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• W2017240991 abstract "Ceramide as central second messenger of the apoptosis-related sphingomyelin signaling pathway is a potential target for the control of cancer. A complex metabolizing network defines cell type and stage-specific final ceramide concentrations. Successful therapeutic control of ceramide levels requires a knowledge of multiple related turnover rates. The metabolism of ceramide and sphingomyelin was studied in keratinocytes under the condition of an unstimulated sphingomyelin signaling pathway. Preparations enriched in plasma membranes contain a neutral Mg2+-dependent sphingomyelinase and a Mg2+-independent sphingomyelin synthase that vigorously preserve balanced ceramide and sphingomyelin levels. Ceramide regulates neutral sphingomyelinase. Inhibition of sphingomyelin synthase by D609 treatment results in temporary loss of intercelluar contacts and in cellular shrinking. It is ineffective for sustained elevation of ceramide levels. Ceramide phosphorylating and deacylating activities are insignificant. Recently, fatty-acid remodeling in sphingomyelin was reported as likely to counteract the membrane-rigidifying effects of cholesterol. Keratinocytes transfer fluorescence labeled acyl-chains between phosphatidylcholine and sphingomyelin. A transferase of that kind would allow rapid adjustment of local lipid composition in response to acutely changed conditions. In addition, this transferase might have a function in the formation of the epidermal permeability barrier. Ceramide as central second messenger of the apoptosis-related sphingomyelin signaling pathway is a potential target for the control of cancer. A complex metabolizing network defines cell type and stage-specific final ceramide concentrations. Successful therapeutic control of ceramide levels requires a knowledge of multiple related turnover rates. The metabolism of ceramide and sphingomyelin was studied in keratinocytes under the condition of an unstimulated sphingomyelin signaling pathway. Preparations enriched in plasma membranes contain a neutral Mg2+-dependent sphingomyelinase and a Mg2+-independent sphingomyelin synthase that vigorously preserve balanced ceramide and sphingomyelin levels. Ceramide regulates neutral sphingomyelinase. Inhibition of sphingomyelin synthase by D609 treatment results in temporary loss of intercelluar contacts and in cellular shrinking. It is ineffective for sustained elevation of ceramide levels. Ceramide phosphorylating and deacylating activities are insignificant. Recently, fatty-acid remodeling in sphingomyelin was reported as likely to counteract the membrane-rigidifying effects of cholesterol. Keratinocytes transfer fluorescence labeled acyl-chains between phosphatidylcholine and sphingomyelin. A transferase of that kind would allow rapid adjustment of local lipid composition in response to acutely changed conditions. In addition, this transferase might have a function in the formation of the epidermal permeability barrier. ceramide N-acetyl-sphingosine tricyclodecan-9-yl-xanthate diacylglycerol fluorescent high-performance thin-layer chromatography lecithin–cholesterol acyltransferase phosphate-buffered saline phosphatidylcholine phosphatidylcholine-specific phospholipase C sphingomyelin sphingomyelinase sphingomyelin synthase The major polar lipid constituents of membranes are the glycerophospholipid phosphatidylcholine (PC) and the phosphosphingolipid sphingomyelin (SM). PC and SM share phosphocholine as their polar head group. PC further consists of glycerol and of two esterified acyl-chains, while SM contains the sphingoid base sphingosine and an amide-linked acyl-chain. In difference to most biological phospholipids, the acyl-chains of SM are mostly long and largely saturated. This acyl-chain structure allows a more dense and ordered packing of molecules likely to result in the formation of specialized functional microdomains like the SM- and cholesterol-rich structures called rafts (Brown and London, 2000Brown D.A. London E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts.J Biol Chem. 2000; 275: 17221-17224Crossref PubMed Scopus (2001) Google Scholar;Holthuis et al., 2001Holthuis J.C.M. Pomorski T. Raggers R.J. Sprong H. van Meer G. The organizing potential