EAR2 and EAR3/COUP-TFI Regulate Transcription of the Rat LH Receptor
Ying Zhang and
Maria L. Dufau
Section on Molecular Endocrinology, Endocrinology and Reproduction
Research Branch, National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, Maryland,
20892
Address all correspondence and requests for reprints to: Dr. Maria L. Dufau, Building 49, Room 6A-36, 49 Covent Drive, MSC 4510, NIH 20892-4510. E-mail: dufau{at}helix.nih.gov
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ABSTRACT
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Our previous studies demonstrated regulation of the human LH
receptor (hLHR) promoter by nuclear orphan receptors EAR2,
EAR3/COUP-TFI (repression), and TR4 (activation) through a
direct-repeat motif (hDR). The current studies investigated the
differential binding of orphan receptors to rat (rLHR) and hLHR
promoters, and their modulation of rLHR gene transcription in rat
granulosa cells. The rLHR DR with one nucleotide difference from hDR at
its core sequence mediated inhibition of the rLHR transcription, to
which EAR2 and EAR3/COUP-TFI but not TR4 bound. The A/C mismatch was
responsible for the lack of TR4 binding and function, but had no effect
on EAR2 and EAR3/COUP-TFI. EAR2 and EAR3/COUP-TF bound to the rLHR DR
with lower affinity than to the hDR, and exhibited lesser inhibitory
capacity. This difference resulted from the lack of a guanine in the
rDR, which is present 3' next to the hDR core. These studies have
identified sequence-specific requirements for the binding of EAR2,
EAR3/COUP-TFI, and TR4 to the DRs that explain their differential
regulation of rat and human LHR genes. In addition, hCG treatment
significantly reduced the inhibition of rLHR gene in granulosa cells
and also decreased EAR2 and EAR3/COUP-TFI protein levels. These results
indicate that hormonally regulated expression of EAR2 and EAR3/COUP-TFI
contributes to gonadotropin-induced derepression of LHR promoter
activity in granulosa cells.
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INTRODUCTION
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THE LH receptor (LHR) is a G
protein-coupled receptor that plays an essential role in gonadal
development and differentiation. It mediates gonadotropin signals and
triggers intracellular responses that participate in the maturation and
function of the gonads as well as the regulation of steroidogenesis and
gametogenesis (1, 2). The LHR expression has also been
identified in several nongonadal tissues, including human nonpregnant
uterus, placenta, fallopian tubes, uterine vessels, umbilical cord,
brain, and lymphocyte, and in rat prostate (reviewed in Ref.
2).
Characterization of the LHR gene promoter regions from different
species has provided insights into transcriptional regulatory
mechanisms of the LHR gene expression (3, 4, 5, 6, 7). Our previous
studies have demonstrated that the minimal promoter domains of the
human (hLHR) and rat LHR genes (rLHR) resides within 5' 180-bp regions
relative to the translation initiation codon (ATG,+1). Both promoters
are TATA-less, which contain multiple transcriptional start sites and
Sp1/Sp3 binding elements of central importance for basal promoter
activity (3, 4, 5, 7). Furthermore, an imperfect DR motif,
which harbors a consensus estrogen response element half-site (EREhs)
and a second degenerated half-site, was identified recently in our
laboratory as a marked inhibitory domain for the human LHR gene
transcription (8). Three nuclear orphan receptors, EAR2,
EAR3/COUP-TFI, and TR4, were subsequently isolated and characterized by
employing yeast one-hybrid screening of a human placenta cDNA library
followed by functional analysis (8). It was shown that
EAR2 and EAR3/COUP-TFI potently repressed the hLHR promoter activity
upon binding to the DR motif, whereas TR4 was a transcriptional
activator through competitive occupancy of the same cognate site. Thus,
the hLHR gene expression could be influenced by the relative
availability of repressors (EAR2 and EAR3/COUP-TFI) and activator (TR4)
at a given physiological state. To address the functional contribution
of these orphan receptors to the LHR gene transcription during gonadal
development, further studies were performed in cultured rat ovarian
granulosa cells, in which LHR expression is induced by the actions of
FSH and E2 and is subsequently up-regulated by LH (9, 10).
This process closely resembles the induction of the LHR gene in human
granulosa cells, which are not readily available for functional
studies. The studies have demonstrated repression of the rLHR gene
transcription by EAR2 and EAR3/COUP-TFI through their binding to an rDR
domain. However, unlike the case for the human gene, no activation by
TR4 was observed. The lack of TR4 binding was attributable to a single
nucleotide difference at the DR core motifs. The lower binding
affinities of EAR2 and EAR3/COUP-TFI for the rLHR promoter were
attributable to species differences in the adjacent 3'-sequences.
Furthermore, our findings have provided evidence for the participation
of gonadotropin in the orphan receptor-modulated transcription of the
rLHR gene in granulosa cells.
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RESULTS
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The Imperfect DR Motif of the rLHR Promoter Is a Binding Site for
EAR2 and EAR3/COUP-TFI, but Not TR4
Figure 1
illustrates the aligned
sequence comparison of the rat and human LHR gene promoter regions, in
which the regulatory cis-elements for the promoter activity
are indicated. It is important to note that the rLHR promoter harbors a
putative direct-repeat (DR) motif 5' to the Sp1/Sp3 activation domains.
