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INTRODUCTION |
In all mammalian species, progesterone plays an essential role in
reproduction. The precise timing of both the synthesis and degradation
of this steroid hormone is crucial for reproductive success. The
expression of enzymes implicated in the synthesis and catabolism of
progesterone, therefore, needs to be accurately regulated during the
different reproductive states of the animal. In rodents, the corpus
luteum, which is the only source of progesterone throughout pregnancy
(1), is also able to express the enzyme 20
-hydroxysteroid
dehydrogenase (20-HSD)1
that converts progesterone into a biologically inactive steroid, thus
playing a key role in the termination of pregnancy and allowing parturition to occur (2). Due to the detrimental effect of 20-HSD on
luteal progesterone secretion, the corpus luteum of pregnancy does not
express 20-HSD until 24 h before parturition (3). Our
laboratory has previously demonstrated that the rapid appearance of
luteal 20-HSD activity in the corpus luteum is due to the
massive increase in 20-HSD gene expression and not to
activation of an already existent enzyme (4).
Prostaglandin F2 (PGF2) administration to
pregnant (5-8) and pseudopregnant (9) rats increases luteal 20-HSD
activity. However, the molecular mechanism involved in 20-HSD
activation is not known. PGF2 is also known to induce
abortion in many species including rodents (5, 10). This last effect
seems to be primarily a luteolytic one since progesterone
administration prevents abortion. In rodents the participation of
PGF2 in luteolysis and the induction of labor was
further demonstrated recently by the finding that mice deficient in the
gene for the PGF2 receptor (PGF2-R) do not show the normal pre-partum drop in progesterone and do not exhibit
parturition (11, 12). However, whether the high levels of progesterone
secreted at the end of pregnancy in knockout mice are due to the
absence of 20-HSD activity and whether PGF2
stimulates the activity of an already present enzyme or
enhances/induces the expression of 20-HSD gene are still unknown.
To understand the molecular basis of PGF2 regulation of
20-HSD enzyme activity, we first examined the effect of
PGF2 administration in vivo on
20-HSD gene expression and the expression of 20-HSD mRNA in PGF2-R knockout mice. To determine whether
PGF2 stimulation involves transcriptional activation, we
used both primary rat ovarian cells and a corpus luteum-derived cell
line (GG-CL) to perform a series of gene transfer studies to examine
the mechanisms mediating the effect of PGF2 on 20-HSD
promoter activity. We have shown that PGF2 is
responsible for the abrupt luteal expression of 20-HSD at the end of
pregnancy. We have mapped a region located between position 
1599 and
1606 containing a NUR77 response element that contributes to
PGF2-mediated activation. We have also provided evidence
that PGF2 stimulates the expression of the NUR77
transcription factor, which transactivates the 20-HSD promoter. In
addition, by using an in vivo approach, we have shown that
inhibition of NUR77 binding to DNA prevents
PGF2-mediated stimulation of 20-HSD gene expression.
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EXPERIMENTAL PROCEDURES |
Chemicals--
Acrylamide and bisacrylamide were obtained from
Accurate Chemical Inc. (Westbury, NY); Kodak NTB-2 liquid emulsion and
Bio-Max AR Film were from Eastman Kodak Co.;
[-32P]dCTP was purchased from Amersham Pharmacia
Biotech; Advantage RT-for-PCR kit was purchased from
CLONTECH (Palo Alto, CA); dNTP, ExTaq
DNA polymerase and ExTaq buffer were purchased from Takara Biomedicals (Shiga, Japan); the nucleotides used as primers in the
RT-PCR analysis were obtained from Life Technologies, Inc.; Western
blotting Luminol Reagent was obtained from Santa Cruz Biotechnology
(Santa Cruz, CA); RPMI 1640 medium, nonessential amino acids, sodium
pyruvate, trypsin-EDTA, antibiotics, and antimycotics were purchased
from Mediatech (Herndon, VA). PGF2,
D-glucose, Tri-Reagent, aprotinin, leupeptin, PMSF, and all
other reagent-grade chemicals were purchased from Sigma.
Animals--
Pregnant Harlan Sprague-Dawley rats (day 1 = sperm-positive) purchased from Sasco Animal Labs (Madison, WI) were
housed at 24 °C with a 14-h light, 10-h dark cycle (lights on
0500-1900 h) and allowed free access to Purina Rat Chow and water.
PGF2 receptor knockout mice with a mixed genetic
background of 129/Ola and C57BL/6 strains were used (11, 12). Wild-type
and PGF2 receptor knockout mice were maintained at
23 °C under a 12-h light cycle. Virgin females (9-12 weeks of age)
housed overnight with males were checked the following morning for
vaginal plug. The day the plug was found was counted as day 1 of
pregnancy. Animal care and handling conformed to the National
Institutes of Health Guidelines for Animal Research. The experimental
protocol was approved by the Institutional Animal Care and Use Committee.
Granulosa-luteinized Cells--
27-28-day-old immature female
Harlan Sprague-Dawley rats (Sasco, Madison, WI) were injected with 15 IU of pregnant mare serum gonadotropin intraperitoneally
followed by 15 IU of human chorionic gonadotropin
intraperitoneally 46 h afterward. Seven hours later ovaries were
isolated and incubated sequentially in 6 mM EGTA in
DMEM/F-12 and 0.5 M sucrose in DMEM/F-12. Cells were
cultured at 37 °C in Dulbecco's modified Eagle's medium/Ham's
F-12 (DMEM/F-12, 1:1). After 3 days of culture, the cells were
transfected by LipofectAMINE (Life Technologies, Inc.) with 0.5 µg of
20-HSD-Luc constructs and 0.5 µg of a control 
-galactosidase
expression vector (Life Technologies, Inc.) following the
manufacturer's protocol. The cells were left overnight and then
treated with PGF2 for 12 h. To harvest cells, each
well was washed twice with ice-cold PBS.
