Activation of the Rat Follicle-Stimulating Hormone Receptor Promoter by Steroidogenic Factor 1 Is Blocked by Protein Kinase A and Requires Upstream Stimulatory Factor Binding to a Proximal E Box Element
Leslie L. Heckert
Department of Molecular and Integrative Physiology The
University of Kansas Medical Center Kansas City, Kansas 66160
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ABSTRACT
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The receptor for the pituitary glycoprotein
hormone FSH (FSHR) and the nuclear hormone receptor steroidogenic
factor 1 (SF-1) play important roles in control of the
hypothalamic-pituitary- gonadal axis. FSHR is essential for
integrating the pituitary FSH signal to gonadal response, while SF-1 is
an important transcriptional regulator of many genes that function
within this axis and is essential for the development of gonads and
adrenal glands. Given the critical role of SF-1 in regulation of the
gonads and the coexpression of FSHR and SF-1 in Sertoli and granulosa
cells, we examined the ability of SF-1 to regulate transcription of the
FSHR gene. We found that SF-1 stimulated rat FSHR promoter activity in
a dose-dependent and promoter-specific manner. Examination of various
promoter deletion mutants indicated that SF-1 acts through the proximal
promoter region and upstream promoter sequences. An E box element
within the proximal promoter is essential for activation of the FSHR
promoter by SF-1. This element binds the transcriptional regulators
USF1 and USF2 (upstream stimulatory factors 1 and 2) but not SF-1, as
shown by electrophoretic mobility shift assays. In addition, functional
studies identified a requirement for the USF proteins in SF-1
activation of FSHR and mapped an important regulatory domain within
exons 4 and 5 of USF2. Cotransfection studies revealed that activation
of protein kinase A leads to inhibition of SF-1-stimulated
transcription of FSHR, while it synergized with SF-1 to activate the
equine LH ß-promoter (eß). Thus, stimulation of the cAMP pathway
differentially regulates SF-1 activation of the FSHR and
eß-promoters.
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INTRODUCTION
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FSH is an important pituitary glycoprotein that serves to regulate
gonadal function (1). This hormone elicits its effects by binding a
cell surface receptor found only in the gonads, where hormone binding
results in a number of cellular changes that assist germ cell
development (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). The cDNA and gene for the FSH receptor (FSHR) have
been cloned and characterized from several different species (14, 15, 16, 17, 18, 19).
FSHR expression studies have revealed that transcription of this gene
occurs only within Sertoli cells of the testis and granulosa cells of
the ovary (3, 20, 21, 22). Thus, elucidation of the transcriptional
mechanisms that regulate the FSHR gene will provide insight into both
cell- specific transcriptional events and mechanisms that control
the response of the gonads to FSH.
Transient transfection analysis of various deletion and block
replacement mutants of the FSHR 5'-flanking region showed that basal
transcriptional activity is controlled predominantly by elements
located within the first 100-bp of the promoter (23, 24). Within this
region, a single E box element, located approximately 30 bp upstream of
the transcriptional start site, is an important control element for
FSHR transcription (23, 24, 25). Additional studies have shown that the
basic helix-loop-helix (bHLH)-ZIP proteins USF1 and USF2 (upstream
stimulatory factors 1 and 2) bind and activate FSHR transcription
through this proximal E box (23, 24, 26). Also within the promoter
region, elements 3' to the transcriptional start site have been
identified as important for full promoter function (23, 27). Although
these studies have contributed significant insight into FSHR gene
regulation, it is important to recognize that our current understanding
of FSHR transcription is primarily limited to the regulatory events
represented by the conditions of experimental cell culture systems.
Thus, functionally important transcription factors not present or
active within the cultured cells remain unrecognized. Directly testing
the regulatory effects of relevant transcription factors on FSHR
promoter function therefore provides an important complementary means
to identify proteins that regulate FSHR gene activity.
The transcription factor steroidogenic factor 1 (SF-1), also known as
adrenal 4-binding protein (Ad4-bp), is a key regulator of endocrine
function and sex determination (reviewed in Ref. 28). Its expression is
limited primarily to cells of the gonads, adrenal, pituitary, and
ventral medial hypothalamus, where it is thought to contribute to
cell-specific properties of genes expressed within these tissues
(29, 30, 31, 32, 33, 34). SF-1 is a member of the nuclear hormone receptor family that
was originally recognized for its role in endocrine regulation, as it
bound to an important regulatory element (AGGTCA) common to promoters
of several key steroidogenic enzymes (35, 36, 37, 38, 39, 40). However, SF-1 not only
regulates genes encoding steroidogenic enzymes but a number of genes,
including the gonadotropin
- and ß-subunits, the GnRH receptor,
and the inhibin
- subunit, that are important for production of
the gonadotropin hormones FSH and LH (39, 41, 42, 43, 44, 45, 46, 47, 48, 49). In addition, gene
ablation studies in mice revealed that SF-1 is essential for adrenal
and gonadal development and for proper function of the
hypothalamic-pituitary-gonadal axis (50, 51, 52, 53).