This rat gene DR motif (rDR) contains a consensus EREhs
(hs1) and an imperfect second half-site (hs2),
which is highly homologous to its human counterpart (hDR)
but possesses a single nucleotide substitution. In contrast, diverse
sequences have been noticed at the 5' and 3' adjacent regions to the DR
core motifs of the two promoters. Thus, it was necessary to determine
whether the rDR motif can serve as a cognate binding site for the
nuclear orphan receptors EAR2, EAR3/COUP-TFI, and TR4. Incubation of
the rDR probe (Fig. 2E
) with in
vitro translated EAR2 resulted in formation of a single
DNA-protein complex when compared with the control lane in which
unprogrammed rabbit reticulocyte lysate (NP) was used (Fig. 2A
, lanes 1 and 2). The complex was eliminated in the presence of a
100-fold excess of unlabeled wild-type competitor (lane 3), and it was
supershifted by the EAR2 antibody but not affected by normal rabbit IgG
(lanes 6 and 7). To address binding specificity of the receptor for
individual half-sites of the rDR domain, 100-fold excess unlabeled
oligonucleotides with mutated hs1 (m1) or hs2
(m2) were used in competition assays. The results showed
that neither m1 nor m2 abolished the specific EAR2 complex (lanes 4 and
5), indicating that both half-sites were required for the binding
interaction.

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Figure 1. Alignment of Nucleotide Sequences of the Human (H)
and Rat (R) LHR Gene Promoter Regions
The DNA sequences of the hLHR and rLHR promoters are numbered relative
to the translation initiation codon (ATG, +1). In vitro
transcription start sites are indicated with arrows and
functional domains are in bold italics. The DR nuclear
orphan receptor-binding sites are indicated with horizontal
arrows placed under the hs1 (EREhs) and hs2.
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Figure 2. EMSAs of in Vitro Translated Orphan
Receptors Binding to the Wild-Type rDR Domain or the A/C Mutant Probe
Wild-type double-stranded DNA oligomers containing hs1, hs2, and
adjacent sequences of the rLHR promoter (-181 to -158) or the same
sequence with an A/C switch mutation (termed as A/C) were used as
probes in EMSA analyses (see panel E). The wild-type probe was
incubated with unprogrammed rabbit reticulocyte lysate (NP) or
in vitro translated EAR2 (panel A), EAR3/COUP-TFI (panel
B), and TR4 (panel C). Incubation with the A/C mutant probe was only
shown here for TR4 (panel D; for EAR2 and EAR3/COUP-TFI, see Fig. 5 ).
The probes were incubated with the nuclear receptors in the absence
(lanes 1, 2, 8, 9, 15, 16, 22, and 23) or presence of
unlabeled 100-fold excess wild-type (lanes 3, 10, and 17) or mutant
oligomer (lanes 4, 5, 11, 12, 18, 19, 24, 25, and 26), or in the
presence of normal rabbit IgG (N-IgG, lanes 7, 14, 21, and 28), or
specific antibodies against EAR2 (lane 6), EAR3/COUP-TFI (lane 13), and
TR4 (lanes 20 and 27). E, Sequences of oligomers used in EMSAs as probe
or unlabeled competitors corresponding to the wild-type (WT) or hs1
(m1), hs 2 (m2), and A/C mutant DNA.
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Similar results were obtained when in vitro translated
EAR3/COUP-TFI was investigated in EMSA (Fig. 2B
). EAR3/COUP-TFI formed
a specific complex with the rDR probe, in contrast to its absence in
the NP control reaction (lanes 8 and 9). The complex was abolished by
100-fold excess unlabeled wild-type competitor but remained unchanged
in the presence of 100-fold excess hs1 or hs2 mutant oligomers (lanes
10, 11, and 12). The EAR3/COUP-TFI antibody caused complete supershift
of this complex (lane 13), whereas no supershift was observed when
normal rabbit IgG was used (lane 14). Therefore, the binding of the
complex is attributed to the expressed EAR3/COUP-TFI receptor. Taken
together, the results have demonstrated that the imperfect rDR motif of
the rLHR promoter served as a binding site for the nuclear orphan
receptors EAR2 and EAR3/COUP-TFI, and the functional requirement of the
two half-sites for the binding is consistent with the notion that both
orphan receptors bind to an array of DR motif as homodimers
(11, 12, 13, 14, 15).
In contrast, EMSA failed to detect any binding of TR4 for the rDR
probe, even after prolonged exposure time (Fig. 2C
). Based on this
evidence, it was of interest to discern whether the minor sequence
difference within the DR core motifs was the cause of the observed lack
of TR4 binding to the rLHR promoter. A weak TR4 binding was detected in
EMSA (Fig. 2D
, lane 23) when TR4 was incubated with a probe in which
the deviant nucleotide adenine (A) of the rDR was mutated to cytosine
(C) of the hDR, while the rest of the core and adjacent sequences were
left unchanged (Fig. 2E
). This band was supershifted by the TR4
antibody (lanes 27) and eliminated specifically by the 100-fold excess
unlabeled A/C switch oligonucleotide but not by the hs1 or hs2 mutants
(lanes 24, 25, and 26). Moreover, the fact that only partial
TR4 binding was regained when compared with its binding for the hDR
probe (data not shown) indicated that the single nucleotide mismatch
per se could not fully account for the differential binding
of TR4 for the rat and human promoters. This was further supported by
functional analyses in cotransfected CV-1 cells that showed TR4 caused
a moderate activation on the rLHR A/C mutant promoter construct
(activation by 46%, P < 0.05), when compared with its
significant action on the hLHR promoter activity (Fig. 3
). In contrast, no activation by TR4 was
observed for the rLHR wild-type promoter. The observed correlation
between the partially restored TR4 binding and its partially resumed
functional activity upon A/C mutation further indicated that the
adjacent sequences to the DR core motif, which bear no apparent
similarity between the two LHR promoters, could contribute to the
binding selectivity of the orphan receptors.

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Figure 3. Analyses of TR4 Function on Promoter Activities of
the hLHR, rLHR, and rLHR A/C Mutant Constructs
TR4 expression vector was cotransfected in CV-1 cells with the
wild-type hLHR or the wild-type rLHR or rLHR A/C mutant
promoter/luciferase reporter gene construct. Relative promoter
activities are indicated as the percentage of the luciferase activity
in the absence of TR4. Results were normalized by ß-galactosidase
activity and are expressed as mean ± SE of three
independent experiments in triplicate wells for each transfection (*,
P < 0.05).
