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Regular Article |
B and Induction of Apoptosis by Tumor Necrosis Factor-
in the Mouse Uterine Epithelial WEG-1 Cell Line1
a OBST 5330 Research Unit, Université Catholique de Louvain Medical School, 1200 Brussels, Belgium
ABSTRACT
In order to better understand how tumor necrosis factor (TNF)-
may contribute to the local regulation of uterine cell death, cultures of mouse uterine epithelial WEG-1 cells were exposed to TNF- and observed at different time intervals. Earliest decrease in cell viability was observed after 31 h of exposure to 50 ng/ml mouse TNF- and was associated with the expression of several markers of apoptosis. Treatment with human TNF- or addition of a neutralizing antibody against TNF- receptor protein 80 to mouse TNF- resulted in attenuated induction of apoptosis, suggesting that coengagement of the two TNF- receptor types is required for maximal impact. Ceramide analogs failed to replicate the effect of TNF- and the stress-activated protein kinase signaling pathway was not activated by the cytokine. Treatment with mouse TNF- resulted in an increase in nuclear factor (NF)
B activity that receded after 24 h. The impact of human TNF- on NFB activation was more moderate. Addition of either one of three different inhibitors of NFB (SN50, PDTC, and A771726) to mouse TNF- sensitized WEG-1 cells to the toxicity of the cytokine. Our data suggest that WEG-1 cells initiate their response to TNF- with an increase in NFB activation that may have transiently biased these cells toward cell death resistance.
apoptosis, cytokines, signal transduction, uterus
INTRODUCTION
Previous observations have shown that tumor necrosis factor- (TNF-) [1] and TNF- receptor protein 60 (Rp60) [2] are expressed at both mRNA and protein levels in the luminal and glandular epithelium lining the mouse uterus. Analysis of variations in TNF- and TNF- Rp60 synthesis in cycling mice revealed that both ligand and receptor expression profiles reached maximal levels during diestrus II. Additional studies on hormonally reconstructed ovariectomized mice confirmed the coordinated regulation of uterine epithelial TNF- and TNF- Rp60 synthesis by ovarian steroids [1, 2]. High TNF- mRNA and protein levels have also been detected in human endometrial glandular cells during the mid to late secretory phase of the menstrual cycle [3, 4]. Epithelial TNF- protein synthesis tended to decrease toward the end of the cycle [5] although elevated concentrations of the cytokine remained detectable in menstrual discharge samples [6] and TNF- mRNA levels were high in endometrial samples collected during menstrual bleeding [7]. Consistent with a role for this cytokine in the regulation of endometrial functions, TNF- Rp60 and Rp80 expression was detected in endometrial glandular cells [8] with peak levels observed during the mid to late secretory phase [9].
Very little experimentation has been done so far to identify the exact role(s) of the TNF-/TNF- receptor system in the uterus. Addition of TNF- to cultures of the human endometrial adenocarcinoma ECC1 cell line has been found to inhibit cell proliferation, to prevent cell aggregation [10], and to induce cell death in a dose-dependent manner [6]. The human endometrial adenocarcinoma AN3CA cell line also responded to TNF- with growth inhibition [11]. Exposure of primary cultures of human endometrial glandular cells to TNF- resulted in increased cell death [6], and this effect was reproduced, albeit to a lesser extent, when primary cultures of mouse uterine epithelial cells were tested [12]. Altogether, these data were found consistent with the hypothesis that the cytokine may contribute to the regulation of endometrial degradation that occurs at the time of menstruation [13].
The following study was conducted to determine the impact of TNF- on cultures of the mouse uterine epithelial WEG-1 cell line. Considering the role of the transcription factor nuclear factor B (NFB) in mediating TNF--induced cell death in numerous cell types [14] and the description of NFB protein expression in the epithelial cells lining the human endometrium [15], particular attention was given to the induction of NFB activation in TNF--treated WEG-1 cells and to the consequence of NFB inhibition by different biochemical compounds on WEG-1 cell viability.
