Regulation of Apoptosis during Neuronal Differentiation by Ceramide and b-Series Complex Gangliosides
2001; Elsevier BV; Volume: 276; Issue: 48 Linguagem: Inglês
10.1074/jbc.m107239200
ISSN1083-351X
AutoresErhard Bieberich, Sarah MacKinnon, Jeane Silva, Robert K. Yu,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoLipid analysis of gestational day E14.5 mouse brain revealed elevation of ceramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramide was coincident with the first appearance of b-series complex gangliosides (BCGs). Expression of BCGs by stable transfection of murine neuroblastoma (F-11) cells with sialyltransferase-II (ST2) resulted in a 70% reduction of ceramide-induced apoptosis. This was most likely due to an 80% reduced expression of prostate apoptosis response-4 (PAR-4). PAR-4 expression and apoptosis were restored by preincubation of ST2-transfected cells with N-butyl deoxinojirimycin (NB-DNJ) or PD98059, two inhibitors of ganglioside biosynthesis or p42/44 mitogen-activated protein (MAPK) kinase, respectively. In sections of day E14.5 mouse brain, the intermediate zone showed intensive staining for complex gangliosides, but only low staining for apoptosis (TUNEL) and PAR-4. Apoptosis and PAR-4 expression, however, were elevated in the ventricular zone which only weakly stained for complex gangliosides. Whole cell patch clamping revealed a 2-fold increased calcium influx in ST2-transfected cells, the blocking of which with nifedipine restored apoptosis to the level of untransfected cells. In serum-free culture, supplementation of the medium with IGF-1 was required to maintain MAPK phosphorylation and the anti-apoptotic effect of BCG expression. BCG-enhanced calcium influx and the presence of insulin-like growth factor-1 may thus activate a cell survival mechanism that selectively protects developing neurons against ceramide-induced apoptosis by up-regulation of MAPK and reduction of PAR-4 expression. Lipid analysis of gestational day E14.5 mouse brain revealed elevation of ceramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramide was coincident with the first appearance of b-series complex gangliosides (BCGs). Expression of BCGs by stable transfection of murine neuroblastoma (F-11) cells with sialyltransferase-II (ST2) resulted in a 70% reduction of ceramide-induced apoptosis. This was most likely due to an 80% reduced expression of prostate apoptosis response-4 (PAR-4). PAR-4 expression and apoptosis were restored by preincubation of ST2-transfected cells with N-butyl deoxinojirimycin (NB-DNJ) or PD98059, two inhibitors of ganglioside biosynthesis or p42/44 mitogen-activated protein (MAPK) kinase, respectively. In sections of day E14.5 mouse brain, the intermediate zone showed intensive staining for complex gangliosides, but only low staining for apoptosis (TUNEL) and PAR-4. Apoptosis and PAR-4 expression, however, were elevated in the ventricular zone which only weakly stained for complex gangliosides. Whole cell patch clamping revealed a 2-fold increased calcium influx in ST2-transfected cells, the blocking of which with nifedipine restored apoptosis to the level of untransfected cells. In serum-free culture, supplementation of the medium with IGF-1 was required to maintain MAPK phosphorylation and the anti-apoptotic effect of BCG expression. BCG-enhanced calcium influx and the presence of insulin-like growth factor-1 may thus activate a cell survival mechanism that selectively protects developing neurons against ceramide-induced apoptosis by up-regulation of MAPK and reduction of PAR-4 expression. N-acetylgalactosaminyltransferase I N-acetylsphingosine high performance thin layer chromatography N-butyl deoxinojirimycin prostate apoptosis response-4 