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J Biol Chem, Vol. 274, Issue 20, 13870-13876, May 14, 1999
From the We have previously shown that early growth
response (Egr) 1-deficient mice exhibit female infertility,
reflecting a luteinizing hormone (LH) Endocrine signals within the female
hypothalamus-pituitary-gonad axis regulate ovarian follicle
development, ovulation, and steroidogenesis. Broadly, these changes in
the hormonal milieu not only regulate sex organ development and
reproductive function but also influence bone formation and
cardiovascular homeostasis. Gonadotropins play a central role in
orchestrating gonadal function. Gonadotropin production is directed by
pulsatile secretion of hypothalamic
GnRH1 and transmitted to
gonadotropes via the pituitary portal system (1-5).
Follicle-stimulating hormone and luteinizing hormone (LH) are
heterodimeric glycoproteins that consist of a common The production of LH In addition to Egr-1 elements, the LH Like SF-1, Egr-1 is also required for normal reproductive development
and function, as Egr-1 Plasmids and Mutagenesis--
The wild type Cell Culture and Transfection--
L Electromobility Shift Assay--
Double-stranded
oligonucleotides (100 ng) that contained the wild type or mutated
binding elements of either 5' Egr-1, 3' Egr-1, 5' SF-1, or 3' SF-1
(detailed above) were end-labeled with 25 µCi of
[ Expression Analysis--
Total RNA was isolated from L
A recombinant adenovirus expressing rat Egr-1 from the CMV promoter in
the Ad5PacIGFP vector was provided by Markus Ehrengruber (Brain Research Institute, University of Zurich, Switzerland) (50).2 Approximately 100,000 L GnRH-dependent Stimulation of LH
It has been previously shown that the LH
To test whether or not GnRH regulates the expression of either Egr-1 or
SF-1, we used Northern blot analysis to determine their expression in
GnRH-stimulated L Other Egr Family Members Synergistically Interact with SF-1 in
Regulation of LH
To assess the role of the cooperativity between SF-1 and the members of
the Egr family of proteins that stimulate the LH DAX-1 Represses Basal- and GnRH-stimulated Activity of LH
To determine whether or not DAX-1 represses the
GnRH-dependent stimulation of the LH Egr-1 is essential for LH Our results are consistent with the finding that changes in the
transcription rate of LH The mechanism of synergy between Egr-1 and SF-1 is currently unknown.
Our results, as well as the results of others (29), indicate that both
proteins must be DNA-bound to synergistically activate LH In addition to Egr-1 and SF-1, other DNA binding elements within the
LH Unlike Egr-1, little is known at the present time about the role of
other Egr proteins in reproductive physiology or pathophysiology. Mice
homozygous for mutant Egr-2 exhibit abnormal hindbrain development and
die within 2 weeks after birth (59, 60). Egr-3-deficient mice have
muscle spindle agenesis, which results in sensory ataxia, and are not
known to have gonadotropin deficiency (61). We used JEG3 cells to
assess cooperativity between Egr proteins and SF-1 in activation of the
LH DAX-1 is a potent repressor of SF-1 activity (36, 37), and this
repression is achieved, at least in part, through recruitment of the
co-repressor N-CoR to DNA-bound SF-1. DAX-1 is expressed in the
anterior pituitary and in Our findings highlight the significance of the synergy between the
immediate early gene product Egr-1 and the gonadotrope-specific SF-1 in
GnRH stimulation of LH We are grateful to Pamela Mellon for
generously providing the L *
This work was supported by Deutsche Forschungsgemeinschaft
Grant DO-653/1 (to C. D.), National Institutes of Health Grant HD-34110, and the Howard Hughes Medical Institute Pilot Research Projects Award (to Y. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
2
J. Svaren, manuscript in preparation.
The abbreviations used are:
GnRH, gonadotropin-releasing hormone;
LH, luteinizing hormone;
Egr, early
growth response;
SF-1, steroidogenic factor-1;
DAX-1, dosage-sensitive
sex reversal adrenal hypoplasia congenital critical region on
chromosome X, gene 1;
PCR, polymerase chain reaction;
CMV, cytomegalovirus.
Activation of Luteinizing Hormone 
Gene by
Gonadotropin-releasing Hormone Requires the Synergy of Early Growth
Response-1 and Steroidogenic Factor-1*
,,¶
Department of Obstetrics and Gynecology, the
§ Department of Pathology, and the ¶ Department of Cell
Biology and Physiology, Washington University School of Medicine,
St. Louis, Missouri 63110
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
deficiency. Egr-1
activates the LH gene in vitro through synergy with
steroidogenic factor-1 (SF-1), a protein required for gonadotrope
function. To test if this synergy is essential for
gonadotropin-releasing hormone (GnRH) stimulation of LH, we examined
the activity of the LH promoter in the gonadotrope cell line LT2.
