Hypoxia Prevents Induction of Aromatase Expression in Human Trophoblast Cells in Culture: Potential Inhibitory Role of the Hypoxia-Inducible Transcription Factor Mash-2 (Mammalian Achaete-Scute Homologous Protein-2)
Bing Jiang,
Amrita Kamat and
Carole R. Mendelson
Departments of Biochemistry (B.J., A.K., C.R.M.) and Obstetrics &
Gynecology (C.R.M.) The University of Texas Southwestern Medical
Center at Dallas Dallas, Texas 75390-9038
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ABSTRACT
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The human placenta has a remarkable capacity
to aromatize C19-steroids, produced by the
fetal adrenals, to estrogens. This reaction is catalyzed by aromatase
P450 (P450arom), encoded by the CYP19 gene. In placenta,
CYP19 gene expression is restricted to the
syncytiotrophoblast layer. Cytotrophoblasts isolated from human
placenta, when placed in monolayer culture in 20%
O2, spontaneously fuse to form
syncytiotrophoblast. These morphological changes are associated with a
marked induction of aromatase activity and CYP19 gene
expression. When cytotrophoblasts are cultured in an atmosphere
containing 2% O2, they manifest increased
rates of DNA synthesis and fail to fuse and form syncytiotrophoblast.
The objective of the present study was to utilize cytotrophoblasts
isolated from midterm human placenta to analyze the effects of
O2 on CYP19 gene expression and the
molecular mechanisms that mediate these effects. We observed that when
trophoblast cells were maintained in 2% O2,
there was only a modest induction of CYP19 expression as a
function of time in culture, and aromatase activity was barely
detectable. However, when cytotrophoblasts that had been maintained in
2% O2 for 3 days were placed in a 20%
O2 environment, there was a rapid onset of cell
fusion and induction of P450arom mRNA and aromatase activity. In
addition, mRNAs for the helix-loop-helix factors Mash-2 (mammalian
achaete-scute homologous protein-2) and Id1 (inhibitor of
differentiation 1) were readily detectable in freshly isolated
cytotrophoblasts and were markedly decreased upon differentiation
to syncytiotrophoblast in 20% O2. By
contrast, when cytotrophoblasts were cultured in 2%
O2, mRNA levels for Mash-2 and Id1 remained
elevated. Interestingly, overexpression of Mash-2 in primary cultures
of human trophoblast cells markedly inhibited cell fusion and the
spontaneous induction of P450arom mRNA levels and caused a marked
decrease in expression of cotransfected fusion gene constructs
containing either 125, 201, 246, or 501 bp of DNA flanking the 5'-end
of the placenta-specific exon (exon I.1) of the human CYP19
gene linked to the human GH (hGH) structural gene, as reporter. In
studies using BeWo, a human choriocarcinoma cell line, overexpression
of Mash-2 also inhibited expression of cotransfected
CYP19I.1:hGH fusion gene constructs. The findings that
Mash-2 had no effect on the expression of a
CYP19I.1-42:hGH fusion gene in
primary cultures of human trophoblast and BeWo cells suggest that
Mash-2 exerts its inhibitory effects directly or indirectly though
CYP19I.1 5'-flanking sequences that lie between -42 and
-125 bp. By contrast, neither Id1 nor Id2 had an effect on
CYP19I.1 promoter activity in the transfected BeWo cells.
These findings suggest that Mash-2 may serve as a hypoxia-induced
transcription factor that prevents differentiation to
syncytiotrophoblast and aromatase induction in human trophoblast
cultured under low O2 conditions.
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INTRODUCTION
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In the human placenta, cytotrophoblasts play a critical role in
implantation and in trophoblast development. Growth of the placenta is
driven by the replication of mononuclear cytotrophoblasts, which is
likely regulated by autocrine and paracrine factors (1). As the
cytotrophoblasts mature, they stop dividing and fuse to form the
terminally differentiated syncytiotrophoblast layer, which covers the
chorionic villi and is bathed by maternal blood that perfuses the
intervillous spaces (2). Throughout pregnancy, the syncytiotrophoblast
layer is the major site for many placental functions required for
maintenance of pregnancy and for fetal growth and development,
including nutrient and gas exchange, and biosynthesis of steroid and
polypeptide hormones (2).
The human placenta is unusual in its capacity to produce very large
quantities of estrogen. Estrogens are formed by aromatization of
C19-steroids secreted by the fetal adrenals (3).
The aromatase reaction is catalyzed by an enzyme complex containing two
polypeptides, the ubiquitous flavoprotein NADPHcytochrome P450
reductase, and a unique form of cytochrome P450 (P450arom), the product
of the CYP19 gene, which is expressed exclusively in
estrogen-producing cells (4, 5, 6).
Human CYP19 is a single-copy gene (7, 8, 9); the protein coding
sequence is contained within nine exons (exons IIX) which span
approximately 35 kb of DNA (7). CYP19 gene expression occurs
at relatively high levels in the ovaries (10), testes (11, 12, 13), and in
discrete nuclei within the brain (14) of vertebrate species from fish
to man. On the other hand, humans also manifest relatively high levels
of aromatase expression in stromal cells of adipose tissue (15), in
fetal liver (16, 17), and in placenta, exclusively in the
syncytiotrophoblast layer (18). Human P450arom mRNAs in ovary/testis,
adipose, and placental cells have distinct 5'-termini encoded by
alternative first exons that are spliced onto a common site that lies
36 bp upstream of the translation initiation codon (19). In placenta,
the majority (>95%) of the P450arom mRNA transcripts contain
sequences encoded by exon I.1 (20), which lies
35,000 bp upstream of
the translation initiation site in exon II.
Isolated human mononuclear cytotrophoblasts, when placed in culture in
a 20% O2 environment, spontaneously aggregate
and fuse to form nonproliferative, multinucleated syncytiotrophoblast,
which produce a variety of polypeptide hormones and steroid
metabolizing enzymes (1). We have observed that syncytiotrophoblast
differentiation is associated with a rapid and dramatic induction of
CYP19 gene expression (21). It is apparent that the
differentiation of cytotrophoblasts to syncytiotrophoblast is
associated with the generation of a cascade of regulatory signals;
however, the molecular events that accompany this differentiation
process are poorly understood. It has been suggested that biochemical
differentiation can occur only after syncytial formation (22). On the
other hand, others have reported that syncytial formation is not
prerequisite to cellular differentiation, but is one of the
consequences of the differentiation process (23).
