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Ovary |
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Departments of Cell Biology3 and Obstetrics
Gynecology,4 University of Connecticut Health Center, Farmington, Connecticut 06030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-specific inhibitor, DT-3, and a dominant negative PKG-1. Further, the type I isoform of PKG was shown to be expressed by SIGCs and activated by P4. P4's antiapoptotic action was not affected by the PKA inhibitor, KT5720. Collectively, these findings indicate that P4 maintains SIGC viability by activating PKG-1. PKG-1-GFP was shown to localize predominantly to the cytoplasm of SIGCs. To identify potential cytoplasmic targets of PKG-1, SIGCs were cultured for 5 h with P4 in the presence or absence of DT-3. Cell lysates were prepared and subjected to two-dimensional electrophoresis. The resulting gels were sequentially stained with ProQ-Diamond Gel Stain and Coomassie Blue to reveal phosphorylated proteins. The two-dimensional gels revealed one major protein, the phosphorylation status of which was abrogated by DT-3. Mass spectrometric analysis identified this protein as 14-3-3
, with 14-3-3 being phosphorylated on tyrosine 19, serine 28, serine 69, serine 74, threonine 90, threonine 98, and serine 116. Finally, difopein, a specific 14-3-3 inhibitor, was shown to induce apoptosis even in the presence of serum. These data suggest that 1) P4 regulates the phosphorylation status of 14-3-3 through a PKG-dependent pathway and 2) 14-3-3 plays a central and essential role in maintaining the viability of SIGCs.
apoptosis, cyclic guanosine monophosphate, granulosa cells, kinases, progesterone, protein kinase G, signal transduction, 14-3-3
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the apoptotic mechanism is conserved, different survival factors activate different pathways to inhibit the apoptotic cascade. For example, gonadotropins increase cAMP, with this increase in cAMP accounting for the antiapoptotic actions of gonadotropins [6, 7]. The antiapoptotic action of gonadotropins can be mimicked by moderate doses of cAMP analogues, although high doses of cAMP-elevating agents often induce granulosa cell apoptosis [6, 8]. It is generally believed that the antiapoptotic action of cAMP is due to the activation of protein kinase A [6]. Other survival factors, such as epidermal growth factor, insulin-like growth factor-1, and basic fibroblastic growth factor, ultimately activate protein kinase C (PKC) [6]. As might be expected, phorbol esters, which directly activate PKC, prevent and PKC inhibitors induce granulosa cell apoptosis [9, 10]. Finally, nitric oxide and other agents that increase cGMP promote granulosa cell viability [11–13]. Cyclic GMP activates PKG with PKG agonists promoting and PKG antagonists inhibiting granulosa cell viability [11–13]. Interestingly, cGMP prevents apoptosis of granulosa cells of both preantral and antral follicles [14]. Although cGMP and PKG promote the granulosa cell viability throughout the course of follicular development, very little is known about the role of PKG in regulating granulosa cell viability.
In nonovarian cells, PKG can activate various kinases, including c-jun, c-myc, ERK 1/2, PI3 kinase, and AKT kinase [15, 16]. The activation of some or all of these signal transduction components is associated with cell survival [15–19]. Conversely, in human colon cells, PKG activates the JNK-1 pathway [20], which ultimately results in these cells undergoing apoptosis [20]. Collectively, these studies demonstrate that the downstream pathways that are activated by PKG are very complex and cell specific. Cell-specific responses to PKG are most likely a function of which PKG targets are expressed and activated (i.e., phosphorylated).
Our previous studies have shown that P4 maintains the viability of both granulosa cells and spontaneously immortalized granulosa cell (SIGCs) [5]. These studies also indicate that P4 does so by activating membrane-initiated events that control the activity of various kinases [21, 22]. In an attempt to identify the kinases that might be involved in maintaining granulosa cell viability, a kinase screen of lysates prepared from SIGCs in the presence or absence of P4 was conducted [21]. This screen suggested that PKG might be involved in P4's actions. Therefore, the present studies were designed to determine whether PKG mediates P4's antiapoptotic actions and, if so, to identify potential PKG targets that are expressed and activated as part of the P4-mediated survival pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The following reagents were used in these experiments. The concentration at which they were used is shown in parentheses. Progesterone (P4, 1 µM), 8-br-cAMP (100 µM) and 8-br-cGMP (20 µM) were purchased from Sigma Chemical Co. (St. Louis, MO). Rp-8-pCPT-cGMP (100 µM) and the PKG-1 inhibitor, DT-3 (0.25 µM) were obtained from Calbiochem (La Jolla, CA). The PKA inhibitor, KT-5720 (25–100 nM) and PKG inhibitor, KT5823 (100–200 µM) was provided by Alomone Labs (Jerusalem, Israel). See Table 1 for more details regarding these inhibitors.
