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Department of Medicine (M.A.C., Q.Z., J.M.H.), The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033; and University of Illinois at Chicago and Chicago Veterans Healthcare System (T.G.U.), Chicago, Illinois 60612
Address all correspondence and requests for reprints to: James M. Hammond, The Pennsylvania State University Hershey Medical Center, 500 University Drive, C6636, Hershey, Pennsylvania 17033. E-mail: jhammond{at}psu.edu
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Introduction |
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Recent evidence that FSH can activate alternative pathways to the classic cAMP/protein kinase A (PKA) pathway has provided a new perspective on FSH-stimulated cell signaling. Data suggest that PKA is not the only downstream effector of FSH-stimulated cAMP but that FSH can more directly stimulate the phosphorylation of Akt/protein kinase B (PKB) in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent but PKA-independent manner (6). The PI 3-kinase pathway is well known to set in motion a coordinated set of events leading to cell cycle progression, cell growth, and cell survival in response to cell stimulation by growth factors and hormones. When PI 3-kinase is activated, protein serine-threonine kinases like Akt are directed to the membrane where they in turn are phosphorylated and activated. Akt activation results in the phosphorylation of a host of other proteins involved in cell cycle regulation and survival, many of which become inhibited by the subsequent phosphorylation event (for review, see Refs. 7 and 8).
In the ovary, the PI 3-kinase/Akt signaling pathway has been implicated in the survival of granulosa cells in response to IGF-I stimulation (9, 10). IGF-I treatment prevented apoptosis in granulosa cells, and this survival response was blocked by treatment with specific PI 3-kinase inhibitors, wortmannin and LY294002. This result was perhaps not surprising because IGF-I is an important modulator of granulosa cell growth and development in vivo, and IGF-I is well known to stimulate the PI 3-kinase pathway in many other systems (for review, see Refs. 11, 12, 13). Whether FSH can prevent apoptosis via the PI 3-kinase/Akt pathway and via what effectors is yet to be determined. During the course of our studies in porcine granulosa cells, FKHR (FoxO1a), FKHRL1 (FoxO3), and AFX (FoxO4) expression were demonstrated in the rat ovary. Using a combined in vivo and in vitro approach, Richards et al. (14) established the physiological regulation of FKHR and its mRNA during the reproductive cycle. Most recently, it was demonstrated that knockout of FoxO3a, a related family member, causes accelerated follicular initiation leading to early depletion of follicles suggestive of premature ovarian failure (14A ). These data suggest FKHR as a possible regulator of follicular development and survival.
The FoxO transcription factors are an important class of physiological targets of PI 3-kinase/Akt signaling. All of the FoxO genes conserve the sequence for three (at least) putative Akt phosphorylation sites. One way in which Akt is known to promote cell survival is via the phosphorylation and cytosolic sequestration of FoxO transcription factors, effectively inhibiting their role as transactivators of cell-cycle arrest and apoptosis-inducing genes, such as p27kip1 and Fas ligand (15, 16).
Historically, the porcine granulosa system has been a useful model for studying the action and interaction of the FSH and IGF systems. However, the nature of intracellular cross-talk between the traditional PKA-dependent FSH signaling pathway and traditional IGF-stimulated pathways was poorly understood. In addition, the role of forkhead family members in these processes (if any) had not been studied. To address these issues, we have examined downstream signaling elements in the PI 3-kinase pathway that might be involved in FSH-mediated granulosa cell survival in porcine granulosa cells. Furthermore, we determined the expression of FoxO1a in these cells, its hormonal regulation, and whether the demonstrated regulation by FSH correlated with FSH-stimulated Akt phosphorylation. We consider these studies to be the first step in determining the potential role of FoxO transcription factors in the survival of granulosa cells in this system.
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Materials and Methods |
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Cell culture
Porcine granulosa cells (GCs) were harvested from medium-sized antral follicles (5–10 mm) as previously described (16A ) and used for all experiments. Briefly, granulosa cells were scraped from follicle walls, resuspended in plating medium containing DMEM/F-10 (1:1) with 10% fetal bovine serum (FBS), and grown to confluence and frozen. After a single passage, GCs were plated in medium containing 10% FBS for 48–72 h (subconfluent) with one medium change. Cultures were then stepped down to serum-free medium for 18 h before hormone or growth factor treatment.
