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Originally published In Press as doi:10.1074/jbc.M409486200 on December 21, 2004

J. Biol. Chem., Vol. 280, Issue 10, 9135-9148, March 11, 2005
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Induction of Cyclin D2 in Rat Granulosa Cells Requires FSH-dependent Relief from FOXO1 Repression Coupled with Positive Signals from Smad*

Youngkyu Park{ddagger}, Evelyn T. Maizels{ddagger}, Zachary J. Feiger{ddagger}, Hena Alam{ddagger}, Carl A. Peters{ddagger}, Teresa K. Woodruff§, Terry G. Unterman¶, Eun Jig Lee||, J. Larry Jameson||, and Mary Hunzicker-Dunn{ddagger}**

From the Departments of {ddagger}Cell and Molecular Biology and ||Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, the §Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208, and the Department of Medicine, University of Illinois College of Medicine and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612

Received for publication, August 18, 2004 , and in revised form, December 15, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ovarian follicles undergo exponential growth in response to follicle-stimulating hormone (FSH), largely as a result of the proliferation of granulosa cells (GCs). In vitro under serum-free conditions, rat GCs differentiate in response to FSH but do not proliferate unless activin is also present. In the presence of FSH plus activin, GCs exhibit enhanced expression of cyclin D2 as well as inhibin-{alpha}, aromatase, steroidogenic factor-1 (SF-1), cholesterol side chain (SCC), and epiregulin. In this report we sought to identify the signaling pathways by which FSH and activin promote GC proliferation and differentiation. Our results show that these responses are associated with prolonged Akt phosphorylation relative to time-matched controls and are dependent on phosphatidylinositol 3-kinase (PI 3-kinase) and Smad2/3 signaling, based on the ability of the PI 3-kinase inhibitor LY294002 or infection with adenoviral dominant negative Smad3 (DN-Smad3) mutant to attenuate induction of cyclin D2, inhibin-{alpha}, aromatase, SCC, SF-1, and epiregulin. The DN-Smad3 mutant also abolished prolonged Akt phosphorylation stimulated by FSH plus activin 24 h post-treatment. Infection with the adenoviral constitutively active forkhead box-containing protein, O subfamily (FOXO)1 mutant suppressed induction of cyclin D2, aromatase, inhibin-{alpha}, SF-1, and epiregulin. Transient transfections of GCs with constitutively active FOXO1 mutant also suppressed cyclin D2, inhibin-{alpha}, and epiregulin promoter-reporter activities. Chromatin immunoprecipitation results demonstrate in vivo the association of FOXO1 with the cyclin D2 promoter in untreated GCs and release of FOXO1 from the cyclin D2 promoter upon addition of FSH plus activin. These results suggest that proliferation and differentiation of GCs in response to FSH plus activin requires both removal of FOXO1-dependent repression and positive signaling from Smad2/3.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ovarian follicles produce hormones and growth factors that regulate not only the hypothalamic-pituitary axis, uterine receptivity, and secondary sex characteristics but also in an autocrine or paracrine manner, granulosa, and theca cells as well as the oocyte contained within a follicle. The initial recruitment of primordial follicles is incompletely understood, but appears to require members of the transforming growth factor {beta} (TGF{beta})1 family including bone morphogenic protein-7 (BMP-7) (1) and activin (2). Subsequent granulosa cell (GC) proliferation to form a secondary follicle requires the oocyte product growth differentiation factor-9 (GDF-9), also a member of the TGF{beta} family (3) as well as BMP-15 (1). However, GC proliferation during these early stages of folliculogenesis proceeds with a doubling time of greater than 7 days (4) and is independent of follicle-stimulating hormone (FSH) (1, 5). Final follicular maturation to a preovulatory phenotype requires FSH (6). In response to an FSH stimulus in the intact animal, GC doubling time is reduced to ~24 h (4), and GCs express proteins that characterize the preovulatory phenotype such as P450 aromatase (aromatase), P450 side chain cleavage (SCC), inhibin-{alpha}, and others (7). Although GCs are the only FSH target in female mammals, rat GCs do not divide in primary culture under serum-free conditions (7). However, rat GCs in the presence of FSH plus activin or GDF-9 do proliferate under serum-free culture conditions (8–11). Additionally, while FSH alone promotes GC differentiation (7), FSH plus activin or GDF-9 leads to a synergistic response by many markers of differentiation, including aromatase and inhibin-{alpha} (11–15).

FSH signals in GCs by activating the heptahelical G protein-coupled FSH receptor (16). This receptor, via the stimulatory G protein Gs, promotes activation of adenylyl cyclase, generating increased cAMP production (7), and consequent activation of protein kinase A (PKA) (17). cAMP/PKA then signals to promote induction of genes, which characterize differentiated GCs via multiple pathways and transcription factors, including the extracellular-regulated kinase (ERK) (18) and phosphatidylinositol 3-kinase (PI 3-kinase) (19–23) pathways. As a result, chromatin structure can be relaxed at FSH-responsive gene promoter loci in response to the phosphorylation and acetylation of histone H3 on Ser10 and Lys14, respectively (24), and transcription factors such as CREB (25, 26), SF-1 (27–29), liver receptor homolog-1 (LRH-1) (27, 30), and hypoxia inducible factor 1 (HIF1) (20) are activated, resulting in the transcriptional activation of genes that characterize the preovulatory phenotype. However, in contrast to this differentiation response to FSH, the cellular mechanisms by which FSH stimulates GC proliferation are poorly understood.

Progression from G1 to the S phase of the cell cycle is mediated by increased expression of the cyclin-dependent kinase (Cdk) 4/6 partner cyclin D followed by increased expression of the Cdk2 partner cyclin E (31, 32). The resulting hyperphosphorylation of the retinoblastoma (Rb) protein catalyzed by Cdk 4/6 and subsequently by Cdk2 relieves inhibition of the transcription factor E2F by Rb, allowing for the expression of genes necessary for DNA replication and S phase entry (31, 32). Cell cycle progression is also regulated by the levels of the Cdk inhibitors p27Kip1, p21Waf1, p130, and others (31, 32).

FSH-stimulated GC proliferation in intact animals requires increased expression of cyclin D2; follicles of cyclin D2-null mice do not proceed beyond the secondary stage of development (33). Similarly, follicles of FSH-null mice do not proceed beyond the secondary stage of follicle development (6). Yet, the addition of FSH alone to serum-free GC cultures promotes at most a modest (9, 34) increase in cyclin D2 protein expression. However, in the presence of activin, cyclin D2 mRNA expression is strongly enhanced by FSH under serum-free culture conditions (9, 10). There is also a recent report that FSH-stimulated proliferation of porcine GCs is associated with exit of the Cdk2 inhibitor p27Kip1 from the nucleus, thereby relieving its inhibition of Cdk2 (22).

