HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rouquette-Jazdanian, A. K. Articles by Aussel, C. Search for Related Content PubMed PubMed Citation Articles by Rouquette-Jazdanian, A. K. Articles by Aussel, C.

Cholera Toxin B-Subunit Prevents Activation and Proliferation of Human CD4⁺ T Cells by Activation of a Neutral Sphingomyelinase in Lipid Rafts¹

Alexandre K. Rouquette-Jazdanian^*, Arnaud Foussat^*, Laurence Lamy^*, Claudette Pelassy^*, Patricia Lagadec, Jean-Philippe Breittmayer^* and Claude Aussel^2,*

^* Institut National de la Santé et de la Recherche Médicale (INSERM) Unit 576, IFR 50, Hôpital de l’Archet I, Nice Cedex 3, France; and INSERM Unit 526, Activation des Cellules Hématopoïétiques, Physiologie de la Survie et de la Mort Cellulaires et Infections Virales, IFR 50 Génétique et Signalisation Moléculaires, Faculté de Médecine Pasteur, Nice Cedex 2, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The inhibition of human CD4⁺ T lymphocyte activation and proliferation by cholera toxin B-subunit (CTB) is a well-established phenomenon; nevertheless, the exact mechanism remained unclear. In the present study, we propose an explanation for the rCTB-induced inhibition of CD4⁺ T lymphocytes. rCTB specifically binds to GM1, a raft marker, and strongly modifies the lipid composition of rafts. First, rCTB inhibits sphingomyelin synthesis; second, it enhances phosphatidylcholine synthesis; and third, it activates a raft-resident neutral sphingomyelinase resembling to neutral sphingomyelinase type 1, thus generating a transient ceramide production. We demonstrated that these ceramides inhibit protein kinase C phosphorylation and its translocation into the modified lipid rafts. Furthermore, we show that rCTB-induced ceramide production activate NF-B. Combined all together: raft modification in terms of lipids, ceramide production, protein kinase C inhibition, and NF-B activation lead to CD4⁺ T cell inhibition.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Cholera toxin B-subunit (CTB)³, produced by the bacterium Vibrio cholerae, exerts profound immunomodulatory effects on blood cell populations in vitro. CTB triggers the polyclonal activation of B cells. This activation occurs in the absence of significant proliferation and involves the up-regulation of a number of important molecules, namely MHC class II (Ia), B7, CD40, ICAM-1, and IL-2R (CD25) (1, 2). CTB induces selective apoptosis of T CD8⁺ cells. This apoptosis is preceded by enhanced expression of CD25 and is independent of Fas (CD95) or TNF-. It involves a NF-B-dependent and caspase-3-dependent pathway (3). A similar effect is exerted by CTB in vivo where oral administration of CTB leads to a demonstrable depletion of CD8⁺ T cells from both the Peyer’s patch and intraepithelial lymphocyte compartments (4). Heat-labile enterotoxin B-subunit (ETB) from Escherichia coli, which is a close homologue of CTB, induces the release of IL-10, IL-6, and TNF- by human monocytes (5). CTB modulates Ag processing and presentation by macrophages (6, 7, 8). CTB inhibits CD3- and PMA/ionomycin-induced murine T cell proliferation (9, 10). Such proliferation is inhibited even if CTB is added hours after the start of culture (10). CTB also inhibits the proliferation of 2.10 cells (a human IL-2-dependent, CD4^– T cell clone) in response to IL-2 produced endogenously on stimulation with anti-TCR or provided exogenously (11). ETB induces nuclear translocation of NF-B in Jurkat cells (12). To conclude, CTB displays pleiotropic effects on human PBMCs (hPBMCs), it inhibits CD4⁺ T cell activation and proliferation, but the exact mechanism remained unclear.

In this study, our results explain the inhibitory effect of rCTB both on the activation and on the proliferation of human CD4⁺ T lymphocytes. rCTB specifically binds to the monosialoganglioside GM1, a raft marker, and strongly alters the lipid composition of rafts of CD4⁺ T lymphocytes. First, rCTB inhibits sphingomyelin (SM) synthesis, secondly it enhances phosphatidylcholine (PtdCho) synthesis, and thirdly it activates a raft-resident neutral sphingomyelinase (SMase) resembling to NSM1, thus generating a transient ceramide production. We demonstrated that these ceramides inhibit protein kinase C (PKC) phosphorylation and its translocation into the modified lipid rafts. We also demonstrated that rCTB as ETB activates the NF-B transcription factor. Furthermore, we also show that rCTB induces NF-B activation via the production of ceramides. Combined all together, raft modification in terms of lipids, ceramide production, PKC inhibition, and NF-B activation lead to CD4⁺ T cell inhibition.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Cells

Citrate anticoagulated venous blood samples of healthy adult volunteers and buffy coats collected from normal donors by the Etablissement Français du Sang were obtained according to institutional guidelines. hPBMCs were isolated from either blood samples or buffy coats by centrifugation on a Ficoll-Hypaque density-gradient (1.077 g/ml). Interface PBMCs were pelleted, washed, and cultured in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FCS, 50 U/ml penicillin G sodium, 50 µg/ml streptomycin sulfate, 2 mM L-glutamine, 1 mM sodium pyruvate, 20 mM HEPES, and 1x minimal essential medium nonessential amino acid solution (Invitrogen Life Technologies). The human leukemic T cell line Jurkat was obtained from American Type Culture Collection. Cells were cloned by limiting dilution. Clone D was selected on the basis of its IL-2 production when activated with PHA and the phorbolester 12-O-tetradecanoylphorbol-13-acetate. Jurkat clone D were grown in the same medium as PBMCs. Cells were maintained at densities between 8 x 10⁵ and 1 x 10⁶/ml in a humidified incubator under 5% CO₂ (Heraeus).

Abs, reagents, and radioactive products

rCTB was a kind gift of Dr. F. Anjuère and Prof. C. Czerkinsky (U721, Nice, France). rCTB was produced in a mutant strain of Vibrio cholerae 01 deleted of its CT genes and transformed with a multicopy plasmid encoding CTB. The rCTB used was purified from the culture medium by a combination of salt precipitation and chromatographic methods, as previously described (13), and has been already used in several studies (14, 15, 16). Rabbit polyclonal Ab anti-GM1, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, HCl (DL-threo-PDMP, hydrochloride/PDMP), and fumonisin B1 (FB1) from Fusarium moniliforme were purchased from Calbiochem. mAb anti-PKC (clone M4, IgG1), rabbit polyclonal Ab anti-phospho-PKC (Ser⁶⁵⁷) (IgG), rabbit polyclonal Ab anti-p56Lck (IgG), and rabbit polyclonal Ab anti-LAT (IgG) were obtained from Upstate Biotechnology. Rabbit polyclonal Ab anti-PLC1 (sc-81) was purchased from Santa Cruz Biotechnology. Anti-CD3 mAb (clone X3, IgG2a) and anti-CD28 mAb (clone 28.2, IgG1) were produced in our laboratory. CD4-PerCP (clone SK3, IgG1), CD69-FITC (clone FN50, IgG1k), CD25-PE (clone M-A251, IgG1k), and apoptosis detection kit (annexin V-PE, 7-aminoactinomycin D (7-AAD)) were purchased from BD Pharmingen. Peroxidase-labeled anti-rabbit IgG and rabbit anti-mouse IgG coupled to peroxidase were obtained from Rockland, and R-PE-Cy5-conjugated streptavidin was obtained from DakoCytomation. Methyl--cyclodextrin (m--CD), CTB, biotin-labeled CTB, monosialoganglioside-GM1, N-acetyl-L-cysteine (NAC), glutathione (GSH), cGMP, PMA, ionomycin, pepstatin, leupeptin, chymostatin, and anti-PMSF were purchased from Sigma-Aldrich. ₂-macroglobulin was purchased from Roche. 1,2(n)-[³H]cholesterol (1.3–1.85 TBq/mmol), methyl-[³H]choline chloride (2.22–3.14 TBq/mmol), 9,10(n)-[³H]palmitic acid (37 MBq/mmol), [methyl-³H]thymidine (740 GBq/mmol), [-³²P]ATP, and [N-methyl-¹⁴C]SM (2.04 Gbq/mmol, 55 mCi/mmol) were purchased from Amersham Biosciences.

In vitro T lymphocyte proliferative responses

Positive selection of CD4⁺ T lymphocytes from freshly isolated hPBMCs was first performed using a fluorescence activated cell sorter (FACStar⁺; BD Biosciences). Reanalysis of the sorted population showed a purity higher than 98%. Purified CD4⁺ T lymphocytes were extensively washed then resuspended in prewarmed culture medium at a cellular concentration of 1 x 10⁶/ml. Cell suspension was cultured in triplicate sets in flat-bottom 96-well plates in the volume of 200 µl/well. Cells pretreated or not with either rCTB (10 µg/ml) and/or others reagents (as detailed in the figure legends) were stimulated or not by either PMA (10 ng/ml) plus ionomycin (100 nM) or soluble anti-CD3 mAb (5 µg/ml) plus anti-CD28 mAb (5 µg/ml) for 72 h. The cultures were pulsed with 1 µCi/well [³H]thymidine during the last 16 h. Cells were harvested with a semiautomatic cell harvester (Skatron Instruments) onto glass fiber filter paper. Filters were dried and counted by liquid scintillation in a Beckman Tricarb scintillation spectrometer. Results are expressed as mean cpm ± SEM of triplicate cultures.