RT-PCR
Total RNA was extracted from granulosa cells using the RNeasy RNA isolation kit (Qiagen, Valencia, CA) according to the manufacturer. Reverse transcription was carried out using Superscript II (Invitrogen/Life Technologies, Inc., Carlsbad, CA). Briefly, 2 µg total RNA were mixed with 1 µM oligo-d(T)16 (Roche/Applied Biosystems, Foster City, CA) and incubated at 70 C for 10 min. Reactions were placed on ice for 5 min and mixed with dithiothreitol, deoxynucleotide triphosphates, 5 x reverse transcription buffer, and incubated at 42 C for 5 min. Superscript II RNase H-reverse transcriptase was then added, and the reaction was carried out at 42 C for 1 h and terminated by incubating at 70 C for 15 min.
The following oligonucleotides were synthesized for use as PCR primers: 5'-TGTCGCAGATCTACGAGTG-3' (FKHR sense); 5'-TCTGGATTGAGCATCCACCAAG-3' (FKHR antisense); 5'-AATGACCCCTTCATTGACCTCC-3' [glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense]; 5'-GCTTCCCATTCTCAGCCTTGAC-3' (GAPDH antisense). After activating Taq for 1 min at 95 C, PCR was carried out for 30 cycles (denaturing at 94 C for 45 sec, annealing at 55 C for 45 sec, and extension at 72 C for 1 min) in a 50-µl reaction containing cDNA, 100 nM of each gene-specific primer, 10 x PCR buffer, deoxynucleotide triphosphates, and JumpStart Taq polymerase (Sigma). The PCR products were electrophoresed on a 1% agarose gel. Preliminary experiments with varying cycle number and/or varying template concentrations showed the result depicted to be on the linear portion of the amplification curve.
Western blot analysis
Lysates were prepared by scraping granulosa cells in 250 µl of cold whole-cell lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1.0% Nonidet P-40, 1.0% deoxycholic acid] with additional protease and phosphatase inhibitors [2 µg/µl leupeptin, 2 µg/µl aprotinin, 2 µg/µl pepstatin, 0.2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM sodium fluoride] and 0.5 mM dithiothreitol. Alternatively, cell fractionation to obtain nuclear and cytosolic extracts was accomplished by first scraping granulosa cells in 500 µl of cold hypotonic buffer [10 mM HEPES (pH 7.9), 1.5 mM MgCl, 10 mM KCl] with the ser/thr phosphatase inhibitor microcystin (Calbiochem) in addition to the inhibitors listed above. The cells were lysed for 10 min on ice, centrifuged for 10 sec at 14,000 rpm, and the supernatant (cytosolic fraction) was removed. The pellet was then resuspended in 60 µl of a high-salt buffer [20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl, 50% glycerol] with inhibitors as above and incubated for 30 min on ice. After a 10-min high-speed spin, the supernatant (nuclear extract) was again removed and protein concentrations for both fractions were determined by a bicinchoninic protein assay (Bio-Rad, Hercules, CA). SDS-PAGE sample buffer was added and the samples were heat denatured. To assess efficiency of the cell fractionation step, control Western blots were performed with anti-CREB (exclusively in nucleus) and anti-PI 3-kinase (exclusively in cytosol). There was no detectable contamination between nuclear and cytoplasmic extracts by this technique.
Protein (40 µg) was separated by SDS-PAGE (4–12% acrylamide running gel) and transferred to nitrocellulose. The membranes were blocked in 5% nonfat milk in TBS-T [10 mM Tris (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] for 1 h at room temperature and probed with primary antibody (1:1000) for 1.5 h at room temperature (or overnight at 4 C). After a series of 5-min TBS-T washes, the blot was incubated with secondary antibody (1:5000) for 1 h at room temperature. The membrane was again washed three times in TBS-T, and antigen-antibody complexes were visualized using enhanced chemiluminescence (Amersham Pharmacia, Piscataway, NY) exposed on Biomax Light film (Kodak, Rochester, NY). Quantitation of bands was performed using a densitometer (Molecular Dynamics, Sunnyvale, CA) and Bio-Rad Quantity One software.
32p labeling of FKHR in intact GCs
Granulosa cells were grown in 100-mm culture dishes to near confluence as described above. Growth media were removed and replaced with phosphate-free DMEM containing 0.3 mCi/ml [32P]orthophosphate (NEN Life Science Products, Boston, MA) for 60 min at 37 C. The cells were then treated with FSH (100 ng/ml) for 10 min, 30 min, or 1 h. The incubations were terminated by aspirating medium, and the cells were washed two times with ice-cold PBS to remove residual radioactive phosphate. The cells were then harvested in 500 µl of cold lysis buffer and incubated for 30 min. The lysate was centrifuged (14,000 rpm, 10 min) and the soluble extract was recovered. A protein assay (Bio-Rad Laboratories, Hercules, CA) was performed and the extracts were normalized for total protein before being immunoprecipitated overnight at 4 C with an anti-FKHR antibody. Immunoprecipitates were incubated with protein A/G agarose (Santa Cruz Biotechnology) for 1 h at 4 C. Agarose beads were recovered by centrifugation and washed two times with cold lysis buffer and once with cold PBS. The immunoprecipitated proteins were mixed with sample buffer, heated, and centrifuged for 10 min at 14,000 rpm. The proteins present in the supernatant fraction were separated and analyzed by 10% SDS-PAGE. The gels were dried and exposed to Biomax Light film (Kodak) at -70 C overnight. Densitometry was performed and data sets were compared using one-way ANOVA and Tukey’s multiple comparison posttest.
