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Departments of Cell Biology (J.J.P., A.P.) and Obstetrics and Gynecology (J.J.P.), University of Connecticut Health Center, Farmington, Connecticut 06030; Faculty of Clinical Medicine (R.L., M.W.), Mannheim, Institute of Clinical Pharmacology, University of Heidelberg, D-68167 Mannheim, Germany; and Department of Medicine/Experimental Medicine (M.W.), AstraZeneca Research and Development, S48183 Molndal, Sweden
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.
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Introduction |
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There are at least two potential membrane P4 receptors that could account for P4’s actions in these ovarian cells. First, one of the three recently identified membrane PGRs (MPR; MPR
, MPRß, or MPR
) could be involved because MPR has been shown to be expressed in human ovary (13, 14). Although the expression pattern of MPRs during follicular development has not been published, all three MPRs are expressed in rat luteal cells (15). Unfortunately, functional studies linking these receptors to any of P4’s intraovarian actions have not been conducted.
A second protein that could mediate P4’s action is a P4 binding protein referred to as PGR membrane component-1 (PGRMC1) (16, 17). PGRMC1 has been detected in mouse granulosa (18), luteal cells (15, 19), and SIGCs (1). PGRMC1 is a relatively small protein (28 kDa) that possesses a short N-terminal extracellular domain, a single transmembrane domain, and a cytoplasm domain (20). The cytoplasmic domain has several potential Src homology 2 and Src homology 3 domains (http://scansite.mit.edu/motifscan_seq.phtml), through which ligand activation could transduce an intracellular signal. Structural analysis revealed similarities with the IL-6 receptor, which belongs to the cytokine/GH/prolactin receptor superfamily (17). Interestingly, PGRMC1 tends to form aggregates that can be as large as approximately 200 kDa, although Western blots often detect PGRMC1 as a 56-kDa dimer or a 28-kDa monomer (17, 21). Finally, PGRMC1 interacts with plasminogen activator inhibitor mRNA binding protein 1 (PAIRBP1) (1). Because PAIRBP1 localizes to the plasma membrane and is involved in transducing P4 membrane-initiated actions (22), the present studies were designed to determine PGRMC1’s ovarian expression pattern and its role in mediating P4’s antiapoptotic actions.
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Materials and Methods |
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SIGC cultures
SIGCs were generously provided by Dr. Robert Burghardt (Texas A & M University, College Station, TX) and cultured as previously described (23). Unless otherwise indicated SIGCs were plated at 1 x 106 cells in 35-mm culture dishes.
Western blot and immunochemical analysis
Freshly isolated granulosa cells and luteal cells as well as SIGCs were lysed in radioimmunoprecipitation assay buffer [50 mM Tris, 150 mM sodium chloride, 1.0 mM EDTA, 1% Nonidet P40, and 0.25% sodium-deoxycholate (pH 7.0)], which was supplemented with complete protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitor cocktail 1 (Sigma Chemical Co., St. Louis, MO) and then centrifuged at 1000 x g at 4 C for 5 min. The supernatant was collected and centrifuged at 100,000 x g at 4 C for 1 h (8). Twenty micrograms of this membrane preparation were run on a 12% acrylamide gel and transferred to nitrocellulose. The nitrocellulose was then incubated with 5% nonfat dry milk overnight at 4 C. The nitrocellulose blot was then incubated with either the chicken PAIRBP1 antibody at a dilution of 1:2000 (22) or the rabbit PGRMC1-NT antibody (1:2000) (24) for 1 h at room temperature. Western blots were processed using a horseradish peroxidase goat antichicken IgY (1:50,000; Aves Labs, Tigard, OR) or a horseradish peroxidase goat antimouse antibody (1:10,000). A LumiGlo detection system (KPL, Gaithersburg, MD) was used to reveal the presence of both proteins. As a negative control, an immunodepleted antibody preparation or rabbit IgG was used in place of the PAIRBP1 antibody and PGRMC1-NT antibody, respectively.