N-(2-hydroxy-1-(hydroxymethyl)ethyl)-oleoylamide,N-oleoyl serinol CMP-NeuAc:GM3 α2–8-sialyltransferase b-series complex gangliosides green fluorescent protein phosphatidylinositol 3-kinase insulin-like growth factor 1 embryoid bodies polyacrylamide gel electrophoresis mitogen-activated protein kinase insulin-transferrin-selenite nerve growth factor neuroprogenitor. Glycosphingolipid structures are abbreviated according to the IUPAC-IUB recommendations (IUPAC Commission on Biochemical Nomenclature, 1977) except gangliosides, which are abbreviated according to Svennerholm (46Svennerholm L. J. Lipid Res. 1964; 5: 145-154Abstract Full Text PDF PubMed Google Scholar) Ceramide and gangliosides are sphingolipids that are abundant in the plasma membrane of neuronal cells and are suggested to have a regulatory function in cellular differentiation and apoptosis (1Goswami R. Dawson G. J. Neurosci. Res. 2000; 60: 141-149Crossref PubMed Scopus (54) Google Scholar, 2Yu R.K. Prog. Brain Res. 1994; 101: 31-44Crossref PubMed Scopus (153) Google Scholar). Ceramide is known to induce differentiation or apoptosis in a variety of different cell types whereas the physiological significance of gangliosides for these processes is still unclear (3Schwarz A. Futerman A.H. J. Neurosci. 1997; 17: 2929-2938Crossref PubMed Google Scholar, 4Toman R.E. Spiegel S. Faden A.I. J. Neurotrauma. 2001; 17: 891-898Crossref Scopus (29) Google Scholar). Recent studies with transgenic mice lacking neutral glucosphingolipids (glucosyltransferase knockout) or complex gangliosides (N-acetylgalactosaminyltransferase I knockout) indicate a high incidence of apoptosis in the nervous system during embryonal development or adulthood (5Sheikh K.A. Sun J. Liu Y. Kawai H. Crawford T.O. Proia R.L. Griffin J.W. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7532-7537Crossref PubMed Scopus (353) Google Scholar, 6Wu G. Xie X. Lu Z.-H. Ledeen R.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 307-312Crossref PubMed Scopus (70) Google Scholar, 7Yamashita T. Wada R. Sasaki T. Deng C. Bierfreund U. Sandhoff K. Proia R.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9142-9147Crossref PubMed Scopus (403) Google Scholar). Most recently, theN-acetylgalactosaminyltransferase I/sialyltransferase II (GalNAcT1/ST2) double knockout mouse has been reported to show spontaneous death and audiogenic seizures (8Kawai H. Allende M.L. Wada R. Kono M. Sango K. Deng C. Miyakawa T. Crawley J.N. Werth N. Bierfreund U. Sandhoff K. Proia R.L. J. Biol. Chem. 2000; 276: 6885-6888Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). These results prompted us to investigate the role of gangliosides as potential anti-apoptotic effectors during early mouse brain development. Cell survival is crucial in this developmental period because about half of the proliferative neurons and glial cells die by apoptosis between gestational days E12 and E18 (9Blaschke A.J. Staley K. Chun J. Development. 1996; 122: 1165-1174Crossref PubMed Google Scholar). This period is coincident with a switch from simple to complex gangliosides in brain tissue at days E14-E16 that has also been found during neuronal differentiation of teratocarcinoma-derived embryonic stem cells (2Yu R.K. Prog. Brain Res. 1994; 101: 31-44Crossref PubMed Scopus (153) Google Scholar,10Liour S.-S. Kapitonov D. Yu R.K. J. Neurosci. Res. 2000; 62: 363-373Crossref PubMed Scopus (37) Google Scholar). The simple gangliosides GM3 and GD3 are converted to the b-series complex gangliosides (BCGs) of the type GD1b, GT1b, and GQ1b by up-regulation of ST2 and GalNAcT. We have established an in vitro model for conversion of simple gangliosides to BCGs in neuronal cell culture by stable expression of ST2 in F-11A cells (11Bieberich E. Tencomnao T. Kapitonov D. Yu R.K. J. Neurochem. 