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Regular Article |
B-Mediated Induction of Flice-Like Inhibitory Protein Prevents Tumor Necrosis Factor 
-Induced Apoptosis in Rat Granulosa Cells1
a Reproductive Biology Unit and Division of Reproductive Medicine, Department of Obstetrics & Gynecology and Cellular & Molecular Medicine, University of Ottawa, Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), Ottawa, Ontario, Canada K1Y 4E9
b Département de Chimie-Biologie, Université du Québec à Trois-Rivières, C.P. 500, Trois-Rivières, Québec, Canada G9A 5H7
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF) in vitro. Granulosa cells from immature rats primed with eCG were cultured in serum-free RPMI in the absence or presence of TNF (20 ng/ml), cycloheximide (CHX, 10 µg/ml), SN50 (a specific inhibitor of nuclear factor 
B [NFB] translocation, 100 or 200 µg/ml), or a combination of these. (SM50, a mutated inactive peptide of SN50, was used as control.) Inhibitor B (IB; total and phosphorylated forms) and NFB binding abilities were measured by Western blot and electrophoretic mobility shift assay, respectively. Apoptosis was assessed by in situ TUNEL assay, whereas FLIP mRNA levels were determined by semiquantitative reverse transcriptase-polymerase chain reaction. TNF alone failed to induce granulosa cell death but significantly increased the apoptotic cell number in the presence of cycloheximide. TNF significantly up-regulated the expression of the short form of FLIP (FLIPS) but not the long form (FLIPL). TNF induced IB phosphorylation and NFB activation. SN50, but not SM50, attenuated TNF-induced FLIPS expression and enhanced TNF-induced apoptosis. Down-regulation of TNF-induced FLIPS by FLIPS antisense expression enhanced TNF-induced apoptosis. A full length of rat FLIPS, with high homology to mouse FLIPS (85%), had been cloned and sequenced. These findings suggest that, in addition to its proapoptotic function, TNF can induce an intracellular survival factor for the maintenance of follicular development. TNF-induced, NFB-mediated FLIPS expression is a determinant of granulosa cell fate.
apoptosis, cytokines, granulosa cells, ovary, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor necrosis factor (TNF) is a multifunctional cytokine with proapoptotic properties, and also induces proliferation and differentiation of many cell types [6]. In the ovary, macrophages, white blood cells, oocytes, and follicular cells produce TNF, and TNF has been shown to suppress follicular cell differentiation via inhibition of gonadotropin-stimulated cAMP [7] and steroid production [8, 9]. TNF plays a key role in follicular development and atresia, and decreases ovarian cell viability and proliferation [6] and stimulates apoptosis in cultured ovarian follicles [10]. However, TNF alone failed to induce apoptosis in murine granulosa cells in vitro but promoted Fas-mediated cell kill [11]. Treatment with cycloheximide (CHX) potentiated Fas-mediated death, suggesting that inhibitors of the Fas death pathway might be present and regulated in granulosa cells [11]. When TNF binds its receptor (TNFR1), capase-8 and caspase-3 are cleaved and activated [12, 13], and induction of IB phosphorylation and degradation activates nuclear factor B (NFB) [14, 15]. Recently, we have demonstrated in rat granulosa cells that XIAP expression is regulated by the TNF pathway via an NFB-mediated mechanism [16].
Flice-like inhibitory protein (FLIP) is an antiapoptotic factor that structurally resembles caspase-8 but lacks proteolytic activity (the cysteine residue within the active site) [17, 18]. FLIP is recruited to the death-inducing signaling complex (DISC) through the adaptor molecule, Fas-associated death domain (FADD), thereby preventing the recruitment of caspase-8 into the complex and subsequent caspase-8 activation, and then suppresses apoptosis [19]. FLIP exists in 2 different spliced isoforms, long (FLIPL) and short (FLIPS), both of which contain 2 death-effector domains (DEDs) within their N-termini [17]. FLIPL isoform includes an additional C-terminal structure resembling the p20 and p10 subunits of caspases, and is closely related to caspase-8 when primary sequences are compared [20].