For immunohistochemical assessments, rat ovaries were sectioned at 5 µm and mounted on glass slides. 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 either PAIRBP1 antibody (1:500 dilution) or PGRMC1-NT (1:100) antibody. The slides were then incubated with either biotinylated goat-antichicken IgY or biotinylated goat-antimouse IgG 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 (DAB)-peroxidase substrate for 5 min. Finally, the slides were counterstained with methyl green for 10 sec, rinsed in distilled water, dehydrated, cleared, and mounted. The presence of PAIRBP1 and PGRMC1 was revealed by the presence of a reddish brown precipitate. Negative controls for the immunohistochemical studies were run as described for the Western blots.
SIGCs grown on glass coverslips within 35-mm culture dishes were used for the confocal studies. These cells were washed and then fixed in 10% formalin as previously described. The coverslips were then incubated overnight at 4 C with the antibodies to PAIRBP1 (1:50), PGRMC1-NT (1:50), or both. After washing to remove the primary antibodies, the coverslips were incubated for 1 h at room temperature in the dark with Alexa Fluor 633-goat antichicken IgG (1:100) and Alexa Fluor 488-goat antirabbit IgG (1:100). The coverslips were again washed and observed under the confocal microscopy. Negative controls were also processed as described above with the exception that the immunodepleted antibody preparation or IgG was used in place of the PAIRBP1 or PGRMC1-NT antibody, respectively.
Biotinylation of proteins at the extracellular surface of the plasma membrane
To determine whether PAIRBP1 and PGRMC1 localize to the extracellular surface of the plasma membrane, the plasma membrane proteins were biotinylated using the EZ-link sulfo-NHS-LC-LC-biotin reagent and protocol provided by Pierce (Rockford, IL) (25). Once these proteins were biotinylated, cell lysates were prepared. A 20-µg sample of this lysate was assessed for the ß-actin expression by Western blot using a monoclonal anti-ß-actin antibody (clone AC-15; 1:1000 dilution; Sigma). The plasma membrane proteins in the remainder of the sample were isolated using the Ultralink immobilized streptavidin protocol provided by Pierce. Twenty micrograms of the avidin-purified material were then run on a 12% acrylamide gel. The presence of PAIRB1 and PGRMC1 at the plasma membrane was determined by Western blot as previously described.
Overexpression of PGRMC1 and P4 binding studies
SIGCs were transfected with either 2 µg /dish of either pcDNA3.1(–) or pcDNA3.1(–)-PGRMC1 (16) as previously described (22). For this procedure, SIGCs were plated at 3.6 x 105 cells/35-mm culture dish and cultured overnight in serum-supplemented medium. The cells were then washed twice in PBS and then incubated at 4 C in 500 µl of 0.1% digitonin in a buffer of 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-progesterone (1 nM 3H-P4, 50,000 cpm, SA = 86 Ci/mmol; Amersham, Arlington Heights, IL), and either vehicle or 1 µM P4 was added and the incubation continued for an additional 60 min. The cells were then washed three times, harvested, and filtered through Whatman glass microfiber filters (Fisher Scientific Inc., Pittsburgh, PA), rinsed four times with 1 ml cold PBS, and then the filter counted in a scintillation counter. Specific binding was determined by subtracting the counts per minute obtained in the presence of 1 µM P4 from the cpm obtained in the absence of P4. Means ± 1 SE of each P4 binding parameter was calculated for each treatment group (9). Lysates were also prepared from cells transfected with either empty vector or pcDNA(3.1)-PGRMC1 lysates and analyzed by Western blot as previously described.
Overexpression of PGRMC1 and SIGC apoptosis
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(–)-PGRMC1 (22). 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 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 epifluorescent optics. Under these conditions only cells with condensed or fragmented nuclei were stained with hydroethidine. These cells were considered to be apoptotic (22).
To determine whether overexpression of PGRMC1 altered the cell’s ability to respond to P4, random areas within each cell culture were sequentially observed under the fluorescein isothiocyanate filter set and the 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 (fluorescein isothiocyanate-green fluorescence) and viability (apoptosis; tetramethylrhodamine isothiocyanate-red fluorescence) of each cell could be determined (22). Approximately 200 transfected cells per culture dish were evaluated for apoptosis. The percentage of transfected apoptotic cells per treatment dish was calculated. In total seven PGRMC1-transfected and six pcDNA-transfected dishes were studied.
Before this study a P4 dose-response study was conducted with nontransfected cells to determine a dose of P4 that would not inhibit SIGC apoptosis. In this study, apoptosis was assessed by in situ DNA staining using hydroethidine as described above.