In this report we sought to evaluate the signaling pathways by which FSH plus activin mediate GC proliferation. Recent studies from various cellular models suggest that the PI 3-kinase pathway plays a prominent role in regulating cell cycle progression. Signals into the PI 3-kinase pathway, traditionally via the insulin or insulin-like growth factor 1 (IGF1) receptors, result in the activation of the serine/threonine kinase Akt (35) leading to the phosphorylation of a number of substrates (direct and indirect), including three of the forkhead transcription factors in the FOXO subfamily (forkhead box-containing protein, O subfamily; FOXO1, -3a, and -4); tuberin, the upstream regulator of mammalian target of rapamycin (mTOR); and glycogen synthase kinase {beta} (GSK3{beta}). Akt has been reported to regulate cell cycle progression by a variety of pathways. Akt can block the action of the cell cycle inhibitors p27Kip1 and/or p21Waf1 by promoting their direct phosphorylation, resulting in their exit from the nucleus (36–38). However, this phosphorylation site on p27Kip1 is not conserved in rats (35, 39). Regulation of the cyclins can occur as a result of Akt-dependent activation of mTOR, which functions to relieve inhibition of translational machinery, resulting in increased expression of cyclins (40, 41). Regulation of the cyclins can also occur at the level of proteasomal degradation. In the absence of signaling down the PI 3-kinase pathway, GSK3{beta} is active and phosphorylates the D-type cyclins, leading to their ubiquitination and degradation (42–45). Regulation of both the cyclins and/or p27Kip1 can also occur at the level of Akt-regulated FOXO transcription factors, which are functional in the absence of PI 3-kinase signals. In response to phosphorylation by Akt, the DNA binding affinity of FOXO1, -3a, and -4 factors is reduced and they exit the nucleus (46–50). FOXO transcription factors have been reported to inhibit cell cycle progression by enhancing transcription of p27Kip1 and other Cdk inhibitors (51–55), by repressing cyclin D transcription (56–58), and/or by increasing transcription of the unconventional cyclin G2, which inhibits the cell cycle (59). Inactivation of FOXO upon Akt-dependent phosphorylation would result in decreased expression of the Cdk inhibitors, increased expression of cyclin D, and decreased expression of cyclin G2, thereby favoring cell cycle progression. Indeed, FOXO3a-null mice exhibit global follicular maturation and consequent follicular depletion as a result of increased GC mitotic activity at the primary stage of follicular development, consistent with a role for FOXO3a to suppress early follicular maturation (60).

While FSH is well known to activate the PI 3-kinase pathway leading to Akt activation (18, 19, 23, 50, 61) and FOXO1 phosphorylation (23, 50) as well as to activate mTOR (20), cell cycle progression in cultured rat GCs requires FSH plus activin or GDF-9 (8–11). It is not known how activin/GDF-9 synergizes with FSH to initiate the cell cycle. TGF{beta} family members including activin and GDF-9 bind to the type II cell surface receptor, the type I receptor is recruited and phosphorylated by the type II receptor, and the type I receptor then phosphorylates the mediatory Smad proteins (Smad2 and -3) (1, 62). Co-Smad4 binds to Smad2 or -3, and the complex translocates to the nucleus where it initiates transcription by complexing with other transcription factors and coactivators (62). Smad3-deficient mice exhibit reduced fertility (63). A recent study showed that the reduced fertility of the Smad3-deficient mice results from slowed follicle growth and low levels of proliferating cell nuclear antigen (PCNA), cyclin D2, and Cdk4 (64).

We report herein that FSH plus activin uniquely promote sustained phosphorylation/activation of Akt 24 h post ligand addition compared with time-matched controls, and that this response is correlated with induction of cyclin D2 and PCNA as well as increased thymidine incorporation into DNA. Prolonged Akt phosphorylation as well as induction of cyclin D2, SF-1, and markers of differentiation are blocked by infection of GCs with an adenoviral dominant negative (DN)-Smad3 mutant, which lacks activin type I receptor phosphorylation sites. FOXO1 appears to function as a repressor of cyclin D2 expression, based on results showing that infection of GCs with an adenoviral constitutively active FOXO1 mutant abrogates the induction of cyclin D2 as well as cyclin D2-luciferase reporter activity stimulated by FSH plus activin in GCs. In vivo association of constitutively active FOXO1 with cyclin D2 in GCs is confirmed by chromatin immunoprecipitation (ChIP) assay. Taken together, these results suggest that FSH plus activin signal to sustain Akt phosphorylation by a pathway that requires Smad2/3 phosphorylation, thereby relieving cyclin D2 from repression, and allowing the cell cycle to progress.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Recombinant human activin A (activin) was purified as described previously (65). The following were purchased: ovine FSH (oFSH-20) from Dr. A. F. Parlow of the NIDDK National Hormone and Pituitary Program (Harbor-UCLA Medical Center, Torrance, CA); enhanced chemiluminescence (ECL) reagents, rainbow molecular weight markers, and Hybond-C nitrocellulose membranes from Amersham Biosciences; SDS-PAGE reagents from Bio-Rad; X-Omat AR film from Eastman Kodak; all culture media from Invitrogen; LY294002, rapamycin, PD98059 from Calbiochem; AG1478 from Alexis Biochemicals (San Diego, CA). All other biochemical reagents were purchased from Sigma, unless otherwise indicated.

Antibodies—The following were purchased: anti-phospho-ERK (Thr202/Tyr204), anti-phospho-AKT (Ser473), anti-phospho-FOXO1 (Ser256), anti-phospho-Smad2 (Ser465/Ser467; also reacts with phospho-Smad3, Ser423/Ser425), anti-AKT, and secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technologies, Beverly, MA); anti-SF-1 (ABR Affinity Bioreagents, Golden, CO); anti-PCNA (Upstate Biotechnology, Lake Placid, NY); anti-cyclin D2, anti-p27, anti-FOXO1, anti-CREB binding protein (CBP), and anti-G{alpha}s (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-Smad2 and anti-Smad3 (Zymed Laboratories Inc. South San Francisco, CA). Phosphorylation sites on FOXO1 correspond to human FOXO1 sequence.

Cell Cultures—For primary GC cultures, female Sprague-Dawley rats were obtained at 15–18 days of age (Charles River Laboratories, Inc., Wilmington, MA) and maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats at 21–24 days of age were injected subcutaneously with 1.5 mg/ml estrogen for 3 days. Ovaries were trimmed to remove the bursa, fat, and oviducts, and incubated for 15–30 min at 37 °C in 6 mM EGTA in Dulbecco's modified Eagle media/Ham's F-12 (DMEM, F-12) media. Ovaries were then incubated for 5–20 min in 0.5 M sucrose in DMEM,F-12. GCs were expressed by penetration of follicles with a 30-gauge needle. Cells were plated on fibronectin (BD Biosciences, San Jose, CA)-coated 60-mm dishes or 12-well plates at a density of ~3 x 106 cells/dish and ~3 x 105 cells/well, respectively, in DMEM/F12 serum-free medium supplemented with 1 nM estradiol-17{beta}, 100 units/ml penicillin G, and 100 µg/ml streptomycin. Cells were treated with hormones (FSH and/or activin) ~20 h after plating (17, 66). When indicated, cells were pretreated for 1 h with LY294002 (12.5 µM in Me2SO), rapamycin (100 nM in Me2SO), PD98059 (50 µM in Me2SO), or AG1478 (250 nM in Me2SO). Treatments were terminated by aspirating medium and rinsing cells once with PBS. Cells were scraped in SDS-PAGE sample buffer (67) followed by heat denaturation for Western blotting or TRIzol solution (Invitrogen) for RNA isolation. Total cells on 12-well plates were lysed for either thymidine incorporation or luciferase assays, as described below. For TSA cell culture, TSA201 cells (human embryonic kidney cell line) were cultured in DMEM/F12 supplemented with 10% fetal bovine serum, penicillin G, and streptomycin at 37 °C in 5% CO2. Cells (2 x 105 cells/well) were plated on 12-well plates for transient transfection experiments.

Electrophoresis and Western Blotting—Protein concentrations were controlled by plating identical cell numbers per plate in each experiment then loading equal volumes of cell extract per gel lane. Proteins in total cell extracts were separated by SDS-PAGE (68), electrotransferred to Hybond C-extra nitrocellulose overnight at 4 °C, and stained using Ponceau-S. The nitrocellulose blots were incubated with primary antibody at 4 °C overnight. Antigen-antibody complexes were detected using ECL. Protein loading was assessed by total Akt Westerns, as indicated. Quantitation was carried out using the Molecular Analyst/PC Image Analysis software (Bio-Rad).