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Evaluation of Binding Parameters of EAR2 and EAR3/COUP-TFI for
the Rat and Human DR Domains
Scatchard analyses of EMSAs were performed to evaluate binding
affinities of EAR2 and EAR3/COUP-TFI for the two DR motifs (rDR and
hDR). It is shown that EAR2 displayed 2.5-fold higher binding affinity
for the hDR probe than that for the rDR with dissociation constant
(Kd) of 0.91 ± 0.06 nM and of
3.5 ± 0.22 nM, respectively (n = 3,
P < 0.01). However, the binding capacities for the two
sequences were not significantly different (0.073
nM for the hDR vs. 0.068
nM for the rDR, Fig. 4A
, top panel). Similar
results were obtained for EAR3/COUP-TFI, which also displayed preferred
binding to the hDR probe over the rDR probe, with
Kd of 0.8 ± 0.04 nM
and 2.39 ± 0.13 nM, respectively (n =
3, P < 0.01) (Fig. 4A
, bottom panel).
Subsequent binding analyses conducted to compare the binding affinities
of EAR2 and EAR3/COUP-TFI for the wild-type and A/C mutant rDR probes
(see Fig. 2E
) showed that EAR2 displayed comparable binding parameters
for the two sequences (Fig. 5
, left
panel). Also, EAR3/COUP-TFI exhibited similar binding affinities
for these probes (Fig. 5
, right panel). Thus, the switch of
adenine to cytosine did not revert the reduced binding of EAR2 and
EAR3/COUP-TFI for the rLHR promoter. These studies indicate that
sequences adjacent to the DR core motifs influence the binding of EAR2
and EAR3/COUP-TFI for the rat and human DR domains.
Evaluation of Rat and Human Sequences Adjacent to the DR Core
Motifs for Their Contribution to the Differential Binding of EAR2
and EAR3/COUP-TFI
Binding-inhibition studies were performed to characterize the
component(s) of sequences adjacent to the DR core motifs that may
contribute to the differential binding affinities of these orphan
receptors between species. The wild-type rDR and hDR as well as four
rat/human hybrid DR sequences (nos. 14), in which the corresponding
5'- or 3'-adjacent region to either rDR or hDR core motif was
interchanged, were used as unlabeled competitors (Fig. 6C
). The inhibition of binding of
EAR3/COUP-TFI to the rDR probe by the wild-type rDR was similar to that
induced by the hybrid no.1 sequence containing the rDR with a
substituted 5'-adjacent region from the hDR (6A, lanes 113). In
contrast, the hybrid no. 2 (rDR with 3'-hDR) showed a more sensitive
displacement of the EAR3/COUP-TFI binding, indicating that the presence
of the 3'-flanking region of the hDR in the rDR hybrid conferred higher
binding affinity for the EAR3/COUP-TFI (panel A, lanes 1319). The
displacement curve of EAR3/COUP-TFI binding to hDR was unaffected when
using a hybrid hDR sequence (no. 3) that contained the 5'-adjacent
sequence of the rDR as competitor. This sequence competed the binding
with comparable efficiency as the wild-type hDR (panel B, lanes
2030). On the other hand, significantly reduced displacement potency
was observed in the presence of hybrid no. 4 due to the introduction of
the 3'-adjacent sequence of the rDR into the hDR (panel B, lanes
3135). These results demonstrated that the 3'-adjacent sequence of
the hDR domain is relevant to the higher binding affinity of
EAR3/COUP-TFI for the human LHR promoter. When binding of EAR2 for
either rDR or hDR domain were examined, similar findings were obtained.
The results from such analyses for both EAR2 and EAR3/COUP-TFI are
shown as binding-inhibition curves for a direct comparison (Fig. 7
). Overall, the changes in potencies
caused by introduction of the 3'-human sequence to the rDR domain (2.8-
to 3.4-fold) were consistent with the differences observed in EAR2,
EAR3/COUP-TF affinities between species. Taken together, the results
demonstrate that the 3'-adjacent sequences to the DR core motifs play a
critical role in the differential binding of EAR2 and EAR3/COUP-TFI
between species. The 5'- flanking regions of the DRs did not contribute
to the observed difference, although no apparent sequence similarity
was observed within these regions in the rat and human LHR
promoters.

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Figure 6. EMSA of Binding Displacement Analyses of
EAR3/COUP-TFI for the Rat and Human DR Domains
In vitro translated EAR3/COUP-TFI was incubated with
labeled wild-type rat DR (A) and human DR (B) probes, respectively.
Binding of the rat DR probe was performed in the absence (lane 1) or
presence of the indicated fold-excess unlabeled wild-type rDR (WT,
lanes 27) or hybrid rDR sequences 1 (lanes 813) and 2 (lanes
1419). Similarly, binding of the human DR probe was performed in the
absence (lane 20) or presence of the indicated fold-excess of unlabeled
wild-type hDR (WT, lanes 2125) or hybrid hDR sequences 3 (lanes
2630) and 4 (lanes 3135). C, DNA sequences for the wild-type rat
and human DR domains containing the DR core motifs and the adjacent
flanking regions, as well as the hybrid sequences 18 used in the
displacement analyses, are shown. The boxed area shows
the sequence shared by the two DR domains at their 3'-adjacent regions.
The guanine residue (g) 3' next to the DR core in the human but absent
in the rDR is indicated with a bullet symbol. Deletion
or insertion of a g residue in corresponding sequences is indicated as
" g" or "±g." The substitution of GGA with ac is indicated
as " GGA/+ac," while replacement of ac by GGA is stated as
" ac/+GGA." 5'H or 5'R represents the 5'-adjacent sequence of
human or rat DR domain, respectively, and 3'H or 3'R stands for the
respective 3'- flanking region of the human or rat DR domain.
