Interleukin-1β Induces Chronic Activation and de Novo Synthesis of Neutral Ceramidase in Renal Mesangial Cells
2001; Elsevier BV; Volume: 276; Issue: 38 Linguagem: Inglês
10.1074/jbc.m102153200
ISSN1083-351X
AutoresRochus Franzen, Andrea Pautz, Lutz Bräutigam, Gerd Geißlinger, Josef Pfeilschifter, Andrea Huwiler,
Tópico(s)Lysosomal Storage Disorders Research
ResumoThe lipid signaling molecule ceramide is formed by the action of acid and neutral sphingomyelinases and degraded by acid and neutral ceramidases. Short-term stimulation of mesangial cells with the pro-inflammatory cytokine interleukin-1β (IL-1β) leads to a rapid and transient increase in neutral sphingomyelinase activity (Kaszkin, M., Huwiler, A., Scholz, K., van den Bosch, H., and Pfeilschifter, J. (1998) FEBS Lett. 440, 163–166). In this study, we report on a second delayed peak of activation occurring after hours of IL-1β treatment. This second phase of activation was first detectable after 2 h of treatment and steadily increased over the next 2 h, reaching maximal values after 4 h. In parallel, a pronounced increase in neutral ceramidase activity was observed, accounting for a constant or even decreased level of ceramide after long-term IL-1β treatment, despite continuous sphingomyelinase activation. The increase in neutral ceramidase activity was due to expressional up-regulation, as detected by an increase in mRNA levels and enhanced de novo protein synthesis. The increase in neutral ceramidase protein levels and activity could be blocked dose- dependently by the p38 MAPK inhibitor SB 202190, whereas the classical MAPK pathway inhibitor U0126 and the protein kinase C inhibitor Ro 318220 were ineffective. Moreover, cotreatment of cells for 24 h with IL-1β and SB 202190 led to an increase in ceramide formation. Interestingly, IL-1β-stimulated neutral ceramidase activation was not reduced in mesangial cells isolated from mice deficient in MAPK-activated protein kinase-2, which is a downstream substrate of p38 MAPK, thus suggesting that the p38 MAPK-mediated induction of neutral ceramidase occurs independently of the MAPK-activated protein kinase-2 pathway. In summary, our results suggest a biphasic regulation of sphingomyelin hydrolysis in cytokine-treated mesangial cells with delayed de novosynthesis of neutral ceramidase counteracting sphingomyelinase activity and apoptosis. Neutral ceramidase may thus represent a novel cytoprotective enzyme for mesangial cells exposed to inflammatory stress conditions. The lipid signaling molecule ceramide is formed by the action of acid and neutral sphingomyelinases and degraded by acid and neutral ceramidases. Short-term stimulation of mesangial cells with the pro-inflammatory cytokine interleukin-1β (IL-1β) leads to a rapid and transient increase in neutral sphingomyelinase activity (Kaszkin, M., Huwiler, A., Scholz, K., van den Bosch, H., and Pfeilschifter, J. (1998) FEBS Lett. 440, 163–166). In this study, we report on a second delayed peak of activation occurring after hours of IL-1β treatment. This second phase of activation was first detectable after 2 h of treatment and steadily increased over the next 2 h, reaching maximal values after 4 h. In parallel, a pronounced increase in neutral ceramidase activity was observed, accounting for a constant or even decreased level of ceramide after long-term IL-1β treatment, despite continuous sphingomyelinase activation. The increase in neutral ceramidase activity was due to expressional up-regulation, as detected by an increase in mRNA levels and enhanced de novo protein synthesis. The increase in neutral ceramidase protein levels and activity could be blocked dose- dependently by the p38 MAPK inhibitor SB 202190, whereas the classical MAPK pathway inhibitor U0126 and the protein kinase C inhibitor Ro 318220 were ineffective. Moreover, cotreatment of cells for 24 h with IL-1β and SB 202190 led to an increase in ceramide formation. Interestingly, IL-1β-stimulated neutral ceramidase activation was not reduced in mesangial cells isolated from mice deficient in MAPK-activated protein kinase-2, which is a downstream substrate of p38 MAPK, thus suggesting that the p38 MAPK-mediated induction of neutral ceramidase