Endocrinology Vol. 142, No. 12 5116-5123
Copyright © 2001 by The Endocrine Society
LH Induces Orphan Nuclear Receptor Nur77 Gene Expression in Testicular Leydig Cells
Kwang-Hoon Song,
Jae-Il Park,
Mi-Ock Lee,
Jaemog Soh,
Keesook Lee and
Hueng-Sik Choi
Hormone Research Center, Chonnam National University
(K.-H.S., J.-I.P., J.S., K.L., H.-S.C.), Kwangju 500-757, and
Department of Bioscience and Biotechnology, Sejong University
(M.-O.L.), Seoul 143-747, Republic of Korea
Address all correspondence and requests for reprints to: Hueng-Sik Choi, Ph.D., Hormone Research Center, Chonnam National University, Kwangju 500-757, Republic of Korea. E-mail: hsc{at}chonnam.ac.kr
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Abstract
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The orphan nuclear receptor Nur77 (NR4A1) is a member of the
nuclear receptor superfamily and plays an important role in the
regulation of genes involved in steroidogenesis and cell death.
Northern blot analysis revealed that the expression of Nur77 mRNA was
increased after puberty in mouse testis, and hCG treatment of
peripubertal animals induced this gene expression in the testis.
Moreover, LH treatment induced a transient increase in Nur77 mRNA, and
this induction was LH dose dependent in mouse Leydig tumor cell line,
K28. Western blot analysis showed that LH transiently induced Nur77
protein. The protein kinase inhibitor H-89, bisindolymaleimide I, and
wortmannin strongly inhibited this inductive effect of LH on Nur77 gene
expression. Transient transfection assay demonstrated that LH
significantly increased the Nur77 promoter-driven luciferase reporter
activity in a dose-dependent manner, and LH also increased the activity
of a luciferase reporter gene driven by a promoter containing multi
copies of a Nur77-responsive element. Moreover, EMSA showed that Nur77
DNA-binding activity was increased in response to LH. Finally,
overexpression of dominant negative Nur77 reduced LH-mediated
progesterone biosynthesis. Taken together, these results demonstrate
that LH induces Nur77 gene expression, and Nur77 may play an important
role in the LH-mediated steroidogenesis in Leydig cells.
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Introduction
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THE NUCLEAR HORMONE receptor
superfamily gene includes genes that encodes structurally related
protein that regulates pivotal gene networks important for eukaryotic
cell growth, development, and homeostasis and includes orphan nuclear
receptor that do not have known ligands (1, 2, 3). Orphan
nuclear receptor Nur77, also known as NGFI-B in rats and TR3 in humans,
is classified as a member of the nuclear receptor superfamily, and
ligand for this receptor has not been reported (1). Nur77
is one of the immediate-early response genes originally identified by
virtue of its rapid activation by nerve growth factor (NGF) in PC12
pheochromocytoma cells (4) and serum in fibroblast
(5). Moreover, Nur77 gene is widely expressed in several
tissues, including testis, ovary, muscle, thymus, adrenal gland, and
brain (1, 6). In addition to its gene regulation at the
transcriptional level, it has been reported that the activity of Nur77
is controlled by posttranslational modification; Nur77 is rapidly
modified via phosphorylation and the extent of phosphorylation is
dependent on the types of stimulus (7, 8, 9). Several lines
of evidence indicated that Nur77 might play an important role in the
organization of neuroendocrine regulation of
hypothalamus-pituitary-adrenal axis (10, 11, 12) and in the
apoptosis of T lymphocytes (13, 14, 15, 16). Recently, it has been
demonstrated that TR3, human homolog of Nur77, can translocate into
mitochondria, where it triggers membrane permeabilization and apoptotic
cell death (17), and that Nur77 is involved in the
induction of 20
-hydroxysteroid dehydrogenase by
PGF2
(18).
Using a genetic selection approach, Nur77 was found to recognize a
specific nucleotide sequence called NGFI-B (Nur77)-responsive element
(NBRE) (19, 20), which contains an E receptor half-site
(AGGTCA) preceded by two adenines. Furthermore, Nur77 binds DNA as a
monomer, and a region outside of the zinc finger domain (A box) was
shown to play an important role in DNA binding specificity
(19). Recently, a novel Nur77 target sequence called Nur
response element (NurRE) was identified in the POMC promoter (21, 22), and Nur77 binds this element as a homodimer and heterodimer
with other Nur family members (23). It has also been
demonstrated that Nur 77 heterodimerizes with RXR and binds to a RAR
element composed of direct repeats separated by five nucleotides (DR5)
in the presence of 9-cis-retinoic acid (24).
Although information regarding Nur77 function has been accumulated, the
physiological role of this orphan nuclear receptor largely remains to
be determined.
The present study demonstrates that LH induces Nur77 gene
expression in mouse Leydig cells, and this LH-mediated induction of
Nur77 gene expression is regulated via diverse cell signaling pathway.
Furthermore, LH increased the Nur77 activity, and inhibition of Nur77
reduced LH-mediated progesterone biosynthesis in Leydig cells. Taken
together, these results suggest that LH-induced Nur77 gene expression
may play an important role in the steroidogenesis of Leydig cells in
the testis.
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Materials and Methods
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Hormones, reagents, and animals
Ovine LH (LH-s-26; 2300 IU/mg) was obtained from the National
Hormone and Pituitary Distribution Program, NIDDK, NIH (Baltimore, MD).
Bisindolylmaleimide I (GF109203X), forskolin, human CG (hCG), and
12-O-tetradecanoyl-phorbol-13-acetate (TPA) were purchased
from Sigma (St. Louis, MO). H-89, wortmannin, and
8-bromo-cAMP were purchased from Calbiochem (San Diego,
CA). Male ICR mice were purchased from Daehan Laboratories (Chungbuk,
Korea). The animals were killed by ethylether and cervical dislocation,
and the testes were removed for Northern blot analysis.
Twenty-two-day-old mice received a single ip injection of 5 IU hCG, and
testes were obtained at different time intervals for Northern blot
analysis.
