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Endocrinology Vol. 148, No. 5 2037-2044
Copyright © 2007 by The Endocrine Society

Progesterone Maintains Basal Intracellular Adenosine Triphosphate Levels and Viability of Spontaneously Immortalized Granulosa Cells by Promoting an Interaction between 14-3-3{sigma} and ATP Synthaseß/Precursor through a Protein Kinase G-Dependent Mechanism

John J. Peluso, Xiufang Liu and Jonathan Romak

Departments of Cell Biology (J.J.P., X.L., J.R.) and Obstetrics and Gynecology (J.J.P.), University of Connecticut Health Center, Farmington, Connecticut 06030

Address all correspondence and requests for reprints to: John J. Peluso, University of Connecticut Health Center, Department of Physiology (MC3505), 263 Farmington Avenue, Farmington, Connecticut 06030-1230. E-mail: peluso{at}nso2.uchc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present studies were designed to 1) describe changes in both the mitochondrial membrane potential and ATP content of spontaneously immortalized granulosa cells as they undergo apoptosis, 2) identify some of the downstream events that are activated by progesterone (P4), and 3) relate these downstream events to changes in mitochondrial function and apoptotic cell death. These studies revealed that in response to serum deprivation, the mitochondrial membrane potential initially hyperpolarizes and ATP content increases. That this increase in ATP is required for apoptosis was demonstrated by the finding that oligomycin inhibited the increase in ATP and apoptosis. Piridoxalphosphate-6-azopeyl-2'-4'-disulfonic acid, an inhibitor of purinergic receptors, which are activated by ATP, also inhibited apoptosis due to serum withdrawal. This study provides additional support for ATP’s causative role in apoptosis. Moreover, 8-Br-cGMP, a protein kinase G (PKG) activator, mimicked P4’s action, whereas a PKG antagonist, DT-3, attenuated P4’s suppressive effect on ATP and apoptosis. Finally, DT-3 treatment was shown to attenuate P4-regulated phosphorylation of 14-3-3{sigma} and its binding partner, ATP synthaseß/precursor and the amount of ATP synthaseß/precursor that bound to 14-3-3{sigma}. Based on these data, it is proposed that P4 prevents apoptosis in part by activating PKG, which in turn maintains the interaction between ATP synthaseß/precursor and 14-3-3{sigma}. In the absence of P4-induced PKG activity, we further propose that some ATP synthaseß precursor dissociates from 14-3-3{sigma}, resulting in its activation and incorporation into the ATP synthase complex, which ultimately results in an increase in ATP and apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROGRAMMED CELL DEATH, or apoptosis, plays a major role in regulating the development and function of the mammalian ovary (1). Granulosa cells are one of the major ovarian cell types that undergo apoptosis with granulosa cell apoptosis accounting in part for the atresia of ovarian follicles. A variety of stimuli, including oxidative stress, activation of CD95 (i.e. FAS), and deprivation of growth factors, induce granulosa cell apoptosis (2, 3, 4, 5). Once initiated, apoptosis proceeds through an evolutionarily conserved pathway that involves the release of cytochrome c and other mitochondrial proteins. The release of these mitochondrial proteins leads to the activation of caspases, which in turn results in the apoptotic death of the granulosa cell (1).

Interestingly, the membrane potential of the mitochondrion also changes during apoptosis with depolarization often associated with a release of cytochrome c and a decrease in ATP (6, 7, 8). Although ATP levels are lower in apoptotic cells, several studies have shown that elevated levels of ATP are required to induce apoptotic cell death (5, 9, 10). Therefore, the first objective of the present study was to assess changes in mitochondrial membrane potential and ATP content in spontaneously immortalized granulosa cells (SIGCs) as they undergo apoptosis.

To inhibit granulosa cell apoptosis, the ovary synthesizes numerous survival factors. One of these survival factors is progesterone (P4), which is synthesized by granulosa cells and inhibits both granulosa cells (11, 12, 13, 14, 15, 16, 17, 18, 19) and SIGCs (20, 21) from undergoing apoptosis in vitro. Moreover, many studies have demonstrated that P4 acts to regulate granulosa cell viability in vivo (22). Among the best in vivo studies is a study conducted by Stouffer’s group (23). In this study, trilostane, a 3ß-hydroxysteroid dehydrogenase inhibitor, was used to deplete P4 levels in monkeys. Under these conditions, the percentage of atretic follicles with numerous apoptotic nuclei increased to 70%. Supplemental P4 significantly reduced the percentage of atretic follicles to control levels.