GG-CL--
Cells derived from rat luteal cells were cultured in
RPMI 1640 medium supplemented with L-glutamate, 1 mM nonessential amino acids, 1 mM sodium
pyruvate, an additional 0.45% glucose, 1× antibiotics and
antimycotics, 10 µg/ml nystatin, and 10% FBS at 33 °C.
Trypsinized cells were suspended in OPTI-MEM I (Life Technologies,
Inc.) at 1 × 106 cells/0.8 ml and transfected by
electroporation with 0.5 µg of 20-HSD-Luc construct and 0.5 µg
of a control -galactosidase expression vector and a different amount
of NUR77 expression plasmid using the gene pulser (Bio-Rad) at a
capacitance setting of 975 microfarads and 280 V. The total amount of
DNA per well was kept constant by the addition of an empty vector.
After each electroporation, cells were pooled and resuspended in RPMI
1640 medium containing 10% FBS and plated into 6-well plates at a
density of 1 × 106 cells/well. Cells were cultured
overnight and then the medium was changed to phenol red-free RPMI 1640 with 1% FBS. Cells were maintained for a maximum of 48 h after
electroporation. To harvest cells, each well was washed twice with
ice-cold PBS.
Luciferase Activity Measurement--
Passive lysis buffer (100 µl) (Promega) was added into each well, and 20 µl of cell lysate
was used to measure both firefly luciferase activity driven by the
20-HSD promoter. 20 µl of cell lysate was also used to measure
-galactosidase activity using Promega's Luciferase Reporter or
-Galactosidase Assay System, respectively, in a Lumat LB 9507 Luminometer (EG & G Berthold). Relative light units were obtained by
dividing the luciferase activity by the -galactosidase activity.
Electrophoretic Mobility Shift Assay--
To prepared whole cell
extract, one 100-mm plate of GG-CL cells transfected with a NUR77
expression plasmid or empty vector were harvested in PBS and
centrifuged for 5 min at 12,000 × g to obtained the
pellet. The pellet were resuspended in 150 µl of 10 mM
Tris-HCl buffer, pH 7.9, containing 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 400 mM KCl, 1 mM vanadate, and leupeptin, aprotinin, and pepstatin (2 µg/ml). Cells and nuclei were lysed by three rapid freeze-thaw cycles
and centrifuged at 12,000 × g, and protein concentration in the supernatant was measured (Bradford method, Pierce). Nuclear extract from corpus luteum of day 19 pregnant rats was
obtained as described by Dignam et al. (13) with slight modifications. Corpora lutea were homogenized in solution A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM
MgCl2, 0.1 mM EGTA, 0.5 mM PMSF,
0.5 mM dithiothreitol), and the cells were broken by
plunging the cells 10-15 times in a Dounce homogenizer. The nuclear
pellet was obtained by centrifugation for 30 min at 4 °C in an
Eppendorf centrifuge and resuspended in solution B, which was similar
to solution A except that it contained 420 mM NaCl and 5%
(v/v) glycerol and no KCl. The solution was rocked for 30 min at
4 °C and then centrifuged at 14,000 × g at 4 °C
for 20 min. The supernatant containing the nuclear extract was divided
into portions and was stored at 80 °C. In vitro
translated NUR77 was produced with reticulocyte lysate kits purchased
from Promega (TNT SP6/T7). The efficiency of protein synthesis was
monitored by [35S]methionine labeling of the reaction
products, and about 10 ng of in vitro synthesized NUR77 was
used. For electrophoretic mobility shift assays, 10 µg of protein
extract were incubated with the NUR77-binding sites found in the rat
20-HSD promoter (distal site, 5'-GCC ATG TGG GTA CTG GAA AAT GAA CAC
AGA-3', or the proximal site, 5'-TAG CCT CTT AAA TGG TCA TTA TAA TTCA
CAA-3') (50,000 cpm, 20 fmol) in 20 µl of EMSA buffer (20 mM Hepes, pH 7.6, 1 mM dithiothreitol, 1.5 mM MgCl2, 10 µM
ZnCl2, 90 mM NaCl, 10% glycerol, NaF 20 mM), plus 2 µg poly(dI-dC) as nonspecific competitor were
incubated for 30 min at 22 °C. Competition assays were carried out
by including 10-, 50-, or 100-fold molar excess of unlabeled oligonucleotide in the reaction mixture. A 30-mer mutant
oligonucleotide 5'-GCC ATG TGG GTA CTG GAcgcT GAA CAC AGA-3' (lowercase
represents NUR77 RE mutations) was also used. For supershift assays,
0.5 or 1 µl of NUR77 antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the reaction mixture and incubated for 30 min at
22 °C before the addition of the labeled probe. The DNA protein complexes were separated from the unbound DNA probe by non-denaturing PAGE (4% gel) at 4 °C, in 0.5× Tris borate EDTA buffer (44 mM Tris-HCl, 44 mM boric acid, 12.5 mM EDTA, pH 7.5).
RNA Isolation and RT-PCR Analysis--
Total RNA from frozen rat
corpus luteum or mouse ovary was isolated using Tri-Reagent following
the manufacturer's instructions. For mRNA analysis by RT-PCR, 1 µg of total RNA was reverse-transcribed at 42 °C using Advantage
RT-for-PCR kit (Promega, Madison). The PCR mixture containing specific
oligonucleotide primers (20 pmol), [-32P]dCTP (2 µCi
of 3000 Ci/mmol), dNTP (150 µM), ExTaq DNA
polymerase (0.8 units), was added to each tube containing 5 µl of
reverse transcription product. Each PCR included primer for rat or
mouse ribosomal protein L19 mRNA used as internal control. Before
proceeding with the semi-quantitative PCR, the conditions were
established such that the amplification of the products was in the
exponential phase, and the assay was linear with respect to the amount
of input RNA. After autoradiography, data were analyzed using a
Molecular Dynamics PhosphorImager and ImageQuant version 3 software
(Molecular Dynamics, Sunnyvale, CA).
