 422400 bp of P450aromI.1 5'-flanking DNA in Primary
Cultures of Human Trophoblasts
Freshly isolated cytotrophoblasts in culture were infected with 1
x 106 recombinant adenoviral particles containing
P450aromI.1-2400-hGH, P450aromI.1-923-hGH,
P450aromI.1-501-hGH, P450aromI.1-246-hGH,
P450aromI.1-201-hGH, P450aromI.1-125-hGH,
P450aromI.1-42-hGH, and P450aromII-952-hGH
fusion genes. Culture media were harvested and replaced with fresh
medium every 24 h over a 5-day period. Shown here are the levels
of hGH that accumulated in the culture medium between days 2 and 3 of
culture. Values are the mean ± SEM of data from three
independent experiments, each conducted in triplicate.
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The 5'-Flanking Sequence of P450aromI.1 between -501 bp and -42
bp Is Sufficient for Placenta-Specific Expression
To delineate the genomic regions required for
syncytiotrophoblast-specific expression of P450arom promoter I.1
activity, expression of these fusion gene constructs also was analyzed
in JEG-3 cells and in a number of other cell lines including a human
lung adenocarcinoma cell line (A549), a rat hepatoma cell line (H4IIE),
the Madin-Darby canine kidney cell line, MDCK, and a rat Leydig tumor
cell line (R2C). In JEG-3 cells, fusion gene expression was
considerably lower than in the primary trophoblast cell cultures (Fig. 6
). As shown in Fig. 2
, JEG-3 cells
express very low levels of aromatase activity and contain low levels of
P450arom mRNA transcripts. Furthermore, in contrast to the pronounced
differences in their relative levels of expression in primary cell
cultures, expression in JEG-3 cells of the -246 bp and -923
bp-containing fusion gene constructs were comparable, whereas
expression of P450aromI.1-501-hGH fusion gene construct
was considerably lower than expression of the
P450aromI.1-923-hGH fusion gene construct. The markedly
reduced levels of aromatase activity, P450arom mRNA levels, and
P450aromI.1-hGH fusion gene expression in JEG-3 cells, as compared with
primary trophoblast cultures, indicate that this choriocarcinoma cell
line may not provide a relevant system for functional mapping of
cis-acting elements and trans-acting factors
involved in syncytiotrophoblast-specific expression of the human
P450arom gene.

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Figure 6. Expression of P450aromI.1-hGH Fusion Genes in
Primary Human Trophoblast Cultures and in Various Cell Lines
The different cell types shown were infected with recombinant
infectious viral particles containing
P450aromI.1-2400-hGH, P450aromI.1-923-hGH,
P450aromI.1-501-hGH, P450aromI.1-246-hGH,
P450aromI.1-201-hGH, P450aromI.1-125-hGH, and
P450aromI.1-42-hGH fusion genes. Shown are the
levels of hGH that accumulated in the medium over a 24-h period between
days 2 and 3 of culture. Values are the mean ± SEM of
data from two to four independent experiments, each conducted in
triplicate. UD, Undetectable.
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To determine whether expression of P450aromI.1-hGH fusion genes is
dependent upon trophoblast-specific factors, the levels of expression
of the P450aromI.1-hGH fusion genes in trophoblast cells in primary
culture also were compared with expression in a number of nonplacental
cell lines, including R2C, H4IIE, A549, and MDCK (Fig. 6
). The R2C
Leydig cell line expresses relatively high levels of aromatase
constitutively using the ovary-specific promoter, which lies just
upstream of exon II (36, 37), whereas the other cell lines do not
express aromatase (our unpublished observations). The different
cell types also were infected with recombinant adenoviruses containing
TK-hGH fusion genes (in which hGH was under the control of the
thymidine kinase promoter) to monitor transfection efficiency, which
was found to be equivalent in all cell types (data not shown). Although
expression levels of fusion genes containing 2400 bp, 923 bp, 501 bp,
and 42 bp of exon I.1 5'-flanking DNA were barely detectable in any of
the above mentioned transfected cell lines, hGH production was
detectable in these cell lines infected with adenoviruses carrying
P450aromI.1-246-hGH, P450aromI.1-201-hGH, and
P450aromI.1-125-hGH fusion gene constructs. In A549 cells
transfected with adenoviruses carrying the
P450aromI.1-246-hGH fusion gene construct, hGH production
was as high as in the primary placental cell cultures transfected with
the same fusion gene. On the other hand, as expected, the fusion gene
containing 952 bp of DNA flanking the 5'-end of ovary-specific exon II
was expressed only in the R2C Leydig cells (data not shown). Although
the levels of hGH produced varied in the different cell lines, it is
clear from these results that the region between -501 bp and
-42 bp upstream of exon I.1 is sufficient for enhanced
activity of P450arom promoter I.1 in primary human trophoblast cultures
and that sequences between -501 and -246 bp mediate inhibition of
expression of this promoter in nonplacental cell types.