Transient transfection
Granulosa cells were seeded on 2-well glass chamber slides (Lab-Tek) coated with poly-D-lysine (20 µg/ml, 10 min at room temperature). Using a liposome-mediated method (Lipofectin, Life Technologies, Inc.), transient transfections were accomplished with 1.5 µg of a GFP-control vector, GFP-FKHR WT vector, or a GFP-FKHR-T/S/S vector in which three putative Akt phosphorylation sites in FKHR were mutated to alanines (20). Specifically, the mutations made were Thr24Ala, Ser256Ala, and Ser319Ala. Subconfluent GCs were transfected in Optimem media (Gibco/Invitrogen, Carlsbad, CA) for 4–6 h and then placed in 10% FBS DMEM/F-10 overnight. The cultures were then stepped down to serum-free media for 6 h before hormone or growth factor treatment. After treatment, cells were washed twice in PBS and fixed in 4% paraformaldehyde. Before GFP visualization on an IX50 inverted system microscope (Olympus, Tokyo, Japan), the cells were stained with Hoechst 33342 (Molecular Probes, Eugene, OR) to examine nuclei. For each construct and treatment group, 100–300 transfected cells were counted and FKHR localization determined.
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).
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).
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Granulosa cells were labeled in culture with [32p]orthophosphate, and FKHR was immunoprecipitated with a polyclonal anti-FKHR antibody overnight. This antibody is isoform specific and therefore does not recognize other FoxO family members. The immunoprecipitated FKHR was then analyzed by SDS-PAGE and autoradiography. When granulosa cells were treated with FSH (100 ng/ml) for 30 min under serum-free conditions, the rabbit polyclonal anti-FKHR antibody immunoprecipitated a phosphoprotein of approximately 70 kDa, which matched the band recognized by anti-FKHR in a concomitant Western blot. Phospho-FKHR was increased more than 9-fold at 30 min after FSH treatment (P < 0.05) in n = 3 experiments. At 1 h post FSH treatment, FKHR was still robustly phosphorylated (P < 0.05). These results demonstrate that the FSH-stimulated signal transduction pathway(s) can directly modify FKHR protein posttranslationally allowing for short-term regulation of this protein by FSH in porcine granulosa cells.
FSH-treated granulosa cells have decreased nuclear FKHR protein and increased cytosolic FKHR protein at early time points
Having demonstrated FSH stimulation of FKHR phosphorylation, we next sought to determine whether FSH might be able to regulate the intracellular localization of FKHR protein in these cells. We performed cell fractionation studies to analyze nuclear and cytosolic levels of total FKHR protein separately. After serum starvation, untreated and FSH-treated granulosa cells were harvested over a 24-h time-course, and nuclear and cytoplasmic extracts were prepared. Western blots of nuclear extracts demonstrated a significant decrease in FKHR protein at 1 and 3 h after FSH treatment, with maximal response to FSH achieved at 1 h (Fig. 4A). At an earlier time point (30 min, data not shown), nuclear FKHR protein was decreased but not maximally. Three separate time-course experiments were performed, each exhibiting the greatest decrease in nuclear FKHR protein after FSH treatment for 1 h (Fig. 4B).
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), suggesting that the PI 3-kinase pathway plays a role in this FSH effect.
When we analyzed cytosolic extracts by Western blots after FSH or FSH+LY treatment for 1 h, we found a consistent increase in cytosolic FKHR protein by approximately 50% (P < 0.01) in FSH-treated cells (Fig. 4D). Because the increase in cytosolic FKHR in response to FSH was blocked by the addition of LY, this again suggested PI 3-kinase involvement.
