A Novel Inhibitor of Ceramide Trafficking from the Endoplasmic Reticulum to the Site of Sphingomyelin Synthesis
2001; Elsevier BV; Volume: 276; Issue: 47 Linguagem: Inglês
10.1074/jbc.m104884200
ISSN1083-351X
AutoresSatoshi Yasuda, H. Kitagawa, Masaharu Ueno, Haruro Ishitani, Masayoshi Fukasawa, Masahiro Nishijima, Shū Kobayashi, Kentaro Hanada,
Tópico(s)Cellular transport and secretion
ResumoCeramide produced at the endoplasmic reticulum (ER) is transported to the lumen of the Golgi apparatus for conversion to sphingomyelin (SM).N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12) is a novel analog of ceramide. Metabolic labeling experiments showed that HPA-12 inhibits conversion of ceramide to SM, but not to glucosylceramide, in Chinese hamster ovary cells. Cultivation of cells with HPA-12 significantly reduced the content of SM. HPA-12 did not inhibit the activity of SM synthase. The inhibition of SM formation by HPA-12 was abrogated when the Golgi apparatus was made to merge with the ER by brefeldin A. Moreover, HPA-12 inhibited redistribution of a fluorescent analog of ceramide,N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C5-DMB-Cer), from intracellular membranes to the Golgi region. Among four stereoisomers of the drug, (1 R,3 R)-HPA-12, which resembles natural ceramide stereochemically, was found to be the most active, although (1 R,3 R)-HPA-12 did not affect ER-to-Golgi trafficking of protein. Interestingly, (1 R,3 R)-HPA-12 inhibited conversion of ceramide to SM little in mutant cells defective in an ATP- and cytosol-dependent pathway of ceramide transport. These results indicated that (1 R,3 R)-HPA-12 inhibits ceramide trafficking from the ER to the site of SM synthesis, possibly due to an antagonistic interaction with a ceramide-recognizing factor(s) involved in the ATP- and cytosol-dependent pathway. Ceramide produced at the endoplasmic reticulum (ER) is transported to the lumen of the Golgi apparatus for conversion to sphingomyelin (SM).N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12) is a novel analog of ceramide. Metabolic labeling experiments showed that HPA-12 inhibits conversion of ceramide to SM, but not to glucosylceramide, in Chinese hamster ovary cells. Cultivation of cells with HPA-12 significantly reduced the content of SM. HPA-12 did not inhibit the activity of SM synthase. The inhibition of SM formation by HPA-12 was abrogated when the Golgi apparatus was made to merge with the ER by brefeldin A. Moreover, HPA-12 inhibited redistribution of a fluorescent analog of ceramide,N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C5-DMB-Cer), from intracellular membranes to the Golgi region. Among four stereoisomers of the drug, (1 R,3 R)-HPA-12, which resembles natural ceramide stereochemically, was found to be the most active, although (1 R,3 R)-HPA-12 did not affect ER-to-Golgi trafficking of protein. Interestingly, (1 R,3 R)-HPA-12 inhibited conversion of ceramide to SM little in mutant cells defective in an ATP- and cytosol-dependent pathway of ceramide transport. These results indicated that (1 R,3 R)-HPA-12 inhibits ceramide trafficking from the ER to the site of SM synthesis, possibly due to an antagonistic interaction with a ceramide-recognizing factor(s) involved in the ATP- and cytosol-dependent pathway. ceramide endoplasmic reticulum sphingomyelin glucosylceramide Chinese hamster ovary N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)alkanamide 6-[N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproyl-d-erythro-sphingosine N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine N-palmitoyl-d-sphingosine phosphate-buffered saline brefeldin A N-acetylneuraminyl lactosylceramide human placental alkaline phosphatase