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Endocrinology, doi:10.1210/en.2004-0067
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Endocrinology Vol. 145, No. 6 3014-3022
Copyright © 2004 by The Endocrine Society

Involvement of an Unnamed Protein, RDA288, in the Mechanism through which Progesterone Mediates Its Antiapoptotic Action in Spontaneously Immortalized Granulosa Cells

J. J. Peluso, A. Pappalardo, G. Fernandez and C. A. Wu

Departments of Cell Biology (J.J.P., A.P., G.F.), Obstetrics and Gynecology (J.J.P.), and Medicine (C.A.W.), University of Connecticut Health Center, Farmington, Connecticut 06030

Address all correspondence and requests for reprints to: John J. Peluso, Ph.D, Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut 06030. E-mail: peluso{at}nso2.uchc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Progesterone (P4) inhibits apoptosis of rat granulosa cells and spontaneously immortalized granulosa cells (SIGCs), which were derived from rat granulosa cells. Defining the mechanism through which P4 mediates its action has been difficult because these cells do not express the classic nuclear P4 receptor. Previous studies have shown that a P4 receptor antibody, C-262, detects a 60-kDa protein that is involved in regulating P4’s antiapoptotic action. Using a C-262 affinity column, this 60-kDa protein was isolated and sequenced by mass spectrometry. This analysis revealed that the C-262-detectable protein is an unnamed protein referred to as RDA288. This protein has several putative hyaluronic acid binding sites. Further hyaluronic acid antagonizes 3H-P4 binding to SIGCs and mimics P4’s action, whereas exogenous hyaluronic acid binding protein attenuates P4’s actions. RT-PCR demonstrated that RDA288 mRNA was present in SIGCs, immature rat ovary, lung, and skeletal muscle but was not present in several other organs. Forced expression of RDA288 increased the capacity of SIGCs to bind and respond to P4. An antibody was also developed against RDA288. Using this antibody in a Western blot protocol, RDA288 expression was confirmed in both SIGCs and granulosa cells. An immunohistochemical study detected RDA288 in the cytoplasm and plasma membrane components of granulosa cells of antral follicles. Immunocytochemical studies on living nonpermeabilized SIGCs revealed that RDA288 was present on the extracellular surface of the plasma membrane. Finally, pretreatment with the RDA288 antibody blocked P4’s antiapoptotic actions. Taken together, these data suggest that RDA288 plays a significant role in mediating P4’s antiapoptotic action in granulosa cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROGESTERONE (P4) IS not only an intermediary in the synthesis of androgens and estrogen but also is an essential regulator of ovulation, atresia, and luteal function (1). It has also been shown to be necessary for the maintenance of pregnancy in mammals (1). P4 is synthesized and secreted by the granulosa cells of follicles of all sizes with the rate of P4 secretion increasing throughout follicular development (2, 3). Although during the rat estrous cycle the follicular fluid concentration of P4 varies in parallel to that of serum P4 levels, follicular fluid P4 concentrations are always in the micromolar range (4). Recent studies have shown that these high P4 levels are important in inducing ovulation (5, 6), preventing the granulosa cells of periovulatory follicles from undergoing apoptosis (7), and regulating luteal function (8, 9).

P4’s role in ovulation coincides with the expression of the classic nuclear P4 receptor (PR), which is induced in granulosa cells by the preovulatory gonadotropin surge (10, 11, 12). However, P4 also regulates mitosis and apoptosis of granulosa cells isolated before the gonadotropin surge (13). These granulosa cells do not express the classic nuclear PR (10, 11, 12); thus, the nuclear PR cannot mediate P4’s actions in these cells.

Fluorescein isothiocyanate (FITC)-BSA-P4 binds to the plasma membrane of granulosa cells (14), which suggests that P4 could function through a nongenomic, membrane-initiated mechanism. The membrane-initiated actions of P4 within the ovary have been observed by several investigators, and these studies have been reviewed by Tony Bramley (1). In addition to membrane-initiated actions in granulosa cells, several studies using spontaneously immortalized granulosa cells (SIGCs), which do not express the classic nuclear PR (15), also indicate that P4 acts through a rapid membrane-initiated action (14, 15, 16).

Several different membrane receptors have been proposed to mediate P4’s actions. At one time, GABAA-like receptors and glucocorticoid receptors were thought to transduce P4’s action in granulosa cells, but ultimately neither of these receptors proved to be involved in P4’s actions (15). Recently, a putative membrane receptor for P4 has been detected in a sea trout cDNA library with the {alpha}-type of the membrane P4 receptor being expressed in human ovaries (17, 18). The expression of this receptor has not been assessed in other mammalian ovaries.

