The Protein Kinase C System Acts through the Early Growth Response Protein 1 to Increase LHß Gene Expression in Synergy with Steroidogenic Factor-1
Lisa M. Halvorson,
Ursula B. Kaiser and
William W. Chin
Division of Genetics (L.M.H., W.W.C.) and
Endocrine-Hypertension Division (U.B.K.) Department of Medicine
Brigham and Womens Hospital and Harvard Medical School Boston,
Massachusetts 02115
Department of Obstetrics and Gynecology
(L.M.H.) Tufts University School of Medicine Boston,
Massachusetts 02111
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ABSTRACT
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Expression of the LHß gene has been shown to
be modulated by both the orphan nuclear receptor, steroidogenic
factor-1 (SF-1), and the early growth response protein 1, Egr-1. It is
also well known that LHß mRNA levels are increased after hormonal
activation of the protein kinase C (PKC) signaling system, for example
by GnRH; however, the mechanisms by which the PKC system exerts this
effect has not been fully characterized. By transient transfection of
the GH3 cell line, we demonstrate that
activation of the PKC system with the phorbol ester, phorbol
12-myristate 13-acetate (PMA), increases activity of region -207/+5 of
the rat LHß gene promoter (
2-fold) and markedly augments
SF-1-induced stimulation (95-fold in the presence of both factors
vs. 13-fold for SF-1 alone). Mutation of the two previously
identified Egr-1 sites not only prevents Egr-1 effects on the LHß
gene promoter, but also eliminates the synergistic response to PMA and
SF-1 together, findings that were confirmed in a longer construct
spanning region -797/+5. In the gonadotrope-derived cell line,
T31, these mutations eliminate the GnRH responsiveness of the
-207/+5 LHß promoter construct. We next show that PMA treatment
(GH3 and
T31 cells) or GnRH treatment
(
T31 cells) induces expression of Egr-1, as detected by Egr-1
interaction with Egr-1 DNA-binding sites in the rat LHß gene promoter
sequence. Furthermore, we demonstrate that PMA increases steady-state
Egr-1 mRNA levels via increased Egr-1 transcription. We conclude that
PMA-induced stimulation of LHß gene expression is achieved, at least
in part, by induction of Egr-1 expression.
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INTRODUCTION
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Gonadotropin gene expression is regulated by the complex
interaction of multiple factors originating from the hypothalamus,
pituitary, and gonads. The hypothalamic decapeptide, GnRH, is believed
to exert its effects, at least in part, by activation of the protein
kinase C (PKC) second messenger-signaling system in the gonadotrope.
GnRH has been shown to increase PKC activity based on the measurement
of endogenous PKC activators, such as diacylglycerol, and by kinase
activity (1, 2, 3, 4). Increased PKC activity, in turn, up-regulates
steady-state LHß mRNA levels and LHß polypeptide biosynthesis, as
demonstrated by treatment of rat pituitary cells with phorbol esters
(5, 6, 7). As phorbol 12-myristate 13-acetate (PMA)-induced increases in
LHß mRNA levels are suppressed by the addition of the transcriptional
inhibitor, actinomycin D, the PKC response has been attributed to an
increase in LHß mRNA biosynthesis, rather than to an alteration in
mRNA stability (8). This result suggests the presence of PMA-stimulated
DNA-regulatory element(s) in the LHß gene promoter.
Recent attempts to identify transcriptional regulators of LHß gene
expression have identified two transcription factors steroidogenic
factor-1 (SF-1) and the early growth response gene 1 product
(Egr-1) with profound effects on LHß gene promoter activity. SF-1,
an orphan member of the nuclear hormone receptor superfamily, is
selectively expressed in the gonadotrope subpopulation of the pituitary
gland, as well as in the adrenal gland and gonads (9). SF-1 binds to a
DNA promoter region called the gonadotrope-specific element, or GSE, as
defined in the common glycoprotein
-subunit by Barnhart and Mellon
(10). Variations of this sequence, alternatively called the Ad4
response element, are present in a wide range of genes that play a role
in steroidogenesis, sexual differentiation, and adult reproductive
function (9, 11). Our studies of the rat LHß gene promoter have
characterized two functional GSE sites, located at positions -127 and
-59 relative to the transcriptional start site in the rat (12, 13).
In vivo model systems have confirmed the importance of SF-1
in the regulation of LHß gene expression. In addition to other
abnormalities, transgenic mice null for the Ftz-F1 gene,
which encodes SF-1, lack detectable LHß mRNA levels (9). In a second
transgenic model, mutation of the 5'-GSE site of the bovine LHß gene
promoter markedly decreased expression of a reporter gene relative to
expression levels in the presence of the wild-type promoter (14).
In studies of other members of the steroid receptor superfamily,
receptor function has been shown to be profoundly altered by
posttranslational modifications, such as phosphorylation, that modulate
receptor stability, DNA-binding affinity, and/or transcriptional
efficiency (15, 16, 17, 18). SF-1 is known to exist as a phosphoprotein.