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Animals and Granulosa Cell Culture
Immature female CF-1 mice (21 days of age) were obtained from Charles River Laboratory (Wilmington, MA) and housed under controlled conditions of temperature, humidity, and photoperiod (12L:12D; lights-on at 0700 h). On the day of the experiment, immature animals were 23 or 25 days of age. The mice were killed (cervical dislocation) between 0930 and 1000 h and the ovaries removed. Granulosa cells were isolated from antral follicles as previously described [23], 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.
The granulosa cells 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 Dulbecco modified Eagle medium (DMEM)/F-12 supplemented with 5% fetal bovine serum for 24 h. The serum-supplemented medium was removed; the cells were rinsed with three changes of serum-free medium and then cultured in serum-free DMEM/F-12 with various reagents for 5 h.
Spontaneously Immortalized Granulosa Cell Culture
SIGCs were generously provided by Dr. Robert Burghardt of Texas A&M University (College Station, TX) and cultured as previously described [10]. Prior to treatment, the serum-supplemented medium was removed; the cells were rinsed with three changes of serum-free medium and then cultured in serum-free DMEM/F-12 as described for the granulosa cell cultures. For most studies involving apoptosis, SIGCs were plated in eight-chamber glass lab-tek slides (Nunc Inc.) as outlined above. Studies requiring a larger amount of cells were plated in 100-mm glass dishes and assigned to treatment when the dishes were approximately 70% confluent.
Detection of Granulosa Cell Apoptosis
Unless stated otherwise, the percentage of cells with apoptotic nuclei was assessed by in situ staining using the nuclear dye YOPRO-1 [10]. To stain apoptotic cells, YOPRO-1 was added directly into each culture chamber at a final concentration of 10 µM. The cells were incubated for 10 min at 37°C and then observed at a magnification of 200x under fluorescent optics using the fluorescein isothiocyanate (FITC) filter set (Omega Optical, Brattleboro, VT). The number of fluorescent cells (i.e., apoptotic cells) in a field was counted. The total number of cells in that field was counted under phase optics. A total of 100 cells per well were counted. The percentage of apoptotic cells was then calculated.
Western Blot Analysis
SIGCs were lysed in RIPA buffer (50 mM TRIS, 150 mM sodium chloride, 1.0 mM EDTA, 1% Nonidet P40, 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 relative centrifugal force at 4°C for 5 min. Protein was determined using the BCA protein assay (Bio-Rad, Hercules, CA). Levels of vasodilator-stimulated phosphoprotein (VASP), VASP-ser239 and PKG-1, were determined by Western blot analysis using antibodies supplied by Calbiochem. The antibodies were used at the dilutions recommended by the supplier.
For Western blot analysis, lysates were run on a 10% acrylamide gel for 2 h at 100 V and transferred to nitrocellulose. The nitrocellulose was stained with Ponceau S and photographed. The nitrocellulose blot was then incubated with 5% nonfat dry milk for 1 h at room temperature. The nitrocellulose blot was incubated with the various antibodies overnight at 4°C and processed for Western blot analysis using a horseradish peroxidase goat anti-mouse IgG and KPL LumiGlo detection system, as previously described [9].
PKG activity was estimated by monitoring the level of serine 239 phosphorylated VASP. VASP is a well-characterized PKG substrate that is preferentially phosphorylated by PKG, with the amount of VASP-serine 239 phosphorylation routinely used as a monitor of PKG activity in intact cells or cell lysates [24–26].
Localization and Effect of a Dominant Negative PKG on P4-Regulated SIGC Viability
SIGCs were plated on a coverglass within a 35-mm culture dish. 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, Rockville, MD) according to the manufacturer's instructions. SIGCs were transfected with 2 µg/dish of either pEGFP-N1 (Clontech, Palo Alto, CA), PKG-1-GFP, or G1R-GFP, a dominant negative PKG [24]. The PKG-1-GFP and G1R-GFP vectors were provided by Dr. Darren Browning (Medical College of Georgia). By 24 h after transfection, the percentage of transfected cells ranged between 30% and 40% for all of the GFP expression vectors. Some of the cultures were observed under the confocal microscope to determine the cellular localization of both the PKG-1-GFP and the dominant negative PKG.