During development, SF-1 expression is first observed in the urogenital
ridge of the embryo and is later found in discrete populations of cells
that give rise to adrenocortical and gonadal cells (29). Shortly after
induction of the testis, SF-1 expression is observed in the
interstitial region and within the seminiferous cords, indicating that
both Leydig (interstitial) and Sertoli (cords) cells express this
protein (29, 30). In the developing ovary, SF-1 expression is
significantly lower than what is observed in the testis but appears to
increase just before the time of birth (29, 31). SF-1 expression
continues after birth in the adrenal gland, testis, and ovary, where it
has been detected in adrenocortical cells, testicular Leydig and
Sertoli cells, and ovarian theca and granulosa cells (29, 32, 54).
In male rats, FSHR expression is first detected just after the testis
begins to form in the embryo [embryonic day 14.5 (e14.5)],
while in females, ovarian FSHR expression is delayed until just before
birth (e20.5) (2). Although FSHR expression is not observed in the
early SF-1-expressing cells of the urogenital ridge, its coexpression
with SF-1 in Sertoli and granulosa cells as gonadogenesis proceeds
suggests that SF-1 may influence FSHR expression. Furthermore, SF-1s
known role in the regulation of the hypothalamic-pituitary-gonadal axis
suggests that its functions may extend beyond hormone production to
that of hormone response through regulation of receptor levels. Studies
presented herein demonstrate that SF-1 regulates transcription of the
FSHR gene via a mechanism that involves regions of the FSHR 5'-flanking
region and requires USF1 and USF2 binding to the proximal E box
element.
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RESULTS
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SF-1 Regulates Expression of the Rat FSHR Promoter
To determine whether SF-1 regulates FSHR, the rat FSHR promoter
(-2,700/+123) driving expression of a luciferase reporter gene was
cotransfected into the placental cell line JEG3 together with an
expression vector for SF-1 (RSV-SF1). JEG3 cells were chosen to examine
SF-1 regulation of FSHR, as these cells have been shown to lack SF-1
and to activate known SF-1 target response elements via transient
transfection (55, 56). Cotransfection with increasing amounts of the
SF-1 expression vector showed a dose-dependent increase in FSHR
promoter activity, as measured by the production of luciferase (Fig. 1A
). RSV-CAT (Rous sarcoma
virus-chloramphenicol acetyl transferase) was used as a control to
equalize the amount of cotransfected DNA in each sample. In addition,
the effects of SF-1 were promoter specific, as luciferase production
from either the pGL3-Control (SV40 promoter) or pGL3-Basic
(promoterless) vectors was only modestly effected by cotransfection
with SF-1 expression vector (Fig. 1B
). The stimulatory effect of SF-1
on rat FSHR promoter suggests that this transcription factor is
important for proper regulation of the FSHR gene.

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Figure 1. SF-1 Regulates Transcription of the Rat FSHR
Promoter
A, One microgram of FSHR(-2,700/+123)Luc vector was transiently
transfected into JEG3 cells together with 200 ng
RSV-ßgalactosidase (a control for transfection efficiency) and
increasing amounts of RSV-SF1. The total amount of cotransfected
expression vector was kept constant by adjusting to 1.5 µg with
RSV-CAT expression vector, which was used as a negative control. B,
FSHR(-2,700/+123)Luc, pGL3-Control, or pGL3-Basic (0.5 µg) was
cotransfected into JEG3 cells with either 1.5 µg RSV-CAT or
RSV-SF1. Each sample also received 200 ng RSV-ß-galactosidase. The
relative activity represents the luciferase/ß-galactosidase activity
of each sample normalized to the luciferase/ß-galactosidase activity
of the promoter transfected with RSV-CAT alone. Error bars
represent the SEM.
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Multiple Regions of the FSHR Promoter Are Important for Regulation
by SF-1
To help delineate regions of the promoter important for response
to SF-1, various deletion mutants of the FSHR promoter were tested for
response to cotransfection with the SF-1 expression vector (RSV-SF1).