Carlone and Richards (19) and Zhang and Mellon (20) have demonstrated
that SF-1 can be phosphorylated by the catalytic subunit of protein
kinase A, resulting in decreased binding to the SF-1-binding site in
the rat P450c17 gene promoter. Interestingly, in the type II
3ß-hydroxysteroid dehydrogenase gene promoter, the maximal PMA
response requires the presence of an intact GSE site, suggesting that
PMA may act through modulation of SF-1 (21). Although it is not known
whether SF-1 is phosphorylated by PKC, these observations raise the
intriguing possibility that the PKC system may increase LHß gene
promoter activity by modulating SF-1 effects.
In vivo and in vitro data have also implicated
the early growth response protein 1, Egr-1, in the transcriptional
regulation of LHß gene expression (12, 22, 23). Egr-1, also known as
zif/268, Krox-24, and NGFI-A, is a member of the immediate early gene
family whose members contain a zinc finger domain with a
Cys2-His2 motif that recognizes GC-rich
nucleotide sequences (24, 25, 26, 27). Within the pituitary gland, Egr-1
expression is limited to the gonadotrope and somatotrope subpopulations
based on colocalization of LHß-subunit protein and X-gal staining
that is conferred by a lacZ transgene inserted 3' to the endogenous
Egr-1 (Krox-24) gene promoter (22). Interestingly, two separate
transgenic models have demonstrated specific loss of LHß gene
expression in Egr-1-deficient mice with maintenance of normal FSHß
gene expression (22, 23). In transient transfection experiments,
the addition of Egr-1 has been shown to increase LHß gene promoter
activity, an effect attributed to Egr-1 binding sites located at
positions -112 and -50 in the rat sequence (12, 23). In a variety of
nonreproductive systems, Egr-1 gene expression has been shown to be
induced by a wide range of stimuli, including growth factors and
phorbol esters (28). Therefore, it is also possible that the observed
PMA response of the LHß gene is mediated via induction of Egr-1
expression in the gonadotrope.
In the results reported here, we determine that activation of the PKC
system with the phorbol ester, PMA, increases rat LHß promoter
activity, both alone and in conjunction with SF-1. Furthermore, we
investigate the role of SF-1 and Egr-1 and their cognate DNA-binding
sites in the generation of this response.
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RESULTS
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PMA Acts Alone and in Conjunction with SF-1 to Increase LHß Gene
Expression
In previous investigations, steady-state LHß mRNA levels had
been shown to be increased by activation of the PKC-signaling system
with the phorbol ester, PMA (5, 6). Nevertheless, it had not been
definitively determined that this PMA response was due to an increase
in transcription rate, rather than altered mRNA stability. We therefore
used transient transfection-luciferase reporter assays to determine
whether PMA could alter LHß gene promoter activity and whether this
effect, if present, acted in concert with the previously described SF-1
response.
While ideally we would have used a gonadotrope-derived cell line for
these functional studies, the only cell line available for study at the
time these experiments were performed was the
T31 cell line.
Although
T31 cells express the endogenous gonadotropin
-subunit
gene, they are unlike normal gonadotropes in that they do not express
either of the ß-subunit genes (28). In addition, it has proven
technically difficult to obtain high levels of expression of LHß
promoter constructs in this cell line, limiting its usefulness for
these studies. We therefore performed these experiments in an
alternative pituitary-derived cell line, the rat somatolactotrope
GH3 cell line. This cell line lacks endogenous SF-1, based
on the failure of GH3 nuclear extracts to produce specific
protein-DNA complexes with a GSE-containing oligonucleotide probe on
electrophoretic mobility shift assay (EMSA) (data not shown). Use of an
SF-1-deficient cell line allowed investigation of PMA effects on basal
as well as SF-1-stimulated LHß gene promoter activity.
GH3 cells were transfected with a reporter construct
containing region -207 to +5 of the rat LHß gene promoter. Treatment
with PMA (100 ng/ml) for 46 h increased luciferase activity by
2.8-fold, demonstrating that the proximal rat LHß gene promoter
can confer PMA responsiveness (Fig. 1A
).
Consistent with previously published results, cotransfection with the
cytomegalovirus (CMV)-driven SF-1 expression vector resulted in a
13-fold increase in the luciferase activity of a reporter construct
containing region -207 to +5 of the rat LHß gene promoter (13).
Interestingly, marked synergy was observed in the presence of both SF-1
and PMA, with a 95-fold increase in luciferase activity in the presence
of both factors compared with a 13-fold response to SF-1 alone.

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Figure 1. SF-1 and PMA Act Both Alone and Synergistically to
Increase LHß Gene Promoter Activity
A, GH3 cells were transiently transfected with a construct
containing region -207/+5 of the rat LHß gene promoter linked to a
luciferase reporter construct, pXP2. Cells were cotransfected with a
CMV-driven SF-1 expression vector and with an RSV-ß-galactosidase
expression vector. Cells were treated with 100 ng/ml PMA or
dimethylsulfoxide for 46 h starting approximately 40 h
after transfection. Luciferase activity was normalized to
ß-galactosidase activity, and promoter activity was calculated as
fold-change over expression in the presence of the untreated control
expression vector. Results are shown as the mean ±
SEM of 30 samples in 10 independent experiments. B,
GH3 cells were transiently transfected with a construct
containing the CMV promoter linked to the cDNA encoding
ß-galactosidase and treated as described above. ß-Galactosidase
activity was measured and expressed as fold-change over expression in
the untreated cells. Results are shown as the mean ±
SEM of nine samples in three independent experiments. *,
P < 0.05 vs. vehicle-treated
control.