Interestingly, the PR receptor antibody, C-262, detects a 60-kDa protein in granulosa cells (13) and SIGCs (16). This C-262-detectable protein is localized to the extracellular surface of the plasma membrane (15), and treatment with the C-262 antibody reduces FITC-BSA-conjugated P4 binding to granulosa cells (14). That this C-262-detectable protein binds P4 is demonstrated by the observation that C-262 immunoprecipitates a 60-kDa protein that binds horseradish peroxidase-labeled P4 (16). In addition, this C-262 blocks P4’s ability to inhibit mitogen-induced mitosis and apoptosis of both granulosa cells and SIGCs (13, 19). These characteristics suggest that this C-262-detectable protein plays a significant role in promoting P4’s actions.

A C-262-detectable protein may also be involved in transducing P4’s actions in other cells. In sperm, treatment with C-262 blocks the P4-induced 1) increase in intracellular free calcium and chloride levels and 2) the acrosome reaction (20). In a murine Leydig tumor cells (21), which do not express the classic nuclear PR but bind 3H-P4 through a low affinity site (Kd {approx} 284 nM), C-262 detects a 57-kDa protein that binds horseradish peroxidase-conjugated P4. Taken together then, these studies suggest that C-262-detectable proteins could be involved in mediating P4’s actions in granulosa cells, sperm cells, and Leydig tumor cells. Therefore, experiments were designed to first identify the C-262-detectable protein and then to confirm its role in P4’s mechanism of action in SIGCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and materials
Unless otherwise indicated, all chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO). P4 was used at a concentration of 1 µM if not stated otherwise. For the blocking antibody studies, C-262 and RDA288-1 were used at 20 µg/ml and 34 µg/ml, respectively. Glial hyaluronic acid binding protein (HABP) was also purchased from Sigma Chemical Co. It was purified from bovine brains and was used at a concentration of 0.05 µM.

Animals
Immature female Wistar rats (22 d of age) were obtained from Charles River Laboratory (Wilmington, MA) and housed under controlled conditions of temperature, humidity and photoperiod (12-h light, 12-h dark; lights on at 0700 h). On the day of the experiment, immature animals were 23 or 25 d of age. To induce preovulatory follicular development, some 23-d-old rats were injected ip with 20 IU of pregnant mare’s serum gonadotropin (PMSG) and autopsied 48 h after injection. The rats were cervically dislocated between 0930 and 1000 h, and the ovaries as well as other tissues were removed and used per the various experimental designs outlined in the subsequent sections. Granulosa cells were isolated from antral follicles of 23- to 25-d-old rats. The protocol to isolate granulosa cells was identical with that described by Peluso and Pappalardo (13) with the exception that the granulosa cells were not separated into small and large granulosa cells by Percoll gradient centrifugation. This protocol was approved by the Animal Care Committee of the University of Connecticut Health Center.

Spontaneously immortalized granulosa cell culture
SIGCs were cultured in DMEM/F-12 supplemented with 5% fetal bovine serum as previously described (22). The SIGCs were routinely maintained in Falcon T-flasks (Becton Dickinson Labware, Lincoln Park, NJ). For most studies involving apoptosis, SIGCs were plated in 0.5 ml of medium at 1.25 x 105 cells/ml in eight-chamber glass Lab-Tek slides (Nunc Inc., Naperville, IL). The cells were initially cultured in DMEM/F-12 supplemented with 5% fetal bovine serum for 24 h. The serum-supplemented medium was removed and the cells cultured in serum-free DMEM/F-12 with various reagents for an additional 5 h. Unless otherwise stated, apoptosis was assessed by YOPRO-1 nuclear staining as previously described (22).

Identification of the C-262-detectable protein
C-262-detectable protein was isolated using a C-262 affinity column. To do this, C-262 (StressGen, Victoria, British Columbia, Canada) was covalently linked to SulfoLink gel/agarose beads using the protocol provided by Pierce (Rockford, IL). Once a C-262-agarose bead column was constructed, it was tested by loading 1 mg of the A isoform of the PR (The Binding Site, San Diego, CA) in 1 ml of sample preparation buffer [0.1 M sodium phosphate, 5 mM EDTA (pH 6.0); Pierce] onto the column. The column was incubated for 1 h at room temperature and then washed with 16 ml of sample buffer. The C-262-detectable protein was eluted from the column by adding 8 ml of elution buffer (pH 2.5–3.0; Pierce) and the collecting elutriate in eight 1-ml fractions. Samples (10 µl) of each fraction were then run on a 10% acrylamide gel and stained with Coomassie blue (CB; Simply Blue SafeStain; Bio-Rad, Hercules, CA).