Although P4’s antiapoptotic action is well defined, very little is known about the mechanism through which P4 inhibits apoptosis. Our recent studies indicate that P4 acts in part through a protein kinase G (PKG)-dependent mechanism (21). This conclusion is based on the observation that an activator of PKG, 8-Br-cGMP, inhibits the rate of apoptosis of granulosa cells and SIGCs. Furthermore, the cGMP antagonist Rp-8-pCPT [Rp-isomer of 3'-5-cyclic monophosphorothioate 8-(4-chlorophenylthio) guanosine]-cGMP, a dominant negative PKG, and the PKG inhibitor DT-3 all attenuate P4’s action (21). Finally, P4 induces PKG activity as judged by an increase in the serine 239 phosphorylation of the PKG substrate vasodilator-stimulated phosphoprotein (21). Although P4 mediates its action through a PKG-dependent mechanism, the components of P4’s pathway downstream of PKG activation are not known. Thus, the second objective of this study was to identify some of these downstream components and attempt to relate changes in these components to changes in mitochondrial function and apoptotic cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
SIGC culture
SIGCs were used in these studies. These cells were derived from rat granulosa cells isolated from preovulatory follicles as described by Stein et al. (24). SIGCs were cultured as previously described (21). Unless otherwise indicated, SIGCs were plated at 2 x 105 cells/ml in either 35-mm culture dishes containing 25-mm circular glass coverslips for studies involving measurements of mitochondrial membrane potential and ATP levels or glass Lab-Tek slides for studies of apoptosis. The cultures were maintained for 24 h in DMEM-F12 supplemented with 5% fetal bovine serum. After 24 h, cells were washed three times in serum-free DMEM-F12 and treated with various reagents as outlined in each experiment. Depending on the experiment, SIGCs were cultured with P4 (1 µM), 8-Br-cGMP (20 µM), oligomycin (13 µM), piridoxalphosphate-6-azopeyl-2'-4'-disulfonic acid (PPADS; 10 µM), or DT-3 (0.25 µM).

Mitochondrial membrane potential and ATP measurements
JC-1 mitochondrial potential sensor (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide) was purchased from Molecular Probes (Eugene, OR) and used to monitor changes in the mitochondrial membrane potential according to the manufacturer’s instructions. JC-1 was added to each dish at a final concentration of 10 µg/ml, and the cells were incubated for 10 min at 37 C. The cells were then washed three times with Krebs-HEPES buffer (18 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 4.2 mM KH2PO4, 11.7 mM D-glucose, 1.3 mM CaCl2, 10 mM HEPES) and the coverslips removed, inverted onto glass microscope slides, and observed under epifluorescence. Color [red, green, blue (RGB)] images of five to 10 random fields of confluent cells on each coverslip were captured and analyzed using IPLab software. Briefly, each RGB image was split into its components and the intensity of the red and green signals determined using the density program within the IPLab software. The ratio of red (polarized mitochondria) to green (depolarized mitochondria) was calculated for each image.

The Bioluminescent Somatic Cell Assay Kit (Sigma Chemical Co., St. Louis, MO) was used to measure intracellular ATP levels. After 24 h of culture in DMEM-F12 supplemented with 5% fetal bovine serum, the cells were washed three times with serum-free medium and then treated as outlined for each experiment. After treatment, the cells were washed twice with warm PBS and exposed to l00 µl of somatic cell ATP-releasing reagent for 10 sec. This solution was collected and assayed for ATP content according to the protocol supplied by the manufacturer. The protein content was determined for each sample using a BCA protein assay. ATP values were then normalized to the amount of protein in each sample.