GnRH markedly stimulated the LH promoter (15-fold). Mutation of
either Egr-1 or SF-1 elements within the LH promoter attenuated this
stimulation, whereas mutation of both promoter elements abrogated GnRH
induction of the LH promoter. Furthermore, GnRH stimulated Egr-1 but
not SF-1 expression in LT2 cells. Importantly, overexpression of
Egr-1 alone was sufficient to enhance LH expression. Although other
Egr proteins are expressed in LT2 cells and are capable of
interacting with SF-1, GnRH stimulation of Egr-1 was the most robust.
We also found that the nuclear receptor DAX-1, a repressor of SF-1
activity, reduced Egr-1-SF-1 synergy and diminished GnRH stimulation
of the LH promoter. We conclude that the synergy between Egr-1 and
SF-1 is essential for GnRH stimulation of the LH gene and plays
a central role in the dynamic regulation of LH expression.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit and a
unique -subunit (4). Follicle-stimulating hormone and LH are
secreted in a cyclic, fluctuating manner. Whereas follicle-stimulating hormone plays a major role in oocyte development and estrogen production during the follicular phase of the cycle, mid-cycle LH surge
promotes luteinization of the dominant follicle and progesterone production (4).
is modulated at several levels, including
mRNA transcription, polyadenylation, and protein glycosylation (2,
6, 7). Whereas diverse signaling pathways converge on the modulation of
LH gene expression (4, 8-15), recent analysis of mice bearing loss
of function mutations in either early growth response-1 (Egr-1, also
known as NGFI-A, Zif268, or Krox24) or steroidogenic factor-1
(SF-1) demonstrated that these two transcription factors play a
critical role in directing LH expression (16-19). Egr-1, a zinc
finger transcription factor, is the prototype of a family of
egr genes (20, 21). These genes are induced in response to a
variety of extracellular stimuli that lead to proliferation,
differentiation, or apoptosis (21). The expression of Egr-1 is
widespread (22, 23). In the pituitary it is expressed in the anterior
lobe, primarily in gonadotropes and somatotropes (24). The DNA binding
domain of Egr-1 is highly homologous among three other members of this
family, including Egr-2 (Krox20), Egr-3, and Egr-4 (nerve growth factor
induced-C) (21, 25, 26). These Egr proteins bind to sites that resemble the consensus site TGCG(T/G)(G/A)GG(C/A/T)G(G/T) (27). The LH promoter contains similar sites at positions 
50 and 113.
proximal promoter also
contains evolution-conserved SF-1 responsive elements located at
positions 127 and 59 (12, 16, 28, 29). SF-1 is an orphan member of
the nuclear receptor superfamily of proteins (30), which binds to its
cognate DNA element as a monomer (31, 32). SF-1 regulates the
expression of most steroidogenic enzymes in both female and male gonads
and adrenal cortex as well as other proteins relevant to reproductive
development and function (31, 32). Importantly, SF-1 is essential for
reproductive development, because mice deficient in this transcription
factor lack gonads and adrenal glands, which consequently leads to
persistence of Mullerian structures even in male embryos as well as
early neonatal death from adrenal insufficiency (17-19).
SF-1-deficient mice also have impaired development of the hypothalamic
ventromedial nucleus (33). Interestingly, the transcriptional activity
of SF-1 is repressed by DAX-1, an orphan nuclear receptor implicated in
the pathogenesis of adrenal hypoplasia congenita and hypogonadotropic hypogonadism (34-37). Although several studies (12, 38) indicated that
SF-1 is absolutely required for LH expression, subsequent analysis
of SF-1 / mice, maintained until maturity by corticosteroid rescue
treatment, revealed that these mice do express LH in response to
GnRH injections (33).
/female mice exhibit arrested uterine
development and infertility, reflecting a specific deficiency in LH
expression in pituitary gonadotropes (16, 24). Importantly, we and
others (16, 29) have recently demonstrated that a synergistic interaction between Egr-1 and SF-1 is essential for the activation of
the LH promoter in vitro, suggesting that SF-1 and Egr-1
may provide a means of directing LH expression in response to
physiological stimuli that orchestrate gonadotrope function. Pulsatile
release of GnRH acting through GnRH receptors plays a central role in dynamic regulation of LH expression (1, 3, 5, 6, 10, 13, 39).