Cells
The K28 cell line was originally subcloned from the LK17 hybrid
clone, which was derived from the fusion between MA-10 mouse Leydig
tumor cells and freshly isolated mouse Leydig cells (25, 26). The K28 mouse Leydig tumor cell line has been characterized
as a suitable cell culture model for steroidogenesis (27, 28).
Plasmids
The mouse Nur77 cDNA, dominant negative Nur77 (DN-Nur77) cDNA,
and Nur77 promoter-luciferase reporter, NBRE-tk-Luc reporter construct
were described previously (14, 15, 18, 21, 29, 30), and
NurRE 3 copy-POMC-Luc reporter construct and Nurr-1 were obtained from
Dr. Jacques Drouin (Institut de Recherches Cliniques de Montréal,
Canada) and Dr. Thomas Perlmann (Institute for Cancer Research,
Sweden), respectively.
Northern blot analysis
K28 cells were grown in DMEM supplemented with 15% FBS. The
cells were serum-starved at 80% confluence in serum-free medium for
24 h. After the culture medium was changed to the serum-free
condition, cells were treated with LH (200 ng/ml) from 30 min to
24 h. Total RNA was isolated using Tri-Reagent
(Sigma). Twenty micrograms of total RNA were fractionated
by electrophoresis on 1.2% agarose gel containing formaldehyde and
were transferred to a nylon membrane (
-probe, Bio-Rad Laboratories, Inc., Richmond, CA) by capillary blotting with
10x SSC. After UV cross-linking and prehybridization, membranes were
hybridized 24 h at 42 C in solution containing 50% formamide,
10% dextran sulfate, 5x SSC, 1 mM EDTA, 10 mg/ml
denatured salmon sperm DNA, and a total of 24 x
106 cpm
-32P-labeled
mouse Nur77 cDNA containing ligand-binding domain and Nurr-1 cDNA.
After hybridization, membranes were washed twice for 5 min at room
temperature in 2x SSC and 0.1% SDS, followed by 1 h at 65 C in
0.5x SSC and 0.1% SDS. Membranes were then exposed using
Kodak RX films (Eastman Kodak Co., Rochester,
NY) for 1224 h at 70 C. The expression of
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as an
internal control. The band intensities were subsequently measured using
a phosphorimager (Bio-Rad Laboratories, Inc., Hercules,
CA), and the signals were normalized to the GAPDH internal control.
Transient transfection and ß-galactosidase assay
Twenty-four hours before transfection, K28 cells were plated in
24-well culture dishes at a density of 2.5 x
104 cells/well. Transfection was performed using
LipofectAMINE Plus reagent (Life Technologies, Inc.,
Gaithersburg, MD) with Nur77 promoter-Luc, NBRE-Luc,
NurRE-Luc, Nur77, DN-Nur77, and internal control
promoter CMV (pCMV)-ß-galactosidase as recommended by the
manufacturer. Twenty-four hours after transfection, the cells were
treated with various concentrations of LH. Thirty-six hours after LH
treatment, cells were lysed with 100 µl 1% Triton X-100, 25
mM GLY-GLY (pH 7.5), 15 mM
MgSO4, and 2 mM EGTA for 15 min.
Twenty microliters of the cell lysates were assayed for luciferase
activity with a dual luciferase reporter assay system (Promega Corp., Madison, WI) and determined with an MLX microtiter
luminometer (Dynex Technologies, Inc., Chantilly,
VA). The lysates were transferred into 96-well microtiter plates
for ß-galactosidase assay by using the
o-nitrophenyl-ß-D-galactopyranoside
(Sigma) as a substrate as described previously
(31). The luciferase activities were normalized to the
ß-galactosidase activity expressed from the cotransfected
pCMV-ß-gal plasmid and reported as the mean ±
SE in relative light units. All transfection
experiments were performed at least five times in duplicate.
Western blot analysis
LH-treated K28 cells were resuspended in lysis buffer [50
mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium
deoxycholate, 150 mM NaCl, 1 mM EGTA, 1
mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1
µg/ml leupeptin, and 1 mM sodium fluoride] and incubated
on ice for 10 min and then lysed by homogenizer. The lysates were
centrifuged to remove cell debris, and the supernatant was collected
and frozen at -80 C until further use. Protein concentrations were
estimated using bicinchoninic acids protein assays (Pierce Chemical Co., Rockford, IL). Protein lysates (50 µg) were
boiled for 5 min in denaturing sample buffer and loaded onto a 12%
continuous gradient SDS-polyacrylamide gel, and proteins were
transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). The membrane was blocked with
TBST buffer [10 mM Tris-buffered isotonic saline (pH 7.0),
0.1% merthiolate, and 0.1% Tween-20] containing 5% nonfat dry milk
for 30 min at room temperature with shaking, followed by incubation
with primary antibody (anti-Nur77 at 0.5 µg/ml dilution;
PharMingen, San Diego, CA) in TBST buffer for 24 h at
4 C with gentle shaking. Membrane was washed twice with TBST for 10 min
each time and incubated with antimouse IgG conjugated to alkaline
phosphatase (1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in TBST for 1 h at room temperature.
Finally, membrane was washed twice with TBST, and Nur77-specific bands
were visualized using a Western-Star chemiluminescent detection system
(Tropix, Inc., Bedford, MA) according to the manufacturers
guidelines.
EMSA
Briefly, cultured K-28 cells were washed twice with cold PBS and
pelleted by centrifugation at 3,000 rpm for 5 min at 4 C. The pellets
were gently resuspended in buffer (20 mM HEPES, 10
mM EDTA, 0.1% Nonidet P-40, 100 mM NaCl, 0.15
M phenylmethylsulfonylfluoride, leupeptin, and pepstatin)
and broken by passing 20 times through a 25-gauge needle. The cells
were kept on ice for 1 h at 4 C and centrifuged at 14,000 rpm at 4
C for 30 min. The supernatants containing the whole cell extracts were
aliquoted and stored at -80 C. Probes used for EMSA experiments were
prepared by labeling 10 pmol double stranded oligonucleotides with T4
polynucleotide kinase (Promega Corp.) at 37 C for 30 min.
