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Endocrinology Vol. 143, No. 7 2732-2740
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Involvement of Inhibitory Nuclear Factor-{kappa}B (NF{kappa}B)-Independent NF{kappa}B Activation in the Gonadotropic Regulation of X-Linked Inhibitor of Apoptosis Expression during Ovarian Follicular Development in Vitro

Yifang Wang, Simon Chan and Benjamin K. Tsang

Reproductive Biology Unit and Division of Reproductive Medicine, Department of Obstetrics and Gynecology and Cellular and Molecular Medicine, University of Ottawa, Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), Ottawa, Ontario, Canada K1Y 4E9

Address all correspondence and requests for reprints to: Benjamin K. Tsang, Ph.D., Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), 725 Parkdale Avenue, Ottawa, Ontario, Canada K1Y 4E9. E-mail: . btsang{at}ohri.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Increased X-linked inhibitor of apoptosis (XIAP) expression and suppressed follicular apoptosis are important determinants in the regulation of follicular development by FSH. The objective of the present study was to examine the role and regulation of nuclear factor-{kappa}B (NF{kappa}B) in the gonadotropic control of granulosa cell XIAP expression and follicular growth in vitro. FSH (100 ng/ml) increased rat granulosa cell XIAP mRNA abundance and protein content. The gonadotropin also induced granulosa cell p65 subunit-containing NF{kappa}B translocation from cytoplasm to nucleus and increased NF{kappa}B-DNA binding activity. Supershift EMSA indicated the FSH-activated NF{kappa}B contained p65 and p50 subunits. Unlike TNF{alpha}, FSH failed to elicit a significant change in granulosa cell phospho- and total-inhibitory NF{kappa}B (I{kappa}B) I{kappa}B contents in vitro and dominant-negative I{kappa}B expression was ineffective in blocking the increase in NF{kappa}B-DNA-binding activity and XIAP protein content induced by the gonadotropin. In contrast, SN50 (a cell permeable inhibitory peptide of NF{kappa}B translocation, 50–200 ng/ml) suppressed FSH-stimulated NF{kappa}B-DNA binding, XIAP expression, and follicular growth. FSH also increased granulosa cell phospho-Akt contents, a response sensitive to the PI-3K inhibitor LY294002 (10 µM). In conclusion, the present studies demonstrate that the FSH-induced XIAP expression is mediated through the NF{kappa}B pathway through activation of phosphatidylinositol 3-kinase rather than the classical I{kappa}B kinase.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PRESENCE OF FSH is critical for the selection and survival of ovarian follicles during development (1, 2, 3, 4). The gonadotropin has been shown to stimulate rodent ovarian follicular growth and facilitate preantral follicle development induced by IGF-I in vitro (5, 6, 7, 8). In addition, FSH is an apoptosis suppressor for granulosa cells in vitro (9, 10). It is well established that the acquisition of FSH receptor(s) during follicular growth and their coupling to signaling pathways are key events in follicular development and dominance. Although cAMP is a well-established second messenger of FSH, other signal pathway(s), such as those of calcium/calmodulin and phospholipase C/protein kinase C/inositol 1,4,5-triphosphate, have also been suggested to play a role in gonadotropin action (11, 12, 13). The recent discovery of different isoforms of FSH receptor (i.e. FSH-R3), which result from hormone-induced receptor gene splicing (14), raises the possibility that FSH may be mediated through different receptors and signaling pathways. Furthermore, studies from our laboratory have demonstrated that equine chorionic gonadotropin (eCG) increases rat ovarian follicular phospho-Akt content in vivo (15), a response sensitive to the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Whether this response is a consequence of the direct action of gonadotropin or mediated through other ovarian factors is unclear.

The inhibitor of apoptosis (IAP) family, first identified in baculovirus, includes X-linked IAP (XIAP or cIAP-3), human IAP-1 (HIAP-1 or cIAP-2), human IAP-2 (HIAP-2 or cIAP-1), neuronal apoptosis inhibitory protein, Survivin, and Livin (16, 17, 18). Although only a few reports to date have addressed the subcellular action of these antiapoptotic proteins, XIAP, HIAP-1, and HIAP-2 have been shown to be direct inhibitors of caspase-3 and caspase-7 (19) and to modulate the Bax/cytochrome C pathway by inhibiting caspase-9 (20). In the ovary, XIAP is up-regulated by gonadotropin and necessary for follicular development in vivo (21) and in vitro (7). Preliminary studies have shown that although down-regulation of XIAP by antisense expression induces follicular cell apoptosis, XIAP overexpression suppresses cell death (22). Although these findings indicate that XIAP plays an important role in follicular cell survival, the signaling mechanism(s) involved in the gonadotropic regulation on XIAP expression is unclear.