Deletion of the -2,700-bp promoter to either -1,300 bp or -743 bp
resulted in a sequential decrease in stimulation by SF-1 (Fig. 2
). Deletion to -743 reduced stimulation
to approximately 3-fold while further deletion to -220 had no further
impact. Furthermore, deletion of the 3'-region of the promoter from
+123 to +79, a region that contains an SF-1-like element
(5'-TGgCCTTG-3'), had no further impact on SF-1 induction (Fig. 2
;
compare -220/+123 and -220/+79). Thus, several regions within the
FSHR promoter (-2,700 to -1,300, -1,300 to -743, and -220 to +79)
are needed for full response to SF-1, suggesting that multiple elements
are required for full response to SF-1.

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Figure 2. SF-1 Stimulation Maps to Several Regions of the
FSHR Promoter
One microgram of the indicated FSHR promoter deletion mutant was
cotransfected into JEG3 cells with either 0.5 µg RSV-CAT or RSV-SF1.
Each sample also received 200 ng RSV-ß-galactosidase. The relative
activity represents the luciferase/ß-galactosidase activity of each
sample normalized to the luciferase/ß-galactosidase activity of
FSHR(-2,700/+123)Luc transfected with RSV-CAT. Error
bars represent the SEM.
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The E Box Is Required for Stimulation by SF-1
Although SF-1 stimulation required regions of the promoter
upstream of -743, an approximate 3-fold induction of the -220/+79
promoter suggested that a SF-1- responsive element(s) reside within
this region. Additional studies further refined this region to between
-31 bp and +79, as a promoter truncated to -31 bp still responded to
SF-1 stimulation (data not shown). Using various block replacement
mutants in the context of the -220/+123 FSHR promoter, we evaluated
regions within the proximal promoter (-31 to +79) that are necessary
for response to SF-1. Although several mutants had somewhat diminished
responses, mutation µ9.2 had the most severe effect on SF-1
transactivation (Fig. 3A
). Interestingly,
the µ9.2 mutation also eliminated SF-1 response when placed in the
context of the 2,700/+123 promoter that contains the upstream
SF-1-responsive regions (Fig. 3B
).

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Figure 3. An E box in the Proximal Promoter Is Required for
SF-1 Regulation of the Promoter
A (top), Sequence of the FSHR-proximal promoter region (top strand) and
various mutations (bottom strand) examined for SF-1 responsiveness. One
microgram of FSHR(-220/+123) Luc wild-type (wt) promoter or various
mutants in the same context (-220/+123) were cotransfected into JEG3
cells with 200 ng RSV-ß-galactosidase and 0.5 µg of either
RSV-CAT or RSV-SF1. The relative activity represents the
luciferase/ß-galactosidase activity of each sample normalized to the
luciferase/ß-galactosidase activity of FSHR(-220/+123)Luc
transfected with RSV-CAT. B, 0.5 µg of either FSHR(-2,700/+123)Luc
or FSHR(-2,700/+123)µ 9.2Luc, which contain a mutation
through the proximal E box, was cotransfected into JEG3 cells with 1.5
µg of either RSV-CAT or RSV-SF1. The relative activity represents the
luciferase/ß-galactosidase activity of each sample normalized to the
luciferase/ß-galactosidase activity of FSHR(-2,700/+123)Luc
transfected with RSV-CAT. Error bars represent the
SEM. C, EMSA. A radiolabeled probe corresponding to
the FSHR E box (5'-TCTTGGTGGGTCACGTGACTTTGCCCGT-3') was
used in EMSA with nuclear extracts from JEG3 cells. Radiolabeled probe
(25 fmol) was incubated with approximately 8 µg of nuclear extract
and resolved on a 4% polyacrylamide gel as described in
Materials and Methods. Where indicated, unlabeled
homologous (WT) or nonspecific (NS;
5'-CTAGAGTCGACCTGCAGGCATGCAAGCTTGGCATTC-3') DNAs were added to the
reaction at a concentration equal to 100x that of the probe. Where
shown, antibodies specific for the bHLH proteins USF1, USF2, or c-Myc
were added to the reactions before the addition of the probe. The major
specific USF complexes are marked by an arrow.