Cell surface receptors staining

hPBMCs or Jurkat cells (1 x 10⁶) were washed in cold PBS supplemented with 0.1% BSA (pH 7.4) then incubated for 30 min in the dark at 4°C with the appropriate fluorochrome-conjugated mAb according to the manufacturer’s instructions. For GM1 indirect-staining, Jurkat cells were first incubated with biotin-labeled CTB (10 µg/ml). Then cells were washed and incubated (30 min at 4°C) in 100 µl of 1/25 dilution of RPE-Cy5-conjugated streptavidin. Cells were washed again and fixed with 0.37% paraformaldehyde.

Cytometric analysis of T cell activation markers

Freshly isolated hPBMCs were washed then resuspended in prewarmed culture medium at a cellular concentration of 1 x 10⁶/ml. Cell suspension was dispensed in triplicate sets into flat-bottom 96-well plates in the volume of 200 µl/well. hPBMCs pretreated or not with the indicated drugs (as detailed in the figure legends) were stimulated or not by soluble anti-CD3 mAb (5 µg/ml) plus anti-CD28 mAb (5 µg/ml) for 20 h. Then, cells were costained with a PE-CD25 mAb, a FITC-CD69 mAb, and a PerCP-CD4 mAb. hPBMCs were gated on lymphocytes according to their forward and side angle light scatter. CD25 and CD69 surface expression on CD4⁺ T lymphocytes was determined flow cytometry after gating lymphocytes on the basis of membrane expression of CD4. CD25 and CD69 up-regulation was also examined on Jurkat cells pretreated or not with the indicated drugs (as detailed in the figure legends) then stimulated or not with PMA (10 ng/ml) plus ionomycin (100 nM) for 20 h. The mean fluorescence intensity of 5000 cells was determined by flow cytometry (FACScan; BD Biosciences).

Viability measurement of treated cells

hPBMCs, treated or not with rCTB (10 µg/ml) for 72 h, were stained with a FITC-CD4 mAb. Then cells were incubated with both annexin-PE and 7-AAD according to the manufacturer’s specifications. 7-AAD can be excited by the 488-nm argon laser line and emits in the far red range of the spectrum; consequently, its spectral emission can be separated from the emissions of FITC and PE. The fluorescence parameters allow characterization of necrotic cells (annexin-PE⁺/7-AAD⁺), apoptotic cells (annexin-PE⁺/7-AAD^–), and viable cells (annexin-PE^–/7-AAD^–) in the chosen subset of FITC⁺ cells. Moreover, viability of FB1- and PDMP-treated Jurkat cells was also compared with control Jurkat using the same technique.

Lipid rafts isolation

Raft isolation was accomplished using a combination of published protocols (17, 18). After radioactive labeling and/or pretreatment with rCTB (10 µg/ml), Jurkat cells (80–100 x 10⁶) were sonicated gently with a Vibracell sonicator (five bursts of 5 s, 5W; Bioblock Scientific) in 1 ml of ice-cold buffer (25 mM HEPES, 150 mM NaCl, 5 mM EDTA, 10 mM sodium pyrophosphate, 5 mM Na₃VO₄, and 10 mM NaF) supplemented with a mixture of protease inhibitors (1 mM -PMSF, 100 U/ml aprotinin,1 mg/ml leupeptin, 1 mg/ml pepstatin, 2 mg/ml chymostatin, and 5 mg/ml ₂-macroglobulin) and centrifuged at 800 g_av at 2°C for 10 min to remove nuclei and large debris. The resulting supernatant called postnuclear supernatant (PNS) was incubated with 0.5% Triton X-100 (PEG(9, 10)-octylphenyl ether) for 30 min at 2°C. The lysate was then adjusted to 1.33 M sucrose by the addition of 2 ml of 2 M sucrose and placed at the bottom of an ultracentrifuge tube (Ultra-Clear; Beckman Instruments). A step sucrose gradient (0.2–0.9 M with 0.1 M steps, 1 ml each) was placed on top. The weight percentage of sucrose was checked at room temperature using an Abbe-3L refractometer (Bioblock Scientific). The tubes were centrifuged at 38 000,rpm (250,000 x g, using the radial distance maximal (r_max: 158.8 mm) for conversion with normogram) for 16–18 h (L8-70M Ultracentrifuge; Beckman Instruments) in a SW41Ti rotor (Beckman Instruments) at 2°C. One-milliliter fractions were harvested from the top. Rafts were recovered from the low-density fractions 2 and 3 while the heavy/H fractions (soluble material) were recovered from the high-density fractions 8 and 9 at the bottom of the ultracentrifuge tube.

Immunoblot analysis

Aliquots (50 µl) of each sucrose density-gradient fraction were solubilized in 50 µl of 2x Hoessli buffer (150 mM Tris-HCl (pH 8.5), 20% glycerol, 5 mM EDTA, 5% SDS, and 10% 2-ME) and then resolved by 10% SDS-PAGE under reducing conditions, and proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore). Membranes were blocked for 2 h at room temperature in a blocking buffer containing 5% (w/v) nonfat dry milk in TBS (10 mM Tris-HCl and 140 mM NaCl (pH 7.4)) and then incubated for 1 h with the appropriate Ab diluted 1000-fold in the same buffer. The membranes were washed extensively in TBS containing 0.4% (v/v) Tween 20. Detection was performed with HRP-conjugated anti-rabbit or anti-mouse and ECL reagents (Amersham Biosciences) according to manufacturer’s instructions.

For phospho-PKC immunoblotting, aliquots of fraction B (fraction 2 + 3) solubilized in the same volume of 2x Hoessli buffer were loaded on 10% SDS-PAGE and then transferred to PVDF membranes as described above. Membranes were blocked for 2 h at room temperature in a blocking buffer containing 5% (w/v) BSA in TBS and subsequently incubated with rabbit polyclonal Ab anti-phospho-PKC (Ser⁶⁵⁷) for 2 h at room temperature. Phospho-PKC signals were detected with HRP-conjugated goat anti-rabbit, followed by ECL. Membranes were then stripped in a buffer (pH 6.7) containing 62.5 mM Tris-HCl, 2% SDS, and 0.7% 2-ME and reblotted with Ab against PKC.

Sphingolipid content manipulation

To reduce cellular sphingolipid content, Jurkat cells were cultured as control cells in a medium supplemented with either FB1 (10 µM final concentration) or PDMP (10 µM final concentration) for 4 days; FB1 and PDMP were included in each medium change. Inhibition of sphingolipid synthesis was monitored by analyzing surface expression of GM1 by flow cytometry. GM1 replenishment of sphingolipid-depleted Jurkat cells was conducted by incubating cells in serum-free RPMI 1640 containing GM1 (0.5 mg/ml for 30 min) at 37°C.

Glycerophospholipids and SM content analysis

Jurkat cells were washed then incubated for 16–18 h in a HEPES saline buffer (HSB) (pH 7.4), containing 137 mM NaCl, 2.7 mM KCl, 1 mM Na₂HPO₄, 12 H₂O, 2.5 mM glucose, 20 mM HEPES, 5 mM MgCl₂, 1 mM CaCl₂, and 0.1% BSA at 37°C in the presence of 4 µCi of either [³H]palmitic acid or [³H]choline chloride. Lipids were extracted and analyzed from either whole cells (a) or fractions obtained after ultracentrifugation (b).

(a) rCTB-treated cells or control cells were rapidly sedimented, supernatants were discarded, and cell lipids were extracted with chloroform/methanol according to Bligh and Dyer (19) then separated by monodimensional thin-layer chromatography on plates LK6D Silica Gel 60 A (Whatman) in a solvent system composed of chloroform/methanol/acetic acid/water (75/45/12/3). Authentic phospholipid standards (Sigma-Aldrich) were run in parallel and detected with iodide vapors. Radioactivity in lipid spots was determined by using an automatic linear radiochromatography analyzer, Tracemaster 20 (Berthold), equipped with an 8-mm window and the integration software supplied by the manufacturer.

(b) An aliquot (50 µl) of each different fraction obtained after ultracentrifugation on sucrose density-gradient were extracted and analyzed as described above.

SM synthesis measurement

Jurkat cells (2 x 10⁶) were maintained in 500 µl of HSB. At time 0, 4 µCi of either [³H]palmitic acid or [³H]choline chloride were added, with or without rCTB, at the end of the treatment, and lipids were extracted and analyzed as described above.

Cholesterol analysis

[³H]Cholesterol in toluene solution was first evaporated under N₂ and dissolved in ethanol just prior its use. Jurkat cells were washed then incubated for 16–18 h in HBS containing 4 µCi of [³H]cholesterol. Raft purification was performed as detailed above. To determine the distribution of [³H]cholesterol, an aliquot (50 µl) of each different fraction obtained after ultracentrifugation on sucrose density-gradient was mixed with Picofluor and counted by liquid scintillation in a Beckman Tricarb scintillation spectrometer.