of sphingolipids in intracellular membrane transport.Phys Rev. 2001; 81: 1689-1723PubMed Google Scholar;Sprong et al., 2001Sprong H. van der Sluijs P. van Meer G. How proteins move lipids and lipids move proteins.Nat Rev Mol Cell Biol. 2001; 2: 504-513Crossref PubMed Scopus (456) Google Scholar). In addition to serving as structural components, the membrane lipids are sources of bioactive derivatives. An SM-based signaling pathway was proposed in 1988 (Kolesnick and Clegg, 1988Kolesnick R.N. Clegg S. 1,2-Diacylglycerols, but not phorbol esters, activate a potential inhibitory pathway for protein kinase C in GH3 pituitary cells. Evidence for involvement of a sphingomyelinase.J Biol Chem. 1988; 263: 6534-6537Abstract Full Text PDF PubMed Google Scholar) with the hydrolyzed SM metabolite ceramide (Cer) as central second messenger (Okazaki et al., 1990Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. Role of ceramide as a lipid mediator of 1alpha,25-dihydroxyvitamin D3-induced HL-60 cell differentiation.J Biol Chem. 1990; 265: 15823-15831Abstract Full Text PDF PubMed Google Scholar). Cer may be degraded to the effector sphingosine. Cer may be phosphorylated either directly to the bioactive lipid ceramide 1-phosphate or to sphingosine 1-phosphate, after deacylation respectively. Alternatively, Cer may either be glycosylated to generate glycosphingolipids or be converted back to SM by transfer of a phosphocholine moiety. If derived from PC, bioactive diacylglycerol arises at the expense of Cer (Hannun et al., 2001Hannun Y.A. Luberto C. Argraves K.M. Enzymes of sphingolipid metabolism: From modular to integrative signaling.Biochemistry. 2001; 40: 4893-4903Crossref PubMed Scopus (430) Google Scholar). Deacylation of SM generates another second messenger, sphingosylphosphorylcholine (Meyer zu Heringdorf et al., 2002Meyer zu Heringdorf D. Himmel H.M. Jakobs K.H. Sphingosylphosphorylcholine—biological functions and mechanisms of action.Biochem Biophys Acta. 2002; 1582: 178-189Crossref PubMed Scopus (121) Google Scholar) (Scheme 1). SM pathway-related lipids show individual signaling functions and may generate even contrasting outcomes. In most cell types, Cer signaling for instance is connected with cell cycle arrest, differentiation, or apoptosis, whereas sphingosine 1-phosphate is more related to proliferation or inhibition of apoptosis (Cuvillier et al., 1996Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate.Nature. 1996; 381: 800-803Crossref PubMed Scopus (1292) Google Scholar). The bioactive SM derivatives also differ in their physical properties. Individual concentration changes may affect membrane characteristics such as local fluidity. SM and its derivatives are substrates and products of interconnected metabolic pathways. Changing the concentration of any sphingolipid will result in changes in concentrations of other metabolites, depending on the activities of the enzymes involved. Apoptosis-related signaling pathways have emerged as key targets for the control of cancer. Of the multitude of SM metabolites, Cer in particular has been shown to stimulate cell death (Kolesnick, 2002Kolesnick R. The therapeutic potential of modulating the ceramide/sphingomyelin pathway.J Clin Invest. 2002; 110: 3-8Crossref PubMed Scopus (367) Google Scholar). The biological effects of potential therapeutic Cer analogs as well as of compounds modulating metabolic activities surrounding Cer depend, however, on cell type or phase-specific adjustments of the turnover rates of multiple molecules related to the SM network (Hannun et al., 2001Hannun Y.A. Luberto C. Argraves K.M. Enzymes of sphingolipid metabolism: From modular to integrative signaling.Biochemistry. 2001; 40: 4893-4903Crossref PubMed Scopus (430) Google Scholar). This study focuses on the turnover of SM and Cer applied to keratinocytes under the condition of an unstimulated SM signaling pathway. Fluorophore-labeled lipid analogs are reasonable substitutes for membrane lipids in terms of packing similarity (Dahim et al., 2002Dahim M. Mizuno N.K. Li X.M. Momsen W.E. Momsen M.M. Brockman H.L. Physical and photophysical characterization of a BODIPY phosphatidylcholine as a membrane probe.Biophys J. 2002; 83: 1511-1524Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and, regarding metabolizing enzymes, they proved to be substrates comparable to radioactive probes (He et al., 1999He X. Li C.M. Park J.H. Dagan A. Gatt S. Schuchman E.H. A fluorescence-based high-performance liquid chromatographic assay to determine acid ceramidase activity.Anal Biochem. 