Once it was established that the column was capable of isolating PR-A, then a SIGC membrane preparation was prepared from 10 confluent 100-mm culture dishes as previously described (16), and 1.7 mg of this membrane preparation was passed through the C-262 column. The C-262-detectable protein was eluted and collected into eight 1-ml fractions as described above. The fractions were concentrated to near dryness in a speed vacuum. The material was then resuspended in approximately 100 µl of lysate buffer, and a 10-µl aliquot of each fraction loaded onto one of two identical acrylamide gels. The gels were run at the same time within the same electrophoresis unit. After electrophoresis, one of the gels was stained with CB and the other transferred to nitrocellulose and probed with C-262 using a Western blot protocol (see Western blot for experimental details).

In a second experiment, 7 mg of membrane preparation was passed through a freshly prepared C-262 affinity column as previously described. Two 15-µl aliquots of fraction 3 were loaded onto the same gel, electrophoresed, and either stained with CB or subjected to C-262 Western blot analysis. The CB-stained band that corresponded to the C-262 band was sequenced using a Finnigan LCQ-DGCA ionTrap Mass Spectrometer. The CB-stained band was in-gel digested with sequencing-grade modified trypsin. The digested peptides were then separated on a high-resolution reverse-phase microcolumn and further analyzed by the LC-MS/MS procedure as described by Han et al. (23). Each of the fragmented tandem mass spectra was independently identified by searching a database containing protein entries from the Swiss Protein database. This work was done by the Molecular Proteomic Core (University of Connecticut Health Center, Farmington, CT).

RT-PCR analysis and cloning of RDA288
RNA from SIGCs and various organs harvested from immature rats was isolated using the RNAzol isolation system following manufacturer’s instructions (Biotecx, Houston, TX). For RT-PCR analysis, 2 µg of total RNA were converted to first-strand cDNA with Superscript II RNaseH– reverse transcriptase and oligodT (deoxythymidine) primer (Life Technologies, Gaithersburg, MD). PCR was performed using 20% of the RT reaction product as template with primers specific for the RDA288. The primers were: sense 5'-TAA CTC GAG TCA TGC CTG GGC ACC TAC AG-3' and antisense 5'-ACC AAG CTT TTA GGC CAG AGC GGG GAA TGC-3'. The sense and antisense primers contained an XhoI and Hind III sites at their respective 5' end, which was added for cloning proposes as outlined below.

The RT-PCR protocol to assess the expression of RDA288 was as follows: in addition to cDNA from SIGCs or other tissues, the PCR also contained 1x PCR buffer (Life Technologies, Gaithersburg, MD), 1.5 mM MgCl2, 1 pmol/µl of each primer, 200 µM of each deoxynucleotide (dATP, dCTP, dGTP, and 2'-deoxythymidine 5'-triphosphate, sodium salt) (Promega, Madison, WI) and 2.5 U of recombinant Taq polymerase (Life Technologies). Primer products were amplified by PCR in a thermal cycler as follows: after 4 min at 94 C, 40 cycles of the following sequence was run—94 C denaturation, 1 min; 55 C annealing 1 min; 72 C extension. An aliquot of each PCR was separated on a 1.5% agarose gel containing ethidium bromide (0.5 µg/ml) and visualized under UV light. RT-PCR to detect moesin was also run as a positive control. Rat moesin primers were as follows: sense 5'-AGTGGGCGCGCAGCCGTTAGGGAC-3' and antisense 5'-GCTATGTTGAATGAGTGTGACAAAG-3' (accession no. AF004811).

To generate a RDA288 expression vector, cDNA was prepared from mRNA isolated from SIGCs. A 1.2-kb fragment, containing the entire RDA288 open reading frame (accession no. XM_216160), was amplified by PCR using the primers described above. This strategy places a XhoI and a HindIII restriction site at the 5' and 3' ends, respectively, allowing the fragment to be cloned in frame into the pcDNA 3.1(–) vector. The identity of the insert was confirmed by DNA sequence analysis.

Western blot and immunochemical analysis
Granulosa cells and SIGCs were lysed in RIPA buffer [50 mM Tris, 150 mM sodium chloride, 1.0 mM EDTA, 1% Nonidet P-40 and 0.25% sodium-deoxycolate (pH 7.0)], which was supplemented with complete protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitor cocktail 1 (Sigma Chemical Co.) and then centrifuged at 16,000 RCF at 4 C for 5 min. Levels of 60-kDa protein were assessed by using the C-262 antibody and the RDA288-1 antibody in a Western blot analysis. The C-262 antibody purchased from StressGen and details regarding its use in Western blot protocol have been published (16).

