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BOR - Papers in Press, published online ahead of print August 10, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.042747
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BIOLOGY OF REPRODUCTION 73, 1126–1134 (2005)
DOI: 10.1095/biolreprod.105.042747
© 2005 by the Society for the Study of Reproduction, Inc.

Research Article

Regulation of the Rhox5 Homeobox Gene in Primary Granulosa Cells: Preovulatory Expression and Dependence on SP1/SP3 and GABP1

James A. MacLean, II 3, Manjeet K. Rao 3, Kari M.H. Doyle 4, JoAnne S. Richards 4, and Miles F. Wilkinson 2 3

Department of Immunology,3 The University of Texas, M.D. Anderson Cancer Center, Houston, Texas 77030 Department of Cell Biology,4 Baylor College of Medicine, Houston, Texas 77030

ABSTRACT

Homeobox genes encode transcription factors that regulate embryonic development and postnatal events. Rhox5 (previously called Pem), the founding member of a homeobox gene cluster that we recently identified on the X chromosome, is selectively expressed in granulosa cells in the ovary and other somatic-cell types in other reproductive organs. In this report, we investigate its regulation in granulosa cells in the rat ovary. We found that Rhox5 expression in the ovary is governed by the Rhox5 distal promoter and is expressed at least as early as Day 5 postpartum. Rhox5 mRNA levels are regulated during the ovarian cycle, peaking before ovulation. Deletion analysis revealed a 25-nt element essential for distal promoter transcription in primary granulosa cells. This distal promoter element contains two ETS and one SP1 transcription-factor family binding sites that mutagenesis analysis indicated were essential for high-level transcription. This element was both necessary and sufficient for transcription, because it activated transcription when placed upstream of a heterologous minimal promoter. Cold competition and electrophoretic mobility shift assay studies demonstrated that SP1, SP3, and the ETS family transcription factor GABP bound this element. Dominant-negative forms of GABP and SP3 repressed distal promoter expression in primary rat granulosa, showing that these factors are crucial for Rhox5 expression. Cotransfection of dominant-negative mutants indicated that Rhox5 expression in granulosa cells is regulated by the c-Jun N-terminal protein kinase (JNK, MAPK8) and RAS pathways, which are known to be upstream of ETS family transcription factors. The discovery that Rhox5 expression in granulosa cells is regulated by MAPK pathways and ETS and SP1 family members provides an opportunity to understand how these regulatory pathways and factors collaborate to regulate gene expression during the ovarian cycle.

gene regulation, granulosa cells, homeobox, ovary, ovulatory cycle

INTRODUCTION

Ovulation is essential for mammalian reproduction. Successful oocyte development depends upon the regulated proliferation and differentiation of granulosa cells. Initially, a single layer of flattened precursor granulosa cells surround each primordial oocyte, and upon stimulation by FSH, they proliferate and differentiate into two distinct populations of granulosa cells: the mural granulosa cells, associated with the follicular wall, and the cumulus granulosa cells, associated with the oocyte [1]. Granulosa-cell proliferation is terminated by a surge in LH that stimulates granulosa cells to undergo a developmental switch to luteal cells and initiates a signaling cascade that ultimately leads to ovulation [2].

In this report we examine the regulation of a homeobox gene that has the potential to control events in granulosa cells during the preovulatory period. Homeobox genes encode transcription factors that contain a 60 amino-acid DNA-binding motif termed a homeodomain [3]. Rhox5 (previously called Pem) is the founding member of the reproductive homeobox X-linked (Rhox) gene cluster that we recently identified [4]. This gene cluster contains 12 genes encoding related homeodomains that are all expressed selectively in male and female reproductive tissues. The Rhox genes are expressed in a cell-type-specific manner, some exhibit hormone-dependent expression, and all exhibit a colinear expression pattern that precisely corresponds with their position within subclusters on the X chromosome. The tightly regulated windows of Rhox gene expression imply that each is important for regulating diverse developmental events in the male and female reproductive tracts.

In postnatal and adult rodents, Rhox5 is expressed in the ovary, testis, and epididymis [5–7]. In the gonads, RHOX5 protein is restricted to the mural granulosa cells of periovulatory follicles within the naturally cycling ovary and Sertoli cells at stages VI–VIII of the spermatogenic seminiferous epithelial cycle in the testis [5, 8, 9]. This expression is quite specific, because RHOX5 is only in mural granulosa cells, not cumulus granulosa cells, and it is undetectable in the granulosa cells of immature follicles, atretic follicles, and postovulatory, luteinized cells of the corpus luteum [8].