For the rat, the 20-HSD primers used were 5'-TAG GGC TGC CAT CTT AGT
ATT CA-3' and 5'-GAA TGC CAT CTT TAT CTC AAC CA-3'. For the
amplification of the NUR77 message the primers used were 5'-TCT GCT CAG
GCC TGG TGC TAC-3' and 5'-GGC ACC AAG TCC TCC AGC TTG-3' (14). For the
rat ribosomal protein L19, the primers used were 5'-GGA CAG AGT CCA AGG
GTC CGC TGC AGTC-3' and 5'-TCC AAG GGT CCG CTG CAG TC-3'. When a
co-amplification of NUR77, 20-HSD, and L19 message was performed the
following primers for 20-HSD were used, 5'-TGT ATC TCT GAG TTC CCA
GG-3' and 5'-ACT CTT CTA GGG AAG AGC AG-3' (15), and the above primer
for NUR77 and L19 with a protocol previously described (16). For mice,
the 20-HSD primers used were 5'-TCT TCG GTA CTT TCC TGC TGAT-3' and
5'-CTG GGG GTG AGT TGC TAAG-3' based on the luteal mouse 20-HSD
sequence (17). For mouse ribosomal protein L19 the primers used were 5'-AGC GCC TCC AGG CCA AGA AGG-3' and 5'-CCA GGC CGC TAT GTA CAG ACA
CGA-3' based on the sequence found in GenBankTM (accession
number AI225402).
Northern Blot Analysis--
Total RNA was fractionated by
electrophoresis in 1% agarose gels and blotted to nylon membranes.
Ethidium bromide staining indicated whether ribosomal RNAs were intact
and whether equal amounts of RNA were loaded in each lane and an equal
rate of transfer. Full-length of rat 20-HSD cDNA (18) or
rat NUR77 cDNA (19) was labeled with [-32P]dCTP
using the random hexamer primer and the Klenow fragment of
Escherichia coli DNA polymerase. Blots were prehybridized
overnight at 42 °C in a solution containing 40% formamide, 6× SSC,
5× Denhardt's, 20 mM Na2HPO4, pH
7.0, 0.2% sodium dodecyl sulfate, and 100 µg/ml heterologous DNA.
Hybridization was completed in the same solution containing
32P-labeled cDNA probe (1 × 106
cpm/ml) at 42 °C overnight. Blots were washed and then exposed to
Kodak X-AR films (Kodak) with intensifying screen at 80 °C.
Western Blotting Analysis--
Ovaries from wild-type or
PGF2 receptor knockout mice were homogenized on ice by
hand using a Potter-Elvejhem homogenizer in ice-cold lysis buffer (10 mM Tris-Cl, pH 8.0; 150 mM NaCl, 1% Nonidet
P-40, 0.5% sodium deoxycholate, 0.1% SDS, 40 µM PMSF, 0.3 µM aprotinin, and 1 µM leupeptin). This
was followed by a 30-min incubation on ice and centrifugation at
10,000 × g for 20 min at 4 °C. The supernatant was
transferred to new tubes, aliquoted, and stored at 70 °C until the
time of electrophoresis. An aliquot of the supernatant was kept for
protein measurement using bovine serum albumin as a standard. Samples
were denatured by adding sample buffer (62.5 mM Tris-HCl,
pH 6.8, 2% SDS, 10% glycerol, 0.01% bromphenol blue) followed by
boiling for 10 min. 30 µg of protein were separated on 10% SDS-PAGE
gels in Tris glycine, 0.1% SDS buffer, and transferred to
nitrocellulose paper in 25 mM Tris, 192 mM
glycine, and 20% methanol buffer at 250 mA for 1.5 h. The blots
were incubated for 2 h at room temperature in 5% non-fat dry milk
to block unspecific binding. The blots were then washed and incubated
with the polyclonal antibody against rat 20-HSD (20) (1:4000
dilution), overnight at 4 °C, and then washed and incubated with a
secondary antibody conjugated to horseradish peroxidase (1:6000
dilution) for 2 h at room temperature. Protein-antibody complexes
were visualized using Western blotting Luminol Reagent following the
manufacturer's protocol. The band densities were determined by digital
analysis using a Kodak Digital Science DC 120 Zoom Digital Camera and
Kodak Digital Science 1D 2.0.2 software (Kodak).
Radioimmunoassays--
Serum progesterone concentrations were
measured using a commercially obtained kit (Diagnostic Products Corp.,
Los Angeles, CA). The sensitivity of the assay was 0.02 ng/ml, and the
inter- and intra-assay coefficients of variations were 5 and 6%,
respectively. Serum 20-OH-progesterone was assayed after hexane
extraction using a highly specific antiserum kindly provided by Dr.
Quadri (Department of Anatomy/Physiology, Kansas State University,
Manhattan, KS). The sensitivity of the assay was 0.01 ng/assay tube,
and the inter- and intra-assay coefficients of variation were less than
10%.
Statistical Analysis--
One-way analysis of variance (ANOVA I)
followed by the Tukey test was used for the statistical analysis of
plasma steroid concentrations, relative mRNA expression, and
luciferase activity data using the Prism software (Graph Pad Software,
Inc., San Diego, CA). Values were considered statistically significant
at p < 0.05.
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RESULTS |
PGF2 Induces 20-HSD Gene Expression in Rats and
Mice--
To determine whether PGF2 can induce
20-HSD gene expression, either PGF2 (400 µg/rat
intraperitoneal) or vehicle (saline solution) was administered to rats
on day 19 of pregnancy 2 days before this gene becomes expressed in the
corpus luteum. Twenty four hours later (day 20 of pregnancy), corpora
lutea were dissected from each rat and pooled independently. Total RNA
was isolated and subjected to RT-PCR using L19 as an internal control.
As expected from previous results (4), no 20-HSD expression could be
detected in the corpus luteum of all vehicle-treated rats (Fig.