1999; 274: 264-269Crossref PubMed Scopus (28) Google Scholar;Loidl et al., 2002Loidl A. Claus R. Deigner H.P. Hermetter A. High-precision fluorescence assay for sphingomyelinase activity of isolated enzymes and cell lysates.J Lipid Res. 2002; 43: 815-823Abstract Full Text Full Text PDF PubMed Google Scholar). SM- and Cer-analogs labeled with the fluorophore BODIPY FL were used to disturb the SM–Cer homeostasis. Provoked SM–Cer imbalances were found to be rapidly levelled off at the plasma membrane already. Further SM- or Cer-derived bioactive lipids were not substantially generated from the fluorescent analogs applied; however, an activity remodeling the acyl-chain composition of SM via transfer from fluorescent PC was detected. Confluent HaCaT keratinocyte cultures were labeled at 4°C with fluorescent BODIPY FL C5-ceramide (fl-Cer) or BODIPY FL C5-sphingomyelin (fl-SM). In the cold, the fluorescent lipids were incorporated into the plasma membranes Figure 1a. A shift to 37°C caused the fluorescent molecules to move to intracellular compartments. Regarding labeling with fl-Cer, the initial diffuse cytoplasmic staining visibly concentrated in the Golgi stacks after 15 min, already Figure 1b. Labeling with fl-SM also stained the Golgi stacks, which was visible after 30 min at 37°C. After 90 min, staining was also detectable inside the nuclei (not shown). The incorporated lipids were quickly and quantitatively metabolized. HPTLC analysis showed, 120 min after temperature shift, a nearly 70% conversion of fl-Cer to fl-SM and a 25% hydrolysis of fl-SM to fl-Cer Figure 1c. Half-maximal turnover rates were achieved at time points below 5 min Figure 1c. At 3 h after the addition of fl-Cer and fl-SM, the disturbed ceramide and sphingomyelin homeostasis was readjusted Figure 1d. Other products of added fl-Cer and fl-SM were limited amounts of glycolipids, which were detectable not until reactions had proceeded for at least 2 h Figure 1d. HaCaT keratinocyte cultures that were treated on alternate days with either 5 μM fl-Cer, 10 μM C2-Cer, or vehicle control 0.1% DMSO grew up to comparable cell numbers (not shown). After application of 10 μM C2-Cer, FACS analysis revealed no increased annexin V staining (not shown). BODIPY C5-phosphatidylcholine (fl-PC) was incorporated into plasma membranes in the cold and was partly taken up intracellularly at 37°C. Staining, however, did not concentrate in specific organelles (not shown). Fl-PC was not found to be metabolized Figure 1d. The BODIPY-labeled PC proved, however, to be a good substrate for phospholipase C type IV from B. cereus and for phospholipase A2 from bee venom (not shown). A considerable fraction of added lipids was metabolized before being transported to intracellular compartments. This pointed to a localization of rapid metabolic activities at the plasma membranes. As a proof, HaCaT keratinocyte membranes were isolated by nitrogen cavitation and differential centrifugation (Watarai et al., 2000Watarai H. Inagaki Y. Kubota N. Fuju K. Nagafune J. Yamaguchi Y. Kadoya T. Proteomic approach to the identification of cell membrane proteins.Electrophoresis. 2000; 21: 460-464Crossref PubMed Scopus (30) Google Scholar). These membrane preparations were negative for the golgi-marker 58k-protein Figure 2. Alkaline phosphatase activity was 4-fold enriched as compared with whole-cell homogenates Figure 2. The membrane preparations contained a strong initial fl-SM hydrolyzing (SMase) activity, which quickly slowed down Figure 3a. SMase activity was Mg2+-dependent Figure 3b and reached a maximum at neutral pH Figure 3c. Neutral SMase activities, in isolated membranes prepared from normal human keratinocytes were comparable to those in HaCaT membrane preparations (not shown).Figure 3Keratinocyte membranes contain a neutral Mg2+-dependent SMase. Membranes of HaCaT keratinocytes isolated by nitrogen cavitation and differential centrifugation hydrolyze fl-SM to fl-Cer. A typical HPTLC plate and, below, the results of densitometrical evaluations of