occurs independently of the MAPK-activated protein kinase-2 pathway. In summary, our results suggest a biphasic regulation of sphingomyelin hydrolysis in cytokine-treated mesangial cells with delayed de novosynthesis of neutral ceramidase counteracting sphingomyelinase activity and apoptosis. Neutral ceramidase may thus represent a novel cytoprotective enzyme for mesangial cells exposed to inflammatory stress conditions. interleukin-1β tumor necrosis factor-α polyacrylamide gel electrophoresis polymerase chain reaction glyceraldehyde-3-phosphate dehydrogenase base pair mitogen-activated protein kinase mitogen-activated protein kinase-activated protein kinase-2 extracellular signal-regulated kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase The mesangial cell is a smooth muscle cell-like pericyte located in the renal glomerulus and is a key player in the glomerular inflammatory response (1Pfeilschifter J. Eur. J. Clin. Invest. 1989; 19: 347-361Crossref PubMed Scopus (100) Google Scholar, 2Pfeilschifter J. News Physiol. Sci. 1994; 9: 271-276Google Scholar, 3Kashgarian M. Sterzel R.B. Kidney Int. 1992; 41: 524-529Abstract Full Text PDF PubMed Scopus (178) Google Scholar). Inflammatory diseases of the renal glomerulus are accompanied by enhanced formation of the pro-inflammatory cytokine interleukin-1β (IL-1β).1 The primary source is the activated macrophage, but IL-1β is also released by many other cell types after exposure to an inflammatory environment. Soluble IL-1β is the predominant form in biological fluids, and it binds to specific receptors in target tissues. IL-1 is an exemplary pro-inflammatory cytokine that is particularly important in the systemic response to inflammation. It synergizes with tumor necrosis factor-α (TNF-α) for many of its actions, and its synthesis is stimulated, in turn, by TNF-α. Furthermore, it is implicated in the pathogenesis of diseases such as rheumatoid arthritis, inflammatory bowel disease, septic shock, and several autoimmune reactions. In the past, it has become clear that sphingolipids exert important roles as signaling molecules under various physiological and pathophysiological conditions (4Huwiler A. Kolter T. Pfeilschifter J. Sandhoff K. Biochim. Biophys. Acta. 2000; 1485: 63-99Crossref PubMed Scopus (377) Google Scholar, 5Perry D.K. Hannun Y.A. Biochim. Biophys. Acta. 1998; 1436: 233-243Crossref PubMed Scopus (296) Google Scholar, 6Riboni L. Viani P. Bassi R. Prinetti A. Tettamanti G. Prog. Lipid Res. 1997; 36: 153-195Crossref PubMed Scopus (189) Google Scholar, 7Levade T. Jaffrézou J.P. Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (283) Google Scholar). Especially ceramide has attracted a lot of interest due to its potential involvement in regulation of programmed cell death, cell growth arrest, and differentiation (4Huwiler A. Kolter T. Pfeilschifter J. Sandhoff K. Biochim. Biophys. Acta. 2000; 1485: 63-99Crossref PubMed Scopus (377) Google Scholar, 5Perry D.K. Hannun Y.A. Biochim. Biophys. Acta. 1998; 1436: 233-243Crossref PubMed Scopus (296) Google Scholar, 6Riboni L. Viani P. Bassi R. Prinetti A. Tettamanti G. Prog. Lipid Res. 1997; 36: 153-195Crossref PubMed Scopus (189) Google Scholar, 7Levade T. Jaffrézou J.P. Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (283) Google Scholar). However, the regulating mechanisms that determine the intracellular ceramide level are still poorly understood. Most studies have focused on the ceramide-generating enzymes, i.e. the acid and neutral sphingomyelinases. Based on activity measurements from cell extracts, activators of acid and/or neutral sphingomyelinases have been determined and include pro-inflammatory cytokines, growth factors, and other environmental stress stimuli (4Huwiler A. Kolter T. Pfeilschifter J. Sandhoff K. Biochim. Biophys. Acta. 2000; 1485: 63-99Crossref PubMed Scopus (377) Google Scholar, 7Levade T. Jaffrézou J.P. Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (283) Google Scholar). However, sphingomyelinases depict only one side of the regulation of ceramide levels. It is equally important to understand the involvement of ceramide-degrading enzymes, the ceramidases, which hydrolyze ceramide to yield sphingosine. Sphingosine on its own can act either in a proliferative (8Merrill Jr., A.H. Schmelz E.M. Dillehay D.L. Spiege S. Shayman J.A. Schroeder J.J. Riley R.T. Voss K.A. Wang E. Toxicol. Appl. Pharmacol. 