a chimera of human placental alkaline phosphatase with influenza hemagglutinin endoglycosidase H sphingomyelinase (1 S,2 R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol bovine serum albumin newborn bovine serum phosphatidylcholine glycosylphosphatidylinositol 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene Sphingolipids are ubiquitous constituents of membrane lipids in mammalian cells and play important roles in cell growth, differentiation, and apoptosis (1Hanada K. Nishijima M. Kiso M. Hasegawa A. Fujita S. Ogawa T. Akamatsu Y. J. Biol. Chem. 1992; 267: 23527-23533Abstract Full Text PDF PubMed Google Scholar, 2Spiegel S. Merrill Jr., A.H. FASEB J. 1996; 10: 1388-1397Crossref PubMed Scopus (636) Google Scholar, 3Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1483) Google Scholar). Moreover, together with cholesterol, sphingolipids in plasma membrane constitute detergent-resistant microdomains termed lipids rafts, which are involved in membrane trafficking and cell signaling (4Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (7948) Google Scholar, 5Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2035) Google Scholar). De novo biosynthesis of sphingolipids in mammalian cells proceeds as follows (6Merrill Jr., A.H. Jones D.D. Biochim. Biophys. Acta. 1990; 1044: 1-12Crossref PubMed Scopus (394) Google Scholar). The first step is the condensation ofl-serine and palmitoyl-CoA, a reaction catalyzed by serine palmitoyl transferase, to generate 3-ketodihydrosphingosine, which is reduced to dihydrosphingosine. Dihydrosphingosine undergoes N-acylation followed by desaturation to generate ceramide (Cer).1 These reactions to produce Cer occur at the cytosolic surface of the endoplasmic reticulum (ER) (7Mandon E.C. Ehses I. Rother J. van Echten G. Sandhoff K. J. Biol. Chem. 1992; 267: 11144-11148Abstract Full Text PDF PubMed Google Scholar, 8Hirschberg K. Rodger J. Futerman A.H. Biochem. J. 1993; 290: 751-757Crossref PubMed Scopus (160) Google Scholar, 9Michel C. van Echten-Deckert G. FEBS Lett. 1997; 416: 153-155Crossref PubMed Scopus (79) Google Scholar). Then, Cer is delivered to the luminal side of the Golgi apparatus and converted to sphingomyelin (SM) by SM synthase catalyzing the transfer of phosphocholine from phosphatidylcholine to Cer (10Futerman A.H. Stieger B. Hubbard A.L. Pagano R.E. J. Biol. Chem. 1990; 265: 8650-8657Abstract Full Text PDF PubMed Google Scholar, 11Jeckel D. Karrenbauer A. Birk R. Schmidt R.R. Wieland F. FEBS Lett. 1990; 261: 155-157Crossref PubMed Scopus (141) Google Scholar). Cer is also converted to glucosylceramide (GlcCer) by GlcCer synthase catalyzing the transfer of glucose from UDP-glucose to Cer. Although the catalytic site of GlcCer synthase appears to be oriented to the cytoplasm (12Futerman A.H. Pagano R.E. Biochem. J. 1991; 280: 295-302Crossref PubMed Scopus (241) Google Scholar, 13Jeckel D. Karrenbauer A. Burger K.N.J. van Meer G. Wieland F. J. Cell Biol. 1992; 117: 259-267Crossref PubMed Scopus (253) Google Scholar), it is controversial whether GlcCer synthase is localized to the Golgi apparatus or more broadly distributed to microsomes. After translocation to the luminal side of the Golgi apparatus, GlcCer is further converted to more complex glycosphingolipids (14Lannert H. Gorgas K. Meissner I. Wieland F.T. Jeckel D. J. Biol. Chem. 1998; 273: 2939-2946Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). We have previously showed that there are two pathways for transport of de novo synthesized Cer from the ER to the site of SM synthesis in various types of cultured cells, including Chinese hamster ovary (CHO) cells, HeLa cells, and human skin fibroblasts (15Fukasawa M. Nishijima M. Hanada K. J. Cell Biol. 