PGRMC1 blocking antibody study
SIGCs were placed at 6 x 104/0.5 ml in eight-chamber glass lab tek slides overnight. The cells were then washed in serum-free medium and cultured for 5 h with either serum-free media supplemented with rabbit IgG (20 µg/ml) or PGRMC1 (20 µg/ml) in the presence or absence of P4 (0.1 µM). After culture the cells were raised in Krebs/HEPES buffer and stained with YOPRO1 to detect apoptotic nuclei (23). One hundred cells in each chamber were counted, and the percentage of apoptotic nuclei determined as previously described.
Statistical analysis
All experiments were repeated at least three times with each experiment yielding essentially identical results. When appropriate, the data were pooled to generate means ± SE and analyzed by either a Student’s t test or a one-way ANOVA followed by a Student-Newman-Keuls test, if more than two treatments groups were being compared. P < 0.05 was considered to be significant.
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Results |
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). The immunohistochemical studies confirmed this and also demonstrated that thecal/stromal cells and ovarian surface epithelial cells expressed both of these proteins (Fig. 2). In addition PGRMC1 was detected in oocytes (Fig. 2). A definite conclusion regarding PAIRBP1 expression in oocytes could not be made because oocytes in the negative controls were DAB positive (Fig. 2A, lower panel).
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, B and C, upper panel). In a few granulosa cells before eCG treatment, PGRMC1 appeared to be concentrated at or near the plasma membrane, with its expression restricted to a limited section of the plasma membrane (arrow in Fig. 2C, upper panel). After eCG treatment the localization dramatically changed such that PGRMC1 expression was almost exclusively at or near select regions of the plasma membrane (Fig. 2D, upper panel). With hCG induction of ovulation and luteinization, PGRMC1 expression increased as assessed by both Western blot (Fig. 1) and immunohistochemical analysis (Fig. 2, E and F, upper panel). Unlike granulosa cells, 100% of the luteal cells expressed high levels of PGRMC1, with PGRMC1 being distributed throughout the cell (Fig. 2, E and F, upper panel).
PAIRBP1 showed a similar pattern of expression in granulosa cells with the exception that PAIRBP1 was always localized at or near the plasma membrane of the granulosa cells (Fig. 2C, lower panel). eCG treatment increased the level and number of granulosa cells expressing PAIRBP1 as assessed by immunohistochemistry but did not change its cellular distribution (Fig. 2D, lower panel). As with PGRMC1, PAIRBP1 was localized throughout the cytoplasm of luteal cells (Fig. 2, E and F, lower panel).
To determine whether these proteins were localized to the extracellular surface of the plasma membrane, the surface membranes of SIGCs were biotinylated and then avidin affinity purified. The affinity-purified proteins were then probed with antibodies to either PGRMC1 or PAIRBP1. This study revealed that both proteins were among the avidin-affinity purified proteins, indicating that they were present at the extracellular surface of the plasma membrane. The failure to detect ß-actin among the biotinylated proteins, confirms the specificity of this approach (Fig. 3). In addition confocal analysis revealed that both proteins colocalized to the plasma membrane and cytoplasm but not to the nucleus (Fig. 4), although in some areas along the plasma membrane, PGRMC1 did not colocalize with PAIRBP1 (Fig. 4).
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). This increase in PGRMC1 expressed correlated with a significant increase in the capacity of SIGCs to specifically bind 3H-P4 (Fig. 5B).
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, P4 induced a dose-dependent decrease in the percentage of apoptotic nuclei observed 5 h after serum withdrawal. This decrease in apoptosis was observed with 10 nM but not 1 nM P4. Cotransfection with pEGFP-C1 DNA revealed that about 40% of the cells expressed green fluorescent protein (GFP). In those cells transfected with GFP and pcDNA3.1-PGRMC1, the percentage of apoptosis after a 5-h exposure to 1 nM P4 was lower that those cells cotransfected with GFP expression vector and pcDNA3.1 (Fig. 6B). Conversely, treatment an antibody that was built against the extracellular domain of PGRMC1 attenuated P4’s antiapoptotic action. This inhibition of P4’s action was not observed in serum-free medium in the presence or absence of IgG (Fig. 7).