2000; 74: 2359-2364Crossref PubMed Scopus (24) Google Scholar). F-11A cells are derived from murine neuroblastoma x rat dorsal root ganglion F-11 cells that were selected for a predominant expression of GM3 (>80% of total gangliosides). The ST2 cDNA was endowed with a C-terminal FLAG epitope and fused with green fluorescent protein (ST2-FLAG-GFP) to monitor the enzyme expression and subcellular localization by immunostaining and fluorescence microscopy. Recently, we have reported that F-11 cells undergo apoptosis upon incubation with novel ceramide analogs that have been synthesized by N-acylation of serinol (12Bieberich E. Kawaguchi T. Yu R.K. J. Biol. Chem. 2000; 275: 177-181Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). These ceramide analogs are highly soluble in water (up to 0.5 mm) and are not degraded by enzymes active in ceramide metabolism. We have used N-oleoyl serinol (S18) in addition to N-acetylsphingosine (C2-ceramide) to induce apoptosis in untransfected, GFP-transfected, or ST2-FLAG-GFP transfected F-11A cells. These cells were analyzed for the degree of apoptosis and signaling pathways known to participate in ceramide-induced cell death, in particular the PKCζ/PAR-4 and PI3K/MEK1 pathways (13Camandola S. Mattson M.P. J. Neurosci. Res. 2000; 61: 134-139Crossref PubMed Scopus (75) Google Scholar, 14Diaz-Meco M.T. Municio M.M. Frutos S. Sanchez P. Lozano J. Sanz L. Moscat J. Cell. 1998; 86: 777-786Abstract Full Text Full Text PDF Scopus (321) Google Scholar, 15Wang Y.M. Seibenhener M.L. Vandenplas M.L. Wooten M.W. J. Neurosci. Res. 1999; 55: 293-302Crossref PubMed Scopus (122) Google Scholar, 16Barradas M. Monjas A. Diaz-Meco M.T. Serrano M. Moscat J. EMBO J. 1999; 22: 6362-6369Crossref Scopus (98) Google Scholar). The significance of BCG expression for a specific cell signaling event was verified by preincubation withN-butyl deoxinojirimycin (NB-DNJ), an inhibitor of glycolipid biosynthesis that does not induce apoptosis by itself (17Bieberich E. Freischütz B. Suzuki M. Yu R.K. J. Neurochem. 1999; 72: 1040-1049Crossref PubMed Scopus (57) Google Scholar). The results from the analysis of apoptosis and protein expression in neuroblastoma cells were compared with those obtained from preimplantation blastocyst-derived murine embryonic stem (ES-J1) cells which were used as in vitro model for neuronal differentiation. The biological significance of ceramide and BCGs for neuronal apoptosis was investigated by analysis of the lipid and regulatory protein expression in mouse brain at different stages of embryonal development. Murine F-11 and ES-J1 cells were kindly provided by Drs. Glyn Dawson (University of Chicago, Chicago, IL) and Levent Keskintepe (Medical College of Georgia, Augusta, GA), respectively. The novel ceramide analog S18 was kindly provided by Drs. Takahisa Kawaguchi, Bin Hu, and Raphael Ottenbrite (Virginia Commonwealth University, Richmond, VA). N-butyl deoxinojirimycin (NB-DNJ) was a generous gift from Dr. Thomas N. Seyfried (Boston College, Chestnut Hill, MA). The epidermal growth factor protein vector was purchased fromCLONTECH (Palo Alto, CA). Rabbit polyclonal antibodies against PAR-4, trkA/B/C, caspase 3, bcl-2, IGF-I-receptor β-subunit, GAP-43, synaptophysin, and mouse monoclonal anti-phospho-Ser490-trkA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal IgG against PKCζ, mouse monoclonal anti-MAP2a/b, horseradish peroxidase-conjugated anti-rabbit IgG, LongR3-IGF-1 peptide analog, tetrodotoxin, glucamine, wortmannin, and nifedipine were from Sigma. Rabbit polyclonal IgG against GFP, anti-rabbit IgG-Alexa Fluor 594, Alexa 594-labeled cholera toxin B, goat anti-rabbit IgG Alexa 