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The µ9.2 mutation disrupts an E box element that has been shown to be
important for FSHR promoter function (23, 24, 25). As has been observed in
other cell types, the E box bound the transcription factors USF1 and
USF2 in JEG3 cells (Fig. 3C
). Although SF-1 has not previously been
shown to bind this element, careful inspection of this region revealed
the presence of an SF-1-like element (gGGTCA) that includes part of the
E box sequence (underlined,
5'-GGGTCACGTGACTT-3'). To determine whether SF-1 regulates
the FSHR promoter by directly binding the region containing the
proximal E box, DNA/protein binding studies were performed. A known
SF-1 binding site from the human
-subunit promoter (h
GSE)
(46) was used as a probe to determine whether the FSHR E box could
compete for SF-1 binding. As a source of SF-1 protein, we used nuclei
isolated from the pituitary gonadotrope cell line
T3 (57). An
electrophoretic mobility shift assay (EMSA) identified several specific
complexes bound to the h
GSE (Fig. 4A
).
One of these complexes cross-reacted with an antibody generated to
SF-1, indicating that SF-1 was present in the bound proteins (Fig. 4A
).
In addition, the SF-1 band was competed with homologous sequence
[gonadotrope-specific element (GSE)] but not with as much as a 400
molar excess of the E box, indicating that the E box does not bind
SF-1. Interestingly, several of the slower migrating complexes did
compete with the E box. The inability of the FSHR E box to bind SF-1
was also indicated by studies in which the E box was used as a probe.
In this case, several specific complexes were observed, but none of the
bands cross-reacted with the SF-1 antibody (Fig. 4A
). However,
antibodies generated to USF1 and USF2 were able to supershift most of
the bound complexes. SF-1 binding to the GSE was also observed when
Sertoli cell nuclear extracts were used and similarly revealed that the
E box was unable to compete for SF-1 binding (Fig. 4B
). To assure that
other complexes did not obscure SF-1 binding to the E box, in
vitro transcribed/translated (TNT) SF-1 was used in an EMSA
together with the E box probe. In these studies, no SF-1 binding was
observed with as much as 10 µl of the SF-1 TNT mixture (Fig. 4C
).
However, SF-1 binding was clearly observable with the GSE probe. Thus,
while the E box is essential for SF-1 regulation of the FSHR promoter,
these studies indicate that regulation does not require the direct
binding of SF-1 to the element. SF-1 regulation may, therefore, occur
via a mechanism involving interactions with the USF proteins.
The USF Proteins Are Needed for Activation of the FSHR Promoter by
SF-1
To determine the role of the USF proteins in SF-1 regulation of
FSHR, several mutant USF proteins that lack different protein domains
were employed. The mutant USF proteins, U1
N and U2
N (Fig. 5A
), efficiently bind DNA but are unable
to transactivate due to the absence of the amino-terminal
transactivation domains, while the U1
B and U2
B mutants lack a
functional DNA binding domain (58, 59). Both sets of mutants inhibit
transactivation by USF1 and USF2. The
N mutants have been shown to
inhibit FSHR transcription by binding to the FSHR E box and interfering
with the ability of endogenous USF to transactivate (26). The
B
mutants inhibit USF transactivation by dimerizing with endogenous USF
proteins and preventing interactions with responsive elements (59).
Cotransfection of either U1
N or U2
N effectively blocked
transactivation of the FSHR promoter by SF-1 (Fig. 5B
). In addition,
both
B mutants were able to significantly reduce SF-1
transactivation (Fig. 5B
). Thus, binding of USF proteins lacking the
transactivation domain to the E box or sequestering endogenous USF
proteins significantly inhibited SF-1 activation of the FSHR promoter.
Further analysis of the regions within the transactivation domain of
USF2 revealed the requirement for amino acids encoded in exons 4 and 5
in SF-1 regulation (Fig. 5
, A and C). Thus, deletion of the first 123
amino acids (
7123), which are encoded for in the first four exons,
significantly diminished SF-1 regulation. Extending the deleted region
to include exon 5 (
199) further diminished SF-1 response.
Additionally, deletion of the amino acids encoded by either exon 4 or 5
alone significantly reduced SF-1 activation. In contrast,
cotransfection with either wild-type USF2 or a mutant in which the USR,
a highly conserved USF-specific region, was deleted had little impact
on SF-1s ability to transactivate the FSHR promoter (Fig. 5C
). These
studies indicate that the amino acids encoded by exons 4 and 5 but not
the USR are important for the regulation by SF-1. Importantly, several
of these mutants only slightly diminished promoter activity by
themselves, suggesting that USF requirements for SF-1 stimulation
differ from the requirements for basal promoter function (Fig. 4
and
Ref. 26).