The RDA288-1 antibody was prepared by Aves Labs, Inc. (Tigard, OR). Briefly, the following amino acid sequence (CZ KQL RKE SQK DRK N) was synthesized and injected into two hens. The resulting antibody was affinity purified and used in the immunological protocols to detect RDA288. For Western blot analysis, lysate was run on a 10% acrylamide gel and transferred to nitrocellulose. The nitrocellulose was then incubated with 5% nonfat dry milk overnight at 4 C. The nitrocellulose blot was incubated with RDA288-1 antibody at a dilution of 1:2000 (0.85 µg/ml) for 1 h at room temperature and processed for Western blot analysis using a horseradish peroxidase goat antichicken IgY (1:50,000; Aves Labs) and KPL LumiGlo detection system. As a negative control, an immunodepleted antibody preparation was used in place of the RDA288-1 antibody.

For immunohistochemical assessments, rat ovaries were removed from 25-d-old PMSG-primed rats. The ovaries were trimmed of fat, fixed in formalin, embedded in paraffin, and sectioned at 5 µm. Endogenous peroxidase activity was quenched by incubating the slides in 0.3% hydrogen peroxide in methanol for 30 min at room temperature. Slides were then incubated in BlokHen (Aves Labs) for 1 h at room temperature to reduce nonspecific staining and then incubated overnight at 4 C with RDA288-1 antibody at 1:500 dilution (3.4 µg/ml). The slides were then incubated with biotinylated goat-antichicken IgY for 30 min at room temperature, washed in PBS, and incubated with avidin-biotin complex reagent for 30 min at room temperature. The slides were developed using a diaminobenzidine-peroxidase substrate for 5 min. Finally, the slides were counterstained with methyl green for 10 sec, rinsed in distilled water, dehydrated, cleared, and mounted. As a negative control, preimmune IgY replaced RDA288-1 in this immunohistochemical protocol. The presence of RDA288 was revealed by the presence of a reddish-brown precipitate.

To determine whether RDA288 was localized to the plasma membrane, living, nonpermeabilized, nonfixed SIGCs were stained as outlined by Peluso et al. (15). This approach takes advantage of the fact that antibodies cannot enter living cells; thus, any staining detects proteins localized to the extracellular surface of the plasma membrane. Briefly, cells were rinsed with PBS and then incubated in the presence of either 34 µg/ml of RDA288-1 antibody (in 8% BSA/PBS) or immunodepleted antibody preparation for 15 min at room temperature. After this incubation, the cells were washed in PBS and incubated with FITC-IgG (1:100 in 8% BSA/PBS) for 15 min in the dark at room temperature. The cells were then washed with PBS and observed under phase and standard epi-fluorescent optics using a FITC filter set.

Ligand binding studies
The protocol used to assess total cellular binding of 3H-P4 to SIGCs has been previously described (16). Briefly, SIGCs were plated at 3.6 x 105 cells/35 mm culture dish and cultured overnight in serum-supplemented medium. The cells were washed twice in PBS and then incubated at 4 C in 500 µl of 0.1% digitonin in TEMGD buffer [10 mM Tris-HCl (pH 7.4), 1.5 mM EDTA, 10% glycerol, 25 mM sodium molybdate and 1 mM dithiothreitol]. After 30 min, 1,2,6,7-3H-P4 (1 nM 3H-P4, 50,000 cpm, specific activity = 86 Ci/mmol, Amersham, Arlington Heights, IL) was added and the incubation continued for an additional 60 min. The cells were then washed several times, harvested, and filtered through Whatman Glass Microfiber filters (GF/F) (Fisher Scientific Inc., Pittsburgh, PA), rinsed twice with 1 ml cold PBS, and then the filter counted in a scintillation counter. For the binding studies (see Fig. 4Go), the values for 3H-P4 bound to SIGCs treated with P4 and hyaluronic acid (HA) were expressed as a percentage of total counts bound to vehicle-treated SIGCs (i.e. controls). The 3H-P4 binding values (see Fig. 8Go) were expressed as a fold increase in 3H-P4 binding compared with control pcDNA3.1(–) values. All 3H-P4 binding experiments were run in duplicate with the entire experiment repeated two to three times.



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  		FIG. 4. The effect of P4 and HA on SIGC apoptosis (A and B) and 3H-P4 binding (C). For the competitive binding studies, 1 mM P4 or HA was competed against 1 nM 3H-P4. Values for 3H-P4 bound to SIGCs treated with P4 and HA were expressed as a percentage of total counts bound to vehicle-treated SIGCs (i.e. controls). In this and subsequent graphs, values are presented as means ± SEM. *, Value is significantly different from control values (P < 0.05).

 


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  		FIG. 8. The effect of forced RDA288 expression on 3H-P4 binding (A) and P4-mediated SIGC survival (C). In panel A, 3H-P4 binding is expressed as a fold increase in 3H-P4 binding compared with control pcDNA3.1(–) values. For the apoptosis study shown in panel C, SIGCs were transfected with pEGFP-C1 and either pcDNA3.1(–) or RDA288 expression vector. Transfected cells were identified by the presence of GFP (green fluorescence) and apoptosis was assessed by hydroethidine staining (red fluorescence). The effect of increasing P4 concentration on the rate of SIGC apoptosis is shown in panel B. Apoptosis was assessed by hydroethidine staining to allow for a direct comparison with the study shown in panel C.