1999; 144: 673-685Crossref PubMed Scopus (147) Google Scholar). The main pathway of the two is ATP-dependent, and the minor pathway is ATP-independent (or less dependent). In contrast to the synthesis of SM, the access of Cer to the site of GlcCer synthesis is largely independent of ATP. Analysis by in vitro assay of Cer transport in semi-intact CHO cells further revealed that the ATP-dependent transport of Cer, from the ER to the site of SM synthesis, requires cytosol but that cytosol is not required for transport of Cer to the site of GlcCer synthesis (16Funakoshi T. Yasuda S. Fukasawa M. Nishijima M. Hanada K. J. Biol. Chem. 2000; 275: 29938-29945Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In addition, that a CHO cell mutant defective in the ATP-dependent pathway of Cer transport exhibits no defect in transport of proteins from the ER to the Golgi apparatus has suggested that the mechanism of Cer transport differs from that of ER-to-Golgi trafficking of proteins (15Fukasawa M. Nishijima M. Hanada K. J. Cell Biol. 1999; 144: 673-685Crossref PubMed Scopus (147) Google Scholar). Inhibitors of de novo sphingolipid biosynthesis are useful tools to investigate the metabolism and functions of sphingolipids in cultured cells and in living animals. Various natural and chemically synthesized compounds have been found to be highly selective, even if not strictly specific, inhibitors of sphingolipid synthesis. For example, sphingofungins and ISP-1 (myriocin) appear to be specific and potent inhibitors of serine palmitoyl transferase (17Zweerink M.M. Edison A.M. Wells G.B. Pinto W. Lester R.L. J. Biol. Chem. 1992; 267: 25032-25038Abstract Full Text PDF PubMed Google Scholar, 18Miyake Y. Kozutsumi Y. Nakamura S. Fujita T. Kawasaki T. Biochem. Biophys. Res. Commun. 1995; 211: 396-403Crossref PubMed Scopus (443) Google Scholar). Fumonisins seem to inhibit (dihydro)sphingosine-N-acyltransferase specifically (19Merrill Jr., A.H. Liotta D.C. Riley R.T. Trends Cell Biol. 1996; 6: 218-223Abstract Full Text PDF PubMed Scopus (132) Google Scholar).d-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol and some of its derivatives selectively inhibit GlcCer synthase (20Inokuchi J. Radin N.S. J. Lipid Res. 1987; 28: 565-571Abstract Full Text PDF PubMed Google Scholar,21Lee L. Abe A. Shayman J.A. J. Biol. Chem. 1999; 274: 14662-14669Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). However, no specific inhibitor for SM synthesis in mammalian cells has been found so far. In addition, no drug that inhibits intracellular trafficking of sphingolipids has been discovered, except for broad-spectrum inhibitors such as brefeldin A and energy poisons. In the present study, we showed that N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12), a novel analog of Cer, inhibits ATP-dependent transport of Cer from the ER to the site of SM synthesis without inhibition of protein trafficking, thereby inhibiting conversion of Cer to SM, but not to GlcCer, in cells. Moreover, a stereoisomer having the 1 R,3 R configuration was found to be the most active among the four stereoisomers of HPA-12. l-[U-14C]Serine (155 mCi/mmol) and [methyl-14C]choline (55.0 mCi/mmol) were purchased from Amersham Pharmacia Biotech.6-[N-(7-Nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproyl-d-erythro-sphingosine (C6-NBD-Cer) and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C5-DMB-Cer) were from Molecular Probes, Inc.d-erythro-[3-3H]Sphingosine (20 Ci/mmol), [palmitoyl-1-14C]N-palmitoyl-d-sphingosine ([14C]C16-Cer) (55 mCi/mmol) and [choline-methyl-14C]sphingomyelin (50 mCi/mmol) were from American Radiolabeled Chemicals, Inc.d-erythro-Sphingosine was from Matraya, Inc. Fumonisin B1, brefeldin A (BFA), and