488-conjugate, and goat anti-mouse IgG Alexa 546-conjugate were from Molecular Probes (Eugene, OR). Mouse monoclonal anti-phospho-Thr202/Tyr204-MAPK, anti-phospho-Ser112-BAD, rabbit polyclonal anti-BAD, and anti-MAPK was purchased from Cell Signaling/New England Biolabs (Beverly, MA). Anti-mouse IgG- horseradish peroxidase conjugate was purchased from Jackson ImmunoResearch (West Grove, PA). The Fluorescein-FragEL TUNEL assay and the Suicide Tracker DNA-laddering assay were from Oncogene (Boston, MA). All cell culture media and supplements including LipofectAMINE, fetal bovine serum, N2 supplement, ITS supplement, murine 2.5 S nerve growth factor, basic fibroblast growth factor, insulin, and human recombinant insulin-like growth factor-1 were from Life Technologies, Inc. (Gaithersburg, MD). Glycolipid standards, including gangliosides, neutral glycolipids, and ceramide were from Matreya (Pleasant Gap, PA). All other chemicals were of analytical grade or higher and solvents were freshly redistilled before use. A cDNA encoding ST2 was amplified from a 15.5-day-old mouse embryo cDNA library as previously described (11Bieberich E. Tencomnao T. Kapitonov D. Yu R.K. J. Neurochem. 2000; 74: 2359-2364Crossref PubMed Scopus (24) Google Scholar). The ST2 cDNA was endowed with a NheI restriction site on its N terminus and with a FLAG-epitope sequence/SalI restriction site on its C terminus using a primer combination of sense 5′-taggtaccgatatcacaccgaggctgcgatgag-3′ and antisense 5′-tagtcgacttgtcatcgtcgtccttgtaatc-3′ for polymerase chain reaction amplification. The polymerase chain reaction product was ligated in-frame into the NheI/SalI restriction sites of the GFP vector. The sequence of the ST2-FLAG-GFP construct was verified by DNA sequencing. Murine neuroblastoma x rat dorsal root ganglion (F-11) cells were separated into single cells and the clones were analyzed for their ganglioside expression. A clone termed F-11A expressed mainly GM3 and was used for stable transfection of the ST2-FLAG-GFP vector following the LipofectAMINE procedure as described by the manufacturer (Life Technologies, Inc.). Clones transfected with the vector were isolated using fluorescence-activated cell sorting. Transfected cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 500 μg/ml geneticin to prevent loss of the vector. The analysis of enzyme activity followed a protocol that has been previously described (11Bieberich E. Tencomnao T. Kapitonov D. Yu R.K. J. Neurochem. 2000; 74: 2359-2364Crossref PubMed Scopus (24) Google Scholar). The novel ceramide analog N-oleoyl serinol (S18) was synthesized from serinol (2-amino-1,3-propanediol) and oleoyl chloride following a previously described procedure (12Bieberich E. Kawaguchi T. Yu R.K. J. Biol. Chem. 2000; 275: 177-181Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Lipid preparation and analysis followed a standard protocol (17Bieberich E. Freischütz B. Suzuki M. Yu R.K. J. Neurochem. 1999; 72: 1040-1049Crossref PubMed Scopus (57) Google Scholar). Analysis of the acidic lipids was performed by HPTLC using the solvent system CHCl3, CH3OH, 0.2% CaCl2 (50:45:10; v/v) whereas the analysis of ceramide was performed using the solvent system CH3OH/HOAc (9:1, v/v). Lipids were stained with 3% cupric acetate in 8% phosphoric acid and compared with standard lipids as described elsewhere (17Bieberich E. Freischütz B. Suzuki M. Yu R.K. J. Neurochem. 1999; 72: 1040-1049Crossref PubMed Scopus (57) Google Scholar, 18Macala L.J. Yu R.K. Ando S. J. Lipid Res. 1983; 25: 1243-1250Google Scholar). Gangliosides were specifically stained for by use of the resorcinol-HCl reagent. Preimplantation blastocyst embryonic stem cells (ES-J1) were grown on γ-irradiated feeder fibroblasts and neuronal differentiation was induced by serum deprivation of embryoid bodies (EBs) as previously described (19Hancock C.R. Wetherington J.P. Lambert N.A. Condie B. Biochem. Biophys. Res. Commun. 