GC Box and Hex Sequences Serve as Enhancers for P450arom Gene
Expression in Primary Cultures of Syncytiotrophoblast
To characterize cis-acting elements present within the
P450arom exon I.1 5'-flanking sequence required for enhanced expression
in human placental cells, we analyzed the expression of a
P450aromI.1-923-hGH fusion gene construct containing a
mutation in a putative GC box at -233 bp
(P450aromI.1-923Sp1 mut-hGH) and of a
P450aromI.1-501-hGH fusion gene construct containing
mutations in two hexameric sequences, present between -191 bp and
-183 bp from the start site of transcription
(P450aromI.1-501Hexmut-hGH). These constructs were
incorporated into the genome of a replication-defective human
adenovirus and introduced into midgestation human placental cells by
infection as described above.
When placental cells were transfected with
P450aromI.1-923Sp1 mut-hGH, there was a 50% reduction in
hGH expression as compared with expression levels observed upon
transfection of P450aromI.1-923-hGH fusion gene containing
the wild-type sequence (Fig. 7
). The
levels of hGH production by trophoblast cells transfected with
P450aromI.1-501Hexmut-hGH fusion genes, in which the
hexameric sequences were mutated, were reduced by
75% when compared
with those of trophoblast cells transfected with
P450aromI.1-501-hGH fusion genes containing the wild type
sequences. These findings suggest that both the GC box and hexameric
sequences serve as enhancers for P450arom gene expression in placental
cells.

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Figure 7. Effect of Mutation of the GC Box and of the Hex
Sequences on Expression of P450aromI.1-923-hGH and
P450arom I.1-501-hGH Fusion Genes, Respectively, in
Trophoblast Cells in Primary Culture
Freshly isolated cytotrophoblasts were infected with 1 x
106 recombinant viral particles containing
P450aromI.1-923-hGH, P450aromI.1-923Sp1
mut-hGH, P450aromI.1-501-hGH, and
P450aromI.1-501Hexmut fusion genes. Shown here are the
levels of hGH that accumulated in the medium over a 24-h period between
days 2 and 3 of culture. Values are the mean ± SEM of
data from three independent experiments, each conducted in
triplicate.
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Sp1 Comprises One of the Proteins Binding to the GC Box in the
5'-Flanking Sequence of Exon I.1
To begin to characterize the proteins binding to the GC box at
-233 bp upstream of the translation start site of exon I.1,
competitive electrophoretic mobility assay (EMSA) was performed. A
fragment spanning a region from -241 bp to -217 bp upstream of exon
I.1 containing the GC box (GCaromI.1) was used as the radiolabeled
probe and either a consensus GC sequence (GCcon) or an oligonucleotide
in which the GC box of P450aromI.1 was mutated (GCaromI.1 mut) was used
as a competitor (Fig. 8A
).