1A, left panel, lanes 1, 3, and 5). In contrast, high expression of 20-HSD mRNA was found in corpora lutea of PGF2-treated rats
(Fig. 1A, left panel; lanes 2, 4 and
6) accompanied by a significant reduction in the circulating
levels of progesterone (Fig. 1A, middle panel) and a rise in
the levels of 20-OH-progesterone (Fig. 1A, right panel).
To examine the time course of PGF2 stimulation, day 19 pregnant rats were injected with PGF2 (400 µg/rat
intraperitoneal) and sacrificed 0.5-24 h thereafter. Total RNA was
isolated and subjected to Northern blot analysis. The results (Fig.
1B) show that PGF2 treatment induces
20-HSD gene expression within 2 h, and mRNA levels
increased progressively for up to 24 h.
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Fig. 1.
Effect of PGF2
treatment on rat luteal 20-HSD mRNA
levels and serum progesterone and
20-OH-progesterone levels. A,
rats were injected with either 400 µg of PGF2
intraperitoneally (+) or vehicle () on day 19 of pregnancy. Luteal
20-HSD mRNA levels by RT-PCR and serum steroid levels by
radioimmunoassay were determined 24 h after administration.
Bars represent the mean ± S.E. (n = five animals). ***, p < 0.001 versus
vehicle (). B, rats on day 19 of pregnancy were treated
with 400 µg of PGF2 intraperitoneally, and corpora
lutea were isolated at 0.5-24 h thereafter. Total RNA was prepared,
and Northern blot analyses were performed by using 20 µg of each
sample and the rat 20-HSD cDNA as probe. Photography of the blot
membrane after transfer is shown as control of loading and
transfer.
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To confirm further the stimulatory effect of PGF2 on
20-HSD expression at the end of pregnancy in rodents, we examined by RT-PCR and Western blot analysis the expression of this gene on days
18-20 of pregnancy in wild-type and PGF2 receptor
knockout mice. 20-HSD mRNA (Fig.
2A) and protein (Fig.
2B) were undetectable on day 18 in corpora lutea of
wild-type mice but became abruptly expressed on day 19 and remained
elevated on day 20, the day of parturition. In sharp contrast, no
20-HSD expression could be detected in corpora lutea of
PGF2 receptor-deficient mice on any day examined,
establishing further the importance of PGF2 in the
induction of 20-HSD at the end of pregnancy. This also provides an
explanation as to why progesterone secretion remains elevated and why
parturition is prevented in PGF2 receptor knockout mice.
Indeed, along with the appearance of 20-HSD protein in wild-type
mice corpora lutea was a rise in the circulating levels of
20OH-progesterone and a drop in progesterone (Fig. 2C).
PGF2-R knockout mice did not show these changes. Serum
progesterone levels remained elevated (11) (Fig. 2C) and
20-OH-progesterone level remained low.
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Fig. 2.
20-HSD expression at
the end of pregnancy in wild-type (+/+) and
PGF2 receptor knockout (/)
mice. A, total RNA was subjected to RT-PCR analysis
using specific primers for mouse 20-HSD and L19 as internal control.
B, whole ovarian proteins were separated by SDS-PAGE
transferred to nitrocellulose and immunoblotted with a specific
20-HSD antiserum. Blots are representative of three different
experiments. C, serum progesterone and 20-OH-progesterone
concentrations in wild-type (+/+) and PGF2 receptor
knockout (/) mice on day 18, 19, and 20 of pregnancy. Values are
expressed as the mean ± S.E. (n = three animals).
*, p < 0.05, and ***, p < 0.001 versus +/+.
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PGF2 Enhances 20-HSD Promoter Activity in
Granulosa-luteinized Cells--
To investigate further the effect of
PGF2 on the expression of 20-HSD gene, we transfected
luteinized granulosa cells with a 2.5-kb 20-HSD promoter, previously
isolated in our laboratory, linked to a luciferase reporter gene
(20-HSD-Luc) (21). The cells were also co-transfected with an
internal reference plasmid expressing -galactosidase, allowing the
transfection efficiencies to be normalized. PGF2
treatment induced a 4-5-fold increase in the activity of 2.5-kb
20-HSD-Luc construct (Fig.
3A). Deletion of the 2467 to
1590 5'-fragment completely blunted responsiveness to
PGF2. PGF2 had no effect on the activity
of constructs carrying further downstream deletions of the 20-HSD
promoter. These results indicate that a region upstream of 1590 in
the promoter confers regulation by PGF2.
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Fig. 3.
PGF2
increases 20-HSD promoter activity in
luteinized granulosa cells. A, luteinized granulosa
cells were transfected with different 20-HSD promoter constructs
(0.5 µg/well) and treated with either PGF2
(10-6 M) or vehicle for 12 h. Transient
expression of the reporter gene was quantified by a standard luciferase
bioluminescence assay and normalized again -galactosidase.
Bars represent means ± S.E. of six experiments. ***,
p < 0.001 versus vehicle. B,
localization of NUR77-binding sites in the 20-HSD promoter.
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Analysis of the 2467/1590 20-HSD promoter region revealed the
presence of one perfect binding site (ACTGGAAAA) for the transcription
factor NUR77 at position 1599 to 1606. Another proximal, less
perfect, putative binding site, with a T insertion (1539)
AAATGGTCA (1531) was found (Fig. 3B).
NUR77-induced Expression of 20-HSD--
nur77
is an immediate-early gene which is known to play a major role in
hormone-induced expression of other steroidogenic enzymes (22-24).