Assessment of apoptosis
Apoptotic cells were detected by in situ YOPRO-1 staining after 5 h of serum-free culture, because this is the optimal time to assess apoptosis in this model system (21). YOPRO-1 was added directly into each culture chamber at a final concentration of 10 µM and the cells incubated for 10 min at 37 C and then observed under fluorescent optics using the fluorescein isothiocyanate filter set. 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 and the percentage of apoptotic cells calculated.

Western blot and immunoprecipitation assays
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-deoxycholate; pH 7.0) that was supplemented with complete protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitor cocktail 1 (Sigma) and then centrifuged at 16,000 RCF (relative centrifugal force) at 4 C for 5 min. Protein was determined using the BCA protein assay (Bio-Rad, Hercules, CA). Levels of 14-3-3{sigma} and ATP synthaseß/precursor were determined by Western blot analysis using antibodies supplied by Upstate (Charlottesville, VA) and BD Transduction Laboratories (Palo Alto, CA), respectively. The antibodies were used at the recommended dilutions. It is important to note that the immunogen used to make the ATP synthaseß/precursor antibody corresponded to amino acids at the C terminus. As such, this antibody detects both ATP synthaseß precursor and active ATP synthaseß. Because the precursor sequence is less than 100 amino acids (25), Western blot analysis cannot distinguish between these two proteins. Because of this, the protein band detected by the ATP synthaseß/precursor antibody will be referred to as ATP synthaseß/precursor to denote both the active and inactive form of ATP synthaseß.

For Western blot analysis, lysates were run on a 10% acrylamide gel, transferred to nitrocellulose, and 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 antimouse IgG and ECL Western Blotting Analysis System by Amersham Biosciences (Piscataway, NJ).

The following immunoprecipitation protocol was used to assess the interaction between 14-3-3{sigma} and ATP synthaseß/precursor. Briefly, 5 µg 14-3-3{sigma} antibody was added to 500 µg SIGC lysate in RIPA buffer. The mixture was placed on ice and gently rocked for 1 h. Rabbit antimouse IgG (5 µg) was added and the mixture vortexed. The incubation was continued for an additional 1 h. Finally, 100 µl of a solution of 50% protein A slurry in prechilled PBS buffer was added and incubated overnight at 4 C with agitation. This mixture was then centrifuged at 13,000 rpm for 5 min, and supernatant was carefully removed. The bead pellet was washed with 500 µl lysis buffer three times. The pellet was resuspended in 20 µl 1x Laemmli sample buffer, boiled for 5–10 min, centrifuged, and loaded onto a 10% polyacrylamide gel. The proteins were then transferred to nitrocellulose and probed with the antibodies to either 14-3-3{sigma} or ATP synthaseß/precursor using the previously described Western blot procedure.

The films from the immunoprecipitation assays were scanned into a computer and the density of each band estimated using IPGel. The amount of ATP synthaseß/precursor bound to 14-3-3{sigma} was determined by dividing the density of the ATP synthaseß/precursor band by the density of the 14-3-3{sigma} band. For each treatment, the mean and SE of this ratio were then calculated.

Two-dimensional electrophoresis and proteomic analysis
This experiment was conducted as previously described (21). Briefly, two 100-mm glass culture dishes were incubated with P4 or P4 plus DT-3 in serum-free medium. After 5 h, the cell lysates were prepared, concentrated using a chloroform/methanol precipitation procedure and the pellets resuspended in Bio-Rad Ready Prep 2D rehydration/sample buffer. The samples were then applied to Bio-Rad ReadyStrips IPG (7 cm, pH 4–7) by passive rehydration, placed in a Bio-Rad Protein IEF cell and focused using the linear program for the 7-cm IPG strips. The strips were then placed on top of a 10% acrylamide gel and electrophoresed for 2 h at 100 V.

Phosphorylated proteins were detected by gel staining with ProQ-Diamond Phosphoprotein Gel Stain and images of the ProQ-Diamond-stained gels acquired using a FluorImager Si. The gels were then washed, stained with Coomassie blue for 1 h, and washed overnight to visualize the protein spots.