The labeled probes were purified by Sephadex G-50 column
chromatography. A sample containing 40,00050,000 cpm of the purified
double stranded oligonucleotides was used for each reaction. EMSA was
performed with 1 µg poly(dI/dC)/sample as a nonspecific competitor.
The DNA-protein complexes were separated from the unbound DNA probe via
6% nondenaturing gel electrophoresis at 4 C in Tris base glacial
acetic EDTA buffer, and the binding reaction was carried out at 25 C
for 30 min. The sequences of oligonucleotides used as probes for NBRE
and mutated SF-1RE were 5'-GGAGTTTAAAAGGTCATGCTC-3' and
5'-CCCATCAATTATATAAAT-3', respectively.
RIA
For LH treatment, the exponentially growing K28 cells in the
100-mm dish were split into an appropriate number of 60-mm dishes
(3.5 x 105 cells/dish) and cultured for 24 h in
DMEM supplemented with 15% FBS. Transfection was performed using
LipofectAMINE Plus reagent (Life Technologies, Inc.) with
DN-Nur77 expression vector (1 µg) or Nur77 expression vector (1 µg)
and internal control pCMV-ß-gal. After 24-h transfection,
cells were washed twice with Dulbeccos PBS, and medium was replaced
with 2 ml DMEM containing 15% FBS and incubated for 24 h. Cells
were washed twice with Dulbeccos PBS and 4 ml serum-free DMEM
containing low density lipoprotein (5 µg/ml) was added to the
cells. After cells were incubated with or without LH (200 ng/ml) for
the designated period, the cell culture medium was obtained for RIA.
Culture media were assayed directly without further purification. The
general assay procedure was used as described previously
(32). The progesterone concentration was calculated with
SecuRIA program (Packard, Downers Grove, IL). Coefficients of variation
for progesterone between and within assay were 9.4% and 9.2%,
respectively. The lower limit of assay sensitivity for progesterone was
6.5 pg. Transfection efficiency was normalized by ß-galactosidase
activity. Each treatment group contained duplicate cultures, and each
experiment was repeated at least twice.
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Results
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Induction of Nur77 gene expression at the pubertal stage during
testis development
To determine whether the Nur77 mRNA level is regulated during
testis development in the mouse, we examined Nur77 gene expression by
Northern blot analysis. In the testis, a low level of Nur77 mRNA
appeared on d 5, and an increase in Nur77 mRNA was detected on d 30,
which persisted in mice up to d 70 (Fig. 1
, A and B). The levels of testis Nur77
expression were 3-fold (n = 2) higher in 30-d-old mice than in
20-d-old mice. This result suggests that Nur77 gene expression is
regulated during the postnatal stage of testis development.

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Figure 1. Nur77 gene expression during postnatal stage of
testis development. Northern blot analysis was performed as described
in Materials and Methods. Twenty micrograms of testis
total RNA were analyzed by Northern blotting using Nur77 cDNA as a
probe. The migration distances of 28S and 18S ribosomal RNA
(left) and the Nur77 transcript (right)
are indicated (A and C). The expression of GAPDH was used as an
internal control. Data are representative of two independently
performed experiments. The Nur77 mRNA was quantified using a
phosphorimager and normalized for GAPDH RNA levels in each sample (B
and D).
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To determine whether Nur77 gene expression is regulated by gonadotropin
treatment in vivo, a 22-d-old peripubertal mouse was
injected with hCG (5 IU/animal). Northern blot analysis demonstrated
that there was a rapid and transient induction of Nur77 mRNA, reaching
a maximal level at 12 h after hCG treatment (Fig. 1C
).
Quantitative analysis showed that hCG treatment resulted in a 4.7-fold
increase in Nur77 mRNA at 12 h (Fig. 1D
), indicating that LH/hCG
could regulate Nur77 gene expression in testis.
LH induces Nur77 gene expression in a time- and dose-dependent
manner
As the secretion of LH is increased during the puberty in testis,
and a large number of studies have shown that LH is the main regulator
of adult Leydig cells (33, 34), we investigated the effect
of LH on the induction of Nur77 gene expression in the mouse testis
Leydig cell line, K28. Northern blot analysis showed that LH treatment
(200 ng/ml) caused a transient increase in Nur77 mRNA expression,
reaching a maximum level, 63-fold higher than the basal level, within
1 h and returning to the basal level after 3 h (Fig. 2
, A and B). Moreover, LH induced
Nur77-related member Nurr-1 mRNA expression with a similar time-course
pattern of Nur77 mRNA expression (Fig. 2A
), and LH induced Nur77 mRNA
in a dose-dependent manner, reaching a saturation level at 100 ng/ml
(Fig. 2C
). To investigate whether LH could also increase Nur77 protein
expression, Western blot analysis with the Nur77-specific antibody was
performed. LH retained its ability to induce Nur77 synthesis (Fig. 2D
),
and the induction of Nur77 protein was dramatically increased within 30
min of LH treatment and remained at its highest level for 2 h
after LH treatment. A broad range of Nur77 protein sizes (70 up to 90
kDa) was observed, suggesting that phosphorylation of Nur77 might be
involved in the response to LH. Taken together, these results suggest
that one of the important roles of LH in Leydig cells might be the
induction of orphan nuclear receptor Nur77 gene expression.

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Figure 2. LH induces Nur77 gene expression in a mouse testis
Leydig cell line, K28. K28 cells were cultured in serum-free conditions
for 24 h. These quiescent cells were then treated with LH (200
ng/ml) for up to 24 h (A) or with different doses of LH for 1
h (C). Total RNA (20 µg) was analyzed by Northern blotting using a
cDNA probe for Nur77 or Nurr-1. The migration distances of 28S and 18S
ribosomal RNA (left) and the Nur77 and Nurr-1 transcript
(right) are indicated (A and C). The expression of GAPDH
was used as an internal control. Nur77 mRNA was quantified using a
phosphorimager and normalized for GAPDH RNA levels in each sample (B).