In order to determine whether the cellular localization of the PKG-1-GFP constructs changed over the 5-h culture period, SIGCs were transfected with pEGFP-N1, PKG-1-GFP, or G1R-GFP as described. After 24 h of culture in serum-supplemented medium, the serum was removed and replaced with serum-free medium. Approximately 10 areas within each culture dish were observed under epifluorescence at a magnification of 400x and then the dishes returned to a 5% CO2, 37°C environment. These same cultures were observed again at 1, 3, and 5 h of culture. This study was repeated on four separate days, with between 50 and 100 individual cells observed for each treatment group at each time point. The fluorescent intensity associated with the cytoplasmic and nuclear components of each cell was determined using image analysis and a nuclear-to-cytoplasmic ratio calculated.
To assess the ability of these transfected cells to respond to P4, the serum-supplemented medium was removed after 24 h of culture and the cells placed in serum-free medium supplemented with 1 µM P4. At 2.5 and 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 epifluorescent optics. Under these conditions, only cells with condensed or fragmented nuclei were stained with hydroethidine. These cells were considered to be apoptotic.
The rate of apoptosis was determined by selecting random areas within each cell culture and sequentially observing these areas under a FITC filter set and a Tetramethylrhodamine isothiocyanate (TRITC) filter set (Omega Optical). 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; TRITC—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. This entire experiment was conducted on 3 different days with all treatments assessed on each day.
Two-Dimensional Electrophoresis and Proteomic Analysis
Two 100-mm glass culture dishes were incubated with P4 or P4 plus DT-3 in serum-free medium. After 5 h, the cells were harvested and lysed in RIPA buffer as described above. All samples were precipitated before two-dimensional gel electrophoresis to minimize nonspecific staining according to the protocol described by Steinberg et al. [27]. Briefly, lysates (150 µg) were concentrated using a chloroform/methanol precipitation procedure [28] and the pellets resuspended in Bio-Rad Ready Prep two-dimensional rehydration/sample buffer. The samples were then applied to Bio-Rad ReadyStrips IPG (7 cm; pH 4–7) by passive rehydration according to the manufacture's instructions. The strips were then placed in a Bio-Rad Protein IEF cell and focused using the linear program for the 7-cm IPG strips as outlined in the manufacturer's instructions. The strip was then placed on top of a 10% acrylamide gel and electrophoresed for 2 h at 100 V.
To detect the phosphorylated proteins, the gels were stained with ProQ-Diamond Phosphoprotein Gel Stain according to the protocol provided by Molecular Probes (Eugene, OR). The ProQ-Diamond stain detects phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins by noncovalently binding to the phosphate moiety of the phosphorylated proteins. This stain has been shown to discriminate between phosphorylated and nonphosphorylated proteins with a high specific-to-nonspecific staining ratio [27, 28]. Images of the ProQ-Diamond-stained gels were acquired using a FluorImager Si with an excitation of 488 nm and a 515-nm bandpass emission filter. The gels were then washed, stained with Coomassie Blue (CB) for 1 h, and washed overnight to visualize the protein spots.
A CB-stained spot that was present in the P4 and P4 plus DT-3 groups but was not stained with the ProQ-Diamond after the P4 plus DT-3 treatment was sequenced using a Finnigan LCQ-DECA ionTrap Mass Spectrometer with the data-dependent mass spectrometry (MS)/MS capability. The CB-stained spot was dissected out of the gel and digested with sequencing-grade modified trypsin. The digested peptide was then separated on a high-resolution reverse-phase microcolumn and further analyzed by the liquid chromatography-MS/MS procedure as described by Han et al. [29]. Each of the fragmented tandem mass spectra was independently identified by searching a database containing protein entries from the Swiss Protein database using the SEQUEST algorithm. To determine which site or sites were phosphorylated, the 14-3-3 spot was dissected from three gels obtained from P4-treated lysates and the spots pooled into a single sample. This sample was then analyzed for phosphorylated amino acids as outlined by Han et al. [30]. This work was done at the Proteomics and Biological Mass Spectrometry Core Facility (University of Connecticut Health Center, Farmington, CT).