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As seen in Fig. 1B
, PMA did not increase expression of a construct
containing the CMV promoter and the cDNA that encodes
ß-galactosidase. This result suggested that the observed interaction
between PMA and SF-1 was not due to increased transcription of SF-1
through stimulation of the CMV promoter.
The GSE Sites of the LHß Gene Promoter Do Not Confer a PMA
Response
SF-1 transactivation efficiency could also be modified through
mechanisms other than increased SF-1 protein levels, such as
alterations in posttranslational processing. We reasoned that if PMA
was directly altering SF-1 functional activity, a PMA response should
be observed in the presence of SF-1 DNA response elements (GSEs).
Therefore, four copies of the rat LHß 5'-GSE sequence (position
-127) were inserted upstream of the GH minimal promoter, GH50, in the
luciferase reporter construct, pXP1. As seen in Fig. 2A
, these sequences conferred an SF-1
response, but failed to confer a response to PMA, either alone or in
conjunction with SF-1. These results suggested that the PMA effect was
generated by sequences outside of the GSE sites and was therefore not
likely to be due to direct effects on the transcription factor, SF-1,
which is known to bind to these regulatory elements.

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Figure 2. The PMA Response Is Not Due to the Presence of GSE
Sites in the Proximal LHß Gene Promoter
A, PMA effects in a heterologous GSE-promoter construct.
GH3 cells were transiently transfected with a construct
containing the GH minimal promoter, GH50, linked to a luciferase
reporter construct, pXP1, or with the addition of four copies of the
5'-GSE site. B, Stimulation of LHß gene promoter activity with loss
of the GSE sites by site-directed mutagenesis. GH3 cells
were transiently transfected with a promoterless luciferase reporter
construct, pXP2, or with the same construct plus region -207/+5 of the
rat LHß gene promoter present as either the wild-type sequence or
with mutations in both GSE sites. Cells were treated (PMA 100
ng/ml x 46 h) and the results calculated as described in Fig. 1A . Results are shown as the mean ± SEM of nine
samples in three independent experiments. n.s., P
0.05. *, P < 0.05 vs.
vehicle-treated control.
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This conclusion was confirmed by transfecting cells with a construct
containing point mutations in both GSE sites within the context of the
native rat LHß gene promoter. These mutations have been shown to
eliminate binding by SF-1 on EMSA (12, 13). Despite marked blunting of
the SF-1 and SF-1/PMA responses, the response to PMA alone was
conserved in the face of these mutations (2.6-fold vs.
2.8-fold in the wild-type construct) (Fig. 2B
). Note that the empty
reporter construct did not respond to either factor, confirming that
the observed responses to SF-1 and/or PMA were due to the presence of
the rat LHß gene promoter sequences.
PMA Acts through the Egr-1-Binding Sites to Increase LHß Gene
Expression
Taken together, the results shown in Fig. 2
indicated the presence
of a non-GSE, PMA-responsive regulatory region in the proximal LHß
gene promoter. Coincident with these experiments, our laboratory had
identified two functional response elements for the immediate early
gene product, Egr-1, a high-affinity site at position -50 and a lower
affinity site at -112 in the rat LHß gene promoter. In these
studies, we further demonstrated that LHß gene promoter activity was
synergistically increased in the presence of both SF-1 and Egr-1
(12), confirming a previous report by Lee et al.
(23).
In a number of nonreproductive systems, activation of the PKC system
has been shown to induce Egr-1 expression (29, 30). Hypothesizing that
PMA-induced stimulation of LHß gene promoter activity may be due to
effects on Egr-1, we cotransfected the wild-type rat LHß gene
promoter-luciferase reporter construct with SF-1 and/or Egr-1 in the
presence or absence of PMA (Fig. 3A
). As
observed previously (Fig. 1
), PMA and SF-1 together produced a
synergistic increase in LHß gene promoter activity. In contrast, PMA
did not significantly increase the response to Egr-1 alone or SF-1 and
Egr-1. The lack of further stimulation by PMA in the presence of both
SF-1 and Egr-1 suggested a mechanism by which PMA induces Egr-1
expression. Egr-1, in turn, would interact with SF-1 to increase LHß
promoter activity. In this model, PMA would not provide further
stimulation in the presence of maximally effective levels of
exogenously introduced Egr-1.