In the present study we hypothesized that TNF induces FLIPS expression and that this response prevents TNF receptor death signaling. We have investigated the ability of TNF to induce granulosa cell apoptosis in the presence and absence of CHX and explored intracellular mechanisms that mediate the TNF action. Specifically, we have studied the involvement of NFB in TNF-induced FLIP expression using SN50 (a specific inhibitor of NFB translocation) and FLIPS antisense cDNA expression vector.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Agarose, Tris, and PMSF were obtained from Sigma Chemical Company (St. Louis, MO). The enhanced chemiluminescence Western blotting detection kit and [
-32P]dATP (30 Ci/mmol) were obtained from Amersham (Arlington Heights, IL). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco-BRL (Burlington, ON, Canada). Moloney murine leukemia virus reverse transcriptase and TRIzol reagent were from Life Technologies (Gaithersburg, MD). HotStar Taq DNA polymerase and Effectene Transfection Reagent were from Qiagen Inc. (Mississauga, ON, Canada). Nitrocellulose membrane, acrylamide (electrophoresis grade), N,N'-methylene-bis-acrylamide, ammonium persulfate, dithiothreitol (DTT), glycine, and the Bio-Rad protein assay kit were purchased from Bio-Rad Laboratories (Hercules, CA). x-Ray films were from Eastman Kodak Company (Rochester, NY). Recombinant rat TNF was from R&D Systems Inc. (Minneapolis, MN). CHX was from BDH Laboratory Supplies (Toronto, ON, Canada). NFB probe and T4 polynucleotide kinase were from Promega (Madison, WI). Rabbit polyclonal anti-human phosphorylated and total IB antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-human FLIP antibody was from Alexis Biochemicals (San Diego, CA). pcDNA3.1/CT-GFP-TOPO expression vector was from Invitrogen Corporation (Carlsbad, CA). SN50 and SM50 were obtained from BIOMOL Research Laboratories, Inc (Plymouth, PA). (SM50, a mutated inactive peptide of SN50, was used as control.)
Preparation and Culture of Rat Ovarian Granulosa Cells
Immature female Sprague-Dawley rats at 24–25 days of age were injected with eCG (15 IU, s.c.) 24 h before the ovaries were removed. Granulosa cells were collected by follicular puncture, washed, and pelleted (200 x g for 5 min). Cells were resuspended and plated for 24 h at a concentration of 5 x 105 cells per 35-mm dish in 2.5 ml of RPMI 1640 with 10% FBS. To effectively block NFB activation, granulosa cells were pretreated with SN50 (15 min; SM50 as control) before the addition of TNF (20 ng/ml). At the end of the incubation period the cells were harvested by trypsinization (0.5% trypsin and 5.3 mM EDTA) and centrifugation (200 x g for 5 min) for further analysis.
Cell number in each treatment group was determined with a hemocytometer. Cell viability before treatment, as determined by a previously described trypan blue dye-exclusion test [21], was about 90%.
Cloning and Sequencing of Rat FLIPS cDNA
Rat FLIPS cDNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using four sets of primers: forward, 5'-GCTTCTCGTGGTTCCCAGAG-3' (2–21); reverse, 5'-CCAATCTCCATCAGCAGGACCC-3' (391–370); forward, 5'-GCCAAGGACAAGAGTTTCTTGG-3' (474–495); reverse, 5'-AGCTTTCCACAGTAGTCATGCC-3' (949–928); forward, 5'-GCCCATTATCTGGGCATGACTA-3' (916–937); reverse, 5'-TCAGGAAGTTTATTTTGCAGCA-3' (1589–1568); forward, 5'-GGCGGTTTGACCTGCTCAAGAG-3' (277–298); reverse, 5'-ACTGGTTCATGCTGATATTCCA-3' (737–716).
The first 3 sets of primers designed were based on the mRNA sequence of mouse CASH beta protein (FLIPS) (GenBank accession number MMY14042) and the last set designed was based on the sequenced rat FLIPS cDNA. The PCR products were subcloned into pcDNA3.1/CT-GFP-TOPO vector and sequenced by automated sequence analysis (GenBank accession number AF244366).
Preparation of FLIPS Sense and Antisense Constructs
The cDNA fragment encoding the open reading frame of rat FLIPS (nucleotides 54–710) was prepared by RT-PCR using the primers 5'-CTGACTTCTGCGGTTCTGAACA-3' (33–54) and 5'-CTGCTGATATTCCACACACTGGCT-3' (706–685). The primers were designed based on the rat FLIPS sequence (GenBank accession number AF244366), and the PCR products were subcloned into pcDNA3.1/CT-GFP-TOPO expression vector. The antisense FLIPS-pcDNA3.1/CT-GFP constructs were verified by automated sequence analysis.