FSH treatment of granulosa cells results in cytosolic sequestration of a FKHR-green fluorescent protein (GFP) fusion protein
To further investigate the possible regulation of FKHR intracellular localization after FSH stimulation, we transfected our granulosa cells with a series of plasmids expressing GFP or GFP-FKHR fusion proteins (20). These include a control GFP-only vector, a GFP-FKHR WT vector, and a GFP-FKHR-T/S/S mutant vector, which has three consensus threonine/serine Akt phosphorylation sites point-mutated. As shown in Fig. 5A, when granulosa cells were transfected with the control GFP-only vector, both control and FSH-treated cells have diffuse fluorescence in both the nucleus and cytoplasm demonstrating that GFP’s cellular localization is not regulated, as expected. When granulosa cells were transfected with a GFP-FKHR plasmid but left untreated, the majority of the transfected cells also have a diffuse pattern of fluorescence with, in some cases, more of the GFP-FKHR fusion protein in the nucleus than cytosol. In contrast, when GFP-FKHR-transfected cells were treated with IGF-I for 10 min, shown to modulate FKHR location in other cells (21), the GFP-FKHR fusion protein became more concentrated in the cytosol than in the nucleus. When the transfected cells were enumerated, greater than 60% of GFP-FKHR transfected cells had evident nuclear exclusion of GFP when treated with IGF vs. approximately 17% of untreated transfected cells (Fig. 5B). Importantly, we found nearly identical results when GFP-FKHR-transfected cells were treated with FSH for 1 h. The majority of cells transfected with GFP-FKHR and stimulated with FSH exhibited cytosolic sequestration of the fusion protein (59% vs. 17% of untreated cells).
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, A and B). This result supports the hypothesis that FSH activation of the PI 3-kinase pathway and subsequent phosphorylation of these critical sites was responsible for the sequestration of FKHR in the cytosol.
To further investigate PI 3-kinase involvement in this FSH response, a series of transfection experiments was performed in the presence of the PI 3-kinase inhibitor LY294002. When these cells were examined microscopically, we found that preincubation of transfected granulosa cells with LY294002 blocked FSH-stimulated nuclear exclusion of FKHR. This effect was even more dramatic than what we had observed in our Western blot studies (Fig 5C). This LY294002 block of FKHR nuclear exclusion supports the hypothesis that the PI 3-kinase pathway is mediating these FSH effects on FKHR protein localization.
FSH stimulates the phosphorylation of Akt/PKB in porcine granulosa cells
To further examine the involvement of the PI 3-kinase pathway in the aforementioned FSH effects on FKHR protein, we performed Western blot analysis of granulosa whole-cell lysates to look for activation of Akt. We found that FSH stimulated the phosphorylation of Akt beginning at 1 h, although this phosphorylation was variable and therefore of borderline significance at this time point (Fig. 6, A and B). However, Akt phosphorylation in response to FSH was consistently increased at later time points tested, up to 24 h (Fig. 6A).
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Discussion |
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The activity of the FKHR (FoxO) family is known to be regulated by PI 3-kinase-activated signal transduction pathways, as has been observed in many systems (for review, see Ref. 22). The PI 3-kinase pathway was initially implicated when cultured cells were treated with growth factors (i.e. epithelial growth factor, IGF-I, insulin) that are known to activate the PI 3-kinase pathway, and FKHR and FKHR-related proteins were shown to be excluded from the nucleus. Additional studies demonstrated that removal of growth factors resulted in the accumulation of FKHR protein in the nucleus. Furthermore, it is well acknowledged that Akt/PKB mediates many of the effects of insulin and other growth factors downstream of PI-3 kinase (for review, see Ref. 23), and several members of the forkhead family of transcription factors are now established substrates of Akt/PKB in this pathway (15, 16, 17, 18).
In our current studies, we demonstrated both FKHR message and protein expression in porcine GCs. Expression of FKHR had previously been shown in GCs of the rat, and evidence that FKHR is transcriptionally regulated by FSH (in addition to estradiol, IGF-I, and other gonadotropins) was put forth (14). These effects have not been excluded in the pig ovary in vivo. However, in our cultured porcine GC system, FSH does not regulate FKHR mRNA or protein levels in the short term. Accordingly, we focused on posttranslational regulation of FKHR by FSH in which we encountered more dramatic effects.
