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Ganglioside GD3 enhances apoptosis by suppressing the nuclear factor-B-dependent survival pathway ¹

ANNA COLELL^*,2, CARMEN GARCÍA-RUIZ^*,2, JUAN ROMAN^*, ANTONIO BALLESTA and JOSÉ C. FERNÁNDEZ-CHECA^*3

^* Liver Unit, Instituto de Malalties Digestives,
Servicio de Bioquímica Clínica, Hospital Clinic i Provincial, and Instituto de Investigaciones Biomedicas August Pi Suñer, Consejo Superior de Investigaciones Científicas, Barcelona, 08036, Spain

³Correspondence: Liver Unit, Hospital Clinic i Provincial Villarroel, 170, 08036-Barcelona, Spain. E-mail: checa{at}medicina.ub.es

SPECIFIC AIM

Glycosphingolipidshave emerged as cell death effectors because of their role in apoptosis signaling. Because cell survival reflects a balance between death and survival pathways and because the nuclear factor-B (NF-B) is known to downregulate apoptosis in response to diverse stimuli, our study examined the role of ganglioside GD3 (GD3) and related structural analogs, including short-chain ceramide analogs—for example, ceramide C2 (C2)—on NF-B regulation, mitochondrial reactive oxygen species (ROS) generation, and survival of cultured rat hepatocytes.

PRINCIPAL FINDINGS

1. Mitochondrial ROS formation is common for both C2 and GD3
Incubation of hepatocytes with C2 and GD3 (0–50 µM) resulted in a similar dose-dependent peroxide formation. It is interesting that, despite this, hepatocytes displayed a selective susceptibility to GD3 treatment, which was reflected by a decreased survival (36±5% vs. 92±7% for GD3 and C2 at 50 µM for 12 h), which was accompanied by apoptotic features. Inhibitors of mitochondrial electron flow at complexes I and II prevented both peroxide formation and the GD3-induced loss of cell viability, which highlights the relevance of the complex III-induced ROS generation in the killing of hepatocytes by GD3. Other cell-permeable, short-chain ceramides, N-hexanoyl (C6)- or N-octanoyl(C8)-sphingosine, were equally effective as C2 in this event. Furthermore, various GD3 analogs, including GluCer, LactCer, and GM1, mimicked the stimulating effect of GD3 on ROS formation; whereas Lyso-GluCer was ineffective, which indicates the requirement of fatty acids for this event. Thus, the N-fatty acyl-sphingosine moiety determines the structural requirement of C2 and GD3 for mitochondrial stimulation of ROS generation, whereas the presence of sugar residues is dispensable for this event.

2. NF-B inactivation is a selective feature of GD3
Because transcription factor NF-B is known to activate survival pathways, we examined the activation of NF-B in nuclear extracts from hepatocytes incubated with C2 and GD3. Whereas C2 dose-dependently enhanced the DNA binding of NF-B, which consisted of p65/p50 and p50/50 dimers, in contrast, the same dose range of GD3 was ineffective (Fig. 1A ). Furthermore, the activating effect of C2 on NF-B was accompanied by proteolytic degradation of IB-. Unexpectedly, however, the levels of IB- from GD3-treated cells were as low as those of C2-treated hepatocytes, which indicates that both C2 and GD3 signaled the degradation of IB-. Moreover, whereas short-chain ceramide analogs (C2 to C8) enhanced the DNA binding of NF-B to a similar extent, the repressing effect of GD3 on NF-B activation was also observed with GluCer, LactCer, GM1, and Lyso-GluCer. Thus, these findings indicate that the presence of sugar residues in the backbone of ceramide is necessary for the failure in activating NF-B, whereas the presence of fatty acids does not modulate this process.

View larger version (68K): [in this window] [in a new window]   Figure 1. GD3 blocks the nuclear translocation of active B members. A) NF-B activation in nuclear and cytosolic fractions from C2 or GD3 (1 µM each) treated hepatocytes. B) Confocal immunofluorescence distribution of NF-B p65 in response to C2 and GD3 (1 µM).

3. GD3 prevents the nuclear translocation of NF-B
Because GD3 failed to activate NF-B in nuclear extracts, despite IB- degradation, we tested whether active NF-B complexes remained in the cytosol. In contrast to C2, which increased NF-B DNA binding in nuclear extracts, the enhanced DNA binding of NF-B induced by GD3 was detected only in cytosolic fractions (Fig. 1A ). Furthermore, confocal microscopic analyses using antibody anti p65 revealed a diffused fluorescence in the cytosol of GD3-treated cells; whereas, in response to C2, p65 was translocated to the nuclei where it could bind to specific DNA regions (Fig. 1B ). Moreover, preincuation of cells with GD3 abolished the C2-induced nuclear translocation of p65 (Fig. 1B ). These data demonstrate that competent DNA-binding NF-B complexes stimulated by GD3 are retained in the cytosol and are unable to be translocated to the nuclei, which suggests that GD3 acts at a late, critical step involved in the nuclear translocation of NF-B members.