Assays for neutral- and acidic-SMase

The activity of neutral- and acidic-SMase was calculated by using a combination of published protocols (20, 21, 22). To prepare a stock solution of 50 µM radioactive SM substrate, 55 µl (1375 µCi, 25 nmol) of [N-methyl-¹⁴C]SM (55 mCi/mmol, 10 µCi/400 µl in toluene/ethanol, 1/1, v/v) were placed in a glass tube, and the organic solvent was removed under N₂. The dried [¹⁴C]SM was solubilized in 500 µl of 1% (w/v) -octylglucoside by brief sonication with a bath-type sonicator. Fifty-microliter aliquots of selected fractions (2 + 3 and 8 + 9) were assayed for the presence of different SMase activities. Reactions were started by adding 50 µl of substrate solution. For the measurement of the neutral-SMase activity, this solution consisted in 10 µl of the stock solution of [¹⁴C]SM (0.5 nmol), 10 µl of a buffer consisting in 250 mM HEPES (pH 7.5), 50 mM MgCl₂, and 0.5% (v/v) Triton X-100 and 30 µl of deionized water. After incubation at 37°C for 3 h, the reaction was stopped by adding 800 µl of chloroform/methanol (2/1, v/v) and 200 µl of deionized water. A 100-µl aliquot of the aqueous upper phase containing [¹⁴C]phosphorylcholine released from [N-methyl-¹⁴C]SM was collected and counted by liquid scintillation. The reaction was linear within this frame, and the amount of [N-methyl-¹⁴C]SM hydrolyzed during an assay did not exceed 10% of the total amount of radioactive SM added. For calculation of the specific activities, values were corrected for volume of the aqueous phase, volume of the sample, protein content, reaction time, and specific activity of the substrate. For the assay of acidic-SMase activity, the substrate solution consisted in 10 µl of the stock solution of [¹⁴C]SM (0.5 nmol) and 10 µl of 0.5 M sodium acetate buffer (pH 4.8), consisting of 10 mM EDTA, 0.5% (v/v) Triton X-100, and 30 µl of deionized water. The initiation and termination of the reaction and the determination of the water-soluble radioactivity released from [N-methyl-¹⁴C]SM was proceed as described above.

Diacylglycerol (DAG) kinase assays

Total cellular ceramide levels were quantified by the DAG kinase assay as ³²P incorporated upon phosphorylation of ceramide to ceramide-1-phosphate (C-1-P) by DAG kinase from Escherichia coli (23). After rCTB treatment (10 µg/ml) for different period of time, Jurkat cells (5 x 10⁶) were washed twice with ice-cold PBS. After centrifugation (1000 x g, 5 min, 4°C), lipids were extracted with 1 ml of chloroform/methanol/hydrochloric acid (1 N) (100/100/1, v/v/v), 170 µl of buffered saline solution (135 mM NaCl, 4.5 mM KCl, 1.5 mM CaCl₂, 0.5 mM MgCl₂, 5.5 mM glucose, and 10 mM HEPES (pH 7.2)), and 30 µl of 100 mM EDTA. The lipids of the organic phase were transferred to a new glass tube and dried under a stream of N₂. Lipid extracts were then subjected to mild alkaline hydrolysis (0.1 M KOH in methanol for 1 h at 37°C) to remove glycerophospholipids. Five hundred microliters of chloroform, 270 µl of buffered saline solution, and 30 µl of 100 mM EDTA were added. After drying the organic phase with N₂, in vitro phosphorylation of extracted ceramides was performed as described by the manufacturer (RPN 200 kit; Amersham Biosciences). A total of 1 µCi of [-³²P]ATP (4000 Ci/mmol) was used to start the reaction. After 30 min at room temperature, the reaction was stopped by extraction of lipids with 1 ml of chloroform/methanol/hydrochloric acid (1 N) (100/100/1, v/v/v), 170 µl of buffered saline solution, and 30 µl of 100 mM EDTA. The lower organic phase was dried under N₂. The samples were resuspended in 30 µl of chloroform/methanol (95/5, v/v) and spotted on plates LK6D Silica Gel 60 A. C-1-P was resolved by TLC using chloroform/methanol/acetic acid (75/25/5, v/v/v) as solvent and migrated as a single spot at R_F = 0.25. Linearity of the assay was established using purified C₁₆-ceramide (Sigma-Aldrich). Radioactivity in lipid spots was determined by using an automatic linear radiochromatography analyzer, Tracemaster 20 (Berthold), equipped with an 8-mm window and the integration software supplied by the manufacturer.

Semiquantitative RT-PCR

After cell treatment, total RNA was isolated from Jurkat cells using TRIzol Reagent (Invitrogen Life Technologies) based on method derived by Chomczynski and Sacchi (24). RNA (150 ng) was then reverse transcribed using the SuperScript II RNAase H^– reverse transcriptase (Invitrogen Life Technologies) following the manufacturer’s instructions and resuspended in 150 µl final volume. cDNAs (5 µl) or water as control were amplified by PCR in a final volume of 25 µl using the Platinum TaqDNA Polymerase (Invitrogen Life Technologies) and 300 nM of forward and reverse primers. RT-PCR was typically performed for 35 cycles (denaturation at 95°C for 20 s, annealing at 68°C for 1 min, extension at 72°C for 1 min). Primers were designed using the PRIMER Express Software 1.5 (Applied Biosystems). The following 5' and 3' primers were as follows: human CD69, 5' primer (5'-CGTAGCAGAGAACAGCTCTTTGC-3') and 3' primer (5'-ACAGGACAGGAACTTGGAAGGA-3'), and human CD25, 5' primer (5'-GGGACTGCTCACGTTCATCA-3') and 3' primer (5'-TTCAACATGGTTCCTTCCTTGTAG-3'). -actin was used as loading control.

EMSAs

Total cellular extracts were prepared in Totex lysis buffer (20 mM HEPES (pH 7.9), 350 mM NaCl, 20% glycerol, 1% Nonidet P-40, 1 mM MgCl₂, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, and 10 µg/ml aprotinin). Supernatants from a 15,000 x g, 10 min centrifugation at 4°C were collected. The NF-B probe used for mobility shift assay was constituted of a synthetic double-stranded oligonucleotide containing the NF-B site of the Ig promoter (5'-GATCCAAGGGACTTTCCATG-3'). The ³²P-end-labeled probe (T4 kinase) was incubated with Totex extract samples (20 µg) for 25 min at room temperature. Complexes were then separated by electrophoresis on a 5% nondenaturating polyacrylamide gel in 0.5 x Tris-borate EDTA. Dried gels were subjected to autoradiography.

Luciferase assays

Jurkat cells were transiently transfected by electroporation (320 V, 960 µF) with 10 µg of a luciferase reporter gene controlled by a minimal thymidine kinase promoter and six reiterated B sites (Bx6 thymidine kinase luc). At 36 h after transfection, cells were stimulated as indicated. Cells were washed twice in PBS (pH 7.2) and lysed in 100 µl of reporter lysis buffer (Promega). Luciferase activity was assayed by luminometry (Lumat; EG&G Berthold) using the Promega luciferase assay system. Normalization of transfection efficiency was done using a cotransfected -galactosidase expression vector. Luciferase activity was determined in triplicate and expressed as fold increase relative to basal activity seen in untreated unstimulated mock-transfected cells.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
GM1-rCTB interaction but not GM1-anti-GM1 Abs interaction inhibits PMA/ionomycin-induced CD4⁺ T proliferation

Previous works have demonstrated that CTB is able to inhibit T cell activation and proliferation induced by either polyclonal mitogens or by specific Ags (9, 10, 11). To see whether GM1 binding alone is sufficient or not to inhibit PMA/ionomycin-induced CD4⁺ T proliferation, CD4⁺ T lymphocytes were pretreated with various concentrations of either purified CTB (pCTB), rCTB, or rabbit polyclonal anti-GM1 Abs, then cells were stimulated with PMA/ionomycin, and proliferation was measured. Fig. 1A unambiguously demonstrates that PMA/ionomycin-induced CD4⁺ T cell proliferation is inhibited by both pCTB and rCTB in a dose-dependent manner. Our result unambiguously demonstrates that the inhibition of the proliferation of CD4⁺ T lymphocytes is not due to cAMP produced by contaminant cholera toxin A-subunit because rCTB gives the same results as pCTB. Furthermore, we investigated whether cGMP inhibit the effects of pCTB. Indeed, if pCTB is contaminated by cholera toxin A-subunit, it will produce cAMP, and it is well known that cGMP and cAMP have antagonistic action on proliferation of T lymphocytes (25). As shown in Fig. 1A, cGMP does not prevent the pCTB-induced inhibition of the PMA/ionomycin-induced proliferation of CD4⁺ T lymphocytes, thus indicating that pCTB does not exert its effect via AMPc. In contrast with rCTB, anti-GM1 Abs have no effects on PMA/ionomycin-induced CD4⁺ T cell proliferation. In conclusion, GM1 binding by specific Abs is not able to inhibit CD4⁺ T cells, whereas GM1-rCTB interaction is necessary to exert inhibitory effect. Furthermore, we questioned whether Ac anti-GM1 would potentiate the effects of rCTB or would block its activity. CD4⁺ T lymphocytes incubated with a combination of rCTB plus Ac anti-GM1 were stimulated by PMA/ionomycin and [³H]thymidine incorporation was measured. As shown in Fig. 1A, Ac anti-GM1 neither potentiates nor blocks the effect of rCTB on PMA/ionomycin-induced proliferation. Fig. 1B clearly shows that the epitopes recognized by either rCTB or Ac anti-GM1 are different. There is no competition between rCTB and Ac anti-GM1 for the binding of GM1. This result may explain why Ac anti-GM1 does not block the action of rCTB. The epitope recognized by Ac anti-GM1 may be not involved in the inhibition of CD4⁺ T lymphocytes because they do not potentiate the effects of rCTB.

View larger version (39K): [in this window] [in a new window]   FIGURE 1. Effect of rCTB and rabbit polyclonal Abs anti-GM1 on PMA plus ionomycin-induced CD4+ T lymphocyte proliferation. A, FACS-sorted CD4+ T lymphocytes were pretreated or not in 96-well flat-bottom plates with either pCTB, rCTB, rabbit polyclonal Abs anti-GM1, pCTB + cGMP, or rCTB + Abs anti-GM1 for 30 min at the indicated concentrations, then CD4+ T lymphocytes were stimulated or not with 10 ng/ml PMA plus 100 nM ionomycin. Proliferation was monitored by [3H]thymidine incorporation during the last 16 h of culture. CD4+ T lymphocytes were harvested for beta scintillation counting. Data represent one of three similar experiments. The values represent means ± SEM. B, Jurkat cells (1 x 106) were first incubated with Abs anti-GM1 (30 min at 4°C), then GM1 receptors were stained with pCTB-biotin + streptavidin-RPE-Cy5 (30 min at 4°C) as indicated in Materials and Methods. GM1 staining was measured by cytometric analysis.