Nuclear factor {kappa}B (NF{kappa}B) is a group of inducible dimeric transcription factors. They are composed of DNA-binding proteins (Rel) that recognize a common sequence motif on the NF{kappa}B-regulated genes. It has been demonstrated that NF{kappa}B activation increases the expression of genes involved in the inflammatory response (23) and prevention of TNF{alpha}-induced apoptosis, such as zinc finger protein A20 (24), and members of the Bcl-2 (25) and IAP (26) families. Other studies have shown that XIAP and HIAP-1 have NF{kappa}B-binding motif at their 5' end nontranslational regions and that IAP expression is NF{kappa}B activation dependent (27, 28, 29). If and how gonadotropic regulation on XIAP expression is mediated through the NF{kappa}B pathway is unknown.

Delfino and Walker (30) reported that FSH increases NF{kappa}B DNA-binding activity in rat Sertoli cells. Previous studies from our laboratory have demonstrated that TNF{alpha} increases rat granulosa cell XIAP content via the NF{kappa}B pathway in vitro (29). However, if and how NF{kappa}B activation plays a role in the gonadotropic control of the granulosa cells fate (survival vs. apoptosis) during follicular development has not been investigated. The objective of the present study was to assess the possible involvement of the NF{kappa}B pathway in the gonadotropic regulation of granulosa cell XIAP expression and follicular growth in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Culture media and fetal bovine serum (FBS), Trizol, deoxynucleotide triphosphate (dNTP), and Moloney murine leukemia virus-reverse transcriptase (RT) were purchased from Gibco Bethesda Research Laboratories (Burlington, Ontario, Canada). Oligo dT and RNase inhibitor were products of Ambion, Inc. (Austin, TX). HotStarTaq DNA polymerase, RNeasy minikit, PCR purification kit, and the Effectene transfection reagent were from QIAGEN Inc. (Mississauga, Ontario, Canada). Low-melting-point agarose, Triton X-100, Tween 20, eCG, Tris, collagenase, Ponceau S, DNase 1, and phenylmethylsulfonyl fluoride (PMSF) were obtained from Sigma (St. Louis, MO). The chemiluminescence ECL Western blotting detection kit and [{gamma}32P]-ATP (30 Ci/mmol) were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Ovine FSH (NIAMDD oFSH-14) was obtained from NIDDK (Baltimore, MD). Nitrocellulose membrane, acrylamide (electrophoresis grade), N,N'-methylenebis-acrylamide, ammonium persulfate, dithiothreitol (DTT), glycine, and protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA), SDS-PAGE prestained molecular weight standards (low range) and antirabbit and antimouse IgG-horseradish peroxidase (HRP)-conjugated products were purchased from Bio-Rad Laboratories, Inc. The x-ray films were from Eastman Kodak Co. (Rochester, NY). Chamber slides were from Nunc Inc. (Naperville, IL). Recombinant human TNF{alpha} was from R&D Systems Inc. (Minneapolis, MN). SM50 and SN50 were from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). NF{kappa}B-oligo probe and T4 polynucleotide kinase were from Promega Corp. (Madison, WI). Fluoresce expression vector pcDNA3.1/CT-GFP, parental vector pCMV, and pCMV-I{kappa}B construct containing dominant negative I{kappa}B{alpha} (serine-to-alanine mutation at residue 32 and 36) were from CLONTECH Laboratories, Inc. (Palo Alto, CA). Multiwell plates were from Becton Dickinson and Co. Labware (Franklin Lakes, NJ). Rabbit polyclonal antibodies against human phosphorylated and total I{kappa}B-{alpha} were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); XIAP antibody was a generous gift from Dr. Eric LaCasse, Ægera Therapeutics Inc. (Ottawa, Ontario, Canada). P65 shift kit was from Geneka Biotechnology Inc. (Montréal, Québec, Canada). NE-PER nuclear and cytoplasmic extraction reagents were from Pierce Chemical Co. Biotechnology (Rockford, IL).

Animal preparation and culture of rat ovarian granulosa cells
Immature female Sprague Dawley rats (24–25 d old) from Charles River Laboratories, Inc. Canada (Montréal, Québec, Canada) were injected with eCG (15 IU, ip) and ovaries were collected 24 h thereafter in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with HEPES (10 mM, pH 7.4) and FBS (10%). Granulosa cells were harvested by follicle puncture as previously described (31), washed, and centrifuged (900 x g, 10 min). Cells were plated for 24 h in RPMI 1640 medium with FBS (10%) under a humidified atmosphere of 95% air and 5% CO2 and cultured in serum-free medium containing FSH, TNF{alpha}, and SN50 (SM50 as control) for various duration.

Transient transfection
Rat granulosa cells were seeded in 60-mm dishes (1 x 106 cell/dish) and transfected the following day with 4 µg of the expression vector pcDNA3.1/CT-GFP alone, pCMV, or pCMV containing mutated I{kappa}B, using the Effectene transfection reagent. Twenty-four hours after transfection, cells were treated with FSH (100 ng/ml) or TNF{alpha} (20 ng/ml) for 30 min and then harvested for further analyses. Transfection efficiency, defined as the percentage of pcDNA3.1/CT-GFP transfected cells with fluorescent signal, was 30%.