Syncytiotrophoblast nuclear proteins interacted with the radiolabeled
GCaromI.1 fragment as three complexes of different mobilities (lane 2);
the two slower mobility complexes were effectively competed by a
200-fold excess of nonradiolabeled GCaromI.1 oligonucleotide (self,
lane 3) and by a 200-fold excess of GCcon (lane 4) but not by a
200-fold excess of GCaromI.1 mut oligonucleotide (lane 5). These
findings indicate that factors that comprise the two slower mobility
complexes are specific for the GC box of P450aromI.1. To determine
whether Sp1 is a component of these complexes, antibody supershift EMSA
was performed using a polyclonal antibody directed against Sp1 and
either the radiolabeled GCaromI.1 or the radiolabeled GCcon sequences
as probes (Fig. 8B
). As can be seen, an identical gel shift pattern was
obtained using syncytiotrophoblast nuclear proteins and either the
GCaromI.1 or GCcon as radiolabeled probes (lanes 3 and 6,
respectively). The addition of Sp1 antibody to the binding reaction
containing the nuclear proteins and the radiolabeled GCaromI.1 probe
supershifted the slowest mobility complex (lane 4). The antibody itself
did not interact with the GCaromI.1 radiolabeled probe in the absence
of nuclear proteins (lane 2). A similar supershifted complex was
observed when the Sp1 antibody was included in the incubation with the
nuclear proteins and the radiolabeled GCcon probe (lane 7). The results
of the competitive EMSA (Fig. 8A
) and the Sp1 antibody supershift EMSA
(Fig. 8B
), together with the finding that an identical gel shift
pattern is obtained using GCaromI.1 or GCcon as probes (Fig. 8B
),
suggest that Sp1 comprises one of the proteins binding to the GC box in
the 5'-flanking sequence of exon I.1.

View larger version (100K):
[in this window]
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|
Figure 8. Competitive EMSA (A) and Supershift EMSA (B) Using
Syncytiotrophoblast Nuclear Protein and Either Radiolabeled GCaromI.1
or GCcon as Probes
A, Syncytiotrophoblast nuclear proteins were incubated with
radiolabeled GCaromI.1 probe in the absence ([-], lane 2) or in the
presence of a 200-fold excess of nonradiolabeled GCaromI.1
oligonucleotide (self, lane 3), a 200-fold excess of nonradiolabeled
oligonucleotide containing a consensus GC box sequence (GCcon, lane 4),
or a 200-fold excess of nonradiolabeled oligonucleotide containing a
mutated GC box in GCaromI.1 (GCaromI.1 mut, lane 5). No protein was
incubated with the probe in the first lane (free probe, lane 1). B,
Syncytiotrophoblast nuclear proteins (3 µg) were incubated for 1
h at 4 C in the absence (Syn, lanes 3 and 6) or presence of antisera
(Ab) to Sp1 (lanes 4 and 7), and poly(dI-dC)-poly(dI-dC), as
nonspecific competitor. The nuclear protein mix was then incubated
either with 32P-labeled GCaromI.1 (lanes 14) or a GC box
consensus (GCcon, lanes 57) probe for an additional hour at 4 C. The
radiolabeled GCaromI.1 probe was incubated with the Sp1 antibody in the
absence of nuclear proteins in lane 2. The DNA-protein complexes were
resolved on a 5% polyacrylamide gel and visualized by autoradiography.
The arrow indicates the supershifted complex. No protein
was incubated with the probe in lanes 1 and 5 (free probe).
|
|
 |
DISCUSSION
|
---|
The syncytiotrophoblast layer of the human placenta, which is
formed upon the fusion of the underlying proliferative, mononuclear
cytotrophoblast cells, synthesizes both steroid hormones and
steroid-metabolizing enzymes, including aromatase. Previous studies
from our laboratory indicate that human syncytiotrophoblast P450arom
mRNA transcripts contain 5'-untranslated sequences encoded by exon I.1,
which lies at least 35 kb upstream of the translation start site in
exon II of the aromatase gene (16). The molecular events that promote
and maintain syncytiotrophoblast differentiation and culminate in
expression of the gene encoding P450arom are not clearly
understood.