fatty acid-free bovine serum albumin were from Sigma Chemical Co. TLC and high performance TLC plates (Silica Gel 60) were from Merck. Dimethyl sulfoxide (Me2SO) was from Wako Pure Chemical Industries, Inc (Osaka, Japan). (1 S,2 R)-d-erythro-2-(N-Myristoylamino)-1-phenyl-1-propanol (d-e-MAPP) was from BIOMOL Research Laboratories, Inc. N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)alkanamides (HPA-3, HPA-8, HPA-12, and HPA-16) were prepared based on three component Mannich-type reactions. For the synthesis of enantiomers, catalytic asymmetric Mannich reactions were performed. Details will be described elsewhere. 2Ueno, M., Kitagawa, H., Ishitani, H., Yasuda, S., Hanada, K., and Kobayashi, S., Tetrahedron Lett. in press. The CHO cell line was obtained from the American Type Culture Cell Collection (ATCC CCL 61). Cells were routinely maintained in Ham's F12 medium supplemented with 10% newborn bovine serum (NBS), penicillin G (100 units/ml), and streptomycin sulfate (100 μg/ml) at 33 °C in a 5% CO2incubator (22Hanada K. Nishijima M. Akamatsu Y. J. Biol. Chem. 1990; 265: 22137-22142Abstract Full Text PDF PubMed Google Scholar). Nutridoma medium (Ham's F12 containing 1% Nutridoma-SP (Roche Molecular Biochemicals) and 25 μg/ml gentamicin) was used as serum-free medium. When the medium was supplemented with Me2SO or drugs dissolved in Me2SO, the concentration of Me2SO in medium was adjusted to 0.01% (v/v). CHO cells were seeded at a density of 1.0 × 106 in 5 ml of F12 medium containing 10% NBS per 60-mm dish and cultured at 33 °C overnight. The cell monolayers were incubated in 1.5 ml of Nutridoma medium supplemented, if necessary, with various drugs at 4 °C for 15 min, and after addition of [14C]serine (0.75 μCi) or [14C]choline (1.0 μCi), incubated at 33 °C for various periods of time. After being washed twice with 2 ml of cold phosphate-buffered saline (PBS), the cells were lysed with 1000 μl of 0.1% cold SDS, and 800 and 20 μl of the lysate was used for lipid extraction and determination of protein concentration, respectively. For analysis of lipids labeled with [14C]serine, lipids were separated on TLC plates with a solvent of methyl acetate/n-propanol/chloroform/methanol/0.25% KCl (25:25:25:10:9, v/v). For analysis of lipids labeled with [14C]choline, lipids were separated by one-dimensional TLC with a solvent of chloroform/methanol/acetate/H2O (25:15:4:2, v/v), or two-dimensional TLC; chloroform/methanol/H2O (65:25:4, v/v) for the first dimension and 1-butanol/acetate/H2O (60:20:20, v/v) for the second dimension. Radioactive lipids separated on the plates were detected with a BAS 2000 image analyzer (Fuji Film, Inc., Tokyo). After the collection of gels from plates by scraping, the radioactivity of each lipid was determined by liquid scintillation counting and normalized to the protein concentration of cells. For metabolic labeling with [14C]serine in BFA-treated cells, cells were preincubated with 1 μg/ml BFA in Nutridoma medium at 33 °C for 30 min, and, then, treated with or without 1 μmHPA-12 in the presence of 1 μg/ml BFA at 4 °C for 15 min prior to addition of [14C]serine. CHO cell monolayers were grown in 60-mm dishes as described above. The cell monolayers were labeled with 1 μm C5-DMB-Cer complexed with 1 μm BSA in 1.5 ml of Nutridoma medium at 4 °C for 30 min. After being washed three times with F12 medium, the cells were incubated in 1.5 ml of Nutridoma medium with or without 2.5 μm HPA-12 at 4 °C for 15 min and then incubated at 33 °C for 15 min. After being washed with PBS, the cells were lysed with 0.1% SDS as described above. Lipids extracted from the cell lysate were separated by high performance TLC with a solvent of chloroform/methanol/H2O (65:25:4, v/v). After