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 or pcDNA3.1/CT-GFP containing rat FLIPS antisense cDNA using Effectene Transfection Reagent. Twenty-four hours after transfection, cells were treated with TNF (20 ng/ml) for 6 h and then harvested for further analysis. The transfection efficiency was approximately 60%.
Quantitation of FLIP mRNA by Semiquantitative RT-PCR
Total RNA was isolated from cultured cells with TRIzol reagent according to the manufacturer's instructions. Five hundred nanograms of total RNA was reversed transcribed for cDNA synthesis using oligo-dT as primer. One tenth (2 µl) of the cDNA synthesized was then amplified with the following primers for mouse FLIPL (GenBank accession number U97076): forward, 5'-CCGTGACAGTCAAAGAACACTG-3' (825–846); reverse, 5'-GGAGAACCCTGAGTGAACTTGA-3' (1173–1152). The primers used for rat FLIPS were forward, 5'-GCCAAGGACAAGAGTTTCTTGG-3' (444–465); reverse, 5'-AGCATTCCACAGTAGTCATGCC-3' (926–905). Human ß-actin (GenBank accession number NM001101) primers were forward, 5'-GAAACTACCTTCAACTCCATC;ch3' and reverse, 5'-CGAGGCCAGGATGGAGCCGCC-3'. PCR cycle conditions for FLIPL and FLIPS were 95°C for 15 min, 94°C for 45 sec, 60°C for 1 min, and 72°C for 1 min for 35 cycles, and 72°C for 10 min. ß-Actin conditions were 95°C for 15 min, 94°C for 45 sec, 55°C for 1 min, and 72°C for 1 min for 25 cycles, and 72°C for 10 min. Samples were resolved on a 2% agarose gel and visualized with ethidium bromide. FLIPS mRNA levels were normalized with its respective ß-actin contents.
Protein Extraction and Western Blot Analysis
Cells were sonicated in a lysis buffer (pH 7.4) containing NaCl (150 mM), SDS (0.1%), sodium deoxycholate (0.5%), NP-40 (1%) in PBS, and protease inhibitors (1 mM PMSF, 10 µg/ml aprotinin, and 1 mM sodium orthovanadate). The sonicates were pelleted (15 000 x g for 20 min) and the supernatant was retained and stored at -20°C. Protein content of the extracts was determined with the Bio-Rad DC Protein Assay Reagent. Samples (50 µg protein) were mixed with loading buffer, resolved by 12% SDS-PAGE, and electrotransferred (30 V, overnight) onto nitrocellulose membranes. After blocking for 1 h with nonfat milk powder (5%) in Tris-buffered saline (TBS; 10 mM Tris and 150 mM NaCl) and 0.05% Tween-20 (TBS-T), membranes were incubated for 3 h with primary antibodies in TBS-T containing 5% nonfat milk powder, and subsequently with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 to approximately 10 000) in TBS-T with milk powder (RT for 45 min). Immunoreactivity was detected by chemiluminescence autoradiography (ECL kit) in accordance with the manufacturer's instructions.
Electrophoretic Mobility Shift Assay
Nuclear extracts of rat granulosa cells were prepared as previously described [22] with minor modifications. Briefly, 3 x 106 cells were pelleted (200 x g for 5 min) and resuspended in 30 µl of buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 1.5 mM MgCl, 0.5 mM DTT, 0.5 mM PMSF, and 0.67% Nonidet P-40). Cells were allowed to swell (0°C for 15 min) and were centrifuged (10 000 x g at 4°C). The supernatant was collected and stored at -80°C. The cell pellet (containing cell nuclei) was resuspended in 30 µl of buffer B (20 mM Hepes pH 7.9, 0.4 M NaCl, 0.2 mM EDTA, 1.5 mM MgCl, 0.5 mM DTT, and 0.5 mM PMSF) and rocked vigorously (4°C for 15 min). The nuclear extract was centrifuged (10 000 x g for 30 min) and stored at -80°C. Double-stranded DNA oligonucleotides containing consensus sequences for NFB were 32P-labeled with [32P]ATP and T4 polynucleotide kinase. Nuclear proteins (8 µg) were incubated with radiolabeled DNA probes (RT for 20 min) in the binding buffer. DNA-protein complexes were resolved on a native 5% polyacrylamide gel in Tris-buffered EDTA (1x pH 8.0) and detected by autoradiography.