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Figure 3. Mutation of Both Egr-1-Binding Sites Eliminates
PMA-Induced Increases in -207/+5 LHß Gene Promoter Activity
A, Stimulation of wild-type LHß gene promoter activity by PMA, SF-1,
and/or Egr-1. GH3 cells were transiently transfected with
region -207/+5 of the rat LHß gene promoter in a luciferase reporter construct, as well as with CMV-driven
expression vectors for SF-1 and/or Egr-1, as indicated. Cells were
cotransfected with an RSV-ß-galactosidase expression vector. Cells
were treated with 100 ng/ml PMA or dimethylsulfoxide for 46 h
starting approximately 40 h after transfection. Luciferase
activity was normalized to ß-galactosidase activity, and promoter
activity was calculated as fold- change over expression in the presence
of the untreated control expression vector. *, P <
0.05 relative to vehicle-treated wells. n.s., P 0.05. B,
Loss of PMA effect with mutation of Egr-1 sites. GH3 cells
were transiently transfected with a luciferase reporter construct
containing region -207/+5 of the rat LHß gene promoter present as
either the wild-type sequence or with mutation of both Egr-1-binding
sites, as indicated. *, P < 0.05 between the PMA
response of the mutated vs. wild-type construct. C,
Mutation of the Egr-1-binding sites eliminates the PMA interaction with
SF-1. Cotransfections, PMA treatments, and data calculations for panels
B and C were performed as in panel A. Results are shown as the
mean ± SEM of at least nine samples in three
independent experiments.
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To confirm the importance of Egr-1 to the PMA response, we next
transfected cells with a reporter construct containing mutations in
both of the Egr-1 DNA-binding sites. We have previously demonstrated
that in vitro translated Egr-1 is unable to bind to an
oligonucleotide probe containing these mutations (12). The presence of
these Egr-1 site mutations eliminated the ability of PMA to stimulate
LHß gene promoter activity (Fig. 3B
). Furthermore, PMA
treatment no longer augmented SF-1-stimulated luciferase activity (Fig. 3C
).
In the context of a longer region of the rat LHß gene 5'-flanking
sequence (-797/+5), synergy between SF-1 and PMA was observed with a
68-fold increase in the presence of both factors compared with an
approximately 2-fold increase in the presence of either factor alone
(Fig. 4
, upper panel). This
synergy was lost with mutation of the Egr-1 binding sites; however, in
contrast to the shorter construct, the PMA alone response was
maintained (Fig. 4
, lower panel).

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Figure 4. Loss of PMA-SF-1 Synergy with Mutation of the
Egr-1-Binding Sites in the Context of Region -797/+5 of the Rat LHß
Gene Promoter
GH3 cells were transiently transfected with a luciferase
reporter construct containing region -797/+5 of the rat LHß gene
promoter, present as either the wild-type or with mutation of both
Egr-1 DNA-binding sites. Where indicated, cells were cotransfected with
a CMV-driven SF-1 expression vector. All cells received an
RSV-ß-galactosidase expression vector. Luciferase activity was
normalized to ß-galactosidase activity, and promoter activity was
calculated as fold-change over expression in the presence of the
untreated control expression vector. Results are shown as the mean
± SEM of nine samples in three independent experiments.
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PMA Induces Egr-1 DNA Binding in Nuclear Extracts
The studies shown in Figs. 3
and 4
suggested that PMA acts through
Egr-1 to stimulate LHß gene expression. This observed relationship is
physiologically relevant only if PMA is able to increase Egr-1 gene
expression and/or functional activity in the gonadotrope. While known
to be widely distributed, only recently has Egr-1 expression been
identified in the gonadotrope subpopulation of the pituitary (22).
Interestingly, in preliminary experiments, we had been unable to detect
Egr-1 binding to the 3'-Egr site (position -50) using nuclear extracts
from either our model cell line, GH3, or the
gonadotrope-derived cell lines,
T31 and LßT2 (data not
shown).
We, therefore, attempted to induce Egr-1 expression in GH3
cells and in the gonadotrope-derived cell line,
T31, by treatment
with PMA and/or a GnRH analog. As shown in Fig. 5A
, nuclear extracts from untreated
GH3 cells formed a single dominant band in the presence of
an oligonucleotide probe that contains both the 3'GSE and 3'Egr-1
DNA-regulatory sites (lane 1). Addition of specific antibodies
demonstrated the presence of Sp1 (lane 3), but not Egr-1 (lane 2) in
this extract. In contrast, nuclear extracts obtained from
GH3 cells that had been treated with PMA for 1 h
demonstrated induction of a protein-DNA complex that contains Egr-1
(lanes 4 and 5). The relative mobilities of in vitro
translated Egr-1 and a purified Sp1 preparation were consistent with
the presence of Sp1 and Egr-1 in the extracts (lanes 7 and 8). Similar
results were obtained in the gonadotrope-derived
T31 cell line
(Fig. 5B
, lanes 14) and LßT2 cell line (data not shown). The
induction of Egr-1 binding could also be detected using an
oligonucleotide probe that spans the lower affinity 5'-Egr-1 site (data
not shown). EMSA did not indicate PMA induction of any proteins other
than Egr-1.