1997; 142: 208-225Crossref PubMed Scopus (564) Google Scholar, 9Coroneos E. Wang Y. Panuska J.R. Templeton D.J. Kester M. Biochem. J. 1996; 316: 13-17Crossref PubMed Scopus (106) Google Scholar, 10Olivera A. Zhang H. Carlson R.O. Mattie M.E. Schmidt R.R. Spiegel S. J. Biol. Chem. 1994; 269: 17924-17930Abstract Full Text PDF PubMed Google Scholar) or pro-apoptotic (11Jarvis W.D. Fornari Jr., F.A. Auer K.L. Freemerman A.J. Szabo E. Birrer M.J. Johnson C.R. Barbour S.E. Dent P. Grant S. Mol. Pharmacol. 1997; 52: 935-947Crossref PubMed Scopus (138) Google Scholar, 12Sweeney E.A. Inokuchi J. Igarashi Y. FEBS Lett. 1998; 425: 61-65Crossref PubMed Scopus (94) Google Scholar, 13Hung W.C. Chang H.C. Chuang L.Y. Biochem. J. 1999; 338: 161-166Crossref PubMed Scopus (80) Google Scholar, 14Sakakura C. Sweeney E.A. Shirahama T. Hagiwara A. Yamaguchi T. Takahashi T. Hakomori S. Igarashi Y. Biochem. Biophys. Res. Commun. 1998; 246: 827-830Crossref PubMed Scopus (38) Google Scholar) manner depending on the cell system, but it can also serve as a substrate for sphingosine kinase to yield sphingosine 1-phosphate (8Merrill Jr., A.H. Schmelz E.M. Dillehay D.L. Spiege S. Shayman J.A. Schroeder J.J. Riley R.T. Voss K.A. Wang E. Toxicol. Appl. Pharmacol. 1997; 142: 208-225Crossref PubMed Scopus (564) Google Scholar), which is a potent mitogen for several cell types (15An S. Goetzl E.J. Lee H. J. Cell. Biochem. 1998; 30–31: 147-157Crossref Google Scholar,16Van Brocklyn J.R. Lee M.J. Menzeleev R. Olivera A. Edsall L. Cuvillier O. Thomas D.M. Coopman P.J. Thangada S. Liu C.H. Hla T. Spiegel S. J. Cell Biol. 1998; 142: 229-240Crossref PubMed Scopus (446) Google Scholar). Due to this equally important role of ceramidases in determining cellular levels of ceramide, which is a pro-apoptotic stimulus, and sphingosine 1-phosphate being a proliferative stimulus, it is essential to understand the regulation of these enzymes. It has become clear that there are at least two subtypes of ceramidases existing in mammalian cells: an acidic form, which is localized in the lysosomes (17Li C.M. Hong S.B. Kopal G. He X. Linke T. Hou W.S. Koch J. Gatt S. Sandhoff K. Schuchman E.H. Genomics. 1998; 50: 267-274Crossref PubMed Scopus (95) Google Scholar), the main organelle involved in lipid degradation, and a neutral/alkaline form (18Tani M. Okino N. Mori K. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 11229-11234Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 19Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), which has only recently been cloned and about which not much is known regarding its localization or activation. It is tempting to speculate that this enzyme plays an equally important role in signal transduction as the sphingomyelinase and it counterbalances ceramide generation by the latter enzyme. Biochemical characterization of this novel neutral ceramidase reveals that it is a 94-kDa enzyme in mouse tissue (18Tani M. Okino N. Mori K. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 11229-11234Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and a 110–120-kDa enzyme in rat tissue (19Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), containing several putative protein kinase C and casein kinase II phosphorylation sites in its primary sequence. Recently, El Bawab et al. (20El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) cloned another human neutral ceramidase that contained an N-terminal mitochondrial signal peptide and therefore was suggested to be a mitochondrial enzyme. In this study, we investigated the effect of the pro-inflammatory cytokine IL-1β on the neutral sphingomyelinase and neutral ceramidase activities in rat mesangial cells. We show that chronic IL-1β treatment of mesangial cells leads to a biphasic activation of the neutral sphingomyelinase and a delayed activation of the neutral ceramidase, ultimately leaving cellular ceramide levels low. The activation of the neutral ceramidase is due to expressional up-regulation of the gene. [14C]Serine (specific activity, 