A Novel Cyclic Adenosine 3',5'-Monophosphate-Responsive Element Involved In the Transcriptional Regulation of the Lutropin Receptor Gene in Granulosa Cells
Shiyou Chen1,
Xuebo Liu1 and
Deborah L. Segaloff
Department of Physiology and Biophysics The University of Iowa
College of Medicine Iowa City, Iowa 52242
 |
ABSTRACT
|
---|
The induction of the lutropin receptor (LHR) in
granulosa cells by FSH is mediated, at least in part, by cAMP. However,
the classic cAMP-responsive element (CRE) is not present in the
5'-flanking region of the rat LHR gene. Previous studies from our
laboratory had shown that three Sp1 sites within the promoter region of
the rat LHR (rLHR) bind Sp1 and Sp3 and are involved in the basal and
cAMP-mediated transcription of the rLHR gene. In the present studies we
show that the rLHR promoter region forms a complex (designated complex
A) with nuclear extracts from rat granulosa cells, and the abundance of
complex A is markedly increased when using cells that had been
pretreated with 8-bromo (Br)-cAMP. We have localized the binding of the
protein(s) in complex A to a DNA sequence immediately upstream and
partially overlapping with the Sp1c binding site. The core site
(designated SAS for Sp1c adjacent sequence) is localized to nucleotide
(nt) -146 to -142 and contains the sequence GGGGG. The consensus
sequence for the core portion of this element appears to be
(G/T)GGGG. Mutations of the SAS site, but not SP1c site, abolish
complex A formation. Experiments utilizing rat granulosa cells
transfected with luciferase reporter genes driven by the 5'-flanking
region of the rLHR gene demonstrate a functional role for the SAS site
in the cAMP responsiveness of the rLHR gene.
 |
INTRODUCTION
|
---|
The induction of the lutropin receptor (LHR) in granulosa cells
during the growth of small antral follicles to preovulatory follicles
is of physiological importance for LH-induced ovulation and corpus
luteum formation (1, 2, 3, 4). Earlier studies have shown that the induction
of the LHR in granulosa cells is mediated by the concerted actions of
both FSH and estradiol (5, 6, 7, 8, 9, 10). Whereas the effects of FSH on LHR
induction are apparently due to its ability to increase intracellular
cAMP levels (11, 12, 13, 14), the role of estradiol in the induction of the LHR
is still unclear.
It has since been shown that the FSH or 8-bromo (Br)-cAMP-mediated
increase in the LHR and LHR mRNA in granulosa cells obtained from
diethylstilbestrol (DES)-treated rats is associated with a marked
increase in LHR gene transcription. (15). However, increases in LHR
gene transcription are not apparent within the first 24 h of
treatment with FSH or 8-Br-cAMP, consistent with there being a 24-h lag
time before increases in LHR mRNA are apparent (15). Interestingly, the
5'-flanking region of the rat LHR (rLHR) gene does not contain any
classical cAMP-responsive elements (16). However, granulosa cells
transfected with a luciferase reporter gene driven by 2.1 kb of the
5'-flanking region of the rLHR gene respond to exogenously added
8-Br-cAMP (15). A further characterization of that region of the gene
has revealed that a number of cis-regulatory elements and
trans-acting factors are involved in the transcriptional
regulation of the rLHR gene in rat granulosa cells (17). Among these
are three Sp1 sites (Sp1a, Sp1b, and Sp1c) in the promoter region,
which we have shown to be functionally important. Thus, each of these
sites forms complexes with Sp1 and/or Sp3 as determined by mobility
shift assays. Furthermore, mutation of each of these sites results in
both diminished basal as well as cAMP-induced transcriptional activity
of the rLHR gene as determined by reporter gene assays (17).
Mobility gel shift assays have shown the marked increase in the
abundance of a complex formed between the promoter region of the rLHR
gene and nuclear extracts from 8-Br-cAMP-induced rat granulosa cells as
opposed to extracts from control cells (17). The current studies were
undertaken to identify the DNA element involved in this complex
formation. We report herein the identification of a unique
cAMP-responsive element in the promoter region of the rLHR gene.
The nature of this site and its role with the Sp1c site in regulating
basal and cAMP responsiveness of the rLHR gene are presented.
 |
RESULTS
|
---|
Delineation of nt -151/-136 as Containing the Region Involved in
Complex A Formation
Previous studies from our laboratory using electrophoretic
mobility shift assays (EMSAs) had examined the ability of probes
corresponding to overlapping regions of the nt -2056/-2 portion of
the rLHR gene to form complexes with nuclear extracts from granulosa
cells that had been untreated or treated with 8-Br-cAMP (17). All but
one probe exhibited several DNA/protein complexes, the patterns of
which were different with the various probes. In many cases, the
intensity of a given complex was increased when the extracts were
derived from 8-Br-cAMP-treated cells. In the present study we chose to
focus on a cAMP-dependent complex that was observed using a probe
corresponding to the promoter region of the rLHR. Figure 1
shows the formation of this complex,
which we have designated complex A, with the -187/-2 region of the
rLHR gene. As seen in Fig. 1
, its abundance is markedly increased by
pretreatment of granulosa cells with 8-Br-cAMP.

View larger version (36K):
[in this window]
[in a new window]
|
Figure 1. Analysis of the Involvement of Consensus Elements
in the Formation of Complex A
Panel A, Mobility shift assays were performed using a
32P-labeled fragment corresponding to nt -187/-2 of the
rLHR gene promoter. The labeled probe was incubated with 2 µg of
nuclear extracts isolated from uninduced (C) or 8-Br-cAMP-induced (I)
rat granulosa cells as described in Materials and
Methods. Competitions between the labeled fragment and
unlabeled fragments containing mutations in consensus sequences at
molar excess ratios of 100 and 200, respectively, are shown for each
element. Panel B, The sequences of the synthetic oligomer sequences
used for competition are shown. The core binding residues for each
corresponding factor are underlined.
|
|
To precisely define the DNA binding region contributing to complex A,
we first examined its relationship with the consensus elements in the
promoter region. The 5'-flanking region of rLHR gene contains three Sp1
sites (Sp1a, Sp1b, and Sp1c), two steroidogenic factor 1 (SF1) sites
(SF1a and SF1b), and one AP2 site (17). Previous studies have shown the
SF1 and AP2 sites are not required for basal or cAMP-induced expression
of hLHR gene transcription. However, mutation of any of the three Sp1
sites decreases both basal and cAMP-induced rLHR gene transcription.