Therefore we hypothesized that GnRH activation of the LH gene
requires the synergistic interaction of Egr-1 and SF-1. We tested our
hypothesis utilizing the gonadotrope cell line LT2, which expresses
Egr-1 and SF-1 and responds to GnRH administration with LH
production (40-42).
EXPERIMENTAL PROCEDURES
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156 to +7 LH
promoter construct, cloned upstream of luciferase in PL(KS)b-Luc vector
(a gift from Stuart Adler, Washington University, St. Louis, MO), was
described previously (16). To generate mutations of either Egr-1 or
SF-1 sites, we subcloned the LH promoter into a pBSKS vector
(Stratagene) and generated the following mutations: Egr-1 at 113 from
CGCCCCCAA to TAGTACTCA, Egr-1 at 50 from CACCCCCAC to GATTCTTAT, SF-1
at 127 from TGACCTTG to TGATCATG, and SF-1 at 59 from TGGCCTTG to
TGGAATTC. For mutagenesis, overlapping mutagenic oligonucleotides were
synthesized and used in PCR performed with Klentaq (43) for high
fidelity and processivity (PCR parameters were 25 cycles of
94 °C × 0.5 min, 56 °C × 2 min, and 72 °C × 4 min). The PCR products were phenol/chloroform, which was extracted
and digested with DpnI (NEB) to select for mutated,
PCR-generated plasmid. The plasmid mixture was amplified in XL-1 blue
bacteria (Stratagene), isolated, and sequenced using the dideoxy method
in an automatic sequencer (Applied Biosystems). All LH promoter
mutants were subcloned back into the PL(KS)b-Luc vector at the
SpeI/XhoI sites. Expression vectors for the
Egr-1, SF-1, DAX-1, and DAX-11-369 deletion mutants were
previously described (37, 44, 45). Expression vectors for Egr-2, Egr-3,
and Egr-4, all cloned in a CMV-Neo expression vector, were kindly
provided by Jeffrey Milbrandt (Washington University, St. Louis, MO).
T2 cells (40) were
generously provided by P. Mellon (La Jolla, CA) and maintained in
monolayer cultures in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum and antibiotics in humidified 10%
CO2, 90% air at 37 °C. For transfection experiments the
cells were plated in 6-well plates at a density of 350,000 cells/well.
We have determined that optimal results were obtained when
transfections were conducted 24 h after plating. The growth medium
was routinely replaced with standard fresh medium 4 h before
transfection. Cells were transfected using the modified calcium-phosphate method described previously (46) using a total of 2.5 µg/well, which included 0.05 µg of CMV--galactosidase plasmid
(to normalize for cell viability and transfection efficiency). After
24 h the medium was changed to medium that contained 1% charcoal/dextran-treated serum (Hyclone). GnRH (Sigma) was added to the
medium at a concentration range of 0.1-100 nM. After 15 min the medium was removed and replaced by the 1% serum containing medium. In some of the experiments, this cycle was repeated every 90 min for a total of four cycles following a previously published protocol (41). Standard luciferase assays were performed 48 h
after transfection as described previously (45). All experiments were
performed in duplicate and repeated at least three times. Results
(mean ± S.D.) normalized to -galactosidase activity were expressed as relative luciferase units. The human choriocarcinoma cell
line JEG3 was maintained and transfected as described previously (45).
-32P]ATP using polynucleotide kinase. Nuclear extract
from LT2 cells was generated as described previously (47). For
positive control proteins, we used a bacterially expressed and purified
His-Egr-129-536 or glutathione
S-transferase-SF-11-106, both previously described (48, 49). For each binding reaction we mixed 0.02 µg of the
proteins or 3 µg of the extracts with 1 ng of a labeled probe in a
binding buffer that contained 50 mM NaCl, 1 mM
EDTA, and 5% glycerol in 10 mM Tris (pH 7.5) and incubated
for 30 min at 25 °C. Each binding reaction was loaded onto a 5%
polyacrylamide gel and run in 0.5× Trisborate/EDTA buffer at 150 V for
2 h. The gel was then dried and exposed to a PhosphorImager screen
or film.
T2
cells using Tri-reagent (Molecular Research Center, Cincinnati, OH).
RNA samples were extracted, precipitated, and resuspended in water. RNA
samples (15 µg) were resolved by electrophoresis using a 1% agarose,
1.5% formaldehyde gel. Specific probes for Northern blot analysis of
each murine Egr transcript as well as SF-1 were generated by PCR using
standard techniques and labeled with [32P]dCTP using a
Prime-It II (Stratagene) labeling kit. RNA was transferred to nylon
membranes (Zeta-Probe, Bio-Rad) and hybridized overnight at 42 °C.
