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*FUMONISIN B1
The Journal of Immunology, 2000, 165: 3564-3570.
Copyright © 2000 by The American Association of Immunologists

Ligation of the WC1 Receptor Induces {gamma}{delta} T Cell Growth Arrest Through Fumonisin B1-Sensitive Increases in Cellular Ceramide1

Paul A. Kirkham2,*, Haru-Hisa Takamatsu*, Eric W.-F. Lam{dagger} and R. M. E. Parkhouse*

* Department of Immunology, Institute for Animal Health, Pirbright, Surrey, United Kingdom; and {dagger} Ludwig Institute for Cancer Research and Section of Virology and Cell Biology, Imperial College School of Medicine at St. Mary’s Hospital, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ceramide is a powerful regulator of cell fate, inducing either apoptosis or growth arrest. We have previously shown that an Ab to the {gamma}{delta} T cell-specific orphan receptor, WC1, is able to induce growth arrest in proliferating IL-2-dependent {gamma}{delta} T cells. We now show that this WC1-mediated growth arrest is associated with an increase in cellular ceramide, in the absence of any measurable changes in acidic/neutral sphingomyelinase activity. Moreover, cell-permeable analogues of ceramide also mimicked WC1-induced growth arrest along with an associated decrease in pocket protein expression and phosphorylation status. An important role for ceramide in WC1-induced growth arrest was confirmed by demonstrating that the specific ceramide synthase inhibitor fumonisin B1 blocked WC1-induced growth arrest and the associated molecular effects on the pocket proteins. Finally, we observed constitutive expression of both antiapoptotic factors bcl-2 and bcl-X, the former having increased expression upon WC1 stimulation. It is therefore proposed that ligation of WC1 leads to an accumulation in cellular ceramide through activation of ceramide synthase. This in turn results in a decreased overall expression of the pocket proteins pRb and p107, their hypophosphorylation, and an eventual growth arrest of the {gamma}{delta} T cell. To our knowledge, these results demonstrate for the first time that cell surface receptor-mediated ceramide synthase activation can affect cell fate through increases in cellular ceramide and provide further evidence that the orphan receptor WC1 regulates {gamma}{delta} T cell biology through a novel signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The orphan receptor WC1 is a large type 1 transmembrane protein (220 kDa) exclusively expressed by {gamma}{delta} T cells with homology to the macrophage scavenger receptor and other members of this superfamily of molecules, which are now known to play an increasingly important role in innate immunity. We have recently shown that ligation of WC1 triggers the induction of G1 growth arrest in proliferating, IL-2-dependent, {gamma}{delta} T cells (1, 2) by regulating key components of the cell cycle machinery, such as p27kip1, the pocket proteins, and the transcription factor E2F1 (3). Thus, it has been proposed that WC1 may play an important role in the regulation of {gamma}{delta} T cell biology (1, 2, 4). As {gamma}{delta} T cells may regulate both the innate and acquired immune responses (5), a greater understanding as to the mechanism of action of WC1 within {gamma}{delta} T cell biology is fundamental to our understanding of immune responses regulated through the {gamma}{delta} T cell.

Sphingolipids, such as ceramide and sphingosine-1-phosphate, have emerged as important second messengers regulating cell fate (6, 7). Serum starvation, cell-cell contact, and stimulation of cells with agonists, for example TNF-{alpha}, IFN-{gamma}, IL-1ß, and vitamin D3, all lead to increases in cellular ceramide as a result of sphingomyelinase activation and subsequent sphingomyelin hydrolysis (8). In all cases, these agonist-induced increases in ceramide precede the antimitogenic effects of these stimuli. Currently, two classes of sphingomyelinase exist, acidic sphingomyelinase and neutral sphingomyelinase. Both can give rise to increases in cellular ceramide levels, but at different subcellular sites, thereby resulting in different functional outcomes (9). Increases in ceramide, however, can also be achieved by synthesis from sphinganine through the action of ceramide synthase to produce dihydroceramide, which is then rapidly oxidized to give ceramide. Such a pathway has been shown to occur after daunorubicin treatment of P388 and U937 cells, thereby triggering apoptosis in these cells (10). Further evidence highlighting the importance of ceramide in regulating cell fate has come from experiments demonstrating that the addition of short chain ceramide analogues can induce antiproliferative effects on various cell types by inducing either apoptosis (11, 12) or cell cycle arrest (13). In general, ceramide has been widely reported to induce apoptosis. However, when in the presence of raised diacylglycerol (DAG)3 levels or protein kinase C (PKC) activation, ceramide will then signal cell cycle arrest (13, 14). This latter outcome appears to be a result of up-regulation in the protein inhibitor of apoptosis, bcl-2, as Whitman et al. (15) have demonstrated that PKC activation through phorbol ester stimulation can up-regulate bcl-2 expression in HL-60 cells. Furthermore, other laboratories have shown that bcl-2 expression can inhibit ceramide-induced cell death (16, 17). As a second messenger, ceramide has been shown to directly regulate both phosphatase and kinase activity through either a ceramide-activated protein phosphatase (CAPP) (18) or a ceramide-activated protein kinase (19). Further downstream, ceramide can affect components of the mitogen-activated protein (MAP) kinase signaling cascade (20) as well as those of the cell cycle, such as the pocket protein retinoblastoma gene product (pRb) (21).