Role of 14-3-3 in Regulating SIGC Viability
To assess 14-3-3's role in regulating granulosa cell viability, a specific 14-3-3 inhibitor, difopein, was used. Difopein binds to all 14-3-3 isoforms with high affinity and specificity without having to be phosphorylated and effectively dissociates 14-3-3 from its binding partners [31]. Because difopein is not cell permeable, an empty vector (control) and difopein green fluorescent protein expression vectors were transfected into SIGCs as previously described. The difopein expression vector (pSCM138) was provided by Dr. H. Fu (Emory University School of Medicine). The transfection rate was approximately 30% for both this expression vector and the empty vector control. After transfection, the SIGCs were maintained in serum-supplemented medium for 24 h. The percentage of apoptotic-transfected cells in the presence of serum was determined as previously described. This experiment was repeated five times on 2 different days.
Statistical Analysis
All experiments were repeated at least three times, with each experiment yielding essentially identical results. When appropriate, the data were pooled and analyzed by either a Student t-test (the difopein experiment) or a one-way ANOVA followed by a Student-Newman-Keuls test. P values of less than 0.05 were considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because 8-br-cGMP is an activator of PKG, the effect of various PKG antagonists on P4's ability to maintain SIGC viability was assessed. Both the PKG antagonist, Rp-8-pCPT-cGMP, and the PKG-1-specific inhibitor, DT-3, attenuated P4's action (Fig. 3, A and B). Similar studies revealed that the PKG inhibitor KT5823 also attenuated the antiapoptotic action of both P4 and 8-br-cGMP when used at 100 and 200 µM, respectively (data not shown). Further, P4 induced PKG activity as shown by an increase in serine239 VASP (Fig. 4).
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Western blot analysis revealed that SIGCs express PKG-1 (Fig. 5A). To confirm that PKG-1 was mediating P4's action, cells were transfected with a GFP vector that also encodes a dominant-negative form of PKG-1 (G1R-GFP). The SIGCs that expressed the dominant-negative form of PKG-1 underwent apoptosis in the presence of P4, while the viability of cells transfected with either the control GFP vector or the PKG-1-GFP vector was preserved by P4 treatment (Fig. 5B).
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Prior to culture, PKG-1-GFP and the dominant negative PKG-GFP fusion proteins were preferentially localized within the cytoplasm (nuclear/cytoplasmic ratios of 0.73 ± 0.02 and 0.71 ± 0.03, respectively; Fig. 6, B and C). Both of these GFP fusion proteins remained within the cytoplasm and did not enter the nucleus during the 5-h culture period in serum-free media (nuclear/cytoplasmic ratios of 0.74 ± 0.02 and 0.75 ± 0.02, respectively). Similarly, P4 treatment did not significantly alter the distribution pattern of either of these GFP-fusion proteins over the 5-h culture period. In contrast, the GFP encoded by pEGF-N1 tended to be localize within the nucleus (nuclear/cytoplasmic ratio of 1.76 ± 0.03; Fig. 6A) and its localization did not change throughout the culture period (nuclear/cytoplasmic ratio of 1.74 ± 0.04).
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Given that most of the PKG-1-GFP localizes within the cytoplasm, it is likely that the protein targets of PKG are within the cytoplasm. An analysis of the two-dimensional gels prepared from whole-cell lysates of SIGCs treated for 5 h with P4 or P4 plus DT-3 revealed that DT-3 treatment did not dramatically alter the number or pattern of intensely CB-stained proteins (compare Fig. 7A with 7B). The major effect of DT-3 was to reduce the number phosphorylated proteins as assessed by ProQ-Diamond staining (compare Fig. 7C with 7D). Importantly, there was one major protein that was readily detectable by CB staining after P4 and P4 plus DT-3 treatments and was phosphorylated in the P4 group but not in the P4 plus DT-3 group (see arrow in Fig. 7, A–D).
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Mass spectrometric analysis identified this protein as 14-3-3. This identification was based on the fact that three peptide sequences that showed 100% homology with 14-3-3 were detected. These three fragments comprised 18% of the entire amino acid sequence of 14-3-3. A subsequent analysis confirmed our initial analysis and identified five fragments that comprised 25% (63 of 248 amino acids) of 14-3-3. This analysis revealed that the following seven amino acids were phosphorylated: tyrosine 19, serine 28, serine 69, serine 74, threonine 90, threonine 98, and serine 116 (Fig. 8).