The monosialoganglioside GM1 is certainly the most commonly used lipid raft marker. Cholesterol extraction by m--CD disrupts cholesterol-rich rafts and raft-resident molecules leave rafts. However, a recent study (26) showed that depletion of 73% of cell cholesterol with m--CD significantly affects the recovery in detergent-resistant membranes (DRMs) of GM1 acetylated or acylated with C₈ or unsaturated fatty acids (FAs) but not of GM1 acylated with C₁₈, C₂₂, or C₂₄ FAs. To see whether cholesterol-rich raft integrity is required for rCTB-induced CD4⁺ T cell inhibition, we first treated hPBMCs with m--CD to disrupt cholesterol-rich rafts. Then cholesterol-depleted hPBMCs were pretreated with rCTB or pCTB and cells were stimulated with PMA/ionomycin. As shown in Fig. 2A, cholesterol depletion of CD4⁺ T lymphocytes with m--CD does not prevent rCTB and pCTB to inhibit PMA/ionomycin-induced CD69 and CD25 up-regulation. The same results were obtained in Jurkat cells (Fig. 2B).

View larger version (24K): [in this window] [in a new window]   FIGURE 2. Cholesterol rich-raft disruption by m--CD does not prevent rCTB to inhibit PMA/ionomycin-induced CD69 and CD25 up-regulation. A, Freshly isolated hPBMCs were treated or not with m--CD (10 mM, 10 min at 37°C). Then cells pretreated or not with rCTB (10 µg/ml) or pCTB (10 µg/ml) for 30 min were stimulated or not by PMA (10 ng/ml) plus ionomycin (100 nM) for 20 h. Then cells were costained with a PE-CD25 mAb, a FITC-CD69 mAb, and a PerCP-CD4 mAb. hPBMCs were gated on lymphocytes according to their forward and side angle light scatter. CD25 and CD69 surface expression on CD4+ T lymphocytes was determined flow cytometry after gating lymphocytes on the basis of membrane expression of CD4. Data represent one of three similar experiments. B, CD25 and CD69 up-regulation was also examined on Jurkat cells pretreated or not with m--CD, pretreated or not with either rCTB or pCTB, and stimulated or not with PMA/ionomycin as in A. Data represent one of three similar experiments. The values represent means ± SEM.

Because cholesterol is not required for rCTB-induced inhibition of CD4⁺ T lymphocytes, we investigated a role for SM in rCTB-induced CD4⁺ T inhibition. To this end, we used Jurkat cells instead of hPBMCs because these cells are more suitable for studying lipid metabolism because their global metabolism is greatly more rapid than those of hPBMCs. Indeed, Jurkat cells do spontaneously proliferate whereas hPBMCs do not. Incorporation of [³H]palmitic acid or [³H]choline is quite equal in Jurkat cells and in hPBMCs. But the kinetic of SM synthesis is completely different. After a 4 h-incubation with tritiated precursors, it is already possible to easily detect SM synthesis in Jurkat cells, whereas it is impossible in hPBMCs. We used two drugs to modulate the level of SM and/or gangliosides: FB1 and PDMP. FB1 prevents the production of both SM and gangliosides (27). PDMP specifically diminishes levels of endogenous gangliosides (28, 29). Note that PDMP treatment also results in a significant accumulation of SM (30).

We studied the surface expression of GM1 both in FB1- and PDMP-treated Jurkat cells. As shown in Fig. 3B, FB1 and PDMP considerably diminish the level of the ganglioside GM1 after a 4-day treatment. Reduction of GM1 is time dependent, but treatments cannot be longer extended because beyond a 4-day incubation, cell viability starts to decline. After a 4-day treatment, FB1-treated cells exhibits only 34% of control GM1 and cell viability is greater than 93%. PDMP-treated Jurkat only possess 29% of GM1, and cell viability is not affected at all. Incubation of GM1-depleted Jurkat with exogenous GM1 (0.5 mg/ml for 30 min) approximately multiplies by 3.5 the basal content.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. SM is necessary for rCTB-induced CD4+ T inhibition. A, FB1 inhibits both SM and gangliosides. PDMP specifically prevents the formation of gangliosides while enhancing SM production. B, Jurkat cells (1 x 106) were cultured with or without FB1 or PDMP (in both case, 10 µM final concentration) for 24, 48, 72, and 96 h. At 96 h, half the culture was supplemented with GM1 (0.5 mg/ml for 30 min). Surface expression of GM1 was measured by flow cytometry. Data represent one of three similar experiments. The values represent means ± SEM. C, Jurkat cells (1 x 106) pretreated or not with either FB1 (10 µM) or PDMP (10 µM) for 4 days were incubated or not with exogenous GM1 (0.5 mg/ml) for 30 min. Then cells pretreated or not with rCTB (10 µg/ml) for 30 min were stimulated or not by PMA (10 ng/ml) plus ionomycin (100 nM) for 20 h. Surface expression of CD69 and CD25 was measured by flow cytometry as in Fig. 2B. Data represent one of three similar experiments. The values represent means ± SEM. D, Jurkat cells were treated as in C, except that they were stimulated by PMA/ionomycin for 6 h. RNA was extracted, and RT-PCR was performed using primers amplifying either CD69, CD25, or -actin. Data represent one of three similar experiments.

To determine whether mRNA correlates with the cell surface expression of CD69 and CD25, RT-PCRs were performed (Fig. 3D). In accordance with the flow cytometry, CTB inhibits mRNA synthesis of CD69 and CD25. FB1 and PDMP treatment prevent the inhibitory effect of CTB on PMA/ionomycin-induced CD69 and CD25 up-regulation. Exogenous GM1-addition in PDMP-treated Jurkat cells allows CTB to inhibit T cell activation. In contrast, exogenous GM1-addition in FB1-treated Jurkat cells does not allow CTB to inhibit T cell activation. It unambiguously demonstrates that if cholesterol is not required, SM is necessary to GM1 signaling via the binding of CTB.

rCTB inhibits SM synthesis and enhances PtdCho synthesis

Because we demonstrated that SM is required for GM1 signaling via the binding of rCTB (Fig. 3), we analyzed the synthesis of SM in control and in rCTB-treated Jurkat cells. We also analyzed the synthesis of PtdCho and phosphatidylethanolamine (PtdEtn) (Fig. 4). The uptake of [³H]palmitic acid and [³H]choline chloride is not affected by rCTB treatment (data not shown). We show that [³H]palmitic acid-labeled SM synthesis and [³H]choline-labeled SM synthesis are both inhibited in rCTB-treated cells compared with control cells (–54 and –56%, respectively). In contrast, [³H]palmitic acid-labeled PtdCho synthesis and [³H]choline-labeled Ptcho synthesis are both enhanced in rCTB-treated Jurkat cells (+122 and + 84% respectively). [³H]Palmitic acid-labeled PtdEtn is not affected by rCTB treatment (data not shown).

View larger version (29K): [in this window] [in a new window]   FIGURE 4. rCTB inhibits SM synthesis and enhances PtdCho synthesis. Jurkat cells (2 x 106) were maintained in 500 µl of HSB. At time 0, 4 µCi of either [3H]palmitic acid (A) or [3H]choline chloride (B) were added, with (10 µg/ml, ) or without rCTB (), at the end of the treatment (120 min); lipids were extracted and analyzed as described in Materials and Methods. Data represent one of three similar experiments. The values represent means ± SEM.

Jurkat cells prelabeled with either [³H]palmitic acid (Fig. 5A, upper graph) or [³H]choline (Fig. 5A, lower graph) were left untreated or incubated with rCTB for different period of time varying from 0 to 30 min. The analysis of lipids extracted from whole cells indicates that rCTB treatment results in a time-dependent decrease of SM (Fig. 5A). The decrease of SM is maximal at time 30 min and reaches 47% for [³H]palmitic acid-labeled SM and 46% for [³H]choline-labeled SM.

View larger version (27K): [in this window] [in a new window]   FIGURE 5. rCTB induces SM hydrolysis. A, Jurkat cells (2 x 106) were first prelabeled until isotopic equilibrium with either [3H]palmitic acid (upper graph) or [3H]choline chloride (lower graph), then washed and treated () or not () with rCTB (10 µg/ml) for different periods of time varying from 0 to 30 min. Lipids were extracted with chloroform/methanol then analyzed by TLC on silicagel plates. These graphs are representative of three different and independent experiments. The values represent means ± SEM. B, A PNS preparation of Jurkat cells (80–100 x 106) metabolically prelabeled for 16 h until isotopic equilibrium with [3H]palmitic acid and stimulated or not by rCTB (10 µg/ml) for 30 min was treated with Triton X-100 at 4°C and fractionated on a sucrose density-gradient as described in Materials and Methods. After ultracentrifugation, 1-ml fractions were harvested from the top. Lipids from each fraction were extracted with chloroform/methanol then analyzed by TLC on silicagel plates. The distribution of [3H]palmitic acid-labeled SM, [3H]palmitic acid-labeled PtdCho, and [3H]palmitic acid-labeled PtdEtn is shown for control () and rCTB-treated cells () from the top to the bottom, respectively. The buoyant fractions (rafts) at the top of the gradient correspond to fractions 2 and 3, while the soluble material at the bottom of the centrifuge tube to fractions 8 and 9. These graphs are representative of three different and independent experiments. The values represent means ± SEM. C, Jurkat cells (80–100 x 106) labeled with [3H]choline chloride as in B were treated by rCTB as in B, then raft purification was performed as in B. The distribution of [3H]choline-labeled SM and [3H]choline-labeled PtdCho is shown for control () and rCTB-treated cells () from the top to the bottom, respectively. These graphs are representative of three different and independent experiments. The values represent means ± SEM.