To determine whether the requirement for the USF proteins in SF-1
transactivation was specific to the FSHR promoter, we examined the
effects of wild-type USF2 and the
1199 mutant on the equine LH
ß- subunit (eß) promoter. Similar to previous reports, we
observed a significant transcriptional increase from the eß promoter
in response to cotransfected SF-1 (Fig. 5D
and Ref. 42). However, in
contrast to our observations on the FSHR promoter, the USF2
1199
mutant did not affect the ability of SF-1 to transactivate the eß
promoter (Fig. 5D
). Thus, the requirement for the USF proteins in SF-1
transactivation is specific to the FSHR promoter and suggests that the
mechanism by which SF-1 activates genes is dependent on promoter
context.
Stimulation of the cAMP Pathway Differentially Affects SF-1
Activation of the FSHR and eß Promoters
Previous studies on the
-inhibin promoter indicated that
stimulation of the cAMP pathway enhanced the actions of SF-1 (41).
While activation of this pathway is known to stimulate inhibin
production, evidence for its regulation of FSHR suggests both positive
and negative regulatory effects on expression (60, 61, 62, 63, 64, 65). To determine
whether the cAMP pathway influences SF-1 activation of FSHR, we
transfected cells with the FSHR promoter and expression vectors for
SF-1 (RSV-SF1) and the catalytic subunit of protein kinase A (RSV-PKA),
which acts as a constitutive activator of this pathway. Cotransfection
with RSV-PKA alone had little impact on FSHR promoter activity, while
in the presence of RSV-SF-1, RSV-PKA blocked the ability of SF-1 to
induce FSHR transcription (Fig. 6
). We
also examined the effects of cotransfections with RSV-PKA and RSV-SF-1
on the promoters for the equine
(e
) and ß (eß) subunits of
LH (Fig. 6
). PKA activated both the eß and e
promoters, while only
the eß promoter was induced by SF-1. Interestingly, cotransfection
with RSV-SF-1 and RSV-PKA resulted in a synergistic effect on the
activity of the eß promoter, while RSV-SF-1 reduced the inductive
effects of PKA on the e
promoter (Fig. 6
). Treatment with the cAMP
analog, 8-bromo-cAMP, had similar effects to those observed with the
RSV-PKA (data not shown). Thus, activation of the cAMP pathway resulted
in differential effects on SF-1- stimulated promoter activity, as it
blocked SF-1-activated transcription from the FSHR promoter and
stimulated it on the eß promoter.

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Figure 6. PKA Differentially Regulates SF-1 Stimulation of
the FSHR and eß Promoters
Rreporter vector (0.5 µg) containing either the promoter for FSHR,
eß, or e was cotransfected in the presence or absence of
RSV-PKA-ver2 (an expression vector for the catalytic subunit of PKA)
and with or without RSV-SF1. RSV-CAT was used to normalize for the
amount of transfected DNA. Fold induction represents the activity of
the promoter in each transfected sample relative to the activity of the
promoter transfected in the presence of RSV-CAT alone. No induction of
the e promoter was observed in the presence of SF-1. Error
bars represent the SEM.
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DISCUSSION
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FSHR and SF-1 are both integral components of the
hypothalamic-pituitary-gonadal axis. SF-1 regulates transcription of
many genes within this axis, while FSHR is essential for integrating
the pituitary FSH signal to gonadal response. The important role of
SF-1 in endocrine regulation is evident from its involvement in the
transcriptional control of a number of genes important for either the
biosynthesis or regulation of steroid hormones (66). Thus, SF-1 has
been shown to regulate genes encoding steroid hormone biosynthetic
enzymes and genes needed for LH and FSH production. With respect to the
latter, SF-1 has been shown to regulate the genes for the gonadotropin
ß-subunit, the GnRH receptor, and the
-subunit for the
FSH-regulatory protein inhibin (39, 41, 42, 43, 44, 45, 46, 47, 48, 49). Here, we show that SF-1
acts at yet another level of the axis to modulate FSH response in the
gonads through transcriptional regulation of the FSHR gene.