Plasmid Construction—Expression vectors of FOXO1 in pcDNA3 (Invitrogen) including FOXO1 wild type (WT) and FOXO1 mutants were kind gifts of Dr. K. L. Guan, University of Michigan, Ann Arbor (69). Expression vectors for Smad2 and -4 in pRK5 (BD Pharmingen) were kind gifts from Dr. Y. Zhang (National Institutes of Health, Bethesda, MD) (70). A 680-bp fragment of the 5'-flanking region of the rat cyclin D2 (–680 to +225) gene was amplified from rat genomic DNA by PCR using 2 primers; antisense 5'-GATCAAGCTTAGCTAGCCGGTCACCACTCGGTCC and sense 5'-GATCCTCGAGGGTCATATTCTACCAGGC. A fragment was excised with XhoI and HindIII digestion and subcloned into pGL3 basic vector (Promega) digested with XhoI and HindIII. Inhibin-{alpha} promoter (2021 bp)-reporter was described previously (29). Epiregulin-reporter plasmid was the generous gift of Dr. Kaoru Miyamoto, Fukui Medical University, Fukui, Japan. The SF-1 proximal promoter-luciferase construct was the kind gift of Dr. Leslie Heckert, University of Kansas Medical Center, Kansas City.

Generation of Transfer Plasmids and Recombinant Adenoviral Vectors—A cassette containing either the WT or mutant FOXO1 and DN-Smad3 cDNAs driven by the cytomegalovirus (CMV) promoter/enhancer with a simian virus (SV) 40 polyadenylation (p(A)) sequence was subcloned into an adenoviral transfer plasmid based on pcDNA3 (71, 72). Smad2 plasmid was the kind gift of Dr. J. Massague, Memorial Sloan-Kettering Cancer Center (73). DN-Smad3 was constructed by the Woodruff laboratory. The resulting plasmids were used to generate recombinant adenoviruses (72), in which the E1 gene has been deleted. Empty (E) recombinant adenoviruses (Ad) or those carrying WT-FOXO1; FOXO1 T24A, S256A, S319A (identified as A3-FOXO1); A3-FOXO1 H215R (identified as A3HR-FOXO1); and a DN-Smad3 were designated Ad-E, Ad-WT-FOXO1, Ad-A3-FOXO1, Ad-A3HR-FOXO1, and Ad-DN-Smad3, respectively. Individual clones of the recombinant adenoviral vectors were purified and titrated by plaque assays. The sequences of the expression cassettes in the adenoviral vectors were confirmed by automated DNA sequencing. Ad-Gal {beta}-galactosidase driven by CMV promoter (74) was used to evaluate the efficiency of gene transduction in the cell culture system.

Adenoviral Infection of Primary GCs—GCs were plated on fibronectin-coated 60-mm dishes at a density of 3 x 106 cells per dish and allowed to attach for 4–6 h. Cells were infected with Ad-Gal to determine transfection efficiency or with Ad-DN-Smad3 overnight (13 h). Cells were then washed with PBS. Fresh medium containing estrogen was added, and cells were either not treated or treated with FSH plus activin for 24 h. For X-gal staining, GCs were fixed with 1.0% glutaraldehyde for 10 min, washed with PBS, and then incubated with X-gal substrate solution (10 mM potassium ferrocyanide, 10 mM potassium ferricyanide, 1 mM MgCl2, 20% Nonidet P-40, and 0.1% X-gal in PBS) at 37 C for 2 h as described previously (72). After washing cells with PBS three times, cells were placed in 75% glycerol solution and photomicrographs were taken using an inverted microscope (Leitz, Wetzlar, Germany). Results are representative of two separate experiments. For infections with FOXO1 mutants, pilot time course studies showed that an overnight infection promoted cell death while a 4-h infection was not cytotoxic. Thus, GCs were infected (4–6 h after plating) for 4 h with FOXO1 mutants, fresh medium containing estrogen was added, and cells were incubated overnight so that all cells were then treated with FSH plus activin at the same time. 24 h post-treatment with FSH plus activin, cells were lysed for Western blotting or RNA isolation, as described above.

RT-PCR—Total RNA was isolated from the primary GCs 24 h after hormone treatments. RT-PCR was performed as described previously (71). Expression levels of aromatase, epiregulin, inhibin-{alpha}, SCC, SF-1, LRH-1, L19, cyclin D2, and tubulin mRNAs were measured using primers identified in Table I.


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  		TABLE I
Primer sequences used in RT-PCR

 
PCR was carried out at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min, for 24 cycles for L19 cyclin D2(product size, 188 bp) and tubulin (406 bp), and 29 cycles for aromatase (334 bp), epiregulin (205 bp), inhibin-{alpha} (370 bp), SCC (545 bp), SF-1 (160 bp), cyclin D2 (299 bp), and LRH-1 (193 bp). Cycle number was chosen based on preliminary studies to identify the linear signal range from 22 to 40 cycles. PCR products were separated on 2% agarose gels, and visualized by ethidium bromide staining on the UV illustrator. Images were taken using Kodak Image software.

Transient Transfection and Luciferase Assays—GCs were plated on 12-well plates for transient transfection experiments. Cells were transfected with luciferase reporter plasmids (500 ng/well) and indicated expression vectors (50 ng/well) using Lipofectamine 2000 (Invitrogen) as described (20). DNA and Lipofectamine were mixed in OPTI-MEM (Invitrogen) and added to cells 20 h after plating. Fresh medium was added 6 h later, and cells were harvested for luciferase assays 30 h after transfection in lysis buffer (25 mM glycyl-glycine, 15 mM MgS04, 4 mM EGTA, 1% (v/v) Triton X-100, and 10 mM dithiothreitol). Hormones were present during the last 6 h of cell culture. Total protein was measured with the Bio-Rad protein assay reagent according to the manufacturer's instructions. Luciferase assays were done using a luminometer, as described previously (29, 71). TSA201 cells were plated in 12-well plates 24 h prior to transfection. Cells were transfected with same amount of DNA described above using calcium phosphate methods (75).

ChIP Assay—Following indicated treatments, ChIP assays were performed, as described previously (24, 29). Briefly, cells were incubated (25 min at room temperature) in 1% formaldehyde to cross-link DNA and proteins and then sonicated in cell lysis buffer. One-tenth of the total lysate was used for purification of total genomic DNA. The rest of the lysate was incubated with indicated antibody at 4 °C for 18 h. Following the collection of immunoprecipitates using protein A/G-agarose, DNA was extracted by phenol/chloroform extraction and ethanol precipitation, and PCR was performed using either total DNA or immunoprecipitated DNA. PCR products were separated on 1.5% agarose gel and visualized by ethidium bromide staining. Primers used for PCR corresponding to sequences within rat cyclin D2 promoter regions (–680 to –659) are 5'-GAGGGTCATATTCTACCAGG-3' and (–280 to –258) 5'-GAACCCTCAAAACCCACGGATT-3'.

Thymidine Incorporation Assay—Cells (3 x 105 cells/well in 12-well plate) were plated overnight, treated with FSH (50 ng/ml) and/or activin (30 ng/ml) for 26 h, and then labeled with [3H]thymidine (1 µCi/well, Amersham Biosciences) during the last 4 h (76). Cells were then washed with PBS and lysed into 0.5 M KOH and genomic DNA was precipitated using 20% trichloroacetic acid. Precipitated DNA was bound to Whatman GF/C filter paper using a Millipore Filter Manifold (Millipore Corp., Bedford, MA), filters were washed with ethanol, and 3H was quantified by liquid scintillation counting. Values (cpm/min) were expressed as a fold induction over control and represent the mean ± S.E. of four independent experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
FSH Plus Activin Stimulates GC Proliferation and Differentiation—We initially confirmed under our serum-free culture conditions that GC proliferation required the addition of FSH plus activin. As shown in Figs. 1, A and B, increased thymidine incorporation into DNA and expression of cyclin D2 as well as S phase marker (77) PCNA proteins, were detected only in GCs treated for 24 h with FSH plus activin and not in cells treated with FSH or activin alone. We did not detect a change in the total levels of the cell cycle inhibitor p27Kip1 in cells treated for 24 h with FSH plus activin as compared with untreated cells (not shown). We also evaluated the protein expression of the orphan nuclear receptor steroidogenic factor-1 (SF-1) in GCs treated for 24 h with FSH and/or activin, based on its established role in the transcriptional activation of FSH target genes (29, 78, 79). Results (Fig. 1B) show that while FSH promotes a modest increase in SF-1 expression, consistent with an earlier report (80), expression of SF-1 was readily detected in cells treated with FSH plus activin compared with FSH alone. Treatment of cells with activin alone for 24 h did not affect the expression of SF-1 protein.