In our next series of studies, we sought to determine whether FSH could direct the phosphorylation of FKHR. As noted previously, FKHR is functionally regulated by multiple extracellular signals (e.g. growth factors) resulting in activation of the PI 3-kinase signal transduction pathway and Akt phosphorylation of FKHR. Recent data suggest that Akt is phosphorylated in response to FSH in a PI 3-kinase-dependent manner in cultured rat granulosa cells (6). In addition, Richards et al. (14) demonstrated FSH-stimulated FKHR phosphorylation via Western blot with phospho-FKHR-specific antibodies. In porcine cells we encountered cross-reactivity among FKHR species when using the phospho-FKHR (Ser256) antibody (data not shown). In addition, and more troublesome, we could not be sure that the antibody was specific for phosphorylated FKHR vs. total FKHR. We also tested phospho-FKHR (Thr24) but found that FSH did not stimulate the phosphorylation of FKHR on this site (data not shown). Consequently, we assessed the direct phosphorylation of FKHR by performing a series of 32p-labeling studies in intact GCs, which demonstrated rapid phosphorylation of FKHR in response to FSH by 30 min (Fig. 3). Our time course also illustrated the transient nature of the effect because phospho-FKHR is diminished by 1 h and nearly gone by 3 h (data not shown).
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Cytosolic accumulation of endogenous FKHR after FSH treatment was recently demonstrated immunohistochemically in the rat GC (14), and it was hypothesized that this regulation of FKHR was the result of phosphorylation of critical sites in the transactivation domain downstream of PI 3-kinase activation. To further investigate the intracellular localization of FKHR after FSH stimulation in these cells, we employed GFP-tagged FKHR expression constructs (20). With the use of a GFP-FKHR triple mutant (T24A, S256A, and S319A), we were able to determine that the FSH effect was dependent on phosphorylation of one or more of these sites. These data provide the first evidence that the phosphorylation sites shown to be critical for growth factor regulation of FKHR are also critical for FSH action on FKHR (15, 17, 21, 23, 24). These sites are known targets of Akt/PKB; thus, these findings implicate the PI 3-kinase pathway in the FSH effect. This conclusion was further supported by the finding that the PI 3-kinase inhibitor LY294002 abrogated the FSH effect. These results suggest a similar mode of action for the inhibitor and mutation strategies: decreased phosphorylation of one or more of the three sites. In the absence of evidence to support FSH stimulation of Thr24 phosphorylation, we will focus our future studies to investigate the other two point-mutated sites of the T/S/S mutant, Ser256 and Ser319, which may be critical sites for FSH regulation.
Next, we investigated whether Akt, the most widely documented FKHR kinase (15, 16, 17, 18, 22, 25), was activated in response to FSH in these cells. Akt was robustly phosphorylated after FSH treatment, but the effect was later and more sustained than the FKHR response. It is possible that our Western blot experiments were not sensitive enough to detect early changes in Akt phosphorylation that may still be sufficient to activate FKHR. Alternatively, it is quite possible that the FSH-dependent effect on FKHR in these cells is mediated by another PI 3-kinase substrate. Gonzalez-Robayna et al. (6) demonstrated FSH activation of Sgk, an Akt-related kinase that has recently been postulated to perform some of the roles originally attributed to Akt. Sgk or another PI 3-kinase/phosphoinositide-dependent kinase 1 (PDK1) substrate could potentially mediate FSH effects on this pathway (Fig. 7). A dominant negative strategy, or other Akt-blocking experiment, needs to be employed to determine whether the observed FSH effects on FKHR are Akt dependent. A recent study by Zeleznik et al. (26) used a dominant negative strategy to demonstrate that Akt does indeed play an important role in at least some aspects of FSH signaling (e.g. FSH-stimulated GC differentiation).
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). Promotion of GC growth and survival by FSH in vivo has long been acknowledged, but demonstrating the mechanism by which these physiologic end points are stimulated has been difficult to achieve in vitro. The evidence for involvement of the forkhead family in cell cycle arrest and apoptosis, and FKHR expression and hormonal regulation in the ovary, suggests FKHR as a potential mediator of these important events in follicular growth and development. Data to date in several cell lines indicate that negative regulation of forkhead family members results in cell-type specific survival effects (15 , reviewed in Ref. 22). It will be exciting to determine the effects of FKHR and FKHR-related transcription factors in GCs. Recently developed expression and dominant negative strategies should make this possible (27, 28). Exploration of the physiologic effects resulting from these agents may provide insight on the potential role for FKHR in normal follicular atresia or perhaps pathological conditions such as idiopathic premature ovarian failure.
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Footnotes |
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Abbreviations: CREB, cAMP response element-binding protein; FBS, fetal bovine serum; FKHR, forkhead homolog in rhabdomyosarcoma (FoxO1a); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, granulosa cell; GFP, green fluorescent protein; PI 3-kinase, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKB, protein kinase B; Sgk, serum- and glucocorticoid-induced kinase; TBS-T, Tris-buffered saline plus Tween 20.
Received May 30, 2003.
Accepted for publication August 28, 2003.
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