EMSAs further showed that S1 and Sp3 proteins bind to each of the three
Sp1 sites in the rLHR gene promoter region. However, it is important to
note, for the purposes of the present studies, that the EMSA conditions
required for the detection of the Sp1 and Sp3-containing complexes with
the Sp1 sites were quite different than those required for the
detection of complex A formation with the -187/-2 rLHR probe. As a
result, experiments designed to detect Sp1/Sp3-containing complexes
with the Sp1 sites cannot detect complex A. Conversely, those designed
to detect complex A cannot detect the Sp1/Sp3-containing complexes with
the Sp1 sites. The experiments shown in Fig. 1
were performed under
EMSA conditions chosen to optimize detection of complex A. Therefore,
the uppermost bands on the gel (which are specific since all the
complexes can be completed by the inclusion of an excess of an
unlabeled -187/-2 fragment) do not correspond to Sp1/Sp3-containing
complexes. Competing DNA fragments containing the SF1, Sp1a, Sp1b,
Sp1c, or AP2 sites were synthesized and added to EMSAs examining
complex A formation to the -187/-2 region of the rLHR gene. As shown
in Fig. 1A
, fragment D, which contains the Sp1c site (nt -151 to
-130), completely inhibited the formation of complex A. Fragment B,
which contains the Sp1a site, also inhibited complex A formation, but
to a smaller extent. Fragments A, C, and E, which contain SF1a, Sp1b,
and AP2 binding sites, respectively, exhibited no competition.
Additional EMSAs were performed using competing fragments of different
lengths to narrow down the region of the rLHR gene contributing to
complex A formation. From these experiments we were able to determine
that the cis-element contributing to complex A formation
lies within nt -151 to -136 of the rLHR gene (data not shown).
Identification of the GGGGG Sequence of nt -146/-142 as the Core
Sequence Required for Complex A Formation
To more precisely define the DNA sequence in the -151 to -136
region of the rLHR gene involved in complex A formation, the following
experiments were performed. Thirteen pairs of complementary
oligonucleotides were synthesized based upon the wild-type sequence of
fragment D in which a single nucleotide at a time spanning the region
of nt -150 to -138 was substituted. These fragments were then used as
competing oligonucleotides in EMSAs to determine whether any decreased
the formation of complex A. The results shown in Fig. 2
were obtained utilizing nuclear
extracts that had been obtained from 8-Br-cAMP-treated granulosa cells.
Each of the fragments containing single substitutions of the
nucleotides GGGGG located between nt -146/-142 prevented the
formation of complex A. In contrast, all the other singly substituted
oligonucleotides did not inhibit complex A formation. These results
suggest that the GGGGG sequence located between nt -146 and -142 is
the core sequence necessary for complex A formation. The same results
were obtained when nuclear extracts from untreated granulosa cells were
used (data not shown).

View larger version (59K):
[in this window]
[in a new window]
|
Figure 2. Identification of the Core DNA Element That
Contributes to Complex A Formation
Shown are the results of a mobility shift assay using a fragment
corresponding to nt -187/-2 of the rLHR gene as the labeled probe.
The labeled probe was incubated with 2 µg of nuclear extracts
isolated from 8-Br-cAMP-induced rat granulosa cells in the absence or
the presence of an unlabeled competing oligonucleotide at a 100-fold
molar excess. The unlabeled competing oligonucleotides corresponded to
nt -151/-130 (fragment D of Fig. 1 ) with either the wild-type
sequence (lane 1) or a sequence in which one residue between -150 and
-138 was substituted (lanes 315). Lanes 1 and 2 show the results in
the presence or absence, respectively, of a competing fragment
corresponding to the wild-type sequence. Lanes 315 show the results
using competing fragments containing a mutation. The five Gs whose
substitution did not cause competition are underlined.
|
|
To further determine the consensus sequence for the element forming
complex A, each of the five Gs was separately substituted by A, C, or
T within the context of the -151/-130 fragment. Each of the
substituted fragments or wild-type fragment was used in EMSAs at
different concentrations to competitively inhibit the formation of
complex A. The intensities of complex A before and after competition by
wild-type or mutant fragments are shown in Fig. 3
. The wild-type oligonucleotide
corresponding to each G completely inhibited complex A formation even
at a 50-fold molar excess. Therefore, the wild-type GGGGG sequence is
the best candidate. When the G corresponding to nt -146 was
substituted with T, complex A formation was also significantly
inhibited. All other substitutions did not show significant inhibition
of complex A formation. We conclude, therefore, that the consensus
sequence for this core element is (G/T)GGGG.

View larger version (32K):
[in this window]
[in a new window]
|
Figure 3. Determination of the SAS Element Consensus Sequence
Mobility shift assays were performed in which a labeled probe
corresponding to nt -187/-2 of the rLHR gene was incubated with 2
µg of nuclear extracts isolated from 8-Br-cAMP-induced rat granulosa
cells in the absence or the presence of an unlabeled competing
oligonucleotide at molar excess ratios of 50, 100, and 200. The
competing oligonucleotides corresponded to nt -151 to -130 of the
rLHR gene in which each of the five consecutive Gs corresponding to
nt -146/142 was left intact or mutated to either C, A, or T. The
complexes were resolved on polyacrylamide gels, which were then exposed
to a phosphor screen for 4 h at room temperature. The screen was
scanned and the intensity of complex A (relative to background) was
quantified.
|
|
As shown by the sequence in Fig. 4
, the
core sequence identified as the cis element forming complex
A with granulosa cell nuclear protein(s) is upstream of and partially
overlapping the Sp1c site. Therefore, we have termed this
cis element SAS for Sp1 adjacent sequence.