MATERIALS AND METHODS
Cell Cultures and Experiments
The WEG-1 cell line originates from a simian virus 40 large T-antigen-transfected primary culture of CF-1 mouse uterine epithelial cells [16]. The WEG-1 cells were obtained at passage 19 (a gift from Dr. D.D. Carson, University of Delaware, Newark) and routinely passaged at 60–70% confluence in order to prevent a progressive decrease in TNF- responsiveness that was observed when cells were allowed to reach confluence between passages (data not shown). The WEG-1 cultures were discarded when they reached the 40th passage. The WEG-1 cells were maintained in DME (Life Technologies NV/SA, Merelbeke, Belgium; cat. 31885) mixed 1:1 with Ham's F-12 (Life Technologies; cat. 21765) and supplemented with 5% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Before experimental incubations, WEG-1 cells were seeded at 50 000 cells per 25-cm2 flasks and brought to confluence by culturing them in the above medium for 24 h. Confluent cells were then incubated in fresh culture medium supplemented with reduced (0.1%) heat-inactivated fetal calf serum and increasing concentrations of mouse recombinant TNF- (Roche Diagnostics-Boehringer, Mannheim, Germany; cat. 1271156). The TNF--containing media were replaced after 24, 72, and 120 h. In other experiments, confluent WEG-1 cells were treated with a neutralizing hamster monoclonal anti-mouse TNF- Rp80 antibody or a neutralizing hamster monoclonal anti-mouse TNF- Rp60 antibody (R&D Systems Europe Ltd., Oxon, UK; cat. 80400301 and 80400501, respectively), human recombinant TNF- (BioSource Europe SA/NV, Nivelles, Belgium; cat. PHC3015), mouse recombinant interleukin (IL)-1 (Genzyme, Genzyme SA/NV, Leuven, Belgium; cat. 192001), mouse recombinant IL-1ß (BioSource; cat. PMC0814), or with cell-permeable synthetic ceramide analogues N-acetyl-D-erythro-sphingosine (C2-ceramide; Calbiochem-EuroBiochem, Bierges, Belgium; cat. 110145) and N-hexanoyl-D-erythro-sphingosine (C6-ceramide, Calbiochem; cat. 376650). Additional experiments involved the use of ceramide synthase inhibitor fumonisin B1 (Calbiochem; cat. 344850), a cell-permeable inhibitor of NFB nuclear translocation SN50 (BioMol-SanverTech SA/NV, Boechout, Belgium; cat. P600), and IB inhibitors PDTC (Sigma-Aldrich NV/SA, Bornem, Belgium; cat. P8765) and A771726 (a gift from R. Schleyerbach; Hoechst Marion Roussel GmBH, Frankfurt, Germany). Concentrations of the last three compounds were based on preliminary toxicity assays with doses ranging from 0.3 to 30 µg/ml for SN50, 1 to 100 µM for PDTC, and 1 to 100 µM for A771726 due to the possibility that constitutive apoptosis may also be enhanced by these drugs (data not shown). In other preliminary experiments, confluent WEG-1 cells were exposed to 10 µM PBS solution, 5 µM dimethyl sulfoxide, or 0.2% methanol to confirm the lack of effect of the vehicles that were used in the preparation of the above stock solutions (data not shown). Experiments were also performed on cultures of mouse fibrosarcoma WEHI-164/13 cells [17] to test the efficiency of mouse TNF- and ceramide analogues on a highly sensitive cell line (a gift from Dr. P. Coulie, Ludwig Institute for Cancer Research, Brussels, Belgium). The WEHI-164/13 cells were maintained routinely in RPMI-1640 medium (Sigma; cat. R6504) supplemented with 5.5 mM arginine, 0.2 mM asparagine, 1.5 mM glutamine, and 5% heat-inactivated fetal bovine serum. Before experiments, WEHI-164/13 cells were seeded at 20 000 cells per 25-cm2 flasks and exposed at confluence.
Messenger RNA Analysis
Reverse transcription was performed on total RNA extracted from adult mouse spleen and from WEG-1 cells with 10 µM poly(dT) primers and 100 units of Moloney murine leukemia virus reverse transcriptase per reaction. Amplification was then carried out during 40 thermal cycles with 400 µM of specific primers and 5 units of Taq DNA polymerase per reaction. The primer pair used to amplify TNF- Rp60 cDNA corresponded to positions 818–846 and 1160–1188 of the mouse sequence (Genbank reference number M58377) and produced a 371-base pair (bp)-long amplicon. The primer pair used to detect TNF- Rp80 cDNA were complementary to positions 177–204 and 785–812 of the mouse sequence (Genbank reference number M59378) and generated a 636-bp-long amplicon. Negative control reverse transcription and amplification reactions were also run without RNA input or cDNA input.