We have previously shown that WC1 stimulation results in cell growth arrest (1) and because of the importance of ceramide in regulating cell fate, we therefore investigated the effect of WC1 stimulation on cellular ceramide levels, the mechanism through which it is produced, and the impact this has on key components involved in cell cycle regulation. Finally, in the absence of antiapoptotic factors, ceramide is a potent inducer of apoptosis, and we therefore determined whether WC1 stimulation of S-59 {gamma}{delta} T cells influenced the expression of any antiapoptotic factors such as bcl-2 or bcl-X.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue culture, cell treatment, and Abs

S-59 {gamma}{delta} T lymphocytes were cultured in Iscove’s modified MEM supplemented with 10 U/ml IL-2, as previously described (2). For treatment of cells with ceramide analogues, the method of Hauser et al. (22) was used. Briefly, 0.5 µmol of either N-acetyl-D-erythro-sphingosine (C2-ceramide), N-hexanoyl-D-erythro-sphingosine (C6-ceramide), N-octanoyl-D-erythro-sphingosine (C8-ceramide), or the negative control analogue dihydro-N-acetyl-D-erythro-sphingosine (C2-dihydroceramide) was dissolved in 10 µl ethanol, then 0.5 ml serum-free Iscove’s medium containing 0.1% (w/v) BSA was added and the mixture was incubated for 1 h at 37°C. Before addition to the cells, the ceramide mixture was vortexed. All ceramide analogues were purchased from Calbiochem (Nottingham, U.K.). Treatment of S-59 cells with fumonisin B1 (Calbiochem) was performed by initially making a 10 mg/ml stock solution of fumonisin B1 in methanol. This solution was then diluted into cell culture medium to give the final fumonisin B1 concentration used in the experiments described below. For mAb treatment, S-59 cells were incubated with either 20 µg/ml mAb anti-WC1 (SC-29; IgG1) or control mAb anti-IgM (ILA-30; IgG1) at 37°C. Abs against WC1 (SC-29) and IgM (ILA-30) were described previously (2). Rabbit anti-Rb (C-15) and anti-p107 (C-18) for Western blotting were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-labeled goat anti-rabbit Ig and goat anti-mouse Ig were obtained from Southern Biotechnology Associates (Birmingham, AL). A mouse anti-bcl-2 mAb (catalogue B46620) and a rabbit anti-bcl-X pAb (catalogue B22630) were supplied by Transduction Laboratories through Affiniti (Exeter, U.K.).

DAG and ceramide measurements

This method is based on that described by Preiss et al. (23), which uses the phosphorylation of DAG or ceramide by DAG kinase and [{gamma}-32P]ATP. Cells (1 x 106) in 0.1 ml Iscove’s medium were dispensed into 2-ml glass vials containing either 2 µg anti-WC1 or anti-IgM and incubated at 37°C. The reaction was stopped by adding 0.375 ml methanol/chloroform (2:1), mixed, and kept on ice for 5 min. Then 0.125 ml chloroform followed by 0.125 ml 1 M NaCl were added, mixed, and centrifuged at 1500 rpm for 2 min for phase separation. The organic phase (100 µl) was removed, dried down under nitrogen, and then reconstituted in 50 µl detergent mix (0.6% v/v Triton X-100, 0.288 mM phosphatidylserine) with gentle vortexing and sonication at 4°C. Standards consisting of DAG (Sigma, Poole, U.K.; catalogue P1293) or ceramide (Calbiochem; catalogue 376650) were also prepared in detergent mix. A series of reagents was then added to the samples in the following order: 20 µl 5x incubation buffer (250 mM Imidazole/HCl, pH6.6, 250 mM NaCl, 62.5 mM MgCl2, 5 mM EGTA), 10 µl 0.1 M DTT, 10 µl 0.2 mg/ml DAG kinase (Calbiochem; catalogue 266724), and 10 µl ATP mix (0.1 M Imidazole/HCl, pH 6.6, 5 mM ATP, 0.125 µCi/µl [{gamma}-32P]ATP), and the reaction was then incubated for 30 min at 30°C. The reaction was stopped by the addition of 470 µl chloroform/methanol/10 mM HCl (150:300:20) and allowed to extract for 10 min. The phases were then separated by the addition of 150 µl chloroform and 150 µl water, followed by centrifugation for 2 min at 1500 rpm. The upper aqueous phase was discarded, and the organic phase was washed with 1 ml water twice. The organic phase was removed, dried down, and then subject to TLC on Merck silica gel 60 TLC plates using a chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1) solvent system. Following overnight autoradiography of the TLC plate, the spots corresponding to radiolabeled DAG and ceramide on the TLC plate were scraped off and scintillation was counted.