Basic-helix-loop-helix (bHLH) transcription factors have been suggested
to serve a role in placental differentiation (24). Interestingly,
expression of the HLH factor, Id (inhibitor of differentiation), which
lacks a basic DNA-binding domain (25), is elevated in trophoblast stem
cells and declines during cell differentiation (26, 27). Similarly,
expression of the gene encoding the bHLH transcription factor
Mash-2 (mammalian achaete-scute homologous protein-2) (28), which is
expressed at high levels in mouse trophoblasts, is diminished as
trophoblasts differentiate into giant cells (29). It is not known,
however, what role these HLH transcription factors may play in the
regulation of CYP19 gene expression in the human
placenta.
It has been reported that hypoxia stimulates cytotrophoblast
proliferation (30, 31), impairs cell fusion (32), and decreases
placental polypeptide hormone production (32, 33). Exposure of human
placental villous explants to a low (2%) O2
environment was found to increase cytotrophoblast proliferation,
decrease differentiation into invasive cells (34), and mimic the
placental defect associated with preeclampsia (34, 35, 36). Preeclampsia, a
disease characterized by placental hypoxia (37), is associated with
reduced serum estradiol and increased progesterone levels (38, 39).
In the present study, we used midgestation human cytotrophoblasts in
primary culture to analyze effects of atmospheric
O2 on CYP19 gene expression. We
observed that culture of cytotrophoblasts in 2%
O2 blocked the induction of P450arom mRNA levels;
this effect of low O2 was rapidly reversible.
Additionally, differentiation of cytotrophoblasts to
syncytiotrophoblast and induction of CYP19 gene expression
in 20% O2 was accompanied by a marked decline in
expression of the bHLH transcription factor Mash-2 and of Id1, which
was prevented by culture of cytotrophoblasts in 2%
O2. The finding that overexpression of Mash-2 in
human trophoblast cells in primary culture markedly reduced syncytia
formation and the spontaneous induction of CYP19 expression,
as well as expression of cotransfected reporter gene constructs
containing 5'-flanking sequences of CYP19 exon I.1, suggests
that Mash-2 may serve as a hypoxia-inducible transcription factor that
promotes human cytotrophoblast proliferation at the expense of
differentiation.
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RESULTS
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Effects of Oxygen Tension on Differentiation and DNA Synthesis in
Cultured Trophoblast Cells
When freshly isolated mononuclear cytotrophoblasts isolated from
midgestation human placenta were placed in monolayer culture in a 20%
O2 environment for 3 days, the cells aggregated
and fused to form multinuclear syncytiotrophoblast (Fig. 1A
). By contrast, when parallel dishes of
cells were cultured in an environment of 2% O2,
they aggregated, but failed to fuse (Fig. 1B
). On the other hand, when
cells that had been maintained in 2% O2 for 3
days were shifted to a 20% O2 environment, they
fused to form a multinuclear syncytiotrophoblast (Fig. 1C
). These
findings support previous observations using term placental cells (32)
and further indicate that the inhibitory effects of hypoxia on
trophoblast fusion are reversible.

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Figure 1. Effects of Oxygen on Differentiation of Human
Trophoblast Cells in Culture
Freshly isolated cytotrophoblasts were suspended in DMEM containing 2%
FCS and plated at a density of 2 x 106 cells per
dish. Cells were maintained either in an atmosphere of 20%
O2 (panel A) or 2% O2 (panel B) for 3 days.
After 3 days, some dishes were shifted from the 2% to the 20%
O2 environment and cultured for an additional 3 days (panel
C). In the studies shown in the lower panels, the
trophoblast cells were incubated for 3 days in either 20% (panel D) or
2% (panel E) O2; BrdU (1 µM) was added
during the last 24 h of culture. The cells were then fixed and
incubated with a fluorescein-labeled antibody to BrdU, followed by
fluorescence microscopy.
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To analyze the effects of oxygen tension on DNA synthesis by cultured
trophoblast, freshly isolated cytotrophoblast cells from midgestation
human placenta were incubated for 72 h in a 20% or 2%
O2-containing environment; bromodeoxyuridine
(BrdU; 1 µM) was added to the medium during the last
24 h of culture. As can be seen in Fig. 1D
, essentially no BrdU
incorporation was detected in trophoblast cultured in 20%
O2. By contrast, BrdU incorporation was readily
detectable in cells cultured in a 2% O2
environment (Fig. 1E
). When cells that had been maintained in 2%
O2 for 3 days were shifted to 20%
O2 and cultured for another 3 days, incorporation
of BrdU added during the last 24 h of culture (between days 5 and
6) was essentially undetectable (not shown). These findings are
correlated with those of [3H]thymidine
incorporation, in which it was observed that incorporation of
radiolabeled thymidine into DNA of trophoblast cells maintained in 2%
O2 was 2.5-fold greater than in cells cultured in
20% O2 (data not shown). Our observations
corroborate previous reports (35), which indicate that under hypoxic
conditions, cytotrophoblast DNA synthesis and cell proliferation are
increased.
Effects of Oxygen on Aromatase Activity and on P450arom mRNA levels
in Human Trophoblast Cells in Culture
To evaluate the effects of O2 tension on
aromatase expression, cytotrophoblasts from midgestation human placenta
were cultured for up to 6 days in 2% or 20% O2.
As observed previously, the midgestation cytotrophoblasts manifest
barely detectable levels of aromatase activity (Fig. 2
) and low or undetectable levels of
P450arom mRNA (Fig. 3
) (21). When the
cells are cultured in a 20% O2 environment,
aromatase activity (Fig. 2
) and P450arom mRNA levels (Fig. 3
) are
rapidly induced, reaching peak levels within 34 days of culture (21).