Western blot analysis of Nur77 protein induced by LH (D). Whole cell
extracts (50 µg/lane) were then analyzed by immunoblotting with Nur77
antibody for indicated time. The positions of protein molecular mass
standards are indicated as 108 (108 kDa), 69 (69 kDa), and 48 (48 kDa)
to the left. The arrowed bracket
indicates the Nur77 protein range to the right. Data are
representative of three independently performed experiments.
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Multiple signaling pathways are involved in LH-mediated Nur77 gene
expression
To determine the signaling pathways involved in LH-mediated Nur77
gene expression, K28 cells were treated with various activators and
inhibitors of diverse cell signaling pathways. Northern blot analysis
indicated that induction of Nur77 by LH treatment was decreased 73% by
the PKA inhibitor H-89 (10 µM) and 76% by the PKC
inhibitor bisindolylmaleimide I (GF109203X; 100 nM),
suggesting that both PKA and PKC pathways are involved in LH-mediated
Nur77 gene induction (Fig. 3
, A and B).
Moreover, adenylate cyclase activator, forskolin (10 µM),
cell-permeable cAMP analog, 8-bromo-cAMP (1 mM), or TPA
(200 nM) alone induced Nur77 gene expression, confirming
that PKA and PKC pathways are involved in Nur77 gene induction.
Finally, wortmannin (10 nM), an inhibitor of PI3 kinase,
inhibited 70% of LH-mediated Nur77 gene induction, suggesting that the
PI3K signaling pathway might also be involved in this induction. Taken
together, these results suggest that the Nur77 gene induction by LH
stimulation is mediated through diverse signal transduction
pathways.

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Figure 3. Induction of Nur77 gene expression is regulated by
diverse signaling. K28 cells were cultured for 1 h in serum-free
conditions under 5% CO2 at 37 C in the absence (control;
C) or presence of H-89 (10 µM), forskolin (FSK; 10
µM), bisindolylmaleimide I (GFX; 100 nM), TPA
(200 nM), wortmannin (WM; 10 nM), and
8-bromo-cAMP (1 mM) with or without LH (200 ng/ml). Twenty
micrograms of total RNA were analyzed by Northern blot analysis. The
migration distances of 28S and 18S ribosomal RNA (left)
and the Nur77 transcript (right) are indicated (A). The
expression of GAPDH was used as an internal control. The Nur77 mRNA was
quantified using a phosphorimager and normalized for GAPDH RNA levels
in each sample (B). Data are representative of three independently
performed experiments.
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LH induces Nur77 gene promoter activity and its DNA-binding
activity
To confirm whether the induction of Nur77 gene expression by LH is
regulated at a transcriptional level, a transient transfection assay
was performed. The response of the Nur77 promoter (bp -336 to +67) to
LH in transient transfection was consistent with the pattern of its
mRNA expression by LH in K28 cells. Nur77 gene promoter was strongly
induced by LH treatment in a dose-dependent manner (Fig. 4A
). Forskolin and TPA also significantly
induced Nur77 promoter activity (Fig. 4B
), suggesting that Nur77 gene
transcription is regulated by both PKA and PKC cell signaling. These
results indicate that the transcriptional regulation of Nur77 gene
expression is associated with LH-mediated Nurr77 gene induction via a
specific cell signaling pathway.

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Figure 4. LH activates Nur77 gene promoter. A, Effects of LH
were tested on Nur77 promoter (bp -336 to 67) fused to luciferase
reporter. K28 cells were transfected with 300 ng of the indicated
luciferase reporter gene. Cells were treated with LH (nanograms per ml)
or without LH (0) and assayed for luciferase activity after 36 h.
Luciferase activity was normalized by ß-galactosidase activity to
determine the transfection efficiency. B, Specific induction of the
Nur77 promoter by forskolin (FSK; 10 µM) and TPA (200
nM). The Nur77 promoter reporter was transfected as
described above and treated with the indicated dose of forskolin and
TPA. Data shown represent the means of five independent experiments.
All promoter activities are shown as fold activation relative to the
control (±SEM).
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To determine whether Nur77 induction by LH treatment causes an increase
in Nur77 DNA-binding activity, EMSA was performed. NBRE-Nur77 protein
complexes were observed after 2 h of LH treatment and were
continuously present up to 24 h after LH treatment (Fig. 5A
). Nur77 DNA-binding activity was
completely abolished by 25- and 50-fold excesses of unlabeled specific
probe. However, a 50-fold excess of oligomer containing the mutated
SF-1RE motif could not competitively inhibit protein binding. To
determine whether Nur77 is present in this complex, whole cell extracts
were preincubated with a monoclonal antibody against Nur77 protein.
Nur77-specific antibody reduced the formation of the LH-induced
Nur77-DNA complex, whereas nonspecific antibody had no significant
effect on Nur77-DNA complex formation (Fig. 5B
). These results clearly
demonstrate that LH induces Nur77 DNA-binding activity.

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Figure 5. NBRE-binding activity of Nur77 in Leydig cells.
EMSA was performed using NBRE as a probe and whole cell extracts (100
µg) from K28 cells stimulated with LH for the indicated time. Whole
cell extracts (100 µg) from each time point were incubated with NBRE
probe (A). Whole cell extracts (100 µg) from either control cells (C)
or cells treated with LH for 2 h (LH) were incubated with the NBRE
probe with no competition or with a 10-, 25-, or 50-fold excess of
unlabeled NBRE competitor ( ) and a 50-fold excess of
nonspecific competitor (N. S.), mutated SF-1 binding sequence, and
1 µg Nur77-specific antibody (Nur77 Ab) or nonspecific antibody,
progesterone antibody (PR Ab), were added to the reaction mixture
before the addition of labeled probe (B). Free, Running of labeled
probe only; arrows, Nur77-DNA complex;
arrowheads, nonspecific binding (N.B.).
