Sp1, Steroidogenic Factor 1 (SF-1), and Early Growth Response Protein 1 (Egr-1) Binding Sites Form a Tripartite Gonadotropin-Releasing Hormone Response Element in the Rat Luteinizing Hormone-ß Gene Promoter: an Integral Role for SF-1
Ursula B. Kaiser,
Lisa M. Halvorson and
Marian T. Chen
Endocrine-Hypertension Division (U.B.K.) and Genetics Division
(M.T.C.) Department of Medicine Brigham and Womens Hospital
and Harvard Medical School Boston, Massachusetts 02115
Department of Obstetrics and Gynecology (L.M.H.) New England
Medical Center Tufts University School of Medicine Boston,
Massachusetts 02111
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ABSTRACT
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Recently, several cis-regulatory
elements that play roles in LHß gene expression, and their cognate
DNA-binding transcription factors, have been identified. These factors
include Sp1, steroidogenic factor-1 (SF-1), and early growth response
protein 1 (Egr-1). Using the GH3 pituitary cell
line (which lacks SF-1) as a model, we demonstrate that expression of
SF-1 or Egr-1 increases rat LHß gene promoter activity but has little
effect on the fold response to GnRH. However, expression of both SF-1
and Egr-1 synergistically enhances LHß gene promoter activity and
prevents further stimulation of activity by GnRH. Mutations in the Sp1
binding sites of the rat LHß gene promoter decrease GnRH
responsiveness, whereas mutations in the SF-1 and/or Egr-1 binding
sites alone have little effect on the GnRH response. Combinatorial
mutations in both the Sp1 and Egr-1 binding elements result in
almost complete loss of the GnRH response. In contrast, in
GH3 cells cotransfected with SF-1, mutations in
the Sp1, SF-1, or Egr-1 binding elements independently decrease GnRH
responsiveness. In LßT2 cells, a gonadotrope-derived cell line that
expresses SF-1 endogenously, mutations in either the Sp1 or Egr-1
binding elements decrease GnRH responsiveness. These data suggest that
the Sp1, SF-1, and Egr-1 binding sites form a tripartite GnRH response
element in the rat LHß gene promoter. Changes in the spacing between
the upstream Sp1 binding sites and the downstream SF-1/Egr-1 binding
elements reduce the response to GnRH. SF-1, while having little direct
effect on GnRH responsiveness, has a critical role in integrating the
effects of Sp1 and Egr-1.
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INTRODUCTION
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The production and secretion of the pituitary gonadotropins, LH
and FSH, are controlled by the complex interaction of multiple factors
originating from the hypothalamus, pituitary, and gonads. The
hypothalamic peptide, GnRH, is one of the most critical factors,
effecting regulation of secretion of LH and FSH as well as of the
transcription and biosynthesis of the gonadotropin subunits,
,
LHß, and FSHß (1). Recent studies have identified several
transcription factors that have profound effects on basal and/or
GnRH-stimulated LHß gene promoter activity.
Steroidogenic factor-1 (SF-1), a member of the nuclear hormone receptor
superfamily, is selectively expressed in the gonadotrope subpopulation
of the anterior pituitary gland, as well as in the adrenal gland and
the gonads (2). SF-1 binding elements have been identified in a range
of genes important for steroidogenesis, sexual differentiation, and
reproductive function (3). An SF-1 binding site was identified in the
glycoprotein
-subunit gene (referred to as the gonadotrope-specific
element, or GSE) and SF-1 binding was shown to result in
transcriptional activation of this gene (4). Studies of the rat LHß
gene have identified and characterized two SF-1 binding elements,
located at positions -127 and -59 relative to the transcriptional
start site (Fig. 1
) (5, 6). The
functional role of SF-1 in trans-activation of the LHß
gene has been demonstrated in vivo and in vitro
(5, 6, 7, 8). However, the role of SF-1 in the regulation of GnRH
responsiveness of the LHß gene has not been fully characterized.
Pituitary SF-1 mRNA and protein levels have been reported by some
to increase in response to GnRH (9), whereas others have not observed
this regulation (10, 11, 12). SF-1 has been found to have no effect on the
fold activation of LHß gene promoter activity by GnRH (11, 13).
Transgenic mice null for the SF-1 gene have reduced levels of
gonadotropins, yet remain capable of responding to GnRH with an
increase in gonadotropin expression (2, 14). Thus, the role of SF-1 in
the regulation of LHß gene expression by GnRH remains
controversial.

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Figure 1. Schematic Diagram of the Organization of the Rat
LHß Gene Promoter
Two Sp1 binding sites are located upstream between positions
-451/-386; two composite SF-1 and Egr-1 binding sites are located
between -127/-106 and -59/-42. All numbering is relative to the
transcriptional start site of the rat LHß gene. GTF, General
transcription factors.