View larger version (12K):
[in this window]
[in a new window]
|
Figure 4. Schematic Representation of the SAS Site and Its
Relationship to the Sp1c Site Shown is the sequence of nt -151/-130
of the rLHR gene. The consensus sequence for the Sp1/Sp3 binding site
corresponding to Sp1c between nt -142/-137 is noted in
bold. The core SAS sequence of nt -146/-142 is noted
by the shaded box. Both sequences are also
overlined. It can be appreciated that nt -142 and -143
are within both the core SAS site as well as the Sp1c site. Residues
that were substituted in the creation of site-selective mutations are
noted by the underlined sequences. For Sp1c(mt), the
GCGG sequence of nt -141/-138 was replaced by AGTC. For SAS(mt1), the
GG sequence of nt -146/-145 was replaced by AT. For SAS(mt2), the
GGGGG sequence of -150/-145 was replaced by TATCAT.
|
|
The SAS Site Is Related to cis Elements Binding G
String Factors
A search of the literature and databases for DNA cis
elements revealed that the SAS core sequence shares the common feature
of a G-rich sequence with some other cis-acting elements.
These include the cis-elements that bind the
transcription factors G-string (18), G-fer (19), BGP1 (20, 21), IF-1
(22, 23), and Zif268 (24). We, therefore, examined whether fragments
corresponding to these G-string and related binding elements could
compete for complex A formation. Figure 5
shows the results of an EMSA using a labeled fragment corresponding to
-187/-2 of the rLHR gene incubated with nuclear extracts from induced
granulosa cells where unlabeled fragments corresponding to the
wild-type or mutated sequences of G-string and related binding elements
were added in excess. Fragments corresponding to the wild-type
sequences that bind G-string, G-fer, BGP1, or IF-1 effectively competed
for complex A formation, and mutations in the G-string, G-fer, and
IF-1-binding fragments abolished their ability to compete for complex A
formation. However, two different mutations of the BGP1-binding DNA
fragment did not diminish the ability of the fragments to inhibit
complex A formation. This could imply that the inhibition of complex A
formation by the wild-type BGP1-binding fragment is mediated by
residues other than those that we mutated. The latter is not an
unlikely scenario given the large tracts of G-rich sequences in the
BGP1-binding DNA fragment. It should also be noted that the wild-type
BGP1-binding sequence, in addition to inhibiting complex A formation,
also inhibited the formation of other complexes in the upper portion of
the gel. This may reflect a nonspecific effect of the BGP1-binding DNA
fragment. Alternatively, this fragment may be blocking the formation of
multimerized complexes. As also shown in Fig. 5
, not all G-rich DNA
elements competed for complex A formation, as evidenced by the
inability of the Zif268-binding fragment to inhibit complex A
formation. These data suggest that the SAS site may be related to some
(e.g. the G-string and G-fer binding sites), but not all,
G-rich DNA binding cis-elements.

View larger version (36K):
[in this window]
[in a new window]
|
Figure 5. DNA Binding Sites for G-String-Related
Transcription Factors Compete for Complex A Formation
Shown are the results of a mobility shift assay using a fragment
corresponding to nt -187/-2 of the rLHR gene as the labeled probe.
The labeled probe was incubated with 2 µg of nuclear extracts
isolated from 8-Br-cAMP-induced rat granulosa cells in the absence (NA)
or the presence of an unlabeled competing oligonucleotide at a 100-fold
molar excess. The unlabeled competing oligonucleotides corresponded to
wild-type or mutated (mt) DNA binding sequences for G-string, G-fer,
BGP1, IF-1, and Zif268. The residues in the wild-type sequence that
were substituted are highlighted and
underlined. The mutated sequences showing the residues
that were inserted in place of the wild-type residues are
highlighted and underlined.
|
|
The SAS Site, but Not Sp1c Site, Is Required for Complex A
Formation
Because the SAS site partially overlaps the Sp1c site, it is
important to determine whether complex A formation is dependent upon
nuclear proteins binding to the SAS site only or the SAS and Sp1c
sites. To address this question, we designed mutations of the SAS and
Sp1c sites that would be site-specific (Fig. 6
). A mutation of the Sp1c site was made
by substituting the GCGG residues immediately downstream of the core
SAS site. Two different mutations of the SAS site were examined. The
SAS(mt1) mutation substituted the two G nucleotides at the
5'-end of the core SAS site. The SAS(mt2) mutation substituted
these same two G nucleotides as well as additional four nucleotides
immediately upstream of the core SAS sequence. Both the mt1 and mt2
mutations of the SAS sites left the Sp1c site intact. EMSAs were
performed examining the ability of the wild-type or mutated -187/-2
fragments to compete with complex A formation to a labeled -187/-2
fragment. As shown in Fig. 6
, the Sp1c(mt1) fragment completely
inhibited complex A formation, demonstrating that an intact Sp1c site
is not required for complex A formation. In contrast, the SAS(mt2)
fragment caused no inhibition of complex A formation. Although a
20-fold excess of SAS(mt1) did not compete, a 50-fold excess of
SAS(mt1) caused a slight inhibition of complex A formation. The ability
of the SAS(mt1) fragment at high concentrations to partially compete
for fragment A formation suggests that nucleotides in addition to the
guanines at -146 and -145 are required for high affinity binding of
complex A. One possibility, of course, is that the other three guanines
in the core sequence are required. Unfortunately, we cannot readily
test this because disruption of guanines -143 and -142 would affect
the Sp1c site as well. Notably, the lack of ability of the SAS(mt2)
fragment to compete for complex A formation implicates residues
upstream of the core SAS site as being involved in complex A formation.
Although the individual substitutions of nt -150 through -147 did not
implicate these residues (see Fig. 2
), the aggregate mutation of these
residues suggests a role for them in the formation of complex A (Fig. 6
). Importantly, these data show that mutation of the Sp1c site does
not affect the formation of complex A to the SAS site. We further
addressed the question of the potential role of Sp1 and Sp3 binding
contributing to complex A formation by examining the effects of
antibodies to Sp1 and Sp3 on complex A formation in EMSAs. As shown in
Fig. 7
, the addition of antibodies to Sp1
and/or Sp3 had no effect on the mobility or the intensity of complex A.
Because the complexes binding to the Sp1c site cannot be visualized
under the EMSA conditions used to detect complex A, controls showing
the effects of antibodies to Sp1 and Sp3 on known Sp1/Sp3-containing
complexes cannot be determined from the experiments shown in Fig. 7
.
However, as shown in Fig. 8
, the
concentrations of antibodies used are indeed effective in inhibiting
the binding of Sp1 and Sp3 to the Sp1c site in the rLHR promoter.
Therefore, the lack of effect of these antibodies on complex A
formation (as seen in Fig. 7
) suggests that complex A does not contain
either Sp1 or Sp3.