five independent experiments (mean±SD) are shown (a). SMase activity strongly depended on the presence of Mg2+ (-, without; +, 5 mM Mg2+) (b). SMase activities in isolated membranes (empty columns) and in whole-cell homogenates (filled columns) of HaCaT keratinocytes in dependence of pH. Reactions were performed in solutions containing 5 μM fl-SM, 5 mM Mg2+, and 50 mM MOPS or 100 mM Na-phosphate with a pH as indicated, and proceeded for 5 min at 37°C. Results represent % of activity relative to the condition that yielded maximal activity in membranes or in homogenates, respectively (mean±SD of three independent experiments) (c).View Large Image Figure ViewerDownload (PPT) No SMase activity was found in membranes at pH 5.0. In whole-cell homogenates of HaCaT keratinocytes, SMase activity was at least 50 times stronger at neutral pH as compared with pH 5.0 when measured after a 5 min reaction time Figure 3c. In homogenates of normal human epidermis, SMase activities at pH 5.0 needed reaction times of 3 h to become detectable, whereas at neutral pH, SMase activities were comparable to HaCaT homogenates (not shown). Epidermal acid SMase activity was abolished by the presence of 1% Triton-X-100 (not shown). BODIPY-labeled SM proved to be a good substrate for neutral SMases from B. cereus and S. aureus as well as for acid SMase prepared from human placenta (not shown). The membrane preparations from HaCaT keratinocytes generated fl-SM from added fl-Cer at metabolic rates in the range of fl-SM hydrolysis Figure 4. SM synthesis was independent of both Mg2+ Figure 4 and Ca2+ (not shown). To confirm the reliability of the SMase and SMS assays used, we determined the turnover rates of the fluorescent lipid analogs at various ratios of fluorescent to natural lipids. For both SM hydrolysis and SM synthesis, the turnover rates of fluorescent lipids decreased linearly according to replacement with their natural counterparts Figure 5. SM synthesis is not a monomolecular reaction like SM hydrolysis. To generate SM from Cer, a phosphocholine moiety is needed. This moiety mainly originates from PC hydrolyzed by a PC-specific phospholipase C (PC-PLC) (Spence, 1989Spence M.W. Vance D.E. Phosphatidylcholine Metabolism. CRC Press, Boca Raton, FL1989: 185-203Google Scholar). The xanthogenate compound D609 is both a supposed potent and specific inhibitor of PC-PLC (Wiegmann et al., 1994Wiegmann K. Schutze S. Machleidt T. Witte D. Kronke M. Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling.Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (662) Google Scholar;Luberto et al., 2000Luberto C. Yoo D.S. Suidan H.S. Bartoli G.M. Hannun Y.A. Differential effects of sphingomyelin hydrolysis and resynthesis on the activation of NF-κB in normal and SV40-transformed human fibroblasts.J Biol Chem. 2000; 275: 14760-14766Crossref PubMed Scopus (64) Google Scholar) (Scheme 1). Confluent HaCaT keratinocyte cultures labeled with fl-Cer were treated either with D609 at a final concentration of 0.1 mM or with vehicle control DMSO at 0.1%. D609 decreased the part of Cer converted to SM from 20% to 10% after a reaction time of 5 min Figure 6a. After 30 min, no difference was detectable between D609 and vehicle control treatment Figure 6a. The influence of D609 on sphingolipid metabolism was investigated in isolated HaCaT keratinocyte membranes also. Either D609 at 0.1 mM or DMSO at 0.1% final concentration was added to the membranes, and reactions were allowed to proceed for 5 min at 37°C. Both the synthesis of SM and its hydrolysis were inhibited by D609 as compared with vehicle control Figure 6b. In additional experiments, HaCaT keratinocyte cultures were treated with D609 as described above, but no fl-Cer was added, and the dishes were incubated at 37°C for up to 5 h. The main lipid classes of the intact whole cells were detected in comparable amounts, regardless of whether they were treated with D609 or with vehicle control Figure 6c. On microscopical examination, corresponding D609-treated cell cultures revealed striking morphological changes. Treated cells shrank and lost their contacts to neighboring cells (Figure 6d a, b). The enlargement of the intercellular spaces was not due to removal of non-adhering cells as nuclear staining revealed the same numbers of nuclei in equal areas of D609- and DMSO-treated keratinocyte cultures (Figure 6d c, d). In contrast to vehicle controls, no mitotically active cells were found in D609-treated cell cultures (Figure 6d c, d). The shrunk cells were viable as observed by fluorescence microscopy. 