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Figure 7. Contribution of the 3'-Region of the Rat and Human
DR Domains to the Differential Binding of EAR2 and EAR3/COUP-TFI
Binding-inhibition curves of EAR3/COUP-TFI (A and B) or EAR2 (C
and D) for the rat or human DR domain, respectively. The amount of
unlabeled DNA competitors used is indicated as a fold-ratio of
unlabeled/labeled, and the specific binding is expressed as relative
percentage of the binding in the absence of unlabeled DNA. The
displacement curves presented were derived from the EMSAs using labeled
wild-type rDR or hDR probe, and wild-type rDR, hDR, or hybrid sequences
14 as unlabeled competitor DNAs.
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Determination of Nucleotide(s) Responsible for Differential Binding
of EAR2 and EAR3/COUP-TFI to Rat and Human DR Domains
The 3'-adjacent sequences to the rat and human DR core motifs
share five nucleotides within the 8-bp region (Fig. 6
, panel C,
boxed area). A guanine residue located 3' next to the hDR core was
noted to be absent in the corresponding position of the rDR. Moreover,
the last two (ac in the hDR) or three nucleotides
(GGA in the rDR) after their respective 3'-conserved region
are also different. Therefore, it was reasonable to speculate that the
cause of the differential binding of EAR2 and EAR3/COUP-TFI could
result from the presence or absence of one or more such deviant
nucleotides. Deletion of the unique G 3' next to the hDR converted the
highly efficient displacement of EAR3/COUP-TFI binding to the hDR
domain by the wild-type hDR to a less potent one comparable to that
caused by the rDR wild-type sequence (Fig. 8
, panel A, no. 5). Furthermore,
examination of EAR3/COUP-TFI binding to the rDR domain showed that
insertion of a guanine 3' next to the rDR core increased significantly
its displacement potency to that observed with the hDR wild-type
competitor (panel B, no. 7). On the other hand, hybrid hDR or rDR
oligomers (nos. 6 and 8) containing a GGA-for-ac or ac-for-GGA
substitution did not have any effect on the binding. Similar results
were obtained when the binding of EAR2 was studied, showing that
the G residue rather than the last few dissimilar nucleotides at the
far 3'-end of the DR domains was responsible for the change in binding
of EAR2 (panels C and D). The changes in potencies (2.86- to 3.9-fold)
in these experiments were consistent with the differences in the
affinities between species. In summary, the results have demonstrated
that the single guanine residue that is present only in the hDR is
responsible for the higher binding affinities of EAR2 and EAR3/COUP-TFI
for the human LHR gene promoter.

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Figure 8. Identification of Nucleotide(s) Within 3'-DR
Domains That Determines the Differential Bindings of EAR2 and
EAR3/COUP-TFI for the Rat and Human LHR Promoters
Binding curves of EAR3/COUP-TFI (A and B) or EAR2 (C and D) for the
human and rat DR domains. The amount of unlabeled DNA competitors used
is indicated as a fold-ratio of unlabeled/labeled, and the specific
binding is expressed as relative percentage of the binding in the
absence of unlabeled DNA. Displacement curves presented were derived
from the EMSAs using labeled wild-type rDR or hDR probe, and wild-type
rDR, hDR, or hybrid sequences 58 as unlabeled competitor DNAs.
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EAR2 and EAR3/COUP-TFI Are Transcriptional Repressors of the rLHR
Promoter
The specific binding of the nuclear orphan receptors EAR2 and
EAR3/COUP-TFI to the rLHR promoter made it necessary to examine their
potential functions in the regulation of the rLHR gene transcription.
For these studies, the nuclear receptor expression vectors were
cotransfected with the rLHR promoter/luciferase reporter constructs
(wild-type, hs1 mutant or hs2 mutant) in CV-1 cells. It was shown that
EAR2 exhibited dose-dependent inhibition on the wild-type rLHR promoter
activity by up to 55% (Fig. 9A
). Similar
results were obtained with EAR3/COUP-TFI that inhibited the wild-type
promoter activity by 45% (P < 0.05) (Fig. 9A
). It was
noted that the repressive strength of EAR2 or EAR3/COUP-TFI on the rat
gene was lower than their ability to suppress the hLHR gene [70% by
EAR2 and 55% by EAR3/COUP-TFI, respectively (8)]. This
could be explained by the observed lower binding affinities of the two
receptors for the rLHR promoter. In contrast, TR4 did not affect the
wild-type rLHR promoter activity regardless of the dose used (Fig. 9A
).
To verify that the EAR2 and EAR3/COUP-TFI-mediated inhibition of the
rLHR gene requires a functionally intact DR element, the nuclear
receptors were cotransfected with the rLHR hs1 or hs2 mutant
promoter/reporter construct (Fig. 9B
). Mutation at either half-site
(m1 or m2) abolished the inhibitory effect of EAR2 or
EAR3/COUP-TFI, indicating that both half-sites are essential for the
orphan receptors to exert their regulation on the rLHR gene. In
summary, the results have shown that EAR2 and EAR3/COUP-TFI are
transcriptional repressors for the rLHR gene.

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Figure 9. Transcriptional Repression of the rLHR Gene by EAR2
and EAR3/COUP-TFI
Top, The wild-type rLHR promoter/luciferase reporter
gene construct (0.5 µg) was cotransfected in CV-1 cells with
increasing dose (0.2, 0.4, and 0.6 µg) of EAR2, EAR3/COUP-TFI, or TR4
cDNAs. Bottom, Fixed amount of EAR2 or EAR3/COUP-TFI
cDNA (0.6 µg) was cotransfected in CV-1 cells with the wild-type
(WT), or hs1 (m1), hs2 (m2) mutated rLHR
promoter constructs (0.5 µg/each). Relative promoter activities are
indicated as the percentage of the luciferase activity from the
wild-type promoter in the absence of coexpression of nuclear orphan
receptors. Results were normalized with ß-galactosidase activity and
are expressed as mean ± SE of three independent
experiments in triplicate wells for each transfection (*,
P < 0.01).