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  		FIG. 1.
Effect of FSH and activin on the induction of markers of GC differentiation and proliferation. A, cells in 12-well plates were not treated (CON) or were treated with 50 ng/ml FSH and/or 30 ng/ml activin (ACT) for 26 h. [3H]thymidine (1 µCi/well) was present during the last 4 h. Incorporated [3H]thymidine levels were measured as described under "Experimental Procedures." Values represent mean of fold induction over control ± S.E. of four independent experiments. B, cells were not treated (CON) or were treated with FSH and/or activin for 24 h, as described in A. Western blots of total cell extracts were probed with indicated antibodies. Akt is used as a loading control. Results are representative of two separate experiments. C, cells were treated with FSH and/or activin for 24 h. RT-PCRs were performed using RNAs isolated from cells and specific primers, as described under "Experimental Procedures." L19 or tubulin, as indicated, is used as a loading control. PCR products were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. Results are representative of two independent experiments.

 
Treatment of cells with FSH plus activin for 24 h also resulted in increased expression of mRNA for cyclin D2 (10.3-fold over levels with FSH alone) as well as for select markers of GC differentiation, including aromatase2 (2.9-fold over levels with FSH alone), epiregulin (3.3-fold), inhibin-{alpha} (1.6-fold), SCC (2.6-fold), and SF-1 (2.6-fold) (Fig. 1C), consistent with earlier reports (12–14, 81–83). Activin alone modestly increased mRNA expression of inhibin-{alpha}, consistent with earlier reports (14, 84, 85), cyclin D2 (9), and SF-1. Taken together, these results confirm that rat GC proliferation as well as the synergistic induction of a number of protein markers of GC differentiation under serum-free conditions requires the presence of both FSH plus activin.

Signaling Pathways Activated by FSH Plus Activin—In the following experiments we sought to evaluate the signaling pathways activated by FSH plus activin that lead to GC proliferation and differentiation. FSH is recognized to promote rapid activation both of the PI 3-kinase pathway leading to the phosphorylation of Akt and its downstream target FOXO1 (18, 19, 23, 50, 61) and of the ERK pathway (18, 24, 86, 87). Results showing activation of the PI 3-kinase and ERK pathways are shown in Fig. 2A for GCs treated with FSH for 1 h. In cells treated with FSH plus activin for 1 h, the phosphorylation of Akt, FOXO1, and ERK was equal to or slightly elevated over that detected with FSH alone (~1.5-fold for Akt and ERK phosphorylations). Both Akt and FOXO1 phosphorylations remain elevated 4 h post-addition of FSH plus activin (not shown). Activin alone as well as FSH plus activin promoted the phosphorylation of Smad2/3 (Fig. 2A). Despite evidence in some cell models that members of the TGF{beta} family can stimulate the activation of Akt (88–90), activin alone did not promote activation of the PI 3-kinase pathway in GCs, as neither Akt nor FOXO1 phosphorylation was detected in cells treated with activin alone.



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  		FIG. 2.
Effect of FSH and/or activin on the phosphorylation of Akt, FOXO1, and ERK at 1 and 24 h post-treatment. A, GCs were not treated (CON) or were treated with FSH and/or activin for 1 h. Western blots of total cell extracts were probed with indicated antibodies. Akt is used as a loading control. Results are representative of four separate experiments. B, GCs were pretreated with and without 12.5 µM LY294002, 100 nM rapamycin, or 250 nM AG1478 for 1 h and then untreated (CON) or treated with FSH and/or activin (ACT) for 24 h. Results are representative of three separate experiments. C, GCs were pretreated with and without 12.5 µM LY294004 for 1 h and then left untreated (CON) or treated with FSH and activin for 24 h. RT-PCRs were performed using specific primers described under "Experimental Procedures." Tubulin is used as a loading control. PCR products were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. Results are representative of three separate experiments.

 
By 24 h post-treatment of GCs with FSH alone, Akt, FOXO1, and ERK phosphorylations were undetectable or barely detected in cells treated with FSH alone (Fig. 2B, lane 2). However, phosphorylation of Akt and ERK was readily detected in cells treated with FSH plus activin compared with control cells at this 24 h time point (lanes 1 versus 4). Time course studies showed that the phosphorylation of Akt in the presence of FSH plus activin remained elevated, relative to time-matched controls, throughout the 24 h time phase (not shown). Phosphorylation of FOXO1 was variably detected at 24 h post-FSH plus activin, consistent with its down-regulation upon phosphorylation (46, 50, 91, 92), and indeed, reduced levels of total FOXO1 were detected at 24 h post-FSH plus activin in many cultures (not shown).

To evaluate the signaling pathway(s) leading to the phosphorylation of Akt and ERK at 24 h post-addition of FSH plus activin, we tested the effects of selective inhibitors of PI 3-kinase (LY294002, Ref. 93), mTOR (rapamycin, Ref. 93), and the epidermal growth factor (EGF) receptor (AG1478, Ref. 94) on the phosphorylation of Akt and ERK as well as on induction of cyclin D2 and SF-1 proteins. Although the PI 3-kinase inhibitor LY294002 modestly reduced total cellular protein levels, as evidenced by the reduced signal of total Akt and Ponceau-stained histone proteins, treatment with LY294002 resulted in a pronounced inhibition of Akt and ERK phosphorylation (97 and 93% inhibition, relative to total Akt, respectively) and inhibition of the induction of cyclin D2 and SF-1 proteins (98 and 89% inhibition, relative to total Akt, respectively) (lanes 4 and 8). These results suggest that indeed the phosphorylation of Akt and ERK and induction of SF-1 and cyclin D2 24 h post-treatment of GCs with FSH plus activin require signaling via PI 3-kinase. While the mTOR inhibitor rapamycin also slightly reduced cellular protein, as evidenced by total Akt levels, rapamycin did not reduce the phosphorylation of Akt but reduced the induction of cyclin D2 (by 52%, relative to total Akt) and SF-1 (by 34%) in cells treated 24 h with FSH plus activin (lanes 12 and 4). However, rapamycin abolished ERK phosphorylation detected 24 h post-addition of FSH plus activin. These results suggest that translation regulated by the mTOR pathway likely contributes to the induction of cyclin D2 and SF-1 and is responsible for ERK phosphorylation detected 24 h post-addition of FSH plus activin. Based on recent evidence that FSH increases expression of the EGF receptor agonist epiregulin (81) as well as our evidence of this response in cells treated with FSH plus activin (see Fig. 1C), we hypothesized that ERK phosphorylation at 24 h was a consequence of the induction of epiregulin. We tested this idea by treating cells with the EGF receptor inhibitor AG1478. AG1478 did not modulate the prolonged phosphorylation of Akt or induction of cyclin D2 or SF-1 but prevented the phosphorylation of ERK in cells treated 24 h with FSH plus activin (lane 16 compared with lane 4). Similar results were obtained with the ERK kinase inhibitor PD98059 (not shown). These results indicate that sustained Akt phosphorylation and induction of SF-1 and cyclin D2 are independent of ERK activation and therefore are not the consequence of autocrine factors that activate the EGF receptor. However, ERK activation at 24 h post-FSH plus activin likely results from expression of epiregulin, the expression of which is inhibited by the PI 3-kinase inhibitor LY294002, as shown below. Based on reports that rat GCs express IGF1 (50, 95), we considered that prolonged Akt phosphorylation in GCs treated with FSH plus activin for 24 h could result from autocrine effects of IGF1. However, treatment of GCs with FSH plus activin for 1 or 24 h did not promote detectable IGF1 receptor phosphorylation while a strong positive signal was seen with addition of IGF1 (not shown), suggesting that prolonged Akt phosphorylation is not the result of enhanced production or transactivation of the IGF1 receptor.