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Early growth response protein 1 (Egr-1) has also been implicated in the
tissue-specific expression of the LHß gene. Egr-1 is a member of the
immediate early gene family characterized by zinc finger domains with
Cys2-His2 motifs that
recognize GC-rich nucleotide sequences (15). In vitro
studies have demonstrated two functional Egr-1 binding sites in the rat
LHß gene promoter, located at positions -112 and -50 relative to
the transcriptional start site (Fig. 1
) (6, 16). Synergy between SF-1
and Egr-1 has been demonstrated in the activation of the LHß gene (6, 11, 12, 17). Transgenic mice null for Egr-1 demonstrated specific loss
of LHß gene expression and failed to respond to GnRH (16, 18). GnRH
is known to activate protein kinase C (PKC) in gonadotropes, and we
have demonstrated previously that activation of PKC increases
Egr-1 expression levels and also activates rat LHß gene
promoter activity, suggesting a role for Egr-1 in mediating the LHß
gene response to PKC (17, 19). Furthermore, GnRH increases Egr-1
expression in pituitary gonadotrope-derived cell lines, and mutations
of the Egr-1 binding sites almost completely prevent stimulation of the
proximal LHß gene promoter by GnRH, implicating Egr-1 as a critical
transcription factor involved in effecting this GnRH response (11, 12, 17, 20).
Our previous studies have identified additional DNA sequences further
upstream, between -490/-352, in the rat LHß gene promoter that are
also important for LHß gene expression and GnRH responsiveness
(13). More detailed analyses demonstrated several Sp1 binding sites
within this region (Fig. 1
). Mutations of these elements, which block
binding of Sp1, reduce basal LHß gene promoter activity as well as
lessen the stimulation by GnRH (21, 22). Like Egr-1, Sp1 is
a member of the Cys2-His2
zinc finger family of transcription factors and recognizes a similar
but distinct GC-rich nucleotide sequence (23). These data suggest that
Sp1 also plays an important role in conferring GnRH responsiveness and
bring into question the requisite role of Egr-1 in this response.
The relative importance of these three transcription factors, Sp1,
Egr-1, and SF-1, and possible interactions among them in mediating GnRH
responsiveness have not been characterized. In this report, we
demonstrate that all three transcription factors contribute to GnRH
responsiveness and that the cognate binding sites form a tripartite
GnRH response element. SF-1, while having little direct effect on GnRH
responsiveness, has a critical role in integrating the effects of Sp1
and Egr-1 on the GnRH response.
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RESULTS
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SF-1 and Egr-1 Independently and Synergistically Increase LHß
Gene Expression and Block GnRH Responsiveness When Present in
Combination
A systematic approach to identifying mechanisms of hormonal
regulation of gonadotropin subunit gene expression has been hampered by
the lack of available cell lines that express either the endogenous or
transfected LHß and FSHß genes in a regulated manner. In our
previous studies, we have used GH3 cells, a well
characterized rat somatolactotropic cell line, as a model for the
analysis of cis-regulatory elements in the rat LHß gene
(5, 13, 21). We, therefore, initially sought to determine the effects
of SF-1 and Egr-1 on GnRH responsiveness of the LHß gene using this
cell model.
GGH3-1' cells (GH3 cells
stably transfected with rat GnRHR cDNA) were transfected with a
reporter construct containing region -797 to +5 of the rat LHß gene
promoter fused to a luciferase reporter (-797/+5LHßLUC) (Fig. 2
). Treatment with a GnRH agonist
(GnRHAg) for 6 h increased luciferase activity 6.6 ±
0.4-fold, consistent with our previous reports. Cotransfection with the
cytomegalovirus (CMV)-driven SF-1 expression vector resulted in a
2.4 ± 0.1-fold increase in luciferase activity. Further
stimulation with GnRHAg in the presence of SF-1 resulted in a 5.7
± 0.2-fold stimulation in -797/+5LHßLUC activity (relative to
vehicle-treated cells expressing SF-1), statistically slightly
decreased from the fold GnRH response in the absence of SF-1
(P < 0.05). Cotransfection with the CMV-driven Egr-1
expression vector also increased luciferase activity by 6.5 ±
0.8-fold. Stimulation with GnRHAg in the presence of Egr-1 resulted in
a 4.4 ± 0.2-fold stimulation of luciferase activity, again
slightly decreased from the fold GnRH response in the absence of SF-1
and Egr-1 (P < 0.001). As previously reported, marked
synergy was observed in the presence of both SF-1 and Egr-1, with a
180 ± 17-fold increase. Interestingly, the fold response to GnRH
was almost completely abrogated (1.5 ± 0.1-fold) in the presence
of both Egr-1 and SF-1. The loss of GnRH responsiveness after
overexpression of both SF-1 and Egr-1 supports the possibility that
these factors may have critical roles in mediating the GnRH response.