The blots were washed three times in 0.2-2× sodium chloride/sodium
citrate buffer (1× buffer is 0.15 M sodium chloride and
0.015 M sodium citrate) with 0.1% sodium dodecyl sulfate
at 65 °C. Blots were exposed to a PhosphorImager screen for 4 h
and to Kodak film at 80 °C overnight. The ImageQuant software for
the PhosphorImager was used for quantitative analysis of the expression
of Egr proteins or SF-1 corrected to glyceraldehyde-3-phosphate dehydrogenase expression.
T2 cells were infected with 3 × 108
plaque-forming units/ml of adenovirus for 2 h, and the medium was
changed twice. Cells were harvested 24 h after infection, and
total RNA was analyzed by Northern blotting using a mouse LH probe
previously described (16).
RESULTS
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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Gene Requires the
Synergistic Interaction of Egr-1 and SF-1--
We used the LT2
gonadotrope line to determine whether or not Egr-1 and SF-1
cooperatively transduce GnRH stimulation of the LH promoter. The
LT2 cell line was derived from a pituitary tumor in transgenic mice
that express the SV40 T antigen driven by the rat LH promoter (40).
In addition to the SF-1 and GnRH receptors, these cells express both
LH and LH subunits, and LH secretion is enhanced by GnRH
stimulation in vitro (40, 41, 51). To test for GnRH
enhancement of LH promoter activity, we transfected the LT2 cells
with a LH reporter construct, which contains nucleotides 156 to +7
of the rat LH promoter, upstream of luciferase (16). As shown in
Fig. 1A, we exposed the cells to either single or multiple pulses of GnRH, which were previously shown to stimulate LH secretion in LT2 cells (41). We found that
GnRH enhanced the activity of the LH reporter in a time- and
concentration-dependent fashion, and two GnRH pulses (100 nM each) given for 24 and 8 h resulted in maximum
enhancement (15-fold) of the LH reporter gene.
View larger version (36K):
[in a new window]
Fig. 1.
GnRH-dependent stimulation of the
LH promoter requires the synergistic
interaction of Egr-1 and SF-1. A, LT2 cells were
transiently transfected with the rat 156 to +7 LH-luciferase
construct and stimulated with GnRH 24 h after transfection. In two
paradigms (depicted at the bottom of the figure) GnRH was
administered in four 90 min cycles 24 h before harvest and one
additional administration 1 h before harvest as described
previously under "Experimental Procedures" (41). Results are
expressed as -fold luciferase activity over baseline (mean ± S.D.) and represent three independent experiments performed in
duplicate. B, LT2 cells were transiently transfected with
wild type or mutated forms of the rat LH-luciferase construct and stimulated with GnRH (100 nM) 24 and
8 h before harvest. Results (mean ± S.D.) are expressed as
relative luciferase units (RLU) and represent three
independent experiments performed in duplicates. TATA, TATA box.
C, mutated Egr-1 or SF-1 elements cannot bind their
respective proteins present in an LT2 nuclear extract.
Electromobility shift assay was performed as described under
"Experimental Procedures." For positive control we used a
bacterially expressed and purified His-Egr-129-536 or
glutathione S-transferase-SF-11-106. The SF-1
fusion protein is truncated at the ligand binding domain and therefore
migrates slightly faster than wild type SF-1. Results represent two
independent experiments.
promoter fragment utilized
in our studies contains two binding elements for Egr-1 and two for
SF-1, which are essential for basal LH production in vivo
(16, 29). To analyze the role of these transcription factors in
GnRH-dependent stimulation of LH expression, we added GnRH to LT2 cells that were transiently transfected with either the
wild type LH promoter reporter or the LH promoter mutated in the
binding elements for Egr-1 or SF-1. We found that mutation of the
higher affinity Egr-1 site at 50 (29) reduced both basal- and
GnRH-stimulated activity of the LH promoter (Fig. 1B).
Promoter activity was further diminished when both Egr-1 sites were
mutated. Similarly, we found that mutation of both SF-1 sites reduced
the basal- and GnRH-induced activity of the LH promoter, albeit to a
lower extent. As expected, mutation of all four Egr-1 and SF-1 sites
abolished basal- as well as GnRH-stimulated promoter activity. We used
an electromobility shift assay to confirm that the mutated promoter
elements for either Egr-1 or SF-1 could not bind their respective
protein when expressed in a nuclear extract from LT2 cells (Fig.