The studies presented in this report support our current hypothesis
that SF-1 stimulates FSHR transcription by binding several low-affinity
binding sites within the upstream portion of the FSHR promoter and
interacting with the USF proteins bound to the E box (Fig. 7
). Cotransfection studies revealed that
SF-1 stimulated FSHR promoter activity and that stimulation required
multiple sites within the 5'-flanking region. Optimal SF-1 response was
observed with the largest FSHR promoter construct (-2,700/+123) and
SF-1 stimulation decreased sequentially as larger regions of the
promoter were deleted from the 5'-end. Furthermore, characterization of
additional deletion mutants (-2,343, -2,049, and 1,023) resulted in
similarly modest changes in SF-1 response (data not shown). The
moderate impact of these deletions on the full stimulation suggested
that multiple weak SF-1 binding sites upstream of -734 are needed for
full response of the FSHR promoter to SF-1. Response within the
proximal promoter region (-220/+79) mapped to an E box element
previously shown to be regulated by the transcription factors USF1 and
USF2 (23, 24, 26). Interestingly, mutation of the E box in the context
of the 2,700/+123 promoter eliminated all response to SF-1,
demonstrating the absolute requirement for this element in SF-1
regulation of FSHR. In the absence of SF-1 binding to this element, the
data suggest a mechanism in which SF-1 interacts with the USF proteins,
either directly or indirectly via a secondary protein, to stimulate
FSHR transcription (Fig. 7
, options 1 and 2, respectively). In support
of this hypothesis, basal promoter activity and SF-1-stimulated
promoter activity had differential requirements for the USF
proteins.

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Figure 7. Model for SF-1 Regulation of the FSHR Promoter
As postulated, SF-1 induces FSHR transcription by binding several sites
within the FSHR promoter that span the region from -2,700 to -743.
After binding to these upstream sites, SF-1 interacts either directly
with the USF proteins (1 ) or with an intermediate regulatory protein
(2 ). Interaction with the E box-bound complex results in a stimulation
of the initiation complex and induction of transcription. While not
shown in the model, the data also suggest that in the absence of these
upstream sites (-2,700 to -743), SF-1 binds sufficiently to the E
box-bound complex to stimulate transcription. PKA interferes with the
ability of SF-1 to transactivate the FSHR promoter at a point likely to
be downstream of SF1 binding.
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Further confirmation of a role for SF-1 in FSHR gene regulation was
recently provided by a study on the mouse FSHR promoter. In this
report, the investigators showed that SF-1 activated the mouse FSHR
promoter through two SF-1 binding sites located between -1,171 and
-1,369 bp relative to the translations start site(67). Examination of
the rat promoter sequence within this region identified two potential
SF-1 binding sites similar to those described for the mouse. The
elements reside at positions -1,213 (5'-TTTCCTTGG-3') and 1,252
(5'-TTTCCTTGA-3') relative to the major transcriptional start site.
Interestingly, removal of both of these sites with the -1,300 to -743
deletion (Fig. 2
) resulted in a decrease in promoter response to SF-1.
However, in addition to a role for these elements, our studies further
suggest that other sites important for SF-1 response reside upstream of
-1,300 and that the USF proteins are important for the overall
response to SF-1.
The involvement of the USF proteins in SF-1 activation of FSHR was
further substantiated by studies in which plasmids expressing various
USF mutants were cotransfected with the FSHR promoter and an expression
vector for SF-1. These studies revealed that the amino-terminal
transactivation domains of USF1 and USF2 are required for SF-1
stimulation and implicated the amino acids encoded by both exons 4 and
5 of USF2 as needed for activation by SF-1. Importantly, the effects of
the USF mutants on SF-1 transactivation were specific to the FSHR
promoter, as neither wild-type USF2 nor the
1199 mutant influenced
SF-1 activation of the eß promoter. Interestingly, many recent
reports have revealed that SF-1 commonly acts in concert with other
transcription factors to regulate gene activity. Thus, SF-1 has been
shown to synergize with the transcriptional regulators RAR (retinoic
acid receptor), GATA-4 (a member of the GATA family of zinc proteins),
Egr-1 (early growth response protein 1), Ptx-1 (pituitary homeobox 1),
and Wt1 (Wilms tumor suppressor) (40, 43, 45, 55, 68, 69, 70).
Therefore, a common mechanism by which SF-1 regulates transcription is
one involving cooperation with other transcription factors, of which
USF1 and USF2 can now be included. Although a role for USF proteins in
SF-1 transcriptional activation has not been previously reported, it is
of interest to note that USF and SF-1 are implicated in the regulation
of both the human
- subunit and SF-1 genes, suggesting
that a similar mechanism may be shared by them as well (46, 71, 72, 73, 74, 75).