Two of the Coomassie blue-stained spots that were present in the P4 and P4 plus DT-3 groups but were not stained with the ProQ-Diamond after the P4 plus DT-3 treatment were sequenced using a Finnigan LCQ-DECA ionTrap Mass Spectrometer with the data-dependent tandem mass spectrometry capability (21). This work was done at the Proteomics and Biological Mass Spectrometry Core Facility (University of Connecticut Health Center, Farmington, CT).

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. The data from experiments with only control and treatment groups were analyzed using a Student’s t test, whereas comparison of three or more groups was assessed using a one-way ANOVA followed by a Fisher’s protected least significant difference post hoc test. P < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
As judged by JC-1 staining, there is considerable variation in the membrane potential of individual mitochondria within SIGC cultured in serum-supplemented medium. Some mitochondria were depolarized (green), whereas others were more polarized as revealed by the intensity of their orange-red fluorescence (Fig. 1Go). Within 1 h of serum withdrawal, the orange-red fluorescent intensity of the mitochondria increased in virtually all the cells (Fig. 1Go). In addition, those cells with the most intense mitochondrial orange-red fluorescence appeared to be rounding up and detaching from the other cells; two characteristics of cells undergoing apoptosis (Fig. 1Go) (26).


Figure 1
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  		FIG. 1. The effect of serum deprivation on mitochondrial membrane potential as assessed by JC-1 staining. In this study, SIGCs were stained with JC-1 to reveal mitochondria that are depolarized (green) or polarized (orange to red). The upper panel is the complete RGB image; the middle panel is the red channel, and the lower panel is the green channel of the same images. Note that after 1 h in serum-free medium, the mitochondrial membrane potential increases as judged by an increase in the intensity of the orange (upper panel) or red fluorescence (middle panel). Note that the green fluorescent intensity remained unchanged (lower panel). The arrow in the image for the 5-h group denotes one of the cells that are detaching from other cells and are rounding up, which is characteristic of cells undergoing apoptosis.

 
A quantitative analysis of the red to green ratio also revealed an increase in the average mitochondrial membrane potential (Figs. 1Go and 2AGo). This increase in mitochondrial membrane potential appeared to be transient, peaking between 1 and 3 h of serum-free culture. In addition, this transient increase in mitochondrial membrane potential preceded the increase in ATP (Fig. 2BGo) and correlated with an increase in the percentage of cells undergoing apoptosis (5 ± 3% for serum vs. 27 ± 2% after 5 h in serum-free culture, n = 12 per group; P < 0.05). Interestingly, by 24 h in serum-free conditions, most of the cells had detached from the culture dish and were presumed to have undergone apoptosis. As previously reported (27), virtually all the cells observed after 24 h of P4 treatment were nonapoptotic, indicating that P4 actually blocked apoptosis (data not shown).


Figure 2
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  		FIG. 2. The effect of serum deprivation on quantitative changes in mitochondrial membrane polarity (red/green ratio after JC-1 staining; A) and ATP levels (B). In this and subsequent figures, values are expressed as means ± 1 SE; *, values that are significantly different from control and 5-h values (P < 0.05); **, value that is greater than 0-h control but less than the other treatments (A). The average number of observations per group was 120. An average of 17 observations was made for each time point in B. The average protein content of each sample was 59 ± 5 µg/ml.

 
To determine whether the increase in ATP was required for the increase in apoptosis, SIGCs were cultured in serum-free medium in the presence or absence of oligomycin, a well-characterized inhibitor of ATP synthase (28). In the present study, oligomycin blocked the serum-withdrawal-induced increase in ATP (Fig. 3AGo) and apoptosis (Fig. 3BGo). To further implicate ATP as an apoptotic agent, the effect of the purinergic receptor inhibitor PPADS on apoptosis was assessed, because ATP is known to activate these receptors (29). Under serum-free culture conditions, PPADS also inhibited apoptosis (Fig. 3CGo).


Figure 3
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  		FIG. 3. A and B, Effect of oligomycin (13 µM) on ATP content (A) and apoptosis (B) in SIGCs cultured in serum-free conditions for 5 h; C, effect of PPADS (10 µM), an inhibitor of purinergic receptors known to be activated by ATP, on SIGC apoptosis. For these studies, SIGCs were exposed to PPADS for 5 h in serum-free culture medium. The means for each treatment group in all of the figures were derived from 12 observations.