1C). These results indicate that Egr-1 and SF-1 sites are
essential for GnRH stimulation of LH gene expression and suggest
that between the two proteins, the influence of Egr-1 is stronger than
that of SF-1 on the GnRH-stimulated LH promoter.
T2 cells. As shown in Fig. 2A, we found that GnRH
stimulated the expression of Egr-1 in a concentration-dependent manner. In contrast, GnRH had no
significant effect on SF-1 expression under the same conditions.
Because it was previously shown that GnRH stimulates the transcription
of the LH gene in LT2 cells (41), we sought to recapitulate the response of LT2 cells to GnRH by directly assessing the influence of
enhanced Egr-1 expression on LH transcription. Using
adenovirus-mediated transfection of LT2 cells, we found that
overexpression of Egr-1 enhanced the transcription of the LH gene
(Fig. 2B). Together, these results indicate that Egr-1 is
not only required for LH expression (16, 24) but is also sufficient
to induce transcription of the LH gene in LT2 cells.
View larger version (33K):
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Fig. 2.
GnRH enhances Egr-1 expression in
L T2 cells. A, GnRH (0-30
nM) was added to the culture medium 24 and 8 h before
harvest. Total RNA was isolated from the cells and hybridized with the
indicated probes as described under "Experimental Procedures."
Results represent three independent experiments. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase. B, quantitative
analysis of the data presented in Fig. 2A presented for each
protein as -fold over control and corrected to
glyceraldehyde-3-phosphate dehydrogenase expression. C,
control. C, enhanced expression of LH in LT2 cells
that overexpress Egr-1. LT2 cells were infected with a recombinant
adenovirus expressing Egr1. Total RNA was isolated 24 h after
infection and analyzed by Northern blotting using a mouse LH probe.
The 18 S ribosomal RNA from these samples demonstrated equal loading of
the samples. The control lane is derived from uninfected
cells. Infection with a control adenovirus demonstrated that adenovirus
infection itself does not up-regulate the LH gene (data not
shown).
Promoter in Vitro--
All four members of the Egr
family of proteins share a similar binding specificity and were
previously shown to bind promoter elements that resemble Egr-1 sites
(at 113 and 50) within the LH promoter (27). Using Western and
Northern blot analyses, we have determined that Egr-2, Egr-3, and Egr-4
are all expressed in the T2 gonadotrope line (Fig. 4) (data not
shown). To determine if Egr proteins are capable of synergizing with
SF-1 in the activation of LH promoter, we expressed each Egr protein
in JEG3 cells, which express a low level of these proteins (data not
shown), and tested for activation of the LH reporter in the presence or absence of SF-1. As shown in Fig. 3,
transcriptional activity of either Egr-3 or Egr-4 was similar to that
exhibited by Egr-1, yet the activity of Egr-2 was higher than that of
the other Egr proteins. Nevertheless, all four members of the Egr
family were capable of synergistically interacting with SF-1 in
activation of the LH promoter. Importantly, the transcriptional
activity of all four Egr proteins as well as their synergy with SF-1
required intact Egr-1 sites in the LH promoter, as a mutation of
these sites abrogated their induction of a LH promoter.
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Fig. 3.
Egr proteins synergistically interact with
SF-1 in activation of LH promoter. JEG3
cells were transiently transfected with plasmids that contain either
the wild type (WT) rat LH promoter (0.5 µg) or the
LH promoter that harbors mutations in the two Egr elements or in the
two Egr elements as well as the two SF-1 elements (see Fig.
1B for promoter structure). Cells were co-transfected with
either control plasmid (CMV-Neo, 0.1 µg) or CMV-SF-1 (0.1 µg) along
with CMV-driven expression vectors for Egr proteins. The following
amounts of plasmid (previously optimized in similar experiments not
shown) were used: Egr-1, 0.1 µg; Egr-2, 0.3 µg; Egr-3, 0.01 µg;
Egr-4, 0.01 µg. Results (mean ± S.D.) are expressed as relative
luciferase units (RLU) and represent three independent
experiments performed in duplicate.
promoter, we sought
to determine the effect of GnRH on the expression of these proteins in
LT2 cells. We found that GnRH administration 24 and 8 h before
harvest enhanced the expression of Egr-1 to a greater extent than its
effect on Egr-2, Egr-3, and Egr-4. A similar result was obtained when
the expression of Egr proteins was measured 1 h after GnRH
administration (Fig. 4). Together, these
results suggest that although all four Egr proteins are expressed in
LT2 and can synergize with SF-1, Egr-1 may play the most prominent
role in GnRH stimulation of the LH gene.