Our laboratory has previously shown that Rhox5 transcripts are derived from two distinct promoters. A distal promoter drives Rhox5 expression in ovary and placenta [7, 9] and an androgen-dependent proximal promoter is responsible for Rhox5 expression in Sertoli cells of the testes and principal cells in the caput region of the epididymis. The regulation of the proximal promoter in testis and epididymis has been extensively characterized by our lab [5, 7], but little is known about Rhox5 distal promoter regulation in female reproductive tissues and placenta. The distal promoter is also aberrantly expressed in a wide variety of tumors from different tissues, including lymphomas, neuroblastomas, retinoblastomas, carcinomas, and sarcomas [10, 11]. We previously established a minimal promoter element required to drive distal promoter transcription in tumor cells [10]. We identified ETS and SP1 family members that bind to this element and transactivate distal promoter transcription in the SL12.4 T-cell lymphoma cell line. Interestingly, SP3 was more potent in its activation of the distal promoter than was SP1, which contrasts with the activity of these factors on most other promoters: SP3 is typically a weak activator or a strong repressor [12, 13].

Although our previous studies defined the minimal promoter elements and factors that regulate the distal promoter's transcription in tumor cells, it was not clear how this promoter is regulated in normal cells. The goal of the present study was to determine the identity and role of cis elements and transcription factors that regulate the distal promoter in normal granulosa cells. To place this regulation in a physiological context, we also examined the timing of distal promoter expression in the rat ovary during postnatal development and the ovulatory cycle in vivo.

MATERIALS AND METHODS

Plasmids

We cloned a 1.3-kb Spe-I/Sal-I rat genomic Rhox5 fragment containing exon 1 and upstream sequences into the eukaryotic expression vector pRL-Null (Promega Corp.) to generate the –304 construct (Pem-128). We made the following deletion constructs from Pem-128: –112 (Pem-147), –104 (Pem-170), –94 (Pem-167), –73 (Pem-148) as described in [10]. The Pem-147 construct served as the parent for site-specific mutagenesis of putative transactivating factor binding sites: Pem-171 (-112/S1), Pem-172 (-112/E1), Pem-173 (-112/S1+E1), Pem-175 (-112/E2). pCMV-DNSP3 (G-313), which encodes the dominant-negative (DN) form of SP3 in pCDNA3.1/His C, was generously provided by Yoshihiro Sowa and Toshiyuki Sakai (Kyoto Prefectural University of Medicine, Kyoto, Japan). The DN GABPA and GABPB expression plasmids (G-300 and G-301, respectively) were provided by Thierry Seroz (Neurobiologie Moleculaire, Institut Pasteur, France). The DN RAS (17N), constitutive RAS (61L), and D56FosdE-luciferase vectors (G-213A, B, and C, respectively) were kindly provided by Crag Hauser (Burnham Institute, La Jolla, CA). Pem-179 contains a single copy of the distal promoter nt –104 to –80 cloned into the SalI-HindIII site of the D56FosdE-luciferase vector, which contains a minimal Fos promoter. Rat cyclophilin in the pSP6S vector (G-99) was obtained from J. Gregor Sutcliffe (Research Institute of Scripps Clinic, La Jolla, CA).

RNA Isolation and Assay

Total cellular RNA was isolated from whole ovaries and primary granulosa cells by using the standard TRIZOL protocol (Life Technologies Inc.). RNase protection analysis (RPA) was performed as previously described [5]. The [32P]UTP-labeled Rat Rhox5 RPA probe was prepared by in vitro transcription, as previously described (210-nt Pem probe B; [9]). The cyclophilin RPA probe was prepared in the same manner; it contained nt 437 to 495 of the rat cyclophilin gene (GenBank accession number m19533). A set of RNA size markers generated from the century ladder template (Ambion, Inc.) was included in each gel.

Real-time PCR was performed by first generating single-stranded cDNA from 500 ng tcRNA template by priming with oligo-dT in a 20 µl reaction including Superscript II (Life Technologies Inc.). For negative controls, we prepared duplicate reaction mixtures without Superscript. There was no detectable genomic DNA contamination, as shown by using primers flanking intronic regions of both Rpl19 and Rhox5. We used real-time RT-PCR to quantify the relative abundance of Rhox5 cDNA (1 µl) by using the standard SYBR Green-I premix protocol in a 25 µl reaction (iCycler iQ Real Time PCR; Bio-Rad). Calibration curves were constructed to confirm equivalence of the reaction efficiencies for primers used in each assay over a wide concentration range of input cDNA or control plasmids. The primer pairs used for PCR are as follows: Rhox5: 5'-GCCTGGGAGTCAAGGAA-3' (MDA-1287) and 5'-CATAGGACCAGGAGCACCA-3' (MDA-1288); Actb: 5'-CTTCACCACCACGGC-3' (MDA-1282) and 5'-CCATCTCTTGCTCGAAG-3' (MDA-1283). Reaction conditions were: 3 min at 95°C to activate the "hot start" taq polymerase, followed by 40 cycles at 95°C for 1 min and 55°C for 45 s. After amplification, the specificity of the PCR was verified by both melt-curve analysis and gel electrophoresis to verify that only a single product of the correct size was present.