If GnRH stimulates LHß gene expression by increasing levels of
functionally active SF-1 and Egr-1, then we would expect that no
further GnRH response could be elicited in the presence of high levels
of both SF-1 and Egr-1.

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Figure 2. Effects of SF-1 and Egr-1 on Rat LHß Gene
Promoter Activity
GGH3-1' cells were transiently transfected with
-797/+5LHßLUC. Cells were cotransfected with expression plasmids
encoding either SF-1, Egr-1, or both, or the appropriate control
expression vector, and with an RSV-ß-Gal expression vector. Cells
were treated with ± 100 nM GnRHAg for 6 h before
harvesting. Luciferase activity was normalized to ß-galactosidase
activity. Results are shown as the mean ± SEM of at
least nine samples in three independent experiments. Fold response to
GnRHAg relative to vehicle-treated controls was calculated for each
transfection group.
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Mutations in the SF-1 or Egr-1 Binding Sites Do Not Decrease GnRH
Responsiveness of -797/+5LHßLUC in GGH3-1'
Cells
If GnRH does stimulate LHß gene expression by increasing levels
of functionally active SF-1 and Egr-1, as hypothesized above, then
mutations in the DNA binding sites for SF-1 and Egr-1 in the rat LHß
gene promoter, which block binding of these factors, would be predicted
to prevent stimulation by GnRH. We generated constructs containing
point mutations in either the two SF-1 DNA binding sites, the two Egr-1
DNA binding sites, or both, in the context of -797/+5LHßLUC.
These mutations have been shown previously to eliminate binding by SF-1
and/or Egr-1 by electrophoretic mobility shift assay (EMSA) (6). These
mutant constructs were first transfected into
GGH3-1' cells. GGH3-1'
cells lack endogenous SF-1, based on the failure of
GH3 nuclear extracts to produce specific
protein-DNA complexes with a GSE-containing oligonucleotide probe by
EMSA (data not shown). However, these cells do have endogenous Sp1 and
Egr-1 (17, 21). The mutations had little effect on basal luciferase
activity. Interestingly, mutations in the SF-1 and Egr-1 binding sites
of the LHß gene promoter also had little effect on the fold response
to stimulation with GnRHAg [-797/+5LHßLUC: 7.1 ± 0.4-fold;
LHSF1MLUC: 6.5 ± 0.3-fold; LHEgr1MLUC: 6.3 ± 0.3-fold;
LHSF1MEgr1MLUC: 6.6 ± 0.6-fold] (Fig. 3
). Therefore, these results do not
suggest a major contribution of the SF-1 and Egr-1 binding sites in the
rat LHß gene promoter in mediating GnRH responsiveness in
GGH3-1' cells.

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Figure 3. Effects of Mutations in the SF-1 and Egr-1 Binding
Sites on the Stimulation of Rat LHß Gene Promoter Activity by GnRH
Transient transfections were performed in GGH3-1' cells
with either wild-type -797/+5LHßLUC or mutant constructs harboring
mutations in the SF-1 and/or Egr-1 binding sites, as indicated. Cells
were cotransfected with an RSV-ß-Gal expression vector. Cells were
treated with ±100 nM GnRHAg for 6 h before
harvesting. Luciferase activity was normalized to ß-galactosidase
activity. The results are expressed graphically as the fold stimulation
by GnRH over control. Results are shown as the mean ±
SEM of at least nine samples in three independent
experiments.
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Combinatorial Mutations in Sp1, SF-1, and Egr-1 Binding Sites in
the LHß Gene Promoter Are Necessary to Block GnRH Responsiveness
Our previous studies suggested that Sp1 is also involved in
mediating the stimulation of the rat LHß gene in response to GnRH
(13, 21). Mutation of the Sp1 binding sites in the rat LHß gene
promoter reduced, but did not entirely eliminate, the GnRH response. We
generated a new panel of constructs containing point mutations in the
SF-1 DNA binding sites, the Egr-1 DNA binding sites, or both, in the
context of LHßSp1MLUC, a mutant variant of -797/+5LHßLUC with
mutations in the Sp1 binding sites (21). Transfections were performed
with these constructs in GGH3-1' cells (Fig. 4
). Similar to our previous findings,
mutations in the Sp1 binding sites decreased the GnRH response
[7.1 ± 0.4-fold to 3.4 ± 0.2-fold (P <
0.001)]. Additional mutations in the SF-1 binding sites had no further
effect on the fold response to GnRH, but mutations in the Egr-1 binding
sites almost completely blocked GnRH responsiveness [2.0 ±
0.2-fold]. In sum, whereas blockade of Egr-1 binding had no effect on
GnRH response in the context of the wild-type LHß gene promoter
in GGH3-1' cells, mutations that prevent the
upstream binding of Sp1 "unmask" a role for Egr-1 in mediating GnRH
responsiveness.