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Fig. 4.
The effect of GnRH on expression of Egr
transcripts in L T2 cells. A,
GnRH was added to the culture medium either 24 and 8 h prior to
harvest at a concentration range of 0-30 nM or 1 h
prior to harvest at a concentration of 30 nM. Total RNA was
isolated from the cells and hybridized with the indicated probes as
described under "Experimental Procedures." Results represent two
independent experiments. B, quantitative analysis of the
data presented in Fig. 4A presented for each protein as
-fold over control (C) and corrected to
glyceraldehyde-3-phosphate dehydrogenase expression.
Promoter--
We and others (36, 37) have previously utilized a
synthetic SF-1 reporter gene to demonstrate that the nuclear receptor DAX-1 represses the transcriptional activation of SF-1. Because GnRH
enhances Egr-1 expression, and Egr-1 synergistically interacts with
SF-1 to induce the activity of LH reporter, we hypothesized that
DAX-1 would diminish the activity of the LH promoter. To test our
hypothesis, we first determined the effect of DAX-1 on the synergy
between Egr-1 and SF-1 in JEG3 cells. We found that unlike its effect
on a synthetic promoter, DAX-1 had a minimal repressive activity on
SF-1-dependent activation of the LH promoter in JEG3
cells (Fig. 5A). In contrast,
DAX-1 markedly diminished the cooperative interaction between all Egr
proteins and SF-1 in activation of the LH promoter. As expected,
DAX-1 had no effect when the Egr proteins were expressed alone. These
results indicate that the repressive effect of DAX-1 on the LH
promoter is SF-1-dependent, yet the ability of DAX-1 to
repress SF-1 activity is context-dependent.
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Fig. 5.
DAX-1 represses the synergistic interaction
of Egr-1 and SF-1 in activation of LH
promoter. A, JEG3 cells were transiently
transfected with plasmids that contain wild type rat LH promoter
(0.5 µg) and with either control plasmid (CMV-Neo) or CMV-SF-1 (both
at 0.1 µg) along with CMV-driven expression vectors for Egr proteins
(see legend to Fig. 3 for plasmid concentrations). Cells were
co-transfected with CMV-DAX-1 vector at 0.01 µg/well previously found
optimal for DAX-1 expression in JEG-3 cells (not shown). B,
the effect of increasing concentration of DAX-1 on basal- or
GnRH-stimulated LH promoter activity in LT2 cells. Cells were
stimulated with GnRH (100 nM) 24 and 8 h before
harvest. 
DAX-1 denotes DAX1-369, which is deficient in
repressive function (36, 37). Results (mean ± S.D.) are expressed
as relative luciferase units (RLU) and represent three
independent experiments performed in duplicate.
gene, we
transfected DAX-1 into LT2 cells. As shown in Fig. 5B,
DAX-1 repressed both basal- and GnRH-dependent stimulation
of the LH reporter. As expected, this effect was attenuated when a
C-terminally truncated form of DAX-1 (DAX-11-369), which
corresponds to naturally occurring mutations that cause adrenal
hypoplasia congenita, was used (35). A similar level of attenuation by
DAX-11-369 was previously observed using a synthetic SF-1
reporter (37). The repressive effect of DAX-1 was diminished when Egr-1
and SF-1 sites were mutated (not shown). Together, these results
indicate that optimal repression of the LH promoter activity by
DAX-1 in LT2 cells requires the cooperative interaction of Egr-1 and
SF-1.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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production in vivo, as
Egr-1 / mice exhibit LH deficiency, resulting in abnormal sexual
development and infertility (16, 24). Similarly, SF-1 / mice are
deficient in LH production (33, 38), which can be restored with GnRH administration (33). GnRH is the most important physiologic regulator
of LH production and stimulates LH production and secretion in
vitro (41). Whereas it is known that the synergy between Egr-1 and
SF-1 is required for basal expression of LH in vitro (16,
29), our results provide the first link between the
GnRH-dependent stimulation of LH and the synergistic
interaction of Egr-1 and SF-1. Maximal diminution of the LH promoter
activity is observed only when both Egr-1 and SF-1 elements are
mutated, providing further support to the role of Egr-1-SF-1 synergy
in directing LH production. It is also evident that mutations in the
Egr-1 binding elements within the LH promoter have a more profound effect on GnRH-stimulated activity of the LH promoter when compared with analogous mutations in the SF-1 elements, suggesting that Egr-1
plays a more important role in the dynamic regulation of the LH gene
by GnRH. This conclusion is supported by the finding that GnRH
regulates the expression of Egr-1 and not SF-1. In addition, we
confirmed that overexpression of Egr-1 alone was sufficient to activate
expression of endogenous LH in LT2 cells even in the
absence of GnRH stimulation. Nevertheless, it is likely that endogenous
SF-1 in LT2 cells supports the ability of Egr-1 to activate the
LH gene.
in vivo are not associated with a concomitant change in SF-1 expression in sheep pituitary (52), although a weak induction (<1.65-fold) of SF-1 expression by pulsed GnRH treatment of GnRH-deficient female rats has been reported (53).