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Transcriptional Repression of the rLHR Gene in Granulosa
Cells
The repression of the rLHR gene by EAR2 and EAR3/COUP-TFI in
cotransfection studies led us to investigate whether a similar
regulation of the rLHR gene by these orphan receptors could be observed
in a physiologically relevant setting. Primary cultures of rat ovarian
granulosa cells, which express the functional LHR gene, were used for
these studies. Reporter gene analyses were initially carried out to
test whether the rDR domain was a functional cis-element in
these cells. Transfection of the wild-type, or hs1, hs2 mutant rLHR
promoter/luciferase constructs demonstrated that mutation of the hs1
(m1) caused the promoter activity to increase by 80%
compared with that of the wild-type promoter, and a comparable
induction was observed when the hs2 mutant construct (m2)
was analyzed (Fig. 10A
). The results
thus identified the rDR motif as an inhibitory domain for the rLHR
promoter activity in granulosa cells, to which putative repressor
protein(s) could bind. Incubation of granulosa cell nuclear extracts
with the rDR probe in EMSA (see Fig. 2E
) resulted in the formation of
three major DNA-protein complexes (Fig. 10B
, lane 1). These complexes
were competed by 100-fold excess unlabeled wild-type DNA but were not
abolished by either hs1 (m1) or hs2 (m2) mutant
oligonucleotides (lanes 2, 3, and 4). This indicated that the binding
of these DNA-protein complexes was dependent on an intact DR motif
containing both half-sites. The results were in agreement with the
binding specificity data of in vitro translated EAR2 and
EAR3/COUP-TFI (see Fig. 2
). Antibody supershift assays were conducted
to elucidate the identity of these DNA-protein complexes using
antibodies against EAR2, EAR3/COUP-TFI, and TR4. Both EAR2 and
EAR3/COUP-TFI antibodies caused supershift of complex 3, and the
supershifted band comigrated with complex 2, significantly increasing
the intensity of this complex (lanes 5 and 6). Moreover, complex 1 was
almost abolished upon addition of EAR2 antibody (lane 6). The ability
of the EAR2 antibody to recognize two DNA-protein complexes (1, 3)
with different mobility indicated the possibility of recruitment of
additional proteins (e.g. corepressors) to complex 1.
Although the EAR2 antibody was not raised against the DNA binding
domain, it could affect the conformational stability of a
multiple-component complex to favor its dissociation and loss of
binding to the rDR domain. In contrast, the TR4 antibody, like normal
rabbit IgG, did not cause any change in DNA-protein binding (lanes 7
and 8), a result consistent with its absent binding and function for
the rLHR (Figs. 2C
and 3
). In addition, it might not be excluded that
complex 2, of unknown identity, participated in rLHR gene regulation.
Therefore, the results have demonstrated that endogenous EAR2 and
EAR3/COUP-TFI, largely if not exclusively, repressed rLHR gene
transcription in granulosa cells through specific binding to the rDR
element.

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Figure 10. Transcriptional Repression of the rLHR Gene in
Granulosa Cells
A, Reporter gene analyses performed in rat ovarian granulosa cells. The
wild-type 181-bp rLHR promoter/luciferase reporter gene
(WT), and its mutant constructs with mutated hs1
(m1) or hs2 (m2) were transiently
expressed in granulosa cells. The promoter-less vector
(Basic) was used as negative control. Relative promoter
activities are indicated as the percentage of the luciferase
activity of the wild-type promoter. The results were normalized
by the ß-galactosidase activity and are expressed as the mean ±
SE of three independent experiments in triplicate wells for
each construct (*, P < 0.005). B,
EMSA of granulosa cell nuclear proteins binding to the rDR domain of
the rLHR promoter. Double-stranded oligonucleotide probe (rDR domain)
derived from the rLHR promoter was used in EMSA (see Fig. 2E ). The
probe was incubated with granulosa cell nuclear extracts in the absence
(lane 1) or presence of 100-fold excess of unlabeled wild-type
(WT, lane 2) or mutated oligonucleotides (m1,
m2, lanes 3 and 4) or in the presence of normal rabbit IgG
(lane 8) or specific antibodies against EAR2 (lane 5), EAR3/COUP-TFI
(lane 6), and TR4 (lane 7). Specific DNA-protein complexes are
indicated with arrows.
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Derepression of the rLHR Gene Transcription by hCG in Granulosa
Cells
Because of the important involvement of gonadotropin in LHR gene
expression during granulosa cell development and differentiation, we
have expanded the current study to address the potential role of
gonadotropin (e.g. hCG) in EAR2- and
EAR3/COUP-TFI-mediated repression of the rLHR gene transcription in
these cells. Reporter gene assays were carried out and the promoter
activities were analyzed in primary cultures of granulosa cells treated
with or without hCG. In the absence of hormone, mutation of the hs1
site to disrupt the rDR motif resulted in an elevated promoter activity
compared with the wild type (Fig. 11A
, left panel), which was consistent with the previous data
(Fig. 10
). After hCG treatment for 24 h, the promoter activity of
either the wild-type or hs1 mutant was significantly increased. This is
consistent with the notion that both human and rat LHR promoter
activity are activated by hCG/cAMP treatment (Refs. 16 and
17 (rat), and our unpublished data (human)]. Moreover, it
is necessary to note that the mutant promoter still showed a minor
increase in activity over the wild type after hCG incubation (by 12%,
P < 0.05); however, it was much less marked than in
parallel cultures in the absence of hormone (by 84%). In granulosa
cells cultured for 48 h without hCG, a 60% increase in promoter
activity was observed due to the hs1 mutation (Fig. 11A
, right
panel). Treatment with hCG for the same period of time resulted in
induction of activities in both the wild-type and mutant promoters.
However, under this condition the negative regulation of the rLHR gene
through the rDR domain was not present since both the wild-type and hs1
mutant promoter displayed comparable activities. Therefore, the results
have demonstrated that hCG treatment released the inhibition of the
rLHR gene in granulosa cells. Since the rDR motif has been shown to be
specifically bound by endogenous EAR2 and EAR3/COUP-TFI in these cells,
examination of whether the expression of the orphan receptors was
subject to hormonal control could provide meaningful insights into the
mechanism of gonadotropin-mediated derepression of the rLHR gene.