Thymidine uptake assays

These assays were performed as described elsewhere (24) using 2 x 104 S-59 {gamma}{delta} T cells/well with or without cell treatment, as detailed above.

Western blotting

Cells (5 x 106) were lysed with 40 µl lysis buffer (1% (v/v) Triton X-100, 1% (v/v) sodium deoxycholate, 0.1% (v/v) SDS, 50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% (w/v) sodium azide, 1 mM EDTA and EGTA, 0.5 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 100 µg/ml aprotonin, 10 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin, 5 µg/ml each of N{alpha}-p-tosyl-L-lysine chloromethyl ketone and N-tosyl-L-phenylananine chloromethyl ketone, and 1 mM PMSF) for 30 min on ice. The lysate was minifuged at 14,000 rpm for 5 min at 4°C, and the supernatant was collected, then assayed for total protein content using the Pierce bicinchoninic acid protein assay (Pierce & Warriner, Chester, U.K.). The lysate (10 µg of protein) was resolved by reducing SDS-PAGE, and the proteins were then transferred and immobilized onto nitrocellulose membrane. The level of total proteins loaded and transferred to the nitrocellulose membrane was again checked by staining with Ponceau S. The membrane was blocked with 10% (w/v) dried milk Marvel in PBS containing 0.05% (v/v) Tween 20 (PBST) for 1 h at room temperature, followed by probing with primary Ab (1/1000 dilution in blocking solution) overnight at 4°C. After extensive washing with PBST (20 min per wash, repeated six times), primary Ab was detected with the appropriate secondary Ab (diluted 1/1000 in blocking solution) for 30 min at room temperature. After three further PBST washes, 20 min each, the blot was visualized with the Pierce Supersignal/Supersignal-ultra chemiluminescence system. Western blots were reprobed with a different Ab after stripping, as previously described (2).

Flow cytometric analysis for WC1 surface expression and apoptosis detection

S-59 {gamma}{delta} T cells (1 x 105 cells/well) were incubated in a 96-U-well plate with either medium alone (Iscove’s modified MEM supplemented with 10 U/ml IL-2, as previously described (2)) or medium containing 400 nM fumonisin B1 (Calbiochem) for 30 min at 37°C. The cells were then washed and stained with anti-WC1 mAb SC-29 or an isotype-matched negative control mAb ILA-30 (anti-IgM) for 30 min on ice in PBA (PBS containing 1% (w/v) BSA and 0.1% (w/v) sodium azide). The cells were washed twice with PBA, followed by incubating with PE-conjugated goat anti-mouse Ig for 15 min on ice. The cells were washed twice more, fixed with paraformaldehyde, and then analyzed on a FACScan (Becton Dickinson, Mountain View, CA).

To detect apoptotic S-59 cells, 2 x 105 cells/sample were examined by FACS after ethanol fixing, then RNase digestion, followed by staining the cellular DNA with 40 µg/ml propidium iodide, as described previously (1).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
WC1 activation increases cellular ceramide levels, but not DAG levels

We initially sought to investigate whether WC1 ligation with an anti-WC1 mAb could induce increases in cellular ceramide or DAG levels in S-59 {gamma}{delta} T cells. In Fig. 1Go, addition of 20 µg/ml anti-WC1 to S-59 {gamma}{delta} T cells resulted in a statistically significant steady increase in cellular ceramide levels within the first 30 min (p < 0.001, as determined by ANOVA) compared with control anti-IgM-treated cells. Over a further 2.5-h period of WC1 stimulation, ceramide levels continued to rise, attaining levels up to 83% above that for control. The experimental design allowed the simultaneous measurement of both DAG and ceramide levels within the same assay, thereby allowing a direct comparison between varying DAG and ceramide levels to be made, as well as allowing the increasing ceramide levels as a result of WC1 stimulation to act as an internal assay control. As such, in contrast to Fig. 1GoA, Fig. 1GoB shows that mAb anti-WC1 treatment did not induce significant increases in DAG formation above that observed for cells treated with the negative control mAb (anti-IgM).