TUNEL Technique
In situ cell death detection by the TUNEL technique was carried out as previously described [23]. Fifty microliters of the cell suspension was placed on the positively charged slides, air-dried, fixed with formaldehyde (10% for 30 min), and immersed in methanol containing H2O2 (0.3%). After a 15-min rinse with distilled water, the slides were incubated in the terminal deoxynucleotidyl transferase (TdT) buffer (25 mM Tris-HCl, 200 mM sodium cacodylate, 5 mM cobalt chloride, and 250 µg/ml BSA pH 6.6 for 15 min) and then in 50 µl of TdT buffer containing 10 U TdT and biotinylated dUTP (1 nmol) in a humidified chamber at 37°C for 60 min. The biotinylated dUTP molecules incorporated into nuclear DNA were visualized with horseradish peroxidase-conjugated streptavidin (1:100; RT for 30 min) and DAB solution (RT for 5 min). At least 500 cells were counted in each experimental group.
Statistical Analyses
Results are expressed as means ± SEM of 3 independent experiments. Statistical analyses were carried out by one-way or two-way ANOVA. Significant differences between treatment groups were determined by the Tukey test. Statistical significance was inferred at P < 0.05. Because FLIPS mRNA abundance (Figs. 3 and 5) and protein content (Fig. 6a) were expressed as folds of control, the data were arcsine square root transformed before ANOVA. Differences in the extent of apoptosis between experimental groups (Table 1 and Fig. 6b) were analyzed with the chi-square test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We cloned the open reading frame of 654 base pairs of rat FLIPS cDNA by the RT-PCR method (Fig. 1; GenBank accession number AF244366). The coding region of rat FLIPS cDNA shows high homology with its mouse counterpart at the nucleotide (88%) and amino acid (85%) levels [24]. Homology to human is 65% in the amino acid [24].
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TNF-Induced Apoptosis in the Presence of CHX
Rat ovarian granulosa cells were cultured for 12 h in serum-free RPMI 1640 medium in the absence or presence of TNF (20 ng/ml), CHX (10 µg/ml), or CHX plus TNF. Treatment of cells with either CHX or TNF alone failed to induce granulosa cell apoptosis as determined by in situ TUNEL assay (Fig. 2, Table 1). In the presence of protein synthesis inhibitor, the cytokine significantly increased the number of TUNEL positive cells in the cultures (P < 0.01).
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TNF Increased FLIPS mRNA Steady-State Levels
To determine whether FLIP is the putative survival factor induced by the cytokine, granulosa cells were incubated with TNF (20 ng/ml) for 1, 3, and 6 h, and FLIPS and FLIPL mRNA abundance were measured by semiquantitative RT-PCR. Whereas TNF has no apparent effect on steady state FLIPL mRNA levels, it rapidly increased FLIPS mRNA abundance. A significant elevation in FLIPS mRNA levels was evident within 1 h of TNF challenge and was sustained for at least 6 h (P < 0.01; Fig. 3, A and B).
NFB Activation Is Involved in the TNF-Modulated FLIPS Expression
To assess whether NFB activation is involved in TNF-induced FLIPS expression, we examined the temporal changes in phosphorylated IB, total IB contents, and nuclear NFB binding ability in rat granulosa cells in response to TNF. Treatment of granulosa cells with TNF (20 ng/ml) resulted in increased phosphorylated IB levels, which reached a maximum at 5 min and subsequently dropped to pretreatment levels (Fig. 4a). Total IB levels remained constant until 5 min following the TNF challenge, after which a significant decrease was observed at 15 min (Fig. 4a). Nuclear NFB binding ability, as measured by electrophoretic mobility shift assay, was markedly increased after TNF challenge, reaching a maximum at 15 min (Fig. 4a).
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To determine whether TNF-induced FLIPS expression is mediated via NFB activation, the influence of the treatment of SN50, an inhibitor of NFB translocation on nuclear NFB binding ability and FLIPS abundance was examined. Addition of SN50 to granulosa cell cultures 15 min before TNF challenge markedly attenuated NFB activation, as evident by suppressed nuclear NFB binding activity in a concentration-dependent manner. However, the same concentrations of the inactive peptide SM50 (100 and 200 µg/ml) were ineffective (Fig. 4b). These responses were coincidental to a marked decrease in TNF-induced FLIPS mRNA expression. FLIPL mRNA abundance was not affected by any of the treatments (Fig. 5).