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Figure 4. Effects of Combinatorial Mutations in the Sp1 and
SF-1 and/or Egr-1 Binding Sites on the Stimulation of Rat LHß Gene
Promoter Activity by GnRH
Transient transfections were performed in GGH3-1' cells
with luciferase reporter plasmids encoding -797/+5 of the rat LHß
gene promoter harboring mutations in the Sp1, SF-1, and/or Egr-1
binding sites, as indicated. Cells were cotransfected with an
RSV-ß-Gal expression vector. Cells were treated with ± 100
nM GnRHAg for 6 h before harvesting. Luciferase
activity was normalized to ß-galactosidase activity. The results are
expressed graphically as the fold stimulation by GnRH over control.
Results are shown as the mean ± SEM of at least nine
samples in three independent experiments. *, P <
0.005 compared with the fold response of the reporter construct
harboring mutations in the Sp1 sites only.
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The Sp1, SF-1, and Egr-1 Binding Sites All Contribute to GnRH
Responsiveness of the LHß Gene Promoter in the Presence of SF-1
As mentioned above, GH3 cells lack SF-1.
Therefore, it is not surprising that mutations in the SF-1 binding
sites of the rat LHß gene had no effect on basal or GnRH-stimulated
luciferase activity. The use of an SF-1-deficient cell line allows
investigation of GnRH effects on LHß gene expression both in the
absence and presence of SF-1. We repeated transfections of the
wild-type and mutant -797/+5LHßLUC constructs in
GGH3-1' cells, this time in the presence of a
cotransfected CMV promoter-driven SF-1 expression vector, and studied
the effects of the mutations on basal and GnRHAg-stimulated luciferase
activity (Fig. 5
). In the presence of
SF-1, mutations in the Sp1, SF-1, or Egr-1 DNA binding elements
independently reduced GnRH responsiveness [-797/+5LHßLUC: 8.8
± 1.5-fold; LHSp1MLUC: 5.6 ± 0.8-fold; LHSF1MLUC: 5.2 ±
1.1-fold; LHEgr1MLUC: 4.1 ± 0.5-fold; LHSF1MEgr1MLUC:
5.4 ± 0.9-fold; P < 0.01 compared with
-797/+5LHßLUC]. In addition, combined mutations in the Sp1 and
either the SF-1 or Egr-1 binding elements almost completely abrogated
GnRH responsiveness [LHSp1MSF1MLUC: 2.6 ± 0.3-fold;
LHSp1MEgr1MLUC: 1.9 ± 0.2-fold;
LHSp1MSF1-MEgr1MLUC: 2.2 ± 0.1-fold]. In the
presence of SF-1, all three elements appear to contribute to mediate
the full GnRH response.

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Figure 5. Effects of Mutations in the Sp1, SF-1, and/or Egr-1
Binding Sites on the Stimulation of Rat LHß Gene Promoter Activity by
GnRH in the Presence of SF-1
Transient transfections were performed in GGH3-1'
cells with either wild-type -797/+5LHßLUC or mutant constructs
harboring mutations in the Sp1, SF-1, and/or Egr-1 binding sites, as
indicated. Cells were cotransfected with an SF-1 expression vector and
with an RSV-ß-Gal expression vector. Cells were treated with ±100
nM GnRHAg for 6 h before harvesting. Luciferase
activity was normalized to ß-galactosidase activity. The results are
expressed graphically as the fold stimulation by GnRH over control.
Results are shown as the mean ± SEM of at least nine
samples in three independent experiments. *, P <
0.01 compared with the fold response of -797/+5LHßLUC; **,
P < 0.05 compared with the reporter construct
harboring mutations in the Sp1 sites only.
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The Roles of the Sp1, SF-1, and Egr-1 Binding Sites in the LHß
Gene Promoter in Mediating GnRH Responsiveness in the LßT2 Cell
Line
Our studies in GGH3-1' cells suggested that
the Egr-1 binding sites played a role in mediating GnRH responsiveness
of the rat LHß gene promoter in the presence of SF-1 or,
alternatively, in the absence of Sp1 binding. However,
GH3 cells lack endogenous SF-1, and
cotransfection of the SF-1 expression vector may not accurately mimic
SF-1 levels or activity present in gonadotropes. Therefore, we turned
to the LßT2 cell line to confirm our findings. LßT2 cells are an
immortalized, gonadotrope-derived cell line generated from a pituitary
tumor occurring in a transgenic mouse expressing an LHß gene
promoter-SV40 virus T antigen fusion gene (24). These cells express
both
- and ß- subunits of LH as well as GnRHR and SF-1 (24, 25).