By contrast, when cytotrophoblasts were maintained in a 2%
O2 environment, aromatase activity (Fig. 2
) and
P450arom mRNA levels (Fig. 3
) remained low. On the other hand, when
cells that had been cultured in 2% O2 for 3 days
were shifted to a 20% O2 environment, aromatase
activity was rapidly increased (Fig. 2
); this was associated with an
increase in levels of P450arom mRNA (Fig. 3
). Conversely, when
trophoblasts that had been cultured in 20% O2
for 3 days were transferred to a 2% O2
environment, aromatase activity declined rapidly (data not shown). It
should be noted that the aromatization reaction requires molecular
O2. To ensure that the assayed aromatase activity
was a reflection of the effects of atmospheric O2
on CYP19 gene expression and on levels of P450arom protein
and not merely due to availability of molecular
O2, the 1-h incubations with
[3H]androstenedione substrate were carried out
in a 20% O2 environment regardless of the
O2 tension of the prior incubation. Since both
aromatase activity and P450arom mRNA levels were markedly reduced under
hypoxic conditions, we consider it likely that the low aromatase
activity was due to effects of hypoxia on CYP19 gene
expression.

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Figure 2. Induction of Aromatase Activity in Human
Trophoblast Cells in Culture Is Prevented by Incubation in a 2%
O2 Environment
Freshly isolated cytotrophoblasts plated at a density of 2 x
106 cells per dish were maintained in either 2% or 20%
O2 environment for up to 6 days. After 3 days, some dishes
that had been maintained in 2% O2 were shifted to 20%
O2 and cultured for 3 additional days. Aromatase activity
(picomoles of androgen metabolized to estrogen/mg/min) was analyzed
every 24 h by assaying the incorporation of tritium from
[1ß-3H]androstenedione into water. Shown are the
means ± SEM of data from three independent
experiments each conducted in triplicate.
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Figure 3. Hypoxia Prevents Induction of CYP19 Gene
Expression in Trophoblast Cells in Culture
Freshly isolated cytotrophoblasts were cultured in 2% or 20%
O2 for up to 6 days. After 3 days, some dishes cultured in
2% O2 were shifted to the 20% O2 environment
and cultured for another 3 days. Aliquots (20 µg) of total RNA
obtained from cytotrophoblasts before and after culture under the
different conditions were analyzed for P450arom mRNA transcripts by
Northern blotting using a 32P-labeled human P450arom cDNA
probe. This experiment was repeated three times with similar results.
Shown is an autoradiogram of Northern blot from a representative
experiment. The blot was reprobed for 28S rRNA as a control for RNA
loading and transfer. In cells cultured for 3 and 6 days in 2%
O2, P450arom mRNA levels were reduced by >85% as compared
with normalized values for cells cultured for those periods in 20%
O2.
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Expression of the HLH Transcription Factors, Mash-2 and Id1 and
Id2, Is Elevated in Freshly Isolated Cytotrophoblasts and Declines in
Association with Syncytiotrophoblast Differentiation: Evidence for
Induction by Hypoxia
To analyze changes in expression of the HLH transcription factors,
Mash-2, Id1, and Id2, during trophoblast differentiation, as well as
the effects of oxygen, RNA was isolated from cytotrophoblasts before
culture and after 3 days of monolayer culture either in a 2% or 20%
O2 environment. The levels of Mash-2 and Id2 mRNA
transcripts were analyzed by Northern analysis of 3 µg
poly(A)+ RNA, whereas the levels of Id1 mRNA were
analyzed by Northern analysis of 20 µg of total RNA because of its
relative abundance (Fig. 4
). As can be
seen, mRNA transcripts for Mash-2, Id1, and Id2 were detectable in
cytotrophoblasts before culture. After 3 days of culture in a 20%
O2 environment, the mRNA levels for all three HLH
proteins declined, as compared with those in the cells before culture.
On the other hand, when trophoblast cells were maintained in a 2%
O2 environment, the levels of Mash-2 and Id1 were
increased as compared with cells cultured in 20%
O2, while Id2 mRNA declined to levels even lower
than those observed in cells cultured in 20% O2
(Fig. 4
). These findings suggest that expression of Mash-2
and Id1 are induced by hypoxia and may mediate the
inhibitory effects of low oxygen on CYP19 gene
expression.

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Figure 4. Effects of Low Oxygen on Expression of bHLH
Transcription Factors
In the Northern blots shown on the left,
poly(A)+ RNA (3 µg/lane) obtained from freshly isolated
cytotrophoblasts (Cyto) and trophoblast cells cultured in atmospheres
containing either 2% O2 or 20% O2 for 3 days
were probed using 32P-labeled cDNAs encoding Mash-2, Id2,
and ß-actin (as a control for loading and transfer). In cells
cultured in 2% O2, the levels of Mash-2 mRNA were
increased by 7.5-fold over the normalized values for cells cultured in
20% O2. In the Northern blots shown on the
right, total RNA (20 µg/lane) obtained from freshly
isolated cytotrophoblasts and trophoblast cells cultured in atmospheres
containing either 2% O2 or 20% O2 for 3 days
were analyzed using 32P-labeled probes for Id1 and 28S rRNA
(control for RNA loading and transfer). This experiment was repeated
twice with similar results. Shown is a representative Northern blot.
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Effects of Mash-2, Id1, and Id2 on CYP19 Promoter Activity in
Transfected Choriocarcinoma Cells
To analyze effects of Mash-2, Id1, and Id2 on CYP19
promoter activity, BeWo choriocarcinoma cells were transfected with
fusion genes containing either 42 (basal promoter), 125, 201, 246, or
501 bp of DNA flanking the 5'-end of placenta-specific exon I.1 of the
human CYP19 gene, linked to the hGH structural
gene, as reporter. In previous studies, we observed that the 125, 246,
and 501 bp-containing fusion genes were expressed at relatively high
levels in human trophoblast cells in primary culture, whereas levels of
expression of the 42 bp-containing construct were barely detectable
(21). In the present study, the choriocarcinoma cells were
cotransfected either with empty pCMV expression vectors, or
with pCMV expression vectors containing Mash-2,
Id1, or Id2. Mash-2 markedly inhibited expression
of the -125, -201, -246, and -501 bp-containing fusion genes after
72 h of incubation, as well as at earlier time points (data not
shown), whereas, no effects on the basal promoter construct (Fig. 5
) or on the promoterless hGH-containing
plasmid (pACsk2.0GH) (data not shown) were
observed. Furthermore, Id1 and Id2 had only a modest effect to inhibit
expression of the
CYP19I.1-501:hGH fusion gene
(Fig. 5
). These findings suggest that the increased levels of
Mash-2 expression observed under hypoxic conditions may
block induction of CYP19I.1 promoter activity in trophoblast
cells either directly or indirectly through exon I.1 5'-flanking
sequences between -42 and -125 bp.