 
Forced expression of RDA288
SIGCs were plated on either 35-mm cultures dishes for 3H-P4 binding studies or coverglass within a 35 mm culture dishes for studies involving apoptosis. The cells were cultured for 24 h in DMEM/F-12 medium that was supplemented with 5% FBS. Transfections were performed with Lipofectamine (Life Technologies) according to the manufacturer’s instructions. For the 3H-P4 binding studies, cells were transfected with 2 µg/dish of either pcDNA3.1(–) or pcDNA3.1(–)-RDA288. 3H-P4 binding was assessed 24 h after transfection using the previously described protocol (16).

For the apoptosis studies, SIGCs were cotransfected with 1 µg/dish of pEGFP-C1 DNA (CLONTECH, Palo Alto, CA) and 1 µg/dish of either pcDNA3.1(–) or pcDNA3.1(–)-RDA288. After 24 h, the serum-supplemented medium was removed and the cells placed in serum-free medium supplemented with 1 nM P4. After 5 h, the cells were rinsed three times in Krebs/HEPES buffer and stained with hydroethidine (3.5 µg/ml of Krebs/HEPES buffer) for 15 min at room temperature in the dark. After staining, the cells were rinsed three times in Krebs/HEPES buffer and observed under an epi-fluorescent optics. Under these conditions, only cells with condensed or fragmented nuclei were stained with hydroethidine. These cells were considered to be apoptotic.

To determine whether forced expression of RDA288 altered the cells ability to respond to P4, random areas within each cell culture were sequentially observed under FITC filter set and tetramethylrhodamine isothiocyanate filter set. Images of each area under each optical condition were captured and stored in a computer. By comparing the images from the same area the transfection status (FITC; green fluorescence) and viability (apoptosis; tetramethylrhodamine isothiocyanate; red fluorescence) of each cell could be determined. Approximately 100 transfected cells per culture dish were evaluated for apoptosis. The percentage of transfected apoptotic cells/treatment dish was calculated. In total, eight RDA288-transfected and six pcDNA-transfected dishes were studied. To observe about 100 transfected cells, 10 fields at x400 magnification were examined. These experiments were conducted on 5 different days.

Statistical analysis
All experiments were repeated at least two to three times with each experiment yielding essentially identical results. When appropriate, the data were pooled and analyzed by either a Student’s t test or a one-way ANOVA followed by a Student-Newman-Keuls test. Regardless of the statistical test, P values of less than 0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Recombinant PR-A was passed through the C-262 affinity column to test the effectiveness of the column. As seen in Fig. 1AGo, the column retained a protein that was the expected size of PR-A. When 1.7 mg of a crude membrane preparation were passed through this column, there was not enough protein in any of the eight fractions to be detected by CB staining (i.e. < 7 ng/ml). However, a Western blot conducted using the C-262 antibody on the gel that was run in parallel with the CB-stained gel revealed the presence of a 60-kDa protein. The majority of this staining was in fraction 3 (Fig. 1BGo). Based on this finding, 7 mg of crude membrane preparation were run through a freshly prepared C-262 affinity column and two 15-µl aliquots of fraction 3 run on the same gel with one lane of the gel ultimately stained with CB and the other subjected to C-262 Western blot analysis. This study revealed the presence of a CB-stained band that corresponded to the band identified by C-262 (Fig. 1CGo).



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  		FIG. 1. Isolation of C-262-detectable protein using a C-262 affinity column. A, A C-262 affinity column was loaded with 1 mg of recombinant PR-A. Eight 1-ml fractions were collected and 10 µl of fractions 2–8 electrophoresed on a 10% acrylamide gel. The gel was stained with a CB-based stain to reveal the presence of a protein in fraction 6 that has the apparent molecular mass of PR-A. A similar study was conducted in which 1.7 mg of crude membrane preparation were loaded onto a C-262 affinity column. Fractions were concentrated, subjected to electrophoresis, and stained with CB, but no proteins were detected. A similar gel was transferred to nitrocellulose and processed for a C-262 Western (B). This Western revealed to presence of a 60-kDa protein in fractions 3–6. C, A freshly prepared C-262 affinity column was loaded with 7 mg of crude membrane preparation and two 15-µl aliquots of fraction 3 run on adjacent lanes separated by molecular mass standard. One lane was stained with CB and the other processed for C-262 Western blot analysis.