LßT2 cells respond to pulsatile GnRH administration with an increase
in GnRHR and LHß mRNA levels (25). We have transfected LßT2 cells
with -797/+5LHßLUC by electroporation and demonstrated a 3.2 ±
0.2-fold increase in luciferase activity in response to GnRHAg (Fig. 6
). In LßT2 cells, mutation of the Sp1
binding elements in the rat LHß gene decreased the fold response to
GnRH [2.3 ± 0.1-fold, P < 0.001]; mutation of
the SF-1 binding elements had no effect on the fold response to GnRH
[2.9 ± 0.3-fold, P = 0.4, NS]; and mutation
of the Egr-1 binding sites almost completely abolished stimulation of
the LHß gene promoter by GnRH [1.4 ± 0.1-fold]. Thus, as
anticipated from our results in GGH3-1' cells,
both the Sp1 and the Egr-1 binding elements appear to contribute to
mediating the full GnRH response in LßT2 cells (i.e. in
the presence of SF-1).

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Figure 6. Effects of Mutations in the Sp1, SF-1, and/or Egr-1
Binding Sites on the Stimulation of Rat LHß Gene Promoter Activity by
GnRH in the LßT2 Cell Line
LßT2 cells were transfected with either wild-type -797/+5LHßLUC or
mutant constructs harboring mutations in the Sp1, SF-1, and/or Egr-1
binding sites, as indicated. Cells were cotransfected with an
RSV-ß-Gal expression vector. Cells were treated with ± 100
nM GnRHAg for 6 h before harvesting. Luciferase
activity was normalized to ß-galactosidase activity. The results are
expressed graphically as the fold stimulation by GnRH over control.
Results are shown as the mean ± SEM of at least nine
samples in three independent experiments. *, P <
0.001 compared with the fold response of -797/+5LHßLUC.
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DNA Spacing between the Sp1 Binding Sites and the SF-1 and Egr-1
Binding Sites Is Important for Full GnRH Responsiveness of the LHß
Gene
The SF-1 and Egr-1 DNA binding sites form two composite elements
in the proximal LHß gene promoter within 130 bp of the
transcriptional start site. In contrast, the Sp1 binding sites are
further upstream, between positions -451/-386. For Sp1 bound to the
LHß gene promoter to interact with downstream SF-1 and Egr-1, DNA
bending or looping and/or interacting proteins need to be hypothesized.
We, therefore, wondered whether the spacing between the upstream Sp1
sites and the downstream SF-1/Egr-1 binding elements had an effect on
the ability of GnRH to stimulate LHß gene expression. We generated a
series of internal deletion constructs of the LHß gene promoter fused
to a luciferase reporter. These internal deletions removed either the
Sp1 binding sites (LH
A, -490/-353 deleted), one of the SF-1/Egr-1
composite elements (LH
B, -207/-83 deleted), or the intervening 135
bp (LH
C, -352/-208 deleted). These internal deletion constructs
were transfected into GGH3-1' cells, along with
either the SF-1 expression vector or the empty control vector (Fig. 7
).
As we would predict from our studies with point mutations, internal
deletion of the Sp1 binding sites decreased the fold response to GnRH
both in the absence and presence of SF-1 [-SF-1: -797/+5LHßLUC:
11.6 ± 1.1-fold, LH
A: 4.2 ± 0.2-fold, P
< 0.001; +SF-1: -797/+5LHßLUC: 7.8 ± 0.9-fold, LH
A:
5.3 ± 0.2-fold, P < 0.005]. Internal deletion
of the SF-1 and Egr-1 binding sites resulted in a small but significant
decrease in the fold response to GnRH in the absence of SF-1, but a
more marked decrease in response to GnRH in the presence of SF-1
[-SF-1: LH
B: 8.1 ± 0.6-fold, P < 0.001
compared with -797/+5LHßLUC; +SF-1: LH
B: 1.6 ± 0.2-fold,
P < 0.001 compared with -797/+5LHßLUC], again
consistent with our observations using point mutations in these
elements (Figs. 3
and 5
). Interestingly, internal deletion of the
intervening sequence (-359/-208) resulted in a decrease in the fold
response to GnRH, both in the absence and presence of SF-1, even though
all of the known binding sequences remain intact [-SF-1: LH
C:
4.9 ± 0.3-fold, P < 0.001 compared with
-797/+5LHßLUC; +SF-1: LH
C: 4.1 ± 0.4-fold,
P < 0.001 compared with -797/+5LHßLUC]. In
contrast, our previous studies have demonstrated that 5'-deletion of
this region has no effect on GnRH responsiveness (13). The
possibility that this deleted segment may contain transcriptionally
functional domains despite the lack of effect of 5'-deletion needs to
be considered. Nonetheless, these findings are consistent with the
hypothesis that the spacing between the Sp1 sites and the SF-1/Egr-1
sites is important for their interaction.
 |
DISCUSSION
|
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The importance of GnRH in the regulation of LH and FSH secretion
as well as of gonadotropin subunit gene expression is firmly
established. The recent development of GnRH-responsive cell lines and
of transgenic animal models has begun to lead to new insights into the
molecular mechanisms of this regulation, particularly for the LHß
gene. Using these approaches, several transcription factors important
for LHß gene expression have been identified. While each of these
factors has been implicated individually in mediating GnRH
responsiveness, their relative importance and possible interactions
among them have not been fully characterized.