Follicular isolation and culture
Ovaries from 22- to 24-d-old rats were cut into small pieces and incubated (37 C, 30 min) in {alpha}-MEM medium containing collagenase (type 1A, 4 mg/ml) and DNase 1 (0.3 mg/ml). The incubation was terminated with the transfer of the ovarian tissues into Leibovitz’s L-15 medium with bovine serum albumin (BSA) (0.1%), and follicles (160–210 µm) were dissected using 281/2-gauge needles. To minimize the experimental variation because of damages incurred during the isolation procedures, only follicles judged to be normal (with oocyte and granulosa cells completely enclosed by the basement membrane and the theca layer) were selected for experiments on both the day of isolation (d 0) and on d 1 of culture (7). Confocal microscopic (M500, Bio-Rad Laboratories, Inc. Ltd., Hertfordshire, UK) examination of the selected follicles [following fixation with paraformaldehyde (4%; 30 min, RT) and staining with ethidium bromide (5 mg/ml; 15 min, RT)] revealed that they were at the preantral (75%) and early antral (25%; as evident by the presence of an antral space as large as an area occupied by about three granulosa cells) stages of development. Selected follicles were cultured individually for 6 d in 96-well plates in 100 µl follicular culture medium [{alpha}MEM medium supplemented with HEPES (10 mM), BSA (0.1%), rat serum (1%), bovine insulin (5 µg/ml), transferrin (10 µg/ml), ascorbic acid (25 µg/ml), sodium selenium (1 ng/ml) (32), nonessential amino acids (1%), streptomycin-penicillin (0.5%), and fungizone (0.25%)] with or without FSH. The follicular size was determined daily before the medium change during the 6-d culture duration. The changes in follicular volume were defined as volume difference between day n and d 0.

Protein extraction and Western blot analysis
Changes in protein [XIAP, total- and phospho-inhibitory NF{kappa}B (T-I{kappa}B, p-I{kappa}B), total- and phospho-Akt (T-Akt, p-Akt)] contents were assessed by Western blot as previously described (21). Granulosa cells attached and detached from the growth surface were harvested (0.25% trypsin, 37 C, 3 min) and pelleted. Whole-cell lysate was extracted by addition of ice-cold lysis buffer [PBS, Nonidet P-40 (1%; vol/vol), sodium deoxycholate (0.05%; wt/vol), SDS (0.1%; wt/vol)] containing protease, phosphatase, and kinase inhibitors [PMSF (10 µM), aprotinin (50 µg/ml), sodium orthovanadate (1 mM), sodium pyrophosphate (Nappi, 10 mM), leupeptin and pepstatin (both 5 µg/ml)]. Nuclear and cytoplasmic fractions from granulosa cells were prepared with the NE-PER kit (Pierce Chemical Co. Biotechnology), according to manufacturer’s instructions. Cells were sonicated (5 sec/cycle, three cycles; 0 C), incubated on ice (30 min), and centrifuged (15,000 x g; 30 min.). The sonicates were pelleted and the supernatant was retained and stored at -20 C. Protein content of the extracts was determined with the DC protein assay reagent (Bio-Rad Laboratories, Inc.). Samples were mixed with loading buffer, boiled for 5 min to denature proteins, resolved by 10% SDS-PAGE, and electrotransferred (30 V, overnight) onto nitrocellulose membranes. Each membrane was stained and scanned for total protein with Ponceau S (0.2%) before immunoblotting. After blocking 1 h in 5% Blotto [Tris-HCl (10 mM; pH 8.0), NaCl (150 mM), Tween-20 (0.05%; vol/vol) (TBS-T) containing skim milk (5%; wt/vol)], membranes were incubated with primary antibodies (RT, 3 h; or 4 C, over night) in TBS-T containing 5% nonfat milk, and subsequently with HRP-conjugated secondary antibody (1:5000~10,000) in TBS-T with 5% milk (RT, 45 min). Peroxidase was visualized with the ECL kit according to the manufacturer’s instructions. The signal of specific protein (e.g. XIAP, Akt, p-Akt, I{kappa}B, and p-I{kappa}B) was scanned and determined by dividing its signal intensity by that of the corresponding total protein to correct for any loading difference between lanes. The intensity of protein of interest was densitometrically determined, using Molecular Analyst software version 1.4 (Bio-Rad Laboratories, Inc. Mississauga, Ontario, Canada), and expressed as fold of control before statistical analyses and presentation.