Because saturated FA carbon chains are known to interact with cholesterol, we investigated the effect of SM degradation on cellular [³H]cholesterol content. In control cells, a clear enrichment of [³H]cholesterol is observed in raft fractions compared with detergent-soluble fractions (Fig. 6). rCTB treatment results in a decrease of cholesterol in agreement with previous reports (31, 32), which demonstrated that the hydrolysis of plasma membrane SM alters cellular cholesterol homeostasis. We observed that this cholesterol decrease specifically occurs in rafts.

View larger version (18K): [in this window] [in a new window]   FIGURE 6. rCTB lowers cholesterol level in rafts. A PNS preparation of Jurkat cells (80–100 x 106) labeled for 16 h until isotopic equilibrium with [3H]cholesterol and stimulated or not by rCTB (10 µg/ml) for 30 min was treated with Triton X-100 at 4°C and fractionated on a sucrose density-gradient as described in Materials and Methods. The nine different fractions obtained were assayed for radioactivity by liquid scintillation. The distribution of [3H]cholesterol in the gradient is shown for control () and rCTB-treated cells (). This graph is representative of three different and independent experiments. The values represent means ± SEM.

Because we observed an important decrease of raft-SM in rCTB-treated Jurkat, we were interested in characterizing the rCTB-induced SMase activity. For that purpose, Jurkat cells were treated or not with rCTB and raft isolation was performed. Raft fractions, and fractions containing the Triton X-100-soluble material were assayed for either neutral or acidic SMase (ASM) activity. As shown in Fig. 7A, a neutral pH optimum SMase activity was found in raft of rCTB-treated Jurkat cells. This result indicates that a raft-resident neutral SMase is involved in GM1 signaling via the rCTB. Furthermore, this neutral SMase activity is almost entirely inhibited by GSH. It indicates that the involved enzyme is probably a NSM1-like one. ASM has not been found implicated in that process (data not shown).

View larger version (22K): [in this window] [in a new window]   FIGURE 7. rCTB activates a neutral SMase. A, Jurkat cells (80–100 x 106) were pretreated or not with rCTB (10 µg/ml) or pCTB (10 µg/ml) for 30 min. Raft isolation was performed as in Fig. 5B, except that RPMI 1640 supplemented with a mixture of protease inhibitors was used in place of the initial buffer. Fifty-microliter aliquots of selected fractions (2 + 3 and 8 + 9) were assayed with or without GSH (3 mM) for the presence of either neutral SMase activity or ASM activity as described in Materials and Methods. This graph is representative of three different and independent experiments. The values represent means ± SEM. B, Jurkat cells (5 x 106) were treated () or not () with rCTB (10 µg/ml) for the indicated times. Lipids were extracted and, after mild alkaline hydrolysis, subjected to phosphorylation by the DAG kinase in the presence of [-32P]ATP as described in Materials and Methods. The resulting C-1-P was separated by TLC. Radioactivity in lipid spots was determined by using an automatic linear radiochromatography analyzer. This graph is representative of three different and independent experiments. The values represent means ± SEM.

GSH and NAC pretreatment inhibits the effects of rCTB

On one hand, we demonstrated that rCTB-GM1 association inhibits CD4⁺ T lymphocyte activation (Figs. 2, A and B, 3, D and E) and proliferation (Fig. 1). In the other hand, we demonstrated that 1) rCTB inhibits SM synthesis (Fig. 4) and 2) activates a NSM1-like enzyme in lipid rafts that produces transient ceramides (Fig. 7). To demonstrate that rCTB-induced CD4⁺ T lymphocyte inhibition is due in part to SM level modifications, we pretreated Jurkat cells with either the antioxidant GSH or NAC. NAC acts as a precursor of reduced GSH biosynthesis and consequently it inhibits neutral SMase. As shown in Fig. 8A, pretreatment with GSH or NAC inhibits the inhibitory effect of rCTB on Jurkat cells. rCTB-treated Jurkat do not up-regulate CD69 and CD25 when they are stimulated by PMA/ionomycin. By contrast, rCTB-treated cells that have been pretreated before with NAC or GSH are able to up-regulate the activation markers CD69 and CD25.

View larger version (33K): [in this window] [in a new window]   FIGURE 8. NAC and GSH reverse the inhibitory effect of rCTB. A, Jurkat cells (1 x 106) pretreated or not with NAC (10 mM) or GSH (3 mM) for 1 h were treated or not with rCTB (10 µg/ml) for 30 min. Then cells were stimulated or not by PMA (10 ng/ml) plus ionomycin (100 nM) for 20 h. Surface expression of CD69 and CD25 was measured by flow cytometry as in Fig. 2B. This graph is representative of three different and independent experiments. The values represent means ± SEM. B, Jurkat cells were treated as in A, except that they were stimulated by PMA/ionomycin for 6 h. RNA was extracted, and RT-PCR was performed using primers amplifying either CD69, CD25, or -actin. Data represent one of three similar experiments. C, FACS-sorted CD4+ T lymphocytes were pretreated or not in 96-well flat-bottom plates with either NAC (10 mM) or GSH (3 mM) for 30 min, then CD4+ T lymphocytes treated or not with rCTB (10 µg/ml) for 30 min were stimulated or not with either 10 ng/ml PMA plus 100 nM ionomycin or soluble anti-CD3 mAb plus anti-CD28 mAb (5 µg/ml each). Proliferation was monitored by [3H]thymidine incorporation during the last 16 h of culture. CD4+ T lymphocytes were harvested for beta scintillation counting. This graph is representative of three different and independent experiments. The values represent means ± SEM.

To link the absence of proliferation of rCTB-treated CD4⁺ T lymphocytes and the neutral SMase activity (i.e., the transient accumulation of ceramides), purified CD4⁺ T lymphocytes were pretreated or not with GSH or NAC before being treated or not with rCTB (10 µg/ml, 30 min). Then cells were stimulated either by CD3/CD28 or by PMA/ionomycin. As expected, rCTB inhibits less the proliferation of GSH- or NAC-pretreated CD4⁺ T lymphocytes than the proliferation of control lymphocytes.

Thus, these results demonstrate that the rCTB-induced SMase activation is responsible for the observed inhibition (activation and proliferation) of CD4⁺ T lymphocytes when they are preincubated with rCTB.

SM hydrolysis is required for rCTB-induced NF-B activation

Heat-labile ETB from Escherichia coli, a close homologue of CTB, is known to activate nuclear translocation of NF-B in Jurkat cells (12). Activation of NF-B by rCTB has never been investigated before. Translocation of NF-B was visualized after 1 h of rCTB (10 µg/ml) stimulation by its binding to a radioactive probe containing B sites from the Ig promoter (Fig. 9A, lane 2 compared with unstimulated cells, lane 1). In PDMP-treated Jurkat cells, rCTB fails to translocate NF-B. In addition, in FB1-treated cells, rCTB also fails to translocate NF-B (Fig. 9A, lane 11 vs lane 2). Furthermore, rCTB is able to translocate NF-B in GM1-restored PDMP-treated cells (Fig. 9A, lane 8 vs lane 2); by contrast, rCTB cannot translocate NF-B in GM1-restored, FB1-treated cells (Fig. 9A, lane 12 vs lane 2), indicating that SM is necessary to NF-B translocation by rCTB. Preincubation with GSH leads to an inhibition of NF-B DNA-binding activity (Fig. 9A, lane 4 vs lane 2), indicating that SM hydrolysis is required for rCTB-induced NF-B translocation.

View larger version (32K): [in this window] [in a new window]   FIGURE 9. SM hydrolysis is required for rCTB-induced NF-B activation. A, Jurkat cells were pretreated or not with either PDMP (10 µM, 4 days), or FB1 (10 µM, 4 days), or GSH (3 mM for 1 h). Sphingolipid-depleted cells were preincubated or not with GM1 (0.5 mg/ml) for 30 min. Then cells were treated or not with rCTB (10 µg/ml) for 1 h. The total cell extracts were then incubated with a radioactive double-stranded oligonucleotide encompassing the B site of the Ig promoter. Complexes were then separated by nondenaturing electrophoresis followed by autoradiography. Data represent one of three similar experiments. B, For sphingolipid depletion experiments, Jurkat cells were pretreated or not with either PDMP (10 µM, 3 days) or FB1 (10 µM, 3 days), then cells were transfected with 10 µg of luciferase reporter gene. At 36 h after transfection (Jurkat cells were maintained for 36 h in a medium containing either PDMP or FB1), cells preincubated or not with GM1 (0.5 mg/ml) for 30 min were treated or not with rCTB (10 µg/ml) for 30 min. For NAC and GSH experiments, 36 h after transfection, Jurkat cells were pretreated or not with NAC (25 mM, 1 h) or GSH (3 mM, 1 h), then cells were treated or not with rCTB (10 µg/ml) for 30 min. Luciferase activity was measured as detailed in Materials and Methods. Data represent one of three similar experiments. The values represent means ± SEM.

rCTB treatment prevents PKC phosphorylation and translocation into modified lipid rafts

Because rCTB treatment strongly modifies the lipid composition of rafts (1) rCTB diminishes SM synthesis (Fig. 4), 2) hydrolyzes SM, and 3) enhances PtdCho synthesis (Fig. 5)), we were interested in studying the distribution of raft-resident proteins highly involved in T cell activation. As shown by Fig. 10A, rCTB treatment modifies neither the distribution of linker for activation of T cells nor Lck. Then, we studied the effect of rCTB on PMA-induced recruitment of PKC into lipid rafts (Fig. 10B, upper panel). As shown in Fig. 10B, upper panel, PMA induces a partial translocation of PKC into lipid rafts. rCTB treatment (i.e., raft modifications in terms of lipids) importantly prevents PKC translocation into lipid rafts. Interestingly, GSH and NAC pretreatment, which prevents rCTB to inhibit Jurkat activation, allows PKC to redistribute itself after rCTB pretreatment and PMA stimulation.