Western blot analyses illustrated that the levels of endogenous EAR2
protein showed a significant decrease in the cells treated by hCG for
24 h when compared with the untreated controls (Fig. 11
, B and C,
left panel). Longer exposure of the cells to hCG for 48
h further decreased the EAR2 signal to 55% of the control. In
contrast, actin levels remained constant during the hCG administration.
Furthermore, 40% reduction of the EAR3/COUP-TFI protein was observed
after 48 h incubation with hCG (Fig. 11
, B and C, right
panel). The reduction was specific since both actin and endogenous
TR4 protein level remained unchanged after hCG treatment (data not
shown). Taken together, the results demonstrated that both EAR2 and
EAR3/COUP-TFI proteins were specifically decreased by hCG treatment in
granulosa cells. The simultaneous reduction of the two proteins could
contribute to the hormone-induced derepression of the rLHR gene
promoter activity in these cells.

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|
Figure 11. Derepression of the Orphan Receptor-Mediated
Inhibition on the rLHR Promoter Activity in Granulosa Cells by hCG
A, Basic (B), wild-type (WT), or hs1 mutant (m1) rLHR promoter/reporter
constructs were transfected into granulosa cells followed by treatment
with/without hCG (final concentration, 0.5 µg/ml) for 24 h
(left) or 48 h (right). Within each
time point group, the relative promoter activities are indicated as the
percentage of luciferase activity of the wild-type promoter at minus
hCG condition. The results were normalized by ß-galactosidase
activity values and are expressed as the mean ± SE of
three independent experiments in triplicate wells for each construct.
B, Western blot analyses of endogenous EAR2 (left) and
EAR3/COUP-TFI (right) proteins extracted from granulosa
cells treated with/without hCG (0.5 µg/ml) for 24 or 48 h. Actin
signals are also shown. C, EAR2 and EAR3/COUP-TFI signals normalized by
actin signals at each time point. Within each experimental group, the
relative intensities of the signals are indicated as percentage of the
receptor/actin ratio at minus hCG condition. Data are expressed as the
mean ± SE of three independent experiments (*,
P < 0.01).
|
|
 |
DISCUSSION
|
---|
These studies have demonstrated that transcription of the rat LHR
gene is modulated by the nuclear orphan receptors, EAR2 and
EAR3/COUP-TFI, but not by TR4. This difference was found to be
attributable to the differential binding characteristics of these
orphan receptors for the DR elements of the rat and human promoters.
The rat DR domain, which bears a single nucleotide mismatch at its core
sequence from its human counterpart and adjacent sequences with low
similarity to the human, was identified as the cognate binding site for
EAR2 and EAR3/COUP-TFI but not for TR4. Moreover, EAR2 and
EAR3/COUP-TFI exhibit significant lower binding affinities for the rat
DR than for the human DR domain. This results solely from a G residue
that is located 3' to the human DR core and is absent in the rat. The
lack of TR4 binding to the rat promoter was attributable to the single
nucleotide difference at the DR core motifs of the genes. Such binding
differences are responsible for the occurrence of an EAR2- and
EAR3/COUP-TIF-mediated repression that is less pronounced than in the
human, and absence of TR4-induced activation of the rLHR promoter.
Furthermore, hCG treatment of granulosa cells released the inhibition
of EAR2 and EAR3/COUP-TFI, an effect that could result from the
significant decrease of EAR2 and EAR3/COUP-TFI proteins observed in the
hormone-treated cells. It is also possible that hCG influences an as
yet unidentified protein that interacts with the orphan receptors to
derepress the LHR promoter.
Partial TR4 binding activity and function were acquired by the rat LHR
promoter construct that contained the A (rat) to C (human) mutation
within its core sequence. However, the TR4 activity/function attained
was about 34% of that elicited on the human LHR promoter. The complete
switch from the absence of TR4 binding for the wild-type rat DR to an
interacting TR4 binding-pair by the single nucleotide switch indicated
that the C nucleotide was directly involved in the TR4-DR interaction,
since the stable TR4 binding complex observed for the hDR was unable to
be formed on the rDR. However, a C (human) to A (rat) substitution did
not cause significant impact on the binding of EAR2 and EAR3/COUP-TFI,
since both receptors displayed comparable binding potency for the
wild-type and A/C mutant rDR domains. This is probably due to the fact
that TR4 shares only 70% and 50% sequence homology with COUP
receptors at the DNA binding and putative ligand domains, respectively,
whereas EAR2 is a subtype of EAR3/COUP-TFI.
Our initial studies indicated that nucleotides(s) of the distinct 5'-
and/or 3'-adjacent sequences to the rDR core element might contribute
to the decreased binding affinities of EAR2 and EAR3/COUP-TFI for the
rLHR promoter. This could be also the case for TR4, binding of which
was not completely restored upon the A-to-C switch. Studies using the
wild-type and hybrid 3'- or 5'-sequences to the core rDR or hDR
identified the 3'-sequence of the rat responsible for the observed
reduced affinity, since comparable affinity to the human was observed
in the rDR hybrid with 3'-human domain sequence. Further studies
identified that the G residue present in the 3'-hDR but not in the
3'-rDR domains was responsible for the higher binding affinities of
EAR2 and EAR3/COUP-TFI to the hLHR gene. However, two dissimilar
nucleotides present in the rat sequence 3' to the rDR and the
5'-flanking regions to the DR domains in both species did not
contribute to the observed binding differences. It is generally
accepted that the binding of a nuclear receptor to a particular target
site is primarily determined by the hormone response element core
sequence composed of hexameric base pairs in single or repeated
configuration; however, recognition that nucleotides flanking the core
motif may have a significant influence in the binding interaction has
emerged. Studies of ER binding to variant ERE has demonstrated that the
affinity of ER binding for a nonconsensus ERE can be greatly altered by
changes in the sequences flanking the core elements, either positively
or negatively (18, 19, 20, 21). Accumulated reports have
demonstrated that specific DNA-protein interactions often result in
conformational change in protein(s), DNA, or both
(22, 23, 24). The lack of the 3'-G next to the rDR core may
cause a conformational change on the EAR2 and EAR3/COUP-TFI that
resulted in a less tight binding of the receptors for the rat LHR gene.