In the present study, we have used a primary cell culture system using
cytotrophoblasts isolated from midgestation human placenta for
functional analysis of the genomic sequences involved in
syncytiotrophoblast-specific expression of aromatase. The levels of
aromatase activity and of P450arom mRNA in freshly isolated
cytotrophoblasts from midterm placenta are very low or undetectable;
however, upon differentiation to syncytiotrophoblast, there is a marked
induction of aromatase activity and P450arom gene expression. This is
the first time that regulatory regions of a human placenta-specific
gene have been mapped using primary cultures of trophoblast cells. To
define the regulatory regions of the P450arom gene that mediate
placenta-specific expression, trophoblast cells in primary culture were
infected with recombinant adenoviruses containing P450aromI.1-hGH
fusion genes comprised of genomic sequences flanking the 5'-end of exon
I.1 of the human P450arom gene. Replication-defective recombinant human
adenoviruses have been used as vehicles for efficient transfer of
exogenous genes into slowly dividing or nonproliferating cells (38, 39). In the present study, we have used recombinant adenoviruses for
transfer of P450arom I.1-hGH fusion gene constructs into midgestation
cytotrophoblast cells immediately after plating. In this manner,
expression of the fusion genes can readily be assessed over time as the
cells differentiate to form syncytiotrophoblast that expresses high
levels of aromatase.
Our findings indicate that expression of P450arom-hGH fusion genes
containing 125-2400 bp of exon I.1 5'-flanking DNA increases in concert
with syncytiotrophoblast differentiation and P450arom gene expression.
By deletional analysis, we observed that expression of fusion genes
containing 2400 bp of exon I.1 5'-flanking sequence was lower than
expression of P450aromI.1-923-hGH fusion genes in the
transfected trophoblast cells. Similarly, hGH production levels in
placental cells infected with adenoviruses carrying the -501 bp
containing fusion gene construct were lower than those of the -246 bp
containing fusion gene construct. These results suggest the presence of
silencer elements between -2400 bp and -923 bp, and also between
-501 bp and -246 bp upstream of P450arom exon I.1. The highest levels
of fusion gene expression were observed in trophoblast cells infected
with P450aromI.1-hGH fusion genes containing -246 bp of exon I.1
5'-flanking sequence. Further deletion of the 5'-flanking region
resulted in a gradual decrease in fusion gene expression, suggesting
the presence of enhancer elements within the 5'-flanking sequence
between -246 bp and -42 bp of exon I.1. These findings support those
of Toda et al. (14), which suggested that 5'-flanking
sequences between -500 bp and -243 bp, and between -242 bp and -138
bp, contained cis-acting elements that repressed and
activated expression, respectively, of P450arom I.1-CAT reporter gene
constructs in BeWo choriocarcinoma cells.
Aromatase mRNA transcripts containing 5'-untranslated sequences encoded
by exon I.1 also have been found in the human choriocarcinoma cell
line, JEG-3 (18). A number of investigators have used this cell line
for gene transfection studies because of its ability to proliferate and
readily take up DNA. However, we have observed that aromatase activity
of JEG-3 choriocarcinoma cells is
0.02 that of the
midgestational trophoblast cells in primary culture (Fig. 2A
). The low
levels of aromatase expression in choriocarcinoma cell lines is not
surprising since choriocarcinomas consist of mitotically active
cytotrophoblast cells with moderate degrees of differentiation (21, 27, 40). Hence, these cells secrete high levels of CG, characteristic of
cytotrophoblasts, and only modest amounts of hormones, characteristic
of syncytiotrophoblast, such as chorionic somatomammotropin (21). In
the present study, the P450arom I.1-hGH fusion genes were found to be
expressed at considerably lower levels in the JEG-3 cells than in
primary human trophoblast cultures. Additionally, the pattern of
expression of the different fusion gene constructs in the JEG-3 cells
was not comparable to that observed in the midgestational placental
cells in culture. Hence, although the JEG-3 cells share many
characteristics of normal trophoblast cells, our studies indicate that
they may not provide a suitable system for mapping of genomic regions
required for placenta-specific expression of the human P450arom
gene.
To assess the importance in placenta-specific expression of
regulatory elements present within -2400 bp of sequence flanking the
5'-end of P450arom exon I.1, we compared the expression of the
P450aromI.1-hGH fusion gene constructs in primary human trophoblast
cultures with expression in cell lines that do not express aromatase
(the human lung adenocarcinoma-derived cell line A549, the canine
kidney cell line MDCK, and the rat hepatoma cell line H4IIE), as well
as in the R2C rat Leydig cell line, which expresses aromatase from a
different promoter. We observed that whereas expression of the
P450aromI.1-hGH fusion genes containing
501 bp of exon I.1
5'-flanking DNA were expressed at relatively high levels in trophoblast
cells in primary culture, they were expressed at extremely low or
undetectable levels in kidney (MDCK), lung (A549), liver (H4IIE), or
Leydig (R2C) cell lines. Interestingly, in addition to being expressed
at relatively high levels in the human syncytiotrophoblast, fusion
genes containing 246 bp, 201 bp, and 125 bp of exon I.1 5'-flanking
sequence were found also to be highly expressed in A549 and MDCK cells.