53 Ci/mol), [14C]sphingomyelin (specific activity, 55 Ci/mol), [35S]methionine and [35S]cysteine pro-mixture (specific activity, >1000 Ci/mmol), [α-32P]dCTP (specific activity, 3000 Ci/mmol), and protein A-Sepharose CL-4B were from Amersham Pharmacia Biotech(Freiburg, Germany). [14C]Ceramide (specific activity, 55 Ci/mol) was from ICN Biomedicals GmbH (Eschwege, Germany). SB 202190, U0126 and Ro 318220 were from Calbiochem-Novabiochem (Schwabach, Germany). All cell culture nutrients were from Life Technologies, Inc. (Karlsruhe, Germany). Interleukin-1β was kindly provided by Novartis Pharma Ltd. TNF-α was a gift of Knoll AG (Ludwigshafen, Germany). An antibody against acid ceramidase was kindly provided by Prof. K. Sandhoff (University of Bonn, Bonn, Germany). A synthetic peptide (ENHKDSGNHWFSTC) based on the N-terminal sequence of the murine neutral ceramidase (GenBankTM/EBI Data Bank accession number AB037111) was synthesized, coupled to keyhole limpet hemocyanin, and used to immunize rabbits. For characterization of the antibody, lysates of IL-1β-stimulated (8 h) rat mesangial cells were separated on a MonoQ column coupled to a BioLogic FPLC® system. The cell lysate was loaded into 25 mm Tris (pH 7.4) and eluted with a linear gradient of 1 m NaCl in 25 mmTris (pH 7.4) at a flow rate of 2 ml/min. The eluted fractions were analyzed by Western blotting and neutral ceramidase activity assay. Rat mesangial cells were cultivated and characterized as described previously (21Pfeilschifter J. Biochem. J. 1990; 272: 469-472Crossref PubMed Scopus (90) Google Scholar). In a second step, single cells were cloned by limited dilution on 96-well plates. Clones with apparent mesangial cell morphology were characterized by positive staining for the intermediate filaments desmin and vimentin (considered to be specific for myogenic cells), positive staining for Thy1.1 antigen, and negative staining for Factor VIII-related antigen and cytokeratin (excluding endothelial and epithelial contaminations, respectively). For the experiments, passages 8–20 were used. Confluent mesangial cells in 60-mm diameter dishes were stimulated for the indicated time periods in Dulbecco's modified Eagle's medium containing 0.1 mg/ml fatty acid-free bovine serum albumin. To stop the reaction, the medium was removed, and the cells were washed with ice-cold phosphate-buffered saline. Cells were then scraped directly into lysis buffer (50 mm Hepes (pH 7.4), 150 mm NaCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 10% glycerol, 1% Triton X-100, 20 mmβ-glycerophosphate, 50 mm sodium fluoride, 1 mm Na3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 μm pepstatin A, and 1 mmphenylmethylsulfonyl fluoride) and homogenized by 10 passes through a 26-gauge needle fitted to a 1-ml syringe. The homogenate was centrifuged for 10 min at 14,000 × g, and the supernatant was taken for protein determination. 100 μg of protein were separated by SDS-PAGE and transferred to nitrocellulose membrane, and Western blot analysis was performed as previously described (22Huwiler A. Wartmann M. van den Bosch H. Pfeilschifter J. Br. J. Pharmacol. 2000; 129: 612-618Crossref PubMed Scopus (50) Google Scholar) using polyclonal antibodies against neutral and acid ceramidases at dilutions of 1: 500 and 1:2000, respectively. Confluent mesangial cells in 100-mm diameter dishes were washed with phosphate-buffered saline and incubated in methionine-free Dulbecco's modified Eagle's medium in the absence or presence of the stimulators for the indicated time periods. For the last 4 h of incubation, [35S]methionine and [35S]cysteine were added (140 μCi/plate). After labeling, cells were washed twice with ice-cold phosphate-buffered saline. Cells were then scraped directly into 1 ml of lysis buffer and homogenized. The homogenate was centrifuged for 10 min at 14,000 × g, and the supernatant was taken for immunoprecipitation. Samples of 1-ml volume containing 250 × 106 cpm of labeled proteins, 5% fetal calf serum, and 1.5 mmiodoacetamide in lysis buffer were incubated overnight at 4 °C with a polyclonal antiserum against the neutral ceramidase at a dilution of 1:100. 