the collection of gels from the plates, C5-DMB-SM was scraped from the plates and extracted in chloroform/methanol/H2O (1:2:0.8). DMB fluorescence was measured with a spectrofluorometer (excitation at 480 nm; emission at 515 nm). The assay of the conversion of [3H]Cer to [3H]SM in cells was performed by a method described previously (16Funakoshi T. Yasuda S. Fukasawa M. Nishijima M. Hanada K. J. Biol. Chem. 2000; 275: 29938-29945Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) with minor modifications. In brief, the cell monolayers were prelabeled with 1.5 ml of Nutridoma medium containing 1 μmd-erythro-[3H]sphingosine (0.5 μCi) complexed with 2 μm BSA at 15 °C for 30 min for pulse reaction. After being washed three times with 2 ml of cold F12 medium, the cells were incubated in 1.5 ml of Nutridoma medium with or without 1 μm HPA-12 at 4 °C for 15 min and then incubated at 33 °C for various periods of time in the presence of 100 μm fumonisin B1 for chase. Lipids extracted from cells were separated on TLC plates with a solvent of chloroform/methanol/H2O (65:25:4, v/v), and the radioactivity of each lipid was determined as described above. CHO cells were seeded at a density of 3.0 × 106 per 150-mm dish in 20 ml of F12 medium containing 10% NBS and cultured at 33 °C overnight. After being washed twice with 10 ml of serum-free F12 medium, the cells were incubated in 20 ml of Nutridoma medium with or without 2.5 μm HPA-12 at 33 °C for 48 h. Each medium was replaced every 24 h. The cells were washed twice with 10 ml of PBS, harvested by scraping, and precipitated by centrifugation. Lipids were extracted from the harvested cells and resuspended in PBS (23Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (41848) Google Scholar). For phospholipids, extracted lipids were separated on TLC plates with a solvent of chlorohorm/methanol/acetate/H2O (25:15:4:2, v/v), and the phosphorous content of the phospholipids was measured by the method of Rouser et al. (24Rouser G. Siakotos A.N. Fleischer S. Lipids. 1966; 1: 85-86Crossref PubMed Scopus (1312) Google Scholar). GlcCer and N-acetylneuraminyl lactosylceramide (GM3) contents were determined by densitometric analysis of lipids stained with Coomassie Brilliant Blue as described previously (25Hanada K. Nishijima M. Methods Enzymol. 2000; 312: 304-317Crossref PubMed Google Scholar). The amounts of Cer were determined by using an sn-1,2-diacylglycerol assay reagent system (Amersham Pharmacia Biotech). The membrane fractions prepared from CHO cells and bovine brain were used as the enzyme sources. CHO cells cultured in spinner bottles were homogenized in buffer A (10 mm Tris-HCl buffer (pH 7.4) containing 0.25 msucrose) with a stainless ball homogenizer as described previously (16Funakoshi T. Yasuda S. Fukasawa M. Nishijima M. Hanada K. J. Biol. Chem. 2000; 275: 29938-29945Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). After the removal of nuclei and unbroken cells by centrifugation (900 × g for 10 min), the post nuclear supernatant of the cell homogenate was centrifuged (105 × gfor 60 min). The precipitate as the membrane fraction was suspended in buffer A (∼7 mg of protein/ml), and stored at −80 °C until use. Bovine brain membranes were prepared as described previously (26Hanada K. Mitamura T. Fukasawa M. Magistrado P.A. Horii T. Nishijima M. Biochem. J. 2000; 346: 671-677Crossref PubMed Scopus (52) Google Scholar). Enzyme assays of SM synthase activity were carried out by the modifications of a previously described method (27Hanada K. Horii M. Akamatsu Y. Biochim. Biophys. Acta. 1991; 1086: 151-156Crossref PubMed Scopus (24) Google Scholar). In brief, CHO cell membranes (400 μg of protein) were incubated in 800 μl of 