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To examine whether the induction of Nur77 gene expression represents
enhancement of Nur77 activity, Nur77 DNA-binding element-driven
luciferase reporter, NurRE 3 copy-POMC-luc, and NBRE-tk-luc were
examined in the presence of LH. LH increased Nur77 activity in a
dose-dependent manner, indicating that the LH-mediated increase in both
Nur77 mRNA and its protein contribute to the increase in Nur77
activity. Interestingly, the NurRE-reporter was 8 times more responsive
than the NBRE reporter in response to LH (Fig. 6
, A and B), suggesting that the monomer
or dimer formation of Nur77 was differentially regulated by LH. These
results indicate that LH regulates Nur77 activity via direct induction
of its gene expression.

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Figure 6. NurRE (A) and NBRE (B) confer higher
responsiveness to LH treatment. K28 cells were transfected with 300 ng
of the indicated luciferase reporter gene. Cells were treated with LH
(nanograms per ml) or without LH (0), and assayed for luciferase
activity after 36 h. Luciferase activity was normalized by
ß-galactosidase activity to determine the transfection efficiency.
The data shown represent the mean of five independent experiments. Data
are reported as fold activation relative to the control
(±SEM).
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Nur77 is involved in steroidogenesis in Leydig cells
To confirm whether Nur77 trans-activation is inhibited
by DN-Nur77 (14, 16, 18) in K28 cells, transient
transfection assays were performed with NBRE-Luc, Nur77, and DN-Nur77
expression vector. As shown Fig. 7A
, DN-Nur77 significantly inhibited Nur77 trans-activation in a
dose-dependent manner. To correlate the LH-mediated induction of Nur77
gene expression with the regulation of steroidogenesis, DN-Nur77 was
transiently expressed in K28 cells, the cell culture media were
collected, and the amount of progesterone was determined by RIA.
Progesterone synthesis was significantly induced after 1 h and
gradually declined up to 6 h in LH-treated (200 ng/ml) K28 cells.
In contrast, overexpression of DN-Nur77 in LH-treated cells was
accompanied by a significant reduction in progesterone (Fig. 7B
).
DN-Nur77 maintained an average transfection efficiency of about 40%
(data not shown). Moreover, overexpression of the active form of Nur77
in K28 cells significantly increased progesterone production in the
absence of LH (Fig. 7C
). These results strongly suggest that
LH-mediated Nur77 expression may be involved in steroidogenesis in
Leydig cells.
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Discussion
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The present study demonstrates that expression of orphan nuclear
receptor Nur77 is regulated by LH, and that LH-mediated induction of
Nur77 gene expression is involved in steroidogenesis in testicular
Leydig cells. Leydig cells in the interstitial compartment of the
testis were capable of steroidogenesis, which is the conversion of
cholesterol to pregnenolone and then to progesterone (35).
Moreover, the major stimulus for the biosynthesis of T in the Leydig
cells is the gonadotropin, LH (34, 36). The current study
demonstrated that the Nur77 mRNA level is increased during postpubertal
development in mouse testis. As LH secretion is increased during
puberty in testis, it is reasonable to speculate that a high level of
LH could be implicated in the induction of Nur77 gene expression in
Leydig cells, the main target cell of LH in the testis. Moreover, this
idea is strongly supported by the finding that Nur77 mRNA was induced
by hCG treatment in peripubertal mouse testis.
It has been shown that ACTH, an anterior pituitary peptide hormone,
rapidly induces Nur77 mRNA synthesis in the mouse adrenocortical tumor
cell, Y1 (10), and more recently that PTH also induces
Nur77 mRNA in primary mouse osteoblasts (37), and LH
induces NGFI-B mRNA in rat ovarian follicle (38). Our
results demonstrated that LH rapidly increased the expression of Nur77
mRNA in the Leydig cell line, K28. Unlike ACTH, which induced Nur77
mRNA up to 16 h, LH-mediated induction of Nur77 mRNA returned to
basal levels after 6 h, suggesting that LH-mediated induction of
Nur77 gene expression is more transient than that of ACTH-mediated
induction. Moreover, LH induced the subfamily member of Nur77, Nurr-1,
suggesting that the normal phenotype in Nur77 null mice
(11) might be due to functional redundancy among the Nur77
family. Western blot analysis showed that LH induces Nur77 protein
ranging from 7090 kDa. It has been well documented that Nur77 is
phosphorylated at multiple sites (7, 8, 9, 39) related to its
trans-activation, and a recent study demonstrates that NGF
induces phosphorylation of Nur77 on Ser105 in
PC12 pheochromocytoma cells, which results in the translocation of
Nur77 from nucleus to cytoplasm (40), and the
phosphorylation on Ser354 decreases Nur77
DNA-binding activity (41, 42). Therefore, LH-mediated
hyperphosphorylation of Nur77 protein may modulate both its DNA-binding
and trans-activation activities, and the potential amino
acid residues of phosphorylation of Nur77 by LH still remain to be
determined.
To investigate the intracellular signaling pathways controlling Nur77
gene expression, we examined several inhibitors and activators of
signaling pathway. In addition to LH, forskolin, TPA, and 8-bromo-cAMP
also induced Nur77 expression in K28 cells. Although it has been well
documented that cAMP is the major second message for LH, there is
considerable evidence to suggest that other intracellular signaling
systems may also be involved in LH action (43). These
include IP3, diacylglycerol, calcium, arachidonic acid, and
various free radicals. This idea is strongly supported by the
observation that LH-mediated Nur77 gene expression was abolished by
H-89, an inhibitor of PKA; bisindolylmaleimide I (GF109203X), an
inhibitor of PKC; and wortmannin, an inhibitor of PI3 kinase, whereas
there was no significant effect of PD98059, an inhibitor of MAPK, on
Nur77 gene expression (data not shown). In contrast to a previous
report on ACTH- and angiotensin II-mediated Nur77 induction in adrenal
cortical cells (44), LH-mediated Nur77 induction in K28
was strongly inhibited by H-89, indicating that Nur77 expression is
differentially regulated in a stimulus- and cell type-specific-manner.
Therefore, it seems reasonable to speculate that LH induces Nur77 gene
expression through diverse cell signaling pathways in Leydig cells.
