Biochemical Properties of Mammalian Neutral Sphingomyelinase2 and Its Role in Sphingolipid Metabolism*

Norma Marchesini, Chiara Luberto, and Yusuf A. HannunDagger

From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425

Received for publication, December 2, 2002, and in revised form, January 21, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neutral sphingomyelinase (N-SMase) is one of the key enzymes involved in the generation of ceramide; however, the gene(s) encoding for the mammalian N-SMase is still not well defined. Previous studies on the cloned nSMase1 had shown that the protein acts primarily as lyso-platelet-activating factor-phospholipase C. Recently the cloning of another putative N-SMase, nSMase2, was reported. In this study, biochemical characterization of the mouse nSMase2 was carried out using the overexpressed protein in yeast cells in which the inositol phosphosphingolipid phospholipase C (Isc1p) was deleted. N-SMase activity was dependent on Mg2+ and was activated by phosphatidylserine and inhibited by GW4869. The ability of nSMase2 to recognize endogenous sphingomyelin (SM) as substrate was investigated by overexpressing nSMase2 in MCF7 cells. Mass measurements showed a 40% decrease in the SM levels in the overexpressor cells, and labeling studies demonstrated that nSMase2 accelerated SM catabolism. Accordingly, ceramide measurement showed a 60 ± 15% increase in nSMase2-overexpressing cells compared with the vector-transfected MCF7. The role of nSMase2 in cell growth was next investigated. Stable overexpression of nSMase2 resulted in a 30-40% decrease in the rate of growth at the late exponential phase. Moreover, tumor necrosis factor induced ~50% activation of nSMase2 in MCF7 cells overexpressing the enzyme, demonstrating that nSMase2 is a tumor necrosis factor-responsive enzyme. In conclusion, these results 1) show that nSMase2 is a structural gene for nSMase, 2) suggest that nSMase2 acts as a bona fide N-SMase in cells, and 3) implicate nSMase2 in the regulation of cell growth and cell signaling.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sphingolipid metabolites are now recognized as important components in signal transduction, not only in mammalian cells but also in yeast where they are implicated in heat stress responses. Ceramide, a major sphingolipid metabolite, has been shown to play important roles in apoptosis, cell cycle arrest, and differentiation (for recent reviews, see Refs. 1-3). As a consequence of this diverse biology, the study and characterization of enzymes that regulate ceramide levels have become essential areas of study.

Sphingomyelinases (SMases)1 are enzymes that cleave the phosphodiester linkage of sphingomyelin into ceramide and phosphocholine, and they are implicated in several pathways of signal transduction and cell regulation. Activation of acid sphingomyelinase (A-SMase) has been observed after treatment with UV-A radiation (4) and stimulation of the p75 neurotropin receptor (5), CD28 (6), TNF receptor, and CD95 (7), although the involvement of A-SMase in apoptosis and/or differentiation induced by tumor necrosis factor-alpha (TNFalpha ) and Fas is somewhat controversial (8, 9). Attention has also focused on the neutral magnesium-dependent SMase (N-SMase) for its role in mediating a variety of cellular processes including differentiation, cell cycle arrest, and programmed cell death (apoptosis) through the generation of ceramide (1, 2). N-SMase activation has been observed after stimulation of the p75 neurotropin receptor (10), after ligation of CD95 and TNF receptor (11, 12) and irradiation (13), and also upon heat stress and serum starvation (14), treatment with vitamin D (15), and CD40 ligation (16). Recently, GW4869 was developed as an inhibitor for N-SMase, and it was shown to inhibit TNF-induced activation of N-SMase in MCF7 cells. GW4869 showed no inhibitory activity on other hydrolytic enzymes, such as A-SMase, and it showed significantly higher activity against the rat brain enzyme compared with the human lyso-PAF PLC (17). Moreover, GW4869 significantly protected from TNF-induced death and growth suppression, indicating that N-SMase activation is an important step for the full development of the signaling program induced by TNF (17). However, the N-SMase target for GW4869 has not been identified molecularly.