To ascertain whether the transcriptional regulation of these targets was via the PI 3-kinase pathway, we tested the effect of FSH plus activin in the absence and presence of LY294002 on target gene mRNA levels by RT-PCR. Results in Fig. 2C show that induction by FSH plus activin of aromatase, inhibin-{alpha}, and cyclin D2 mRNA is dependent on signaling via the PI 3-kinase pathway as LY294002 inhibited mRNA expression greater than 80% relative to control tubulin levels. However, the induction of mRNA for SF-1, LRH-1, epiregulin, and SCC was only partially dependent on PI 3-kinase activity; LY294002 treatment resulted in a ~40% decrease in mRNA for these genes, relative to total tubulin levels, compared with mRNA levels in control cells not treated with LY294002 (lanes 2 and 4). These results suggest that the PI 3-kinase pathway is essential for the regulation of cyclin D2, aromatase, and inhibin-{alpha} expression by FSH plus activin at the level of transcription. While expression of epiregulin, SCC, SF-1 and LRH-1 in response to FSH plus activin is also regulated at the level of transcription in a PI 3-kinase-dependent manner, expression at least of SF-1 also appears to be regulated in a PI 3-kinase-dependent manner at the level of translation and/or protein stability.

Taken together, these results establish that induction of the proliferative and accompanying differentiation responses in GCs in response to FSH plus activin is dependent in large part on PI 3-kinase activity and correlates with prolonged phosphorylation of Akt and appears to be independent of ERK activation. The molecular basis for the prolonged phosphorylation of Akt in cells treated with FSH plus activin is not known, but it does not appear to be attributable to the generation of an autocrine factor, such as epiregulin (81) or IGF1 whose effects are exerted via the EGF or IGF1 receptors, respectively.

Smad2/3 Is Necessary for the Prolonged Phosphorylation of Akt and Induction of Cyclin D2 and SF-1 Proteins—To evaluate the contribution of the activin mediators Smad2/3 in GCs to the proliferation and differentiation responses in cells treated with FSH plus activin, we constructed an adenoviral DN-Smad3 vector. The C-terminal phosphorylation sites on Smad3 which are phosphorylated by the activin type I receptor (62) were deleted, resulting in a protein which should be retained in the cytoplasm and function as a dominant negative (Fig. 3A). Transduction efficiency of adenoviral vectors into primary GCs was tested using adenovirus carrying {beta}-galactosidase (Ad-Gal). {beta}-galactosidase expression was detected in 95% of GCs ~40 h after infection at 2.5 plaque-forming units (pfu) per cell (Fig. 3B). Therefore, subsequent experiments were done using 2.5 pfu of recombinant adenoviral vectors. Results (Fig. 3C) confirm overexpression of the DN-Smad3 in adenoviral-infected GCs versus those infected with an empty adenoviral construct.



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  		FIG. 3.
Construction and expression of adenoviral vectors carrying {beta}-galactosidase, Smad3, and FOXO1 in GCs. A, schematic illustration of recombinant adenoviral vectors carrying DN-Smad3, FOXO1, and {beta}-galactosidase. ITR{psi} and packaging sequences are located on the far left-hand side (1–1.2 map unit). Mutated sites on indicated genes are indicated and are described in the text. DN-Smad3 and {beta}-galactosidase genes were subcloned into E1A-deleted transfer vector, while FOXO genes were subcloned into E1B- and E1A-deleted transfer vector (72). One map unit (m.u.) refers to 360 base pairs. B, the expression of {beta}-galactosidase in GCs infected with 2.5 pfu/cell Ad-Gal is shown (multiplicity of infection, i.e. number of viral particles per cell, of 1). Cells infected with Ad-GAL were washed with PBS ~17 h post-infection and were not treated (CON, left panel) or treated with FSH and activin (FSH ACT, right panel) for 24 h. Cells were fixed with 1.0% glutaraldehyde for 10 min, washed with PBS, and then incubated with X-gal substrate solution at 37 °C for 2 h. Images were taken using an inverted microscope. C, expression of Smad3, total FOXO1, and phospho-FOXO1 proteins in GCs, which were not infected (NoV) or infected with 2.5 pfu/cell Ad-E (empty adenoviral vector), Ad-WT-FOXO1, Ad-A3-FOXO1, Ad-A3HR-FOXO1, and Ad-DN-Smad3 is shown. GCs were infected, as detailed under "Experimental Procedures," washed with PBS, and fresh medium was added. After 17 h, GCs were not treated (CON) or treated with indicated hormones for 24 h.

 
We then evaluated the effect of the adenoviral Smad3 mutant versus control empty viral vector on signaling in GCs treated 1 and 24 h with FSH plus activin. Results (Fig. 4A) show that the Smad3 mutant reduced the phosphorylation of Smad2/3 in cells treated 1 h either with activin alone or with FSH plus activin but did not affect the phosphorylation of Akt or FOXO1. These results confirm that this Smad3 construct indeed functions in a dominant negative manner but is not cytotoxic since Akt and FOXO1 phosphorylations are unaffected. By 24 h, expression of adenoviral DN-Smad3 mutant inhibited the prolonged phosphorylation of Akt and the induction of cyclin D2 and SF-1 protein by 90–100%, relative to total Akt levels, in cells treated with FSH plus activin (Fig. 4B). Infection with the adenoviral DN-Smad3 mutant inhibited the induction by FSH plus activin of epiregulin, inhibin-{alpha}, and SCC mRNA levels by 80–100%, relative to the L19 control, compared with levels of induction with empty adenoviral vector, while SF-1, aromatase, and cyclin D2 mRNA levels were reduced ~50% by the DN-Smad3 mutant (Fig. 4C, compare lanes 2 and 4). Infection with the adenoviral DN-Smad3 mutant did not affect p27 protein expression levels (not shown). Taken together, these results indicate that phospho-Smad2/3 either increases a signal into the PI 3-kinase pathway to prolong Akt phosphorylation or that phospho-Smad2/3 blocks the dephosphorylation of Akt. These results also suggest that activation by FSH plus activin of epiregulin, inhibin-{alpha}, and SCC as well as of aromatase, SF-1 and cyclin D2 genes requires signaling via the Smad2/3 pathway.



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  		FIG. 4.
Effect of DN-Smad3 on signaling to downstream targets 1 and 24 h post-treatment of cells with activin plus FSH and on induction of gene markers for cell proliferation and differentiation. A and B, GCs were infected with 2.5 pfu/cell Ad-E or Ad-DN-Smad3 for 13 h. Cells were then not treated (CON) or treated with FSH and/or activin for 1 h (A) or 24 h (B and C). Western blots in A and B of total cell lysates were probed with indicated antibodies. Akt is used as a loading control. Results are representative of three separate experiments. C, RT-PCRs were performed using specific primers described in Table I. L19 is used as a loading control. Results are representative of two separate experiments.

 
These results led us to test whether or not activin via Smads could activate promoter-reporter activities for cyclin D2, epiregulin, and inhibin-{alpha}. Because of their high transfection efficiency, we used an embryonic kidney TSA201 cell line grown in the presence of 10% serum to conduct these experiments. Co-transfection of TSA201 cells with expression vectors containing Smad2 and co-Smad4 as well as indicated luciferase reporters resulted in a 2–3-fold increase in reporter activities for cyclin D2, epiregulin and inhibin-{alpha} (Fig. 5, compare lanes 1 and 3). These results suggest that Smad2/3 can indirectly or directly activate promoters for cyclin D2, epiregulin, and inhibin-a.