Although the mechanisms by which SF-1 and USF cooperate to regulate
FSHR are not fully understood, the finding that these transcription
factors act in concert to activate transcription provides important
insights into FSHR gene regulation, transactivation function of SF-1,
and the expanding physiological roles of SF-1 in the
hypothalamic-pituitary-gonadal axis. Within this axis, SF-1 appears to
regulate both FSH production and response. Thus, through its regulation
of the
-subunit of the FSH-regulatory protein inhibin, SF-1 can
directly influence FSH production in the pituitary, and through its
regulation of FSHR, SF-1 can influence FSH response. Interestingly,
activation of the inhibin
subunit by SF-1 has been shown to
dramatically increase in the presence of activated PKA, the major
downstream regulator of FSH action in the gonads (41). Thus, rising
levels of FSH in the presence of SF-1 should dramatically stimulate
inhibin levels produced by the gonads, which would then feed back on
the pituitary to specifically decrease FSH production. Remarkably, our
studies revealed that activated PKA had the opposite impact on
SF-1-stimulated transcription of the FSHR promoter. Thus, with SF-1 in
position to regulate these two genes, FSH stimulation can lead to
opposite transcriptional effects resulting in a rise in
inhibin and
a fall in FSHR, which would result in a dramatic decrease in FSH
signaling in the gonads. Together these observations provide a
compelling molecular argument for SF-1 as a central regulator in the
integrated control of FSH action in the reproductive system.
In support of our observations, several studies have demonstrated
inhibitory effects of cAMP on the expression of FSHR. In the ovary,
treatment with hormones such as PMSG or human CG (hCG), which stimulate
the cAMP pathway, can result in either an increase or decrease in FSHR
expression (65, 76). Treatment with PMSG to stimulate follicle growth
results in an increase in FSHR mRNA, whereas, treatment with hCG to
induce ovulation and luteinization results in a marked decrease in FSHR
expression. It is therefore tempting to speculate that the differential
effects of cAMP on FSHR expression in the ovary may reflect the
expression level of SF-1 in granulosa cells at the time of hormone
stimulation. In the testis, FSHR mRNA in Sertoli cells is significantly
reduced in response to cAMP induction (62, 63). Thus, our studies
suggest that SF-1 in granulosa and Sertoli cells is an important
modulator of FSHR regulation by the gonadotropin hormones FSH and
LH.
The mechanisms by which PKA influences SF-1-activated transcription are
not fully understood. Our data, and data with the
inhibin
promoter, demonstrate the importance of promoter context in the
integration of SF-1 and cAMP transcriptional signals. Studies
with the
inhibin promoter suggested that the synergistic effects of
SF-1 and activated PKA were the result of direct interaction between
SF-1 and the cAMP response element binding protein, CREB, which are
bound to different but closely spaced response elements on the promoter
(41). Our studies also demonstrate synergistic effects between
activated PKA and SF-1 but on a different promoter, the eß promoter.
This is a novel finding and the mechanism has not been fully explored.
While studies have demonstrated a role for SF-1 in the regulation of
eß, information regarding cAMP regulation is lacking (42). In the
horse, this gene is expressed in both the placenta and pituitary,
where, in the latter, it is under the control of the hypothalamic
peptide GnRH. Although not extensively studied, the cAMP pathway has
been implicated in GnRH stimulation of pituitary gonadotrope cells.
Thus the adenylate cyclase inhibitor 2',3'-dideoxyadenosine blocked
GnRH-induced LHß mRNA, and GnRH was shown to induce phosphorylation
of CREB, a known downstream target of PKA (77, 78). In the placenta,
cAMP is known to regulate the human ß-subunit of CG, which is
functionally similar to the equine ß subunit in these cells (79).
These observations, together with our findings on the effects of SF-1
and activated PKA, suggest that the cAMP pathway is an important
regulator of the LH and CG ß genes and SF-1 is involved in modulating
the effects.
With respect to the FSHR promoter, PKA stimulation had the opposite
effect on transcriptional induction by SF-1, providing a molecular
explanation for the reported negative changes in FSHR mRNA in response
to cAMP induction (41, 80, 81, 82, 83, 84). While our current understanding of how
these two signals converge to regulate FSHR is limited, our favored
hypothesis is that PKA activation leads to modification of SF-1 and
prevents its interaction with the USF proteins (Fig. 7
). Alternatively,
the transcriptional repressor ICER (inducible cAMP early
repressor), which is induced by FSH in testicular Sertoli cells,
may bind the FSHR promoter and interfere with SF-1-activated
transcription. Previous reports provide evidence for ICER regulation of
FSHR (85). Further analysis of the protein interactions and domains
required for regulation of transcription will provide additional
insight into the mechanisms required for SF-1 activation and PKA
repression of FSHR promoter activity.