2000; 271: 418-421Crossref PubMed Scopus (45) Google Scholar). Protein was prepared from undifferentiated fibroblast-free ES-J1 cells, EBs, and differentiated neuroprogenitor cells at day 4 post-attachment of EBs on poly-l-ornithin/laminin-coated tissue culture dishes. Forebrains were prepared from mouse embryos extracted at days E12.5 to E18.5 by axial incision above the eye anlage followed by careful removal of the outer skin layers from the embryo head. The forebrains (5 to 20, depending on age) were taken up in 1 ml of deionized water and subjected to protein analysis and lipid extraction. For immunofluroescence microscopy, whole embryos were fixed by immersion in 10% formalin followed by gelatin embedding and saggital Vibratome sectioning at 30 μm thickness. The embryo slices were first permeabilized for 5 min with 0.5% Triton X-100 and subjected to the TUNEL assay as described by the manufacturer (Oncogene). For identification of single cells, the slices were stained for 30 min with 1 μg/ml Hoechst 33258 before immunostaining. The identification of complex gangliosides was performed by first incubating the embryo slices with 100 milliunits/ml Vibrio cholerae sialidase for 2 h at 37 °C which resulted in the conversion of a- and b-series complex gangliosides into GM1. The desialylation reaction was followed by staining of GM1 with 1 μg/ml Alexa 596-labeled cholera toxin subunit B (cholera toxin B) for another 2 h at 37 °C. The degree of cell death was determined by fluorescent staining of living and dead cells by the use of the Live/Dead Viability/Cytoxicity assay with cells cultivated on 96-well tissue culture dishes following the protocol given by the manufacturer (Molecular Probes). A quantitative analysis of apoptosis was performed using a TUNEL-based fluorescein-FragEL assay (Oncogene) by counting of TUNEL-positive cells in 5 areas with an average cell number of 200 cells that were grown on coverslips. Apoptosis was verified by visualization of fragmented DNA that was separated by agarose gel electrophoresis following the procedure of the Suicide Tracker DNA-laddering assay (Oncogene). The recording of whole-cell calcium currents was performed as described elsewhere (20Hamill O.P. Marty E. Neher E. Sakmann B. Sigworth F.J. Pflugers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15138) Google Scholar). Calcium influx was monitored using a bath solution composed of 20 mm BaCl2, 20 mm CsCl2, 110 mm tetraethylammonium chloride, 1.8 mmMgCl2, 15 mm glucose, and 0.1 μmtetrodotoxin in 10 mm HEPES-buffer adjusted to pH 7.4 with CsOH. The pipette solution contained 120 mm N-methyl-d-glucamine, 20 mmtetraethylammonium chloride, 11 mm EGTA, 1 mmCaCl2, 4 mm MgATP, 0.1 mmNa2GTP, and 14 mm phosphocreatine in 10 mm HEPES buffer adjusted to pH 7.2 with methanesulfonic acid. A holding potential of −80 mV was employed with depolarization currents evoked at −90 to 50 mV and digitized at 180 μs per point using low-pass filtering at 5 kHz (−3 dB). Analysis of the data was performed using a Power Macintosh 8600/200 computer supplied with PCI-16 Host Interface card. It was connected to an ITC-16 Data Acquisition Interface (Instrutech Corp, Port Washington, NY) utilizing Pulse Control 5.0 XOPs (obtained from Richard Bookman, Jack D. Herrington, and Kenneth R. Newton, University of Miami, FL) with Igor software (WavesMetrics, Lake Oswego, OR). The amount of protein was analyzed using a modification of the Folin phenol reagent (Lowry) assay as described (21Wang C.-S. Smith R.L. Anal. Biochem. 