Animals and Granulosa Cell Culture

For transfection experiments, immature Holtzman Sprague Dawley rats (less than 25 days old and less than 55 g) were induced to superovulate with eCG and hCG as described previously [14]. Rats were injected s.c. with 10 IU eCG (G4877; Sigma Chemical Co.) followed by s.c. injection of 10 IU hCG (CG-5; Sigma) 48 h later. Ovarian granulosa cells were isolated as described previously [15]. Briefly, the ovaries were punctured multiple times with a 22-gauge needle to isolate granulosa cells. The granulosa cells were pooled and treated with 20 µg/ml trypsin for 1 min, and then 300 µg/ml of soybean trypsin inhibitor and 160 µg/ml Dnase I were added to remove necrotic cells. The cells were washed twice with serum-free Dulbecco modified Eagle medium (DMEM):F12 containing penicillin and streptomycin in multiwell dishes that were coated with serum. Cells were cultured in DMEM:F12 medium at 37°C in 95% air and 5% CO2 for 16 h before transfection.

For the timed ovarian time course study, we used primary granulosa cells prepared from these animals as described above. RNA was extracted from purified granulosa cells at intervals of 0, 2, 4, 8, 12, and 16 h after hCG administration. Ovulation is known to occur around 12 h following hCG and is followed by luteinization [14]. Therefore, RNA was extracted from whole luteinized ovaries at 24, 48, and 72 h.

All rats were obtained from Harlan Sprague Dawley and were housed under a 16L:8D schedule in the Smith Research Building at the M.D. Anderson Cancer Center. The animals were provided food and water ad libitum, and were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All procedures were approved by the institutional committee on animal care of the M.D. Anderson Cancer Center.

Transfection of Primary Rat Granulosa Cells

For transient transfection, DNA concentrations were independently determined by using a fluorometer and by analytical gel electrophoresis. After overnight culture, the granulosa cells were transfected by the calcium phosphate precipitation method, as previously described [10]. Eight hours later, the cells were washed thoroughly in fresh medium and incubated another 12–24 h, and cell lysates were prepared and used to measure luciferase activity according to standard protocols (Dual Luciferase Assay System; Promega Corp.). Relative light units were normalized to internal control plasmid expression or to total protein.

Statistical Analysis

Data are presented in the figures as the mean ± SEM of three independent transfection experiments performed with at least three different pools of granulosa cells using separate batches of ovaries to confirm reproducibility of results. Data from transient transfection assays were analyzed by ANOVA and differences between individual means were tested by a Tukey multiple-range test using Prism 3.0 (GraphPad Software). Differences were defined as significant at P < 0.05.

Electrophoretic Mobility Shift Assay

Total cell extracts purified from rat granulosa cells were prepared as previously described [16]. Electrophoretic mobility shift assays (EMSAs) were performed by using a 32P-labeled blunt-end double-stranded probe generated by annealing two complementary oligonucleotides; 5'-GTGGAAGGAATAGGCGGGACTTCCGGATC-3' (MDA-397) and 5'-GTGGAAGGAATAGGCGGGACTTCCGGATC-3' (MDA-398). The probe (5 x 104 cpm) was incubated for 30 min at room temperature in 20 µl of binding buffer (15 mM Tris [pH 7.5], 100 mM KCl, 5 mM dithiothreitol, 1 mM EDTA, 12% glycerol, and 1 µg of poly dI:dC) containing 2 µg of purified granulosa whole-cell extract. For competition assays, mutant oligo pairs (50 µM) were denatured at 95°C and allowed to anneal overnight as they cooled to room temperature in a heat block. For antibody supershift and blocking assays, the reaction mixtures were preincubated with monoclonal antibodies or polyclonal antisera at room temperature for 20 min before addition of the 5' 32P-labeled blunt-ended double-stranded oligonucleotide. The DNA-protein complexes were resolved in 4.5%–5% nondenaturing polyacrylamide gels at 150 V for 3–4 h at 4°C. The autoradiographs presented for each EMSA (distal promoter binding, cold competition, and supershift) are representative of the outcome from at least three experiments.

RESULTS

Rhox5 mRNA Expression Is Regulated During the Ovarian Cycle

We began our investigation of Rhox5 regulation in the ovary by performing a time-course study. We showed previously, by RNase protection analysis, that Rhox5 transcripts in the adult rat ovary originate exclusively from the distal promoter [7]. Here we examined distal promoter transcript levels in the rat ovary from shortly after birth to sexual maturity. We found that distal promoter transcripts were detectable at 5 days postpartum and remained at a low level until Day 25 postpartum, when distal promoter transcript levels increased by 3- to 5-fold (Fig. 1A).