 |
MATERIALS AND METHODS
|
---|
DNA Constructs
Rat FSHR promoter/luciferase constructs FSHR(-2,700/+123)Luc,
FSHR(-1,300/+123)Luc, FSHR(-763/+123)Luc, FSHR(-220/+123)Luc, and
FSHR(-220/+79)Luc are described elsewhere (23). For
FSHR(-2,700/+123)µ9.2, the 343-bp EcoRV/XhoI
fragment from -220 to +123 was removed and replaced with the analogous
region generated from FSHR(-220/+123)µ9.2. Expression vectors for
the USF were kindly provided by Dr. Michele Sawadogo (59). PGL3-Control
and pGL3-Basic were purchased from Promega Corp. (Madison,
WI). The cDNA for mouse SF-1 was generously provided by Dr. Keith
Parker and subcloned downstream of the Rous Sarcoma viral promoter as
described (42). RSV-CAT is described elsewhere (86). The eß and e
promoters were a gift from Dr. Michael Wolfe and are described
elsewhere (42, 87). The expression vector for the catalytic subunit of
PKA (RSV-PKA-ver2) was generously provided by Dr. Richard Maurer and is
described elsewhere (88). Plasmid DNAs were prepared from overnight
bacterial cultures using QIAGEN DNA plasmid columns
according to the suppliers protocol (QIAGEN, Chatsworth,
CA).
Transfection and Enzyme Analysis
The choriocarcinoma cell line JEG3 was cultured in DMEM
supplemented with 10% FBS. For transfection, cells were seeded onto
six-well plates (35 mm/well) at a density of 110,000 cells per well.
Unless otherwise stated, cells were transfected with 1 µg of
FSHR-luciferase construct, 0.2 µg RSV-ß-galactosidase, and
0.5 µg of expression vector using 5 µl of lipofectamine. The
lipofectamine/DNA complex was replaced after 16 h with JEG3 media
(see above). Cells were harvested and assayed approximately 60 h
after transfection according to previously described procedures (89).
To control for transfection efficiency, luciferase activity generated
from the FSHR promoter was normalized to the ß-galactosidase activity
from the cotransfected RSV-ß-galactosidase vector. Data were averaged
over a minimum of three independent experiments.
Electrophoretic Mobility Shift Analysis
The gonadotrope cell line
T3 was grown in culture as
indicated elsewhere (57, 89) and nuclei were isolated as described
(90). Nuclear extracts from primary Sertoli cells and JEG3 cells were
prepared as described (73). In vitro
transcription/translation reactions were done according to the
manufacturers recommendation (Promega Corp., Madison.
WI) using cDNA vectors encoding human USF1 and mouse SF-1 as indicated
above. EMSAs were performed as previously described using approximately
3 x 105
T3 nuclei or 510 µg of
nuclear proteins from JEG3 cells and 25 fmol radiolabeled
double-stranded oligodeoxynucleotide probe (91). End-labeled
oligodeoxynucleotide probes were generated using T4 kinase. DNA
sequences of the human
- subunit GSE and FSHR E box
oligodeoxynucleotides are 5'-TTTCATGGGCTGACCTTGTCGTCACCATCAC-3' and
5'-TCTTGGTGGGTCACGTGACTTTGCCCGT-3', respectively. All reaction
components were incubated at room temperature for 30 min in the absence
of probe. Competitors were added at the indicated molar ratios, and 1
µl of antibody was used for supershift analysis. After the addition
of probe, reactions were incubated an additional 15 min on ice and then
resolved on a 4% nondenaturing polyacrylamide gel. Antibodies for USF1
(C-20)X and USF2 (C-20)X were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the SF-1 antibody was
purchased from Upstate Biotechnology, Inc. (Lake Placid,
NY).
 |
ACKNOWLEDGMENTS
|
---|
I would like to thank the Center of Reproductive Sciences at the
University of Kansas Medical Center for imaging, cell culture, and DNA
sequencing services. I also thank Dr. Michael Wolfe for generously
providing
T3 nuclei and Daren Rice and Jiang-kai Chen for their
technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Leslie Heckert, Department of Molecular and Integrative Physiology, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160. E-mail:
lheckert{at}kumc.edu
This work was supported by NIH Grant R29HD-3521701A1 (to L.L.H.).
Received for publication March 27, 2000.
Revision received January 24, 2001.
Accepted for publication February 6, 2001.
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