In conjunction with the deletional analysis in primary cultures of
trophoblast cells, these findings suggest that 501 bp of exon I.1
5'-flanking DNA contains binding sites for transcription factors that
mediate enhanced levels of trophoblast-specific expression of the
P450arom gene. The finding that P450aromI.1-hGH fusion genes containing
246 bp of exon I.1 5'-flanking DNA also were expressed at relatively
high levels in other cell types suggests that the 5'-flanking region
between -501 bp and -246 bp contains silencer elements that may bind
inhibitory transcription factors expressed in other cell types.
By use of site-directed mutagenesis, we have identified two potential
enhancer elements present within 501 bp of P450arom exon I.1
5'-flanking DNA: a GC box (5'-GGCGGG-3') at -233 bp and two Hex
sequences (5'-ATTCCAGAGGAGGTCATGC-3', shown in
bold) between -191 bp and -183 bp. Mutation of either the
GC box or Hex sequences reduced fusion gene expression, suggesting
their functional significance in mediating high levels of aromatase
gene expression in placenta. Sp1 is known to bind to a consensus GC box
(GGGCGG) and direct tissue-specific, developmental, and hormonal
regulation of a number of genes (41, 42). Although Sp1 is a ubiquitous
transcription factor, it is likely that it does not function alone but
acts in concert with other regulatory factors (43). In electrophoretic
mobility shift assays using human syncytiotrophoblast nuclear proteins
and Sp1 antibody, we have observed that Sp1 comprises one of the
components that binds to the GC box at -233 bp. It should also be
noted that GC-rich sequences serve as binding sites for other members
of the Krüppel family of transcription factors, several of which
are expressed in a tissue-selective manner (44). Studies are now in
progress to characterize the other placental nuclear proteins that bind
to the GC box in the 5'-flanking sequence of P450arom exon I.1.
The transcription factor, Ad4BP/SF-1, which binds to the sequence
PuPuAGGTCA and regulates gonadal and adrenal development (30, 31, 45),
has been reported to participate in the transcriptional regulation of
P450arom in the ovary of humans (46) and rodents (36, 47). However, the
findings that SF-1 is expressed at very low levels in human placenta
(48) and that the placenta develops and functions normally in SF-1
knock-out mice (49) suggest that this transcription factor may not
play an important role in placenta-specific gene expression. It is
possible that another member(s) of the nuclear receptor superfamily
could bind to the Hex sequence upstream of exon I.1 and activate
expression of P450arom promoter in the human placenta. Recently, Sun
et al. (50) have found that an imperfect palindromic
sequence between -183 bp and -172 bp upstream of P450arom exon I.1,
which contains the downstream Hex sequence (5'-GGAGGTCA-3')
binds a heterodimer of RXR
and VDR in JEG-3 cells.
A unique combinatorial interaction of ubiquitously expressed and
cell-selective transcription factors has been reported to mediate
placenta-specific expression of a growing number of eukaryotic genes,
including those encoding the
-subunit of the glycoprotein hormones
(51), leptin (52), and placental lactogen I (53). Expression of the
-subunit of the glycoprotein hormones in placental cells requires
the cooperative interaction of proteins bound to five different
regulatory elements, including a TSE, an
-activating element
(
-ACT), a tandem pair of cAMP response elements (CREs), a junctional
regulatory element (JRE), and a CCAAT box (54). Our findings that
P450arom gene expression is induced in concert with syncytiotrophoblast
differentiation suggest that transcription factors involved in
trophoblast differentiation may also serve a role in induction of
P450arom expression. In a number of recent reports, it has been
suggested that trophoblast differentiation is regulated by changes in
expression of both positive and negative transcriptional regulators
that belong to basic helix-loop-helix, POU domain, zinc finger, and
nuclear receptor transcription factor families (55, 56, 57, 58, 59).