 
P4 as well as 8-Br-cGMP inhibited the increase in ATP (Fig. 4AGo) and apoptosis (Fig. 4BGo) that is observed after serum withdrawal. In addition, an inhibitor of PKG, DT-3, attenuated P4’s effect on ATP (Fig. 5AGo) and apoptosis (Fig. 5BGo). These studies not only confirmed that PKG activation is an essential component of P4’s antiapoptotic pathway but also indicated that PKG activation is somehow linked to the regulation of ATP synthesis.


Figure 4
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  		FIG. 4. The effect of P4 (1 µM) and 8-Br-cGMP (20 µM) on ATP content (A) and apoptosis (B) of SIGCs cultured for 5 h in serum-free culture medium. For the ATP study, an average of 15 observations per group were made, whereas the apoptosis study had eight observations per group.

 

Figure 5
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  		FIG. 5. The effect of P4 (1 µM) and the PKG inhibitor DT-3 (0.25 µM) on ATP content (A) and apoptosis (B) of SIGCs cultured for 5 h in serum-free culture medium. For the ATP study, an average of six observations per group were made, whereas the apoptosis study had 12 observations per group.

 
To gain insight into the mechanism through which P4-regulated PKG activity influences ATP content, attempts were made to identify downstream events that are regulated in response to PKG activation. As seen in Fig. 6Go, numerous proteins were observed whose expression was maintained but whose phosphorylation status was reduced in response to DT-3 treatment. One of these proteins was previously identified as 14-3-3{sigma} (21). The present mass spectrometric analysis identified ATP synthaseß/ precursor (ATP5b; NM_134364) as a P4-PKG-regulated protein. This mass spectrometric analysis detected 146 of the 529 amino acids of ATP synthaseß/precursor (~28%). In total, 11 fragments with amino acid sequences that matched the exact sequences of ATP synthaseß/precursor were identified (Fig. 7Go).


Figure 6
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  		FIG. 6. A two-dimensional analysis of Coomassie blue-stained (A and B) and phosphorylated (C and D) proteins after treatment for 5 h in serum-free medium supplemented with either P4 (1 µM; A and C) or P4 (1 µM) plus PKG inhibitor DT-3 (0.25 µM; B and D) as previously described (21 ). Two proteins are identified in each panel. Arrow 1 marks the protein 14-3-3{sigma} whereas arrow 2 marks the protein ATP synthaseß/precursor. This study was run four times.

 

Figure 7
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  		FIG. 7. The amino acid sequence of ATP synthaseß/precursor (ATP5b; NM_134364). The amino acids shown in black were detected by the mass spectrometry analysis. The amino acid sequence shown in brackets represents the 14-3-3 binding motif.

 
Interestingly, ATP synthaseß/precursor has been shown to possess a phosphorylated 14-3-3{sigma} binding site and to bind to 14-3-3 (30, 31). The present Western blots demonstrated that both ATP synthaseß/precursor and 14-3-3{sigma} are expressed in SIGCs (Figs. 8Go, A and B, and 9AGo), whereas the immunoprecipitation studies confirmed that these two proteins interact in SIGCs (Fig. 9BGo). Finally, the amount of ATP synthaseß/precursor that was bound to 14-3-3{sigma} was significantly reduced in the presence of P4 plus DT-3 compared with P4 alone (Fig. 9Go).


Figure 8
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  		FIG. 8. Western blot analysis of 14-3-3{sigma} (A) and ATP synthaseß/precursor (B) in SIGCs cultured in serum-supplemented medium. The positive control (Pos. Cont) for 14-3-3{sigma} was provided by Upstate and lysate from Jurkat cells was used as a positive control for ATP synthaseß/precursor as suggested by BD Transduction Laboratories. Western blots using lysates from SIGCs were conducted using either primary and secondary antibodies (+) or IgG and the secondary antibody (–; i.e. negative control). SIGCs were immunoprecipitated using the 14-3-3{sigma} antibody. Western blots were then conducted in the presence of either the primary antibody or IgG.