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  		FIGURE 1. WC1 ligation increases cellular ceramide levels. S-59 {gamma}{delta} T cells were treated with 20 µg/ml mAb SC-29; anti-WC1 ({diamondsuit}) or mAb IL-A30; anti-IgM ({square}) at 37°C. Cellular ceramide (A) and DAG (B) levels were measured by a DAG kinase-based assay using [{gamma}-32P]ATP. Radiolabeled lipids were separated by TLC and then quantitated by scintillation counting. Further details are as described in Materials and Methods. Mean basal DAG and ceramide levels for untreated control cells were 224 ± 6 pmol/106 cells and 537 ± 19 pmol/106 cells, respectively. The results are expressed as a percentage of control (untreated cells) and represent the mean ± SEM for three separate experiments.

 
Fumonisin B1 inhibits WC1-induced growth arrest

Besides sphingomyelinase activity, increases in cellular ceramide levels can also be generated through the action of ceramide synthase on sphinganine. The fungus fusarium monoliforme produces a mycoprotein called fumonisin B1, which has structural similarities to sphinganine and sphingosine. Consequently, it has been shown that fumonisin B1 is a specific inhibitor of ceramide synthase (10, 25, 26). To study the effect of blocking de novo ceramide synthesis by ceramide synthase on WC1-induced growth arrest, we looked at tritiated thymidine uptake in S-59 cells treated for 24 h with fumonisin B1 with or without WC1 stimulation. Fig. 2Go showed that treatment with 20 nM fumonisin B1 alone did not affect the ability of S-59 cells to proliferate. As previously described, WC1 treatment dramatically reduced thymidine uptake in these cells, indicative of growth arrest (1). However, up to 30-min pretreatment of the {gamma}{delta} T cells with 20 nM fumonisin B1 before anti-WC1 mAb SC-29 addition blocked the effects of anti-WC1 treatment alone on reducing thymidine uptake. This effect could be observed when the addition of fumonisin B1 was as little as 1 min before the addition of anti-WC1, resulting in a significant increase in thymidine uptake when compared with the WC1 only-treated cells (p < 0.001; as determined by Student t test). Moreover, this rise in thymidine uptake increased as the fumonisin B1 pretreatment time increased before the addition of anti-WC1. In complete contrast, when fumonisin B1 was added to the cells together with anti-WC1 mAb SC-29, no inhibition in the reduction of thymidine uptake was observed.



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  		FIGURE 2. The ceramide synthase inhibitor fumonisin B1 inhibits WC1-induced growth arrest. S-59 {gamma}{delta} T cells were pretreated with 20 nM fumonisin B1 for the times indicated before incubation for an additional 24 h with 20 µg/ml mAb SC-29 (anti-WC1). Cells were also left untreated or treated for 24 h with either 20 nM fumonisin B1 or 20 µg/ml anti-WC1 only. Proliferation was measured as a function of [3H]thymidine uptake, as detailed in Materials and Methods. Each assay point was determined in triplicate and represents the mean ± SEM.

 
To remove the possibility that fumonisin B1 treatment of cells may affect the ability of anti-WC1 to bind to the WC1 receptor or alternatively down-regulate WC1 cell surface receptor expression, FACS analysis was employed. In Table IGo, S-59 {gamma}{delta} T cells left untreated or pretreated with fumonisin B1 for 30 min, followed by cell surface staining for WC1 with mAb SC-29 (anti-WC1), showed no change in mean intensity staining for WC1. This implied that fumonisin B1 neither affected WC1 surface expression nor interfered with mAb SC-29 binding to WC1.


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  		Table I. Fumonisin B1 treatment has no effect on either anti-WC1 binding or WC1 surface expression1

 
Ceramide analogues mimic WC1 stimulation at both the biochemical and cell biological levels

In view of the finding that WC1 stimulation raises cellular ceramide levels and inhibition of ceramide synthase can block WC1-induced growth arrest, we explored the possibility that raising ceramide levels through the addition of exogenous ceramide analogues could mimic the effects of WC1 stimulation. Fig. 3Go illustrates this point by looking at thymidine uptake in S-59 {gamma}{delta} T cells. The ceramide analogues C2-, C6-, and C8-ceramide all show a titratable effect in reducing thymidine uptake. At 50 µM concentration, thymidine uptake is almost completely abolished, whereas at 12.5 µM there is approximately 30% reduction in thymidine uptake with all three analogues. At a 25 µM ceramide concentration, the C6-ceramide analogue is the most potent at reducing thymidine uptake, followed by the C2- and then the C8-ceramide analogues. As a negative control, the closely related structural analogue to C2-ceramide, C2-dihydroceramide, had very little effect on thymidine uptake over the same concentration range. In contrast, the positive control, WC1 stimulation with 20 µg/ml mAb SC-29 (anti-WC1), resulted in an 80% decrease in thymidine uptake.



