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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Imperfect DR Motif of the rLHR Promoter Is a Binding Site for EAR2 and EAR3/COUP-TFI, but Not TR4
Figure 1Go 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. 2EGo) 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. 2AGo, 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. 5Go). 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.

 
Similar results were obtained when in vitro translated EAR3/COUP-TFI was investigated in EMSA (Fig. 2BGo). 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. 2CGo). 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. 2DGo, 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. 2EGo). 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. 3Go). 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).

 
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. 4AGo, 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. 4AGo, 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. 2EGo) showed that EAR2 displayed comparable binding parameters for the two sequences (Fig. 5Go, left panel). Also, EAR3/COUP-TFI exhibited similar binding affinities for these probes (Fig. 5Go, 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.



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Figure 4. Scatchard Analysis of EAR2 and EAR3/COUP-TFI Binding to the Human and the Rat DR Domains

Binding of a constant amount of in vitro translated EAR2 (top) or EAR3/COUP-TFI (bottom) to various doses of 32P-labeled hDR ({bullet}{bullet}) or rDR ({blacksquare}{blacksquare}) probe were resolved in EMSAs. The binding parameters were obtained by Scatchard analyses.

 


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Figure 5. Comparison of Binding Affinities of EAR2 and EAR3/COUP-TFI for the Wild-Type vs. the A/C Mutant rDR Sequences

Top, Binding of in vitro translated EAR2 (left) and EAR3/COUP-TFI (right) to various amounts of the wild-type rDR probe (WT) or A/C mutant probe (A/C) was resolved in EMSA. Bottom, Scatchard analyses of the steady-state binding of EAR2 (left) and EAR3/COUP-TFI (right) to the rDR wild-type ({bullet}{bullet}) and A/C mutant ({blacksquare}{blacksquare}) probes.

 
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. 1–4), in which the corresponding 5'- or 3'-adjacent region to either rDR or hDR core motif was interchanged, were used as unlabeled competitors (Fig. 6CGo). 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 1–13). 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 13–19). 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 20–30). 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 31–35). 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. 7Go). 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 2–7) or hybrid rDR sequences 1 (lanes 8–13) and 2 (lanes 14–19). 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 21–25) or hybrid hDR sequences 3 (lanes 26–30) and 4 (lanes 31–35). 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 1–8 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 "{Delta}g" or "±g." The substitution of GGA with ac is indicated as "{Delta}GGA/+ac," while replacement of ac by GGA is stated as "{Delta}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 1–4 as unlabeled competitor DNAs.

 
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. 6Go, 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. 8Go, 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 5–8 as unlabeled competitor DNAs.

 
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. 9AGo). Similar results were obtained with EAR3/COUP-TFI that inhibited the wild-type promoter activity by 45% (P < 0.05) (Fig. 9AGo). 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. 9AGo). 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. 9BGo). 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).

 
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. 10AGo). 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. 2EGo) resulted in the formation of three major DNA-protein complexes (Fig. 10BGo, 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. 2Go). 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. 2CGo and 3Go). 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. 2EGo). 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.

 
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. 11AGo, left panel), which was consistent with the previous data (Fig. 10Go). 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. 11AGo, 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. 11Go, 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. 11Go, 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 10Go) 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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, [{gamma}-32P]ATP end-labeled oligonucleotide probe (5–10 x 104 cpm) was incubated with 1–5 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 30–60 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 (0.1–4 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. 2Go and 6Go. 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.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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