View larger version (52K):
[in this window]
[in a new window]
|
Figure 6. The SAS Site, but Not the Sp1c Site, Is Required
for Complex A Formation
Shown are the results of a mobility shift assay using a fragment
corresponding to nt -187/-2 of the rLHR gene as the labeled probe.
The labeled probe was incubated with 2 µg of nuclear extracts
isolated from 8-Br-cAMP-induced rat granulosa cells in the absence or
the presence of unlabeled competing oligonucleotides at 20- or 50-fold
molar excess. The competing oligonucleotides correspond to nt
-164/-130 with the wild-type sequence or with Sp1c(mt1), SAS(mt1), or
SAS(mt2) mutations as described in Fig. 4 .
|
|

View larger version (52K):
[in this window]
[in a new window]
|
Figure 7. Complex A Is Not Composed of Sp1-Binding Proteins
Shown are the results of a mobility shift assay using a fragment
corresponding to nt -187/-2 of the rLHR gene as the labeled probe.
The labeled probe was incubated with 2 µg of nuclear extracts
isolated from 8-Br-cAMP-induced rat granulosa cells in the absence or
the presence of antibodies to Sp1 and/or Sp3. In some cases, the
antibodies were added to the reaction mixture with all the other
components (lanes 24). In other cases (lanes 68), the antibodies
were preincubated with the nuclear extracts for 30 min at 0 C before
the addition of labeled probe.
|
|

View larger version (55K):
[in this window]
[in a new window]
|
Figure 8. The SAS Site Is Not Required for Sp1 and Sp3
Binding to the Sp1c Site
Shown are the results of a mobility shift assay using a fragment
corresponding to nt -165/-131 of the rLHR gene as the labeled probe.
Results of the labeled probe incubated with 2 mg of nuclear extracts
isolated from 8-Br-cAMP-induced rat granulosa cells are shown in
lane 4. Lanes 13 show the results when antibodies to Sp1 binding
proteins were added to the reaction mixture. Lanes 68 show the
results when competing oligonucleotides were added at a 70-fold molar
excess. The competing oligonucleotides correspond to nt -165/-131
corresponding either to the wild-type sequence or the Sp1c(mt1),
SAS(mt1), or SAS(mt2) mutations as described in Fig. 4 . Note that the
optimal conditions for observing Sp1/Sp3-containing complexes are
different than those for detecting complex A and are described in
Materials and Methods.
|
|
Conversely, we also examined whether or not the SAS site is
required for Sp1 and Sp3 binding to the Sp1c site. To optimally
visualize the binding of Sp1 and Sp3 to the Sp1c site, it was necessary
to perform EMSAs using a smaller labeled fragment, -156/-131. Also,
as noted before, EMSA conditions different than those used to visualize
complex A are required to detect Sp1 and Sp3 proteins binding to the
Sp1 sites. Under these EMSA conditions, complex A is not detected. As
we have previously shown (17) and is shown herein in Fig. 8
, two
complexes are discerned binding to the Sp1c site. The upper complex I
is composed of both Sp1 and Sp3 as its intensity is decreased by the
addition of either anti-Sp1 or anti-Sp3, and the lower complex II is
composed of Sp3 as its intensity is decreased by anti-Sp3. The
simultaneous addition of antibodies to Sp1 and Sp3 prevented the
formation of both complexes I and II. We then examined the ability of
unlabeled oligonucleotides corresponding to either the wild-type
-156/-131 or those containing mutations of the Sp1c or SAS site (as
shown in Fig. 4
) to compete for complex I and II formation. Whereas the
wild-type fragment, SAS(mt1), and SAS(mt2) competed for complexes I and
II, the Sp1c(mt) fragment was ineffective in competing. These data
demonstrate that the residues mutated in the Sp1c(mt) are sufficient to
disrupt Sp1 and Sp3 binding to the Sp1c site. In contrast, mutations of
the SAS site had no effect on competition for binding of Sp1 or Sp3
to the probe. These data demonstrate that the mt1 and mt2
mutations of the SAS site do not disrupt the Sp1c site.
Taken altogether, our data support the conclusion that the
mutations chosen to disrupt the SAS vs. the Sp1c
sites are indeed site-selective. Furthermore, the cAMP-dependent
complex A, which arises from the binding of nuclear proteins to the SAS
site, does not require Sp1 or Sp3 binding. Thus, although the SAS and
Sp1c sites overlap, they appear to behave independently.
The SAS Site Is Required for cAMP-Induced Transcription of the rLHR
Gene
The data thus far have identified a cis-element in the
5'-flanking region of the rLHR, termed SAS, which binds a granulosa
cell nuclear protein(s) giving rise to a complex termed complex A. The
observation that the abundance of complex A increases when the
granulosa cells have been exposed to 8-Br-cAMP suggests that the SAS
site may be involved in the cAMP-mediated induction of the rLHR gene.
To address this question, rat granulosa cells were transiently
transfected with luciferase reported gene constructs driven by the
wild-type 5'-flanking rLHR gene sequence or by sequences
containing SAS(mt1), SAS(mt2), or Sp1c(mt1) mutations. As shown in Fig. 9
, mutation of the Sp1c site inhibits
both basal as well as cAMP-mediated transcription of the rLHR gene.
Although the SAS(mt1) mutation inhibited basal transcription slightly,
the larger SAS(mt2) mutation had no effect on basal activity. Both the
mt1 and mt2 mutations of SAS, however, markedly reduced
8-Br-cAMP-induced transcription of the rLHR gene. These data clearly
demonstrate that mutation of the SAS site significantly attenuates the
cAMP-mediated induction of rLHR gene transcription. These results show
that the SAS site is required, but not necessarily sufficient, for the
cAMP-mediated increase in rLHR gene transcription.