The above results underlie the need to define the gene(s) encoding N-SMase, and various strategies have been employed for this purpose. By using a monospecific polyclonal antibody against the human urinary N-SMase, a transcript encoding a putative N-SMase was identified from a kidney library (18). Recently, cloning approaches based on homology with bacterial SMases identified two mammalian enzymes, nSMase1 (19) and nSMase2 (20), as putative N-SMases capable of hydrolyzing SM in vitro. Although there is concordance on the ability of nSMase1 to hydrolyze SM in vitro (19, 21-25), the current evidence shows that it is unable to modulate SM metabolism in cells (19, 21, 22). In fact, we demonstrated that, in cells, nSMase1 functions as a lysophospholipase C and not as a SMase (21), and this is supported by multiple studies showing that the enzyme shows similar activity in vitro toward SM and lysophosphatidylcholine or lyso-platelet-activating factor (lyso-PAF) (21, 23-25). Thus, nSMase1 is unlikely to function as a bona fide SMase. The only remaining point is what is the real metabolic function of nSMase1. Studies on the nSMase1 knock out mouse (26) in which cells were labeled with acetate or lyso-PC did not show changes in sphingomyelin or other lipids. Further studies are required to determine conclusively the metabolic function of nSMase1 and its role in lyso-PAF metabolism.

On the other hand, the only published study on nSMase2 demonstrates that it is regulated by PS, and it is abundant in brain (20), consistent with the basic properties of the partially purified N-SMase from rat (27) and bovine brain (28).

In light of these studies, we investigated in this work the hypothesis that nSMase2 functions as an endogenous SMase in cells. The goals of this work are as follows: (a) to establish that nSMase2 is a structural gene for sphingomyelinase; (b) to determine the biochemical properties of this enzyme; (c) to investigate the endogenous and physiological substrates of nSMase2 through in vitro and cell studies; (d) to determine whether nSMase2 is a target for GW4869; and (e) to begin defining biological roles for this enzyme.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- [choline-methyl-14C]SM and [acetyl-14C]C2-ceramide were provided by Dr. Alicja Bielawska (Medical University of South Carolina, Charleston, SC). [methyl-3H]Choline chloride (75 Ci/mmol), [9,10-3H]palmitic acid (43 Ci/mmol), 1-O-octadecyl-9,10-3H-PAF (160 Ci/mmol), [choline-methyl-14C]dipalmitoyl-PC (159 mCi/mmol), and [gamma -32P]ATP (3000 Ci/mmol) were from PerkinElmer Life Sciences. 1-O-octadecyl-3H-Lyso-PAF (161 Ci/mmol) was from Amersham Biosciences. All lipids were purchase from Avanti Polar Lipids (Alabaster, AL). TNF was from PeproTech (Rocky Hill, NJ). EN3HANCETM spray was from PerkinElmer Life Sciences. Silica Gel 60 thin layer chromatography plates were from Whatman. Scintillation mixture Safety Solve was from Research Products International. Other chemicals were from Sigma. Oligodeoxynucleotides were purchased from IDT, Inc.

Yeast Strains and Culture Media-- The Saccharomyces cerevisiae strain JK9-3d (MATa/alpha trp1 leu2-3 his4 ura3 ade2rme1) (29) was used as wild-type cells. Yeast extract and peptone were from Difco. Synthetic minimal medium (SD), SD/Gal, and Ura dropout supplement were purchased from Clontech.

Plasmids-- The pYES2 yeast expression vector containing a galactose-inducible promoter was purchased from Invitrogen. cDNA of the mouse nSMase2 (GenBankTM accession number AJ250461) was generously provided by Drs. Stephan Tomiuk and Kay Hofmann. The FLAG-tagged nSMase2 was generated by PCR (forward primer, 5'-GGCGTACCATGGTTTTGTACACGACCCCCTTTCCT-3', and reverse primer containing the sequence for the FLAG tag, 3'-TCCGCTCGAGCTACTTATCATCGTCGTCCTTGTAGTCCGCCTCCTCTTCCCCTGCAGACAC-5'). pYES2 and the PCR product were digested by the restriction enzymes KpnI and XhoI and ligated. The sequence of the PCR product was analyzed by an ABI377 DNA sequencer. Plasmids were transfected into yeast cells as described (29), and the expression of nSMase2 was induced by incubating the cells in synthetic complete-Ura medium containing 2% galactose overnight.