Qualitative and Quantitative Analysis of Cell Death
Cell viability was assessed after treating each culture flask with 0.05% trypsin and 0.02% EDTA. Each cell suspension was counted using a Bürker hemocytometer after staining with a 0.4% stock solution of trypan blue, a hydrophilic dye that is excluded from plasma membrane-intact cells. Each cell preparation was counted for the numbers of (viable) trypan blue-negative cells and (dead) trypan blue-positive cells. Three independent series of WEG-1 cultures were tested in duplicate for each experimental condition. Cell viability was expressed as a percentage (mean ± SEM) of the average number of viable cells counted in corresponding control flasks.
In order to visualize TNF--induced internucleosomal DNA cleavage, genomic DNA was isolated from WEG-1 cells by lysis in the presence of 10 µg proteinase K and 10 µg ribonuclease A per extraction. After incubation at 37°C for 2 h, DNA preparations were purified by phenol-chloroform-isoamyl alcohol extraction, ethanol precipitation, and resolubilization in DNAse-free water. Equal amounts of DNA (2 µg) from control and TNF--treated cells were then analyzed by means of a ligation-mediated amplification assay (Clontech Laboratories-Westburg, Leusden, The Netherlands; cat. K2021) aimed at detecting the presence of a characteristic 180-bp nucleosomal DNA laddering pattern in the samples. Ligation to special dephosphorylated adaptors was performed with 200 units of T4 DNA ligase per reaction overnight at 16°C, and subsequent amplification was done on 150 ng of ligated DNA with 5 units of Taq-Pwo DNA polymerase (Roche; cat. 1732641) per reaction during 25 thermal cycles. Amplification products were visualized by gel electrophoresis and ethidium bromide staining. The WEG-1 cells were also analyzed by the TUNEL method that identifies apoptotic cells by virtue of the fact that their nucleus contains abnormally abundant 3'-hydroxyl termini [18]. The WEG-1 cells were fixed in 4% paraformaldehyde, treated with 0.3% hydrogen peroxide, permeabilized in 0.1% Triton X-100, prestained in 25 µg/ml bis-benzimide, and then incubated with 50 U/ml of terminal transferase (Roche; cat. 1684817) and 15 µM fluorescein-conjugated dUTP for 35 min at 37°C in a specific reaction buffer. Following exposure to a sheep anti-fluorescein antibody conjugated to peroxidase for 30 min at room temperature, TUNEL staining was developed in a solution of diaminobenzidine and nickel chloride. Each culture was examined under combined UV and visible lights for the presence of cells with either chromatin degradation (positive TUNEL staining, karyolysis), nuclear fragmentation (densely bis-benzimide-stained cluster of nuclear material, karyorhexis), or a typical metaphasic figure (mitosis). Three independent series of WEG-1 cultures were tested in duplicate for each experimental condition. Data were expressed as percentages (mean ± SEM) of cells displaying either one of these three nuclear markers relative to the total number of cells counted per culture.
Stress-Activated Protein Kinase Activity and NFB Activity
The TNF--induced activation of the stress-activated protein kinase (SAPK) signaling pathway was measured using a reporter protein consisting of the N-terminal region of c-Jun (which contained the high affinity site for SAPK as well as the Ser63 residue whose phosphorylation is important for transcriptional activity) linked to the glutathione-binding domain of glutathione-S-transferase (GST; New England Biolabs, Schwalbach, Germany; cat. 9810). The WEG-1 cell lysates (250 µg proteins) were extracted at different time intervals (1, 7, 24, and 31 h of incubation in either control medium or 50 ng/ml mouse TNF-) with 2 µg of c-Jun/GST fusion protein coupled to glutathione-sepharose beads overnight at 4°C. The pulled-down SAPK activity was assayed by performing a kinase reaction onto the pulling-down fusion protein itself in the presence of 100 µM ATP for 30 min at 30°C followed by Western immunoblotting with a rabbit anti-human phospho-c-Jun primary antibody (1:1000 dilution) overnight at 4°C and a peroxidase-conjugated anti-rabbit IgG secondary antibody (1:3000 dilution) for 1 h at room temperature. Peroxidase activity was then detected by chemiluminescence and autoradiography (New England Biolabs; cat. 7071). The SAPK activation data are expressed as arbitrary intensity units (mean ± SEM) based on three independent WEG-1 cultures that were tested for each experimental condition.