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Figure 5. PMA Induces Egr-1 DNA Binding in Nuclear Extracts
from GH3 and T31 Cell Lines
A, EMSA was performed using nuclear extracts from the rat
somatolactotrope cell line, GH3 (lanes 16), in
vitro translated Egr-1 (lane 7), or purified Sp1 (lane 8)
protein. Nuclear extracts were derived from cells maintained in the
absence or presence of PMA (100 ng/ml for 1 h). Region -67/-35
of the rat LHß gene promoter, which contains the 3'Egr-1-binding
site, was [32P]-labeled and used as a probe. Where
indicated, antiserum specific to Egr-1 (lanes 2 and 5) or Sp1 (lanes 3
and 6) was added 2 h before electrophoresis. B, DNA-protein
complexes formed by nuclear extracts from the mouse gonadotrope-derived
cell line, T31, maintained in control medium (lanes 1 and 5) or in
the presence of 100 ng/ml PMA (lanes 24) or 100 nM GnRH
analog (lanes 68) for 1 h before harvest. Antiserum-specific to
Egr-1 (lanes 3 and 7) or Sp1 (lanes 4 and 8) was added as indicated.
NE, nuclear extract.
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Noting that GnRH effects are partially transduced through activation of
the PKC system, we tested the ability of a GnRH analog to increase
Egr-1 gene expression. As shown in Fig. 5B
(lanes 58), GnRH induces
detectable Egr-1 binding in the gonadotrope-derived
T31 cell
line.
Egr-1 mRNA Levels Are Increased By PMA Treatment
Northern analysis was performed to determine whether the
PMA-induced increase in Egr-1-DNA complex formation was due to an
increase in Egr-1 biosynthesis as opposed to increased DNA-binding
affinity. As shown in Fig. 6
, A and B,
treatment of GH3 cells or
T31 cells with PMA markedly
increased steady-state Egr-1 mRNA levels by 16-fold and 34-fold,
respectively. Egr-1 mRNA levels in
T31 cells were also increased
after exposure to a GnRH analog (50-fold, Fig. 6B
).

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Figure 6. Northern Analysis Demonstrating Induction of Egr-1
mRNA Expression by PMA and GnRH Analog
A, Total RNA (10 µg) from GH3 cells was subjected to
Northern analysis using a full-length mouse Egr-1 cDNA
[32P]-labeled probe. The same blot was reprobed with
cyclophilin to control for differences in RNA loading. Cells were
cultured in control medium (lanes 1 and 2) or in the presence of 100
ng/ml PMA (lanes 3 and 4) for 1 h before harvest. B, Total RNA (7
µg) from gonadotrope-derived T31 cells was probed with the mouse
Egr-1 probe and with cyclophilin. Before harvesting, cells were
cultured for 1 h in medium with the appropriate vehicle (lanes 1,
2, 5, and 6), 100 ng/ml PMA (lanes 3 and 4), or 100 nM GnRH
analog (lanes 7 and 8). C, PMA increases Egr-1 gene promoter activity.
GH3 cells were transiently transfected with a construct
containing 1.2 kb of the 5'-flanking region of the mouse Egr-1 gene
linked to the luciferase reporter plasmid, pXP2. An
RSV-ß-galactosidase vector was cotransfected to control for
transfection efficiency. Cells were treated with vehicle or 100 ng/ml
PMA for 4 h before harvesting approximately 48 h after
transfection. After normalization to ß-galactosidase activity,
promoter activity was calculated as fold-change in luciferase activity
in treated vs. untreated wells. Results are shown as the
mean ± SEM of nine samples from three independent
experiments.
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Steady-state Egr-1 mRNA levels could potentially be altered by
changes in either transcription rate and/or stability. To distinguish
between these two alternatives, GH3 cells were transiently
transfected with a luciferase reporter construct containing 1.2 kb of
the Egr-1 gene promoter. PMA treatment increased luciferase activity in
this construct by approximately 50-fold, suggesting that PMA
up-regulates Egr-1 mRNA levels, at least in part, via increases in
transcription of the Egr-1 gene (Fig. 6C
).
Loss of GnRH Responsiveness with Mutation of the Egr-1 Sites in the
LHß Gene Promoter
The PKC system is known to be a major signaling system for GnRH
(1, 3). Furthermore, in the present studies, we have demonstrated that
GnRH induces Egr-1 mRNA levels and DNA binding in the gonadotrope-like
T31 cell line (Figs. 5
and 6
). We, therefore, investigated the
role of the Egr-1-binding sites in conferring GnRH responsiveness by
transfecting
T31 cells with the -207/+5 LHß promoter construct
containing mutations in both of the Egr-1-binding sites. As shown in
Fig. 7
, induction of LHß gene promoter activity by
GnRH is lost in this mutated construct.

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Figure 7. Fig. 7. Loss of the GnRH and PMA Response with Mutation of
Both Egr-1-Binding Sites in the T31 Cell Line
T31 cells were transiently transfected with luciferase reporter
constructs containing region -207/+5 of the rat LHß gene promoter
present as the wild-type sequence (upper panel) or with
mutation of both Egr-1-binding sites (lower panel).