Since the region in the 20-HSD promoter that confers responsiveness
to PGF2 contains a perfect NUR77 RE, we examined whether
this transcription factor may mediate the stimulatory effect of
PGF2. To test this possibility we examined first whether the overexpression of NUR77 protein affects the 20-hsd
gene expression in a cell line derived from the rat corpus luteum,
named GG-CL cells (25). We have previously demonstrated that the rat
20-HSD promoter is active in this cell line (21). We have also
observed that this cell line does not express NUR77 under basal
conditions (25). To assess whether NUR77 could affect the endogenous
expression of 20-HSD, we transfected GG-CL cells with either a NUR77
expression plasmid or with a dominant negative NUR77 (DN NUR77), which
encodes a NUR77 protein lacking the N-terminal transactivation domain (26). Controls were transfected with an empty plasmid. As a member of
the steroid/thyroid hormone receptor superfamily, NUR77 is composed of
a non-conserved N-terminal domain that plays a role in the
transcriptional regulation, a highly conserved central zinc finger
DNA-binding domain, and a moderately conserved C-terminal "ligand-binding" domain (27, 28). The mutant NUR77 protein forms
specific DNA-protein complexes (26).
Transfection of NUR77 into GG-CL cells enhanced the endogenous
expression of 20-HSD (Fig.
4A, compare lanes 1 and 2). No stimulation of 20-HSD mRNA levels was
observed with the dominant negative NUR77 (Fig. 4A, lane 3).
Transfection of equal amounts of either NUR77 or DN NUR77 constructs
results in approximately equivalent levels of protein expression
(26).
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Fig. 4.
Effect of NUR77 on
20-HSD expression and on
20-HSD promoter activity. A,
GG-CL cells were transfected with NUR77 expression vector
(Nur77) or with a dominant negative NUR77 expression vector
(DN-Nur77). Control was transfected with empty vector
(pSG5). 20-HSD mRNA levels were analyzed by RT-PCR 48 h
after transfection. B, the 2.5-kb 20-HSD-Luc was
co-transfected (0.5 µg/well) into GG-CL cells with either different
amounts of NUR77 or with DN Nur77. Bars represent
means ± S.E. of three replicates. Columns with different
letters differ significantly, a c; ab,
p < 0.01; bc, p < 0.05 (ANOVA I). C, different deletion of the 20-HSD-Luc
promoter constructs or a mutant 2.5-kb 20-HSD promoter (0.5 µg/well) were co-transfected together with 4 pmol/well of either
Nur77 or empty vector (EV) into GG-CL cells.
Transient expression of the reporter gene was quantified by a
standard luciferase bioluminescence assay and normalized again
-galactosidase. Bars represent means ± S.E. of six
experiments. ***, p < 0.01 versus EV (ANOVA
I).
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To determine the effect of NUR77 on 20-HSD promoter activity, we
co-transfected the 2.5-kb 20-HSD promoter luciferase construct with
different concentrations of NUR77 expression vector or with DN NUR77
expression vector into GG-CL cells. The total amount of DNA per well
was kept constant by the addition of empty plasmid. The cells were also
co-transfected with an internal reference plasmid expressing
-galactosidase, allowing the transfection efficiencies to be
normalized. The results (Fig. 4B) showed a dose-related
stimulation of the 2.5-kb 20-HSD-Luc activity by NUR77, whereas no
stimulation was observed with DN NUR77.
Deletion or Mutation of the Distal NUR77-binding Site Abolishes the
NUR77 Stimulation of 20-HSD Promoter Activity--
As mentioned
before, the 2.5-kb 20-HSD promoter contains two putative
NUR77-binding sites. The 5' distal putative NUR77 RE is a perfect
consensus NUR77-binding site, 5'-ACTGGAAAA-3', whereas the proximal
putative NUR77-binding site is imperfect with an insertion of a T,
5'-AAATGGTCA-3'. As shown in Fig. 4C, the 1590/+49 construct lacks the distal site but retains the proximal NUR77-like binding-like sequence at 1531, whereas both sites were deleted in the
289/+49 construct. Deletion of the distal NUR77 consensus-binding site abolished the NUR77-induced stimulation of 20-HSD promoter activity. NUR77 did not affect the activity of the construct lacking either the distal or both putative NUR77-binding sites (Fig.
4C). In order to confirm the participation of the distal
NUR77-binding site in the regulation of 20-HSD, we mutated the
ACTGGAAAA sequence to ATCGGAcgc. This mutation has been shown to
prevent NUR77 DNA binding (27). The activity of the mutated promoter
was evaluated in a transient co-transfection assay and compared with
that of the wild-type promoter. Mutation of the distal NUR77 RE
prevented the stimulation of the 20-HSD promoter activity induced by
NUR77 (Fig. 4C).
NUR77 Binds to the Distal NUR77 RE Found in the 20-HSD Promoter
Regions--
To test NUR77 interaction with the 20-HSD promoter, we
examined whether whole-cell extracts from GG-CL cells transfected with
the NUR77 expression vector binds the putative NUR77-binding sites
found in this promoter. Gel shift analysis demonstrated that the distal
site, which is a consensus NUR77-binding sequence, forms a major
protein-DNA complex with extracts from GG-CL cells transfected with the
NUR77 expression vector (Fig. 5,
lane 2) and with an in vitro translated NUR77
protein (lane 4). This complex was not detected when labeled
probe was incubated with extracts from cells transfected with an empty
plasmid (lane 1). The addition of unlabeled probe to the
reaction mixture inhibited the formation of the DNA-protein complex
(lane 3). In contrast, no protein-DNA complex was detected
when the proximal site was used (Fig. 5, lanes 6 and
7). This experiment demonstrates clearly that the distal
NUR77 RE binds NUR77, whereas the imperfect proximal NUR77 RE does
not.
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Fig. 5.
NUR77 binds to the distal NUR77 RE found in
the 20-HSD promoter region. EMSA were
carried out using 32P-labeled double-stranded
oligonucleotide probes representing the distal (lanes 1-4)
or the proximal (lanes 5-7) putative NUR77-binding site.
Lanes 1 and 5, whole-cell extracts of GG-CL cells
transfected with empty plasmid. Lanes 2 and 6,
whole-cell extracts of GG-CL cells transfected with a NUR77 expression
plasmid. Lanes 3 and 7, whole-cell extracts of
GG-CL cells transfected with a NUR77 expression plasmid plus 50 molar
excess of the homologous unlabeled probe. Lane 4, EMSA was
performed using in vitro translated NUR77 protein.