SF-1 and Egr-1 were each able to transactivate the rat LHß gene
promoter but individually had little effect on the fold stimulation by
GnRH. In contrast, when both factors were present, basal LHß activity
was markedly and synergistically increased, and the response to GnRH
was almost completely abrogated (Fig. 2
). Although squelching cannot be
ruled out, these results suggest that the GnRH stimulation of LHß
gene expression may be effected by increased levels of functionally
active SF-1 and Egr-1. To explore this observation further, we used the
approach of mutagenesis of the cognate binding sites.
Mutagenesis of the Egr-1 binding elements had no effect on the GnRH
response in GGH3-1' cells in the absence of SF-1
(Fig. 3
). This was initially quite surprising in view of previous
reports. The Egr-1-/- mouse exhibits LHß
deficiency and fails to respond to gonadectomy with an increase in
LHß mRNA, suggesting a role for Egr-1 in mediating the
transcriptional regulation of the LHß gene by GnRH (16, 18). This
role was confirmed by in vitro studies (11, 12, 17, 20). In
these studies, mutations of the Egr-1 binding sites reduced or almost
completely abrogated GnRH responsiveness of the LHß gene promoter.
Closer evaluation reveals that the LHß promoter constructs used in
these studies did not include the upstream Sp1 binding sites.
Disruption of Sp1 binding to the LHß gene promoter, either by
mutation (Fig. 4
), by internal deletion (Fig. 7
), or by 5'-deletion (11, 12, 17, 20)
restores the importance of Egr-1 binding for the mediation of GnRH
responsiveness. These findings reconcile previous reports of the roles
of Sp1 and Egr-1 and indicate a novel functional relationship between
Sp1 and Egr-1 in mediating the GnRH response.
The role of SF-1 in the regulation of GnRH responsiveness of the LHß
gene has been controversial. While SF-1-/- mice
are deficient in LHß production (3), they remain responsive to GnRH
with an increase in LHß levels (2, 14). In contrast, mutation of the
SF-1 binding sites in the bovine LHß gene promoter prevented GnRH
stimulation in transgenic mice (7). Using GGH3-1'
cells that lack SF-1, we demonstrated previously that exogenous SF-1
increased both basal and GnRH-stimulated LHß gene promoter activity;
however, the fold response to GnRH was unaffected (13). Interestingly,
as shown here, in the presence of SF-1, both Egr-1 and Sp1 binding
elements contribute to GnRH responsiveness, whereas only the Sp1 site
appears important in the absence of SF-1. Thus, it appears that SF-1
plays an important indirect role by integrating the effects of Sp1 and
Egr-1. One could speculate that levels of SF-1 in gonadotropes may
modulate the relative roles in vivo of Sp1 and Egr-1 in
mediating the GnRH response.
In LßT2 cells, mutation of the SF-1 sites somewhat surprisingly did
not affect the GnRH response (Fig. 6
). The reason for this result is
not entirely clear. It may be due to differences in SF-1 levels between
GGH3-1' and LßT2 cells, other differences
between the two cell lines, or possibly due to the technical
limitations imposed by the comparatively small GnRH response observed
in LßT2 cells, making it more difficult to detect reductions in
responsiveness.
In the studies presented, we have not distinguished between the 5'
(-127/-106) and 3' (-59/-42) SF-1/Egr-1 composite elements. The
internal deletion studies in Fig. 7
indicate that the more 5'-site does
contribute to the GnRH response, but these studies do not
examine the 3' site in isolation. We have individually mutated
each of the SF-1 and Egr-1 binding sites and shown that each element
contributes to the overall GnRH response (data not shown). The 3' Egr-1
binding site appeared to have a slightly greater effect than the 5'
Egr-1 site, consistent with observations that the 3'-site has a higher
binding affinity for Egr-1 (L. Halvorson, unpublished data).
Our studies indicate that Sp1, SF-1, and Egr-1 act and interact to
contribute to GnRH responsiveness. Our results have led us to propose a
model in which these factors may interact with a common, as yet
unidentified, transcriptional cofactor (Fig. 8
). According to our proposed model, Sp1,
when bound to its cognate elements in the LHß gene promoter, is able
to interact with this putative cofactor to mediate GnRH responsiveness.