100 μl of a 50% slurry of protein A-Sepharose CL-4B in phosphate-buffered saline were then added, and the mixture was rotated for 1 h at room temperature. After centrifugation for 5 min at 3000 × g, immunocomplexes were washed three times with low salt buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 0.2% Triton X-100, 2 mm EDTA, 2 mm EGTA, and 0.1% SDS), three times with high salt buffer (50 mm Tris-HCl (pH 7.5), 500 mm NaCl, 0.2% Triton X-100, 2 mm EDTA, 2 mm EGTA, and 0.1% SDS), and once with 10 mm Tris. Pellets were boiled for 5 min in Laemmli dissociation buffer and subjected to SDS-PAGE. After fixing in 25% isopropyl alcohol and 10% acetic acid, the gels were dried and exposed on a Bio-Imaging Analyzer (Fuji). Total RNA was isolated using guanidinium isothiocyanate solution. 1.5 μg of RNA were used for reverse transcriptase-PCR (first strand cDNA synthesis kit, MBI Fermentas, St. Leon-Rot, Germany). PCR was carried out as follows (Taq DNA polymerase, recombinant, MBI Fermentas, St. Leon-Rot, Germany): 94 °C for 5 min (one cycle); 94 °C for 1 min, 52 °C for 1.5 min, and 72 °C for 1 min (with a variable number of cycles); and a final extension at 72 °C for 7 min. The number of cycles was 30 for murine neutral ceramidase, 35 for rat neutral ceramidase, and 25 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of the primers for analysis of mRNA were as follows: mouse neutral ceramidase, TTC AAT TCG GGA CTT CAG TGG (forward) and CAA GAA TGT TGG GTG ACA CG (reverse); rat neutral ceramidase, TGA AGA CGT GTA AAG CCG C (forward) and TGC GAT AAC GAC AGT CAT ATC C (reverse); and GAPDH, AAT GCA TCC TGC ACC ACC AA (forward) and GTC ATT GAG AGC AAT GCC AGC (reverse). PCR products (793-bp length for mouse neutral ceramidase, 377-bp length for rat neutral ceramidase, and 470-bp length for GAPDH) were run on a 1.5% agarose gel containing 0.5 μg/ml ethidium bromide. The identities of amplicons were confirmed by sequencing using a Model 310 genetic analyzer (PerkinElmer Life Sciences). Total RNA was isolated using guanidinium isothiocyanate solution. 25 μg of RNA were separated by electrophoresis on formaldehyde-containing 1% agarose gels. RNA was transferred to a nylon membrane by vacuum blotting for 2 h at 55 millibars and cross-linked by UV light. Blots were hybridized with a 540-bp reverse transcriptase-PCR product (forward primer, CCA GTG GGT GAA CAT GAC AG; and reverse primer, GAT GTA TGC AGA CAG GGT GT) for the rat neutral ceramidase and a 1206-bp reverse transcriptase-PCR product (forward primer, GGG GTA CCT GGG AAG ATG GGG GGC CAA AGT CTT CTC; and reverse primer, GAC TAC TGC TCA CCA GCC TAT ACA AG) for the acid ceramidase, which were labeled with [α-32P]dCTP using the Multiprime DNA labeling System (Amersham Pharmacia Biotech). Hybridization was carried out at 42 °C for 16 h, and the membranes were exposed on Bio-Imaging Analyzer (Fuji). To correct for variations in RNA amounts, blots were finally rehybridized with a32P-labeled GAPDH cDNA probe. Confluent mesangial cells in 30-mm diameter dishes were labeled for 24 h with [14C]serine (0.2 μCi/ml) and stimulated as indicated. Lipids were extracted (23Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1953; 37: 911-917Crossref Scopus (42689) Google Scholar), and ceramide was resolved by sequential one-dimensional thin-layer chromatography using chloroform/methanol/ammonium hydroxide solution 25% (65:35:7.5, v/v), followed by chloroform/methanol/acetic acid (9:1:1, v/v). Spots corresponding to ceramide were analyzed and quantified using a Bio-Imaging Analyzer. Alternatively, ceramide was quantitated by liquid chromatography-tandem mass spectrometry. The liquid chromatography unit consisted of a Jasco DG-1580-53 degasser, a Jasco LG-1580-02 ternary gradient unit, a Jasco PU-1585 pump, and a Jasco AS-1550 autosampler (Jasco, Gross-Umstadt, Germany). The mass spectrometer consisted of a PE Sciex API 3000 triple quadrupole mass spectrometer (Applied Biosystems, Langen, Germany) equipped with a turbo ion spray interface. Nitrogen (high purity) was supplied by a Whatman nitrogen generator (Whatman GmbH, Göttingen, Germany). Chromatographic separation of extracted samples was performed in isocratic mode with a Nucleosil C18 column (30 × 2.0-mm inner diameter, 5-μm