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nur77 Expression Is Increased in Pregnant Rats by Treatment with a
Luteolytic Dose of PGF2--
Since our present results
have demonstrated that both PGF2 and NUR77 stimulated
20-HSD mRNA expression and promoter activity, we thought that
the PGF2 stimulation of 20-HSD may be mediated by
NUR77. To examine this possibility, we first analyzed the effect of
PGF2 on luteal NUR77 expression. Northern blot analysis
(Fig. 6A) shows that
administration of PGF2 to rats on day 19 of pregnancy increased NUR77 mRNA expression within 0.5 h. Like many
immediate-early response genes, NUR77 mRNA reached maximal levels
after 30 min of treatment and diminished with time. This pattern of
nur77 expression has been also observed in Y1 adrenocortical
tumor cells where NUR77 mediated the increase of
P450C21 gene expression induced by ACTH (22).
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Fig. 6.
PGF2
induce a rapid increase in nur77 gene expression
in rat corpus luteum at the end of pregnancy. A, rats
on day 19 of pregnancy were treated with 400 µg of
PGF2 intraperitoneally and sacrificed at 0.5-24 h
thereafter. Total RNA was prepared, and Northern blotting analyses were
performed by using 20 µg of each RNA sample and a rat NUR77 cDNA
as a probe. Photography of the blot membrane after transfer is shown as
control of loading and transfer. B, RT-PCR analysis was
performed using plasmids that allow the co-amplification of the NUR77
and 20-HSD messages. Normalized mRNA levels are graphically
represented in the bottom panel. Bars represent
means ± S.E., n = 3.
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To compare the temporal expression of NUR77 and 20-HSD
mRNA levels after PGF2 administration, we determined
their levels by RT-PCR using primer that allows the co-amplification of
these mRNAs. As shown in Fig. 6B, the rapid induction of
nur77 gene expression by PGF2 in the rat
corpus luteum precedes that of the 20-HSD supporting the possible
participation of this transcription factor in the
PGF2-induced 20-HSD expression.
To confirm the stimulatory effect of PGF2 on NUR77, we
examined the expression of this transcription factor in corpora lutea of wild-type and PGF2-R knockout mice. An increase in
nur77 expression was seen at the end of pregnancy in the
wild-type mouse (Fig. 7). Similar to
20-HSD (see Fig. 2A), an increase in nur77
expression did not take place in corpora lutea of mice lacking
PGF2 receptor (Fig. 7).
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Fig. 7.
NUR77 expression at the end of pregnancy in
wild-type or PGF2 receptor
knockout mice. Total RNA was subjected to RT-PCR analysis using
specific primers for mouse NUR77 and L19 as internal control. Data were
quantified by densitometry and corrected using L19 as an internal
standard. Normalized mRNA levels are graphically represented in the
right panel as the means ± S.E., n = 3. ***, p < 0.01 versus the previous day of
gestation.
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PGF2 Induces Binding of NUR77 to the 20-HSD
Promoter--
We next examined whether PGF2-induced
NUR77 binds to the functional NUR77 RE found in the 20-HSD promoter.
EMSA analysis showed that nuclear extracts from corpora lutea of
PGF2 treated rats formed two prominent complexes, I and
II (Fig. 8, lane 2), as
compared with the control rat, where only one major shifted complex is
formed (complex I) (Fig. 8, lane 1). Complexes I and II were
inhibited by 10, 50, and 100 molar excess of unlabeled probe
(lanes 3-5). Oligonucleotides containing the mutated distal NUR77 RE ACTGGAAAA motif to ACTGGAcgc did not competitively inhibit protein binding (lane 6), indicating that a perfect NUR77 RE
sequence is essential for the protein/DNA interaction. Furthermore,
formation of complex II was not inhibited by an oligonucleotide
containing the proximal imperfect NUR77 response (lane 7).
The formation of a broad shift band by NUR77, observed also by Okabe
et al. (29), might be due to post-transcriptional
modification (phosphorylation in multiple sites), which has been
identified as the cause of the heterogeneity in NUR77 protein size (26,
30, 31).
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Fig. 8.
PGF2-induced NUR77 binds to
the distal NUR77-binding site found in the
20-HSD promoter. Rats on day 19 of
pregnancy were treated with 400 µg of PGF2
intraperitoneally, and luteal nuclear proteins were isolated 3 h
after. An oligonucleotide containing the distal NUR77-binding site was
used to perform EMSA analysis. Lane 1, luteal nuclear
extract from vehicle-treated rats; lanes 2-9, luteal
nuclear extract from PGF2-treated rats; lanes
3-5, unlabeled distal site probe in 10-, 50-, or 100-fold molar
excess; lane 6, 50-fold molar excess of unlabeled mutated
distal NUR77 RE; lane 7, 50-fold molar excess of unlabeled
proximal NUR77 RE; lanes 8 and 9, 1 or 0.5 µg
of a NUR77 antibody was added to the reaction mixture prior to the
addition of labeled probe.
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To determine whether NUR77 is present in either of these complexes, we
preincubated luteal nuclear extracts with a monoclonal antibody that
specifically recognizes NUR77 and inhibits DNA-NUR77 complex formation
(29). The addition of 0.5 µl of this antibody (Fig. 8, lane
9) prevented the formation of the PGF2-induced complex II but had no effect on complex I, indicating the participation of NUR77 protein in the formation of this complex. When 1 µl of NUR77
antibody was added, complex I was also decreased, indicating that NUR77
may be part of this complex found in corpus luteum of untreated rats,
which agrees with basal labels of NUR77 expression found during
gestation in rats (data not shown). These results demonstrated that the
PGF2-stimulated NUR77 binds to its response elements in
the 20-HSD promoter.