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Figure 5. Effects of Overexpression of the HLH Factors
Mash-2, Id1, and Id2 on Expression of Cotransfected Human
CYP19I.1:hGH Fusion Genes in Human BeWo
Choriocarcinoma Cells
BeWo cells were transfected with either
CYP19I.1-42:hGH, CYP19I.1-125:hGH,
CYP19I.1201:hGH,
CYP19I.1-246:hGH, or
CYP19I.1-501:hGH fusion gene
constructs (4.5 µg each). The cells were cotransfected either with
empty expression plasmid or with expression plasmid containing a cDNA
encoding Mash-2 (2.0 µg). In parallel, BeWo cells transfected with
the CYP19I.1-501:hGH fusion gene construct
(4.5 µg) were cotransfected either with Id1, or Id2 cDNAs (2.0 µg
each). Each dish also was transfected with ß-galactosidase expression
vector (0.5 µg). After 72 h of incubation, the media were
collected for assay of hGH and of ß-galactosidase activity.
ß-Galactosidase values were used to correct for transfection
efficiency. Shown are the corrected means ± SEM
of data from three independent experiments each conducted in
triplicate.
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Effects of Mash-2 on Syncytiotrophoblast Differentiation and CYP19
Gene Expression in Human Trophoblast in Primary Culture
To determine the effects of overexpression of Mash-2 on
trophoblast differentiation and CYP19 gene expression, we
generated recombinant adenoviruses containing CMV/Mash-2 and
used these to infect human cytotrophoblast cells maintained in a 20%
O2 environment. Freshly isolated
cytotrophoblasts, plated at a density of 2 x
106 cells per 35-mm dish in 2% FBS, were
infected with the recombinant adenoviruses containing either
CMV/Mash-2 or CMV/ß-gal at a
multiplicity of infection (m.o.i.) of 5.0. After 72 h of culture,
the cells were stained with hematoxylin and eosin and viewed by light
microscopy. When cells were incubated either in the absence of
recombinant adenovirus (Fig. 6A
) or in
the presence of adenovirus containing CMV/ß-gal
at a m.o.i. of 5.0 (Fig. 6B
), there was clear evidence of syncytia
formation. By contrast, when the cells were incubated with adenovirus
containing CMV/Mash-2 (m.o.i. = 5.0), syncytia formation was
markedly inhibited (Fig. 6C
).

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Figure 6. Mash-2 Inhibits Differentiation of Human
Trophoblasts in Primary Culture
Freshly isolated human cytotrophoblasts plated on 35-mm dishes at a
density of 2 x 106 cells per dish were infected with
recombinant adenoviruses containing either CMV/Mash-2 or
CMV/ß-gal at a m.o.i. of 5.0. Some
dishes were not infected. After 72 h of incubation, the cells were
stained with hematoxylin and eosin and viewed by light microscopy.
Shown are micrographs (x200) of uninfected cells (panel A), cells
infected with CMV/ß-gal (panel B) or
CMV/Mash-2 (panel C). This experiment was repeated three
times with similar results.
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When CYP19 gene expression in the virus-infected primary
human trophoblast cells was analyzed by Northern analysis of P450arom
mRNA, we observed that CMV/Mash-2 caused a dose-dependent
inhibition of P450arom mRNA induction. An inhibitory effect of
CMV/Mash-2 was observed at a m.o.i. as low as 0.5. On the
other hand, no effect on P450arom mRNA induction was observed in cells
infected with CMV/ß-gal at comparable m.o.i.
(Fig. 7
). It should be noted that the
levels of P450arom mRNA in trophoblast cells infected with
Mash-2-expressing adenovirus were similar to those of cells cultured
for 72 h in a 2% O2-containing
environment.

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Figure 7. Overexpression of Mash-2 Inhibits the Induction of
P450arom Gene Expression in Trophoblast Cells in Culture
Freshly isolated cytotrophoblasts were infected by overnight incubation
with recombinant adenoviruses expressing CMV/Mash-2 or
CMV/ß-gal (m.o.i. = 0.55.0). After
72 h of further incubation in a 20% oxygen-containing
environment, RNA was isolated and analyzed for mRNAs encoding P450arom,
Mash-2, and ß-actin (as an index of loading and transfer). RNA also
was isolated from an aliquot of the cytotrophoblast cells before
culture (Cyto) and from uninfected trophoblast after culture for
72 h in a 20% O2- (Syn) or 2% O2- (2%
O2) containing environment. Aliquots (20 µg) of total RNA
obtained from cytotrophoblasts before and after culture under the
different conditions were analyzed by Northern blotting using
32P-labeled cDNAs encoding human P450arom, rat Mash-2, and
human ß-actin. Shown is an autoradiogram of a representative Northern
blot of an experiment that was repeated three times with comparable
results. In trophoblasts infected with Mash-2 expressing adenovirus at
a m.o.i. = 5.0, P450arom mRNA levels were reduced by 85% as compared
with corrected values in cells infected with comparable amounts of
adenovirus expressing CMV/ß-gal.