Transient transfection experiments with Nur77 promoter (bp -336 to
+67)-Luc reporter indicate that LH induces Nur77 promoter activity.
Although we have not ruled out the possible implication of the further
upstream region of Nur77 promoter, this promoter region was sufficient
to confer LH-mediated Nur77 gene induction in K28 cells. Multiple
cis-acting elements, such as GC-rich/SP-1 site,
activating protein-1-like elements, and RSRF elements in this promoter
region, were characterized previously (15, 41, 45, 46, 47, 48).
Therefore, multiple transcription factors may also be involved in the
LH-mediated Nur77 induction in Leydig cells.
It has been reported that Nur77 recognizes a specific sequence called
the NBRE, AAAGGTCA (19). We showed that Nur77 constitutes
NBRE-binding activity in LH-stimulated Leydig cells. Interestingly,
Nur77 DNA-binding activity was observed from 224 h, indicating that
the Nur77-DNA complex is continuously maintained in LH-treated
testicular Leydig cells. This pattern of Nur77-binding activity is
reminiscent of a previous report (49) of the induction of
immediate-early response gene c-fos/c-jun gene
expression by antioxidant. This immediate-early response gene
expression is transient, but the activating protein-1 binding activity
is continuously maintained, and this delayed-type DNA-binding activity
does not correlate with transient induction of
c-fos/c-jun. Therefore, we concluded that the
delayed and continuous DNA-binding activity of Nur77 by LH treatment
might be due to the intrinsic characteristics of the orphan nuclear
receptor Nur77.
To identify the Nur77 target gene in Leydig cells, we examined the
cholesterol side-chain cleavage enzyme and steroidogenic acute
regulatory protein gene promoter activity by Nur77 in K28 cells.
Consistent with previous report (50), Nur77 did not show
any specific effect on steroidogenic acute regulatory protein or
cholesterol side-chain cleavage enzyme promoter activity (data not
shown). Although we could not determine the putative Nur77 target gene
that is involved in response to LH, identification of specific genes
regulated by Nur77 in Leydig cells is currently under investigation in
our laboratory.
A recent study has demonstrated that Nur77 is the sole transcription
factor that mediates PGF2
stimulation of
20
-hydroxysteroid dehydrogenase (20
HSD), which converts
progesterone into biologically inactive steroid (18). In
contrast to this report, there was no significant change in 20
HSD
mRNA after LH treatment in K28 cells (data not shown), indicating that
LH-mediated Nur77 induction may not be implicated in the regulation of
20
HSD gene expression. However, the existence of NBRE sequence in
several steroidogenic enzyme gene promoters (51, 52)
suggests that Nur77 may participate in the regulation of
steroidogenesis in Leydig cells. Interestingly, we observed that
overexpression of DN-Nur77 suppressed LH-induced progesterone
biosynthesis. Moreover, overexpression of the active form of Nur77
significantly increased progesterone biosynthesis, suggesting that
LH-mediated Nur77 may play an important role in the regulation of
steroidogenesis in Leydig cells. However DN-Nur77 could not completely
abolish LH-mediated progesterone biosynthesis, suggesting that the
induction of steroidogenic enzymes by other transcription factors may
also be implicated in LH-mediated steroidogenesis. To our knowledge,
this is the first report that Nur77 is directly linked to
steroidogenesis in testicular Leydig cells.
In summary, we have shown that orphan nuclear receptor Nur77 gene
expression is regulated by LH in a testis Leydig cell line. LH
treatment induces Nur77 gene expression via diverse signaling pathway,
and this induction is regulated at a transcriptional level.
Furthermore, LH-mediated Nur77 gene expression is involved in
steroidogenesis in testicular Leydig cells. Identification of the
target genes regulated by Nur77 will be necessary to understand the
detailed function of Nur77 responding to LH in testis development.
 |
Acknowledgments
|
|---|
The authors thank Drs. Sang-Young Chun and Jaewoon Lee for
critical reading of this manuscript, Drs. Jacques Drouin and Thomas
Perlmann for providing the NurRE 3 copy-POMC-Luc reporter constructs
and Nurr-1, Dr. C. Finaz for providing the K28 mouse Leydig tumor
cells, and Dr. Ryun Sup Ahn for technical assistance.
 |
Footnotes
|
|---|
This work was supported by the Genetic Engineering Research Fund
(GE98-019-D00115) and Hormone Research Center Grant 99G0201, Republic
of Korea (to H.S.C.).
Abbreviations: CMV-ß-gal,
Cytomegalovirus-ß-galactosidase; DN-Nur77, dominant
negative Nur77; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase;
hCG, human CG; 20
HSD, 20
-hydroxysteroid dehydrogenase; NBRE,
NGFI-B (Nur77)-responsive element; NGF, nerve growth factor;
NurRE, Nur response element; TBST, 10 mM Tris-buffered
isotonic saline (pH 7.0), 0.1% merthiolate, and 0.1% Tween-20; TPA,
12-O-tetradecanoyl-phorbol-13-acetate.
Received April 6, 2001.
Accepted for publication August 1, 2001.
 |
References
|
|---|
-
Giguere V 1999 Orphan nuclear receptors: from
gene to function. Endocr Rev 20:689725[Abstract/Free Full Text]
-
Mangelsdorf DJ Thummel C, Beato M, Herrlich P, Schutz
G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily; the second decade. Cell 83:835839[CrossRef][Medline]
-
Evans RM 1988 The steroid and thyroid hormone
receptors superfamily. Science 240:889895[Abstract/Free Full Text]
-
Milbrandt J 1988 Nerve growth factor induces a
gene homologous to the glucocorticoid receptor gene. Neuron 1:183188[CrossRef][Medline]
-
Hazel TG, Nathans D, Lau LF 1988 A gene inducible
by serum growth factors encodes a member of the steroid and thyroid
hormone receptor superfamily. Proc Natl Acad Sci USA 85:84448448[Abstract/Free Full Text]
-
Law SW, Conneely OM, DeMayo FJ, OMalley BW 1992 Identification of a new brain-specific transcription factor, Nurr1. Mol
Endocrinol 6:21292135[Abstract]
-
Davis IJ, Hazel TG, Chen RH, Blenis J, Lau LF 1993 Functional domains and phosphorylation of the orphan receptor Nur77.