The loss of Sp1 binding to the LHß gene promoter therefore results in
a decrease in the GnRH responsiveness of this gene. The decrease in
GnRH responsiveness observed upon deletion of intervening sequences
could be explained by disruption of interaction of Sp1 with the
putative cofactor caused by alterations in spacing.

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Figure 8. Model for the Interaction of Sp1, SF-1, and Egr-1
with a Putative Transcriptional Cofactor in the Regulation of Rat LHß
Gene Expression in Response to GnRH
A, In the absence of SF-1, GnRH induces Egr-1 binding to the LHß gene
promoter, but Egr-1 is unable to interact with the transcriptional
cofactor, so mutations in the Egr-1 binding sites have no effect on
GnRH responsiveness. It is the interaction of Sp1 with this cofactor
that mediates the GnRH response. B, If mutations are introduced into
the rat LHß gene promoter that prevent Sp1 binding, then the cofactor
is free to interact with Egr-1, and this interaction mediates the
residual GnRH response. C, In the presence of SF-1, all three cofactors
interact with the transcriptional cofactor to mediate the full response
to GnRH.
|
|
We hypothesize that SF-1 is necessary for Egr-1 interaction with the
putative transcriptional cofactor in the context of the wild-type rat
LHß gene promoter. In the absence of SF-1, GnRH induces Egr-1 levels
and Egr-1 binding to the cognate sites in the LHß gene promoter, but
Egr-1 is unable to interact with the putative transcriptional cofactor
(Fig. 8
). As a result, mutation of the Egr-1 binding elements has no
effect on the GnRH response in the absence of SF-1 (Fig. 3
). However,
if Sp1 binding is prevented, then the transcriptional cofactor is able
to interact with Egr-1 even in the absence of SF-1, enabling Egr-1 to
mediate GnRH responsiveness under these conditions (Fig. 4
). We
postulate that the stoichiometry of the interaction is such that in the
absence of SF-1, the cofactor can interact with only Sp1 or Egr-1, but
not both, with a preferential interaction with Sp1 (Fig. 8
). Such a
preferential interaction is not unprecedented. For example, NCoR
(nuclear receptor corepressor) preferentially binds to thyroid hormone
receptor when it is a homodimer rather than when it forms a heterodimer
with retinoid X receptor (26).
In our model, Sp1, Egr-1, and SF-1 are shown interacting with a
non-DNA-binding (classical) cofactor. These three transcription factors
are known to interact variously with the coactivators SRC-1 and
CBP/p300 (27, 28, 29, 30, 31), which were therefore tested for their role in
mediating the GnRH response. In preliminary experiments in
GGH3-1' and LßT2 cells, no effect on basal or
GnRH-stimulated LHß gene promoter activity was observed with the
addition of either cofactor (data not shown). While the interpretation
of these experiments must be cautious, these results raise the
intriguing possibility that a novel, possibly gonadotrope-specific or
GnRH-regulated transcriptional coactivator may be involved.
Efforts to identify such a factor are underway.
The cofactor depicted in the model could also be a transcription
factor, binding directly to an as-yet- unidentified element in the
LHß gene, or there may be more than one cofactor involved.
Alternatively, it is possible that no cofactor is necessary, but rather
Sp1, SF-1, and Egr-1 interact directly. Sp1 and SF-1 have been shown to
interact directly to modulate CYP11A gene transcription (32), and
direct protein-protein interaction between SF-1 and Egr-1 has also been
reported (6, 11).
We have not yet addressed the role of Ptx1, a homeobox-containing
transcription factor affecting the expression of multiple
pituitary hormone genes, in the regulation of LHß gene expression
(33). Ptx-1 has been shown to enhance LHß gene expression and to act
in synergism with SF-1 and Egr-1 through direct protein-protein
interaction in the stimulation of LHß gene expression (11). The role
of Ptx-1 in GnRH responsiveness remains to be studied.
Physiologically, all three of these transcription factors are present
in gonadotropes and are likely to interact to mediate the response to
GnRH. Therefore, the cognate binding sites for these factors can be
considered to form a tripartite GnRH response element. It is possible
that such a composite GnRH response element provides a level of
redundancy to protect such a critical point of regulation of
reproductive function.
 |
MATERIALS AND METHODS
|
---|
Materials
The GnRH agonist des-Gly10,
[D-Ala6]-GnRH ethylamide
(GnRHAg) was purchased from Sigma (St. Louis, MO).
Reporter Plasmids and Expression Vectors
The wild-type reporter construct used for these studies
contained 797 bp of the 5'-flanking sequence of the rat LHß gene and
the first 5 bp of the 5'-untranslated region, cloned into the pXP2
luciferase reporter vector (-797/+5LHßLUC) (34, 35). The
Transformer site-directed mutagenesis kit (CLONTECH Laboratories, Inc., Palo Alto, CA) was used to introduce point mutations into
the Sp1, SF-1, and Egr-1 binding sites in -797/+5LHßLUC.