10 mm Hepes-NaOH buffer (pH 7.5) containing 2 mmEDTA, and 1 μm of C6-NBD-Cer or C5-DMB-Cer complexed with BSA at 33 °C for 10 min. Because exogenous [14C]C16-Cer is a poor substrate for SM synthase due to its water insolubility, 0.1% Triton X-100 was added to the reaction buffer and the reaction period was prolonged to 1 h when [14C]C16-Cer was used as the substrate. The radioactivity of [14C]SM that formed was determined after TLC of extracted lipids. Assays of acid and neutral sphingomyelinase (SMase) activities were performed as described previously (26Hanada K. Mitamura T. Fukasawa M. Magistrado P.A. Horii T. Nishijima M. Biochem. J. 2000; 346: 671-677Crossref PubMed Scopus (52) Google Scholar). Briefly, for the assay of acid SMase activity, CHO cell membranes (5 μg of protein) were incubated in 50 μl of 0.1m sodium acetate buffer (pH 4.8) containing 10 μm [3H]SM, 0.2% β-octylglucoside, and 0.1% Triton X-100 at 37 °C for 30 min. For the assay of neutral SMase activity, bovine brain membranes (5 μg of protein) were incubated in 50 μl of 50 mm Hepes-NaOH (pH 7.5) containing 10 μm [3H]SM, 0.2% β-octylglucoside, 0.1% Triton X-100, 10 mmMgCl2 and 1 mm phosphatidylserine at 37 °C for 30 min. Then, the amount of radioactive [14C]phosphocholine liberated was determined. Serine palmitoyl transferase activity was determined as described previously (28Hanada K. Hara T. Nishijima M. J. Biol. Chem. 2000; 275: 8409-8415Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Sphingosine N-acyltransferase activity was determined by a modification of a previously described method (29Wang E. Merrill Jr., A.H. Methods Enzymol. 2000; 311: 15-21Crossref PubMed Scopus (28) Google Scholar). In brief, CHO cell membranes (50 μg of protein) were incubated in 400 μl of 25 mm Hepes-NaOH buffer (pH 7.5) containing 1 μmd-erythro-[3H]sphingosine, 50 μm palmitoyl-CoA, and 0.5 mm dithiothreitol at 33 °C for 30 min. After extraction, lipids were separated on high performance TLC plates with a solvent of chloroform/methanol/acetate (94:5:5, v/v), and the amount of radioactive [3H]Cer separated on the plates was analyzed with a BAS1800 image analyzer (Fuji Film, Inc., Tokyo). CHO cells grown on glass coverslips (22-mm diameter) in 35-mm dishes were incubated in 1 ml of F12 medium containing 1 μm of C5-DMB-Cer or C6-NBD-Cer complexed with 1 μm BSA at 4 °C for 30 min, washed with 1 ml of F12 medium three times and incubated in 1 ml of Nutridoma medium in the presence or absence of 2.5 μm HPA-12 at 4 °C for 15 min. Then, prelabeled cells were incubated at 33 °C for 15 min, washed with PBS and fixed with 0.125% glutaraldehyde solution in PBS for 5 min at 4 °C. For zero-time control, cells were fixed after the HPA-12 treatment without a shift in temperature. The specimens were observed and photographed under a fluorescence microscope (Axiovert S100TV, Carl Zeiss, Tokyo) soon after fixation. CHO transfectants expressing PLAP or PLAP-HA (30Hanada K. Nishijima M. Akamatsu Y. Pagano R.E. J. Biol. Chem. 1995; 270: 6254-6260Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) were seeded at a density of 3.0 × 105 cells per 35-mm dish in 1.5 ml of F12 medium containing 10% NBS and cultured at 33 °C overnight. The cells were incubated in 1 ml of medium A (methionine, cysteine, and glutamine-free RPMI 1640 medium (ICN Pharmaceuticals, Inc.) supplemented with 1% Nutridoma-SP, 2 mml-glutamine, and 25 μg/ml gentamicin) for 1 h at 33 °C and then in 200 μl of medium A containing 50 μCi of EXPRESS 35S-protein labeling mix (PerkinElmer Life Sciences) for 15 min at 33 °C. The labeled cells were incubated in 1 ml of Nutridoma medium containing 10 mm methionine, 10 mm cysteine, and 10 mm Hepes-NaOH (pH 7.4) in the absence or presence