View larger version (25K):
[in this window]
[in a new window]
|
Figure 9. The Effects of the Sp1c and SAS Sites on Promoter
Activity of the rLHR Gene in Rat Granulosa Cells
Rat granulosa cells were transfected with luciferase reporter gene
constructs containing either nt -2056/-1 of the rLHR gene (2056(wt)),
or that region of the rLHR gene containing mutations within the Sp1c
site or the SAS site. The mutations corresponding to Sp1c(mt1),
SAS(mt1), and SAS(mt2) are described in Fig. 4 . The cells were
incubated without (C) or with (I) 8-Br-cAMP and then assayed for
luciferase activity. Results shown are the mean ± SEM
of three independent experiments.
|
|
 |
DISCUSSION
|
---|
Previous studies from this laboratory have suggested that
numerous cis elements within the 5'-flanking region of the
rLHR gene contribute to the cAMP responsiveness of the gene (17). Two
lines of evidence support this conclusion. First, when granulosa cells
were transfected with luciferase reporter genes driven by increasing
lengths (up to 2.1 kb) of the 5'-flanking region of the rLHR gene, a
gradual increase in cAMP responsiveness was observed (17). Second, in
EMSAs utilizing labeled fragments corresponding to overlapping portions
spanning nt -2 to -2056 of the rLHR gene, numerous complexes were of
greater intensity when nuclear extracts from 8-Br-cAMP-induced
granulosa cells were used as compared with extracts from noninduced
cells (17). These observations suggest that there are likely to be
numerous transcription factors binding to several
cis-elements within the 5'-flanking region of the rLHR gene
that act in a combinatorial manner to elicit the cAMP-mediated increase
in rLHR gene transcription in granulosa cells.
To begin to dissect this potentially complex system, we focused in the
present study on one particular EMSA complex whose formation was
increased when using nuclear extracts from 8-Br-cAMP-treated granulosa
cells.2 This complex (which
we have designated complex A) was initially observed in EMSAs using a
labeled probe corresponding to nt -187/-2 of the rLHR gene. As
presented herein, the core sequence required for protein A complex is a
string of five Gs at positions -146/-142. Thus, in EMSAs using
nt -187/-2 as the labeled probe and competing with unlabeled
fragments corresponding to the wild-type sequence of nt -149/-137 or
mutations thereof containing only one substitution at a time it was
found that mutations of any of these five guanine nucleotides abolished
the ability of the competing fragment to inhibit complex A formation.
Interestingly, nt -146/-142 are situated overlapping with and
extending upstream of one of three Sp1 sites (designated Sp1c) in the
5'-flanking region of the rLHR gene (see Fig. 4
). Accordingly, we have
named the -146/-142 core sequence which forms complex A the SAS site
(Sp1c adjacent site). By performing competitive EMSAs using competing
oligonucleotides in which each of the five Gs in the core SAS site
was mutated to A, T, or C, it was determined that the core consensus
sequence for complex A formation is (G/T)GGGG. However, other
experiments suggest that additional nucleotides upstream of the core
SAS site may also be involved in complex A formation (cf. Figure 6
).
Presumably, they may be interacting with lower affinity as only
disruption of the aggregate, rather than the individual, nucleotides
suggests their role in complex A formation. Although the SAS site
overlaps the Sp1c site, SAS- vs. Sp1c-specific mutations
show that complex A formation on EMSAs is dependent only upon the
integrity of the SAS site. Furthermore, antibodies to Sp1 and Sp3 do
not affect complex A formation, further suggesting that Sp1c-binding
proteins are not required for complex A formation (cf. Figs. 6
and 7
).
It is not uncommon for Sp1 sites to overlap other
cis-elements. Whereas in some cases a concerted action
between the two sites is required for gene regulation (25, 26, 27), this is
not always the case. For example, the binding of BGP1 to a G-rich DNA
cis-element in the ß-globin gene promoter does not require
Sp1 binding to the overlapping Sp1 cis-element (20).
Interestingly, there are several other transcription factor-binding
elements which, like the SAS site, are known to be G-rich. These
include the cis-elements which bind the G-string-related
transcription factors G-string (18), G-fer (19), BGP1 (20, 21), and
IF-1 (22, 23), as well as Zif268 (24). These transcription factors have
been shown to regulate genes in different ways. For example, G-string
positively regulates the LpS1ß gene in Lytechinus pictus
embryos (18), G-fer inhibits the expression of human
H-ferritin-encoding gene (19), BGP1 appears to destabilize or exclude a
positioned nucleosome, allowing access of transcription factors to
their cognate sites (20, 21), IF-1 negatively regulates the mouse
1(I) and
2(I) collagen genes (22, 23), and Zif268 is thought to
modulate the transcription of genes regulated by growth factors (24).
It was of interest, therefore, to determine whether the transcription
factor binding the SAS site was related to any of these previously
described factors. To address this, we asked whether oligonucleotides
corresponding to the binding sequences of these transcription factors
could compete for complex A formation in EMSAs. Whereas binding
sequences for G-string, G-fer, and IF-1 appear to specifically inhibit
complex A formation, Zif228 does not, and the results for BGP1 are
ambiguous. Taken altogether, our data suggest that the SAS-binding
protein may be related to the G-string family of transcription factors.
A more definitive assessment cannot be made until the cDNA encoding the
transcription factor binding to the SAS site is cloned.
Two of the most widely characterized cAMP-responsive elements are
the cAMP-responsive element (CRE) (28) and AP2 (29). Recently, it has
been shown that some other elements, for example SF1 (30, 31, 32) and
Sp1/Sp3 (26, 27, 33, 34), can also mediate cAMP effects on gene
expression. Although the consensus binding sequence for the classical
CRE is not present in the 5'-flanking region of rLHR gene (16),
reporter gene assays using 5'-deletion constructs of nt -2100/-1 have
suggested numerous cAMP-responsive elements within this region (17).
Recent studies from our laboratory have shown that the three Sp1/Sp3
binding sites in the 5'-flanking region of the rLHR gene contribute
both to the basal and the cAMP-stimulated expression of this gene (17).
It should be noted that the earlier studies were performed before the
identification of the SAS site, which is also involved in the cAMP
responsiveness of the rLHR gene. Unfortunately, the Sp1c site in the
prior study was mutated in such a way that both the Sp1c and SAS sites
would be predicted to be disrupted. However, as shown herein, a more
selective mutation of the Sp1c site still results in decreased basal
and cAMP-inducible transcriptional activity. Therefore, the conclusions
regarding the functional role of the three Sp1 sites remain valid.