 
The mass spectrometric analysis revealed that the C-262-detectable protein was an unnamed rat protein, referred to as RDA288 (accession no. XM_216160). A homologous unnamed protein was also detected in mouse lung (accession no. AK004678). This mass spectrometric analysis identified 11 peptide fragments with each fragment having 100% homology to a segment within RDA288 (Fig. 2Go). Furthermore, the protein coverage for this analysis was 132 of 407 amino acids (32%). A domain analysis revealed that RDA288 does not possess a transmembrane domain but has several potential HA binding sites (Fig. 2Go).



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  		FIG. 2. Amino acid sequence of RDA288 (rat) and its mouse homolog. The amino acid fragments that were sequenced are shown in bold type. The difference amino acids in the rat and mouse proteins are in italics. The putative HA binding sites are shown in boxes, and the amino acid sequence used to produce the RDA288-1 antibody is underlined.

 
RT-PCR analysis confirmed that RDA288 was expressed by SIGCs and revealed that RDA288 mRNA was present in immature and PMSG-primed rat ovaries (Fig. 3AGo). RDA288 mRNA was also detected in rat lung and skeletal muscle but not in several other rat tissues (Fig. 3BGo).



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  		FIG. 3. RT-PCR analysis of RDA288 and moesin expression in SIGCs, nonprimed and PMSG-primed immature ovaries (A) and various other tissues harvested from immature rats (B).

 
Because the mass spectrometric analysis indicated that RDA288 has HA binding sites and HA inhibits human granulosa/luteal cell apoptosis (24), studies were conducted to determine whether HA could mimic P4’s antiapoptotic action in SIGCs. These studies demonstrated that HA suppressed SIGC apoptosis in the same dose range as P4 (compare Fig. 4Go, A with B). Furthermore, HA reduced the capacity of SIGCs to bind 3H-P4 (Fig. 4CGo). Moreover, C-262 antibody treatment blocked both P4’s and HA’s antiapoptotic action (Fig. 5AGo). Similarly, HABP attenuated the antiapoptotic effects of both P4 and HA (Fig. 5BGo).



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  		FIG. 5. The effect of C-262 antibody (20 µg/ml) (A) and HABP (B) on P4-and HA-mediated SIGC survival. In panel A, the IgG and C-262 controls were pooled and shown as a single black bar.

 
To assess the cellular localization of RDA288, an antibody was built against RDA288. This antibody (RDA288-1) detected a doublet with molecular masses of 55 and 60 kDa in both SIGCs and granulosa cells (Fig. 6AGo). Immunohistochemical analysis detected RDA288 in the cytoplasm of granulosa cells of antral follicles with RDA288 often being concentrated at the plasma membrane (Fig. 6BGo).



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  		FIG. 6. The expression of RDA288 in SIGCs and granulosa cells as assessed by Western blot analysis (A). In this figure as well as in Fig. 7Go, a negative control (Neg) is also shown, which was conducted using an immune depleted antibody preparation. In this Western blot, 5 µg of protein were used. Immunohistochemical study was also conducted on adjacent sections taken from an immature PMSG-primed rat ovary stained using either preimmune IgY (negative control) or RDA288-1 antibody (B).

 
RDA288 was then cloned into a pcDNA3.1(–) expression vector. Transfection of the RDA288 expression vector increased the amount of protein that was detected by RDA288-1 (Fig. 7Go). Cotransfection with a green fluorescent protein (GFP) expression vector indicated that the transfection efficiency was approximately 30% for both empty vector controls and RDA288. Compared with empty vector controls, transfection with RDA288 expression vector increased the capacity of SIGCs to bind 3H-P4 (Fig. 8AGo) and respond to P4’s antiapoptotic action (compare Fig. 8Go, B with C). Immunocytochemical studies on living SIGCs revealed the presence of RDA288 on the extracellular surface of the plasma membrane (Fig. 9Go, A–D). This suggested that pretreatment with RDA288-1 antibody might be able to block P4 actions. As shown in Fig. 10Go, RDA288-1 pretreatment significantly attenuated P4’s antiapoptotic effects compared with the depleted antibody preparation.



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  		FIG. 7. Forced expression of RDA288 in SIGCs. SIGCs were either transfected with either pcDNA3.1(–) or pcDNA3.1(–)-RDA288. The expression of RDA288 was assessed by Western blot analysis using the RDA288-1 antibody. Note that the amount of protein used in this Western blot was much less than used in the Western shown in Fig. 6Go.

 


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  		FIG. 9. The localization of RDA288 on the extracellular surface of SIGCs. RDA288 was localized to the extracellular surface of the plasma membrane by conducting the immunofluorescent staining protocol on living nonpermeabilized, nonfixed cells. Phase (A and C) and fluorescent (B and D) images of cells stained with either RDA288 depleted antibody preparation (A and B) or RDA288-1 antibody (C and D) are shown.