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  		FIGURE 3. Addition of exogenous ceramides (cer) induces growth arrest in S-59 {gamma}{delta} T cells. {gamma}{delta} T cell proliferation was measured as a function of [3H]thymidine uptake. S-59 {gamma}{delta} T cells were incubated with different concentrations of the ceramide analogues; C2-dihydroceramide, C2-ceramide, C6-ceramide, and C8-ceramide for 18 h at 37°C, then pulse labeled with [3H]thymidine for an additional 6 h. As controls, the {gamma}{delta} T cells were either left untreated or incubated with 20 µg/ml mAb SC-29 (anti-WC1). Further details are as described in Materials and Methods. The raw value for control untreated cells was 33,153 ± 1,558 cpm. Each assay point is expressed as the percentage of control (untreated cells) and represents the mean ± SEM (n = 3).

 
At a biochemical level, regulation of pocket protein phosphorylation and expression is tightly associated with cell cycle control (27). In addition, accumulating evidence has shown that ceramide induces growth suppression through regulation of the pocket proteins, in particular the retinoblastoma gene product (pRb) (7, 11, 13, 21, 28). Fig. 4Go shows that increases in ceramide impact on pocket protein regulation, which in turn will impact upon cell cycle control. Using Western blot to detect the pocket proteins pRb and p107, the effect of WC1 stimulation, both in the presence and absence of fumonisin B1 pretreatment, and the addition of ceramide analogues were investigated. When compared with control untreated cells, 25 µM C2- and C6-ceramide caused an overall decrease in pocket protein expression. This included the slower migrating hyperphosphorylated form. In contrast, 25 µM C8-ceramide showed no effect on pocket protein expression. This would be in agreement with the data in Fig. 3Go showing that 25 mM C8-ceramide did not significantly reduce thymidine uptake. The negative control C2-dihydroceramide analogue did not reduce pocket protein expression, but did appear to increase pRb expression relative to p107. Consistent with our previous data (3), Fig. 4Go shows that WC1 stimulation with 20 µg/ml mAb SC-29 gave decreased pRb and p107 expression, including the hyperphosphorylated form. However, 20 nM fumonisin B1 pretreatment of S-59 {gamma}{delta} T cells completely blocks the WC1-induced reduction in pocket protein expression. These results therefore indicate that in S-59 {gamma}{delta} T cells, ceramide has an effect on pocket protein expression and phosphorylation status. Moreover, the inhibition of ceramide generation by fumonisin B1 acting on ceramide synthase blocks the effect of WC1 stimulation on pocket protein regulation, thereby allowing cell cycling to continue, as shown by thymidine uptake in Fig. 3Go.



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  		FIGURE 4. Ceramide (cer) and the ceramide synthase inhibitor fumonisin B1 (Fum) directly impact on pocket protein expression and phosphorylation status. A, S-59 {gamma}{delta} T cells were treated with either C2-dihydroceramide, or C2-, C6-, or C8-ceramide at a concentration of 25 µM for 24 h. B, S-59 {gamma}{delta} T cells were also treated with 20 nM fumonisin B1 alone, 20 µg/ml mAb SC-29 (anti-WC1) alone, or 30-min fumonisin B1 pretreatment, followed by anti-WC1 for 24 h. The pocket proteins pRb and p107 were detected by Western blot, having been resolved by 5% SDS-PAGE. This is a representative result of the experiment repeated twice. Further details are described in Materials and Methods.

 
S-59 {gamma}{delta} T cells are protected from ceramide-induced apoptosis through WC1 stimulation