In order to monitor the activation of the NFB signaling pathway after exposure to TNF-, WEG-1 cells were transiently cotransfected with an experimental reporter coding for the firefly luciferase protein under the control of the herpes simplex virus thymidine kinase promoter coupled with the NFB-binding GCCCTTAAAG sequence acting as upstream enhancer (pNF-kB-LUC, Clontech; cat. 6053-1) and a constitutively expressed reporter encoding the Renilla luciferase protein under the control of the same promoter (pRL-TK; Promega Benelux BV, Leiden, The Netherlands; cat. E2241). After incubation in either control medium or 50 ng/ml TNF- for different time intervals, cells were harvested and activities of Renilla and TNF--induced firefly luciferases were measured sequentially in each cell extract by means of a dual reporter assay (Promega; cat. E1910) that was based on the abilities of the enzymes to oxidize distinct luciferin substrates. Extensive preliminary experiments were performed to optimize the efficiency of the lipofectamine-mediated transfection procedure (Life Technologies; cat. 10964013), the total amount of plasmid DNA, and the relative ratio of experimental reporter to control reporter to introduce into the WEG-1 cells (data not shown). Values shown are averages (mean ± SEM) of three independent series of transfected WEG-1 cultures that were tested in duplicate for each experimental condition.
RESULTS
Cytotoxicity of TNF-
The effect of mouse TNF- on confluent WEG-1 cells was examined as a function of concentration and exposure time. When cells were treated with the highest dose tested (50 ng/ml), a period of 31 h was needed before a significant decrease in cell viability could be observed against control cultures (Fig. 1A). Concentrations of 5 and 0.5 ng/ml of the cytokine required 94 and 168 h of incubation, respectively, to induce significant cell loss. In contrast, 0.5 ng/ml of TNF- reduced the viability of WEHI-164/13 cells by 60% within 3 h of exposure. Additional WEG-1 cultures were incubated with increasing concentrations of either IL-1 or IL-1ß in order to test the toxicity of these two cytokines. Previous experiments have shown that WEG-1 cells express receptors for IL-1 [19]. No sign of decrease in cell viability was found when concentrations of up to 25 ng/ml of IL-1 or IL-1ß were added to the culture medium for 120 h, suggesting that these cytokines do not induce cell death in WEG-1 cells (data not shown).
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To determine the cause underlying the mouse TNF--induced reduction in WEG-1 cell viability, cultures were exposed to 5 ng/ml of the cytokine for 120 h and then analyzed for the induction of genomic DNA degradation. Compared with control cultures, the occurrence of internucleosomal DNA cleavage was markedly increased following mouse TNF- treatment (Fig. 1B). Application of the TUNEL staining technique confirmed the induction of DNA breakdown by mouse TNF-. After an incubation period of 96 h, a dose-dependent relationship was found between the concentration of mouse TNF- and the proportion of TUNEL-positive WEG-1 cells (Fig. 1C). Nuclear fragmentation, in contrast to DNA degradation, remained very low irrespective of the concentration of mouse TNF- in the culture medium. The frequency of mitotic figures in WEG-1 cells was unaffected by the addition of TNF-. Additional experiments were performed to confirm that exposure to mouse TNF- for 72 h also resulted in a marked increase in the externalization of phosphatidylserine residues at the cell surface compared with control cultures (data not shown).