The participation of adjacent sequences in the binding interaction
between the orphan receptors and the LHR DR domains may also explain
that only partial TR4 binding and function was conferred upon the
single base change at the core motif. However, this aspect was not
further pursued due to the lack of function of TR4 on the rat LHR
gene.
Despite their lower binding affinities for the rLHR promoter, EAR2 and
EAR3/COUP-TFI exhibited significant repression of the rLHR gene
transcription. EMSA of granulosa cell nuclear extracts revealed that a
single DNA-protein complex was supershifted by antibodies of EAR2 and
EAR3/COUP-TFI, indicating that the complex probably contains a
heterodimeric form of the two receptors. Recent studies have
demonstrated that members of the COUP-TF family, including
EAR3/COUP-TFI, Arp-1/COUP-TFII, and EAR2, that have been shown to bind
DR element as homodimers, could also form stable heterodimer with each
other at their cognate sites (25). This combinatorial
process was proposed to provide the COUP-TF receptors an additional
mechanism for the fine tuning of target gene expression
(27). Moreover, the observation in our study that EAR2
antibody blocked formation of a DNA-protein complex with distinct
migrating property from the band supershifted by the same antibody (see
also Results and Fig. 10
) indicates that recruitment of
other cofactors (26, 27) via protein-protein interaction
may participate in the regulation of the rLHR gene transcription. EAR2-
and EAR3/COUP-TFI-mediated repression of target genes could be enhanced
through their interaction with two nuclear corepressors, nuclear
receptor corepressor and silencing mediator of retinoid acid and
thyroid hormone receptor (28, 29) as well as some other
corepressor proteins (30). The receptors can also interact
with basal transcriptional machinery components such as TFIIB
(31). Furthermore, direct interaction between
EAR3/COUP-TFI and Sp1 has been shown to potentiate activation of some
target genes (32, 33). Thus, it is conceivable that these
orphan receptors may perturb Sp1 function through protein-protein
interaction to induce repression of the LHR gene. Therefore, repression
of the rLHR promoter activity by EAR2 and EAR3/COUP-TFI may be achieved
through complex protein-protein interactions involving repressors,
coregulators, basal transcription factors, or Sp1.
Gonadotropin plays an obligatory function in ovarian follicle
maturation and function and significantly activates LHR gene expression
in granulosa cells. Our studies in primary granulosa cells have
revealed a role of hCG in reducing the expression of EAR2 and
EAR3/COUP-TFI with consequent enhancement of LHR promoter activity. The
abolition of the orphan receptor-mediated inhibition of the rLHR upon
hCG treatment via derepression may contribute to the elevated LHR
expression required for progression of granulosa cell maturation. These
observations have provided insights into hormonal regulation of the
orphan receptor-mediated repression of the rLHR promoter activity in
cultured ovarian granulosa cells.
 |
MATERIALS AND METHODS
|
---|
Animals
Immature (24-d-old) female rats (Charles River Laboratories, Inc., Wilmington, MA) were injected sc daily for
3 d with 1.5 mg/day of 17ß-E2 (dissolved in propylene glycol)
(Sigma, St. Louis, MO). On the fourth day, the animals
were killed (CO2 asphyxiation), and ovaries were
removed for preparation of ovarian granulosa cells. The study was
approved from the NICHD Animal Care and Use Committee (protocol
97049).
Cultures of Cell Lines and Primary Rat Ovarian Granulosa
Cells
CV-1 cells (African green monkey kidney cells, American Type Culture Collection, Manassas, VA) were maintained in MEM
with 10% FBS and L-glutamine (Life Technologies, Inc. Inc., Gaithersburg, MD). Rat ovarian granulosa cells were
prepared from E-primed rat ovaries as described previously
(34). The isolated granulosa cells were suspended in
DMEM/F12 (Life Technologies, Inc.) supplemented with 1%
FBS, and with 15 ng/ml of ovine FSH (oFSH 20, National Pituitary
Program, NIDDK) and 10 ng/ml of testosterone (Sigma). The
cells were plated at a density of 1.5 x
106/well in six-well plates and cultured in a
CO2 incubator for 48 h before transfection
or nuclear protein extraction was carried out.
Reporter Gene Constructs and Expression Vectors
All plasmids were constructed by standard recombinant DNA
techniques. A MluI/BglII fragment harboring the
rLHR gene promoter region (-181 to +1) was cloned into promoterless
pGL2 basic vector (Promega Corp., Madison, WI) upstream of
luciferase reporter gene. Mutant constructs generated by PCR were
designed to mutate the DR motif sequences of the rLHR (hs1 and hs2)
with putative orphan receptor binding activity, or to substitute the
dissimilar nucleotide in the rLHR hs2 for the one present in the human
sequence (A/C). All mutants were verified by sequencing analyses.
Construction of expression vectors for the nuclear orphan receptors was
described previously (8). Briefly, the full-length cDNAs
for hEAR2 (1.2 kb), hEAR3/COUP-TFI (1.27 kb), and hTR4 (1.79 kb) were
cloned into pcDNA3.1 vector (Invitrogen, Carlsbad, CA),
respectively. The fidelity of the clones was verified by sequencing
analyses using Thermo Sequenase Radiolabeled Terminator Cycle
Sequencing kit (United States Biochemical Corp.,
Cleveland, OH).