Although Keri and Nilson (12) suggested that SF-1 sites are required
for GnRH stimulation of the bovine LH promoter in transgenic mice,
Ikeda et al. (33) have demonstrated that SF-1 is not
obligatory for GnRH effect, because GnRH can at least partially restore
LH expression even in SF-1-deficient mice. These inconsistencies may
be partly explained by a different promoter or cellular contexts or by
compensation by other nuclear proteins for the absence of functional
SF-1 in the knock-out mouse model. Interestingly, a cooperative
interaction has also been demonstrated between the pituitary
transcription factors Ptx1 and Pit1 in stimulation of the
prl gene (54, 55) as well as between Ptx1 and SF-1 in regulation of LH (56). However, unlike Egr-1, the action of Ptx1 is
not specific to LH, as Ptx1 is also required for expression of the
gonadotropin -subunit (56).
gene
expression. It is possible that in addition to enhancement of Egr-1
expression, GnRH-dependent signals modulate the functional
interaction between Egr-1 and SF-1. Previous studies utilizing a
protein-protein interaction assay in vitro (29) suggest that
Egr-1 and SF-1 physically interact with each other, and this
interaction requires the zinc finger domain of Egr-1. Current studies
are underway to test for the presence and significance of this
interaction in LT2 cells.
gene may modulate GnRH stimulation of LH expression. Using the
rat somatolactotropic cell line GH3, Kaiser et al. (14) have demonstrated that another zinc finger protein, Sp1, binds to the
rat LH promoter at 2 sites located between 451 and 386 and may
play a role in GnRH-stimulated expression of the LH gene. Although
the Sp1-dependent GnRH effect was markedly weaker
(2-3-fold) when compared with the Egr-1-SF-1-dependent
effect in our experiments, these results suggest that GnRH stimulation
of the LH promoter involves an integrated response of multiple
transcription factors binding to discrete promoter elements. Indeed
estradiol, acting via estrogen receptors, modulates LH expression
(8), and estrogen receptors were shown to interact with SF-1
in regulation of the salmon gonadotropin II subunit (57). Other sex
hormones also alter LH expression (9, 11). It is likely that
signaling by these promoter-bound transcription factors is modulated by diverse signal transduction pathways that participate in the
transmission of GnRH stimuli from its surface receptors to the LH
gene (2, 4, 6, 13, 15, 39, 58). Whereas potential cross-talk between
these second messenger pathways and Egr-1-SF-1 synergy remains to be
established, such coordinated interaction is likely to contribute
to the fine tuned LH response to GnRH pulses under diverse
physiological conditions.
promoter, because these cells do not express an appreciable level
of either Egr proteins or SF-1, although they are all expressed in
LT2 cells. All four Egr proteins synergistically interact with SF-1
in transcriptional activation of the LH promoter. Interestingly,
whereas GnRH stimulates the expression of all Egr proteins to some
extent, Egr-1 exhibits the largest and most prolonged response to GnRH
stimulation. This selective effect of GnRH may provide at least a
partial explanation for the fact that the profound phenotype of
Egr-1-deficient mice was not compensated by any of the other proteins
from the Egr family. Studies that focus on the expression of Egr
proteins in wild type and Egr-1-deficient mice are required to further
elucidate these findings.