1975; 63: 414-417Crossref PubMed Scopus (632) Google Scholar). Protein precipitation was performed according to the Wessel and Flügge method (22Wessel D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3163) Google Scholar). SDS-PAGE was carried out using the Laemmli method followed by immunoblotting as described (23Gershoni J.M. Palade G.E. Anal. Biochem. 1983; 13: 1-15Crossref Scopus (646) Google Scholar). Primary antibodies were used at a concentration of 1 μg/ml for immunoblotting and 5 μg/ml for immunofluorescence staining. Forebrains were removed from mouse embryos and analyzed for the expression of gangliosides and ceramide. Fig.1 A shows that the predominant gangliosides at day E12.5 were GM3 and GD3 (lane 2). BCGs were detectable at day E14.5 (lane 3), which was followed by the decline of GM3 and GD3 at day E16.5 (lane 4). The expression of GM1 and GD1a was initiated between days E16.5 (lane 4) and E18.5 (lane 5) and then intensified during post-natal development (P7, lane 6; adult, lane 7). The expression of ceramide was already found at day E12.5 (Fig. 1 B, lane 1) and increased by about 40% at day E14.5 as determined by densitometry (lane 2). This was followed by a decline by more than 50% from day E14.5 to E18.5 (Fig.1 B, lanes 2–4). The ceramide composition resolved into three bands, the middle band of which was intensified during post-natal development (P7, lane 5; P21, lane 6; adult, lane 7). The structural composition of the different ceramide species has not been characterized yet, but the separation of these bands may be a result of the differences in the fatty acid chain length (17Bieberich E. Freischütz B. Suzuki M. Yu R.K. J. Neurochem. 1999; 72: 1040-1049Crossref PubMed Scopus (57) Google Scholar). A quantitative HPTLC with stearoyl-sphingosine as standard (Fig. 1 B, lanes 8–11) revealed that the ceramide level at peak time day E14.5 was about 0.6 ± 0.1 μg of ceramide/mg of cell protein which is equivalent to a tissue concentration of ∼25 ± 5 μm (based on brain wet weight). F-11A-cells were transfected with a cDNA construct composed of a 1341-base pair sequence encoding ST2 and a C-terminal linked FLAG-epitope followed by the sequence of epidermal growth factor protein. Several independent clones were isolated by fluorescence-activated cell sorting and were found to have a 4-fold higher specific ST2 activity than that of untransfected or GFP transfected control cells. As shown in Fig.2 A, expression of ST2-FLAG-GFP resulted in an altered cell morphology revealing bipolar process formation and completely obliterated clustering. Fig. 2 Bshows that ST2-FLAG-GFP was localized in the Golgi apparatus which was identical to the subcellular distribution found with wild-type ST2 (11Bieberich E. Tencomnao T. Kapitonov D. Yu R.K. J. Neurochem. 2000; 74: 2359-2364Crossref PubMed Scopus (24) Google Scholar). SDS-gel electrophoresis and immunoblotting of a protein preparation from transfected cells showed the expression of a 75-kDa protein corresponding to the molecular mass of ST2-FLAG-GFP (Fig.2 B). Sphingolipids were isolated from transfected cells and analyzed by high-performance thin-layer chromatography (HPTLC). As shown in Fig. 2 C, the neutral sphingolipid fraction of ST2-FLAG-GFP-transfected cells (lanes 7 and10) revealed no difference in the expression of glycosphingolipids, sphingomyelin, or ceramide as compared with F-11A cells that were only transfected with GFP (lanes 6 and9). Upon stable expression of ST2-FLAG-GFP the major gangliosides changed from GM3 found in GFP-transfected cells (lane 3) to GT1b and GQ1b (lane 4) which amounted to 50% of the total gangliosides. GD3, GD2, and GD1b were expressed in smaller amounts (40% of total gangliosides) whereas GM3 was only