Figure 1
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  		FIG. 1. Temporal regulation of Rhox5 mRNA expression during ovarian development. A) RNase protection and real-time RT-PCR analyses of total cellular RNA (5 µg) from rat ovaries at the age indicated (five animals per time point). B) Real-time RT-PCR quantification of relative Rhox5 expression in rats induced to superovulate with eCG and hCG. Shown is the mean fold change ± SEM in Rhox5 mRNA levels normalized against ActB mRNA. Equivalent results where obtained when transcripts for the L19 ribosomal protein were used as the internal control (data not shown). Different letters denote time points that have statistically significant (P < 0.001) differences in mean expression levels. The expression of Rhox5 mRNA in untreated rats was arbitrarily given a value of 1

Because Rhox5 expression increased around the time ovaries first become responsive to gonadotropins, we examined Rhox5 expression during synchronous follicular development induced by gonadotropins. Immature rats were subjected to a superovulation protocol involving administering 10 IU eCG (a functional analog of FSH) followed by a single injection (10 IU) of hCG (an analog of LH). This treatment regimen stimulates the maturation and ovulation of multiple follicles within 12 h after hCG exposure [14]. The relative expression of Rhox5 was determined by real-time RT-PCR and the level of input cDNA was normalized by parallel amplification with primers specific for ActB. We found that Rhox5 mRNA levels increased 2 h after hCG treatment, reaching a maximum 4 h post-hCG treatment, when follicles are in the initial stages of the ovulatory process. Rhox5 mRNA levels then sharply declined, with levels falling below the basal level at the time corresponding to ovulation (12 h). Rhox5 mRNA was nearly undetectable in the postovulatory corpora lutea (Fig. 1B). The burst of Rhox5 transcripts in granulosa cells from developing follicles in this superovulation model (Fig. 1B) is consistent with the timing of increased Rhox5 transcripts from nonsynchronized rats (Fig. 1A).

A 25-nt Element Within the Distal Promoter is Necessary and Sufficient for Rhox5 Transcription

After establishing that Rhox5 mRNA peaks transiently just before ovulation, we examined the mechanism responsible for Rhox5 expression in granulosa cells. We first examined whether the same promoter sequences responsible for tumor cell expression were also required for distal promoter transcription in primary granulosa cells. Promoter activity was determined by comparing the relative luciferase levels from constructs containing different lengths of distal promoter 5'-flanking sequences in transiently transfected rat primary granulosa cells. The longest reporter construct contained 304-nt sequences upstream of the Rhox5 exon 1/intron 1 junction (this junction is 21 to 41 nt downstream of the multiple distal promoter transcription start sites we previously defined [7]). This –304 construct expressed ~45-fold more luciferase activity than did the empty vector, indicating that it had high transcriptional activity (Fig. 2A). Shortening of the 5'-flanking sequence to –104 did not significantly decrease luciferase levels. In contrast, deletion of the sequences between –104 and –94 greatly reduced the levels of luciferase, and further deletion to –73 reduced luciferase activity to baseline levels. These data suggest that the sequences between –104 and –73 are crucial for distal promoter transcription in granulosa cells.


Figure 2
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  		FIG. 2. ETS and SP1 consensus sites in the distal promoter crucial for Rhox5 transcription. Relative luciferase activity from distal promoter (PD) region constructs (0.9 µg) transiently transfected into primary rat granulosa cells. The values shown are the ratios of Renilla luciferase activity normalized against firefly luciferase activity from the internal control vector pGL2-CMV. Assays were performed with three independent preparations of granulosa cells, which were split into three independent transfection wells for each treatment, and means ± SEM for nine experiments are indicated. The results are fold increase in luciferase activity above empty vector, which was given an arbitrary value of 1. Different letters denote constructs that have statistically significant (P < 0.001) differences in mean expression levels. A) Expression from constructs containing deletions of the distal promoter 5'-flanking sequence. The numbers indicate the nucleotides upstream of the exon 1/intron 1 junction (the multiple transcription start sites are between approximately –41 and –21 [7]; the gray box indicates Rhox5 (5' UTR) exon 1. The results are from three independent transfection experiments. B) The –101 to –73-nt region contains two consensus ETS family protein-binding sites and one consensus SP1 family protein-binding site. C) Site-directed mutagenesis of the ETS or SP1 binding sites abrogates distal promoter activity, as determined in three independent transfection experiments. D) A single copy of the distal promoter element (shown in B) containing only the ETS and SP1 binding sites is sufficient to drive transcription from a heterologous minimal promoter ({Delta}Fos), as determined in two independent transfection experiments (P < 0.001)

This region contains two putative ETS family-binding sites flanking one SP1 family-binding site (between –101 and –77; Fig. 2B). Mutation of the downstream ETS site, the SP1 site, or both resulted in a severe decline in luciferase activity in granulosa cells. Mutation of the upstream ETS site produced a marked, but not as dramatic, decline in luciferase levels (Fig. 2C).

To determine whether the 25-nt region containing ETS and SP1 binding sites is sufficient for transcription in granulosa cells, we assessed whether a single copy could drive transcription from a heterologous minimal Fos promoter. Indeed, we found that a single copy of this 25-nt element produced a 5-fold increase in luciferase activity over that of empty vector (Fig. 2D). This confirmed its importance and, because only a single copy was sufficient for transcription, indicated that it is a relatively strong regulatory element.