7-Amino-actinomycin D and annexin V-PE hardly ever stained cells or plasma membranes, respectively (not shown). The morphological changes had a maximum at 5 h after D609 application. Thereafter, the keratinocyte cultures recovered to normal appearances. At 18 h after application, D609-treated cell cultures were microscopically indistinguishable from controls (not shown). To study the influence of PC on the homeostasis of Cer and SM more directly, PC in combination with 5 μM fl-Cer was added to HaCaT membranes. The addition of 200 μM unlabeled PC revealed only a minor increase in the rate of fl-SM synthesis (not shown). In contrast, 5 μM fl-PC strongly increased the fl-SM synthesis Figure 7. The increase in fl-SM, however, was not accompanied by the appearance of fluorescent diacylglycerol (fl-DG) (not shown). The addition of 5 μM BODIPY-diacylglyerol (fl-DG) did not affect the fl-SM synthesis Figure 7. The fl-PC-induced increase in fl-SM synthesis was not caused by an improved availability of the phosphocholine moiety. A fluorescent product that comigrated exactly with fl-C5-SM on HPTLC appeared when isolated HaCaT keratinocyte membranes were allowed to react solely with fl-C5-PC to which no fl-Cer was added Figure 8. Such an fl-SM-resembling band appeared also when fl-PC was added to homogenates as well as to isolated membranes of normal human keratinocytes (not shown). Cultured intact HaCaT keratinocytes, however, did not convert any of the added fl-PC to the putative fl-SM Figure 1d. As the fluorophores resided in the acyl-chains of the lipid analogs, the appearance of the fluorescent product must be based on acyl-chain transfer. To elucidate the nature of fl-PC's reaction and degradation products, HaCaT keratinocyte membranes were incubated with 1,2-bis BODIPY FL C11-phosphatidylcholine (bis-fl-PC), which is fluorescence labeled in both its acyl-chains Figure 9. The products detectable on HPTLC were a double band with a mobility between fl-C5-SM and fl-C5-PC, probably representing an fl-C11-SM species Figure 9a. In addition, a band migrating slightly slower than bis-fl-PC was discernible at lower intensifications Figure 9b. This latter band might correspond to the chromatographical behavior of a PC containing one fluorescent fl-C11-chain and one unlabelled acyl-chain. The equal band intensities of bis-fl-PC and PC containing only one fl-C11-chain do not represent a comparable amount of the individual lipid species, because emission of bis-fl-PC is reduced by self-quenching of its adjacent fluorophores. No fluorescent free-fatty-acids and no PC deacylated to fluorescent lyso-phosphatidylcholine could be detected Figure 9. The fluorescent reaction product of fl-C5-PC was tested for being a substrate of SMase from B. cereus to further identify it as a true SM analog. B. cereus SMase hydrolyzed the putative fl-C5-SM to fl-Cer Figure 10. This study relied on sphingo- and phospholipid analogs containing fluorophore labeled short acyl-chains as they allow studies on compartmentalization and metabolism in parallel. Short-chain sphingolipids exist in mammalian tissues (Karasawa et al., 1999Karasawa K. Qiu X. Lee T. Purification and characterization from rat kidney membranes of a novel platelet-activating factor (PAF)-dependent transacetylase that catalyzes the hydrolysis of PAF, formation of PAF analogs, and C2-ceramide.J Biol Chem. 1999; 274: 8655-8661Crossref PubMed Scopus (34) Google Scholar), and their most relevant functional difference is increased freedom of intermembrane movement. Fl-Cer and fl-SM were applied in only double amounts as compared with keratinocyte endogenous contents (Pillai et al., 1999Pillai S. Mahajan M. Carlomusto M. Ceramide potentiates, but sphingomyelin inhibits, vitamin D-induced keratinocyte differentiation: Comparision between keratinocytes and HL-60 cells.Arch Dermatol Res. 1999; 291: 284-289Crossref PubMed Scopus (12) Google Scholar). The analogs were incorporated and metabolized and did not provoke alterations regarding morphology and rates of