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To determine the effects of Mash-2 on CYP19I.1
promoter activity in human trophoblast cells in primary culture,
freshly isolated cytotrophoblasts were coinfected with recombinant
adenoviruses containing
CYP19I.1-501:hGH (m.o.i. = 0.5)
alone or in combination with either CMV/Mash-2 or with
CMV/ß-gal (as control) at a m.o.i. of 1.0, 5.0,
10.0, or 20.0. As can be seen, coinfection with Mash-2-expressing
adenovirus caused a dose-dependent inhibition of CYP19I.1
promoter activity; pronounced inhibition was found at m.o.i. values
5.0. Only a modest inhibitory effect on
CYP19I.1-501:hGH expression was
observed upon infection of control CMV/ß-gal
expressing adenovirus at a m.o.i. as high as 20 (Fig. 8A
).

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Figure 8. Mash-2 Causes a Dose-Dependent Inhibition of
Expression of Cotransfected CYP19I.1:hGH Fusion Genes in
Human Trophoblast Cells in Primary Culture
A, Freshly isolated human trophoblast cells in primary culture were
plated at a density of 2 x 106 cells per 35-mm dish
and infected overnight with recombinant adenovirus containing the
CYP19I.1-501:hGH fusion gene (m.o.i. =
0.5) in the absence (Control) or presence of coinfection with
recombinant adenoviruses containing either
CMV/ß-gal or CMV/Mash-2
at m.o.i. values of 1, 5, 10, or 20. Media were collected and changed
daily for a 72-h period. Shown is the accumulation of hGH in the media
between 48 and 72 h of incubation. Data are the means ±
SEM of triplicate values from a single experiment. B,
The freshly isolated trophoblast cells were coinfected with recombinant
adenoviruses containing either
CYP19I.1-42:hGH,
CYP19I.1-125:hGH, CYP19I.1201:hGH,
CYP19I.1-246:hGH, or
CYP19I.1-501:hGH fusion genes (m.o.i. = 0.5) and
with recombinant adenoviruses containing
CMV/ß-gal (m.o.i. = 10.0) or
CMV/Mash-2 (m.o.i. = 10.0). Culture media were changed
daily. After 72 h of incubation, the media were assayed for hGH.
Shown is the accumulation of hGH in the media between 48 and 72 h
of incubation. Data are the means ± SEM of
triplicate values from a single experiment.
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To define the CYP19I.1 5'-flanking sequences that
mediate the inhibitory effects of Mash-2 on CYP19 promoter
I.1 activity in primary human trophoblast cells, freshly isolated
cytotrophoblasts were coinfected with recombinant adenoviruses
containing CYP19I.1:hGH fusion genes comprised of 42,
125, 201, 246, and 501 bp of exon I.1 5'-flanking sequence
(m.o.i. = 0.5) and recombinant adenoviruses containing either
CMV/ß-gal or CMV/Mash-2 (m.o.i. =
10.0). The relative levels of expression of the various fusion gene
constructs in the trophoblast cells were similar to that reported
previously (21); the
CYP19I.1-246:hGH fusion gene was
expressed at the highest level, while expression of the basal
promoter construct
(CYP19I.1-42:hGH) was barely
detectable. In those studies, we had observed that, whereas expression
of the CYP19I.1-501:hGH fusion gene
was specific for the primary cultures of human placental cells,
the CYP19I.1-125:hGH,
CYP19I.1-201:hGH, and
CYP19I.1-246:hGH fusion
genes also were expressed at relatively high levels in lung and kidney
cell lines (21). As we had observed in the transfected BeWo cells (Fig. 5
), Mash-2 had a pronounced inhibitory effect on expression of all
CYP19I.1:hGH fusion genes except the basal promoter
construct (Fig. 8B
).
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DISCUSSION
|
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When cytotrophoblasts from midgestation or term human
placenta are maintained in primary monolayer culture in a 20%
O2 environment, they rapidly fuse to form
syncytia (21, 40). These morphological changes are temporally
associated with a marked induction of aromatase activity and
CYP19 gene expression (21). On the other hand, when
cytotrophoblasts are cultured in a hypoxic environment, cell fusion is
prevented (32). In the present study, we observed that the induction of
aromatase activity and CYP19 expression that occurs in
association with spontaneous differentiation of human cytotrophoblasts
to syncytiotrophoblast in culture is oxygen dependent. When
trophoblasts were incubated in a 2% O2
environment, induction of aromatase activity and of CYP19
gene expression were prevented in association with an inhibition of
cell fusion and increased incorporation of BrdU into DNA. These effects
of hypoxia on aromatase expression, DNA synthesis, and syncytia
formation were rapidly reversed when the trophoblasts were transferred
to a 20% O2 environment.
Oxygen has been proposed to serve an important role in the modulation
of trophoblast differentiation vs. proliferation (32, 34).
During human placental development, cytotrophoblast stem cells follow
two alternate pathways of differentiation (41). Along one pathway,
cytotrophoblasts detach from their basement membranes and aggregate to
form columns. The cells within these columns rapidly proliferate and
invade the uterine endometrium, part of the myometrium, and spiral
arterioles, which are remodeled and enlarged. In the other pathway of
differentiation, cytotrophoblasts within the floating chorionic villi
also detach from their basement membranes and fuse to form
multinucleated syncytiotrophoblast, which are bathed in maternal blood.
The remodeling of the spiral arterioles results in a pronounced
increase in blood flow to the intervillous space and concomitant
increase in O2 availability to
syncytiotrophoblast cells of the floating chorionic villi, which
produce polypeptide hormones, as well as steroid hormones and
steroid-metabolizing enzymes, including aromatase P450 (35).
Interestingly, estrogen biosynthesis by the human placenta increases
markedly after the ninth week of gestation (3), coinciding with the
time that cytotrophoblast invasion, remodeling, and enlargement of the
uterine arterioles are initiated. The findings of the present study
suggest that placental vascularization and increased
O2 availability are critical for the increase in
placental estrogen production during pregnancy. In the
pregnancy-associated disease preeclampsia, in which the placenta is
relatively hypoxic, cytotrophoblast proliferation is increased and
differentiation into invasive cells is reduced. Thus, fewer arterioles
are invaded and O2 availability to the
hormone-producing syncytiotrophoblast is reduced (37). Such pregnancies
are frequently associated with increased synthesis of progesterone,
decreased synthesis of estrogens and insulin-like growth factor-1, as
well as fetal growth retardation (39). In a recent epidemiological
study, it was found that women who either were offspring of a
preeclamptic pregnancy, or who experienced preeclampsia in their own
pregnancies, have a reduced breast cancer risk (39).