Although the rLHR 5'-flanking sequence also contains an AP2 site and
two SF1 sites, these do not appear to be involved in the cAMP-induced
transcription of this gene in rat granulosa cells (17). The studies
presented herein show that the novel SAS site is functionally important
for the cAMP inducibility of the rLHR gene since disruption of this
site attenuates the ability of 8-Br-cAMP to stimulate transcription of
a reporter gene (cf. Fig. 9
). Experiments performed thus far testing
the ability of the SAS element to confer cAMP responsiveness to a
heterologous promoter have been negative. These negative results may
reflect that the experiment has not been performed optimally. However,
they may also result if the SAS site is necessary, but not sufficient,
for cAMP inducibility. Such a conclusion would be consistent with the
observation that the three Sp1 sites in the 5'-flanking region also
mediate cAMP induction of the rLHR gene (17) and the data suggestive of
several sites throughout the 5'-flanking region acting in a
combinatorial manner to allow for cAMP induction of the rLHR gene
(17).
Using reporter gene assays, we have shown that the selective mutation
of the SAS site has a marked inhibitory effect on the cAMP induction of
rLHR gene transcription. Given that cAMP increases the abundance of
complex A formation at the SAS site, it is reasonable to postulate that
the increase in complex A formation plays a role in the cAMP-mediated
transcription of the rLHR gene, most likely in concert with other
factors bound to other cis-elements as well. The effects of
complex A on rLHR gene transcription may be affected by cAMP by one or
more of several mechanisms. Thus, it is possible that cAMP treatment
increases the steady state levels of the transcription factor binding
to the SAS site. Alternatively, or additionally, phosphorylation of the
transcription factor by activation of protein kinase A may increase its
association with the SAS element (35). These are clearly important
issues that will need to be addressed in future experiments but will
first require the cloning of the factor(s) binding to the SAS site in
the rLHR gene.
 |
MATERIALS AND METHODS
|
---|
Reagents
DMEM, Hams F-12 nutrient mixture, Waymouths MB 752/1 medium,
and oligonucleotides were obtained from Life Technologies, Inc. (Gaithersburg, MD). 8-Br-cAMP, DES, BSA, and corticosterone
were purchased from Sigma (St. Louis, MO). ITS was
purchased from Collaborative Research Inc. (Bedford, MA).
Restriction endonucleases MscI, NcoI,
BstNI, and BstXI, and T4 polynucleotide kinase
were obtained from New England Biolabs, Inc. (Beverly,
MA). [32P]ATP was purchased from NEN Life Science Products (Boston, MA). Poly(dI-dC) was obtained from
Pharmacia Biotech (Piscataway, NJ). Luciferin was from
Analytical Luminescence Laboratory (Ann Arbor, MI).
Antibodies to Sp1 and Sp3 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Primary Cultures of Granulosa Cells
Immature Sprague Dawley female rats (2021 days of age, from
Harlan Sprague, Indianapolis, IN) were primed subcutaneously with
Silastic capsules (18 mm) containing 25 mg DES (a nonsteroidal
estrogen) for 5 days. The ovaries were then excised and the granulosa
cells were released as previously described (15). The granulosa cells
were plated on fibronectin-coated tissue culture plates and cultured in
growth medium (DMEM/Hams F-12 (1:1) supplemented with 10
mM HEPES, pH 7.4, 50 mg/ml gentamycin, 0.1% BSA, 1 mg/ml
ITS, and 18 ng/ml corticosterone) at 37 C in a 5%
CO2 incubator.
Nuclear Extracts and Gel Mobility Shift Assays
Granulosa cells released from the ovaries of DES-pretreated rats
were plated on 60 mm fibronectin-coated plates at a cell density of
4 x 106 cells per dish and incubated with or without 3
mM 8-Br-cAMP for 40 h. The nuclear extracts were then
prepared as previously described (36). The protein concentrations
were determined by Bradford assay (37). Typically, the yield was
approximately 612 mg nuclear protein/dish.
For EMSAs examining the formation of complex A, the following
conditions were used. Nuclear proteins (2 mg) were incubated 60 min on
ice with 10 fmol (
10,000 cpm) of a 32P-labeled
probe corresponding to nt -187/-2 of the rLHR in a total volume of 25
ml of binding buffer (10 mM Tris, pH 7.6, 1 mM
EDTA, 1 mM dithiothreitol, 10% glycerol, 100
mM KCl) also containing 35 mg poly (dI-dC). For
competition experiments, unlabeled competitor DNA was added
simultaneously with the labeled fragments at molar excess ratios as
indicated. The resulting DNA-protein complexes were resolved by
electrophoresis on 5% polyacrylamide gels run in 20 mM
Tris-acetate, 1 mM EDTA, pH 8.0, for approximately 2 h
at 180 V. Under these conditions, complex A can be observed in the
lower portion of the gel. These conditions, however, do not permit
detection of Sp1 and/or Sp3 binding to the Sp1 sites.
For optimal visualization of Sp1 and Sp3 binding to the Sp1c site,
different EMSA conditions were used. In this case the labeled probe
corresponded to nt -156/-131. For competition experiments, unlabeled
competitor DNA was added simultaneously with the labeled fragments at
molar excess ratios as indicated. The resulting DNA-protein complexes
were resolved by electrophoresis on 5% polyacrylamide gels run in 25
mM Tris, 190 mM glycine, 1 mM EDTA,
pH 8.4 for approximately 4 h at 165 V. EMSAs run under these
conditions permit detection of the complex formed between the Sp1c site
with Sp1 and/or Sp3, but not complex A formation.
Gels were either developed by autoradiography or exposed to a Cyclone
storage phosphor screen (Packard Instrument Co., Meriden, CT) for
4 h at room temperature. The screen was scanned by Scan control
software and quantified using OptiQuant software (Packard Instrument
Company, Meriden, CT).
DNA Reporter Gene Constructs and Mutagenesis
Because the rLHR gene contains multiple transcriptional start
sites, the numbering of the 5'-flanking region of the gene is based
relative to the translation initiation codon (16). The rLHR-2056-luc
construct, which contains the luciferase reporter gene and the fragment
from -2056 to -2 of the 5'-flanking region of rLHR gene was a gift
from Dr. Mario Ascoli (University of Iowa, Iowa City, IA). The
promoterless pLLV3-luciferase plasmid was originally provided by Dr.
Richard Maurer (Oregon Health Science University, Portland, OR).