Cells were treated for 46 h with PMA (100 ng/ml), GnRH analog (100
nM), or appropriate vehicle starting approximately 20
h after transfection. Results are shown as the mean ±
SEM of nine samples in three independent experiments.
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DISCUSSION
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Normal reproductive function is dependent upon the precise
regulation of gonadotropin gene expression by numerous factors that
activate an array of signaling systems, including the PKC system. Prior
studies by our laboratory and by others have shown that SF-1 and Egr-1
act cooperatively to increase LHß gene promoter activity (12, 23). In
the results reported here, activation of the PKC system with the
phorbol ester PMA is shown to increase activity of the rat LHß gene
promoter, both alone and in synergy with SF-1. We then demonstrate that
PMA induces Egr-1 biosynthesis in gonadotrope-like cells and that
intact Egr-1-binding sites are required for the synergistic effects of
PMA and SF-1 on the LHß gene promoter. Taken together, these results
strongly suggest that PMA activation of the PKC-signaling system may
increase LHß gene expression via increases in Egr-1 expression.
Mutation of the Egr-1-binding sites eliminated the ability of PMA to
stimulate the -207/+5 region of the LHß gene promoter, supporting
the role of Egr-1 in mediating the PMA response (Fig. 3
). Surprisingly,
mutation of these sites did not alter the PMA effect in the longer
construct, perhaps suggesting the presence of additional PMA-responsive
sequences (Fig. 4
). Nonetheless, this result does not undermine the
importance of the Egr-1- binding sites for PMA-induced activation in
the presence of SF-1 in both constructs.
Results utilizing an Egr-1 promoter construct indicate that PMA induces
Egr-1 gene expression by increasing the transcription rate of this gene
(Fig. 6C
). Nevertheless, our data do not exclude the possibility that
mRNA stability and/or posttranslational modifications further modulate
Egr-1 functional activity.
Our data also suggest that neither SF-1 nor the GSE sites are required
for the PMA response in the LHß gene promoter. This result differs
from that observed in the type II 3ß-HSD (3ß-hydroxysteroid
dehydrogenase) gene in which mutation of the GSE site blunts
PMA-induced increases in promoter activity, even in the absence of
SF-1. Interestingly, 5'-deletion of the GSE site restores the PMA
response, further complicating interpretation of these results (21). It
should be noted that our results do not exclude effects of the PKC
system on SF-1. Based on amino acid sequence, SF-1 contains multiple
potential PKC phosphorylation sites, and GnRH has been shown to
increase SF-1 mRNA levels in vivo (31, 32). Nevertheless,
while SF-1 expression may be modulated by PMA, any functional effect,
if present, appears to be overwhelmed by the effects of Egr-1 and the
Egr-1 DNA-binding sites.
For the functional studies reported here, we used a heterologous system
in which a reporter construct containing LHß gene promoter sequences
and expression vector(s) for SF-1 and/or Egr-1 were transiently
transfected into a pituitary-derived somatolactotrope cell line,
GH3. While use of a gonadotrope cell line would be
preferable, there is precedent for the use of GH3 cells as
a model system for study of the LHß gene. GH3 cells have
been shown to support transcription initiation from the authentic start
site of the LHß gene and to allow cAMP-mediated increases in LHß
gene promoter activity (33). Furthermore, when transfected with the
GnRH receptor, GH3 cells demonstrate GnRH-mediated
regulation of gonadotropin subunit promoter activity that closely
parallels the regulation observed in primary pituitary cells (34).
To confirm the results obtained in GH3 cells, we have
repeated selected studies using the gonadotrope-derived cell line,
T31 (28). These cells resemble normal gonadotropes as they express
endogenous SF-1 and the glycoprotein
-subunit; however, neither the
LHß nor the FSHß subunit genes are expressed. Results in this cell
line confirm that LHß gene promoter activity is increased by
PMA and is dependent on the presence of intact Egr-1-binding sites
(Fig. 7
).
A number of laboratories have documented that treatment of pituitary
cells with phorbol esters mimics GnRH effects on LHß biosynthesis and
secretion (4). Our results in the
T31 cell line clearly
demonstrate that treatment with a GnRH analog increases Egr-1 DNA
binding, Egr-1 mRNA levels, and Egr-1-dependent LHß promoter
activity, paralleling the observed effects of PMA in this cell line and
in GH3 cells. Thus, our results are consistent with a
mechanism in which GnRH stimulates LHß gene transcription via PKC
induction of Egr-1 gene expression.
Of note, GnRH-induced activity of a longer region of the LHß gene
5'-flanking sequence (-797/+5) exceeds the response of the shorter
segment (35). We have identified a GnRH-responsive region from -490 to
-352 in the rat LHß gene promoter and have implicated the
transcription factor Sp1 in the generation of this response (36).
Interestingly, the nucleotide regions that include the Egr-1-binding
sites have also been found to bind Sp1 on EMSA (12). However, unlike
Egr-1, Sp1 binding is not altered by PMA or GnRH treatment (Fig. 5
).