HLH transcription factors have been suggested to serve important roles
in trophoblast differentiation (27). This is of particular interest in
consideration of the role of bHLH transcription factors, including
MyoD, myogenin, myf-5, and mrf-4, in the differentiation of skeletal
myoblasts to form differentiated multinucleated myotubes (42). These
skeletal muscle-specific transcription factors bind to so-called E-box
sequences in DNA as obligate heterodimers with the ubiquitously
expressed E-factors E12 or E47 (43, 44). On the other hand, the HLH
factor Id (inhibitor of differentiation), which lacks a basic
DNA-binding domain, acts as a negative regulator of differentiation
because of its ability to sequester E12 and E47, preventing their
interaction with and DNA binding of muscle-specific bHLH proteins (25).
In the present study, we observed that mRNAs for the bHLH factors
Mash-2, Id1, and Id2 were readily detectable in freshly isolated
cytotrophoblasts from midgestation human placenta and were markedly
decreased upon differentiation to syncytiotrophoblast in 20%
O2. By contrast, when cytotrophoblasts were
cultured in 2% O2, mRNA levels for Mash-2 and
Id1 remained elevated.
Intriguingly, in the present study we observed that overexpression of
Mash-2 in primary cultures of human trophoblast cells
mimicked the effects of culture in low (2%) O2,
in that Mash-2 prevented cell fusion and markedly inhibited the
induction of expression of the endogenous CYP19 gene. Both
in BeWo choriocarcinoma cells and in human trophoblast cells in
primary culture transfected with CYP19I.1:hGH reporter
gene constructs, cotransfection of CMV/Mash-2 markedly
reduced expression of fusion genes containing 125, 201, 246, or 501 bp
of CYP19 exon I.1 5'-flanking sequences; however, no effect
of Mash-2 was observed on expression of a basal promoter construct
(CYP19I.1-42:hGH). Expression
levels of CYP19I.1:hGH fusion genes containing 125501 bp
of CYP19 exon I.1 5'-flanking DNA also were reduced upon
culture in 2%, as compared with 20% O2
(our unpublished observations). In contrast to our findings with
Mash-2, overexpression of Id1 or Id2 in the cotransfected BeWo
cells had little effect on CYP19 promoter I.1 activity. The
lack of effect of Id1 and Id2 on CYP19 promoter expression
may possibly be explained by the recent finding that E12 and E47 mRNAs
are undetectable in human trophoblasts before or after differentiation
in culture (41).
Mash-2 protein was reported to bind to an E-box of a muscle-specific
enhancer as a heterodimer with E12 (45). Our findings in deletion
mapping studies using human trophoblast in primary culture, as well as
BeWo cells, suggest that a Mash-2-responsive element(s) may be
localized between -42 and -125 bp. This 83-bp region contains an
E-box consensus sequence that could possibly serve as a binding site
for Mash-2 or another bHLH transcription factor with which Mash-2
interacts. However, mutation of this E-box in the context of a
CYP19I.1:hGH fusion gene containing 246 bp of exon I.1
5'-flanking region had no effect on basal expression or on the action
of Mash-2 to inhibit fusion gene expression. Additionally, in
electrophoretic mobility shift assays utilizing in vitro
transcribed/translated Mash-2 in the absence or presence of coexpressed
E12, we were unable to demonstrate direct binding of Mash-2 to this
site (our unpublished observations). As noted above, E12 and E47
are not expressed in human trophoblast; thus, Mash-2 may interact with
and prevent the binding and/or activity of a stimulatory transcription
factor(s) that binds to this region. On the other hand, Mash-2 may not
bind to a response element within the 5'-flanking sequence of the
CYP19 gene, but may rather block the program of trophoblast
differentiation and prevent the expression of one or more stimulatory
transcription factors that bind to response elements upstream of
promoter I.1 and are essential for developmental induction of
CYP19 gene expression.
In addition to its effects on CYP19 gene expression in the
human trophoblast, Mash-2 likely affects other processes involved in
trophoblast differentiation. In mice, targeted mutation of the
mash-2 gene resulted in death of embryos at 10 days post
coitum due to placental failure (29). The mutant placentas lacked
spongiotrophoblasts and had an increased number of giant cells
(nonproliferative differentiated cells) (29). It is apparent that
Mash-2 is required for generation and maintenance of spongiotrophoblast
precursors and could possibly block differentiation of giant cells.
Mash-2 expression also was found to be dramatically
decreased during differentiation of a rat choriocarcinoma cell line
(RCHO-1 cells), into giant cells in culture (27).
Unlike the human, the mouse placenta does not synthesize estrogens or
express P450arom (46). However, in studies using transgenic mice, we
found that human CYP19I.1-501:hGH fusion genes
were highly expressed in a developmental and placenta-specific manner,
exclusively within the labyrinthine trophoblast (47). The labyrinthine
layer contains syncytial cells that are analogous to the
syncytiotrophoblast of human placenta. These findings suggest that the
transcription factors that mediate placenta-specific expression of the
CYP19 gene are highly conserved among species, although the
response elements that bind these factors within the mouse
cyp19 gene are not. Moreover, the activating transcription
factors are likely restricted to the mouse labyrinthine trophoblast,
further suggesting its analogy to the human syncytiotrophoblast.
Interestingly, transgene expression is first detected in the mouse
placenta at E10.5, a time when Mash-2 expression is greatly
diminished (48). The decrease in endogenous Mash-2
expression in association with elevated expression of the
CYP19I.1-501:hGH transgene suggests that the
human homolog of this transcription factor may normally block
CYP19 gene expression in proliferating cytotrophoblasts.