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  		FIG. 5.
Effect of A3-FOXO1 constitutively active mutant and Smads on cyclin D2, epiregulin, and inhibin-{alpha} promoter-reporter activities. TSA201 cells were cotransfected with 50 ng/well of indicated expression vectors (pRK, pRK-Smad2, pRK-Smad4, pcDNA, pcDNA-A3FOXO1) and 500 ng/well of reporter plasmids (cyclin D2 promoter-LUC, epiregulin promoter-LUC, or inhibin-promoter-LUC) using calcium phosphate precipitation, as described under "Experimental Procedures." Cells were harvested 48 h after transfection and assayed for luciferase activity. Relative light units (RLU) were calculated as a percent inhibition or induction over control group. Values represent mean ± S.E. of three independent experiments. Each separate experiment is replicated in triplicate.

 
Constitutively Active FOXO1 Mutant Abrogates the Induction of Cyclin D2 and SF-1 Proteins by FSH Plus Activin—In the following experiments we determined whether the PI 3-kinase dependence of prolonged Akt phosphorylation and induction of cyclin D2, SF-1, and other differentiation markers, was mediated by the Akt substrate, FOXO1. FOXO1 is an active transcription factor in the absence of PI 3-kinase signaling. It binds to an insulin response element (IRE) on DNA of target genes as a monomer, primarily through helix 3 of the winged helix domain (46, 96) and functions to activate or repress target genes (46, 58). To evaluate the potential role of FOXO1 in the proliferative and differentiation responses to FSH plus activin, we developed adenoviral constructs (Fig. 3A) of WT FOXO1, FOXO1 in which the three Akt phosphorylation sites were mutated to Ala (Ad-A3-FOXO1) and which is thus constitutively active and cannot exit the nucleus (69), and FOXO1 which has the three phosphorylation sites mutated to Ala and the conserved His in the DNA binding domain of helix 3 muted to Arg (Ad-A3HR-FOXO1), a mutation which abrogates DNA binding of FOXO1 but retains FOXO1 in the nucleus (58, 69). Results (Fig. 3C) confirm overexpression of WT FOXO1, A3-FOXO1, and A3HR-FOXO1 in adenoviral-infected GCs versus those infected with an empty adenoviral construct or those not infected and show that FOXO1 phosphorylation is not detected in cells treated with the phosphorylation-deficient FOXO1 mutants.

We then evaluated the effect of infection of FOXO1 constructs on induction of cyclin D2, SF-1, LRH-1, aromatase, epiregulin, SCC, and inhibin-{alpha}. Results show that infection with constitutively active FOXO1 mutant (Ad-A3-FOXO1) abrogated the ability of FSH plus activin to induce cyclin D2 and SF-1 proteins (Fig. 6A, compare lane 8 with lanes 2, 4, and 6). Neither total CBP (Fig. 6A) nor GSK3{beta} (not shown) protein levels are reduced by adenoviral infection, showing that treatments are not toxic to cells. Moreover, induction of cyclin D2 in GCs infected with Ad-WT FOXO1 was not different from untreated cells (no virus), confirming that adenoviral infection per se is not deleterious to GCs. Constitutively active FOXO1 similarly inhibited induction of aromatase, epiregulin, LRH-1, cyclin D2, SF-1, and SCC mRNAs by ~85%, relative to loading controls, while inhibin-{alpha} was less inhibited (60%) (Fig. 6B, compare lanes 6 and 2). Infection with the constitutively active FOXO1 mutant containing an additional mutation that prevents binding of constitutively active FOXO1 to DNA (Ad-A3HR-FOXO1) largely reversed the inhibitory effects of A3-FOXO1 in cells treated with FSH plus activin (Fig. 6B, compare lanes 8 and 2). This result suggests that the DNA binding ability of FOXO1 is crucial for its ability to repress induction of these genes. However, at least for cyclin D2 and SF-1, protein remained suppressed in cells treated with the constitutively active FOXO1 mutant that cannot bind DNA (Fig. 6A, lanes 9 and 10), suggesting regulation by FOXO1 independent of its DNA binding ability of translation and/or protein stability for these two proteins.



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  		FIG. 6.
Effect of constitutively active FOXO1 (A3-FOXO1) on induction by FSH plus activin of markers of GC proliferation and differentiation. A and B, GCs were not infected (NoV) or were infected with 2.5 pfu/cell of Ad-E, Ad-WT-FOXO1, Ad-A3-FOXO1, or Ad-A3HR-FOXO1 for 4 h, washed, and then placed with fresh medium for 9 h. 13 h post-infection, cells were not treated (CON) or were treated with FSH plus activin (FSH ACT) for 24 h. A, Western blots were probed with indicated antibodies. Akt is used as a loading control. Results are a representative of three separate experiments. B, RT-PCRs were performed using specific primers described in Table I. Tubulin is used as a loading control. Results are representative of two or three separate experiments.

 
These results suggest that active FOXO1 repressed transcription of a subset of FSH target genes. We further tested this supposition by evaluating the effect of the constitutively active FOXO1 mutant introduced into GCs by transient transfection on reporter activities of cyclin D2, epiregulin, and inhibin-{alpha} promoters in cells treated without and with FSH plus activin. Results in Fig. 7A, expressed relative to total cellular protein (97), show that FSH plus activin activated reporter activities for cyclin D2, epiregulin, and inhibin-{alpha} by 2–7-fold, depending on the promoter. Co-transfection with an expression vector containing constitutively active FOXO1 mutant reduced basal activity of the cyclin D2 and epiregulin promoters, and abolished activation by FSH plus activin of all three reporter constructs. Similarly in TSA201 cells in the presence of serum, expression of the constitutively active FOXO1 mutant suppressed promoter-reporter activities for cyclin D2, epiregulin, and inhibin-{alpha} (Fig. 5, compare lanes 3 and 4).



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  		FIG. 7.
Effect of constitutively active FOXO1 on basal and FSH plus activin-stimulated activities of cyclin D2, epiregulin, and inhibin-{alpha} promoters and on the in vivo association of the cyclin D promoter with FOXO1. A, GCs were cotransfected with 50 ng/well of A3-FOXO1 or empty expression vectors and 500 ng/well of reporter plasmid using Lipofectamine 2000, as described under "Experimental Procedures." Cells were washed 4 h post-transfection and were harvested 32 h after transfection and assayed for luciferase activity. Cells were treated with FSH plus activin during last 6 h of treatment. RLU were calculated as a percent over control group (empty vector, no hormone) relative to total protein levels in each well. Values represent mean ± S.E. of three triplicate determinations and are representative of three separate experiments. Results in B, C, and D represent ChIP assays. B, cells were not treated (CON) or were treated with FSH plus activin for 1 h. Following DNA and protein cross-linking with formaldehyde, an aliquot was removed for purification of total DNA, and immunoprecipitations (IP) were conducted using anti-FOXO1 and control anti-G{alpha}s antibodies. DNA was extracted from immunoprecipitates, and PCR (29 cycles) was conducted on total DNA and immunoprecipitated DNA with primers to promoter region of cyclin D2 gene. C, cells were not treated or treated with FSH, activin or FSH plus activin for 1 h. Immunoprecipitations were conducted with anti-FOXO1 antibody, PCR (40 cycles) was conducted on total and immunoprecipitated DNA, as described above. D, cells were infected with Ad-E or Ad-A3-FOXO1, then not treated (CON) or treated with FSH plus activin for 1 h.