proliferation and apoptosis. As reported for unlabeled short-chain analogs, only higher concentrations of fl-Cer and fl-SM would presumably affect keratinocyte proliferation (Geilen et al., 1996Geilen C.C. Bektas M. Wieder T. Orfanos C.E. The vitamin D3 analogue, calcipotriol, induces sphingomyelin hydrolysis in human keratinocytes.FEBS Lett. 1996; 378: 88-92Abstract Full Text PDF PubMed Scopus (55) Google Scholar). This argues for a compatible embedding of the fluorescent sphingolipids and for the physiological relevance of results derived. Fluorescent lipid analogs are increasingly used in studies on metabolism and trafficking. They proved to be reasonable substitutes for membrane lipids in terms of packing similarity. Regarding metabolizing enzymes, they proved to be comparable to radioactive probes (Loidl et al., 2002Loidl A. Claus R. Deigner H.P. Hermetter A. High-precision fluorescence assay for sphingomyelinase activity of isolated enzymes and cell lysates.J Lipid Res. 2002; 43: 815-823Abstract Full Text Full Text PDF PubMed Google Scholar). To confirm the reliability of our results, we studied inhibitory competition by natural Cer and SM species. The turnover rates of both fl-Cer and fl-SM decreased linearly according to replacement with their natural counterparts Figure 5. This indicates that the enzymes involved do not discriminate against the fluorophores used. The Cer–SM homeostasis disturbed by incorporation of exogenous lipid analogs was rapidly readjusted. This occurred already within the plasma membrane because a considerable fraction of applied lipids was metabolized before moving to intracellular compartments. In addition, enzymatic activities consuming fluorescent sphingolipids were detected in preparations enriched in plasma membranes. The turnover rates of fl-Cer to fl-SM and vice versa were balanced. Evidence for the reverse action of sphingomyelin synthase (SMS) was reported (Van Helvoort et al., 1994Van Helvoort A. van't Hof W. Ritsema T. Sandra A. van Meer G. Conversion of diacylglycerol to phosphatidylcholine on the basolateral surface of epithelial (Madin–Darby canine kidney) cells.J Biol Chem. 1994; 269: 1763-1769Abstract Full Text PDF PubMed Google Scholar), but the differential need of Mg2+ in the SM hydrolyzing versus synthesizing activities points more to the presence of a Mg2+-dependent SMase and of a Mg2+-independent SMS in keratinocytes. It is unclear why neutral SMase activity was dominant not only in membrane preparations but also in whole-cell extracts. Fluorescent SM analogs and radiolabeled [3H]SM proved to be comparable substrates for acid SMase (Loidl et al., 2002Loidl A. Claus R. Deigner H.P. Hermetter A. High-precision fluorescence assay for sphingomyelinase activity of isolated enzymes and cell lysates.J Lipid Res. 2002; 43: 815-823Abstract Full Text Full Text PDF PubMed Google Scholar). In a preparation containing the entire membrane population of HaCaT keratinocytes, the activity of acid SMase was reported to be 7-fold higher than that of neutral SMase (Shimizu et al., 1999Shimizu H. Banno Y. Sumi N. Naganawa T. Kitajima Y. Nozawa Y. Activation of p38 mitogen-activated protein kinase and caspases in UVB-induced apoptosis of human keratinocyte HaCaT cells.J Invest Dermatol. 1999; 112: 769-774Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Under the condition of an unstimulated sphingomyelin cycle, neutral SMase could provide the prevailing activity in the regulation of keratinocyte membrane SM–Cer homeostasis, whereas induction of the SM pathway could activate acid SMase. There were no Cer phosphorylating or deacylating activities detectable. These would have generated fl-Cer species with altered mobility on HPTLC or fluorescent free-fatty acids. Technically, however, ceramidase activity could be determined with high sensitivity by using fluorescent ceramide (He et al, 1999). In keratinocytes, the basal activities of both Cer kinase and ceramidases appear insignificant for the regulation of Cer concentration. Using ceramidase inhibitors alone may not result in elevated Cer levels. D609 is regarded as a selective inhibitor of a PC-specific phospholipase C and is also proposed to be a direct inhibitor of SMS (Luberto and Hannun, 1998Luberto C. Hannun Y. Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation.J Biol Chem. 1998; 273: 14550-14559Crossref PubMed Scopus (262) Google Scholar). The rate of synthesis of fl-SM from fl-Cer was inhibited by D609 only for short reaction times. When SMS activity was downregulated by D609, SM hydrolysis in membrane preparations was inhibited at a comparable degree, at the same time. As SMS and SMase activities are obviously exerted by different proteins, keratinocyte neutral SMase may be inhibited by Cer. Thus, an initial D609-caused elevation of Cer concentration would be balanced by decreased SMase activity to result in restored relative levels of Cer and SM. Actually, D609 left the overall phospholipid levels of keratinocytes quantitatively unaltered. In leukemia cells and fibroblasts, on the contrary, a positive feedback control of neutral SMase by Cer was found (Jaffrezou et al., 1998Jaffrezou J.P. Maestre N. de Mas-Mansat V. Bezombes C. Levade T. Laurent G. Positive feedback control of neutral sphingomyelinase activity by ceramide.FASEB J. 1998; 12: 999-1006Crossref PubMed Scopus (67) Google Scholar). Such a self-stimulation, once initiated, ensures Cer-induced apoptotic destruction. A negative feedback control of neutral SMase by Cer would more likely prevent apoptotic destruction. In keratinocytes, Cer is additionally synthesized to become a constituent of the epidermal lipid barrier. As a result, they already contain an increased Cer to SM ratio as compared to the leukemia cells (Pillai et al., 1999Pillai S. Mahajan M. Carlomusto M. Ceramide potentiates, but sphingomyelin inhibits, vitamin D-induced keratinocyte differentiation: Comparision between keratinocytes and HL-60 cells.Arch Dermatol Res. 1999; 291: 284-289Crossref PubMed Scopus (12) Google Scholar). Moreover, the dose of exogenous Cer required to induce a maximal apoptotic effect in the leukemia cells proved ineffective in keratinocytes (Geilen et al., 1996Geilen C.C. Bektas M. Wieder T. Orfanos C.E. The vitamin D3 analogue, calcipotriol, induces sphingomyelin hydrolysis in human keratinocytes.FEBS Lett. 1996; 378: 88-92Abstract Full Text PDF PubMed Scopus (55) Google Scholar). Morphological alterations in response to D609 treatment were, however, obvious. D609 blocks DNA fragmentation during apoptosis in endothelial cells (Miao et al., 1997Miao J.Y. Kaji K. Hayashi H. Araki S. Suppression of apoptosis by inhibition of phosphatidylcholine-specific phospholipase C in vascular endothelial cells.Endothelium. 1997; 5: 231-239Crossref PubMed Scopus (20) Google Scholar). In lymphocytes, it prevents cell spreading and attenuates sodium influx (Wooten et al., 1999Wooten D.K. Teague T.K. McIntyre B.W. Separation of integrin-dependent adhesion from morphological changes based on differential PLC specificities.J Leukoc Biol. 1999; 65: 127-136PubMed Google Scholar;Nofer et al., 2000Nofer J.R. Junker R. Seedorf U. Assmann G. Zidek W. Tepel M. D609-phosphatidylcholine-specific phospholipase C inhibitor attenuates thapsigargin-induced sodium influx in human lymphocytes.Cell Signal. 2000; 12: 289-296Crossref PubMed Scopus (14) Google Scholar). Ceramides can modify the permeability barrier of cell membranes and can induce the efflux of ions and even of large molecules (Montes et al., 2002Montes L.R. Ruiz-Arguello M.B. Goni F.M. Alonso A. Membrane restructuring via ceramide results in enhanced solute efflux.J Biol Chem. 2002; 277: 11788-11794Crossref PubMed Scopus (119) Google Scholar). The 1.5 d half-life time of D609 in tissue culture is longer than keratinocytes needed to recover from shrinking. An initial D609-caused elevation of Cer concentration could possibly have induced efflux and shrinking. Recovery might be based on resynthesis. Calcium chelators are used in studies on the effects of the release of Ca2+ dependent intercellular con" @default.
• W2017240991 created "2016-06-24" @default.
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• W2017240991 modified "2023-10-03" @default.
• W2017240991 title "Keratinocytes Rapidly Readjust Ceramide–Sphingomyelin Homeostasis and Contain a Phosphatidylcholine–Sphingomyelin Transacylase" @default.
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• W2017240991 doi "https://doi.org/10.1111/j.0022-202x.2004.22340.x" @default.
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