Identification of Granulosa-Cell Nuclear Proteins that Interact with the Distal Promoter Element

We next sought to determine which specific transcription factors bind to the distal promoter element by using the EMSA. A double-stranded probe containing the distal promoter element produced two major species of protein-DNA complexes (labeled I and II) when incubated with cell lysates from granulosa cells purified from eCG-primed, hCG-treated ovaries (Fig. 3A). The specificity of each complex was determined by competition with a 50-fold molar excess of cold competitor. Complex I was likely to contain SP1 family factors, because an oligonucleotide (oligo) containing a consensus SP1 binding sequence completely abolished complex I (Fig. 3B), and an oligo with a mutation in the SP1-binding site (M1) was not able to compete with complex I but did compete with complex II (Fig. 3A). Conversely, oligos that had mutated ETS sites (M2 and M4) were able to compete only with complex I, not complex II, suggesting that complex II contains one or more ETS family members. An oligo with both the ETS sites and the SP1 site mutated (M3) was not able to compete away any of the complexes, indicating that binding was confined to the E1, E2, and S1 sites. Finally, the wild-type distal promoter oligo competed with both complex I and complex II, demonstrating they were both sequence-specific complexes (data not shown).


Figure 3
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  		FIG. 3. ETS and SP1 family transcription factors bind to the element crucial for distal promoter transcription. A) EMSA of granulosa whole-cell protein extracts (1 µg) incubated with a 32P-labeled distal promoter (PD) element probe (-101 to –77-nt) produced two major complexes: I and II. Cold-competition experiments were performed with a 50-fold molar excess of unlabeled double-stranded oligos containing mutations in either SP1, ETS, or combinations, as indicated (M1-M4). B) Complex I contains SP1 and SP3, as revealed by this complex being supershifted by anti-SP1 and anti-SP3 monoclonal antibodies. The major component of complex II was the ETS factor GABP, as shown by EMSAs with rabbit polyclonal antisera against GABP {alpha}/ß. Complex II was resolved into two bands after extended electrophoresis. The arrows indicate the relevant complexes that are inhibited or supershifted by antibody preincubation. The GABP supershift was very near to, but distinguishable from, the loading area of the well indicating a large complex was formed upon addition of antibody

To identify the protein factors binding to the distal promoter, we used specific antibodies against ETS and SP1 family members (Fig. 3B). Monoclonal antibodies against SP1 and SP3 partially altered the migration of a portion of complex I, and both antibodies together shifted the migration of virtually all of complex I, indicating that SP1 and SP3 are the transcription factors in complex I. We used a panel of ETS antibodies to directly determine whether any ETS family members were interacting with complex II. We found that the addition of antibody directed against the ETS factor GABP produced a shift of complex II (Fig. 3B). Antibodies against ETS1 and ETS2 did not alter the migration of any of the complexes (data not shown).

Regulation of the Distal Promoter by SP1, SP3, and GABP

To determine whether the factors we identified by EMSA regulate distal promoter transcription in granulosa cells, we transiently cotransfected DN mutants of GABP (comprised of a 1:1 mixture of plasmids expressing mutant GABPA and GABPB subunits) and SP3 (DN-GABP and DN-SP3, respectively) with the distal promoter-luciferase reporter plasmid. These DN proteins lack functional transcription activation domains but have intact DNA-binding domains, and thus they compete with the endogenous transcription factors for binding to the target sites. We found that DN-GABP and DN-SP3 DN mutants repressed distal promoter activity in a dose-dependent manner (Fig. 4, A and B), whereas DN mutants corresponding to unrelated transcription factors, CREB and AP2, did not (Fig. 4C and data not shown).


Figure 4
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  		FIG. 4. ETS and SP1 family members essential for distal promoter transcription in rat granulosa cells. Granulosa cells were cotransfected with the –112-nt construct shown in Fig. 2A (2 µg in 6-well plate [A]; 0.9 µg in 12-well plate [B and C]) and the indicated amount of DN expression construct. Shown is the average luciferase activity determined as in Fig. 2 from 4 independent experiments. Assays were performed with three independent preparations of granulosa cells, which were split into three independent transfection wells for each treatment, and the mean ± SEM for nine experiments are indicated. Different letters denote constructs that have statistically significant (P < 0.001) differences in mean expression levels. The DN-CREB construct had no effect on distal promoter activity and therefore served to indicate the specificity of the inhibitory effects observed with the DN-GABP (comprised of a 1:1 mixture of DN-GABPA and DN-GABPB) and DN-SP3 constructs

To further examine the role of these factors in distal promoter transcription in granulosa cells, we determined whether overexpression of SP1, SP3, and GABP (comprised of a 1:1 mixture of plasmids expressing both normal GABPA and GABPB subunits) leads to increased luciferase production from the –112 distal promoter construct. As shown in Figure 5, low amounts of plasmids encoding these transcription factors increased the transcriptional activity of the distal promoter. The effect was more pronounced for GABP than for the SP1 family members. Higher doses of transfected plasmid did not further increase promoter activity, suggesting that the endogenous level of these factors in granulosa cells is already nearly saturating. GABP stimulated distal promoter transcription more than did SP1 or SP3 (Fig. 5). We do not know whether this indicates that GABP has higher intrinsic transcriptional stimulatory activity on the distal promoter than these other transcription factors, or merely that SP1 and SP3 are already at saturating levels in granulosa cells.