 
To prove that FOXO1 binds in vivo to the cyclin D2 promoter, we conducted ChIP assays. Cells were treated with and without FSH plus activin for 1 h, protein and DNA were cross-linked, then subjected to immunoprecipitation with anti-FOXO1 antibody or a control anti-G{alpha}s antibody. DNA cross-linked to immunoprecipitated FOXO1 and G{alpha}s was subjected to PCR using a primer for the rat cyclin D2 promoter (–680 to –285). Results in Fig. 7B show that cyclin D2 is complexed with FOX01 in control GCs and that treatment with FSH plus activin largely abrogates this association (lanes 3 and 4). Cyclin D2 signal is not detected in control G{alpha}s immunoprecipitates (lanes 5 and 6). We then determined whether FSH or activin alone was sufficient to release FOXO1 from the cyclin D2 promoter. Results (Fig. 7C) show that FSH alone was as effective as FSH plus activin in reducing the binding of FOXO1 to cyclin D relative to total DNA; activin alone was ineffective. Finally, we evaluated the association of FOXO1 and the cyclin D2 promoter in cells that were infected with empty adenovirus or with constitutively active FOXO1 mutant, treated with and without FSH plus activin for 1 h. Results (Fig. 7D) show that in cells infected with adenoviral constitutively active FOXO1 mutant, a strong signal for cyclin D2 promoter DNA is detected in control cells and this signal is not reduced upon treatment with FSH plus activin (lanes 4 and 5). Taken together, these results suggest that FOXO1 functions to repress cyclin D2 expression, and that upon phosphorylation of FOXO1, its repressive actions are reversed.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Proliferation of rat GCs under serum-free culture conditions requires FSH plus activin or GDF-9 (8–11). We show in this report that both activin-regulated Smad2/3 activity as well as FSH-dependent PI 3-kinase-regulated FOXO1 activity contribute to the ability of FSH plus activin to initiate GC proliferation, as depicted in the model shown in Fig. 8. The proliferative response requires release of cyclin D2 from inhibition by FOXO1, resulting in the expected export of phosphorylated FOXO1 (46, 49, 50, 98), as well as stimulation by active Smad2/3. Moreover, synergistic differentiation responses elicited by FSH plus activin also require removal of the suppressive effect of FOXO1 and Smad2/3 signaling, as modeled in Fig. 8.



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  		FIG. 8.
Schematic model depicting signaling by FSH and activin to promote GC proliferation and synergistic differentiation. Model shows that FSH signals to activate PI 3-kinase to promote Akt phosphorylation and consequently phosphorylation of FOXO1, resulting in release from promoters of cyclin D2 and, potentially, epiregulin, aromatase, and SF-1, and exit of phosphorylated FOXO1 from the nucleus. Also necessary for GC proliferation and synergistic activation of differentiation genes is positive signaling through Smad2/3 via activin or a related TGF{beta} ligand, resulting in transactivation of indicated target genes.

 
Activation by FSH of the PI 3-kinase pathway is necessary but not sufficient for initiation of the proliferative response. GC proliferation stimulated by FSH plus activin is distinguished from FSH-stimulated differentiation (in the absence of exogenous activin) by the prolonged phosphorylation of Akt,3 detected 24 h post-addition of FSH plus activin, compared with controls at 24 h. We do not know the molecular basis for prolonged Akt phosphorylation in cells treated with FSH plus activin. Prolonged Akt phosphorylation could result from persistent signaling down the PI 3-kinase pathway or from inhibition of an Akt-phosphatase. We considered the possibility that an autocrine factor generated in response to FSH plus activin could mediate this response. While epiregulin, a recently identified FSH-inducible gene (81) whose protein product binds to the EGF receptor, is a good candidate to promote proliferation, induction of cyclin D2 was unaffected by pretreatment of cells with the EGF receptor inhibitor AG1478. We also considered the possibility that FSH plus activin stimulated expression of IGF1 to prolong Akt phosphorylation, based on results in Sertoli cells that FSH activates Akt via expression of IGF1 (61) as well as evidence in bovine GCs that IGF1 promotes proliferation (99). However, we could not detect increased IGF1 receptor phosphorylation in cells treated with FSH or FSH plus activin at 1 or 24 h post-treatment, while IGF1 alone stimulated robust IGF1 receptor phosphorylation (not shown). IGF1 is also an unlikely candidate in rat GCs as the co-addition of FSH plus IGF1 does not induce cyclin D2 expression in rat GCs (not shown). Similarly, expression of constitutively active Akt in cells treated with FSH does not result in increased expression of PCNA or cyclin D2 (21). However, we cannot rule out the possibility that other unidentified autocrine factors generated in GCs treated with FSH plus activin result in prolonged Akt phosphorylation. It is interesting that prolonged Akt phosphorylation was also reported in porcine GCs under conditions that stimulated proliferation (23).

Infection of GCs with DN-Smad3 mutant prevented both prolonged Akt phosphorylation as well as induction of cyclin D2. Based on the high degree of homology between Smad2 and -3 (62), the DN-Smad3 mutant also functions in a DN manner toward Smad2 (100). There are reports that TGF{beta} and related ligands can signal in a PI 3-kinase-dependent manner to activate Akt to regulate transcription of target genes (88–90). Yet in GCs, activin alone did not promote Akt phosphorylation. There is also recent evidence that phospho-Akt sequesters Smad3, thereby inhibiting Smad3 phosphorylation by activin/TGF{beta} (101). However, in GCs the DN-Smad3 mutant abolished prolonged phosphorylation of Akt. Thus, while we also detect collaboration between PI 3-kinase and Smad signaling pathways, the response in GCs is synergistic rather than antagonistic.

FOXO family members are established regulators of the cell cycle, cell death, metabolism, and oxidative stress (96). FOXO transcription factors bind DNA as a monomer at the IRE consensus sequence TT(A/G)TT(T/G)(A/G)(T/C) on target genes (96) and function both as activators and repressors of gene transcription. FOXO3a appears to function as a suppressor of GC mitosis in very early follicular development, based on the global follicular maturation and resulting follicle depletion seen in FOXO3a-null mice (60). While the most abundant expression of FOXO1 mRNA is detected in the ovary (102), the FOXO1-null mouse dies on embryonic day 10.5 (103), precluding analysis of ovarian functions at later times.

Transcriptional activities of the FOXO1, -3a, and -4 are regulated in part by phosphorylations at key residues catalyzed by Akt (46, 96). Three phosphorylated residues reside within the Akt consensus phosphorylation domain. Phosphorylation of Ser256 diminishes DNA binding and is necessary for phosphorylation of Thr24 and Ser319 (48). Phosphorylation of Thr24 disrupts association of FOXO with CBP and stimulates binding to 14-3-3 proteins, and phosphorylation of Ser319 stimulates nuclear export (46, 49, 98). Phosphorylation-deficient A3-FOXO mutant is thus constitutively active and retained in the nucleus. FOXO transcription factors can also function as cofactors of other transcription factors without binding to DNA. For example, constitutively active DNA binding-deficient FOXO mutant, in which a conserved His in helix 3 is mutated to Arg, still induces expression of superoxide dismutase-2 to the same extent as A3-FOXO, as evidenced by expression profiling (58). Thus, FOXO transcription factors can also interact with promoter elements of target genes indirectly either by binding to another transcription factor or via co-activators or co-repressors. Indeed, physical interactions have been documented between FOXO transcription factors and steroid hormone receptors as well as hepatocyte nuclear factor-4, a member of the steroid nuclear receptor superfamily, resulting in either stimulation or inhibition of nuclear receptor transactivation (104–106).

Our results show that infection of GCs with an adenovirus expressing the constitutively nuclear FOXO1 mutant (Ad-A3-FOXO1) suppressed expression of epiregulin, aromatase, SF-1, LRH1, and SCC and moderately suppressed induction of inhibin-{alpha}. Regulation of these target genes by FOXO1 appears to require primarily direct binding of FOXO1 to their promoter regions, based on the ability of constitutively active FOXO1 mutant containing an additional mutation which prevents binding of FOXO1 to DNA to largely rescue target gene expression by FSH plus activin. Consistent with this conclusion, constitutively active FOXO1 in transient transfection experiments in GCs abolished induction by FSH plus activin of epiregulin and inhibin-{alpha} promoter reporter activities. The constitutively active DNA binding-deficient FOXO1 mutant, however, appears to repress expression of SF-1 protein via an unidentified mechanism. The effects of A3-FOXO1 mutant on expression of inhibin-{alpha}, SCC, and aromatase might also result from a reduction in the expression of SF-1 and/or LRH-1, as it is well established that SF-1 and/or LRH-1 directly regulate FSH-induced expression of these genes (27, 29, 30, 78). Thus, we cannot rule out the possibility that the ability of constitutively active FOXO1 mutant to repress expression of these genes in response to FSH plus activin is secondary to repression of SF-1 and/or LRH-1. While the SF-1 promoter-luciferase construct extending from –734 to +160 (107) contains a putative consensus FOXO response element, identified by computer search, reporter activity was not changed upon expression of the A3-FOXO1 mutant in TSA201 cells (not shown). Thus, the mechanism by which FOXO1 regulates expression of SF-1 remains to be defined.