The signaling mechanism(s) by which GnRH exerts its effects is
currently an area of active investigation. Earlier studies in primary
pituitary cells indicated a role for the PKC system in modulating LHß
mRNA levels (1, 5). Recent work in our laboratory confirmed the
importance of this signaling system in a GH3-derived cell
line (37). In contrast, using a variety of cell types, Weck et
al. (38) concluded that induction of the LHß gene is dependent
on calcium influx, rather than the PKC pathway. Further studies will be
required to resolve the apparent discrepancy of these results. The data
presented here clearly imply a critical role for Egr-1 in transduction
of the PMA response and support a role for the PKC system in the
mediation of GnRH effects. Nevertheless, these data do not exclude a
contribution by calcium-signaling pathways. Full GnRH responsiveness is
likely to involve complex interplay among a myriad of sites, including
those binding SF-1, Sp1, and Egr-1. Some of these may respond to PKC,
whereas others may respond to non-PKC-signaling systems.
In conclusion, our results demonstrate that PMA acts synergistically
with SF-1 to increase LHß gene promoter activity and that this effect
is mediated via the immediate early gene product, Egr-1. We propose
that the magnitude of the PMA-SF-1 cooperative effect and the rapidity
of the PMA-induced increase in Egr-1 gene expression may provide one
mechanism by which dynamic regulation of LHß gene expression is
achieved.
 |
MATERIALS AND METHODS
|
---|
Oligonucleotides
The oligonucleotides used for mutagenesis, EMSAs, and PCRs are
shown in Table 1
. The nucleotide sequence
of the rat LHß gene promoter is based on newly obtained sequencing
data (available at GenBank accession number AF020505) that differs
slightly from that of Jameson et al. (39). The -797LH-S and
-207LH-S primers introduced BamHI restriction sites, while
the +5LH-AS primer introduced a HindIII site (restriction
sites not shown).
In Vitro Translated Proteins and Antisera Used in
EMSA
In vitro translated Egr-1 was generated from a
plasmid containing 3.2 kb of the mouse Egr-1 cDNA (provided by D.
Nathans, Johns Hopkins University, Baltimore, MD) using the TNT
Coupled Reticulocyte Lysate System (Promega, Madison, WI). The
resultant product was determined to be of appropriate size by
comparison with [35S]methionine-labeled protein markers
by SDS-PAGE. Human Sp1 was purchased from Promega (Madison, WI). Egr-1
and Sp1 polyclonal antisera were obtained from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA).
EMSAs
A double stranded oligonucleotide spanning region -67/-35 of
the rat LHß gene promoter was produced by annealing the sense
oligonucleotide indicated in Table 1
with the corresponding antisense
oligonucleotide (not shown). Probes were created by T4 polynucleotide
kinase end-labeling with [
-32P]ATP followed by
purification over a NICK column (Pharmacia Biotech, Uppsala,
Sweden).
Protein samples were incubated with 50,000 cpm of oligonucleotide probe
in DNA-binding buffer [20 mM HEPES (pH 7.9), 60
mM KCl, 5 mM MgCl2, 10
mM phenylmethylsulfonylfluoride, 10 mM
dithiothreitol, 1 mg/ml BSA, and 5% (vol/vol) glycerol] for 30 min on
ice. Where indicated, antiserum (1 µl) was added 30 min after the
addition of probe and the incubation continued for 2 h.
Protein-DNA complexes were resolved on a 5% nondenaturing
polyacrylamide gel in 0.5x Tris-borate-EDTA buffer and subjected to
autoradiography and/or quantification using a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA).
Plasmids Used in Transfection Studies
The wild-type reporter constructs used for these studies contain
either 797 or 207 bp of the 5'-flanking sequence of the rat LHß gene
and the first 5 bp of the 5'-untranslated region. These constructs were
created by subcloning the PCR product generated by primers +5LH-AS and
either -797LH-S or -207LH-S into the pXP2 vector using
BamHI/HindIII restriction sites that were
introduced by the primers (40).
Mutations in the LHß gene promoter reporter constructs were
introduced using the Transformer Site-Directed Mutagenesis Kit
(CLONTECH Laboratories, Inc., Palo Alto, CA). Generation of multiple
mutations was performed sequentially and, therefore, required the use
of two selection primers, one that converted a unique
HindIII restriction site to a unique MluI site
(pXP2) and the other that reversed this mutation (pXP2-rev). The 5'-GSE
mutagenic primer eliminated a TthIII1 restriction site in addition to
introducing the desired mutation, as described previously (13). The
mutagenic primers for the 3'-GSE, 5'-Egr, and 3'-Egr sites introduce
EcoRI, ScaI, and PstI restriction
sites, respectively. All reporter constructs were confirmed by
dideoxysequencing.