Mol Endocrinol 7:953964[Abstract]
-
Fahrner TJ, Carroll SL, Milbrandt J 1990 The
NGFI-B protein, an inducible member of the thyroid/steroid receptor
family, is rapidly modified posttranslationally. Mol Cell Biol 10:64546459[Abstract/Free Full Text]
-
Hazel TG, Misra R, Davis IJ, Greenberg ME, Lau LF 1991 Nur77 is differentially modified in PC12 cells upon membrane
depolarization and growth factor treatment. Mol Cell Biol 11:32393246[Abstract/Free Full Text]
-
Wilson TE, Mouw AR, Weaver CA, Milbrandt J, Parker
KL 1993 The orphan nuclear receptor NGFI-B regulates expression of
the gene encoding steroid 21-hydroxylase. Mol Cell Biol 13:861868[Abstract/Free Full Text]
-
PA Crawford, Sadovsky Y, Woodson K, Lee SL, Milbrandt
J 1995 Adrenocortical function and regulation of steroid
21-hydroxylase gene in NGFI-B-deficient mice. Mol Cell Biol 15:43314336[Abstract]
-
Fernandez PM, Brunel F, Jimenez MA, Saez JM, Cereghini
S, Zakin MM 2000 Nuclear receptors Nor1 and NGFI-B/Nur77 play
similar, albeit distinct, roles in the hypothalamo-pituitary-adrenal
axis. Endocrinology 141:23922400[Abstract/Free Full Text]
-
Liu ZG, Smith SW, McLaughlin KA, Schwartz LM, Osborne
BA 1994 Apoptosis signals delivered through the T-cell receptor of
a T-cell hybrid require the immediate-early gene nur77. Nature 367:281284[CrossRef][Medline]
-
Woronicz JD, Calnan B, Ngo V, Winoto A 1994 Requirement for the orphan steroid receptor Nur77 in apoptosis of
T-cell hybridomas. Nature 367:277281[CrossRef][Medline]
-
Woronicz JD, Lina A, Calnan BJ, Szychowski S, Cheng L,
Winoto A 1995 Regulation of the Nur77 orphan steroid receptor in
activation-induced apoptosis. Mol Cell Biol 15:63646376[Abstract]
-
Cheng LE, Chan FK, Cado D, Winoto A 1997 Functional
redundancy of Nur77 and Nor-1 orphan steroid receptors in T-cell
apoptosis. EMBO J 16:18651875[CrossRef][Medline]
-
Li H, Kolluri SK, Gu J, Dawson MI, Cao X, Hobbs PD, Lin
B, Chen G, Lu J, Lin F, Xie Z, Fontana JA, Reed JC, Zhang X 2000 Cytochrome c release and apoptosis induced by mitochondrial
targeting of nuclear orphan receptor TR3. Science 289:11591164[Abstract/Free Full Text]
-
Stocco CO, Zhong L, Sugimoto Y, Ichikawa A, Lau LF,
Gibori G 2000 Prostaglandin F2
induced
expression of 20
-hydroxysteroid dehydrogenase involves the
transcription factor NUR77. J Biol Chem 275:3720237211[Abstract/Free Full Text]
-
Wilson TE, Fahrner TJ, Johnston M, Milbrandt J 1991 Identification of the DNA binding site for NGFI-B by genetic selection
in yeast. Science 252:12961300[Abstract/Free Full Text]
-
Wilson TE, Fahrner TJ, Milbrandt J 1993 The orphan
receptors NGFI-B and steroidogenic factor 1 establish monomer binding
as a third paradigm of nuclear receptor-DNA interaction. Mol Cell Biol 13:57945804[Abstract/Free Full Text]
-
Philips A, Lesage S, Gingras R, Maira MH, Gauthier Y,
Hugo P, Drouin J 1997 Novel dimeric Nur77 signaling mechanism in
endocrine and lymphoid cells. Mol Cell Biol 17:59465951[Abstract]
-
Philips A, Maira M, Mullick A, Chamberland M, Lesage S,
Hugo P, Drouin J 1997 Antagonism between Nur77 and glucocorticoid
receptor for control of transcription. Mol Cell Biol 17:59525959[Abstract]
-
Maira M, Martens C, Philips A, Drouin J 1999 Heterodimerization between members of the Nur subfamily of orphan
nuclear receptors as a novel mechanism for gene activation. Mol Cell
Biol 19:75497557[Abstract/Free Full Text]
-
Perlmann T, Jansson L 1995 A novel pathway for
vitamin A signaling mediated by RXR heterodimerization with NGFI-B and
NURR1. Genes Dev 9:769782[Abstract/Free Full Text]
-
Ascoli M 1981 Characterization of several clonal
lines of cultured Leydig tumor cells: gonadotropin receptors and
steroidogenic responses. Endocrinology 108:8895[Abstract]
-
Finaz C, Lefevre A, Dampfhoffer D 1987 Construction
of a Leydig cell line synthesizing testosterone under gonadotropin
stimulation: a complex endocrine function immortalized by cell
hybridization. Proc Natl Acad Sci USA 84:57505753[Abstract/Free Full Text]
-
Lefevre A, Rogier E, Astraudo C, Duquenne C, Finaz
C 1994 Regulation by retinoids of luteinizing hormone/chorionic
gonadotropin receptor, cholesterol side-chain cleavage cytochrome
P-450,3ß-hydroxysteroid
dehydrogenase/
54-isomerase and
17
-hydroxylase/C1720lyase cytochrome P-450 messenger ribonucleic
acid levels in the K9 mouse Leydig cell line. Mol Cell Endocrinol 106:3139[CrossRef][Medline]
-
Lee H-K, Yoo M-S, Choi H-S, Kwon H-B, Soh JM 1999 Retinoic acids up-regulate steroidogenic acute regulatory protein gene.