View larger version (32K): [in this window] [in a new window]   FIGURE 10. rCTB inhibits the phosphorylation and the translocation of PKC into rafts. A, Jurkat cells (80–100 x 106) were left untreated or incubated with CTB (10 µg/ml) for 30 min. Raft purification was achieved as in Fig. 5B. The nine fractions, usually harvested from the top of the ultracentrifuge tube, were pooled as follows: lane E, (9 + 8); lane D, (7 + 6); lane C, (5 + 4); lane B, (3 + 2); and lane A, 1. The pooled fractions (lanes A–E) were subjected to SDS-PAGE, transferred onto PVDF membranes, and immunoblotted with the indicated antibodies. Data represent one of three similar experiments. B, Jurkat cells (80–100 x 106) pretreated or not with NAC (25 mM) for 1 h or GSH (3 mM) for 1 h were pretreated or not with pCTB or rCTB (10 µg/ml) for 30 min. Then cells were stimulated or not by PMA (10 ng/ml) plus ionomycin (100 nM) for 1 h. Raft isolation was achieved as in Fig. 5B. Upper panel, Fractions B and E were subjected to SDS-PAGE, transferred onto PVDF membranes, and immunoblotted with anti-PKC mAb. Lower panel, Twenty-five microliters of fraction B "control" and 65 µl of fraction B "rCTB-treated cells," to have the same quantity of PKC, were subjected to SDS-PAGE, transferred onto PVDF membranes, and immunoblotted with anti-phospho-PKC (Ser657) Ab. Then membrane was stripped and reblotted with anti-PKC mAb. Data represent one of three similar experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
In the present article, we propose a mechanism for the inhibitory effect of rCTB both on the activation and on the proliferation of human CD4⁺ T lymphocytes. rCTB specifically binds to GM1, a raft marker, and strongly modifies the lipid composition of rafts. First, rCTB inhibits SM synthesis; second, it enhances PtdCho synthesis; and third, it activates a raft-resident neutral SMase, thus generating a transient ceramide production. These ceramides inhibit PKC phosphorylation and its translocation into the modified lipid rafts. Furthermore, ceramides activate NF-B. We hypothesize that combined all together, raft modification in terms of lipids, ceramide production, PKC inhibition, and NF-B activation lead to T cell inhibition.

Gangliosides and glycosylphosphatidylinositol-anchored proteins are frequently used as positive controls for raft purification. GM1 is certainly the most commonly used raft marker. It is to note that gangliosides and glycosylphosphatidylinositol-anchored proteins are almost ever used indiscriminately as if they were equal. Nevertheless, Vyas et al. (34) demonstrated that GM1 does not cocluster with GD3 on intact neurons. Gomez-Mouton et al. (35) show that the acquisition of a motile phenotype in T lymphocytes results in the asymmetric redistribution of GM3- and GM1-enriched raft domains to the leading edge and to the uropod, respectively. Furthermore, Millán et al. (36) have demonstrated that CD59 and GM1 cluster in different membrane subdomains of Jurkat cells. Furthermore, m--CD extracts cholesterol from Triton X-100-resistant membranes without affecting the buoyant properties of Thy-1 and GM1 (37, 38). The occurrence of GM1 in DRMs depends on its ceramide moiety. Depletion of 73% of cellular cholesterol with m--CD does not affect the recovery in DRMs of GM1 acylated with C18-, C22-, or C24-saturated FAs (26). Combined all together, these data suggest that GM1 resides in a subset of lipid raft that is insensitive to cholesterol depletion by m--CD. Our data support and extend this earlier observation because we show that m--CD-treatment does not affect the inhibitory effect of rCTB via GM1.

CTB and ETB are known to modulate leukocyte function. This property is attributed to the ability of the B-subunit to bind GM1. Nevertheless, it has been demonstrated that GM1 binding alone, contrary to expectations, is not sufficient to initiate toxin action. Fraser et al. (12) described the properties of ETB (H57S), a mutant B-subunit with a HisSer substitution, at position 57. The mutant still binds to GM1 but is found to be severely defective in inducing leukocyte signaling. For example, it fails to trigger caspase-3-mediated T CD8⁺ lymphocyte apoptosis. It does not activate NF-B in Jurkat cells. It fails to induce a potent anti-B-subunit response in mice, and it also fails to serve as mucosal adjuvant. In the same manner, CTB (H57A) binds GM1 but lacks immunomodulatory or toxic activity (39). In the present study, we demonstrate that GM1 ligation alone, via rabbit polyclonal Abs anti-GM1, is not able to inhibit CD4⁺ T lymphocyte activation and proliferation. We demonstrate that GM1 ligation by Abs does not lead to SMase activation and SM hydrolysis (data not shown). Aman et al. (39) hypothesize that CT may require interaction, not only with GM1, but also with another molecule to exert its biological activity. For Aman et al. (39), it is conceivable that rCTB binding to GM1 in lipid rafts would position it to interact with signaling molecules that participate in toxin-mediated immune cell modulation. In respect with our experimental data, it is quite conceivable that the intermediary molecule involved in the toxin action would be a raft-resident neutral SMase.

The isoform of SMase (acidic or neutral), thus the topology of ceramide formation, dictates its outcome (33). The activation of ASM is believed to theoretically allow the hydrolysis of the main pool of SM from the outer plasma membrane leaflet, thus resulting in the formation of ceramide-rich domains (40), while the activation of neutral SMase would rather allow the hydrolysis of the small pool of SM from the inner leaflet resulting, this time, in the generation of ceramides that will act as second messengers. In this study, we demonstrated that rCTB hydrolyzes SM in a time-dependent manner via the activation of a neutral SMase that is inhibited by GSH. Thus, this enzyme involved in that process resembles to NSM1. However, the quantity of hydrolyzed SM by rCTB reaches 47% for [³H]palmitic acid-labeled SM and 46% for [³H]choline-labeled SM. It probably represents more than the minor pool of SM located in the inner leaflet. Thus, what remains unclear is how neutral SMase gains access to the major pool of SM. It has been hypothesized that neutral SMase would hydrolyze SM after its flip-flop from the outer leaflet to the inner leaflet (33), but we did not observe any flip-flop of phospholipids as studied by the externalization of phosphatidylserine (data not shown).

In this study, we demonstrated that rCTB regulates SM at two levels. First, rCTB inhibits SM synthesis, and second, rCTB activates SM hydrolysis. These two complementary actions both contribute to cell cycle arrest of CD4⁺ T lymphocytes. Indeed, the inhibition of SM synthesis 1) causes the inhibition of DAG synthesis and 2) increases the level of ceramides; furthermore, SM hydrolysis generates ceramides. Consequently, rCTB enhances ceramides content by two different manners. Ceramides:DAG ratio plays an important role in cell proliferation, and numerous targets of ceramides are known to negatively regulate cell proliferation.

The biochemical synthesis of SM occurs via the action of a PtdCho:ceramide choline phosphotransferase (SM synthase (SMS)), which transfers the phosphorylcholine (phosphocholine) moiety from PtdCho onto the primary hydroxyl of ceramide, thus producing SM and DAG (41, 42). Thus, the inhibition of SMS reduces the intracellular level of DAG. It has been published that DAG generated during SM synthesis is involved in PKC activation and cell proliferation (43). It constitutes the first way by which rCTB inhibits PKC in CD4⁺ T lymphocytes. Furthermore, the inhibition of SMS also increases the level of ceramides (44, 45). It may constitute a supplementary way, in addition to SM hydrolysis, to elevate ceramide levels. Flores et al. (46) studied the changes in the balance between DAG and ceramides during cell proliferation, cell arrest, and apoptosis in T lymphocytes. Accordingly, augmentation of ceramides and diminution of DAG favor cell arrest.

Ceramides also target specific proteins inducing cell cycle arrest. Ceramides induce a G_o-G₁ cell cycle arrest, and this was mechanistically shown to be due to the induction of dephosphorylation of the retinoblastoma gene product (Rb) (47). Furthermore, it has been also reported that the treatment of NIH 3T3 cells with a specific inhibitor of glucosylceramide synthase, which induces ceramide accumulation, causes a G₂-M cell cycle arrest, possibly mediated by ceramide-induced inhibition of the cyclin-dependent p34^cdc2 and Cdk2 kinases (48). Another study has shown that ceramides specifically inactivates the cyclin-dependent kinase Cdk2 through activation of PP1 and PP2A phosphatases (49). Ceramides also inactivates protein kinase B/Akt (50). In addition, it has been demonstrated that endogenous ceramides, produced either by overexpression of bacterial SMase or by daunorubicin treatment, inhibit mRNA synthesis of telomerase RT and telomerase activity via inactivation of c-Myc transcription factor (51, 52).