4. GD3 blocks B-dependent gene expression and sensitizes hepatocytes against apoptosis stimuli
To examine whether the divergence of NF-B regulation by C2 and GD3 is reflected in the B-controlled gene expression, we used a luciferase reporter gene construct controlled by four B binding sites. Although both C2 and TNF- stimulated luciferase expression and their combination resulted in an additive induction, GD3 was ineffective in this process (Fig. 2A ). Furthermore, preincubation (1 h) of hepatocytes with GD3 blocked the TNF-inducible B-dependent gene regulation, whereas the addition of GD3 following that of TNF- did not prevent luciferase expression (Fig. 2A ). Furthermore, the survival of hepatocytes was determined by following treatment with a sublethal dose of GD3 in the presence or absence of TNF-. GD3 (1 µM) or TNF- did not cause cell killing, which is consistent with previous reports. However, the preincubation (1 h) with GD3 (1 µM) sensitized hepatocytes to TNF- treatment (Fig. 2B ). Moreover, GluCer and LactCer mimicked the ability of GD3 to abolish the TNF--induced luciferase expression, and, consequently, these analogs unmasked the apoptotic potential of TNF-, thus sensitizing hepatocytes to TNF-.

View larger version (55K): [in this window] [in a new window]   Figure 2. A) NF-B-controlled luciferase expression and survival of hepatocytes. Hepatocytes were transfected with the reported plasmid pNF-B-Luc along with PSV-g-galactosidase as control to monitor transfection efficiency, followed by incubation with C2 (1 µM), GD3 (1 µM) with or without TNF- (36 ng/ml) for 6 h. Alternatively, luciferase expression was determined after preincubation with GD3 for 1 h (open bar). B) Survival of hepatocytes in response to C2 and GD3 (1 µM each) with or without the combination of TNF- was determined at 6 h of incubation. Results are the mean±five different experiments. *P < 0.05 versus control.

CONCLUSIONS AND SIGNIFICANCE

Our study has compared the role of sphingolipids (e.g, short-chain ceramides) versus glycosphingolipids (e.g., gangliosides) in NF-B regulation and survival of cultured hepatocytes. Both C2 and GD3 act commonly at the mitochondrial level and stimulate a burst of ROS formation, which, as demonstrated in previous studies, initiates a cell death cascade. Yet hepatocytes were killed only by GD3, which suggests the involvement of a survival factor that counteracted the induced ROS formation and downstream death effectors. Consistent with this hypothesis are our observations that NF-B is selectively inactivated by GD3, as opposed to short-chain ceramides (C2-C8), which renders hepatocytes unable to induce B-dependent gene expression, including the upregulation of critical survival factors. Hence, the present findings provide evidence that the efficiency of GD3 in promoting hepatocyte cell death involves the lethal combination of mitochondrial ROS generation followed by the subsequent release of apoptotic factors and the suppression of NF-B-dependent survival pathway.

The biosphysical properties of the exogenous, short-chain ceramide analogs differ from endogenous or natural ceramides, hence limiting the interpretation and extrapolation of our findings described with the former as to the NF-B regulation and cell survival. Furthermore, natural ceramide can be generated in cells by the activation of different sphingomyelinases that display distinct pH optima located in distinct cellular sites, which release ceramide pools in specific cell locations. However, it has been reported that acidic sphingomyelinase is necessary for GD3 accumulation and efficient apoptosis of lymphoid cells caused by Fas ligand and that acid pH-optimum sphingomyelinase is required in the CD95-mediated apoptosis in vivo.. Our reports that cultured rat hepatocytes are sensitive to the treatment with exogenous acidic sphingomyelinase but resistant to neutral pH-optimum bacillus cereus sphingomyelinase are in line with these observations. Furthermore, the pharmacological inhibition of glucosylceramide synthetase, the enzyme responsible for GluCer synthesis, which is the early precursor in the synthesis of complex glycosphingolipids, downregulates the levels of GD3 and prevents the acidic sphingomyelinase-induced ROS formation and hepatocyte apoptosis. Consistent with this selective cytotoxicity of acidic sphingomyelinase, this enzyme does not signal NF-B activation because it has been reported that TNF- activates NF-B in embryonic fibroblasts from acidic sphingomyelinase-deficient mice. In contrast, the neutral,Mg ²⁺-dependent sphingomyelinase has been reported to activate NF-B in various cell types. Thus, based on these considerations, the present findings suggest a functional link between endogenous ceramide produced specifically by acidic sphingomyelinase that serves as precursor for ganglioside synthesis (e.g., GD3) and that the dual combination of ROS formation from mitochondria and suppression of the NF-B-dependent survival pathway featured by GD3 works very efficiently in the induction of cell death (Fig. 3 ).

View larger version (31K): [in this window] [in a new window]   Figure 3. Schematic diagram depicts the bifunctional mechanism whereby GD3 elicits apoptosis in hepatocytes. GD3 synthesized from ceramide released by the acidic sphingomyelinase exerts a dual mechanism in cell killing, which combines the mitochondrial-dependent apoptosome activation and the suppression of the NF-B-dependent survival pathway.

In conclusion, our work defines a novel role of gangliosides in apoptosis, which act as repressors of B-dependent gene expression by preventing the translocation of active B complexes to the nuclei. This feature, combined with their ability to recruit a mitochondrial-dependent caspase activation, leads to hepatocyte cell death in the absence of any other sensitizing factor.

Our results illustrate the potential therapeutic use of this class of lipid effectors in conditions aimed to overcome the resistance to apoptosis, such as in cancer therapy. However, although previous studies with the adenoviral delivery of a mutant IB- at serine residues have noted that, to prevent IB- degradation and consequently NF-B activation, one must consider that the dissociation of NF-B from its inhibitory moiety, IB, may occur in the absence of IB degradation following its phosphorylation at tyrosine residues. Even in this event, glycosphingolipids (e.g., GD3) may still block NF-B-dependent induction of survival genes as they prevent the nuclear translocation of active NF-B complexes.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0574fje ; to cite this article, use (February 5, 2001) FASEB J. 10.1096/fj.00-0574fje

² These authors contributed equally to the work.

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