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Discussion |
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As with PAIRBP1, PGRMC1 is expressed in virtually 100% of the thecal/stromal cells. Based on expression alone, it is tempting to speculate that P4 acting through these proteins could influence the biology of thecal/stromal cells. However, no studies of which we are aware directly assess the effect of P4 on thecal/stromal cell biology. The presence of PGRMC1 in these cells indicates that assessing P4’s actions in thecal/stromal cells may be warranted.
P4 influences ovarian surface epithelial cells as well as ovarian epithelial cancers (26). In vitro studies have shown that P4 inhibits proliferation of several human ovarian surface epithelial cell lines (27). Because these cells express the PGR (28), it has been assumed that P4’s actions are mediated via these receptors. However, P4 exhibits antimitotic action only at micromolar doses (27). These dose-response characteristics make it difficult to attribute P4’s actions to the PGR, given that the dissociation constant (Kd) for the PGR is 1–5 nM (29). Interestingly the Kd for PGRMC1 is in the 0.20–0.3 µM range (21). Because ovarian surface epithelial cells express PGRMC1 and PAIRBP1, the possibility exists that PGRMC1 transduces P4’s antimitotic action in these cells. This concept is consistent with our previous observation that at micromolar doses, P4 inhibits granulosa cell and SIGC mitosis (8).
Our immunohistochemical study detected PGRMC1 in the oocytes. PGRMC1 mRNA has also been detected in monkey oocytes (Zelinski, M., unpublished observation), thereby supporting the conclusion that PGRMC1 is expressed in mammalian oocytes. It has been known for a long time that P4 acts on amphibian and fish oocytes to induce maturation (i.e. resumption of meiosis) (30). Independent evidence provided by the laboratories of Bayaa et al. (31) and Ruderman and colleagues (32), respectively, suggest that a PGR-like protein mediates P4’s action in amphibian oocytes, whereas data from the laboratory of Thomas and colleagues (13, 14) suggest that the MPR is responsible for the maturation inducing activity of P4 in fish oocytes. Unlike its action in amphibian and fish oocytes, P4 does not induce oocyte maturation in mammals. Rather it acts synergistically with cAMP-elevating agents to inhibit oocyte maturation (33, 34). Because PGRMC1 is expressed in mammalian oocytes, it could be part of the mechanism through which P4 inhibits the resumption of meiosis. This action would be similar to P4’s antimitotic action in granulosa cells (35), SIGCs (8), and ovarian surface epithelial cells (27). However, PGRMC1’s involvement in regulating oocyte maturation remains to be tested.
Unlike thecal/stromal and ovarian surface epithelial cells and oocytes, PARIBP1 and PGRMC1 expression is induced during the differentiation of granulosa cells into luteal cells (present study and Ref.18). Sasson et al. (19) have shown that cAMP-elevating agents increase the mRNA levels of PGRMC1 in cultured human granulosa/luteal cells, suggesting that the increased expression in vivo is due to a gonadotropin-induced increase in intracellular cAMP. Structural studies of the promoter region of the human PGRMC1 gene indicate that it contains several cis-regulatory DNA motifs such as activator protein 2, nuclear factor of activated T cells, aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator, and CCAAT/enhancer-binding protein (36). However, it has not been established that cAMP promotes the transcription of PGRMC1 through any of these DNA sites.
What is clear is that the levels of PGRMC1 are not only greater in luteal cells than granulosa cells but also this protein is present throughout the cytoplasm of luteal cells. These expression studies imply that PGRMC1 is involved in enhancing steroidogenesis, which characterizes granulosa cell luteinization. There are two experimental observations that support a role for PGRMC1 in steroidogenesis. These include the observation that an antibody to PGRMC1, which is also known as inner zona-specific antigen, inhibits the in vitro conversion of P4 into deoxycorticosterone in rat adrenal inner zone microsomal fractions (37). The second finding is that overexpression of PGRMC1 increases the 21-hydroxylation of P4 and deoxycorticosterone production (20).