detectable in trace amounts. This result indicated that the expression of ST2-FLAG-GFP shifted the ganglioside composition from a- to b-series and enhanced the amount of complex gangliosides (b-series complex gangliosides or BCGs). As shown in Figs. 1 A (lane 4) and 2C (lane 4), the composition of gangliosides in ST2-FLAG-GFP-transfected F-11A cells was almost identical to that in mouse brain at day E16.5 of embryonal development. The effect of ganglioside expression on ceramide-induced apoptosis was investigated by overnight incubation of untransfected, GFP, or ST2-FLAG-GFP-transfected cells with 30 μm N-acetylsphingosine (C2-ceramide) or 80 μm of the novel ceramide analog S18. GFP-transfected cells did not reveal any difference in the degree of apoptosis as compared with untransfected cells. As shown in Table I, however, ST2-FLAG-GFP expressing cells revealed up to a 70% reduction in apoptosis depending on the concentration and type of the pro-apoptotic agent used for the incubation reaction. This result was verified by a DNA fragmentation assay that showed no appearance of 200-base pair fragments (DNA laddering) upon incubation of ST2-FLAG-GFP-transfected cells with S18 (Fig. 3 A, lane 4) whereas GFP-transfected cells revealed intensive DNA laddering (Fig. 3 A, lane 2). It should be noted, however, that the degree of apoptosis was critically depending on cell density as previously reported (11Bieberich E. Tencomnao T. Kapitonov D. Yu R.K. J. Neurochem. 2000; 74: 2359-2364Crossref PubMed Scopus (24) Google Scholar). All of the experiments were performed at exactly 50% confluence, a cell density that previously revealed the highest degree of apoptosis induced with ceramide analogs in untransfected F-11A cells.Table IC2-ceramide or S18-induced cell death in GFP or ST2-FLAG-GFP transfected F-11A cellsGFP-F-11AST2-FLAG-GFP-F-11A+ Serum− Serum+ Serum− Serum20 h35 h48 h20 h35 h48 h20 h35 h48 h20 h35 h48 hControl< 5< 5< 5< 51255< 5< 5< 5< 51676C2-ceramide (30 μm)9598294360335280S18 (80 μm)839898273848324891GD1b + S18 (80 μm)417385GT1b + S18 (80 μm)7595GQ1b + S18 (80 μm)657895NBdNJ (250 μm)< 5< 5< 5< 5< 5< 5NBdNJ + C2-cer9398NBdNJ + S18 (80 μm)9895PD98059 (50 μm)< 588< 578PD98059 + C2-cer9898PD98059 + S189398NGF (100 ng/ml)< 5< 57< 5< 532NGF + S18 (80 μm)98223582NGF + C2-cer (30 μm)98357998N2 supplement< 5< 58< 5< 512N2 + S18 (80 μm)98112348ITS supplement< 5< 510< 5< 515Insulin (5 μg/ml)< 51120< 51325Insulin + S18 (80 μm)98152961Insulin (100 ng/ml)< 51845< 51550IGF-1 (100 ng/ml)< 5< 58< 5818IGF-1 + S18 (80 μm)98102552R31GF-1 (100 ng/ml)< 5< 810< 5< 515Cells were cultivated in 96-well dishes and incubated with 30 μm N-acetylsphingosine (C2-cer) or 80 μm N-oleoylserinol (S18) in serum-supplemented or serum-free medium for times indicated. Supplements included the addition of different BCGs (40 μm), N2 or ITS-supplement (1:100), or single growth factors at a concentration of 100 ng/ml (NGF, IGF-1, LongR3-IGF-1 peptide analog, insulin) or 5 μg/ml (insulin). The effect of different inhibitors was analyzed by preincubation for 5 h with 50 μm PD98059 (MEK1-inhibitor) or for 72 h with 250 μm NB-DNJ (glycolipid biosynthesis inhibitor). The number of living cells was quantified by the determination of calcein-esterase activity, and the number of dead cells was determined by diethidium bromide staining (Dead/Alive assay, Molecular Probes). All measurements were done with a sample size ofn = 6. The mean standard variation was 15% of the average value as given in the table. Open table in a new tab Cells were cultivated in 96-well dishes and incubated with 30 μm N-acetylsphingosine (C2-cer) or 80 μm N-oleoylserinol (S18) in serum-supplemented or serum-free medium for times indicated. Supplements included