Ceramides can induce both growth arrest and apoptosis through two distinct pathways (28). Therefore, the effect of exogenous ceramides on the pocket proteins (see Fig. 4Go), which can induce cell cycle arrest, does not discount the possibility that these same exogenous ceramides may also trigger an apoptotic pathway in S-59 {gamma}{delta} T cells, and therefore the reduction in thymidine uptake seen in Fig. 3Go may in fact be due to apoptosis. In addition, it is well established that WC1 stimulation leads to growth arrest (1, 2, 3), and yet it induces increases in cellular ceramide (see Fig. 1Go). Consequently, WC1 stimulation may induce expression of a factor that protects S-59 {gamma}{delta} T cells from ceramide-induced apoptosis or alternatively, these cells may already constitutively express such a factor. To address these questions, flow-cytometric analysis was used for identifying cellular apoptosis in conjunction with Western blotting for bcl-2 and bcl-X expression. As judged by propidium iodide staining of the DNA (Fig. 5Go), none of the ceramide analogues at a 50 µM concentration (Fig. 5Go, B–E) triggered a significant increase in apoptosis over the 24-h time period of the experiment when compared with cells in media only, showing that only 6% of cells had entered apoptosis (Fig. 5GoA). Similarly, and as expected, WC1 stimulation (20 µg/ml mAb SC-29) also failed to stimulate apoptosis, as again shown by only 6.2% of cells entering apoptosis (Fig. 5GoF), although increased apoptosis did occur in the staurosporine-treated positive control, as indicated by 80% of the cells entering apoptosis (Fig. 5GoG). In Fig. 6Go, Western blots for bcl-2 and bcl-X expression are shown. In particular, Western blotting for bcl-2 apparently detects two bands when S-59 cells were stimulated with anti-WC1. The basis of this is unclear; however, it is likely that they represent different phosphorylated forms of bcl-2, as bcl-2 has been shown to be a phosphorylated protein (29). It can be seen that S-59 cells, when grown in the presence of IL-2 only, express both bcl-2 and bcl-X. Treatment with 20 µg/ml SC-29 mAb (anti-WC1) produces a slight decrease in bcl-X, but an increase in bcl-2 expression. As a control, the S-59 cells were starved of IL-2 for 24 h, and this resulted in the abolition of bcl-X expression and a noticeable drop in bcl-2. Therefore, expression of the antiapoptotic factors bcl-2 and bcl-X in S-59 cells could explain why increases in ceramide through either WC1 stimulation or the addition of exogenous ceramides result in growth arrest and not apoptosis.



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  		FIGURE 5. Ceramide fails to induce apoptosis in S-59 {gamma}{delta} T cells. S-59 {gamma}{delta} T cells were either left untreated (A) or treated for 24 h with either 50 µM C2-dihydroceramide (B), 50 µM C2-ceramide (C), 50 µM C6-ceramide (D), 50 µM C8-ceramide (E), 20 µg/ml mAb SC-29 (anti-WC1) (F), or 0.1 µM staurosporine as a positive control (G). The cells were then analyzed by FACS after DNA staining with propidium iodide, as detailed in Materials and Methods. FL2-H, Fluorescence.

 


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  		FIGURE 6. WC1 stimulation increases bcl-2 expression in S-59 {gamma}{delta} T cells constitutively expressing bcl-2 and bcl-X in the presence of IL-2. S-59 {gamma}{delta} T cells grown in the presence of 10 U/ml IL-2 were either left untreated, incubated with 20 µg/ml mAb SC-29 (anti-WC1), or incubated in the absence of IL-2 for 24 h at 37°C. The proteins bcl-2 and bcl-X were then detected by Western blot, having resolved 10 µg of cell lysate per lane by 10% SDS-PAGE. This is a representative blot of the experiment repeated twice. Further details are described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The experiments described in this work provide evidence that ligation of WC1 triggers the generation of intracellular ceramide, and this results in G1 growth arrest in IL-2-dependent {gamma}{delta} T cells. The increase in ceramide was attributable to de novo ceramide synthesis by fumonisin B1-sensitive enzymes such as ceramide synthase. The increase in ceramide was selective as DAG levels failed to concomitantly rise. Thus, the ceramide synthase-specific inhibitor fumonisin B1 blocked WC1-induced growth arrest. Furthermore, ceramide synthase activation appeared to be proximal and downstream to WC1 stimulation, and indeed, addition of ceramide analogues mimicked WC1 stimulation by inducing growth arrest and not apoptosis. This correlation was confirmed at the molecular level, with both WC1 stimulation and the addition of ceramide analogues resulting in decreased expression of the pocket proteins pRb and p107, a biochemical change that was blocked by fumonisin B1. Failure of ceramide to induce apoptosis in these IL-2-dependent {gamma}{delta} T cells can be explained by the observation of constitutive expression of the antiapoptotic factors bcl-X and bcl-2 and, consistent with this, WC1 stimulation actually increased bcl-2 expression.