Figure 5
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  		FIG. 5. SP1, SP3, and GABP transactivate distal promoter transcription. Granulosa cells were cotransfected with the –112-nt construct shown in Fig. 2A (0.9 µg) and the indicated amounts of pCMV-SP1, pCMV-SP3, and a 1:1 mixture of pCMV-GABPA and pCMV-GABPB plasmids. Shown are the average luciferase activities, calculated as described in Fig. 2. Assays were performed with three independent preparations of granulosa cells, which were split into three independent transfection wells for each treatment, and the mean ± SEM for nine experiments are indicated. Different letters denote constructs that have statistically significant (P < 0.001) differences in mean expression levels. Addition of higher amounts of expression plasmids than that shown typically reduced relative luciferase values (data not shown), presumably because of toxicity

The JNK, MAPK, and RAS Signaling Pathways Are Essential for Distal Promoter Transcription

Because the c-Jun N-terminal protein kinase (JNK, MAPK8) and RAS signaling pathways are known to act upstream of ETS and SP1 family members [17, 18], we examined the role of these pathways in distal promoter transcription. Primary rat granulosa cells were transiently cotransfected with distal promoter-luciferase reporter plasmid and DN mutants corresponding to JNK1, JNK2, and RAS (DN-JNK1, DN-JNK2, and DN-RAS, respectively).

As shown in Figure 6, A–C, the JNK and RAS DN molecules repressed distal promoter-promoter activity in a dose-dependent manner. In agreement with a role for RAS in distal promoter transcription, we also found that a constitutively active RAS mutant (RAS 61L) increased the transcriptional activity of the distal promoter (Fig. 6D). The magnitude was similar to the transactivation ability of SP1 family members and the ETS factor GABP (Fig. 6, A–C).


Figure 6
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  		FIG. 6. Suppression of the RAS and JNK MAPK signaling pathways dramatically inhibits distal promoter transcription. Rat granulosa cells were transfected with the –112-nt distal promoter construct (2 µg in A–C; 0.9 µg in D) and the indicated amounts of the DN-JNK1, DN-JNK2, DN-RAS, or RAS 61L (constitutively active RAS) plasmids. Shown are the average luciferase activities, calculated as described in Fig. 2. Assays were performed with three independent preparations of granulosa cells, which were split into three independent transfection wells for each treatment, and means ± SEM for nine experiments are indicated. Letters denote significantly different means (P < 0.001)

DISCUSSION

Homeobox genes were initially identified as master control genes that determine the identity and spatial arrangement of body segments during Drosophila melanogaster embryonic development [3]. Subsequently, homeobox genes have been characterized in nearly every eukaryotic organism, where they regulate not only embryonic events but also postnatal and adult functions [19, 20]. The subject of this report is Rhox5, which is the founding member of a 12-member homeobox gene cluster that we recently identified on the mouse X chromosome [4]. Rhox5 is selectively expressed in somatic cells in male and female reproductive tissues, including Sertoli cells in the testis and granulosa cells in the ovary [6, 8]. Although its regulation in male reproductive tissues has been well studied [5, 7, 10, 18, 21–23], little is known about the regulation of Rhox5 in the female reproductive tract. Herein, we show that Rhox5 expression is transiently induced in rat granulosa cells treated with hormones to simulate ovulation. In this model, Rhox5 mRNA is induced following treatment with eCG and hCG and reaches a peak shortly before ovulation, whereupon it rapidly declines (Fig. 1). We provide evidence that transcription of Rhox5 requires the SP1, SP3, and GABP transcription factors working in concert with the JNK and RAS signaling pathways (Figs. 2–6).

GABP is a unique member of the ETS transcription factor family that acts as a heterotetramer ({alpha}2ß2, subsequently referred to as GABP for simplicity, unless a specific subunit is specified) to stimulate the transcription of genes that govern the cell cycle, apoptosis, and cellular differentiation [24]. Whereas the actions of GABP are well characterized in liver, muscle, and hematopoietic cells, our study is the first to establish a target gene for GABP in the ovary. In particular, we found that GABP binds a site crucial for Rhox5 transcription (Figs. 2 and 3) and positively regulates Rhox5 transcription in granulosa cells, as determined by transient transfection studies with both wild-type and DN forms of GABP (Figs. 4 and 5). Interestingly, in placenta, where the Rhox5 distal promoter is also expressed, GABP is known to enhance the expression of folate receptor 1 (Folr1), a membrane-bound folate transporter protein that controls trophoblast development and maintains proper maternal-fetal communication in species with hemochorial placentae [25–27]. GABP appears to have a conserved role in female reproduction, because mutation of the D. melanogaster GABP ortholog, D-ELG, results in tiny-egg syndrome [28].