The constitutively active FOXO1 mutant also suppressed expression of cyclin D2 in GCs. Binding of FOXO to suppress cyclin D2 expression has been reported to be both indirect (58) and direct (56, 108). Expression of cyclin D2 in a PTEN-null cell line is reported to be suppressed by both A3-FOXO and A3HR-FOXO mutants, consistent with both DNA-dependent and DNA-independent actions of FOXO (58). FOXO3a has recently been shown in a lymphoid cell line to suppress cyclin D2 transcription by binding directly to the signal transducer and activator of transcription 5 site on the human cyclin D2 promoter by ChIP and mobility shift analyses (108). We have shown that expression of A3-FOXO1 mutant not only represses cyclin D2 promoter-reporter activity in transfected GCs but also binds in vivo to the rat cyclin D promoter, as revealed in ChIP assays, potentially to an IRS-like site located from –315 to –308. Results in GCs thus support the notion that actions of FOXO1 on the cyclin D2 promoter result primarily from direct DNA binding of FOXO1 to the cyclin D2 promoter. This conclusion is further strengthened by results showing that the repressive effect of constitutively active FOXO1 requires DNA binding and is largely reversed with Ad-A3HR-FOXO1. However, we cannot rule out the possibility that with the ChIP assay we are detecting both direct binding of FOXO1 to IRS-like sites as well as protein/protein interaction, since formaldehyde treatment may result in both protein/protein and protein/DNA cross-linking. We also observed that constitutively active DNA binding-deficient FOXO1 mutant suppressed expression of cyclin D2 protein, suggesting additional complexity in this response. Taken together, these results indicate that in GCs, FOXO1 suppresses cyclin D2 expression primarily via direct binding to the cyclin D2 promoter and that treatment of GCs with FSH plus activin removes FOXO1 from this promoter.

Infection of GCs with the DN-Smad3 mutant similarly suppressed induction not only of cyclin D2 but also of SF-1, aromatase, epiregulin, inhibin-{alpha}, and SCC. Activin alone did not promote induction of aromatase, SCC, or epiregulin mRNA in GCs but did promote modest induction of inhibin-{alpha}, SF-1, and cyclin D2 mRNAs. Inhibin-{alpha} and cyclin D2 both contain putative Smad2/3 response elements (5'-CAGACA-3') identified by computer search, and thus might be directly regulated by Smad2/3. While the epiregulin promoter does not contain a computer-recognized consensus Smad2/3 response element, transient transfections of expression vectors containing Smad2 with co-Smad4 in serum-treated TSA201 cells resulted in modest activation of promoter-reporter activities for cyclin D2 and inhibin-{alpha} as well as for epiregulin, suggesting the presence of functional Smad response elements on these promoters. However, the mechanism by which Smad2/3 regulates FSH target gene expression remains to be defined.

The ability of the FSH-dependent PI 3-kinase signal and the activin-dependent Smad2/3 signal to regulate expression of cyclin D2, SF-1, aromatase, SCC, inhibin-{alpha}, and epiregulin indicates that these two pathways converge at one or more points. While an outcome of this convergence is the prolonged phosphorylation of Akt in GCs treated with FSH plus activin, it is expected that there is also integration of these pathways in the nucleus at the promoters for these regulated genes. It has recently been shown that FOXO1, -3a, and -4 factors can complex with phospho-Smad2/3 on the p21Cip1 promoter, leading to transcription of the cell cycle inhibitor p21Cip1 (109). In this instance, FOXO and phospho-Smad function together to inhibit cell proliferation. This FOXO-phospho-Smad2/3 complex is disrupted by activation of the PI 3-kinase pathway (109). However, in GCs our results show that phospho-Smad2/3 functions to antagonize the effect of FOXO1 and to promote cell proliferation. Thus, there is also cross-talk in GCs between FOXO and phospho-Smads, but the outcome is to stimulate rather than to inhibit cell proliferation.

We have focused on the actions of FSH to relieve FOXO1-mediated repression of GC cyclin D2 coupled with positive signals to promote cyclin D2 transactivation in response to Smad activation. However, it is likely that additional FSH-directed pathways contribute to the proliferative response. Our results show that FOXO1 represses expression of epiregulin, aromatase, SCC, and SF-1 and, to a more variable extent inhibin-{alpha} genes in the absence of signaling via the PI 3-kinase pathway. It is well established that expression of all of these genes except for SF-1 additionally necessitates positive signals emanating from FSH. Expression of aromatase requires SF-1 and CREB (78, 79); inhibin-{alpha} expression requires SF-1, CREB, and CBP (26, 29) as well as HIF1 (20) and is associated with both phosphorylated and acetylated histone H3 (24); SCC expression requires LRH-1 (27), SF-1 (28), and CBP (110); and epiregulin expression requires Sp1 and Sp3 transcription factors (81). We have shown herein that Smad2/3 transcription factors also contribute to the expression of aromatase, epiregulin, inhibin-{alpha}, SF-1, and SCC genes. Thus, regulation of each of these genes is quite complex and requires not only release from repression by FOXO1 but also positive signals. We conclude that FOXO1 represses proliferation of GCs at least in part by binding the cyclin D promoter, and that release from repression requires not only phosphorylation of FOXO1 and inhibition of its repression in response to FSH-stimulated activation of the PI 3-kinase pathway but also positive signaling from activin-stimulated phosphorylation of Smad2/3.


   FOOTNOTES
 
* This work was funded by National Institutes of Health Grants P01 HD21921 (to M. H.-D. and J. L. J.), R01 HD044464 (to T. K. W.), and R01 DK41430 (to T. G. U.) and by the Department of Veterans Affairs Merit Review Program (to T. G. U.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312-503-8940; Fax: 312-503-0566; E-mail: mhd{at}Northwestern.edu.

1 The abbreviations used are: TGF{beta}, transforming growth factor {beta}; ERK, extracellular signal-regulated kinase; FSH, follicle-stimulating hormone; SF-1, steroidogenic factor-1; PCNA, proliferating cell nuclear antigen; SCC, P450 side chain cleavage enzyme; Cdk, cyclin-dependent kinase; PI 3-kinase, phosphatidylinositol 3-kinase; RT-PCR, reverse transcriptase polymerase chain reaction; DN, dominant negative; EGF, epidermal growth factor; IGF1, insulin-like growth factor 1, LRH-1, liver receptor homolog-1; mTOR, mammalian target of rapamycin; GC, granulosa cell; ChIP, chromatin immunoprecipitation; WT, wild type; pfu, plaque-forming unit; X-gal, 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside; PBS, phosphate-buffered saline; FOXO, forkhead box-containing protein, O subfamily. Back

2 RT-PCR was used to detect changes in gene expression when high affinity/specific antibodies for gene products were not available. Back

3 In untreated and FSH-treated cells (without activin) at 24 h, Akt phosphorylation is equivalent and barely detected (see Fig. 2, lanes 1 and 2) albeit elevated over time 0 control (detected on longer exposure of Fig. 2A, not shown). The previous report of elevated Akt phosphorylation in cells treated with FSH alone for 24 h did not include a 24 h time-matched control (19); therefore, it is likely that this result is in agreement with our findings that show that treatment of GCs with FSH alone for 24 h does not prolong Akt phosphorylation over levels seen with time-matched controls. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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