To confirm, in vivo, that the binding of NUR77 to the
20-HSD promoter induced by PGF2 is responsible for
the increase in the expression of this enzyme, we used cyclosporin A
(CsA), which has been shown to prevent NUR77 binding to its response
element (30). CsA was locally administered into the ovarian bursa 30 min prior to intraperitoneal injection of PGF2 or
vehicle. Corpora lutea isolated 2 h after treatment were used to
obtain nuclear protein for gel mobility shift assay, whereas total RNA
was isolated 24 h after treatment to measure 20-HSD mRNA
levels. As shown in Fig. 9A
(lane 1), nuclear extract from vehicle-treated rats resulted
in a retarded radiolabeled complex I. PGF2 treatment caused a second prominent shift in complex II, containing NUR77 as show
in Fig. 8, to form (Fig. 9A, lane 2) and induced high levels
of 20-HSD mRNA expression (Fig. 9, B and
C, compare lanes 1 and 2). CsA
pretreatment prevented the formation of the NUR77-DNA complex II
induced by PGF2 (Fig. 9A, lane 3) and reduced
markedly the PGF2-induced 20-HSD mRNA expression
(Fig. 9, B and C, lane 3). Administration of CsA
to vehicle treated rat did not affect 20-HSD expression (Fig. 9,
B and C, lane 4).
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Fig. 9.
Cyclosporin A, an inhibitor of NUR77 action,
prevents the PGF2-induced
NUR77-DNA complex formation and the increase in
20-HSD expression. Rats on day 19 of
pregnancy were injected with cyclosporin A (3 µg/ovary) or vehicle
(metil cellulose) locally into the ovarian bursa 30 min prior to
the administration of 400 µg of PGF2
intraperitoneally. A, luteal nuclear extract, from either
control rats (lane 1), PGF2-treated rats
(lane 2), or CsA plus PGF2-treated rats
(lane 3) were isolated 2 h after PGF2
administration. An oligonucleotide containing the distal NUR77-binding
site was used to performed EMSA analysis. B, total RNA was
prepared 24 h after the administration of PGF2, and
RT-PCR analysis was performed using primers for rat 20-HSD and L19
as internal control. C, densitometry analysis of 20-HSD
expression normalized against ribosomal L19 mRNA expression.
Bars represent means ± S.E., n = 3. Columns with different letters differ significantly; a-b,
p < 0.01 (ANOVA I).
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Finally, to examine further whether the activation of 20-HSD
promoter by PGF2 is mediated by NUR77, we determined
whether dominant negative NUR77 can prevent the induction of 20-HSD
promoter activity by PGF2. Luteinized granulosas cells
were co-transfected with the 2.5-kb 20-HSD-Luc promoter together
with 1 or 2 µg of the mutant NUR77 (DN NUR77) or empty plasmid as
control. Cells were then treated with either PGF2 or
vehicle. As shown before in Fig. 3, PGF2 caused a
severalfold increase in the 20-HSD promoter activity (Fig.
10). Increasing amounts of DN NUR77
inhibited this stimulation. 2 µg of the mutant NUR77 prevented
totally the stimulation on 20-HSD promoter activity induced by
PGF2 confirming that the stimulation of promoter
activity by PGF2 is mediated by the transcription
factor, NUR77.
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Fig. 10.
Overexpression of a dominant negative
NUR77 protein prevents the increase in 20-HSD
promoter activity induced by
PGF2. Luteinized granulosas
cells were transfected with 0.5 µg/well of the 2.5-kb 20-HSD-Luc
construct together with two concentrations of the N-terminal truncated
NUR77 expression vector (DN- Nur77). The amount of DNA per
well was kept constant by addition of an empty plasmid
(pSG5). 24 h after transfection the cells were treated
with either PGF2 (10-6 M) or
vehicle for 12 h. Transient expression of the reporter gene was
quantified by a standard luciferase bioluminescence assay and
normalized against -galactosidase. Bars represent
means ± S.E. of three replicates. Columns with different letters
differ significantly; ac and ab,
p < 0.01; bc, p < 0.05 (ANOVA I).
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DISCUSSION |
The stimulatory effect of PGF2 on the activity of
the 20-HSD enzyme in the rat corpus luteum is well recognized
(5-8). The corpus luteum expresses PGF2 receptor
(32-34) and is able to produce the ligand (35, 36). However, whether
PGF2 activates an already pre-existing enzyme or whether
it induces/increases the expression of the 20-HSD gene is unknown.
In addition, the cellular signals linking the PGF2
receptor to the 20-HSD gene remains uninvestigated. In this report,
we have identified the 20-HSD gene as a novel target for the
transcription factor NUR77, and we have demonstrated that
PGF2 stimulates rapidly the expression of NUR77, which
binds to a specific response element in the 20-HSD promoter and
stimulates its transcriptional activity. Moreover, we have shown that
NUR77 and 20-HSD become expressed in the corpus luteum only at the
end of pregnancy and that these expressions are totally obliterated in
PGF2 receptor knockout mice. These results together with
our ability to prevent (i) the PGF2-induced expression
of 20-HSD in vivo by inhibiting NUR77 DNA binding activity and (ii) the PGF2-induced increase in 20-HSD
promoter activity in vitro by overexpressing a dominant
negative NUR77 protein give evidence for the involvement of NUR77 in
the PGF2-mediated functional transcriptional activation
of 20-HSD gene at the end of pregnancy.
In contrast to transcription factors that are constitutively present
and ready to be activated by post-translation modification or by
interaction with allosteric effectors, NUR77 is encoded by an
immediate-early gene, whose expression is tightly regulated by
extracellular signals. Classified as an orphan nuclear receptor, NUR77
displays the tripartite domain structure of members of the steroid
receptor family but binds no known ligand. However, when synthesized,
NUR77 is constitutively active under all conditions examined (19) and
binds as a monomer to a specific response element (27, 37) which
consists of the classical estrogen receptor half-site preceded by two
adenines (5'-AAAGGTCA-3'). In contrast to other orphan receptors that
bind rather promiscuously to their response elements and other response
elements (38), NUR77 is highly selective for its response element.