Mol Cell Endocrionl 148:110[CrossRef][Medline]
-
Wu Q, Li Y, Liu R, Agadir A, Lee M-O, Liu Y, Zhang
X 1997 Modulation of retinoic acid sensitivity in lung cancer
cells through dynamic balance of orphan receptors nur77 and COUP-TF and
their heterodimerization. EMBO J 16:16561669[CrossRef][Medline]
-
Kang H-J, Song M-R, Lee S-K, Shin E-C, Choi Y-H, Kim
S-J, Lee J-W, Lee M-O 2000 Retinoic acid and its receptors repress
the expression and transactivation functions of Nur77: a possible
mechanism for the inhibition of apoptosis by retinoic acid. Exp Cell
Res 256:545554[CrossRef][Medline]
-
Chen JD, Umesono K, Evans RM 1996 SMART isoforms
mediate repression and anti-repression of nuclear receptor
heterodimers. Proc Natl Acad Sci USA 93:75677571[Abstract/Free Full Text]
-
Kwon HB, Schuetz AW 1986 Role of cAMP in modulating
intrafollicular progesterone levels and oocyte maturation in amphibians
(Rana pipiens). Dev Biol 117:354364[CrossRef][Medline]
-
Benton L, Shan LX, Hardy MP 1995 Differentiation of
adult Leydig cells. J Steroid Biochem Mol Biol 53:6168[CrossRef][Medline]
-
Lejeune H, Habert R, Saez JM 1998 Origin,
proliferation and differentiation of Leydig cells. J Mol Endocrinol 20:125[CrossRef][Medline]
-
Hall PF, DC Irby, DE Kretser DM 1969 Conversion of
cholesterol to androgens by rat testes: comparison of interstitial
cells and seminiferous tubules. Endocrinology 84:488496[Medline]
-
Odell WD, Swerdloff RS, Bain J, Wollesen F, Grover
PK 1974 The effect of sexual maturation on testicular response to
LH stimulation of testosterone secretion in the intact rat.
Endocrinology 95:13801384[Medline]
-
Tetradis S, Bezouglaia O, Tsingotjidou A, Vila A 2001 Regulation of the nuclear orphan receptor Nur77 in bone by
parathyroid hormone. Biochem Biophys Res Commun 281:913916[CrossRef][Medline]
-
Park J-I, Park H-J, Choi H-S, Lee KS, Lee W-K, Chun
S-Y 2001 Gonadotropin regulation of NGFI-B messenger ribonucleic
acid expression during ovarian follicle development in the rat.
Endocrinology 142:30513059[Abstract/Free Full Text]
-
Li Y, Lau LF 1997 Adrenocorticotropic hormone
regulates the activities of the orphan nuclear receptor Nur77 through
modulation of phosphorylation. Endocrinology 138:41384146[Abstract/Free Full Text]
-
Katagiri Y, Takeda K, Yu Z-X, Ferrans VJ, Ozato K,
Guroff G 2000 Modulation of retinoid signaling through NGF-induced
nuclear export of NGFI-B. Nat Cell Biol 2:435440[CrossRef][Medline]
-
Davis IJ, Lau LF 1994 Endocrine and neurogenic
regulation of the orphan nuclear receptors Nur77 and Nurr-1 in the
adrenal glands. Mol Cell Biol 14:34693483[Abstract/Free Full Text]
-
Hirata Y, Kiuchi K, Chen H-C, Milbrandt J, Guroff G 1993 The phosphorylation and DNA binding of the DNA-binding domain of
the orphan nuclear receptor NGFI-B. J Biol Chem 268:2480824812[Abstract/Free Full Text]
-
Cooke BA 1996 Transduction of the luteinizing
hormone signal within the Leydig cells. In: Payne AH, Hardy MP, Russell
LD, eds. The Leydig cell. Vienna: Cache River Press; 352363
-
Enyeart JJ, Boyd RT, Enyeart JA 1996 ACTH and AII
differentially stimulate steroid hormone orphan receptor mRNAs in
adrenal cortical cells. Mol Cell Endocrinol 124:97110[CrossRef][Medline]
-
Ryseck R-P, Macdonald-Bravo H, Mattei M-G, Ruppert S,
Bravo R 1989 Structure, mapping and expression of a growth factor
inducible gene encoding a putative nuclear hormonal binding receptor.
EMBO J 8:33273335[Medline]
-
Yoon JK, Lau LF 1994 Involvement of JunD in
transcriptional activation of the orphan receptor gene nur77 by nerve
growth factor and membrane depolarization in PC12 cells. Mol Cell Biol 14:77317743[Abstract/Free Full Text]
-
Williams GT, Lau LF 1993 Activation of the
inducible orphan receptor gene nur77 by serum growth factors:
dissociation of immediate-early and delayed-early responses. Mol Cell
Biol 13:61246136[Abstract/Free Full Text]
-
Liu X, Chen X, Zachar V, Chang C, Ebbesen P 1999 Transcriptional activation of human TR3/nur77 gene expression by human
T-lymphotropic virus type I Tax protein through two AP-1-like elements.
J Gen Virol 80:30733081[Abstract/Free Full Text]
-
Choi H-S, Moore DD 1993 Induction of
c-fos and c-jun gene expression by phenolic
antioxidants. Mol Endocrinol 7:15961602[Abstract]
-
Sugawara T, Holt JA, Kiriakidou M, Strauss III JF 1996 Steroidogenic factor 1-dependent promoter activity of the human
steroidogenic acute regulatory protein (StAR) gene. Biochemistry 35:90529059[CrossRef][Medline]
-
Rice DA, Mouw AR, Bogerd AM, Parker KL 1991 A
shared promoter element regulates the expression of three steroidogenic
enzymes. Mol Endocrinol 5:15521561[Abstract]
-
Morohashi K-I, Honda S-I, Inomata Y, Handa H, Omura
T 1992 A common trans-acting factor, Ad4-binding protein, to the
promoters of steroidogenic P-450s. J Biol Chem 267:1791317919[Abstract/Free Full Text]
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