particle size, and 100-Å pore size; Macherey-Nagel, Düren, Germany). The mobile phase consisted of methanol containing 5 mm ammonium acetate. The flow rate was set at 0.2 ml/min. The injection volume was 10 μl, and the run time was 3 min. The turbo ion spray interface was operated in the positive ion mode at 5200 V and 200 °C and was supplied by an auxiliary gas flow of 4500 ml/min. The nebulizer gas was set at 1.23 liters/min (setting 10); the curtain gas flow was set at 1.08 liters/min (setting 9); and the collision gas was set at 3.7 × 10−6 hectopascals (3.02 × 1015molecules/cm2; setting 4). Nitrogen was used for all gases. C16:0 ceramide standards and cellular lipid extracts were resuspended in 1000 μl of 5 mm ammonium acetate/methanol buffer just prior to mass spectrometric analysis. Standards were analyzed at concentrations ranging from 25 nm to 10 μm. Quantitation was performed by multiple reaction monitoring (dwell time, 200 ms) of the protonated precursor ion and related product ions. The mass transition used for quantification was m/z 538.4 → 264.2 (collision energy, 33 eV). The mass transitions used as qualifier were m/z 538.4 → 82.1 (collision energy, 77 eV) and 538.4 → 252.1 (collision energy, 39 eV). The analytical data were processed by Analyst software (Version 1.1). Confluent mesangial cells in 60-mm diameter dishes were incubated with the indicated stimuli in Dulbecco's modified Eagle's medium containing 0.1 mg/ml fatty acid-free bovine serum albumin for the indicated time periods. Neutral and acid sphingomyelinase activities were measured according to Liu and Hannun (24Liu B. Hannun Y.A. J. Biol. Chem. 1997; 272: 16281-16287Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar) and Quintern et al. (25Quintern L.E. Weitz G. Nehrkorn H. Tager J.M. Schram A.W. Sandhoff K. Biochim. Biophys. Acta. 1987; 922: 323-336Crossref PubMed Scopus (113) Google Scholar) with some modifications as previously described (26Huwiler A. Pfeilschifter J. van den Bosch H. J. Biol. Chem. 1999; 274: 7190-7195Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Confluent mesangial cells were stimulated as described above and homogenized in lysis buffer containing 50 mm sodium acetate (pH 4.5), 0.5% Triton X-100, 5 mm MgCl2, 1 mm EDTA, and 5 mmd-galactonic acid γ-lactone for the acid ceramidase and 50 mm Tris (pH 7.5), 0.5% Triton X-100, 5 mm MgCl2, 1 mm EDTA, and 5 mmd-galactonic acid γ-lactone for the neutral ceramidase. Activity assays were performed according to Mitsutake et al. (27Mitsutake S. Kita K. Okino N. Ito M. Anal. Biochem. 1997; 247: 52-57Crossref PubMed Scopus (46) Google Scholar) with some modifications as previously described (26Huwiler A. Pfeilschifter J. van den Bosch H. J. Biol. Chem. 1999; 274: 7190-7195Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Statistical analysis was performed by one-way analysis of variance. For multiple comparisons with the same control group, the limit of significance was divided by the number of comparisons according to Bonferroni. Previously, we have shown that the pro-inflammatory cytokine IL-1β causes a rapid (within minutes) and transient activation of neutral sphingomyelinase activity in rat mesangial cells (28Kaszkin M. Huwiler A. Scholz K. van den Bosch H. Pfeilschifter J. FEBS Lett. 1998; 440: 163-166Crossref PubMed Scopus (23) Google Scholar), which leads to increased ceramide formation (28Kaszkin M. Huwiler A. Scholz K. van den Bosch H. Pfeilschifter J. FEBS Lett. 1998; 440: 163-166Crossref PubMed Scopus (23) Google Scholar, 29Huwiler A. Brunner J. Hummel R. Vervoordeldonk M. Stabel S. van den Bosch H. Pfeilschifter J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6959-6963Crossref PubMed Scopus (184) Google Scholar). We now have extended these studies and found that prolonged treatment of mesangial cells with IL-1β resulted in a delayed second peak of neutral sphingomyelinase activation that was first detectable after 2 h of stimulation and reached a maximum after 4 h (Fig. 1 A). The acid sphingomyelinase also showed a time-dependent delayed activation after IL-1β treatment (Fig. 1 B). Surprisingly, when the level of ceramide was measured by tandem mass spectrometry after IL-1β stimulation, no