In this study, we observed that rCTB inhibits PKC phosphorylation and prevents its translocation into rafts. PKC is a progrowth cellular regulator, and its inactivation can explain cell cycle arrest. PKC inhibition by rCTB can be easily explained by three ways. First, PKC is activated by DAG, and because of the inhibition of SM synthesis, DAG level is diminished in rCTB-treated lymphocytes. Consequently, PKC is less activated in rCTB-treated cells. Second, ceramides are known to inactivate PKC via a phosphatase (53, 54). Third, rafts are markedly modified in terms of lipids. Indeed, rCTB-treated lymphocytes possess rafts with less SM, less cholesterol, but more PtdCho than control cells. It is quite conceivable that these lipid modifications strongly alter the anchoring of PKC into these modified rafts because ceramides do not inhibit PMA-induced translocation of PKC by themselves (53). Collectively, all of these evidences can explain the inhibition of PKC by rCTB.

Finally, we observed that rCTB induces the activation of NF-B. rCTB-induced NF-B activation is inhibited in FB1-treated cells, suggesting that SM is required. Furthermore, NAC and GSH prevent rCTB-induced NF-B activation, it means that SM hydrolysis i.e., ceramide production is responsible for the rCTB-induced NF-B activation. Ceramides are well known to activate NF-B (55), but the genes involved remain to be elucidated.

	Acknowledgments

 
We thank Dr. Laurence Lamy, Dr. Isabelle Foucault, Romain Gallais, and Frank Leporati. We also thank Prof. Cecil Czerkinsky and Dr. Fabienne Anjuère for providing us the rCTB. We are also grateful to Alexis de la Daudaf for helpful discussion.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Institut National de la Santé et de la Recherche Médicale. A.K.R.-J. is a recipient of a doctoral fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche and from the Association pour la Recherche sur le Cancer.

² Address correspondence and reprint requests to Dr. Claude Aussel, Institut National de la Santé et de la Recherche Médicale Unit 576, IFR 50, Hôpital de l’Archet I, 151 Route de Saint Antoine de Ginestière, B.P. 79, 06202 Nice Cedex 3, France. E-mail address: aussel{at}unice.fr

³ Abbreviations used in this paper: CTB, cholera toxin B-subunit; ETB, enterotoxin B-subunit; hPBMC, human PBMC; SM, sphingomyelin; PtdCho, phosphatidylcholine; SMase, sphingomyelinase; PKC, protein kinase C; 7-AAD, 7-aminoactinomycin D; m--CD, methyl--cyclodextrin; NAC, N-acetyl-L-cysteine; GSH, glutathione; PDMP, 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol; FB1, fumonisin B1; PNS, postnuclear supernatant; PVDF, polyvinylidene difluoride; HSB, HEPES saline buffer; DAG, diacylglycerol; C-1-P, ceramide-1-phosphate; pCTB, purified CTB; DRM, detergent-resistant membrane; FA, fatty acid; PtdEtn, phosphatidylethanolamine; ASM, acidic SMase; SMS, SM synthase.