 

Figure 9
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  		FIG. 9. Western blot analysis (A) of lysates obtained from SIGCs cultured for 5 h in serum-free medium supplemented with P4 (1 µM) or P4 plus DT-3 (0.25 µM). Note that equal amounts of 14-3-3{sigma} and ATP synthaseß/precursor (ATP5b) were present in both treatment groups. In B, SIGCs were treated as above and then lysates were immunoprecipitated using the 14-3-3{sigma} antibody. The immunoprecipitates were then probed in a Western blot analysis using antibodies to either 14-3-3{sigma} or ATP synthaseß/precursor. In this study, equal amounts of 14-3-3{sigma} were detected, but only about half as much ATP synthaseß/precursor was present after P4 plus DT-3 treatment compared with P4 treatment alone as evidenced by a decrease in the ratio of ATP synthaseß precursor to 14-3-3{sigma} (n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mitochondria play several key roles in mediating apoptosis (32, 33). Their most characterized role in apoptosis is the release of cytochrome c and other mitochondrial proteins, which in turn activates the caspase cascade (32, 33). It is the activation of the caspase cascade that ultimately triggers apoptosis in SIGCs (26) and granulosa cells (34, 35, 36). In addition to this role, mitochondria also regulate ATP production (37). Most studies involving ATP and apoptosis indicate that ATP levels are lower in apoptotic cells, and this correlates with a reduction of mitochondrial membrane potential (6, 7, 8, 9). However, most of these studies assess cells in the later stages of apoptosis. In contrast, the present study demonstrates that in the early stages of SIGC apoptosis, both mitochondrial membrane potential and ATP concentrations actually increase. At first glance, this may seem counterintuitive, but a transient increase in both mitochondrial membrane potential and ATP production have been shown to be essential components of the apoptotic cascade (5, 6, 10, 38).

To assess whether the observed increase in ATP is required for serum-withdrawal-induced apoptosis in SIGCs, studies were conducted using oligomycin, a well-known inhibitor of ATP synthesis (28). These studies reveal that in the absence of serum, oligomycin prevents both the increase in ATP and apoptosis. Although limited by the specificity of the oligomycin, the findings from this pharmacological-based study fit well with those made in human granulosa/luteal cells that show that exogenous ATP induces apoptosis (39, 40). The mechanism by which ATP mediates its apoptotic action appears to involve the activation of purinergic receptors. These receptors are expressed by granulosa cells (29), and their activation leads to an increase in intracellular free calcium (29, 41). In SIGCs maintained in serum-free medium, it is likely that an increase in intracellular ATP leads to a release of ATP into the extracellular space and that this ATP activates the purinergic receptors. ATP activation of the purinergic receptors would account for the increase in intracellular free calcium that ultimately results in granulosa and SIGC death. This conclusion is based on the fact that a 2- to 3-fold increase in intracellular free calcium precedes the apoptotic death of granulosa cells (11), SIGCs (20), and rat ovarian surface epithelial cells (42). This increase in intracellular free calcium is essential for apoptosis to proceed because chelating the increase in intracellular free calcium with BAPTA prevents apoptosis (42). The finding that an inhibitor of purinergic receptors, PPADS (43), also prevents serum-withdrawal-induced apoptosis is consistent with the idea that these receptors are activated early in the apoptotic cascade. However, like all pharmacological studies, this study must be confirmed by more detailed biological, biochemical, and molecular studies before this conclusion can be drawn.

It is well known that P4 inhibits granulosa cells and SIGC apoptosis with recent studies demonstrating that the activation of PKG is an essential component of P4’s antiapoptotic signal transduction pathway (21). The present studies confirm this and also demonstrate that these PKG activators prevent the increase in ATP that is observed after serum withdrawal. Similarly, DT-3, a specific inhibitor of PKG (44), attenuates the effect of P4 on both ATP and apoptosis. Taken together, these findings suggest that P4 activation of PKG regulates downstream events that maintain basal levels of ATP and thereby cell viability.