of 2.5 μm(1 R,3 R)-HPA-12 and then chase-incubated at 33 °C for various periods of time. According to a method described previously (15Fukasawa M. Nishijima M. Hanada K. J. Cell Biol. 1999; 144: 673-685Crossref PubMed Scopus (147) Google Scholar), proteins of cell lysates were immunoprecipitated with anti-PLAP antibody (Biomeda Corp.) and protein A-Sepharose CL-4B (Amersham Pharmacia Biotech), digested with endoglycosidase H (Endo H), and separated by SDS-polyacrylamide gel electrophoresis. Cells were seeded at 2 × 104 per well of 48-well plates in 500 μl of F12 medium supplemented with 10% NBS and cultured at 33 °C overnight. The cell monolayers were incubated in 500 μl of Nutridoma medium containing various concentrations of HPAs at 33 °C for 24 h. Then, the viability of cells was determined by using 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide as described previously (31Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Protein concentrations were determined with the Pierce BCA assay reagent kit, using BSA as the standard. A series of N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)alkanamides (HPAs), novel analogs of Cer, were chemically synthesized (Fig.1 A). To examine the effects of these drugs on the de novo biosynthesis of sphingolipids in mammalian cells, CHO cells were incubated with [14C]serine in the presence or absence of 2.5 μm of each drug for 2 h at 33 °C. HPAs inhibited the de novo synthesis of sphingolipids, depending on the length of the N-acyl chains of the drugs. HPA-12, the dodecanoyl derivative, inhibited [14C]SM formation to ∼10% of the drug-free control level (Fig. 1 B). Other HPAs, in which the carbon atom numbers of the acyl chain is 3, 8, and 16, had far less of an inhibitory effect than HPA-12 (Fig.1 B). At 2.5 μm, these drugs did not affect the viability of cells (data not shown, see also below). HPA-12 significantly inhibits the formation of [14C]SM even at 0.1 μm, and the inhibitory effect reaches a plateau around 1 μm (Fig.2 A). HPA-12 at more than 10 μm was highly toxic to CHO cells (data not shown), not allowing us to carry out metabolic labeling experiments at more than 10 μm of this drug. The time course of metabolic labeling up to 5 h showed that 1 μm HPA-12 inhibited formation of [14C]SM to <30% of the drug-free control level throughout the incubation period (Fig. 2 B). HPA-12 did not affect the formation of [14C]phosphatidylserine (PS) or [14C]phosphatidylethanolamine (PE), both of which are metabolically labeled with [14C]serine via a pathway distinct from sphingolipid synthesis (Figs. 1 B and 2 B), indicating that the inhibition of sphingolipid synthesis by HPA-12 was not due to a nonspecific dysfunction of lipid metabolism. HPA-12 moderately inhibited the formation of [14C]Cer and [14C]GlcCer, although its inhibitory effect on GlcCer and Cer synthesis was weaker than that on SM synthesis (Figs. 1 Band 2 B). To examine effects of HPA-12 on the turnover of de novosynthesized SM, we pulse-labeled CHO cells with [14C]serine for 2 h at 33 °C, and chased for 24 h at 33 °C in the presence of 10 mmnon-radioactive serine with or without 1 μm HPA-12. The level of cell-associated [14C]SM was not affected by HPA-12 throughout the chase (Fig.3 A). In addition, HPA-12 did not affect the activities of acid or neutral sphingomyelinases in vitro at 20 μm (Fig. 3, B and C). These results eliminated the possibility that HPA-12 accelerated degradation of SM. When conversion of Cer to SM is inhibited, de novo synthesis of sphingoid bases seems to be suppressed, as suggested previously (15Fukasawa M. Nishijima M. Hanada K. J. Cell Biol. 1999; 144: 673-685Crossref PubMed Scopus (147) Google Scholar). Thus, the moderate inhibition of the