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To assess 14-3-3's role in regulating granulosa cell viability, the specific 14-3-3 inhibitor, difopein, was transfected into SIGCs. After 24 h in serum-supplemented medium, the percentage of transfected apoptotic nuclei averaged 30% ± 2% (n = 5 with 1131 cells examined). This rate of apoptosis was significantly greater than that observed for the empty vector control cells (n = 5, 11% ± 2%; 1180 cells examined; P < 0.05; Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast, P4's antiapoptotic action is mediated by a PKG-dependent mechanism. This conclusion is based on several different types of evidence. First, PKG-1 is expressed by SIGCs. Second, P4 induces PKG activity as judged by the serine 239 phosphorylation of the PKG substrate, VASP. Third, a cell-permeable PKG activator, 8-br-cGMP, mimics P4's action. Fourth, the antiapoptotic action of P4 is attenuated by three different PKG antagonists, Rp-8-pCPT-cGMP, KT5823, and DT-3, with each inhibitor having a different site of action. Finally, transfection of a dominant-negative PKG-1 attenuates P4's ability to maintain SIGC viability. This dominant-negative PKG-1 construct is basically the nucleotide-binding region of PKG. It appears to act as a dominant negative by increasing the availability of an inhibitory pseudosubstrate motif and/or by sequestering intracellular cGMP [24]. Thus, these biochemical, pharmacological, and genetic studies implicate PKG-1 as an intermediary in P4's action.
Interestingly, there are very few granulosa cell studies that assess the effect of steroids on intracellular levels of cyclic nucleotides [33–35]. A study by Sirotkin et al. [35] reveals that P4 increases intracellular levels of cAMP and cGMP within human granulosa/luteal cells. Specifically, these studies show that, over a 12-h culture period, P4 induces a 2.5- to 4-fold increase in cAMP and a 1.5- to 2-fold increase in cGMP. Because both cGMP and cAMP can activate PKG [25], a P4-induced increase in either or both of these cyclic nucleotides could account for P4's ability to activate PKG. However, whether P4 increases the concentrations of either of these cyclic nucleotides in SIGCs remains to be determined.
Although the present observations, as well as other published studies (see review by LaPolt [13]), implicate PKG as an important intermediary in the survival pathway of granulosa cells, a detailed study of PKG expression throughout the course of follicular development has not been undertaken. The PKG family is composed of two types. Type I has two forms, and ß, while only one form of PKG II has been identified [36]. The present study demonstrates that SIGCs express PKG-1. This is consistent with the observation that PKG-1 is detected within the rat ovary [34]. Unfortunately, which isoform of PKG-1 is expressed by SIGCs cannot be discerned because the antibody recognizes both PKG-1 and ß. The major difference between these two isoforms is at the N-terminus [36], and this structural difference appears to account for Iß being 10-fold less sensitive to cGMP stimulation [37].
Some studies have shown that PKG-1 regulates cellular function by moving from the cytoplasm into the nucleus and phosphorylating various transcription factors such as CREB [38], NF-kB [39], and TFII-I [40]. The present studies reveal that both the PKG-1-GFP and the dominant negative PKG-1-GFP fusion proteins localize principally to the cytoplasm and this distribution pattern does not change throughout the entire culture period. This suggests that, in SIGCs, the molecular targets of PKG-1 are mainly localized within the cytoplasm.
As previously indicated, the potential cytoplasmic targets of PKG are very numerous and include ion channels, ion pumps, kinases, heat shock proteins, and receptors [13]. Further, the effect of PKG activation on cell viability is cell specific and is probably a function of which downstream targets are expressed and phosphorylated by PKG. Our proteomic approach utilized two-dimensional gel electrophoresis and staining with CB and ProQ-Diamond to identify potential PKG targets. This analysis revealed one protein that is expressed by P4- and P4 plus DT-3-treated SIGCs but of which the phosphorylation status is reduced by DT-3. This protein is 14-3-3. Of the seven isoforms of 14-3-3, only 14-3-3 is selectively expressed in epithelial cells [41], which is consistent with it being detected in SIGCs. This study also demonstrates that, in viable SIGCs, 14-3-3 is phosphorylated on seven amino acid residues. It is important to appreciate that this study only demonstrates that the phosphorylation status of 14-3-3 is affected by a specific PKG inhibitor and does not demonstrate that PKG directly phosphorylates 14-3-3. Subsequent biochemical studies must be conducted to determine whether PKG directly phosphorylates 14-3-3.
There is very little known about how phosphorylation influences the functional capacity of 14-3-3 [42]. Recently, Woodcock et al. [43] examined the effect of sphingosine-dependent kinase phosphorylation on the ability of 14-3-3
to form a functional dimer. These studies revealed that 14-3-3 is phosphorylated on serine 58 and phosphorylated 14-3-3 exists as a monomer. This is important because it is generally believed that only 14-3-3 dimers bind and functionally interact with their binding partners [44], although phosphorylated 14-3-3 was able to bind some binding partners such as Raf-1 [45].