Role of TNF- Rp60 and Rp80
Total cDNA was prepared from WEG-1 cells and amplified for transcripts encoding different TNF- receptor isoforms. Both TNF- Rp60 and Rp80 were found to be expressed at the mRNA level in this cell line (Fig. 2A). To determine the contribution of TNF- Rp80 to the cytotoxic effect of the cytokine, WEG-1 cells were cotreated with 5 ng/ml mouse TNF- and a 200-fold molar excess of a neutralizing anti-TNF- Rp80 antibody for 120 h (Fig. 2B). Addition of the antibody attenuated (but failed to suppress completely) the impact of the cytokine on WEG-1 cells, suggesting that costimulation of the two TNF- receptors is required for maximal induction of TNF- cytotoxicity. Addition of a 200-fold excess of a neutralizing anti-TNF- Rp60 antibody to 5 ng/ml mouse TNF- produced similar results (data not shown). Previous observations that human TNF- does not activate mouse TNF- Rp80 [20] were exploited to confirm the collaborative effect of TNF- Rp60 and Rp80. The 50% effective dose (ED50) concentration of human TNF- at 120 h was about 15-fold higher than the mouse TNF- ED50 value (Fig. 2B and data not shown), suggesting that engagement of Rp60 alone is weakly effective in triggering WEG-1 cell death.
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Ceramide and SAPK Activation
Experiments were performed to examine whether ceramide was involved in the signal transduction cascade induced by mouse TNF- in WEG-1 cells. Exposure to high concentrations (5 µM) of either C2-ceramide or C6-ceramide for up to 168 h failed to decrease the viability of WEG-1 cells compared with control cultures (Fig. 3A). In contrast, exposing WEHI-164/13 cells to the same concentration of C6-ceramide induced a 65% reduction in cell viability within 6 h of incubation. Consistent with the lack of ceramide cytotoxicity on WEG-1 cells, addition of high concentrations of fumonisin B1 (5 µM), an inhibitor of ceramide synthase with an IC50 value of 100 nM, failed to protect WEG-1 cells against the effect of 5 ng/ml mouse TNF- after 120 h (Fig. 3B). Cell extracts were prepared from WEG-1 at different time intervals during culture in the presence or absence of 50 ng/ml mouse TNF- and analyzed for their content in SAPK activity. No difference in SAPK activation was found between the control and TNF--treated cultures, indicating that this signaling pathway was not triggered by the cytokine within the first 31 h of treatment (Fig. 4A).
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Nuclear Factor B Activation
The WEG-1 cells were transfected transiently with a dual-luciferase reporter assay aimed at monitoring the activation of the NFB signaling pathway by TNF-. Compared with untreated control cultures, WEG-1 cells exposed to 50 ng/ml of mouse TNF- showed a biphasic increase in NFB activity that reached a first peak after 3 h of incubation and a second peak at 24 h (Fig. 4B). The NFB activation was different upon exposure to 50 ng/ml of human TNF-, with the first peak appearing after 24 h of culture (Fig. 4C).
Three independent inhibitors of NFB were used to test the possibility that blocking TNF--induced activation of NFB may sensitize the WEG-1 cells to the toxicity of the cytokine. Upon addition at the final concentration of 30 µg/ml in the culture medium, the inhibitor of NFB nuclear translocation SN50 [21] was found to enhance significantly the deleterious impact of 50 ng/ml mouse TNF- on WEG-1 cells after 24 and 31 h of exposure (Fig. 5A). The PDTC, an inhibitor of the release of inhibitor B (IB) from the cytoplasmic IB-NFB complex [22], was found to have a similar sensitizing effect at 24 and 31 h when used at 1 µM in combination with 50 ng/ml mouse TNF- (Fig. 5B). When used at 1 µM, A771726, a compound known to block the degradation of IB indirectly [23], was also found to sensitize WEG-1 cells to the cytotoxicity of 50 ng/ml mouse TNF- (Fig. 5C). None of these inhibitors affected the viability of the WEG-1 cell cultures when tested alone for the time periods indicated. Cultures were pretreated for 30 min with SN50, for 90 min with PDTC, and for 2 h with A771726 before supplementation of the culture medium with mouse TNF-.