Received for publication July 29, 2004. Accepted for publication August 8, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Francis, M. L., J. Ryan, M. G. Jobling, R. K. Holmes, J. Moss, J. J. Mond. 1992. Cyclic AMP-independent effects of cholera toxin on B cell activation. II. Binding of ganglioside GM1 induces B cell activation. J. Immunol. 148:1999.-2005. [Abstract]
2. Nashar, T. O., T. R. Hirst, N. A. Williams. 1997. Modulation of B cell activation by the B subunit of Escherichia coli enterotoxin: receptor interaction up-regulates MHC class II, B7, CD40, CD25 and ICAM-1. Immunology 91:572.-578. [Medline]
3. Nashar, T. O., N. A. Williams, T. R. Hirst, T. O. Nahar. 1996. Cross-linking of cell surface ganglioside GM1 induces the selective apoptosis of mature CD8⁺ T lymphocytes. Int. Immunol. 8:731.-736. [Abstract/Free Full Text]
4. Elson, C. O., S. P. Holland, M. T. Dertzbaugh, C. F. Cuff, A. O. Anderson. 1995. Morphologic and functional alterations of mucosal T cells by cholera toxin and its B subunit. J. Immunol. 154:1032.-1040. [Abstract]
5. Turcanu, V., T. R. Hirst, N. A. Williams. 2002. Modulation of human monocytes by Escherichia coli heat-labile enterotoxin B-subunit; altered cytokine production and its functional consequences. Immunology 106:316.-325. [Medline]
6. Bromander, A., J. Holmgren, N. Lycke. 1991. Cholera toxin stimulates IL-1 production and enhances antigen presentation by macrophages in vitro. J. Immunol. 146:2908.-2914. [Abstract]
7. Matousek, M. P., J. G. Nedrud, C. V. Harding. 1996. Distinct effects of recombinant cholera toxin B subunit and holotoxin on different stages of class II MHC antigen processing and presentation by macrophages. J. Immunol. 156:4137.-4145. [Abstract]
8. Millar, D. G., T. R. Hirst. 2001. Cholera toxin and Escherichia coli enterotoxin B-subunits inhibit macrophage-mediated antigen processing and presentation: evidence for antigen persistence in nonacidic recycling endosomal compartments. Cell. Microbiol. 3:311.-329. [Medline]
9. Woogen, S. D., W. Ealding, C. O. Elson. 1987. Inhibition of murine lymphocyte proliferation by the B subunit of cholera toxin. J. Immunol. 139:3764.-3770. [Abstract]
10. Woogen, S. D., K. Turo, L. A. Dieleman, K. W. Beagley, C. O. Elson. 1993. Inhibition of murine T cell activation by cholera toxin B subunit is not mediated through the phosphatidylinositol second messenger system. J. Immunol. 150:3274.-3283. [Abstract]
11. Marmor, M. D., M. Julius. 2001. Role for lipid rafts in regulating interleukin-2 receptor signaling. Blood 98:1489.-1497. [Abstract/Free Full Text]
12. Fraser, S. A., L. de Haan, A. R. Hearn, H. K. Bone, R. J. Salmond, A. J. Rivett, N. A. Williams, T. R. Hirst. 2003. Mutant Escherichia coli heat-labile toxin B subunit that separates toxoid-mediated signaling and immunomodulatory action from trafficking and delivery functions. Infect. Immun. 71:1527.-1537. [Abstract/Free Full Text]
13. Lebens, M., S. Johansson, J. Osek, M. Lindblad, J. Holmgren. 1993. Large-scale production of Vibrio cholerae toxin B subunit for use in oral vaccines. Biotechnology 11:1574.-1578. [Medline]
14. George-Chandy, A., K. Eriksson, M. Lebens, I. Nordstrom, E. Schon, J. Holmgren. 2001. Cholera toxin B subunit as a carrier molecule promotes antigen presentation and increases CD40 and CD86 expression on antigen-presenting cells. Infect. Immun. 69:5716.-5725. [Abstract/Free Full Text]
15. Anjuere, F., A. George-Chandy, F. Audant, D. Rousseau, J. Holmgren, C. Czerkinsky. 2003. Transcutaneous immunization with cholera toxin B subunit adjuvant suppresses IgE antibody responses via selective induction of Th1 immune responses. J. Immunol. 170:1586.-1592. [Abstract/Free Full Text]
16. Anjuere, F., C. Luci, M. Lebens, D. Rousseau, C. Hervouet, G. Milon, J. Holmgren, C. Ardavin, C. Czerkinsky. 2004. In vivo adjuvant-induced mobilization and maturation of gut dendritic cells after oral administration of cholera toxin. J. Immunol. 173:5103.-5111. [Abstract/Free Full Text]
17. Montixi, C., C. Langlet, A. M. Bernard, J. Thimonier, C. Dubois, M. A. Wurbel, J. P. Chauvin, M. Pierres, H. T. He. 1998. Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains. EMBO J. 17:5334.-5348. [Medline]
18. Rouquette-Jazdanian, A. K., C. Pelassy, J. P. Breittmayer, J. L. Cousin, C. Aussel. 2002. Metabolic labelling of membrane microdomains/rafts in Jurkat cells indicates the presence of glycerophospholipids implicated in signal transduction by the CD3 T cell receptor. Biochem. J. 363:645.-655. [Medline]
19. Bligh, E. G., W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911.-917.
20. Wiegmann, K., S. Schutze, T. Machleidt, D. Witte, M. Kronke. 1994. Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78:1005.-1015. [Medline]
21. Hanada, K., T. Mitamura, M. Fukasawa, P. A. Magistrado, T. Horii, M. Nishijima. 2000. Neutral sphingomyelinase activity dependent on Mg²⁺ and anionic phospholipids in the intraerythrocytic malaria parasite Plasmodium falciparum. Biochem. J. 346:(Pt. 3):671.-677.
22. Grazide, S., N. Maestre, R. J. Veldman, C. Bezombes, S. Maddens, T. Levade, G. Laurent, J. P. Jaffrezou. 2002. Ara-C- and daunorubicin-induced recruitment of Lyn in sphingomyelinase-enriched membrane rafts. FASEB J. 16:1685.-1687. [Abstract/Free Full Text]
23. Brenner, B., K. Ferlinz, H. Grassme, M. Weller, U. Koppenhoefer, J. Dichgans, K. Sandhoff, F. Lang, E. Gulbins. 1998. Fas/CD95/Apo-I activates the acidic sphingomyelinase via caspases. Cell Death Differ. 5:29.-37. [Medline]
24. Chomczynski, P., N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156.-159. [Medline]
25. Diamantstein, T., A. Ulmer. 1975. The antagonistic action of cyclic GMP and cyclic AMP on proliferation of B and T lymphocytes. Immunology 28:113.-119. [Medline]
26. Panasiewicz, M., H. Domek, G. Hoser, M. Kawalec, T. Pacuszka. 2003. Structure of the ceramide moiety of GM1 ganglioside determines its occurrence in different detergent-resistant membrane domains in HL-60 cells. Biochemistry 42:6608.-6619. [Medline]
27. Merrill, A. H., Jr, G. van Echten, E. Wang, K. Sandhoff. 1993. Fumonisin B1 inhibits sphingosine (sphinganine) N-acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ. J. Biol. Chem. 268:27299.-27306. [Abstract/Free Full Text]
28. Inokuchi, J., N. S. Radin. 1987. Preparation of the active isomer of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, inhibitor of murine glucocerebroside synthetase. J. Lipid Res. 28:565.-571. [Abstract]
29. Nagafuku, M., K. Kabayama, D. Oka, A. Kato, S. Tani-ichi, Y. Shimada, Y. Ohno-Iwashita, S. Yamasaki, T. Saito, K. Iwabuchi, et al 2003. Reduction of glycosphingolipid levels in lipid rafts affects the expression state and function of glycosylphosphatidylinositol-anchored proteins but does not impair signal transduction via the T cell receptor. J. Biol. Chem. 278:51920.-51927. [Abstract/Free Full Text]
30. Naslavsky, N., H. Shmeeda, G. Friedlander, A. Yanai, A. H. Futerman, Y. Barenholz, A. Taraboulos. 1999. Sphingolipid depletion increases formation of the scrapie prion protein in neuroblastoma cells infected with prions. J. Biol. Chem. 274:20763.-20771. [Abstract/Free Full Text]
31. Ohvo, H., C. Olsio, J. P. Slotte. 1997. Effects of sphingomyelin and phosphatidylcholine degradation on cyclodextrin-mediated cholesterol efflux in cultured fibroblasts. Biochim. Biophys. Acta 1349:131.-141. [Medline]
32. Fukasawa, M., M. Nishijima, H. Itabe, T. Takano, K. Hanada. 2000. Reduction of sphingomyelin level without accumulation of ceramide in Chinese hamster ovary cells affects detergent-resistant membrane domains and enhances cellular cholesterol efflux to methyl--cyclodextrin. J. Biol. Chem. 275:34028.-34034. [Abstract/Free Full Text]
33. van Blitterswijk, W. J., A. H. van der Luit, R. J. Veldman, M. Verheij, J. Borst. 2003. Ceramide: second messenger or modulator of membrane structure and dynamics?. Biochem. J. 369:199.-211. [Medline]
34. Vyas, K. A., H. V. Patel, A. A. Vyas, R. L. Schnaar. 2001. Segregation of gangliosides GM1 and GD3 on cell membranes, isolated membrane rafts, and defined supported lipid monolayers. Biol. Chem. 382:241.-250. [Medline]
35. Gomez-Mouton, C., J. L. Abad, E. Mira, R. A. Lacalle, E. Gallardo, S. Jimenez-Baranda, I. Illa, A. Bernad, S. Manes, A. C. Martinez. 2001. Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization. Proc. Natl. Acad. Sci. USA 98:9642.-9647. [Abstract/Free Full Text]
36. Millan, J., M. Qaidi, M. A. Alonso. 2001. Segregation of co-stimulatory components into specific T cell surface lipid rafts. Eur. J. Immunol. 31:467.-473. [Medline]
37. Ilangumaran, S., D. C. Hoessli. 1998. Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane. Biochem. J. 335:(Pt. 2):433.-440.
38. Marwali, M. R., J. Rey-Ladino, L. Dreolini, D. Shaw, F. Takei. 2003. Membrane cholesterol regulates LFA-1 function and lipid raft heterogeneity. Blood 102:215.-222. [Abstract/Free Full Text]
39. Aman, A. T., S. Fraser, E. A. Merritt, C. Rodigherio, M. Kenny, M. Ahn, W. G. Hol, N. A. Williams, W. I. Lencer, T. R. Hirst. 2001. A mutant cholera toxin B subunit that binds GM1-ganglioside but lacks immunomodulatory or toxic activity. Proc. Natl. Acad. Sci. USA 98:8536.-8541. [Abstract/Free Full Text]
40. Gulbins, E., R. Kolesnick. 2003. Raft ceramide in molecular medicine. Oncogene 22:7070.-7077. [Medline]
41. Ullman, M. D., N. S. Radin. 1974. The enzymatic formation of sphingomyelin from ceramide and lecithin in mouse liver. J. Biol. Chem. 249:1506.-1512. [Abstract/Free Full Text]
42. Marggraf, W. D., J. N. Kanfer. 1984. The phosphorylcholine acceptor in the phosphatidylcholine:ceramide cholinephosphotransferase reaction: is the enzyme a transferase or a hydrolase?. Biochim. Biophys. Acta 793:346.-353. [Medline]
43. Cerbon, J., R. del Carmen Lopez-Sanchez. 2003. Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells. Biochem. J. 373:917.-924. [Medline]
44. Schutze, S., K. Potthoff, T. Machleidt, D. Berkovic, K. Wiegmann, M. Kronke. 1992. TNF activates NF-B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell 71:765.-776. [Medline]
45. Luberto, C., Y. A. Hannun. 1998. Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation: does sphingomyelin synthase account for the putative phosphatidylcholine-specific phospholipase C?. J. Biol. Chem. 273:14550.-14559. [Abstract/Free Full Text]
46. Flores, I., D. R. Jones, I. Merida. 2000. Changes in the balance between mitogenic and antimitogenic lipid second messengers during proliferation, cell arrest, and apoptosis in T lymphocytes. FASEB J. 14:1873.-1875. [Abstract/Free Full Text]
47. Dbaibo, G. S., M. Y. Pushkareva, S. Jayadev, J. K. Schwarz, J. M. Horowitz, L. M. Obeid, Y. A. Hannun. 1995. Retinoblastoma gene product as a downstream target for a ceramide-dependent pathway of growth arrest. Proc. Natl. Acad. Sci. USA 92:1347.-1351. [Abstract/Free Full Text]
48. Rani, C. S., A. Abe, Y. Chang, N. Rosenzweig, A. R. Saltiel, N. S. Radin, J. A. Shayman. 1995. Cell cycle arrest induced by an inhibitor of glucosylceramide synthase: correlation with cyclin-dependent kinases. J. Biol. Chem. 270:2859.-2867. [Abstract/Free Full Text]
49. Lee, J. Y., A. E. Bielawska, L. M. Obeid. 2000. Regulation of cyclin-dependent kinase 2 activity by ceramide. Exp. Cell Res. 261:303.-311. [Medline]
50. Schubert, K. M., M. P. Scheid, V. Duronio. 2000. Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine 473. J. Biol. Chem. 275:13330.-13335. [Abstract/Free Full Text]
51. Ogretmen, B., J. M. Kraveka, D. Schady, J. Usta, Y. A. Hannun, L. M. Obeid. 2001. Molecular mechanisms of ceramide-mediated telomerase inhibition in the A549 human lung adenocarcinoma cell line. J. Biol. Chem. 276:32506.-32514. [Abstract/Free Full Text]
52. Ogretmen, B., D. Schady, J. Usta, R. Wood, J. M. Kraveka, C. Luberto, H. Birbes, Y. A. Hannun, L. M. Obeid. 2001. Role of ceramide in mediating the inhibition of telomerase activity in A549 human lung adenocarcinoma cells. J. Biol. Chem. 276:24901.-24910. [Abstract/Free Full Text]
53. Lee, J. Y., Y. A. Hannun, L. M. Obeid. 1996. Ceramide inactivates cellular protein kinase C. J. Biol. Chem. 271:13169.-13174. [Abstract/Free Full Text]
54. Lee, J. Y., Y. A. Hannun, L. M. Obeid. 2000. Functional dichotomy of protein kinase C (PKC) in tumor necrosis factor (TNF-) signal transduction in L929 cells: translocation and inactivation of PKC by TNF-. J. Biol. Chem. 275:29290.-29298. [Abstract/Free Full Text]
55. Colell, A., O. Coll, M. Mari, J. C. Fernandez-Checa, C. Garcia-Ruiz. 2002. Divergent role of ceramide generated by exogenous sphingomyelinases on NF-B activation and apoptosis in human colon HT-29 cells. FEBS Lett. 526:15.-20. [Medline]

This article has been cited by other articles:

				K. Miyamoto, K. Takada, K. Furukawa, K. Furukawa, and S. Kusunoki Roles of complex gangliosides in the development of experimental autoimmune encephalomyelitis Glycobiology, May 1, 2008; 18(5): 408 - 413. [Abstract] [Full Text] [PDF]

				G. Dixit, C. Mikoryak, T. Hayslett, A. Bhat, and R. K. Draper Cholera Toxin Up-Regulates Endoplasmic Reticulum Proteins That Correlate with Sensitivity to the Toxin Experimental Biology and Medicine, February 1, 2008; 233(2): 163 - 175. [Abstract] [Full Text] [PDF]

				L. E. Mansson, P. Kjall, S. Pellett, G. Nagy, R. A. Welch, F. Backhed, T. Frisan, and A. Richter-Dahlfors Role of the Lipopolysaccharide-CD14 Complex for the Activity of Hemolysin from Uropathogenic Escherichia coli Infect. Immun., February 1, 2007; 75(2): 997 - 1004. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rouquette-Jazdanian, A. K. Articles by Aussel, C. Search for Related Content PubMed PubMed Citation Articles by Rouquette-Jazdanian, A. K. Articles by Aussel, C.