increase was observed up to 24 h after stimulation (Fig. 1 C), thus pointing toward additional compensatory mechanisms that regulate the ceramide content of the cell. In contrast, 1 h of stimulation with a bacterial sphingomyelinase led to an 8–10-fold increase in ceramide levels (Fig.1 C). To investigate the effect of IL-1β on the ceramide-degrading enzymes, rat mesangial cells were stimulated for different time periods with the cytokine, and ceramidase activity was measured. As shown in Fig. 2(A and B), IL-1β caused a chronic activation of acid and neutral ceramidases, with maximal stimulation occurring 4 h after cytokine exposure and subsequently remaining at high levels. To test whether the increase in neutral ceramidase activity is due to an increased amount of neutral ceramidase protein, Western blot analysis was performed using a polyclonal antiserum raised against a peptide comprising the N terminus of the murine neutral ceramidase. The antiserum recognized a double band of ∼110–120 kDa. This size is in agreement with the recently described size of rat kidney neutral ceramidase (19Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). To determine whether the recognized protein does indeed show neutral ceramidase activity, cell lysates from IL-1β-stimulated mesangial cells were separated on a MonoQ column, and fractions were analyzed by Western blotting (Fig.3 A, upper panel) and for neutral ceramidase activity (lower panel). Earlier fractions (fractions 9 and 10) showed an ∼94-kDa protein of still unknown identity that was recognized by the neutral ceramidase antibody. Fractions 11 and 12 showed exclusive expression of a 110–120-kDa protein, the predicted size of rat neutral ceramidase (19Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The neutral ceramidase activity was highest in fractions 11 and 12, which also showed the highest protein amounts, thus suggesting that this band is indeed a neutral ceramidase. Furthermore, we investigated whether the antibody could immunoprecipitate a fully active enzyme. As shown in Fig. 3 B, Western blot analysis of the supernatant after immunoprecipitation of neutral ceramidase revealed a disappearance of the protein that was dependent on the antibody dilution used (Fig. 3 B, upper panel). Preimmune serum did not deplete the protein from the supernatant. In parallel, a reduction of neutral ceramidase activity was observed in the supernatant (Fig. 3 B, lower panel). Consistent with a depletion of the enzyme in the supernatant, an increased amount of enzyme was observed in the immunoprecipitates by Western blotting (Fig. 3 C). However, no increased neutral ceramidase activity was recovered in the immunoprecipitates (data not shown). These data suggest that binding of the antibody to its antigen leads to a neutralization of the enzyme activity. Stimulation of mesangial cells with IL-1β led to a marked and time-dependent up-regulation of the neutral ceramidase protein (Fig. 4 A). In contrast, the acid ceramidase protein, which runs at 55 kDa as a holoenzyme under nonreducing conditions (30Bernardo K. Hurwitz R. Zenk T. Desnick R.J. Ferlinz K. Schuchman E.H. Sandhoff K. J. Biol. Chem. 1995; 270: 11098-11102Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), was not significantly changed (Fig. 4 B). We further investigated whether the up-regulation of neutral ceramidase is due to increased de novo synthesis. For this purpose, mesangial cells were stimulated with IL-1β for different time periods, and [35S]methionine and [35S]cysteine were included in the culture medium for the last 4 h of stimulation. Thereafter, the cells were lysed, and the neutral ceramidase was immunoprecipitated and analyzed by SDS-PAGE. Fig. 5 shows that IL-1β triggered increased de novo synthesis of the neutral ceramidase. A similar increase was also observed with another pro-inflammatory cytokine, TNF-α (Fig. 5). In contrast, the degradation of the neutral ceramidase was not affected by IL-1β treatment (data not shown) as analyzed by pulse-chase experiments. In a next step, we tested whether there is also an induction of the mRNA coding for the neutral ceramidase. Based on the mouse
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