Ceramide is an important regulator of cell fate. Its pleiotropic biological effects have been reported to range from inducing apoptosis and growth arrest (30) to stimulation of cell differentiation (31). Our findings showing that ligation of the WC1 receptor induced increased cellular ceramide highlight the importance of the WC1 receptor in regulating {gamma}{delta} T cell biology. The mechanism through which this is achieved, however, is different from that observed with other receptors. In the case of stimulated receptors for TNF-{alpha}, IFN-{gamma}, and nerve growth factor, ceramide is produced through the action of sphingomyelinase on sphingomyelin, although recently there has been a report indicating that TNF-{alpha} can also stimulate ceramide synthase activity (32). In contrast, ligation of the WC1 receptor activated a fumonisin B1-sensitive enzyme ceramide synthase in the absence of any measurable changes in either acidic or neutral sphingomyelinase activity (data not shown), similar, but not identical, to that reported for daunorubicin-induced ceramide generation (10). Furthermore, the optimal concentration of fumonisin B1 (20 nM) used in our experiments was lower than that used in other studies, and it may well be that our {gamma}{delta} T cells are particularly sensitive to this inhibitor. Nevertheless, WC1-mediated increases in ceramide levels could be seen as early as 5 min post-WC1 ligation and were still steadily rising after 3 h (Fig. 1Go). This is in contrast to daunorubicin-induced ceramide generation, which was not apparent until after 4-h incubation with the drug (10). This suggests that different mechanisms of ceramide synthase activation may operate within these two systems. Increased ceramide synthase activity resulting from daunorubicin treatment was proposed to be as a result of de novo synthesis or posttranscriptional regulation located at the endoplasmic reticulum (33, 34). Our experiments with fumonisin B1 (Fig. 2Go), on the other hand, suggest that ceramide synthase activity is rapid in its onset and, as such, probably takes place at the plasma membrane. Addition of fumonisin B1 as little as 1 min before WC1 stimulation begins to reverse WC1-induced growth arrest, whereas addition of fumonisin B1 together (Fig. 2Go) with WC1 ligation did not reverse the anti-WC1-mediated growth arrest. The possibility that fumonisin B1 interferes with mAb SC-29 (anti-WC1) binding or it down-regulates WC1 cell surface expression was ruled out (see Table IGo). Activation of ceramide synthase distal to WC1 receptor stimulation is also an unlikely explanation as 1) ceramide generation was immediate in response to WC1 stimulation, and 2) fumonisin B1 addition together with WC1 stimulation did not reverse WC1-induced growth arrest. Therefore, while it may be concluded that WC1-induced fumonisin B1-sensitive ceramide synthase activity must be proximial to the WC1 receptor, it is possible that only the initial ceramide increase within the first several minutes is all that is necessary for triggering WC1-induced growth arrest. For example, the simultaneous addition of fumonisin B1 with anti-WC1 had little affect on WC1-induced growth arrest (see Fig. 2Go), as WC1 stimulation could have triggered enough ceramide generation before fumonisin B1 could have gotten to and then competetively inhibited any ceramide synthase activity. Indeed, 5-min pretreatment with fumonisin B1 still allowed WC1 stimulation to give a 60% reduction in thymide uptake, whereas by 30-min pretreatment this has been reduced to a 28% reduction in thymidine uptake. Moreover, WC1 activation could have many other pleiotropic effects on ceramide metabolism, stimulating ceramide generation through ceramide synthase activity as well as blocking ceramide-dependent catabolic pathways, such as ceramidases, thereby allowing ceramide levels to rise and to remain elavated. Therefore, any increases in ceramide induced by WC1 activation would remain, even after fumonisin B1 inhibition of ceramide synthesis. In support of such a hypothesis for WC1, a recent study has shown that a neutral ceramidase located within plasma membranes can catalyze both the hydrolysis and synthesis of ceramide (35). Consequently, WC1 stimulation could promote ceramide synthase activity while preventing ceramidase activity within the same enzyme, and therefore the ceramide synthase activity inhibited by fumonisin B1 in our experiments may also be a ceramidase, and we cannot exclude this possibility.