Preparation of Lysates of Yeast Cells-- Yeast cells were suspended in buffer containing 25 mM Tris (pH 7.4), 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 4 µg/ml each chymostatin, leupeptin, antipain, and pepstatin A. Cells were disrupted with glass beads as described (29). Glass beads and cell debris were removed by centrifugation at 2,000 × g for 10 min, and the supernatant was centrifuged at 100,000 × g to obtain the microsomal and cytosolic fractions. For the studies on cation and lipid effects, and substrate requirements of nSMase2, membranes were delipidated by incubation in lysis buffer in the presence of 1% Triton X-100 and incubated at 4 °C for 1 h. The suspension was centrifuged at 100,000 × g for 90 min, and the supernatant was used for enzymatic determinations. Protein concentration was determined using Bio-Rad protein assay reagent.

Cell Culture-- MCF7 cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 incubator.

Transfection-- nSMase2 cDNA cloned into the eukaryotic expression vector pRc/CMV (Stratagene) was transfected into MCF7 cells by using the Superfect reagent from Qiagen (30). For the selection of stable transfectants, 800 µg/ml G418 (Invitrogen) was added to the medium. Independent colonies were picked and cultured in separate wells, and three clones (1, 5, and 30) from single cells expressing the highest N-SMase activity in vitro were used as nSMase2 overexpressors. Reproducible results were obtained with the three clones analyzed for all the assays.

Preparation of Cell Lysates-- Cells were lysed by syringe passage in buffer containing 25 mM Tris (pH 7.4), 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml each of chymostatin, leupeptin, antipain, and pepstatin A, and post-nuclear lysate (800 × g for 5 min) and supernatant were used for the assay.

N-SMase Activity-- Proteins (0.5 or 10 µg) from yeast or MCF7 overexpressors were added to 100 µl of reaction mixture containing 100 mM Tris (pH 7.4), 10 mM MgCl2, 0.2% Triton X-100, 10 mM dithiothreitol, 100 µM [choline-methyl-14C]SM (10 cpm/pmol). PS was used in the assay at the indicated concentrations. The final volume was adjusted to 200 µl with 50 mM Tris buffer (pH 7.4). After 30 min of incubation at 37 °C, the reaction was terminated by the addition of 1.5 ml of chloroform/methanol (2:1); the phases were separated by addition of 200 µl of water, and 400 µl of the upper phase was mixed with 4 ml of Safety Solve (Research Products International) for liquid scintillation counting.

A-SMase Assay-- The assay was performed as described for the N-SMase assay except that the reaction mixture contained 100 mM sodium acetate (pH 5.0).

PC-PLC Assay-- The assay was performed as described for the N-SMase assay except that the reaction mixture contained no phosphatidylserine and 100 µM [choline-methyl-14C]PC (10 cpm/pmol) instead of SM.

Lyso-PAF-PLC Assay-- Cell lysate (1-5 µg) was added to 100 µl of reaction mixture containing 100 mM Tris (pH 7.4), 10 mM MgCl2, 10 mM dithiothreitol, and 100 µM 1-O-octadecyl-3H-lyso-PAF (20 dpm/pmol) in a final volume of 200 µl. After 30 min of incubation at 37 °C, the lipids were extracted by the method of Bligh and Dyer (31) and separated by TLC in solvent system A (chloroform, methanol, 2 N NH4OH, 60:35:5). The TLC plates were sprayed with EN3HANCE (PerkinElmer Life Sciences) and exposed to Biomax MR autoradiographic (Eastman Kodak Co.) films at 80 °C for 4 days. The band corresponding to monoalkylglycerol was identified by comparison to standard and scraped from the TLC plate for liquid scintillation counting.

PAF-PLC Assay-- The assay was performed as described for the lyso-PAF-PLC assay except that the reaction mixture contained 100 µM 1-O-octadecyl-3H-PAF (20 dpm/pmol) instead of lyso-PAF.

SDS-PAGE and Western Blot Analysis-- SDS-PAGE was performed as described (32). Equal amounts of protein were resolved by a 7% SDS-PAGE for analysis of nSMase2. The proteins were transferred onto a nitrocellulose membrane and blocked with PBS, 0.1% Tween 20 (PBS-T) containing 5% dried milk. Proteins were then identified by incubating with a 1:5000 dilution of anti-FLAG M2 antibody (Sigma) for 1 h and 1:3000 dilution of anti-mouse horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 45 min. The signal was visualized by enhanced chemiluminescence (ECL) (Amersham Biosciences) with exposure to Biomax MR film.