The findings presented herein, therefore, suggest for the first
time that Mash-2 may function as a "hypoxia-induced" transcription
factor that blocks differentiative function of the syncytiotrophoblast
layer. A number of HLH transcription factors that contain a PAS
(per-ARNT-Sim) domain have been implicated as mediators of the hypoxic
response. These include hypoxia-inducible factor (HIF)-1
, HIF-1ß
(ARNT), and human endothelial PAS protein-1 (EPAS-1). HIF-1
and
EPAS-1 protein levels have been found to be markedly increased during
hypoxia and to increase transcription of a number of
hypoxia-induced genes (49). Surprisingly, expression levels of both
HIF-1
and ARNT were found to be markedly increased during human
trophoblast differentiation in 20% O2, while
EPAS-1 expression remained relatively unchanged (41). When the cells
were cultured under hypoxic conditions, the levels of HIF-1
and ARNT
decreased slightly, while EPAS-1 levels were relatively unaffected
(41). Since HIF-1
(50) and EPAS-1 (51) are primarily regulated at
the level of protein stability, their roles as hypoxia-regulated
transcription factors in trophoblast differentiation remain to be
defined.
In summary, we have found that hypoxia prevents the induction of
CYP19 gene expression that occurs in association with
differentiation of human trophoblast cells in culture. Culture of
trophoblast cells in a hypoxic environment prevented the decline in
Mash-2 expression that normally occurs in association with human
cytotrophoblast differentiation. The finding that Mash-2
overexpression inhibited fusion of cytotrophoblasts to form
syncytiotrophoblast and the associated induction of endogenous
CYP19 gene expression, and markedly reduced expression
CYP19 promoter activity in transfected placental cells,
suggests that Mash-2 may play an important role as a hypoxia-inducible
transcription factor in human trophoblast. We suggest that with
increased vascularization of the placenta and increased oxygen
availability to the cytotrophoblast cells of the chorionic villi, there
is decreased expression of Mash-2. This removes the brakes on the
cascade of cellular differentiation and placental hormone production.
Studies are in progress to define the mechanisms whereby Mash-2 acts to
regulate syncytiotrophoblast differentiation and the induction of
CYP19 gene expression.
 |
MATERIALS AND METHODS
|
---|
Primary Culture of Human Trophoblast Cells and Maintenance of
Choriocarcinoma Cell Lines
Midtrimester human placental tissues were obtained in accordance
with the Donors Anatomical Gift Act of the State of Texas after written
consent had been 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 primary
culture system developed by Kliman et al. (40) was modified
for isolation and culture of cytotrophoblasts from midgestation human
placenta (21). The placental tissues were washed with HBSS, pH 7.4
(Life Technologies, Inc., Gaithersburg, MD), and then
finely minced and digested with 0.125% trypsin in HBSS at 37 C for 30
min. This procedure was repeated three times. At the end of each
digestion step, the supernatant was collected, layered over 10 ml
serum, and then briefly centrifuged. The pellet was suspended in DMEM
(Life Technologies, Inc.), filtered, and then layered over
a Percoll gradient (70%5%). The gradients were centrifuged at
1,200 x g for 20 min at room temperature, and cells in
the middle layer (density 1.0451.062) were collected, washed, and
counted. The cells were resuspended in DMEM supplemented with 10% FBS
and 1% antibiotic/antimycotic solution (Life Technologies, Inc.) and plated at a density of 2 x
106 cells per dish in 35-mm culture dishes coated
with extracellular matrix (ECM). The cells were then incubated at 37 C
in a humidified atmosphere of either 95% air/5%
CO2 (20% O2) or placed in
a modular incubator chamber (Billups-Rothenberg, Inc., Del Mar, CA) in
which they were maintained in an atmosphere containing 2%
O2, 93% N2, 5%
CO2. Cells were cultured overnight and the medium
was then changed to 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 0.5%
deoxycholate for 5 min. The ECM dishes were washed three times with
HBSS and stored at 37 C until used. BeWo cells were purchased from ATCC
and grown in F-12K medium (Life Technologies, Inc.)
containing 15% FBS.
Tritiated Water Assay of Aromatase Activity in Placental
Cells
Aromatase activity was assayed in placental cells in monolayer
culture using a tritiated water assay as described previously (52). The
[1ß-3H]androstenedione (NEN Life Science Products, Boston, MA) was added to the culture medium
for 1 h. Culture medium was then removed and placed in 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. Aromatase
activity is expressed as picomoles of
[1ß-3H]androstenedione metabolized to
estrogen/min/mg protein. The adherent cells were analyzed for protein
by the method of Lowry et al. (53).
Northern Analysis of Placental Cell mRNAs
Total RNA was isolated from placental cells by the method of
Chirgwin et al. (54). Briefly, cells in monolayer culture
were washed with PBS, lysed in 4 M guanidinium
isothiocyanate, followed by centrifugation for 16 h at 42,000 rpm
through a cesium chloride cushion (5.7 M). The
RNA samples were then ethanol precipitated, resuspended in water, and
quantitated at 260 nm. Cytotrophoblast and syncytiotrophoblast
polyadenylated RNA was isolated using Poly(A) Quick mRNA Isolation
Kit (Stratagene, La Jolla, CA) following the
manufacturers protocol. Total RNA (20 µg) or poly
(A)+ RNA (3 µg) was size-fractionated on a
7.4% formaldehyde/0.9% agarose gel, transferred to Zeta-Probe
Blotting Membrane (Bio-Rad Laboratories, Inc. Hercules,
CA), and then hybridized overnight at 65 C to a
32P-labeled human aromatase cDNA probe (55) made
by Prime-It RmT Random Primer Labeling Kit (Stratagene). A
1.5-kb rat Mash-2 cDNA insert (kindly provided by Dr. Jane Johnson,
University of Texas Southwestern) and cDNA inserts (1.1 kb) for human
Id1 and Id2 (kindly provided by Dr. Judith Campisi, University of
California, Berkeley) were used as probes for detecting mRNAs encoding
the human homolog of Mash-2, Id1, and Id2 expression, respectively. The
membrane was then washed in 0.1% SDS-0.1x NaCl-sodium citrate (SSC)
solution, and the relative levels of mRNA were assessed by
autoradiography. Northern blots were reprobed with ß-actin cDNA
(ATCC, Manassas, VA) or 28S rRNA to assess loading and
transfer of RNA. Autoradiograms were scanned by densitometry, and
relative levels of specific mRNA were normalized either to ß-actin
mRNA or 28S rRNA to correct for RNA loading and transfer.