EMSA
Nuclear extracts of rat granulosa cells were prepared as previously described but with minor modifications (33). Briefly, 3 x 106 cells were pelleted (200 x g; 5 min) and resuspended in 30 µl cold buffer A [HEPES (10 mM), pH 7.9; KCl (10 mM); MgCl2 (1.5 mM); DTT (0.5 mM); PMSF (0.5 mM); Nonidet P-40 (0.67%)]. Cells were vortexed (15 sec) and placed on ice to swell (15 min), and then centrifuged (16,000 x g, 4 C, 20 min). The supernatant was collected and stored at -80 C. The cell pellet (containing cell nuclei) was resuspended in 30 µl buffer B [HEPES (20 mM), pH 7.9; NaCl (0.4 M); EDTA (0.2 mM); MgCl2 (1.5 mM); DTT (0.5 mM); PMSF (0.5 mM)] and vortexed vigorously (4 C, 15 min). The nuclear extract was centrifuged (10,000 x g, 30 min) and stored at -80 C. Double-stranded DNA oligonucleotides containing consensus sequences for the NF{kappa}B binding site was 32P-labeled with [{gamma}-32P]-ATP and T4 polynucleotide kinase. Nuclear proteins (8 µg) were incubated with radiolabeled DNA probes (RT, 20 min) in the binding buffer. For supershift assays, nuclear proteins were incubated (RT; 1 h) with antimouse p65 and p50 polyclonal antibodies (4 µg) before the addition of the labeled DNA probes. Nuclear acid-protein complexes were resolved on a native 5% polyacrylamide gel in Tris-buffered EDTA (1x; pH 8.0) and detected by autoradiography.

Quantification of XIAP mRNA by RT-PCR
Rat granulosa cells were plated in RPMI 1640 with 10% FBS for 24 h and subsequently incubated with FSH at various concentrations (25–100 ng/ml) for another 6 h. Total RNA was isolated from cultured cells with Trizol reagent or RNeasy minikit (QIAGEN Inc.), according to manufacturer’s instructions. One microgram total RNA was reverse transcribed for cDNA synthesis, using oligo-dT as primer. One-tenth of the cDNA synthesized was then amplified with the following primers: rat XIAP [forward: 5'-GGTGGACAAGTCCTATTTTCAA-3' (228–249), reverse: 5'-TCCTGATTACTTAAAGTCGATTCACA-3' (628–602)]; ßactin [forward: 5'-GAAACTACCTTCAACTCCATC-3', reverse: 5'-CGAGGCCAGGATGGAGCCGCC-3']. The samples were denatured (95 C for 15 min), amplified for 30 cycles (XIAP) or 25 cycles (ß-actin) at 94 C for 45 sec, 56 C for 1 min, and 72 C for 1 min, with the last cycle at 72 C extended for 15 min. A different number of cycles (15, 20, 25, 30, and 35) was tested for XIAP RT-PCR, and 25 and 30 cycles were found to produce PCR products in the linear range of the analysis. Samples were resolved on a 2% agarose gel and visualized with ethidium bromide. The fluorescent image of ethidium bromide-stained PCR products were captured using the Gel Doc 1000 system (Bio-Rad Laboratories, Inc.) and densitometrically quantified by the Molecular Analyst Program (version 1.4). XIAP mRNA abundance was normalized against its respective ß-actin mRNA and expressed as fold of control before statistical analysis.

Immunocytochemistry
Rat granulosa cells, cultured for 6 h on chamber slides in the absence and presence of FSH (100 ng/ml), were fixed (RT, 20 min) with paraformaldehyde (4%), permeabilized with Nonidet P-40 (0.5%) in PBS [NaCl (137 mM), Na2HPO4 (8.10 mM), KCl (2.68 mM), KH2PO4 (1.47 mM)] and quenched in ammonium acetate (50 mM). They were incubated with monoclonal p65 antibody (1:50) in PBS containing BSA (0.1%; RT, 1 h) and subsequently with HRP-conjugated secondary antibody (RT, 1 h) and stained with diaminobenzidine mix.

Statistical analysis
Results are expressed as the mean ± SEM of three experiments. Statistical analysis was carried out by one- or two-way ANOVA. Ratio data (mRNA or protein content defined as fold of control) were Acrisine square root transformed before ANOVA. Significant differences between treatment groups were determined by the Tukey test. Statistical significance was inferred at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FSH significantly increased both granulosa cell XIAP mRNA abundance and protein content (P < 0.01; Fig. 1AGo) in a concentration-dependent manner, reaching a plateau at 100 ng/ml. Because SN50 is known to bind to the nuclear localization signal of NF{kappa}B and consequently blocked its translocation from the cytoplasm to the nucleus (34), the influence of this cell-permeable peptide on XIAP expression induced by FSH was assessed (Fig. 1BGo). Pretreatment of the cells with SN50 resulted in a marked decrease in FSH-induced XIAP expression, whereas the control peptide SM50 was ineffective. Two-way ANOVA indicated a significant concentration and treatment effects on XIAP mRNA abundance (P < 0.01 for both) and protein content (P < 0.05 and P < 0.001, respectively) as well as a significant interaction between these factors in the message (P < 0.05) and protein (P < 0.01) levels. In the presence of SN50, the decrease in XIAP protein content appeared to be greater than that in mRNA abundance (P < 0.05; Fig. 1BGo).