The SF-1 expression vector contained 2.1 kb of the mouse SF-1 cDNA
driven by cytomegalovirus promoter sequences in the vector, pCMV5
(provided by K. L. Parker, Southwestern University School of
Medicine, Dallas, TX)(41). The Egr-1 expression vector was
created by cloning 3.2 kb of the mouse Egr-1 cDNA into pCMV5 at
BamHI and HindIII restriction sites (Egr-1 cDNA
provided by D. Nathans, Johns Hopkins University) (25). The
pCMV5-ßgal construct was obtained by transferring the
EcoRI/SalI ß-galactosidase cDNA fragment from
pNASSß (CLONTECH, Palo Alto, CA) into the empty pCMV5 vector. The
Egr-luc reporter construct contained 1.2 kb of the mouse Egr-1 gene
promoter sequence cloned into the SalI site of pXP2 (V.
Sukhatme, Harvard Medical School, Boston, MA) (42).
Transfection Experiments
Rat somatolactotrope GH3 cells or mouse
gonadotrope-derived
T31 cells were cultured to 5070% confluence
in DMEM supplemented with 10% FCS. For the GH3 cell line,
approximately 5 x 106 cells were suspended in 0.4 ml
of Dulbeccos PBS plus 5 mM glucose with the DNA to be
transfected. The cells received a single electrical pulse of 240 V at a
total capacitance of 1000 µFarads using an Invitrogen Electroporator
II apparatus (Invitrogen, San Diego, CA). GH3 cells
received 1.5 µg/well of the reporter constructs. The
T31 cell
line was transfected using the calcium phosphate precipitation method
and 2 µg/well of reporter construct. Where appropriate, cells also
received SF-1 and/or Egr-1 expression vectors or an equivalent amount
of the empty pCMV5 expression vector in amounts of 1 µg/well
(GH3) or 0.1 µg/well (
T31). For both cell lines,
cotransfection with an rous sarcoma virus
(RSV)-ß-galactosidase plasmid (1 µg/well) allowed correction for
differences in transfection efficiency between wells in all experiments
except Fig. 1B
, in which ß-galactosidase activity was used as the
primary reporter. Cells were treated with vehicle, PMA (100 ng/ml) (LC
Laboratories, San Diego, CA), or GnRH agonist des-Gly10,
[D-Ala6]-GnRH ethylamide (100 nM)
(Sigma, St. Louis, MO) for 46 h starting approximately 40 h
(GH3) or 20 h (
T31) after transfection. Cells
were then harvested and the cell extracts were analyzed for luciferase
and/or ß-galactosidase activities (43, 44). Luciferase activity was
normalized to the level of ß-galactosidase activity, where
appropriate. Results were then calculated as fold-change relative to
expression in the presence of the vehicle-treated control wells. Data
are shown as the mean ± SEM.
Northern Blot Analysis
Total RNA was prepared from GH3 or
T31 cells at
approximately 50% confluence using the RNeasy MiniKit (Qiagen Inc.,
Chatsworth, CA). Before extraction, cells were treated for 1 h
with vehicle, PMA (100 ng/ml), or the GnRH agonist (100
nM). Seven micrograms (
T31 cells) or 10 µg
(GH3 cells) of total RNA were separated by electrophoresis
in denaturing agarose gels (2.2 M formaldehyde and 1.5%
agarose), transferred to nylon membranes by diffusion (Nytran NY 12 N,
Schleicher & Shuell, Dassel, Germany), and cross-linked by UV
irradiation. Hybridizations were performed under high stringency
conditions [42 C, 16 h: in 50% formamide, 0.5% SDS, 100 mg
salmon DNA, 0.9 M NaCl, 12 mM EDTA, and 0.09
M sodium phosphate (pH 7.4)] with 50 ng cDNA fragments
randomly labeled with [32P]dCTP. The following cDNA
fragments were used as probes: a 3.1-kb insert of the mouse Egr-1 cDNA
released with EcoRI from pUC13 (provided by V. Sukhatme,
Harvard Medical School) and, as a standard, a 0.7-kb fragment of the
rat cDNA encoding cyclophilin (42, 45). The membranes were washed at a
final stringency of 0.2 x SSPE-0.3% SDS at 42 C [0.2 x
SSPE = 30 mM NaCl, 2 mM sodium phosphate,
and 0.2 mM EDTA (pH 7.4)] and then subjected to
autoradiography and quantification using a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA).
Statistical Analysis
Data were combined across transfection experiments to determine
the mean ± SEM of the corrected luciferase activity.
Two-way analysis of variance followed by comparisons with Students
t test were used to assess whether promoter activity was
statistically different between the indicated groups. Statistical
significance was set at the P < 0.05 level.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Lisa M. Halvorson, M.D., Division of Reproductive Endocrinology, Box 36, New England Medical Center, 750 Washington Street, Boston, Massachusetts 02111. E-mail:
lisa.halvorson{at}es.nemc.org
This work was supported in part by NIH Grant R03-HD-34692 (L.M.H.),
R29-HD-33001 (U.B.K.), and R01-HD-19938 (W.W.C.), as well as an
American Society for Reproductive Medicine-Ortho Pharmaceutical
Research Grant in Reproduction (L.M.H.) and an American Society for
Reproductive Medicine-Serono Research Grant (U.B.K.).
Received for publication February 23, 1998.
Revision received June 16, 1998.
Accepted for publication September 22, 1998.
 |
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