T3 cells, a gonadotrope cell line similar
to LT2 (62). It is also expressed in LH-expressing human pituitary
adenomas (63). Interestingly, whereas DAX-1 has only a marginal effect
on SF-1-mediated activation of the LH promoter in the absence of Egr
proteins, it markedly represses the synergistic activation of LH by
Egr-1 and SF-1. These data provide more insight into the range of the
repressive function of DAX-1 and suggest that the ability of DAX-1 to
repress SF-1-mediated regulation of LH expression depends on the
interaction with the synergistic factor Egr-1. A similar effect was
recently observed by Nachtigal et al. (64) who found that
WT1, an Egr-1-related zinc finger protein, synergistically interacts
with SF-1 to promote Mullerian inhibitory substance expression. DAX-1
antagonizes this synergy, but only when both WT1 and SF-1 are
co-expressed (64). Whereas our results demonstrate an analogous
repressive effect of DAX-1 on SF-1 and Egr-1 in the context of the
LH promoter, versions of the promoter harboring mutations to the
SF-1 and/or Egr-1 binding sites remain partially repressible by DAX-1
(data not shown). SF-1-independent repression by DAX-1 also occurs on the human steroidogenic acute regulatory protein (StAR) promoter via
direct DNA binding by DAX-1 to hairpin structures (65). Comparison of
the rat LH promoter to the human StAR promoter failed to elicit an
identical hairpin-forming sequence, but it remains possible that a
component of DAX-1 repression of LH occurs through direct DNA
binding. Nevertheless, the repressive effect of DAX-1 is more dramatic
when the LH promoter is stimulated by GnRH and is diminished when
Egr-1 and SF-1 sites are mutated. These results support the notion that
optimal repression of the LH promoter activity by DAX-1 relies
heavily upon the synergistic interaction of Egr-1 and SF-1.
expression. A fine modulation of the degree
of synergy between these proteins may provide a means of modulating
LH expression during the estrous cycle and throughout reproductive life.
ACKNOWLEDGEMENTS
T2 cells, Stuart Adler for providing the
PL(KS)b-Luc plasmid, Markus Ehrengruber for the Ad5PacIGFP
adenovirus vector, and Brad Sevetson for the recombinant Egr-1 protein.
We are also grateful to Jeffrey Milbrandt for providing the expression
vectors for the Egr family members and for helpful discussions. We
thank Elena Sadovsky and Lori Rideout for technical assistance.
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Obstetrics and Gynecology, Washington University School of Medicine, P. O. Box 8064, 4911 Barnes-Jewish Hospital Plaza, St. Louis, MO
63110. Tel.: 314-747-0937; Fax: 314-362-8580; E-mail:
sadovskyy{at}msnotes.wustl.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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R. A. Keri, D. J. Bachmann, A. Behrooz, B. D. Herr, R. K. Ameduri, C. C. Quirk, and J. H. Nilson An NF-Y Binding Site Is Important for Basal, but Not Gonadotropin-releasing Hormone-stimulated, Expression of the Luteinizing Hormone beta Subunit Gene J. Biol. Chem., April 21, 2000; 275(17): 13082 - 13088. [Abstract] [Full Text] [PDF]
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J. Weck, A. C. Anderson, S. Jenkins, P. C. Fallest, and M. A. Shupnik Divergent and Composite Gonadotropin-Releasing Hormone-Responsive Elements in the Rat Luteinizing Hormone Subunit Genes Mol. Endocrinol., April 1, 2000; 14(4): 472 - 485. [Abstract] [Full Text]
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B. R. Sevetson, J. Svaren, and J. Milbrandt A Novel Activation Function for NAB Proteins in EGR-dependent Transcription of the Luteinizing Hormone beta Gene J. Biol. Chem., March 24, 2000; 275(13): 9749 - 9757. [Abstract] [Full Text] [PDF]
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E. Barnea and Y. Bergman Synergy of SF1 and RAR in Activation of Oct-3/4 Promoter J. Biol. Chem., February 25, 2000; 275(9): 6608 - 6619. [Abstract] [Full Text] [PDF]
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M. Ito, Y. Park, J. Weck, K. E. Mayo, and J. L. Jameson Synergistic Activation of the Inhibin {alpha}-Promoter by Steroidogenic Factor-1 and Cyclic Adenosine 3',5'-Monophosphate Mol. Endocrinol., January 1, 2000; 14(1): 66 - 81. [Abstract] [Full Text]
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M. W. Wolfe The Equine Luteinizing Hormone {beta}-Subunit Promoter Contains Two Functional Steroidogenic Factor-1 Response Elements Mol. Endocrinol., September 1, 1999; 13(9): 1497 - 1510. [Abstract] [Full Text]
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T. Yokoi, M. Ohmichi, K. Tasaka, A. Kimura, Y. Kanda, J. Hayakawa, M. Tahara, K. Hisamoto, H. Kurachi, and Y. Murata Activation of the Luteinizing Hormone beta Promoter by Gonadotropin-releasing Hormone Requires c-Jun NH2-terminal Protein Kinase J. Biol. Chem., July 7, 2000; 275(28): 21639 - 21647. [Abstract] [Full Text] [PDF]
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