Oligonucleotides used have been described previously (17, 21) and are
summarized in Table 1
. Generation of
multiple mutations was performed either simultaneously or sequentially
and required the use of two selection primers (mpXP2 and mpXP2Rev), as
described previously (17). All mutagenic constructs were confirmed by
dideoxysequencing.
Constructs with internal deletions within the rat LHß gene promoter
were generated by PCR using primers with ends modified to cause
deletion of the indicated sequences during amplification. LH
A
(-490/-353 deleted) and LH
B (-207/-83 deleted) were generated as
previously reported (13). Additional primers, LHß56S
(5'-GTGTTTAAAGCAAATTTGAGCCAATGTCAGTTAAGCTCAG-3') and LHß65AS
(5'-CTGAGCTTAACTGACATTGGCTCAAATTTGCTTTAAACAC-3'), were used to generate
LH
C (-359/-208 deleted). All constructs were confirmed by
dideoxysequencing.
The SF-1 expression vector contained 2.1 kb of the murine SF-1 cDNA
driven by CMV promoter sequences in the vector, pCMV5 (kindly provided
by K. L. Parker, Southwestern School of Medicine, Dallas,
TX) (36). The Egr-1 expression vector was generated by cloning
the murine Egr-1 cDNA (provided by D. Nathans, John Hopkins University,
Baltimore, MD) (37) into pCMV5 at BamHI and
HindIII restriction enzyme sites. The F-SRC-1 expression
vector, encoding the full-length human SRC-1 cDNA in pcDNA1/Amp, was
generously provided by Dr. Akira Takeshita (Brigham and Womens
Hospital, Harvard Medical School, Boston, MA) (38). An expression
vector containing p300 cDNA under the control of CMV promoter sequences
was the generous gift of Dr. D. M. Livingston (Dana Farber Cancer
Institute, Harvard Medical School, Boston, MA) (39). An expression
vector expressing ß-galactosidase driven by the Rous sarcoma virus
promoter (RSV-ß-Gal) was used as an internal standard and control
(40).
Cell Culture and Transfection
GGH3-1' cells were prepared by stably
transfecting GH3 cells with the rat GnRHR cDNA,
as described previously (41). LßT2 cells were generously provided by
Dr. P. L. Mellon (University of California, San Diego, CA) (24, 25). GGH3-1' and LßT2 cells were maintained in
monolayer culture in DMEM (GGH3-1' cells in low
glucose media and LßT2 cells in high glucose media) supplemented with
10% (vol/vol) FBS at 37 C in humidified 5%
CO2/95% air. For transient transfection studies,
cells were cultured to 5070% confluence and transfected by
electroporation. In each experiment, approximately 5 x
106 cells were suspended in 0.4 ml of Dulbeccos
PBS plus 5 mM glucose containing the DNA to be transfected.
The cells received a single electrical pulse of 240 V from a total
capacitance of 1000 microfarads, using an Electroporator II apparatus
(Invitrogen, San Diego, CA). Cells received 2 µg/well of
the reporter constructs. Where appropriate, cells also received 1
µg/well of SF-1, Egr-1, F-SRC-1, and/or p300 expression vectors or an
equivalent amount of the empty pCMV5 expression vector. Cells were
cotransfected with RSV-ß-Gal (1 µg/well). After electroporation,
cells were plated in serum-containing medium. Medium was replaced after
24 h, and cells were harvested 48 h after transfection. Cells
were treated with 100 nM GnRHAg or vehicle for 6 h
immediately before harvesting. Cell extracts were prepared and analyzed
for luciferase and ß-galactosidase activities as described previously
(40, 42). Luciferase activity was normalized to the level of
ß-galactosidase activity. Data are shown as mean ±
SEM.
Statistical Analysis
Transfections were performed in triplicate and repeated at least
three times. Data were combined across transfection experiments to
determine the mean ± SEM of the corrected luciferase
activity for basal and GnRHAg-treated cells, and fold stimulation in
response to GnRH was calculated. Two-way ANOVA followed by post hoc
comparisons with Fishers protected least significant difference test
was used to assess whether changes in GnRH responsiveness were
statistically significant between the indicated groups. Significant
differences were established as P < 0.05.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Dr. William W. Chin for thoughtful
and helpful discussions throughout the course of these
investigations.
 |
FOOTNOTES
|
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Address requests for reprints to: Dr. Ursula B. Kaiser, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail:
ukaiser{at}rics.bwh.harvard.edu
This work was supported in part by NIH Grants R29-HD-33001 and
R01-HD-19938 (U.B.K.) and R01-HD-38089 and R03-HD-34692 (L.M.H.) and by
the George W. Thorn Center (U.B.K.).
Received for publication December 13, 1999.
Revision received April 25, 2000.
Accepted for publication May 1, 2000.
 |
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