 


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  		FIG. 10. The effect of RDA288-1 antibody treatment (34 µg/ml) or immune depleted antibody preparation (IgY) on P4’s ability to prevent SIGC apoptosis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study used the C-262 antibody to identify a protein that is involved in mediating P4’s actions in granulosa cells (13, 14, 15, 16, 19). The C-262 antibody was built against the amino acids, which encode the three-dimensional P4 binding site of the classic nuclear PR (25). Although granulosa cells of immature rat ovaries and SIGCs do not express the classic PR (10, 11, 16), the C-262 antibody recognizes a 60-kDa protein in both of these cells. This antibody also detects similar molecular mass proteins in sperm and murine Leydig tumor cells (21, 26). These unidentified proteins bind P4 as judged by ligand blot assays (21, 26). Further treatment with the C-262 antibody attenuates P4’s ability to bind and regulate granulosa cell function. By using a C-262 affinity column, a sufficient amount of protein was isolated so that the C-262 detectable 60-kDa protein could be sequenced. This approach identified an unnamed protein referred to as RDA288 (accession no. XM_216160). Although RDA288 does not contain the linear amino acid sequence of the P4 binding site of the PR, it is likely that RDA288 possesses an unknown amino acid sequence that results in a three-dimensional P4 binding site that is recognized by the C-262 antibody. However, this cannot be confirmed until a structural analysis of RDA288 is conducted and compared with the three-dimensional structure of the classic nuclear PR.

The RT-PCR studies suggest that RDA288 has a limited tissue distribution with the tissue distribution of RDA288 and the classic nuclear PR being rather exclusive. For example, cells within the aorta (27), uterus (28), kidney (29), and heart (27) express functional nuclear PRs but not RDA288. The lung expresses both PR (30, 31) and RDA288. However, detailed in situ hybridization studies will have to be conducted to determine whether the same cell within the lung expresses both the classic nuclear PR and RDA288. It is also interesting that cells within the intestines do not express either the nuclear PR or RDA288 (32) but are responsive to P4 (33). The mechanism through which P4 acts on the cells within the gastrointestinal tract remains to be determined.

The present study clearly shows that RDA288 is detected within the lung and skeletal muscle. Because the mouse homolog to RDA288 was cloned from mouse lung cDNA library, detecting RDA288 in rat lung was not surprising. Moreover, P4 promotes lung development as assessed by the levels of surfactant-associated protein and decreases epithelial proliferation (see Ref.30 and references therein). P4 also stimulates skeletal muscle growth possibly by increasing protein synthesis (34) and altering glucose metabolism (35). Whether RDA288 is involved in mediating any of P4’s actions in lung or skeletal muscle remains to be determined.

A structural analysis of RDA288 revealed the presence of several potential HA binding sites. Although HA is not expressed in ovarian follicles until the preovulatory period (36, 37), it was theorized that HA might be able to bind to RDA288 and thereby activate P4’s antiapoptotic pathway. In fact, the present studies demonstrate that HA mimics P4’s antiapoptotic actions. Moreover, the antiapoptotic action of both P4 and HA is attenuated by C-262 but not IgG treatment. Similarly, the antiapoptotic action of both these agents is blocked by exposure to a glial HABP. Finally, HA competes with P4 for a P4 binding site on SIGCs. These observations lend credence to the hypothesis that this putative HABP, RDA288, is involved in mediating P4’s actions. These findings are also consistent with the observations of other investigators that HA mimics P4’s actions, which include 1) stimulating transient increases in [Ca2+]i and ultimately inducing the acrosome reaction in sperm (38) and 2) preventing apoptosis of human granulosa-luteal cells (24).

The putative physiological interaction between RDA288 and HA and/or P4 is interesting. During the preovulatory period, the P4 concentrations within follicular fluid of rat antral follicles increases from 12 µM to 50 µM (4). However, the amount of P4 present within antral follicles at the other stages of the estrous cycle is in the micromolar range, which is sufficient to interact with RDA288 (4). In contrast, HA is only synthesized by the cumulus cells during the periovulatory period (37). Thus, it is likely that P4, not HA, interacts with RDA288 throughout the course of antral follicle development to regulate the rate of granulosa cell apoptosis.

The RDA288-1 antibody, which was developed against one of its potential HA binding sites, detects a 55- and a 60-kDa protein in SIGCs and granulosa cells. The presence of both the 55- and 60-kDa forms could indicate that RDA288 undergoes some type of posttranslational modification such as phosphorylation. This possibility is currently being assessed.