In Vitro Transcription and Translation of Nuclear
Orphan Receptors
T7 promoter upstream of the cloned genes in pcDNA3.1 constructs
was used to synthesize the nuclear orphan receptor proteins in an
in vitro coupled transcription and translation system (TNT,
Promega Corp.). The reaction was carried out in the
presence or absence of [35S]-methionine
(Amersham Pharmacia Biotech, Piscataway, NJ). The
molecular weights of the in vitro translated products were
confirmed by electrophoresis/autoradiography.
Nuclear Extract Preparation and EMSA
Nuclear extracts from hCG hormone-treated or vehicle-treated
(PBS) granulosa cells were prepared using NE-PER nuclear and
cytoplasmic extraction reagents (Pierce Chemical Co.,
Rockford, IL) according to the protocols recommended by the
manufacturer. EMSAs were performed as described previously
(8). Briefly, [
-32P]ATP
end-labeled oligonucleotide probe (510 x
104 cpm) was incubated with 15 µl of EAR2,
EAR3/COUP-TFI, or TR4 cDNA programmed rabbit reticulocyte lysate or 5
µg of granulosa cell nuclear proteins in a 20-µl binding reaction
on ice for 15 min. As a control, the probe was also incubated with the
same amount of unprogrammed TNT lysate. In the competition and
displacement assays, indicated doses of excess unlabeled DNA oligomers
were added to the reaction 15 min before addition of the probe. In
supershift assays, specific antibodies or normal rabbit IgG
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were
preincubated with proteins for 3060 min at 4 C before the probe was
added. DNA-protein complexes were resolved by electrophoresis on 5%
native polyacrylamide gel.
Specific rabbit polyclonal antibodies against peptides from EAR2,
EAR3/COUP-TFI, and TR4 were generated and purified as previously
described (8).
Western Blot
The nuclear proteins (30 µg/lane) isolated at 24-h or 48-h
time points from the hCG treated- (0.5 µg/ml) or vehicle-treated
granulosa cells were separated on 12% polyacrylamide gels and
transferred to nitrocellulose membranes. The membranes were first
incubated with specific rabbit polyclonal antibodies against EAR2,
EAR3/COUP-TFI, or TR4 followed by incubation with goat horseradish
peroxidase-conjugated antirabbit IgG second antibody (Life Technologies, Inc.). The immunosignals of relevant orphan
receptors were detected using SuperSignal West Pico Chemiluminescent
Substrate (Pierce Chemical Co.). The same blots were then
used to detect actin signals with a mouse monoclonal antiactin antibody
(Chemicon International, Temecula, CA) after the primary orphan
receptor antibody was stripped off using Western Re-Probe Solution
(Geno Technology Inc., St. Louis, MO). The second goat horseradish
peroxidase antimouse IgG antibody was also purchased from Life Technologies, Inc. The signal intensity was determined by
scanning densitometry, and the specific signals of the nuclear orphan
receptors were normalized by the corresponding actin signals.
Binding Studies of Nuclear Orphan Receptors for the rDR or hDR
DNA Domains
EMSAs were used to evaluate the binding parameters of EAR2 and
EAR3/COUP-TF for either the wild-type or A/C mutant DR elements. For
these studies, constant amounts of in vitro translated
nuclear orphan receptors (1 µl) were incubated with different doses
of 32P-labeled oligonucleotide probe (0.14 ng).
For binding displacement analyses, a constant amount of the orphan
receptor (2 or 5 µl) and fixed amounts of the labeled probe were used
in the binding reaction. An increasing dose of unlabeled competitor DNA
was incubated with the protein for 15 min before addition of the probe.
The DNA sequences for the wild-type rDR or hDR domains containing the
DR core motifs and the adjacent regions, as well as the sequences for
the mutant A/C probe or the eight hybrid competitors used in the
displacement assays, are shown in Figs. 2
and 6
. DNA-protein binding
complexes were resolved by EMSA. Both specific and total binding was
quantified using PhosphorImager (Molecular Dynamics, Inc.,
Sunnyvale, CA). Experiments were performed three times, and the binding
parameters were determined by Scatchard analysis using the Ligand
Program issued from NIH (35); statistical comparisons were
made by t test.
Transient Transfection
LipofectAMINE Plus reagents (Life Technologies, Inc.) were used to transfect both CV-1 cells and primary
cultures of rat ovarian granulosa cells. For CV-1 cells, 2 x
105 cells per well were seeded on 6-well culture
plates 24 h before transfection. The culture medium was replaced
before transfection with 1 ml of MEM without serum and antibiotics.
CV-1 cells were transfected with 0.5 µg of the rLHR
promoter/luciferase construct and different doses (0.2, 0.4, and 0.6
µg) of the orphan receptor expression vectors. pCMV·SPORT-ß gal
reference plasmid DNA (0.3 µg; Life Technologies, Inc.)
was also cotransfected as an internal control to normalize the
transfection efficiency. The final DNA concentration was adjusted using
empty vector DNA. Cells were replaced 3 h later with normal
culture medium and collected 40 h after transfection. For primary
cultures of granulosa cells, 5 µg of reporter gene construct and 0.5
µg of pCMV·SPORT-ß gal reference plasmid were used for each
individual transfection. After 5 h incubation, the medium was
replaced with normal culture medium supplemented with or without 0.5
µg/ml hCG, and the cells were cultured for another 24 or 48 h
before termination. The cell lysates were extracted, and the luciferase
activity was measured by luminometry and normalized by
ß-galactosidase activity. All the experiments were performed at least
three times in triplicate. Statistical significance was evaluated by
variance test analysis with computer programs Statview (Abacus
Concepts, Berkeley, CA) and Superanova (Abacus Concept).
 |
ACKNOWLEDGMENTS
|
---|
Acknowledgements
 |
FOOTNOTES
|
---|
Abbreviations: COUF-TF, Chicken ovalbumin-transcription factor;
EREhs, estrogen response element half-site; hDR, human direct repeat;
hLHR and rLHR, human and rat LH receptors.
Received for publication June 7, 2001.
Accepted for publication July 11, 2001.
 |
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