GABP and some other members of the ETS family are proto-oncoproteins that promote cell growth [24]. This is consistent with fact that normal granulosa cells are rapidly dividing cells, particularly during the mid to late stages of follicular development, when they acquire FSH responsiveness and are cyclin-D2 dependent [29]. However, whether GABP actually directs the growth of granulosa cells and whether its ability to upregulate Rhox5 transcription contributes to this growth response remains to be tested.

SP1 family members are widely known to participate in the regulation of a variety of genes in both normal and cancerous tissues [30]. While studies of tumor cell lines suggest that SP1 expression is ubiquitous and constitutive, there are large differences in the levels of SP1 factors in different normal tissues in vivo, in part because of dramatic differences in SP1 protein turnover in different cell types [31, 32]. In ovary, SP1 is expressed at high levels and is known to regulate many genes, including steroidogenic factor-1 (SF-1, Nr5a1), cholesterol side chain cleavage cytochrome P450 (P450scc, Cyp11a1), glucocorticoid-inducible kinase (Sgk), the transcription factor Egr1, tissue-type plasminogen activator (Plat), progesterone receptor (Pgr), and a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif (Adamts1) [33, 34]. Our demonstration that SP1 family members are essential for Rhox5 transcription could partially explain why Rhox5 is highly expressed in granulosa and trophoblast cells [8, 10], because SP1 is strongly expressed in both of those cell types [35, 36].

Several lines of evidence suggest that ETS and SP1 transcription factors work in concert to activate Rhox5 distal promoter transcription in granulosa cells. First, mutations in the distal promoter that prevented the binding of any one of these transcription factors almost completely abrogated transcription in transfected granulosa cells (Fig. 2). Second, the two ETS sites and single SP1 binding site that we showed are crucial for distal promoter transcription are in close proximity (< ~20 nt) to one another. Third, GABPA and SP1 have recently been shown to physically interact in D. melanogaster Schneider cells and myeloid cells [24]. Lastly, many studies have demonstrated functional interactions between ETS and SP1 family members. For example, SP1 and GABP functionally interact to regulate the transcription of Folr1 gene in placenta, as well as the integrin beta 2, utrophin, and tenascin C genes in other cell types [27, 37, 38]. Interestingly, integrin beta 2 is regulated during myelopoiesis by retinoic acid in an RAR-independent manner by GABP and SP1 [39]. This is of particular interest because Rhox5 is upregulated 35-fold by RA in ES cells [40]. Thus, the RA-mediated regulation of Rhox5 could be through a similar mechanism involving the coordinated efforts of GABP and SP1.

Our DN and transactivation studies indicated that the Ras proto-oncogene is required for Rhox5 distal promoter transcription (Fig. 6). RAS is known to induce the phosphorylation and subsequent activation (but not DNA binding) of several ETS family members, [17] and thus RAS may drive distal promoter expression in granulosa cells as a result of its ability to stimulate the activity of GABP. Phosphorylation of the {alpha} subunit of GABP facilitates neuregulin-mediated transcriptional activation of the nicotinic acetylcholine receptor promoter [41]. RAS may also stimulate distal promoter transcription through its ability to activate SP1. Evidence suggests that several genes are upregulated as a result of RAS-induced SP1 activation, including serum response factor, apolipoprotein A-1, and the cyclin-dependent kinase inhibitor 1A [42–44].

Our DN inhibitor studies suggest that the MAPK pathway is crucial for transcriptional activation of the Rhox5 distal promoter in granulosa cells (Fig. 6). The functional relevance of the MAPK pathway in granulosa cells is currently unclear. Only a few molecules have been shown to be activated or increased in levels by the MAPK pathway in granulosa cells, including progesterone receptor, low density lipoprotein receptor, and the Jun proto-oncogene related gene d1 transcription factor (JUND1) [45–48]. Interestingly, JUND1 is a potential regulator of Rhox5, because it is expressed in terminally differentiating granulosa cells by a MAPK-dependent pathway [16] and it is also overexpressed in tumor cell lines [49], just as Rhox5 is [10, 11]. The MAPK pathway may also regulate Rhox5 via direct effects on transcription factors that we showed here are essential for distal promoter transcription. Most notably, the JNK and ERK MAPK pathways activate GABP [10, 50, 51], which we showed here is essential for Rhox5 transcription (Figs. 3–6).