A subsequent immunohistochemical study using the RDA288-1 antibody confirmed that RDA288 is present in granulosa cells. Furthermore, RDA288 is not detected within the nucleus but rather in the cytoplasm with it often being concentrated at the plasma membrane. To determine whether RDA288 was localized to the extracellular surface of the plasma membrane, an immunocytochemical study was conducted on living nonpermeabilized, nonfixed cells. This approach of using living, nonpermeabilized, nonfixed cells is a standard method to localize proteins at the extracellular surface of the plasma membrane (39) because antibodies cannot enter these living cells. Thus, specific fluorescence staining, shown Fig. 9DGo, indicates that the RDA288 localizes to the extracellular surface of the plasma membrane. Finally, the observation that the RDA288-1 antibody blocks P4’s antiapoptotic action is consistent with RDA288 being localized to the extracellular surface of the plasma membrane, because the RDA288-1 antibody could only interact with surface proteins and not proteins within the cytoplasm. This membrane localization is consistent with the concept that P4’s action is mediated through a membrane-initiated mechanism (15, 19).

Forced expression of RDA288 increases 3H-P4 binding to SIGCs by 20–30%. This increase in 3H-P binding is likely greater than observed because all cells and not just transfected cells were assessed for their capacity to bind 3H-P4. Regardless, the increased binding is due to either RDA288 directly binding to P4 or RDA288 interacting with an unknown surface protein, which in turn enhances its ability to bind 3H-P4. This issue cannot be conclusively resolved until the ability of partially purified RDA288 to directly bind P4 is assessed. However, it is likely that RDA288 directly binds P4, because the C-262-detectable protein (i.e. RDA288) binds P4 in a ligand blot assay (16) and C-262 reduces the amount of FITC-BSA-P4 binding (14).

Not only does transfection with RDA288 increase 3H-P4 binding, it also increases the ability of SIGCs to respond to P4. In nontransfected and pcDNA3.1(–) transfected SIGCs, 1 nM of P4 is not sufficient to suppress apoptosis. However, if the cells are transfected with RDA288 then 1 nM suppresses SIGC apoptosis. Based on these observations, it is possible that forced expression of RDA288 increases the affinity of SIGCs to bind P4. Conversely, if the SIGCs are pretreated with the RDA288-1 antibody, then P4’s ability to suppress apoptosis is attenuated. Taken together, these studies strongly suggest that RDA288 is involved in mediating P4’s actions in SIGCs. However, the exact mechanism through which RDA288 acts remains to be determined.

Although it is unconventional to propose that a HABP mediates P4’s action, there are two known types of HABPs that act as receptors. The best-described HA receptor is CD44 (40, 41). This protein possesses a large extracellular domain that contains a HA binding site and a single transmembrane domain. However, because RDA288 does not have a transmembrane domain, it cannot function in a manner similar to CD44. The second type of HA receptor is referred to as RHAMM (receptor for HA-mediated motility). This protein is secreted and then localizes to the extracellular surface membrane where it can interact with either the receptor for platelet-derived growth factor or nerve growth factor (41). Binding of HA to RHAMM activates the growth factor receptor, resulting in the activation of protein tyrosine kinases, Src, Erk kinases, and protein kinase C (41, 42). It is possible that RDA288 functions in a manner similar to RHAMM.

Although our proposed coreceptor model is not the typical mechanism through which steroid hormones are thought to act, some of estradiol’s actions appear to be mediated by a similar coreceptor mechanism. For example, estradiol binds sex hormone-binding globulin, which in turn binds a sex hormone-binding globulin receptor in breast cancer cells (43, 44). Binding of all three components is required in order for estradiol to mediate its effects (43, 44). Therefore, the proposed RDA288-based model to explain P4’s actions is plausible and merits further evaluation.

In summary, the present studies reveal that RDA288 is selectively expressed by granulosa cells and SIGCs. This protein localizes to the extracellular surface of the plasma membrane where it either directly or indirectly increases the capacity of SIGCs to bind and respond to P4. Thus, RDA288 appears to play an essential role in mechanism by which P4 regulates granulosa cell viability.


   Acknowledgments
 
The authors would like to thank Dr. Robert Burghardt of Texas A&M University (College Station, TX) for providing the SIGC cells and Dr. Bruce A. White (University of Connecticut Health Center, Farmington, CT) for his advice and comments throughout the course of these studies. We would also like to acknowledge Dr. David Han (University of Connecticut Health Center) for his work in sequencing and identifying RDA288 by mass spectrometric analysis.


   Footnotes
 
This work was supported by grants from the National Institutes of Health (HD 334667) and University of Connecticut Health Center Foundation.

Abbreviations: CB, Coomassie blue; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; HA, hyaluronic acid; P4, progesterone; PMSG, pregnant mare’s serum gonadotropin; PR, P4 receptor; RHAMM, receptor for HA-mediated motility; SIGC, spontaneously immortalized granulosa cells.

Received January 21, 2004.

Accepted for publication February 17, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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