The mechanism through which PGRMC1 could modulate ovarian steroidogenesis might be related to its potential role in regulating cholesterol metabolism (38). It is well documented that most of the PGRMC1 localizes to the endoplasmic reticulum (16, 17, 21). Three proteins that ultimately regulate cholesterol metabolism also assemble within the endoplasmic reticulum. These proteins are sterol regulatory element binding protein (SREBP), SREBP cleavage-activating protein (SCAP), and insulin-induced gene-1 (INSIG1). INSIG1 binds to SCAP and SCAP binds to SREBP. This localizes SREBP to the endoplasmic reticulum. In sterol-depleted cells, SCAP escorts SREBP to the Golgi complex in which it is cleaved. This releases the N-terminal segment of SREBP, which allows SREBP to enter the nucleus where it induces the expression of various genes involved in cholesterol synthesis including steroidogenic acute regulatory protein (StAR) (39). It is likely that this system is present in human granulosa-luteal cells because the StAR promoter is up regulated by SREBP-1a (40).
With the observation that PGRMC1 binds to both INSIG1 and SCAP (38), it is possible that this complex could function as an intracellular P4 binding site, with the binding of P4 ultimately activating SREBP. This mechanism would account for P4’s ability to induce StAR expression in MA-10 (41) and promote cholesterol and P4 synthesis in human and rat granulosa cells (42). Because PGRMC1 also binds to PAIRBP1, how this interaction is influenced by P4 and how this might affect the formation of the PAIRBP1-INSIG1-SCAP complex remains an open question.
In addition to the capacity to promote its own synthesis, P4 has antiapoptotic effects in both granulosa and luteal cells (1). This action is rapid, occurring within minutes; is initiated at the plasma membrane; and involves the activation of protein kinase G and the suppression of intracellular free calcium (10, 43). The present studies used several different approaches to demonstrate that PGRMC1 is an essential component of P4’s membrane-initiated antiapoptotic action. First, PGRMC1 and PAIRBP1 were shown to localize to the extracellular surface. This was demonstrated by avidin purification of biotinylated surface proteins and was confirmed by confocal microscopy. Second, overexpression of PGRMC1 increased both P4 binding and the responsiveness of SIGCs to P4’s antiapoptotic actions. Finally, an antibody directed against the extracellular domain of PGRMC1 blocked P4’s capacity to inhibit apoptosis.
In summary, the present expression studies reveal that PGRMC1 is coexpressed with PAIRBP1 within the rat ovary in a cell-specific manner. Although PGRMC1 is expressed in thecal/stromal cells, ovarian surface epithelial cells and oocytes, its expression of these ovarian cells is not dependent on gonadotropins. Gonadotropins do induce the expression of PGRMC1 as part of mechanism through which granulosa cells differentiate into luteal cells. The high level of expression of both PAIRBP1 and PGRMC1 observed throughout the cytoplasm of steroidogenically active luteal and thecal/stromal cells suggests an involvement of these proteins in steroidogenesis. Moreover, overexpression and blocking antibodies demonstrate that PGRMC1 is an essential component in the mechanism by which P4 mediates its antiapoptotic action, which is observed in both granulosa and luteal cells. Taken together these findings indicate that PGRMC1 and PAIRBP1 play important roles in regulating P4’s intraovarian actions. Future studies must now be directed toward defining their precise mechanism of action.
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Acknowledgments |
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Footnotes |
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J.J.P., A.P., R.L., and M.W. have nothing to declare. M.W. is currently employed by AstraZeneca R&D Molndal.
First Published Online March 2, 2006
Abbreviations: DAB, Diaminobenzidine; eCG, equine chorionic gonadotropin; GFP, green fluorescent protein; hCG, human chorionic gonadotropin; INSIG1, insulin-induced gene-1; MPR, membrane PGR; P4, progesterone; PAIRBP1, plasminogen activator inhibitor RNA binding protein-1; PGR, progesterone receptor; PGRMC1, PGR membrane component-1; SCAP, SREBP cleavage-activating protein; SIGC, spontaneously immortalized granulosa cell; SREBP, sterol regulatory element binding protein; StAR, steroidogenic acute regulatory protein.
Received January 26, 2006.
Accepted for publication February 22, 2006.
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. Biol Reprod 71:1870–1878
-dependent pathway. Endocrinology 141:4209–4217 and -ß, progesterone receptor, and androgen receptor mRNA in normal and malignant ovarian epithelial cells. Proc Natl Acad Sci USA 96:5722–5727This article has been cited by other articles:
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