To identify some of the PKG-dependent downstream events, SIGCs were cultured with P4 in the presence or absence of DT-3. Our previous two-dimensional electrophoretic analysis revealed that one PKG-regulated protein is 14-3-3{sigma} (21), which is an important protein in that it functions by binding to over 200 different proteins (31, 45, 46, 47). Although the previous study demonstrated a role for 14-3-3{sigma}, it did not identify any of its binding partners that are involved in the apoptotic process. Our present analysis detected a P4-PKG-dependent change in the phosphorylation status of ATP synthaseß/precursor. Interestingly, ATP synthaseß/precursor possesses a 14-3-3 binding site (rflsqp) (30, 48), which must be phosphorylated on the serine residue to bind 14-3-3 (30, 31). The present study revealed not only that these two proteins interact in SIGCs but also that their interaction is regulated by P4-PKG activation. Importantly, inhibiting PKG activity reduced by approximately 50% the amount of ATP synthaseß/precursor bound to 14-3-3{sigma}. The conclusion that P4-PKG activation regulates the amount of ATP synthaseß/precursor that is bound to 14-3-3{sigma} must be tempered somewhat in that the antibody to ATP synthaseß/precursor also detects ATP synthaseß. Therefore, some ATP synthaseß may be bound to 14-3-3{sigma}, but this is unlikely because the precursor sequences are generally required for interactions with cytosolic chaperones such as 14-3-3 (25).

Based on these studies, it appears that the degree to which ATP synthaseß/precursor is bound to 14-3-3{sigma} plays an important role in regulating ATP production and ultimately cell viability. Binding to 14-3-3 is thought to affect the stability and/or activity of its binding partner (45). In fact, studies by Bunney et al. (30) demonstrate that the activity of ATP synthase is reduced by the presence of recombinant 14-3-3. This finding is consistent with the concept that the activity of ATP synthaseß is suppressed through an interaction with 14-3-3.

Previous studies have shown that in mammalian cells, 14-3-3 can direct proteins to mitochondria (49). Once within the matrix of the mitochondria, an N-terminal sequence of ATP synthaseß precursor is cleaved almost immediately, leaving the active ATP synthaseß (25). This allows ATP synthaseß to be incorporated into the ATP synthase complex and facilitate the production of ATP (30, 48, 50).

Based on these previous studies and our findings, we propose that an interaction with 14-3-3{sigma} allows ATP synthaseß precursor to be delivered to the mitochondria but prevents it from being converted into active ATP synthaseß and/or incorporated into the ATP synthase complex. By maintaining the interaction between 14-3-3{sigma} and ATP synthaseß precursor, P4 regulates the amount of active ATP synthaseß within the ATP synthase complex and thereby ATP production. In the absence of P4, PKG activity is reduced and ATP synthaseß precursor is dephosphorylated, causing ATP synthaseß precursor to dissociate from 14-3-3{sigma}. We propose that this would lead to the cleavage of ATP synthaseß precursor and ultimately to an increase in ATP and the initiation of the apoptotic death cascade. Although consistent with the present data, considerably more work must be done to test each element of this proposed concept.

In summary, the present studies show that in response to serum deprivation, an increase in both mitochondrial membrane potential and ATP occur. This increase in ATP presumably activates the purinergic receptors to trigger the apoptotic cascade and subsequently cell death. P4 prevents the increase in ATP through a PKG-dependent mechanism. This mechanism involves the phosphorylation of 14-3-3{sigma} and ATP synthaseß/precursor, which promotes their interaction. It is further proposed that by regulating the interaction between 14-3-3{sigma} and ATP synthaseß/precursor, P4 controls the amount of active ATP synthaseß within the ATP synthase complex and thereby maintaining ATP homeostasis and cell viability.


   Acknowledgments
 
We thank Ms. Anna Pappalardo for conducting the two-dimensional gel experiment and Dr. David Han for the proteomic analysis of 14-3-3{sigma} and ATP synthaseß/precursor.


   Footnotes
 
This work was supported by a grant from the National Institutes of Health (HD 047205).

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 15, 2007

Abbreviations: P4, Progesterone; PKG, protein kinase G; PPADS, piridoxalphosphate-6-azopeyl-2'-4'-disulfonic acid; RGB, red, green, blue; SIGC, spontaneously immortalized granulosa cell.

Received November 30, 2006.

Accepted for publication February 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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