In summary, in the present study we have observed that sequences
between 42 and 501 bp upstream of exon I.1 of the human aromatase gene
contain an enhancer(s) that mediates elevated levels of expression in
primary cultures of human trophoblast cells. Furthermore, we have found
that sequences between -246 and -501 bp suppress P450arom promoter
I.1 expression in nonplacental cell types. Studies currently are in
progress to further define the roles of the GC box, the Hex elements,
and other cis-acting regulatory elements within the 501 bp
5'-flanking region involved in the enhanced levels of promoter I.1
activity in placental trophoblasts and in inhibition of promoter I.1
activity in other cell types. In parallel, transgenic mice are being
used to define the minimal genomic region and the cis-acting
elements that are required to mediate appropriate placenta-specific,
developmental, and regulated expression of human P450arom gene.
 |
MATERIALS AND METHODS
|
---|
Isolation and Culture of Human Placental Cells
Placental tissues from midtrimester human abortuses were
acquired in accordance with the Donors Anatomical Gift Act of the State
of Texas after written consent was obtained. In all cases, consent
forms and protocols were approved by the Human Research Review
Committee of the University of Texas Southwestern Medical Center at
Dallas. A placental cell culture system developed by Kliman et
al. (25) was modified for isolation and culture of cytotrophoblast
cells from human midgestation placenta. Briefly, the placental tissues
were washed with HBSS, pH 7.4 (GIBCO, Grand Island, NY) before being
finely minced and digested in HBSS containing 0.125% trypsin at 37 C
for 30 min. The trypsin digestion was repeated three times; at the end
of each 30-min digestion period, the supernatant was collected, layered
over serum, and then briefly centrifuged at 1000 x g for 10
min. The pellet was suspended in DMEM (GIBCO), filtered, and then
layered over a Percoll gradient (70%5%). The gradients were
centrifuged at 1200 x g for 20 min at room temperature; the
cells in the middle layer (density 1.0451.062), which comprised the
cytotrophoblasts, were collected, washed, and counted. The cells were
resuspended in DMEM supplemented with 10% FBS and 1%
antibiotic/antimycotic solution, plated at a density of 2 x
106 cells/2 ml on dishes coated with extracellular matrix
(ECM) and incubated overnight at 37 C in a humidified atmosphere of
95% air-5% CO2. After 24 h, the medium was aspirated
and replaced with DMEM containing 2% FBS. The ECM-coated dishes were
prepared from confluent monolayers of Madin-Darby canine kidney cells
(ATCC CRL 6253) (25 x 106 cells per dish) that were
treated with 1% deoxycholate for 5 min. The dishes were then washed
three times with HBSS and stored at 37 C until use.
Preparation and Maintenance of Cell Lines
The human choriocarcinoma cell line, JEG-3 (ATCC HTB-36), was
maintained in RPMI medium supplemented with FBS (10% vol/vol), while
the rat R2C Leydig cell line (ATCC CCL-97) was maintained in Hams
F-10 medium containing horse serum (15% vol/vol) and FBS (2.5%
vol/vol). The human lung adenocarcinoma cell line, A549 (ATCC CCL-185),
the canine kidney cell line, MDCK (ATCC CRL-6253), and the rat hepatoma
cell line, H4IIE (ATCC CRL-1548) were maintained in Waymouths medium
supplemented with FBS (10% vol/vol). The adenovirus-transformed human
embryonic kidney cell line, 293 (ATCC CRL-1573), was cultured in DMEM
containing FBS (10% vol/vol). The various media used contained
antibiotic/antimycotic solution (1%).
Tritiated Water Assay of Aromatase Activity in Placental and
JEG-3 Cells
Aromatase activity was assayed in placental and JEG-3 cells in
monolayer culture by the tritiated water assay as described previously
(60). Briefly, [1ß-3H]androstenedione (New England
Nuclear, Boston, MA) was added to the culture media for 1 h; 2-ml
aliquots were then removed and combined with 1 ml of ice-cold 30%
(wt/vol) trichloroacetic acid. The incorporation of tritium from
[1ß-3H]androstenedione into water was assayed in
aqueous scintillation fluid after extraction with 4 volumes of
chloroform and 1 volume of dextran-charcoal suspension. The cell
monolayers were rinsed twice in PBS and stored at -20 C for subsequent
assays of protein content by the method of Lowry et al.