the addition of different BCGs (40 μm), N2 or ITS-supplement (1:100), or single growth factors at a concentration of 100 ng/ml (NGF, IGF-1, LongR3-IGF-1 peptide analog, insulin) or 5 μg/ml (insulin). The effect of different inhibitors was analyzed by preincubation for 5 h with 50 μm PD98059 (MEK1-inhibitor) or for 72 h with 250 μm NB-DNJ (glycolipid biosynthesis inhibitor). The number of living cells was quantified by the determination of calcein-esterase activity, and the number of dead cells was determined by diethidium bromide staining (Dead/Alive assay, Molecular Probes). All measurements were done with a sample size ofn = 6. The mean standard variation was 15% of the average value as given in the table. The involvement of gangliosides in the reduction of apoptosis was analyzed by preincubation of GFP or ST2-FLAG-GFP-transfected cells with 250 μm of the glycolipid biosynthesis inhibitor NB-DNJ prior to the addition of S18 or C2-ceramide. As shown in Fig.2 C (lane 5), ST2-FLAG-GFP-transfected cells were completely deprived of gangliosides. NB-DNJ did not affect the degree of ceramide-induced apoptosis in GFP-transfected control cells but restored that of ST2-FLAG-GFP-transfected cells to control level (TableI). This was verified by the appearance of DNA laddering that was induced by S18 after preincubation with NB-DNJ (Fig. 3 A, lane 3). The expression of the pro-apoptotic protein PAR-4 was analyzed by means of indirect immunofluorescence microscopy in combination with a TUNEL assay in untransfected F-11A cells upon incubation with S18. As shown in Fig. 3 A (right panel), more than 80% of the TUNEL-positive cells were strongly stained for PAR-4 indicating that its expression is necessary for the induction of apoptosis. The number of strongly stained cells, however, exceeded that of TUNEL-positive cells. ST2-FLAG-GFP-transfected cells expressed about 80% less PAR-4 and showed no TUNEL staining (not shown). The overall expression of PAR-4 was further analyzed by SDS-PAGE and immunoblotting of protein extracts from detergent-solubilized cells. Fig. 3 B shows that the amount of immunostained PAR-4 in ST2-FLAG-GFP-transfected cells (lane 2) was only 20% of that in GFP-transfected control cells (lane 1) consistent with the results from immunofluorescence microscopy. Incubation of ST2-FLAG-GFP-transfected cells with NB-DNJ or the MEK1-inhibitor PD98059 increased PAR-4 expression to the level of control cells (lanes 3). The expression of the anti-apoptotic protein bcl-2 was analyzed by immunoblotting as described before and was found to be about 3–4-fold elevated in ST2-FLAG-GFP-transfected cells (lane 2) as compared with control cells (lane 1). ST2-FLAG-GFP-transfected cells preincubated with NB-DNJ (lane 3) or PD98059 (lane 4), however, showed restoration of the bcl-2 expression level to that of control cells (lane 1). The effects of growth factors on cell proliferation and apoptosis were analyzed by incubation of GFP or ST2-FLAG-GFP-transfected F-11A cells with N2, ITS (insulin-transferrin-selenite), NGF, bFGF/FGF-2, insulin, or IGF-1 supplemented serum-free medium. Removal of serum resulted in an increased cell death in GFP or ST2-FLAG-GFP-transfected cells, even without the addition of C2-ceramide or S18 to the medium (Table I). Cell death resulted from apoptosis as revealed by punctate Hoechst staining of the nuclei. Apoptosis, however, could be prevented by the addition of 0.5% FBS, N2, or ITS supplement to the cell culture medium. Among single growth factors, it was found that NGF (100 ng/ml), insulin (5 μg/ml), IGF-1 (100 ng/ml), or its peptide analog LongR3-IGF-1 (100 ng/ml) could reduce cell death by at least 70% (Table I). Ins
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