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  		Figure 1. In vitro effects of FSH and SN50 on granulosa cell XIAP mRNA abundance and protein content. A, Rat ovarian granulosa cell from eCG-primed immature rats were cultured in the presence of FSH (0–200 ng/ml). B, Granulosa cells were pretreated with SN50 (cell-permeable NF{kappa}B translocation inhibitor) or SM50 (mutated inactive peptide, as control) and cultured with FSH (100 ng/ml). XIAP mRNA (6 h) and protein content (24 h) were determined by RT-PCR and Western blot, respectively. Representative images (top) and densitometric analysis (bottom; expressed as fold of control) are shown. Mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01, compared with control.

 
To confirm the involvement of NF{kappa}B translocation in this process, the subcellular localization of p65 subunit-containing NF{kappa}B was assessed by immunocytochemistry (Fig. 2Go) and cell fractionation-Western blotting (Fig. 3Go), using an anti-p65 antibody. Although p65 was predominantly found in cytoplasm in control cells, addition of FSH to the granulosa cell incubations markedly increased the immunoreactivity of p65 in the nucleus. SN50, but not its control peptide SM50, effectively blocked this response (Fig. 2Go). Western blot on nuclear and cytoplasmic extracts from cells treated for different durations with FSH (Fig. 3Go) indicated that the gonadotropin induces time-dependent and concomitant changes in p65 contents in cytoplasmic (P < 0.05) and nuclear fractions (P < 0.01), resulting in a rapid increase in the nuclear/cytoplasmic ratio of the protein (P < 0.01). Taken together, the above findings suggest that FSH-induced granulosa cell XIAP expression is mediated through NF{kappa}B translocation from cytoplasm to nucleus.



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  		Figure 2. FSH-induced p65 containing-NF{kappa}B translocation. Granulosa cells were incubated with or without FSH, FSH + SN50, or FSH + SM50 for 30 min in chamber slides. Cells were fixed by 10%-neutralized formalin, probed with anti-p65 antibody, and stained with diaminobenzidine. Three independent experiments were performed and represented images are shown. N, Nucleus; C, cytoplasm.

 


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  		Figure 3. Effects of FSH on changes in p65 contents in granulosa cell cytoplasmic and nuclear extracts. Granulosa cells were incubated for varies duration (0–30 min) with FSH (100 ng/ml). Proteins from cytoplasmic and nuclear fractions were extracted and p65 contents were measured by Western blot. Representative images (top) and densitometric analysis (bottom) of p65 contents (normalized against total protein and expressed as fold of control) are shown. Mean ± SEM of three independent experiments. *, P < 0.05, compared with control.

 
To determine whether FSH-induced translocated NF{kappa}B is indeed biologically active, the effects of FSH on NF{kappa}B-DNA-binding activity were tested by EMSA (Fig. 4Go). FSH increased the binding of NF{kappa}B to DNA in a concentration- (Fig. 4AGo) and time-dependent (Fig. 4BGo) manner, with a maximum and earliest observable response at 100 ng/ml and 10 min, respectively. When granulosa cells were pretreated with SN50 or SM50 (50–200 ng/ml) before the gonadotropin challenge, the increased NF{kappa}B-DNA-binding activity was suppressed by the active peptide (but not by SM50) in a concentration-dependent manner. Incubation of the nuclear extracts with anti-p65 or p50 antibody before EMSA resulted in the appearance of slower mobility signals (supershift; Fig. 4DGo). In contrast, incubation of the nuclear extracts with IgG (as negative binding control) was ineffective and resulted in NF{kappa}B-DNA-binding activity similar to that observed with extracts incubated without antibody or IgG (control). These findings are consistent with the hypothesis that up-regulation of XIAP by FSH is mediated through the activation of NF{kappa}B, which involves the p65 and p50 subunits.



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  		Figure 4. Concentration (A) and time course (B) studies on the influence of FSH on NF{kappa}B-DNA-binding activity. Granulosa cells were cultured in the absence and presence of FSH (A, 50–200 ng/ml, 30 min; B, 100 ng/ml, 0–30 min). Nuclear proteins were extracted and NF{kappa}B-binding activity was assessed by EMSA. Effects of SN50 on FSH-stimulated NF{kappa}B-DNA binding activity (C) and p65 and p50 subunits involved in FSH-activated NF{kappa}B based on supershift assay (D). Granulosa cells were exposed to different concentrations (0–200 ng/ml) of SN50 or SM50 (as control) for 15 min before FSH challenge (100 ng/ml) and were incubated for an additional 30 min. Nuclear proteins were extracted and NF{kappa}B-binding activity was assessed by EMSA. Representative images (top) and densitometric analysis (bottom) of NF{kappa}B-binding activity (as fold of control) are shown. The subunits of the FSH-activated NF{kappa}B were assessed by supershift EMSA, using p65 and p50 antibodies, and IgG in the place of an irrelevant antibody. CTL represents sample not incubated with any antibody. The arrow indicates the supershift bands. Mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01, compared with control.