Morphological Analysis
Cells were cultured on glass coverslips in DMEM containing 2%
FBS in atmospheres of 20% or 2% O2, as
described above. After culture, the cells were rinsed with PBS and
fixed in 75% ethanol. Hematoxylin and eosin Y were used to stain
nuclei and cytoplasm, respectively. Morphology was analyzed by light
microscopy.
BrdU Incorporation
Cells were cultured on glass coverslips in DMEM containing
2% FBS in atmospheres of 20% or 2% O2. After
48 h, the culture medium was aspirated, and fresh medium
containing 1 µM BrdU (Sigma, St. Louis, MO)
was added (34). After 24 h of further incubation, the cells were
rinsed with PBS and fixed in absolute methanol at 4 C. Cellular DNA was
denatured by incubating the coverslips in 2 M HCl for 60
min at 37 C. The acid was neutralized by immersing the coverslips in
0.1 M borate buffer, pH 8.5. The cells were then incubated
with anti-BrdU-fluorescein (Roche Molecular Biochemicals,
Indianapolis, IN). Cells were counterstained with hematoxylin. The
coverslips were then dehydrated and mounted on slides.
Plasmid Constructs
A cDNA containing the protein coding sequence of rat Mash-2 (783
bp) (provided by Dr. Jane Johnson, University of Texas Southwestern)
was subcloned into the multiple cloning site of the expression plasmid
pCMV5 (provided by Dr. David W. Russell, University of Texas
Southwestern). pCMV1-Id1 and pCMV1-Id2 were
provided by Dr. Judith Campisi (University of California, Berkley).
Fusion genes comprised of 501, 246, 201, 125, and 42 bp of 5'-flanking
sequence and 103 bp of untranslated exon I.1 of the human
CYP19 gene fused to the human GH structural gene
(hGH) as reporter were constructed as described previously
(21).
Cell Transfection Assays
Some of the cotransfection assays were carried out using BeWo
choriocarcinoma cells, since human trophoblast cells in primary culture
cannot be transfected with plasmid DNA using standard techniques (21).
The day before transfection, 4 x 105 cells
were plated onto 60-mm culture dishes and maintained in 4 ml of growth
medium overnight. All cells were transfected with the same amount of
plasmid DNA (2 µg reporter plasmid, 2 µg expression plasmid or
corresponding empty expression vector, and 0.5 µg ß-gal plasmid).
The plasmid DNAs, to be added to each culture dish, were combined in
150 µl of F-12K medium that was devoid of FBS or antibiotics. Thirty
microliters of SuperFect transfection reagent (QIAGEN,
Valencia, CA) were added to each tube containing plasmid DNA, and the
samples were incubated for 510 min to allow complex formation. One
milliliter of cell growth medium containing serum and antibiotics was
added to each reaction tube containing the plasmid DNA and transfection
reagent; the total content of each tube was then added to each dish of
cultured cells. Cells were then incubated for 3 h at 37 C in an
atmosphere of 95% air/5% CO2. The media were
then removed by gentle aspiration, and the cells were washed once with
4 ml of PBS. F-12K medium containing 15% FBS (2 ml) was added to each
dish. Media were then collected every 24-h for RIA of hGH using a kit
(Nichols Institute Diagnostics, San Juan Capistrano, CA).
ß-Galactosidase assays were performed using Galato-light kit (Tropix,
Inc., Bedford, MA) for normalization of transfection efficiency.
Preparation of Recombinant Adenovirus and Infection of Placental
Cells
To generate a Mash-2 recombinant adenovirus, we used a protocol
involving in vivo recombination in bacteria (56). In short,
the coding sequence of the rat Mash-2 cDNA was subcloned into the
pShuttle vector containing a subcloned CMV promoter to
generate pShuttle-CMV/Mash-2. The recombinant adenoviral
CMV/Mash-2 was generated by cotransformation of
pShuttle-CMV/Mash-2 and pAdEasy-1 into electrocompetent
BJ5183 bacteria. 293 cells, a permissive human embryonic kidney cell
line that expresses adenoviral E1A, were transfected with
PacI-digested recombinant adenoviral CMV/Mash-2
DNA for recombinant adenoviral packaging and propagation. Viral plaques
were isolated and propagated to produce a lysate containing infectious
recombinant virus. Viral DNA was analyzed to confirm the presence of
CMV/Mash-2 by restriction endonuclease digestion, PCR, and
DNA sequencing (ABI 377 automated sequencer). The recombinant virus was
then titered in 293 cells three times to determine the number of
infectious particles (plaque-forming units).
CMV-ß-gal adenovirus was kindly provided by Dr.
Joseph Alcorn (University of Texas Medical School, Houston).
Recombinant adenoviruses containing fusion genes comprised of 42, 125,
201, 246, and 501 bp of sequence flanking the 5'-end of exon I.1 of the
human CYP19 gene and 103 bp of exon I.1 fused upstream of
the hGH structural gene, as reporter, were constructed as described
previously (21).
Freshly isolated cytotrophoblasts plated at a density of 2 x
106 cells per 35-mm dish in DMEM containing 10%
FBS were infected with recombinant adenoviral particles at a m.o.i. of
0.520.0. After an overnight incubation, the media were removed and
replaced with fresh DMEM containing 2% FBS. The cells were incubated
for up to 72 h; media were collected and replaced daily and
analyzed for hGH by RIA.
 |
ACKNOWLEDGMENTS
|
---|
The authors gratefully acknowledge the technical expertise of
Vickey Chau, Margaret Smith, and Barbara Murry, and thank Drs. Margaret
Hinshelwood and Linda Margraf for their helpful discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Carole R. Mendelson, Ph.D., Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9038. E-mail: cmende{at}biochem.swmed.edu
This research was supported by NIH Grant R01 DK-31206.
Received for publication January 3, 2000.
Revision received June 9, 2000.
Accepted for publication July 7, 2000.
 |
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