(61).
Northern Blot of Placental mRNA
Total RNA was isolated from cells by the method of Chirgwin
et al. (62). Briefly, monolayer cultures were washed with
PBS, lysed in 4 M guanidinium isothiocyanate, followed by
centrifugation through a cesium chloride cushion (5.7 M).
The RNA samples were then extracted with phenol/chloroform and
precipitated with ethanol, and the pelleted RNA was resuspended in
water. Total RNA (20 µg) was electrophoresed, transferred to
nitrocellulose, and probed using a 32P-labeled human
aromatase cDNA probe (63). The relative levels of aromatase mRNA were
assessed by autoradiography.
Construction of P450aromI.1-hGH Fusion Genes and Preparation of
Recombinant Adenoviruses
Genomic sequences comprised of 2400, 923, 501, 246, 201, 125,
and 42 bp of 5'-flanking DNA and 103 bp of untranslated exon I.1 of the
human aromatase gene (P450aromI.1) were subcloned into the
Sal/BamHI sites of the plasmid pACsk2OGH to
obtain various P450aromI.1:pACsk2OGH plasmids.
pACsk2OGH contains the promoterless hGH structural gene and
the left 17% of human adenovirus 5 genome (39, 64). In this manner,
various amounts of exon I.1 5'-flanking DNA and the first 103-bp
segment of untranslated exon I.1 of the aromatase gene were fused to
the first exon of the hGH structural gene. A fusion gene containing 952
bp of 5'-flanking DNA and +29 bp of ovary-specific exon II of human
aromatase gene linked to the hGH structural gene also was constructed
and used as a control. As an alternative control, a TK-hGH fusion gene
was constructed in which the hGH structural gene was ligated to the
herpes simplex virus thymidine kinase promoter.
Fusion genes containing mutations in the GC box at -233
bp [5'-CCCAAGGCGGGACTCT-3' (wild-type
sequences are shown with the GC box in bold and
underlined)] or the hexameric (Hex) sequences at -183 bp and
-191 bp
[5'-ATTCCAGAGGAGGTCATGC-3'
(wild-type sequences are shown with the Hex sequences in bold and
underlined)] were created by site-directed mutagenesis according
to the method out-lined in Bio-Rad Mutagene Kit (Bio-Rad
Laboratories, Richmond, CA). Briefly,
pBlaromI.1-501 [pBluescript II KS vector
(Stratagene) containing 501 bp of sequence flanking the 5'-end of
P450arom exon I.1 and 103 bp of exon I.1] and helper phage, M13K07,
were used to transform CJ236 Escherichia coli
strain deficient in dUTPase and
uracil-N-glycosylase. The isolated single-stranded
uracil-containing phage DNA was used as a template for site-directed
mutagenesis using oligonucleotide Sp1 mutrev
[5'-ATCAGATTATAGAGAAAGATCTTGGGTGGATGAA-3' (mutated
nucleotides in italics and bold)] or
Hexmutrev
[5'-GGTATGGGGCATGGGGTCGACCGGGATGAGGGTCTTATG3'
(mutated nucleotides in italics and bold)] as
primers. The resulting plasmids, pBlaromI.1-501Sp1 mut
and pBlaromI.1-501Hexmut, were used to obtain
P450aromI.1-501Sp1 mut: pACsk2OGH and
P450aromI.1-501Hexmut:pACsk2OGH plasmids,
respectively. The SacI/EcoRI fragment (-923 bp
to -501 bp) from P450aromI.1-923:pACsk2OGH
plasmid was then subcloned into
P450aromI.1-501Sp1 mut:pACsk2OGH to obtain
P450aromI.1-923Sp1 mut:pACsk2OGH plasmid.
Sequences of each fusion gene construct were confirmed by
double-stranded sequencing using the dideoxy chain termination method
and a Sequenase kit (US Biochemical, Cleveland, OH).