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DISCUSSION
Previous studies have shown that human TNF- induces cell death in primary cultures of human endometrial glandular cells [6, 7], inhibits cell proliferation in human endometrial adenocarcinoma cell lines [6, 11], and causes moderate levels of cell death in primary cultures of mouse uterine epithelial cells [12]. Other reports have shown that mouse TNF- is detrimental to the development of mouse blastocysts [24] and mouse embryonic stem cells [25, 26]. In addition, human TNF- has been shown to decrease the rate of DNA biosynthesis in rat trophoblastic cell lines [27] and to stimulate cell death in primary cultures of human term trophoblasts [28]. Mouse TNF- has been shown to drastically decrease cell viability in cultures of rat trophoblastic R8RP-3 cells [29]. In keeping with these previous observations, the present paper shows that mouse TNF- can significantly decrease cell viability and trigger the expression of several markers of apoptosis in cultures of the mouse uterine epithelial WEG-1 cell line. Interleukin-1 and IL-1ß, two cytokines for which WEG-1 cells express specific receptors [19], did not influence the viability of these cultures when tested under similar conditions.
Tumor necrosis factor- is known to mediate its numerous effects by binding to two distinct receptors that, due to large differences in their cytoplasmic domains, are thought to transmit their signals via separate intracellular signaling cascades [30]. Recent reports have suggested that cooperativity between the two receptors is required for TNF- to induce cell death in many cell types [31]. The failure of human TNF- to cross-react with the mouse Rp80 [20] has previously been used to show that in cultures of adipogenic TA1 cells and fibroblastic NIH 3T3 cells, two murine cell lines that coexpress the two TNF- receptor types, engagement of p60 alone was not sufficient to elicit cell death [32]. In the present study, WEG-1 cells were found to coexpress Rp60 and Rp80 transcripts and appeared only weakly susceptible to the induction of cell killing when exposed to human TNF-. When compared at the same incubation time, the half-maximal effect with human TNF- occurred at a concentration that was about 15-fold higher than that of mouse TNF-. Blocking the binding of mouse TNF- to either Rp80 or Rp60 by means of neutralizing antibodies against these receptor isoforms also markedly attenuated the impact of the cytokine on WEG-1 cells. In contrast, human trophoblasts were highly sensitive to the cell killing effect of TNF- via activation of Rp60 alone, despite coexpressing both receptor types [28, 33]. Previous reports have shown that TNF- binds to fibronectin [34], an important component of the subepithelial extracellular matrix within the uterine stroma [35], and that solid phase-associated TNF- preferentially engages Rp80 [36] as opposed to soluble TNF- that has a higher affinity for Rp60 [37]. Based on these observations, it is tempting to speculate that the requirement for Rp80 and Rp60 coactivation in WEG-1 cells is integral to the modulation of TNF--induced cell death in the uterine epithelium. Preliminary experiments indicate that culturing WEG-1 cells on a fibronectin matrix resulted in a 2.5-fold enhancement of the impact of mouse TNF- on WEG-1 cell viability when compared with TNF- treatment on a standard plastic substrate (data not shown).
Ceramide has recently emerged as an important mediator of the effects of cytokines such as TNF- and IL-1/ß on cell death and other cellular events [38]. For instance, both TNF- and cell-permeable ceramide analogs induce apoptosis in human histiocytic lymphoma U937 cells [39], in mouse fibrosarcoma WEHI-164/13 cells (this report and [40]), and in rat ovarian granulosa cells [41]. In the present study, however, C2-ceramide and C6-ceramide failed to recapitulate the impact of TNF- on WEG-1 cells.
Instances of ceramide-independent responses to TNF- have been reported previously, with examples including the inability of ceramide analogs to induce apoptosis in human myelogenous leukemia ML-1a cells [42] and adhesion protein expression in human umbilical vein endothelial cells [43]. Experiments were performed to test whether ceramide production [44] may be necessary, but not sufficient, to induce cell death in WEG-1 cells. To investigate the role of ceramide synthase in TNF--mediated cell death signaling, the specific inhibitor fumonisin B1 [45] was used at 50-fold its IC50 concentration value. Cell death was not prevented by the addition of this inhibitor, suggesting that activation of ceramide synthase is not involved in the toxic impact of the cytokine on WEG-1 cells. Ceramide synthase-independent induction of cell death by TNF- has been found in other cell types, such as in U937 cells [46]. Preliminary attempts have also been made to determine the contribution of sphingomyelin breakdown by using the acidic sphingomyelinase-specific inhibitor indolizin sulfone SR33557 [47]. This compound was highly toxic on WEG-1 cells when present in concentrations exceeding 3 µM, however, thus preventing the addition of sufficient compound to the culture medium to achieve the complete blocking of the enzyme. When WEG-1 cells were exposed to a combination of 5 ng/ml TNF- and 3 µM SR33557 for 72 h, no change was observed in the impact of the cytokine on cell viability (data not shown).