FLIPS Modulated TNF-Induced Apoptosis in Granulosa Cells
To confirm the role of FLIPS in the suppression of TNF-induced apoptosis, the influence of FLIPS down-regulation by FLIPS antisense expression before treatment with the cytokine was assessed. Expression of FLIPS antisense cDNA in granulosa cells blocked the TNF-induced increase in FLIPS protein content (Fig. 6a). Down-regulation of FLIPS expression significantly increased TNF-induced apoptosis (P < 0.001, Fig. 6b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in granulosa cells and its molecular cloning in the rat species. The rat FLIPS sequence proved to have substantial homology with its mouse counterpart (88% in nucleotide and 85% in amino acid) and less homology with human ß-CASH (hFLIPS, 65% in amino acid) [24]. The role of FLIP in the regulation of apoptosis is controversial because it has been described as both a proapoptotic molecule [24–26] and an antiapoptotic molecule [17, 24, 27, 28] in different cell types. It has been demonstrated that the transgenic c-FLIP-/- mouse embryo rarely survived past Day 11 of embryogenesis and that their fibroblasts were highly sensitive to FasL- or TNF-induced apoptosis, showing rapid induction of caspase activity [29].
In the present study, we have shown that TNF up-regulates both FLIPS mRNA and protein. In the presence of TNF alone, rat granulosa cells do not undergo apoptosis. This may be explained by the up-regulation of FLIPS. Our hypothesis is that increased expression of FLIPS by TNF prevents TNF receptor downstream death signaling. We have also demonstrated a similar pattern of FLIPS regulation in a human ovarian epithelial cancer cell line (OV2008) by TNF (unpublished data). The importance of FLIPS in the blockade of TNF-induced apoptosis in granulosa cells is further supported by the experiments with CHX, an inhibitor of protein synthesis. In the presence of CHX, TNF was able to induce apoptosis. Because treatment of granulosa cells with TNF stimulates the transcription of FLIPS mRNA and its protein synthesis, it is not surprising to see a decrease in FLIPS protein and an increase of apoptosis in the presence of both CHX and TNF.
To further demonstrate the importance of FLIPS in the prevention of apoptosis induced by TNF, we designed and constructed an expression vector containing FLIPS antisense cDNA and transfected the granulosa cells with the construct to down-regulate FLIPS expression in granulosa cells. TNF was able to induce apoptosis even in the absence of CHX after FLIPS down-regulation, supporting our hypothesis that TNF could not induce apoptosis alone because of the increased expression of FLIPS.
The intracellular death domain of TNF receptor (TNFR1) is coupled to both apoptotic [30] and NFB cell activation [31, 32] pathways. It was important to determine whether the activation of NFB pathway was responsible for the up-regulation of FLIPS mRNA gene expression. Our results have shown that TNF induced a rapid phosphorylation of IB and an increased nuclear NFB binding ability. These findings were supported by the observation the nuclear localization sequence (SN50), which inhibited TNF-induced NFB translocation from cytoplasm into nucleus, significantly attenuated TNF-induced FLIPS mRNA and protein expression. In this context, the TNF-induced NFB pathway acts as a survival pathway, because activation leads to up-regulation of the survival factor FLIPS. These results are consistent with the recently published evidence showing that TNF-induced FLIP expression is mediated through activation of the NFB signaling pathway [33, 34]. However, our results show that FLIPL expression in rat ovarian granulosa cells is not regulated by TNF and NFB activation, and probably plays a minor role in the regulation of TNF-induced apoptosis.
In conclusion, we have demonstrated that FLIPS plays a key role in conferring granulosa cell resistance to the cytotoxic action of TNF. The regulation of the survival factor FLIPS in granulosa cells during follicular development and atresia may be critical in determining the fate of these cells. Further studies are necessary to precisely determine the regulation of FLIPS in vivo.
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FOOTNOTES |
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1 This work was supported by grant MOP-15691 from the Canadian Institutes of Health Research. C.W.X. and E.A. were recipients of Canadian Institute of Health Research Postdoctoral Fellowships. 
2 Correspondence: Benjamin K. Tsang, Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), 725 Parkdale Avenue, Ottawa, ON, Canada K1Y 4E9. FAX: 613 761 4403; btsang{at}ohri.ca
Accepted: February 27, 2002.
Received: November 20, 2001.
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