Indeed our results show clearly that despite the presence of two
putative binding sites in the 20-HSD promoter, NUR77 associates only
with the perfect response element at 1606/1599. NUR77 was shown
previously to regulate the transcriptional activity of two hydroxylase
genes, the rat P450C21 (22, 23) and the mouse
P450C17 (24), leading to increased synthesis of
cortisol and androgen, respectively. Results of the present
investigation have revealed a role for NUR77 in the transcriptional
activity of another steroidogenic gene, which encodes an enzyme with
dehydrogenase activity and causes the catabolism of progesterone
synthesized in the corpus luteum. NUR77 is a homologue to the
steroidogenic factor 1 that is constitutively active in many
steroidogenic tissues and binds to an element similar to but distinct
from NUR77 (38). In contrast to steroidogenic factor 1, NUR77 is
present at low levels under basal conditions and becomes rapidly and
highly expressed in response to external stimuli (39, 40). In addition
to its effect on steroidogenic genes, NUR77 has been implicated as
modulator of the retinoic acid signaling pathway (41, 42) and in the
apoptotic process in T-lymphocytes. Indeed, the induction of NUR77 is
required for the negative selection of thymocytes by apoptosis (30,
43-46). Interestingly, PGF2 was also shown to initiate
programmed cell death in the corpus luteum (47, 48). The possibility
that the induction of nur77 expression by
PGF2 leads to the initiation of an apoptotic process in
the corpus luteum remains to be investigated.
PGF2-induced luteolysis is believed to be initiated
through ligand receptor activation of the phospholipase C that in turn induces production of inositol 1,4,5-trisphosphate and
diacylglycerol, causing an increase in intracellular calcium
mobilization and activation of Ca2+/calmodulin kinase and
protein kinase C (for review, see Ref. 49). Interestingly, either
activation of PKC by phorbol ester or increase in intracellular levels
of calcium with a calcium ionophore leads to the induction of
nur77 expression (50, 51) suggesting that
PGF2-induced expression of nur77 in the
corpus luteum may result from the activation of either one of these
signaling pathways.
Despite the well established role of NUR77 in many processes, mice
deficient in NUR77 have no apparent phenotype suggesting that
biological alternative pathways complement the functional defects (52).
Indeed NURR1 may compensate for the loss of NUR77. NURR1 is also an
immediate-early gene whose DNA and ligand-binding domain are similar to
NUR77 (53). NUR77 was shown to mediate ACTH stimulation of
P450C21 in adrenal cells (22), yet the expression of this
gene is normal in mice deficient in NUR77 (52); however, NURR1 becomes
highly expressed after ACTH treatment in the adrenals of those animals
and appears to compensate for NUR77.
The development of mice lacking the PGF2 receptor
revealed that this molecule plays a key role in the induction of
parturition and that the lack of delivery in these knockout mice is due
to the persistent production of progesterone. Indeed progesterone levels did not decline around day 20, the day of parturition in mice,
whereas ovariectomy of PGF2 receptor-deficient mice led to parturition within 24 h (11, 12). Our results indicate that
these high levels of progesterone are due to the lack of luteal
20-HSD expression in the PGF2 receptor knockout mice. This together with the results showing that administration of PGF2 to pregnant rats induces premature expression of
20-HSD indicates that PGF2 is crucial for the massive
increase in the luteal expression of this gene at the end of pregnancy,
and that it is the expression of 20-HSD that gives the signal for
parturition in rodent.
Because PRL and PRL-related hormones silence the expression of
20-HSD throughout pregnancy (4, 54-56) and because a marked decline
in the level of PRL receptor takes place at a time when 20-HSD
levels rise (57), we previously suggested that this reduction in the
PRL-R levels renders the corpus luteum less responsive to circulating
PRL and PRL-related hormones allowing 20-HSD expression before
parturition. However, the results presented herein do not support such
a conclusion. Indeed, PGF2 can induce the expression of
20-HSD when administered on day 19 of pregnancy, a time when circulating levels of rat placental lactogen-2 are elevated (58, 59)
and when luteal PRL receptor is still highly expressed (57).
Although it has been suggested that PGF2 can affect the
synthesis of progesterone by reducing cholesterol transport to the inner mitochondrial membrane (60, 61), it appears clear that at least
in rodent, the main effect of PGF2 is to enhance the catabolism rather than inhibit the synthesis of progesterone. Results
of this investigation indicate that levels of the progesterone metabolites, 20-OH-progesterone secreted by the ovaries of rats treated with PGF2, exceed the levels of progesterone
produced by non-treated rats. In addition to its ability to decrease
luteal progesterone production, PGF2 was also shown to
affect its own expression in ovine large luteal cells (34) and that of
its own receptor in rat ovary (32).
Our studies have focused on the role of NUR77 in the control of
20-HSD promoter activity. However, the 20-HSD promoter
contains two AP-1 sites (21), which associate with two subunits encoded by the nuclear oncogenes c-fos and c-jun (62,
63). PGF2 treatment in vivo increases the
expression of C-JUN mRNA in bovine corpus luteum (64) and that of
c-JUN and c-FOS in cultured bovine luteal cells (65) and is also shown
to increase the level of DAX-I expression (66). However, our findings
that PGF2 stimulation of 20-HSD mRNA expression
and promoter activity are respectively obliterated by cyclosporin
A, which prevents NUR77 DNA binding, and by a dominant negative NUR77,
suggesting that NUR77 is the sole transcription factor that mediates
PGF2 stimulation of 20-HSD.
-induced Expression of
20
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence and requests for reprints should be
addressed: Dept. of Physiology and Biophysics (M/C 901), University of
Illinois, 835 S. Wolcott Ave., Chicago, IL 60612-7342. Fax: -312-413-0159; E-mail: ggibori@uic.edu.