 
It is well established that NF{kappa}B activation is mediated by I{kappa}B kinase (IKK) activation and subsequent I{kappa}B-phosphorylation and degradation (34). To test whether the FSHinduced NF{kappa}B activation is also I{kappa}B dependent, changes in granulosa cell total I{kappa}B and phospho-I{kappa}B contents following FSH challenge as well as the influence of a dominantnegative I{kappa}B on NF{kappa}B activation and XIAP content were assessed. FSH (100 ng/ml) had no effects on both p-I{kappa}B and total I{kappa}B contents during the experimental period (0–30 min; Fig. 5AGo). In addition, overexpression of the dominant negative I{kappa}B (as confirmed by T-I{kappa}B Western blot) failed to suppress the FSH-induced NF{kappa}B-DNA-binding activity and XIAP expression (Fig. 5BGo). In contrast, TNF{alpha} (20 ng/ml), an established inducer of IKK-mediated NF{kappa}B activation (P < 0.01, Fig. 5BGo), markedly increased the p-I{kappa}B level and decreased total I{kappa}B content after 5 and 15 min of exposure but with both responses returning to basal level at 30 min (Fig. 5AGo). Whereas extracts from granulosa cells transfected with the mutated I{kappa}B construct suppressed TNF{alpha}-induced NF{kappa}B-DNA-binding activity and XIAP contents (P < 0.01 and P < 0.05, respectively, Fig. 5BGo), the dominant-negative I{kappa}B has no significant effect on these responses induced by FSH.



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  		Figure 5. Influence of TNF{alpha} and FSH on phospho-I{kappa}B, total I{kappa}B (A). Granulosa cells were incubated with FSH (100 ng/ml) or TNF{alpha} (20 ng/ml) for different duration (0–60 min). Phospho-I{kappa}B, total-I{kappa}B contents were measured by Western blot. Effects of overexpression dominant-negative I{kappa}B on NF{kappa}B-binding activity induced by TNF{alpha} or FSH (B). Granulosa cells were transfected with pCMV or pCMV-I{kappa}B-DN (4 µg, 24 h) before TNF{alpha} (20 ng/ml) or FSH (100 ng/ml) treatment. Nuclear proteins were extracted (30 min after TNF{alpha} or FSH) and NF{kappa}B-binding activity was assessed by EMSA. XIAP and total I{kappa}B contents were measured by Western blot from whole-cell lysate (24 h after TNF{alpha} and FSH). Representative images (top) and densitometric analysis (bottom) of XIAP protein contents and NF{kappa}B-DNA-binding activity (as fold of control) are shown. Mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01 (compared with control); +, P < 0.05; 2+, P < 0.01 (compared with cells transfected with pCMV-I{kappa}B-DN and treated with TNF{alpha}).

 
Gonadotropins have been shown to activate granulosa cell PI3K/Akt pathway in vivo (15) and in vitro (35). PI3K/Akt signaling pathway is known to play a role in IGF-1-mediated granulosa cell survival (36, 37), oocyte maturation (38, 39), and fetal oogenesis (40). IGF-1 and TGF{alpha} induce rapid Akt phosphorylation in hen granulosa cells of preovulatory follicles, the inhibition of which by the PI3K inhibitor LY292004 leads to apoptosis (41). To determine whether this signal pathway is involved in the FSH-induced NF{kappa}B activation, the influence of FSH on phospho- and total-Akt contents (Fig. 6Go) and the effect of the PI3K inhibitor LY292004 (Fig. 7AGo) or wortmannin (Fig. 7BGo) on FSH-induced NF{kappa}B-DNA-binding activity and XIAP protein content were assessed. Concentration-response studies indicated that, although ineffective on granulosa cell total Akt content, FSH significantly increased phospho-Akt levels (P < 0.05; Fig. 6Go). Pretreatment of granulosa cells with LY292004 (1–20 mM) or wortmannin (25–150 nM) before FSH (100 ng/ml), suppressed the FSH-activated NF{kappa}B-DNA-binding activity (P < 0.01) and XIAP content (P < 0.01) in a concentration-dependent manner (Fig. 7Go).



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  		Figure 6. FSH increases phospho-Akt but not total Akt contents in granulosa cells in vitro. Granulosa cells were cultured for 30 min with different concentrations of FSH (0–150 ng/ml). Whole-cell lysates were extracted (30 min after FSH) and Akt (total- and phospho-Akt) contents were measured by Western blot. Representative images (top) from three independent experiments and densitometric analysis (bottom) of Akt contents (defined as fold of control) are shown. Mean ± SEM of three independent experiments. *, P < 0.05, compared with control.