Ceramide can induce growth arrest through its affect on components of the cell cycle, namely the pocket proteins such as pRb (21). Moreover, as reported by us previously (3), growth arrest during G1 phase of the cell cycle is linked to dephosphorylation and reduced expression of the pocket proteins pRb and p107. Our experiments in this study showed that exogenous ceramide, like WC1 stimulation, reduced thymidine uptake (Fig. 3Go) and induced dephosphorylation as well as reduced expression for pRb and p107 (Fig. 4Go). Moreover, fumonisin B1 inhibited the effects of WC1 stimulation on these pocket proteins, indicating that increased ceramide levels through ceramide synthase activation by WC1 impact on the pocket proteins. These results are in agreement with a previous report indicating that pRb is a downstream target of ceramide (21). It is, however, still unclear which molecular mechanisms link ceramide accumulation to pRb dephosphorylation. One possible candidate may be a CAPP belonging to the PP2A family, which can regulate downstream targets of ceramide through serine/threonine dephosphorylation (36). However, it remains to be ascertained as to whether pRb is indeed a physiological substrate for CAPP. Other downstream signaling targets for ceramide include PKC-{zeta} (37), the stress-activated protein kinases, and the MAP kinases extracellular signal-related kinase 1 (ERK1) and ERK2 (28, 38). Unlike PKC-{zeta} and stress-activated protein kinases, which are both activated by ceramide (37, 38), Westwick et al. showed that MAP kinase activity was inactivated by ceramide in a concentration-dependent manner (38). This observation might therefore provide another link by which ceramide can influence pRb phosphorylation, as MAP kinase activation has been linked to pRb phosphorylation (39) through inactivation of the cyclin kinase inhibitor p27kip1 (40). Consequently, inactivation of MAP kinase by ceramide would allow active p27kip1 levels to rise, thereby inactivating cyclin kinase activity and leading to pRb dephosphorylation. This sequence of events might therefore provide a mechanism (see Fig. 7Go) by which the increases in ceramide reported in this work could be linked to our previously published observations that first, WC1 stimulation inactivates ERK2 activity through its dephosphorylation (2), and second, WC1 up-regulates p27kip1 expression with the resultant pRb dephosphorylation and growth arrest that occur (3).



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  		FIGURE 7. A putative mechanism for WC1 signaling {gamma}{delta} T cell growth arrest. MAPK, MAP kinase.

 
The effects of pocket protein dephosphorylation on cell cycle arrest are well established (27). However, apoptosis can still occur in the presence of pocket protein dephosphorylation. We have shown in this work that increases in ceramide through WC1 stimulation or by the addition of exogenous ceramide analogues in our {gamma}{delta} T cell system result in growth arrest. However, in other cell systems, ceramide induces apoptosis (10, 11, 12), presumably as a result of ceramide-induced activation of the proapoptotic protease prICE (caspase 3) (17). On the other hand, several factors have been shown to be protective against ceramide-induced apoptosis. To date, these include elevation of cellular DAG levels, PKC activation, and bcl-2 expression (13, 14, 15, 16, 30). The expression of bcl-2 being able to inhibit ceramide induced prICE (caspase 3) activation (17). In the absence of any significant increases in cellular DAG levels in WC1-stimulated {gamma}{delta} T cells (Fig. 1Go), other factors must be present to protect these cells from apoptosis. Consequently, we demonstrated that in the presence of IL-2, there was expression of both the antiapoptotic factors bcl-2 and bcl-X (Fig. 6Go). This would account for these cells entering growth arrest and not apoptosis upon the addition of short chain ceramide analogues (Figs. 5Go and 7Go). Moreover, WC1 stimulation actually increased bcl-2 expression levels, further enhancing the protection against apoptosis when in the presence of increasing proapoptotic ceramide levels. Interestingly, upon withdrawal of IL-2, these cells enter growth arrest (1), which corresponds to a loss of bcl-x and a reduction in bcl-2 expression, as shown in this work (Fig. 6Go). This is in contrast to other IL-2-dependent cell lines, in which, for example, depletion of IL-2 for 24 h from mouse TS1{alpha}ß cells results in reduced bcl-2 expression and apoptotic cell death (41).

In conclusion, we have shown that WC1 stimulation, through activation of a fumonisin B1-sensitive ceramide synthesis pathway, leads to increases in cellular ceramide, which in turn, results in decreased hyper- and hypophosphorylated pocket protein expression and subsequent G1 growth arrest. Moreover, these results are the first example, to our knowledge, of cell surface receptor-mediated ceramide generation by fumonisin B1-sensitive enzymes, such as ceramide synthase, and its implications in inducing cell cycle arrest.


   Acknowledgments
 
We thank Dr. Yusuf Hannun for his critical reading and helpful comments during the preparation of this manuscript.


   Footnotes
 
1 This research was supported by Biotechnology and Biological Sciences Research Council Grant AI 201/439. Back

2 Address correspondence and reprint requests to Dr. Paul A. Kirkham, Novartis Horsham Research Centre, Wimblehurst Road, Horsham, West Sussex, RH12 5AB. Back

3 Abbreviations used in this paper: DAG, diacylglycerol; CAPP, ceramide-activated protein phosphatase; ERK, extracellular signal-related kinase; MAP, mitogen-activated protein; PKC, protein kinase C; pRb, pocket protein retinoblastoma gene product. Back

Received for publication December 13, 1999. Accepted for publication July 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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