Activation of NFB has been recently proposed as the leading intracellular determinant underlying cell death protection against TNF- and other cytotoxic effectors [14, 48]. Indications that NFB is involved in cytoprotection derive from observations that mouse fetuses lacking the gene encoding the RelA/p65 subunit of NFB die in utero from massive cell death in the liver [49] and that several TNF--resistant cell lines become highly sensitive to the cytokine upon ablation of the RelA/p65 gene [50] or transfection with a construct coding for a dominant-negative IB mutant protein [51, 52]. Our study indicates that WEG-1 cells responded to mouse TNF- with a biphasic increase in NFB activity that tended to recede after 24 h of exposure to the cytokine (present study and data not shown), when signs of cell death appeared in the cultures. The mechanism responsible for the decline in NFB activity has not been identified. It is possible that sustained exposure to TNF- may trigger the delayed activation of SAPK signaling or the activation of another (non-SAPK) signal transduction pathway that would over-ride the initial NFB protective reaction and, eventually, force WEG-1 cells into committing cell suicide. Interestingly, apoptosis-associated caspases have been described as being able to accelerate the inactivation of NFB either by truncating its RelA/p65 subunit, thereby disabling the transactivating capacity of the nuclear factor [53], by cleaving its cytoplasmic inhibitor IB into a less degradable form [54, 55] or by cleaving and neutralizing the upstream NFB regulator RIP [56]. Using human TNF- instead of mouse TNF- led to a marked change in the NFB response profile, in support of the combined importance of both TNF- receptor isoforms. Investigating whether binding to Rp60 alone fails to induce the first NFB activation peak (observed at 3 h with mouse TNF-) or delays its appearance until 24 h will require additional experiments.
To gain more insight into the possible relationship between NFB activation and cell death induction, experiments were performed with three different inhibitors of the transcription factor. The first inhibitor was SN50, a cell membrane-permeable fusion protein containing the hydrophobic region of the K-fibroblast growth factor signal peptide sequence coupled with the nuclear localization sequence of the p50 subunit of NFB [21]. This peptide, which competes with endogenous NFB for nuclear translocation, was previously found to sensitize murine endothelial LE-II cells and human monocytic THP-1 cells to the cytotoxicity of TNF-. The second inhibitor was PDTC, a compound that has been shown to stimulate the cytotoxic impact of TNF- on human Jurkat T-cells by suppressing the release of IB from latent NFB, due to a combination of antioxidant and heavy metal chelating properties [22]. And finally, A771726 is a metabolite of the anti-inflammatory drug Leflunomide whose action on de novo pyrimidine synthesis leads to the blocking of IB phosphorylation and degradation via a still unclear indirect mechanism [23]. This compound has previously been shown to sensitize Jurkat cells to TNF- [23]. Our data show that the three compounds tested increased the cytotoxicity of the cytokine on WEG-1 cells, in support of the hypothesis that the first intracellular signaling pathway triggered by TNF- in these cultures is NFB-based and protective against cell death.
ACKNOWLEDGMENTS
We thank Cécile Marchand for her expert technical assistance.
FOOTNOTES
First decision: 20 March 2000.
1 Research supported by the Fonds de la Recherche Scientifique Médicale (convention 3.4527.99), the Fonds Suzanne and Jean Pirart de l'Association Belge du Diabète, and the Commission des Relations Internationales de l'Université Catholique de Louvain (grant CGLSF524). S.P. is a Chercheur Qualifié with the Fonds National de la Recherche Scientifique. 
2 Correspondence: Serge Pampfer, OBST 5330 Research Unit, Université Catholique de Louvain Medical School, 53 Avenue Mounier, 1200 Brussels, Belgium. FAX: 32 2 7645396; pampfer{at}obst.ucl.ac.be
3 Current address: Institute of Animal Physiology, Slovak Academy of Sciences, Kosice, Slovakia.
Accepted: April 28, 2000.
Received: February 22, 2000.
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