 


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  		Figure 7. PI3K inhibitors (LY294002 and wortmannin) suppress FSH-induced NF{kappa}B-DNA-binding activity and XIAP contents in granulosa cells in vitro. Granulosa cells were cultured and pretreated (15 min) with different concentrations of LY294002 (0–20 µM) or wortmannin (0–150 nM) before the addition of FSH (100 ng/ml). Nuclear proteins were extracted (30 min after FSH) and NF{kappa}B-binding activity was assessed by EMSA. XIAP contents were measured by Western blot from whole-cell lysate (24 h after FSH). Representative images (top) and densitometric analysis (bottom) of NF{kappa}B-DNA-binding activity (defined as fold of control) are shown. Mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01, compared with control.

 
Preliminary studies from our laboratory have shown that FSH stimulates XIAP expression and the growth of rat ovarian follicles in culture (22). To assess whether FSH-induced granulosa cell NF{kappa}B activation is important in the gonadotropic regulation in follicular growth, preantral and early antral follicles were cultured with SN50 (100 ng/ml; SM50 as control) in the absence and presence of FSH (100 ng/ml). Although follicles cultured in the absence of FSH but presence of SM50 showed a minimum growth in a 6-d culture period, addition of FSH significantly increased follicular volume (P < 0.001; Fig. 8Go). Pretreatment of follicles with SN50 before FSH addition resulted in a significant decrease in FSH-induced follicular growth (P < 0.01, FSH + SN50 vs. FSH + SM50 by repeated measurement two-way ANOVA; Fig. 8Go).



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  		Figure 8. FSH-stimulated follicular growth is mediated through NF{kappa}B activation. Follicles (160–210 µm in diameter, 20 follicles per group) were cultured individually in 96-well plate and pretreated with SN50 or SM50 (100 ng/ml) and cultured in the absence or presence of FSH (100 ng/ml) for up to 6-d period. Follicular diameter was measured daily and changes in follicular volume is defined as the differences in volume between day n and day 0. The follicular volume at d 0 was 2.2 ± 0.25 nl (n = 20). Mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01, compared with control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although the NF{kappa}B family of transcription factors is an important intracellular mediator of extracellular signals in a number of biological systems, whether they play a role in the gonadotropic regulation of ovarian follicular development, is not known. The present study demonstrated for the first time that FSH up-regulates XIAP expression and follicular growth through NF{kappa}B activation. FSH induced p65-containing NF{kappa}B translocation from cytoplasm to nucleus and increased NF{kappa}B-DNA-binding activity shortly after gonadotropin challenge, a phenomenon that could be attenuated by SN50, a cell-permeable inhibitory peptide of NF{kappa}B translocation. Based on supershift EMSA results, the FSH-activated NF{kappa}B contains p56 and p50 subunits. The gonadotropin-induced XIAP expression was also suppressed by SN50 (but not SM50, its inactive peptide), indicating that NF{kappa}B is the mediator of this response. Interestingly, FSH also increased NF{kappa}ß-DNA-binding activity in Sertoli cells, which exhibited higher basal levels of NF{kappa}ß-stimulated reporter gene expression. The activated NF{kappa}B appeared to involve the p50 and p65 subunits and was specific to the stage of spermatogenesis (30). However, unlike the present studies, these investigations did not demonstrate the physiological function of FSH-activated NF{kappa}B, particularly in relation to regulation of a specific gene transcription in this testicular cell type.

There has been considerable progress in the current understanding of the signaling mechanism in NF{kappa}B activation by cytokines, such as TNF{alpha} and IL-1ß (42). TNF{alpha} activates IKK, which is responsible for I{kappa}B phosphorylation at serine 32 and 36. I{kappa}B{alpha} is consequently ubiquitinated and degraded by 26S proteasome, resulting in the unmasking of the nuclear localization signal of NF{kappa}B and its translocation to the nucleus. TNF{alpha} increases nuclear NF{kappa}B-DNA-binding activity via this classical pathway. In contrast, FSH increases NF{kappa}B translocation and DNA binding without detectable changes in phospho-I{kappa}B or total I{kappa}B contents and degradation. Furthermore, overexpression of dominant-negative I{kappa}B (mutation at the phosphorylation sites) also failed to suppress FSH-induced NF{kappa}B activation and XIAP expression but effectively attenuated the responses induced by TNF{alpha}. These findings demonstrate, for the first time, that FSH activates NF{kappa}B through a pathway independent of I{kappa}B phosphorylation and degradation.