Mass Measurement of SM and PC-- Cellular lipids were extracted by the method of Bligh and Dyer (31), and aliquots of 200 µl were used for phosphorous determination (33). For the measurement of SM, the lipids in chloroform were mixed with the same volume of 0.2 N NaOH in methanol and incubated at 37 °C for 1 h. The phases were separated by the addition of 0.45 volume of 0.2 N HCl. Lipids were separated by TLC in solvent system B (chloroform, methanol, 15 mM CaCl2, 60:35:8) and visualized with iodine vapor, and the bands corresponding to SM and PC were scraped. Lipids were extracted from the silica gel, and the mass of lipid phosphate was determined as described (33).

Sphingomyelin Measurement-- Cells were seeded at 0.3 × 106 cells/10-cm dish in 8 ml of complete growth medium. The next day, the cells were labeled with [methyl-3H]choline chloride (0.5 µCi/ml final concentration in 10 ml of growth medium/plate) for ~60 h. The cells were then chased with 10 ml of complete medium, and at the indicated time points, the medium from each plate was collected, and the cells were washed once with 2 ml of ice-cold PBS. Cells were scraped on ice in 2 ml of PBS, and each plate was washed with an additional 2 ml of PBS. Cells and washes were pooled with the medium and centrifuged for 5 min at 2,000 × g (4 °C). Lipids were extracted by the method of Bligh and Dyer (31), and aliquots of 250 µl were used for phosphorous and SM determination as described by Andrieu et al. (34).

Measurements of Mass Levels of Ceramide-- Cells were harvested in methanol, and lipids were extracted by the method of Bligh and Dyer (31). The chloroform organic phase was divided into aliquots (in duplicates) and dried down for ceramide and phosphate measurements. Ceramide levels were evaluated using the Escherichia coli diacylglycerol kinase assay. Ceramide was quantitated using external standards and normalized to phosphorous content (33).

Radiolabeling Experiments of the Cells-- For labeling with [3H]palmitic acid, 2 × 106 cells were incubated with 5 µCi of [3H]palmitic in 10 ml of medium for 24 or 48 h. For labeling with 1-O-octadecyl-3H-lyso-PAF, medium was replaced by serum-free medium, and then cells were incubated with either 1 µCi/ml 3H-lyso-PAF or 3H-PAF for the indicated times. Lipids were extracted by the method of Bligh and Dyer (31) and separated by TLC in solvent system B.

MTT Assay-- Cells (3 × 103/well) were seeded in a 96-well plate in 100 µl of RPMI containing 10% fetal bovine serum. At the indicated time points, 25 µl of MTT stock solution (5 mg/ml in PBS) was added to each well and incubated at 37 °C in 5% CO2 for 3 h. Subsequently, cells were solubilized by the addition of 100 µl of lysis buffer (10% SDS in 0.01 mol/liter HCl) to each well and incubated at room temperature overnight. The production of the formazan dye was quantitated by measuring the absorbance at 595 nm with a multiwell plate reader.

Trypan Blue Exclusion Methods-- The effects of nSMase2 overexpression on cell growth were determined by the trypan blue exclusion method as described (35).

Analysis of Growth by [3H]Thymidine Incorporation-- Cell proliferation was examined by detection of trichloroacetic acid-precipitable [3H]thymidine incorporation as described (36) with modifications. Briefly, cells were seeded at 50 × 103 cells/well in 6-well plates. After 12 h in serum-free media and 12 h in 0.5% serum, the cells were grown for 96 h in 10% SFB RPMI and pulsed with 1 µCi/ml [3H]thymidine in the growth medium for 1 h. The cells were washed with phosphate-buffered saline and incubated with 2 ml of 0.5% trichloroacetic acid on ice for 20 min. The trichloroacetic acid precipitates in the wells were then dissolved in 0.5 ml of 1 M NaOH, and after neutralization with 1 M HCl, 200 µl were counted in a scintillation counter.