To generate recombinant adenoviruses, 293 cells, a permissive human
embryonic kidney cell line, were cotransfected with recombinant
pACsk2OGH plasmids containing the various fusion gene
constructs and with pJM17. The pJM17 plasmid is too large to be
packaged into viral particles, since it contains the entire adenovirus
genome and a 4.3-kb insert of pBR322 plasmid. Infectious viral
particles of appropriate size for packaging are obtained by in
vivo homologous recombination of the plasmids that results in the
formation of a recombinant viral genome with the release of the pBR322
insert (35, 65). Viral DNA was analyzed to confirm the presence of the
fusion genes by restriction endonuclease digestion, Southern analysis,
and DNA sequencing (Sequenase 2.0, U.S. Biochemical). The recombinant
viruses were then titered in 293 cells at least twice to determine the
number of infectious particles (plaque-forming units).
Infection of Trophoblasts in Primary Culture and Different Cell
Lines with Recombinant Adenoviruses
Freshly isolated cytotrophoblast cells, plated at a density of
2 x 106 cells per dish in DMEM containing 10% FBS,
were infected within 1 h of plating with 1 x 106
recombinant infectious viral particles, resulting in a multiplicity of
infection of
0.5. In this manner, the same number of cells (1
x 106) was infected in each experiment. After an overnight
incubation, media were collected and replaced with DMEM containing 2%
FBS. Media from infected cells were then collected at 24-h intervals
and replaced with fresh media containing 2% FBS. The collected media
were assayed for hGH by RIA, using an Allegro hGH kit (Nichols
Institute Diagnostics, San Juan Capistrano, CA).
Different cell lines used in this study were also infected with 1
x 106 recombinant adenoviral particles containing various
P450aromI.1-hGH fusion genes. hGH secreted into the media was assayed
by RIA, as described above for primary trophoblast cultures.
EMSA
Nuclear extracts were prepared from placental cells on day 4 of
culture by the method of Dignam et al. (66). Protein
concentrations were determined by a modified Bradford assay (Bio-Rad,
Richmond, CA). Double-stranded oligonucleotides corresponding to a
region from -241 bp to -217 bp upstream of the translation start site
in P450arom exon I.1 containing a GC box [GCaromI.1,
5'-CCACCCAAGGCGGGACTCTATAATC-3' (wild-type
sequences are shown in bold and underlined)] or a mutated
GC box [GCaromI.1 mut,
5'-CCACCCAATATGTTTCTCTATAATC-3' (mutated
sequences are shown in bold and italics)] were synthesized.
An oligonucleotide containing a consensus GC box (GCcon,
5'-ATTCGATCGGGGCGGGGCGAG-3') was also synthesized. For competition
EMSAs, double stranded oligonucleotides were end-labeled with T4 kinase
and [
-32P]ATP, incubated with syncytiotrophoblast
nuclear proteins (3 µg) for 30 min at room temperature in binding
buffer (20 mM HEPES, pH 7.6, 12% glycerol, 100
mM KCl, 1 mM EDTA, 1 mM
dithiothreitol) in the absence or presence of nonradiolabeled
double-stranded oligonucleotides and 1 µg poly(dI-dC)-poly(dI-dC)
(Pharmacia, Piscataway, NJ) as nonspecific competitor. The DNA-protein
complexes were resolved on a 5% polyacrylamide gel and visualized by
autoradiography. For supershift EMSA, the nuclear proteins were
incubated for 1 h at 4 C in binding buffer in the absence or
presence of Sp1 antibody (1 µg) (Santa Cruz, Biotech, Santa Cruz, CA)
and poly(dI-dC)-poly(dI-dC). The nuclear protein mix was then incubated
with 32P-labeled probe for an additional hour at 4 C before
separation by PAGE.
 |
ACKNOWLEDGMENTS
|
---|
The authors are grateful to Margaret Smith for her expert help
with Northern blot analysis and cell culture.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Carole R. Mendelson, Ph.D., Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9038. E-mail:
cmende{at}biochem.swmed.edu
This research was supported by NIH Grant 5 RO1 DK-31206. A.K. was
supported in part by NIH Training Grant 5-T32-HD-0719016.
Received for publication May 4, 1998.
Revision received June 24, 1998.
Accepted for publication July 16, 1998.
 |
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