Mutants were generated by the PCR using the overlap extension method
(38, 39). The template (fragment -187/-2) for mutagenesis of the Sp1c
site and the SAS site was created from the LHR-2056-luc construct by
digestion with NcoI and MscI. The Sp1c and SAS
mutations are shown in Fig. 4
. The entire PCR region for each mutant
was verified by sequence analysis (40).
Transient Cell Transfection and Luciferase Assays
Granulosa cells were plated in 60-mm dishes at 4 x
106 cells per dish for 16 h and then were
transiently transfected by the calcium-phosphate precipitation method
(41) using 24 µg of plasmid/60-mm dish. After 4 h of exposure to
the DNA precipitation, the cells were washed three times with
Waymouths MB752/1 medium supplemented with 0.1% BSA, refed with
fresh growth medium, and cultured in the absence or presence of 3
mM 8-Br-cAMP. In earlier experiments it was determined that
maximal cAMP-inducible luciferase activity was observed 4048 h after
transfection. For the experiments shown, cells were lysed 40 h
after transfection with 250 µl of lysis buffer (25 mM
glycylglycine hydrochloride, 15 mM
MgSO4, 4 mM EGTA, 1 mM
dithiothreitol, 0.5% NP-40) for 15 min at room temperature. The cell
debris was removed by brief centrifugation, and the supernatant was
transferred to a clean tube. The luciferase assay was performed as
described (42). Protein concentrations of the lysates were determined
by the Bradford assay (37). The data for luciferase assays were
normalized and reported as light units per mg protein. Earlier studies
had shown that similar results were obtained regardless of whether or
not the data were further standardized to ß-galactosidase activity as
determined by cotransfection with the cDNA for ß-galactosidase.
Therefore, in the experiments shown this was not performed.
Data Presentation
The gel shift assays and competition assays were repeated at
least three times with different preparations of nuclear extracts, and
the representative experiments are included shown.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Deborah L. Segaloff, Ph.D., Department of Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52242. E-mail:
deborah-segaloff{at}uiowa.edu
These studies were supported by NIH Grants HD-22196 and HD-33931 (to
D.L.S.). While these studies were in progress, D.L.S. was a recipient
of a Research Career Development Award HD-00968. The services and
facilities of the University of Diabetes and Endocrinology Research
Center, supported by DK-25295, are also acknowledged.
1 S.C. and X.L. contributed equally to this study. 
2 The detection of complex A in uninduced
granulosa cells may at first seem contradictory to the observations
that under these conditions neither rLHR mRNA nor rLHR binding activity
is observed (10 15 ). However, uninduced granulosa cells do exhibit a
detectable level of basal rLHR gene transcription, as measured by
nuclear run-on assays (15 ). 
Received for publication September 1, 1999.
Accepted for publication May 18, 2000.
 |
REFERENCES
|
---|
-
Channing C 1970 Influences of the in vivo and
in vitro hormonal environment upon luteinization of
granulosa cells in tissue culture. Recent Prog Horm Res 26:589622[Medline]
-
Hsueh AJW, Adashi EY, Jones PBC, Welsh Jr TH 1984 Hormonal
regulation of the differentiation of cultured ovarian granulosa cells.
Endocr Rev 5:76127[Medline]
-
Richards JS 1980 Maturation of ovarian follicles: actions and
interactions of pituitary and ovarian hormones on follicular cell
development. Physiol Rev 60:5189[Free Full Text]
-
Richards JS, Jahnsen T, Hedin L, Lifka J, Ratoosh SL, Durica
JM, Goldring NB 1987 Ovarian follicular development: from physiology to
molecular biology. Recent Prog Horm Res 43:231276[Medline]
-
Uilenbroek JTJ, Richards JS 1979 Ovarian follicular
development during the rat estrous cycle: gonadotropin receptors and
follicular responsiveness. Biol Reprod 20:11591165[Medline]
-
Zeleznik AJ, Midgley Jr AR, Reichert Jr LE 1974 Granulosa
cell maturation in the rat: Increased binding of human chorionic
gonadotropin following treatment with follicle-stimulating hormone
in vivo. Endocrinology 95:818825[Medline]
-
Erickson GF, Wang C, Hsueh AJW 1979 FSH induction of
functional LH receptors in granulosa cells cultured in a chemically
defined medium. Nature 279:336338[Medline]
-
Richards JS, Ireland JJ, Rao MC, Bernath GA, Midgley Jr AR 1976 Ovarian follicular development in the rat: Hormone receptor
regulation by estradiol, follicle stimulating hormone and luteinizing
hormone. Endocrinology 99:15621570[Abstract]
-
Richards JS, Kersey KA 1989 Changes in theca and granulosa
cell function in antral follicles developing during pregnancy in the
rat: gonadotropin receptors, cyclic AMP, and estradiol-17ß. Biol
Reprod 21:11851201[Medline]
-
Segaloff DL, Wang H, Richards JS 1990 Hormonal regulation of
LH/CG receptor mRNA in rat ovarian cells during follicular development
and luteinization. Mol Endocrinol 4:18561865[Abstract]
-
Nimrod A 1981 The induction of ovarian LH-receptors by FSH is
mediated by cyclic AMP. FEBS Lett 131:3133[CrossRef][Medline]
-
Erickson GF, Wang C, Casper R, Mattson G, Hofeditz C 1982 Studies on the mechanism of LH receptor control by FSH. Mol Cell
Endocrinol 27:1730[CrossRef][Medline]
-
Segaloff DL, Limbird LE 1983 Luteinizing hormone receptor
appearance in cultured porcine granulosa cells requires continual
presence of follicle-stimulating hormone. Proc Natl Acad Sci USA 80:56315635[Abstract]
-
Knecht M, Catt KJ 1982 Induction of luteinizing hormone
receptors by adenosine 3',5'-monophosphate in cultured granulosa cells.