In the present studies, we have demonstrated that FSH increases granulosa cell phospho-Akt content and NF{kappa}B-DNA-binding activity, the latter response being readily suppressed by the presence of LY294002 or wortmannin. Likewise, FSH-induced XIAP expression is sensitive to these PI3K inhibitors. Taken together, these findings suggest that the NF{kappa}B activation and subsequent XIAP gene expression in response to FSH is PI3K dependent. Recently Koul et al. (43) also reported that the tumor suppressor PTEN, an endogenous antagonist of PI3K, inhibits IL-1ß-induced NF{kappa}B activation in glioma cells without interfering with the I{kappa}B phosphorylation and degradation pathway. Furthermore, IL-1 increases phosphorylation of p65 and p50 subunits of NF{kappa}B and subsequent activation of NF{kappa}B pathway, which was blocked by LY294002 (44). These findings not only are consistent with our concept that FSH-induced XIAP expression is mediated through PI3K-dependent NF{kappa}B pathway but also raise the possibility that the increase of granulosa cell PI3K activity in response to the gonadotropin may involve NF{kappa}B phosphorylation. Whether NF{kappa}B phosphorylation is a result of the direct action of an activated Akt or secondary to the phosphorylation and activation of an as-yet-unknown downstream kinase(s) remains to be investigated.

Using a follicle culture system, we have previously demonstrated that FSH induces follicular XIAP expression, suppresses granulosa cell apoptosis, and stimulates follicular growth in vitro (7). The present studies suggest that although FSH activates a granulosa cell PI3K-dependent NF{kappa}B pathway, addition of SN50 to follicle cultures attenuates FSH-induced follicular growth, supporting our hypothesis that FSH-induced follicular growth involves suppression of apoptosis mediated by a cell survival factor (e.g. XIAP) and NF{kappa}B activation by PI3K. Consistent with this concept is the recent demonstration that the PI3K pathway is involved in NF{kappa}B-mediated Bcl-XL gene expression during CD40 signaling (45). In contrast, evidence also exists in Theileria-transformed leukocytes that activation of the PI3K-Akt pathway is not directly linked to NF{kappa}B (46). The PI3K-Akt pathway does not contribute to the persistent induction of I{kappa}B{alpha} phosphorylation as well as NF{kappa}B and transcriptional activation. In human endothelial cells, TNF{alpha} and IL-1 activate a PI3K/Akt pathway and that the antiapoptotic effect of Akt is also independent of NF{kappa}B (47). These results raise the possibility that the NF{kappa}B dependence of the PI3K cell survival pathway may be agonist and cell type specific.

The role of cAMP in NF{kappa}B activation in granulosa cells is not known. It is well established that FSH increases the granulosa cell cAMP level and activates protein kinase A (PKA) (48). PKA is known to directly phosphorylate NF{kappa}B p65, promote its association with coactivators and consequently increase NF{kappa}B transcriptional activity in LPS-challenged 70Z/3 cells (49, 50). In addition, activation of NF{kappa}B in HeLa and B cells by TNF{alpha} is associated with phosphorylation of I{kappa}B{alpha}, NF{kappa}B precursors, and p65 subunit and is modulated by the presence of shrimp alkaline phosphatase or potato acid phosphatase (51). Stimulation of Sertoli cells with activators of the cAMP-PKA signaling pathway (e.g. forskolin or FSH) also increases NF{kappa}B-DNA-binding activity. Although preliminary data from our laboratory have shown that cAMP can mimic FSH in XIAP up-regulation in rat granulosa cells, whether and how the cAMP/PKA pathway is involved in the FSH-induced, NF{kappa}B-mediated XIAP expression in granulosa cells remains to be determined.

In conclusion, our findings support our hypothesis that the binding of FSH to granulosa cell receptor induces NF{kappa}B activation and translocation to the cell nucleus and increases XIAP gene transcription. This process is mediated through the PI3K/Akt pathway and is I{kappa}B phosphorylation and degradation independent. However, whether the increase in XIAP mRNA observed in the present studies was indeed a consequence of increased gene expression and/or increased mRNA stability requires further experimentation. Moreover, whether and how the cAMP/PKA pathway is involved in this regulation remains to be investigated.


   Footnotes
 
Y.W. was a recipient of the Natural Science and Engineering Research Council of Canada Scholarship (NSERC, PGSB-222413-1999).

This work was supported by a grant from the Canadian Institutes of Health Research (CIHR, MOP-10369 to B.K.T.).

Abbreviations: BSA, Bovine serum albumin; DTT, dithiothreitol; eCG, equine chorionic gonadotropin; FBS, fetal bovine serum; HIAP, human IAP; HRP, horseradish peroxidase; IAP, inhibitor of apoptosis; I{kappa}B, inhibitory NF{kappa}B; IKK, I{kappa}B kinase; NF{kappa}B, nuclear factor {kappa}B; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PMSF, phenylmethylsulfonyl fluoride; RT, reverse transcriptase; TBS-T, tris-buffered saline Tween-20; XIAP, X-linked IAP.

Received December 11, 2001.

Accepted for publication March 19, 2002.


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