formation of [14C]serine-derived Cer and GlcCer by HPA-12 might be due to a secondary effect of this drug on inhibition of Cer-to-SM conversion. To examine effects of HPA-12 on the step of Cer-to-SM conversion specifically, we employed another protocol for metabolic labeling. When CHO cells are incubated with [3H]sphingosine at 15 °C, [3H]sphingosine is efficiently N-acylated to generate [3H]Cer without further conversion of [3H]Cer to [3H]SM in intact cells (16Funakoshi T. Yasuda S. Fukasawa M. Nishijima M. Hanada K. J. Biol. Chem. 2000; 275: 29938-29945Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). After pulse labeling with [3H]sphingosine at 15 °C for 30 min, cells were treated with 1 μm HPA-12 at 4 °C for 15 min and further incubated at 33 °C for up to 1 h for chase in the presence of fumonisin B1, an inhibitor of sphingosine N-acyltransferase. Under these pulse and chase conditions, 1 μm HPA-12 inhibited the formation of [3H]SM by ∼50%, whereas it did not inhibit but rather slightly increased the formation of [3H]GlcCer (Fig.4). The level of [3H]Cer gradually decreased concomitant with the formation of [3H]SM and [3H]GlcCer during chase, and the rate of decrease was slower in HPA-12-treated cells than in drug-free control cells, although the initial levels of [3H]Cer at the start of the chase were almost identical between the two (Fig. 4). These results indicated that HPA-12 inhibits conversion of Cer to SM, but not to GlcCer, in intact cells. SM is synthesized by the transfer of phosphocholine from phosphatidylcholine (PC) to Cer, a reaction catalyzed by SM synthase (19Merrill Jr., A.H. Liotta D.C. Riley R.T. Trends Cell Biol. 1996; 6: 218-223Abstract Full Text PDF PubMed Scopus (132) Google Scholar). Metabolic labeling experiments with [14C]choline showed that treatment of cells with 1 μm HPA-12 did not inhibit formation of [14C]PC but that the level of [14C]SM in drug-treated cells was about half of the control level (Fig. 5). Therefore, inhibition of Cer-to-SM conversion by HPA-12 was not due to inhibition of PC synthesis. HPA-12 itself was unlikely to be converted to choline-containing metabolites, because HPA-12-dependent generation of radioactive lipids was not detected in TLC patterns of [14C]choline-labeled lipids of one-dimensional (Fig. 5) or two-dimensional (data not shown) analysis. We determined chemical amounts of various lipids in cells cultured in a sphingolipid-free medium in the presence or absence of 2.5 μm HPA-12 at 33 °C for 2 days (Table I). HPA-12 did not affect levels of total phospholipids: The contents of total phospholipids in untreated and drug-treated cells were 288 ± 3 and 315 ± 2 nmol/mg of protein, respectively. However, the content of SM in HPA-12-treated cells was lower by ∼30% than the drug-free control level, and the reduction in SM in HPA-12-treated cells seemed to be compensated by an increase in the amount of PC (Table I). HPA-12 did not influence contents of other major glycerophospholipid types. In contrast, HPA-12 treatment increased the content of GlcCer and GM3 to ∼150% and ∼120%, respectively, of the untreated control level (Table I). There was no significant difference in the content of Cer between the two groups (Table I). These results were another line of evidence that the primary target of HPA-12 is the step of conversion of Cer to SM.Table IEffects of HPA-12 on phospholipid composition and sphingolipid contents in CHO cellsHPA-12Phospholipid composition1-aPC, phosphatidylcholine; PI/PS, phosphatidylinositol and phosphatidylserine; PE, phosphatidylethanolamine.GM3GlcCerCerSMPCPI/PSPE% of total phospholipids recoverednmol/mg proteinNone12.0 ± 0.143.3 ± 0.113.2 ±
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