Endocrinology 111:11921200[Medline]
-
Shi H, Segaloff DL 1995 A role for increased
lutropin/choriogonadotropin receptor (LHR) gene transcription in the
follitropin-stimulated induction of the LHR in granulosa cells. Mol
Endocrinol 9:734744[Abstract]
-
Wang H, Nelson S, Ascoli M, Segaloff D 1992 The
5'-flanking region of the rat luteinizing hormone/chorionic
gonadotropin receptor gene confers Leydig cell expression and negative
regulation of gene transcription by 3',5'-cyclic adenosine
monophosphate. Mol Endocrinol 6:320326[Abstract]
-
Chen S, Shi H, Liu X, Segaloff DL 1999 Multiple elements and
protein factors coordinate the basal and cAMP-induced transcription of
the lutropin receptor gene in rat granulosa cells. Endocrinology 140:21002109[Abstract/Free Full Text]
-
Xiang M, Lu S, Musso M, Karsenty G, Klein WH 1991 A G-string
positive cis-regulatory element in the LpS1 promoter binds two distinct
nuclear factors distributed non-uniformly in Lytechinus
pictus embryos. Development 113:13451355[Abstract]
-
Barresi R, Sirito M, Karsenty G, Ravazzolo R 1994 A negative
cis-acting G-fer element participates in the regulation of expression
of the human H-ferritin-encoding gene (FERH). Gene 140:195201[CrossRef][Medline]
-
Lewis CD, Clark SP, Felsenfeld G, Gould H 1988 An
erythrocyte-specific protein that binds to the poly(dG) region of the
chicken b-globin gene promoter. Genes Dev 2:863873[Abstract]
-
Clark SP, Lewis CD, Felsenfeld G 1990 Properties of BGP1, a
poly(dG)-binding protein from chicken erythrocytes. Nucleic Acids Res 18:51195126[Abstract]
-
Karsenty G, DE Crombrugghe B 1990 Two different negative and
one positive regulatory factors interact with a short promoter segment
of the a1(I) collagen gene. J Biol Chem 265:99349942[Abstract/Free Full Text]
-
Karsenty G, DE Crombrugghe B 1991 Conservation of regulatory
elements in the promoters of the coordinately expressed a2(I) and a1(I)
collagen genes. Biochem Biophys Res Commun 177:538544[Medline]
-
Christy B, Nathans D 1989 DNA binding site of the growth
factor-inducible protein Zif268. Proc Natl Acad Sci USA 86:87378741[Abstract]
-
Grimaldi P, Piscitelli D, Albanesi C, Blasi F, Geremia R,
Rossi P 1993 Identification of 3',5'-cyclic adenosine
monophosphate-inducible nuclear factors binding to the human urokinase
promoter in mouse Sertoli cells. Mol Endocrinol 7:12171225[Abstract]
-
Schanke JT, Durning M, Johnson KJ, Bennett LK, Golos TG 1998 Sp1/Sp3-binding sites and adjacent elements contribute to basal and
cyclic adenosine 3',5'-monophosphate-stimulated transcriptional
activation of the rhesus growth hormone-variant gene in trophoblasts.
Mol Endocrinol 12:405417[Abstract/Free Full Text]
-
Sanchez H, Yieh L, Osborne TF 1995 Cooperation by sterol
regulatory element-binding protein and Sp1 in sterol regulation of low
density lipoprotein receptor gene. J Biol Chem 270:11611169[Abstract/Free Full Text]
-
Montminy MR, Sevarino KA, Wagner JA, Mandel G, Goodman RH 1986 Identification of a cyclic-AMP-responsive element within the rat
somatostatin gene. Proc Natl Acad Sci USA 83:66826686[Abstract]
-
Imagawa M, Chiu R, Karin M 1987 Transcription factor
AP-2 mediates induction by two different signal -transduction pathways:
protein kinase C and cAMP. Cell 51:251260[Medline]
-
Parissenti AM, Parker KL, Schimmer BP 1993 Identification of
promoter elements in the mouse 21-hydroxylase gene that require a
functional cyclic adenosine 3',5'-monophosphate-dependent protein
kinase. Mol Endocrinol 7:283290[Abstract]
-
Morohashi K, Lida H, Nomura M, Hatano O, Honda S, Tsukiyama T,
Niwa O, Hara T, Takakusu A, Shibata Y, Omura T 1994 Functional
difference between Ad4BP and ELP, and their distributions in
steroidogenic tissues. Mol Endocrinol 8:643653[Abstract]
-
Liu Z, Simpson ER 1997 Steroidogenic factor-1 and Sp1 are
required for regulation of bovineCYP11A gene expression in bovine
luteal cells and adrenal Y1 cells. Mol Endocrinol 11:127137[Abstract/Free Full Text]
-
Kaiser UB, Sabbagh E, Chen MT, Chin WW, Saunders BD 1998 Sp1
binds to the rat luteinizing hormone ß (LHß) gene promoter and
mediates gonadotropin-releasing hormone-stimulated expression of the
LHß subunit gene. J Biol Chem 273:1294312951[Abstract/Free Full Text]
-
Darrow AL, Rickles RJ, Pecorino LT, Strickland S 1990 Transcription factor Sp1 is important for retinoic acid-induced
expression of the tissue plasminogen activator gene during F9
teratocarcinoma cell differentiation. Mol Cell Biol 10:58835893[Medline]
-
Zhang P, Mellon S 1996 The orphan nuclear receptor
steroidogenic factor-1 regulates the cyclic adenosine
3'-5'-monophosphate-mediated transcriptional activation of rat
cytochrome p450c17(17-
-hydroxylase/c1720 lyase). Mol Endocrinol 10:147158[Abstract]
-
Nelson S, Liu X, Noblett L, Fabritz J, Ascoli M 1994 Characterization of the functional properties and nuclear binding
proteins of the rat luteinizing hormone/chorionic gonadotropin receptor
promoter in Leydig cells. Endocrinology 135:17291739[Abstract]
-
Bradford MM 1976 A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal Biochem 53:304308
-
Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR 1989 Site-directed mutagenesis by overlap extension using the polymerase
chain reaction. Gene 77:5159[CrossRef][Medline]
-
Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR 1989 Engineering hybrid genes without the use of restriction enzymes: gene
splicing by overlap extension. Gene 77:6168[CrossRef][Medline]
-
Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with
chain-terminating inhibitors. Proc Natl Sci USA 74:54635467[Abstract]
-
Sirois J, Richards JS 1993 Transcriptional regulation of the
rat prostaglandin endoperoxide synthase-2 gene in granulosa cells.
J Biol Chem 268:2193121938[Abstract/Free Full Text]
-
Lynch JP, Lala DS, Peluso JJ, Luo W, Parker K, White BA 1993 Steroidogenic factor 1, an orphan nuclear receptor, regulates the
expression of the rat aromatase gene in gonadal tissues. Mol Endocrinol 7:776786[Abstract]