The downstream genes that are regulated by RHOX5 in the ovary are unknown. At least 7 genes which are differentially regulated during folliculogenesis (reviewed in [33] display an expression pattern similar to that of Rhox5: epiregulin, Egr1, G-protein signaling-2 (Rgs2), {gamma}-glutamylcystein synthetase, prostaglandin-endoperoxide synthase 2 (Ptgs2), pituitary adenylate cyclase-activating polypeptide (Adcyap1), and tumor necrosis factor alpha induced protein-6 (Tsg6, Tnfaip6). Two of these genes, Ptgs2 and epiregulin, are expressed at peak levels during precisely the same time window as Rhox5 (4 h post-hCG). PTGS2 is the rate-limiting enzyme in the prostaglandin synthesis pathway; it is believed to stimulate local acute inflammatory reactions within the granulosa cells, which in turn spurs ovulation [52]. Epiregulin is a member of the epidermal growth factor family that has been suggested to be induced in preovulatory granulosa cells to mediate, in part, the LH signal in a paracrine manner throughout the entire follicle [53]. EGR1 is a zinc-finger transcription factor expressed at high levels between 2 and 8 h post-hCG that regulates the production of several key factors that are thought to be involved in ovulation, including p53 and several proteinases, cytokines, and extracellular matrix adhesion proteins. RGS2 is a highly expressed GTPase-activating protein expressed at high levels between 4 and 8 h post-hCG whose function in ovary is not yet known. The other three genes coexpressed with Rhox5 during the preovulatory period are potent inhibitors of inflammatory reactions or protect from unwanted side effects of inflammatory reactions. ADCYAP1 is a member of the secretin superfamily that attenuates inflammatory responses by inhibiting the production of cytokines and nitric oxide. TSG6 covalently binds hyaluronan and neutralizes the inflammatory stress that occurs within the cumulus cell-oocyte complex when the extracellular matrix is remodeled during ovulation [54]. {gamma}-Glutamylcystein synthetase is responsible for the synthesis of glutathione, which protects cells against oxidative stress during the acute phase of inflammation. Other candidate RHOX regulated genes are those that are induced after RHOX5: the proteinases Cathepsin L and Adamts1 and the steroid biogenesis enzymes steroid acute regulatory protein (Star) and 3{alpha}-hydroxysteroid dehydrogenase [34, 55, 56].

To date, the function of RHOX5 in the ovary has remained elusive. Despite being fertile, Rhox5-null females do appear to have subtle reproductive defects. Rederivation of the null line by standard techniques was not successful, because follicles extruded from Rhox5-null mice had fragile and fragmented cumulus granulosa layers and were deemed unacceptable for in vitro fertilization (J.A.M. and M.F.W., unpublished observations). We suspect that the negative impact on fertility resulting from loss of Rhox5 is probably tempered by compensatory expression of related homeobox genes. Rhox5 is part of a 12 homeobox-gene cluster that is conserved in mouse [4] and rat (J.A.M. and M.F.W., unpublished observations). In mouse, 5 of these Rhox genes are expressed in adult ovary [4] and at least four (Rhox2, 3, 7, and 8) are expressed in granulosa cells during the same postnatal time points as Rhox5 (J.A.M. & M.F.W., unpublished observations). In addition to the cooperative actions of homeobox genes, the complex process of ovulation is likely to require the coordinated effort of additional transcription factors. Candidates to work with RHOX5 to regulate gene expression in the ovary are the previously mentioned EGR1 and two members of the GATA family (4 and 6) which have well characterized roles in androgen-dependent regulation of genes in the testes but have only recently been shown to be expressed in the ovary [57].

In summary, the present study is the first to identify a downstream target of the ETS factor GABP in the ovary. Future studies will be directed toward identifying the factors upstream and downstream of GABP and RHOX5 in granulosa cells. We propose that upstream is the JNK (MAPK8) pathway and possibly other MAPK signal transduction pathways, based on our evidence on Rhox5 distal promoter transcription in granulosa cells (Fig. 6) and studies on other promoters showing that ETS factors are downstream of MAPK pathways [24, 51]. It remains for future studies to determine what extracellular stimuli (if any) activate Rhox5 expression; candidates are factors upstream of the MAPK pathways, including fibroblast growth factors, LH, FSH, epidermal growth factor, insulin, insulin-like growth factor I, gonadotropin-releasing hormone, prostaglandin F2{alpha}, and transforming growth factor {alpha} [45–48, 58–64].

ACKNOWLEDGMENTS

The authors would like to thank Dr. Michael H. Melner (Vanderbilt University), Dr. Jonathan Green (University of Missouri–Columbia) and Maureen Goode (M. D. Anderson Cancer Center) for their assistance in the interpretation of our data and preparation of this manuscript.

FOOTNOTES

1 Supported by NIH grants HD-45595 to M.F.W. and HD-SCCPRR-07495 to J.S.R. Back

2 Correspondence: Miles Wilkinson, Department of Immunology, Unit 902, The University of Texas, M.D. Anderson Cancer Center, P.O. Box 301402, Houston, TX 77030. FAX: 713 563 3357; mwilkins{at}mdanderson.org Back

Received: 11 April 2005.

First decision: 2 May 2005.

Accepted: 18 July 2005.

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