Unlike 14-3-3, 14-3-3 does not have a serine at position 58 and sphingosine-dependent kinase does not phosphorylate 14-3-3 [46]. Our proteomic analysis of phosphorylated 14-3-3 indicates that it is phosphorylated on the following residues: tyrosine 19, serine 28, serine 69, serine 74, threonine 90, threonine 98, and serine 116. The tyrosine 19 and the serine 69 and 74 are within the sequences that are likely to be involved in dimerization [44]. It is possible that whether all or some of these sites are phosphorylated determines whether 14-3-3 exists as a monomer or dimer. Moreover, phosphorylation of specific combinations of residues could allow for 14-3-3 dimerization but change the three-dimensional structure of the ligand binding pocket and, in turn, affect 14-3-3's affinity for various binding partners. Therefore, the regulation of specific phosphorylation sites could be an important aspect of the physiology of 14-3-3.
As indicated, 14-3-3 functions as a scaffolding protein, binding to specific phosphorylation sites within several different proteins [41, 42, 44]. To date, there are 200 known 14-3-3 binding partners [47]. That the interaction between 14-3-3 and its binding partner is essential for SIGC survival is supported by the difopein study. Difopein binds to all 14-3-3 isoforms with high affinity and specificity without having to be phosphorylated. As a result, difopein is effective in dissociating 14-3-3 from its binding partners [48]. In the present study, difopein induces a 3-fold increase in apoptotic SIGCs that were cultured in serum (i.e., in the presence of numerous growth factors). This confirms previous studies, which showed that difopein induced a 4-fold increase in the number of apoptotic COS-7 cells that were also grown in serum [31]. Taken together, these data suggest that 14-3-3 plays a central and essential role in regulating granulosa cell apoptosis. However, the binding partners that interact with 14-3-3 to control SIGC viability are not presently known.
One likely binding partner for 14-3-3 that is involved in regulating granulosa cell viability is the Bcl-2 family member, Bad [41, 48]. In viable cells, Bad is bound to 14-3-3 and this prevents Bad from binding Bcl-2. Once the 14-3-3/Bad interaction is disrupted, Bad binds with Bcl-2 and displaces Bax from the Bcl-2/Bax dimer. This allows Bax to translocate to the mitochondria, triggering the release of cytochrome c and ultimately causing apoptosis [41, 48]. Because Bad seems to play a role in regulating granulosa cell apoptosis [49–51], it is possible that P4 activation of PKG results in the phosphorylation of 14-3-3, which could facilitate the 14-3-3/Bad interaction. However, because Bad null mice seem to have normal fertility [52] and Bad-deficient cells undergo apoptosis [52], the ability of 14-3-3 to bind Bad may not be the sole mechanism through which 14-3-3 regulates granulosa cell viability.
Because Bax is required for granulosa cell apoptosis [53, 54], another mechanism must exist that sequesters Bax within the cytoplasm in the absence of Bad. A recent study has shown that 14-3-3 forms a complex with Bax, which prevents Bax from moving into the mitochondria and inducing apoptosis [55]. Based on this observation, it is proposed that, in granulosa cells, Bax is held within the cytoplasm through its interaction with 14-3-3. It is further proposed that only phosphorylated 14-3-3 binds Bax and the loss or a site-specific reduction of phosphorylation of 14-3-3 could disrupt the interaction between Bax and 14-3-3, allowing Bax to translocate to the mitochondria and trigger cytochrome c release, caspase activation, and apoptosis. This concept is currently being assessed.
In summary, the present studies show that P4's antiapoptotic action involves a PKG-dependent mechanism. There are many downstream targets of PKG that could mediate P4's actions. One of these downstream targets is 14-3-3. Although it is not known whether PKG directly phosphorylates 14-3-3, the loss or reduction in the phosphorylation of 14-3-3 is associated with SIGC apoptosis. It is likely that the reduction in the phosphorylation status of 14-3-3 influences its three-dimensional structure and in turn its capacity to bind various partners. These putative alterations in 14-3-3 binding could reduce 14-3-3's interaction with the Bcl-2 proteins, Bax and Bad, which would trigger the caspase cascade and ultimately granulosa cell apoptosis. This proposed central role for 14-3-3 is consistent with the finding that the 14-3-3 inhibitor, difopein, induces SIGC apoptosis even in the presence of serum.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondences: John J. Peluso, Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030. FAX: 860 679 1269; peluso{at}nso2.uchc.edu
Received: 10 May 2004.
First decision: 3 June 2004.
Accepted: 22 July 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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