Specificity of GW4869 on Enzyme Inhibition-- Delipidated or non-delipidated microsome fractions from yeast overexpressing nSMase2 were incubated in the absence or presence of GW4869 at the indicated concentrations. SM hydrolysis was determined as described above, in the presence of the indicated concentrations of PS. The suspension was mixed and warmed at 37 °C until clear. Control membranes were treated with Me2SO containing 5% methanesulfonic acid, similarly to the samples receiving the highest dose of GW4869 solution. In all assays, 30 min of preincubation at 37 °C with the membranes preceded the addition of the substrate.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Studies of nSMase2 in Yeast

Expression of nSMase2 in S. cerevisiae-- In order to establish that nSMase2 is a structural gene for neutral sphingomyelinase (N-SMase), we expressed nSMase2 into the JK9-3d yeast (MATa/alpha trp1 leu2-3 his4 ura3 ade2rme1) strain deleted in ISC1 that encodes for an endogenous inositol phosphosphingolipid phospholipase C (Isc1p), as Isc1p shows robust SMase activity and its deletion removes SMase activity from yeast (37). As shown in Table I, overexpression of nSMase2 in the deletion strain showed higher activity than the vector-transfected cells. SMase activity was greatly reduced at acidic pH (0.57 nmol/µg/h), compared with neutral pH (Table I), indicating a neutral pH optimum. Phospholipase activity on PC was very low in lysates of both vector transfectant and nSMase2-overexpressing cells, demonstrating that PC is not a substrate of this enzyme (Table I). In addition, the yeast inositol phosphosphingolipids (inositol phosphorylceramide, mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide) were not utilized by the enzyme as substrates.2 When cell lysates from the overexpressors were fractionated by centrifugation at 100,000 × g, in the presence of 50 µM PS (3.3 mol %) the highest specific activity was detected in the pellet fraction (Fig. 1A), and only 20% of the total activity was found in the supernatant. The presence of the FLAG-tagged nSMase2 was confirmed in the microsomal fraction at ~78 kDa by Western blot analysis using anti-FLAG antibody (Fig. 1B). Indeed, analysis of the amino acid sequence of the nSMase2 revealed that it contains two putative transmembrane domains. In the absence of PS, N-SMase activity in the microsomal fraction of vector transfectant or nSMase2-overexpressing cells was negligible (0.4 nmol/µg/h). In the presence of 100 µM (6.7 mol %) PS in the assay, the Km and Vmax for SM were 1.8 mol % (27 µM) and 15.8 nmol/µg/h, respectively.


                              
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Table I
Enzymatic activity in lysates of vector transfectant and pYES2 FLAG tag nSMase2 transfectant cells
The results are averages ± S.D. of at least six experiments (nmol/µg/h). PC, lyso-PAF, and PAF were assayed as substrates of nSMase2. N-SMase activity was measured as described under "Experimental Procedures," and in the presence of PS 6.7 mol % (100 µM). PC and lyso-PAF activity were measured in presence of PC and lyso-PAF as substrates and in the absence of PS. When PAF was used as a substrate, activity was not detected (ND).


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Fig. 1.   Overexpression of FLAG-tagged nSMase2 in S. cerevisiae. A, phospholipase activity on SM in the lysates, cytosol, and microsome fractions from deletion mutant of ISC1 transformed with nSMase2 was assayed as described under "Experimental Procedures" and in the presence of 50 µM PS (3.3 mol %). B, Western blot analysis with anti-FLAG antibody. Equal amounts of proteins (2 µg) were loaded from the 100,000 × g pellet of the control cells and the overexpressors. Western blot was performed as described under "Experimental Procedures." The arrow indicates the overexpressed 78-kDa protein. The results are from one experiment representative of three different experiments.

Characterization of the Overexpressed nSMase2 Activity-- N-SMase activity in the microsomal fraction of nSMase2-overexpressing cells was dependent on Mg2+, and maximum activity was found at 5 mM Mg2+ (10 nmol/µg/h). Mn2+ stimulated N-SMase activity at 1-2 mM; however, higher concentrations of Mn2+ slightly suppressed activity. Ca2+, zinc, or copper did not support N-SMase activity (Fig. 2A). nSMase2 activity was dependent on Triton X-100, and in the assay the final concentration of Triton X-100 was adjusted to 0.1%. The dependence of nSMase2 on PS was examined next. At 5 µM (0.33 mol %), PS only slightly induced activity, and 100-150 µM PS increased activity around 40 times (Fig. 2B). In the presence of cardiolipin (100 µM), the activity was similar to that found in the presence of PS (Fig. 2C). Other anionic phospholipids such as phosphatidylinositol, phosphatidylcholine, phosphatidylethanol, and phosphatidic acid only slightly increased activity.


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Fig. 2.   Characterization of nSMase2 expressed in yeast. A, cation effects were assayed using 3.3 mol % of SM (50 µM) and 6.7 mol % of PS (100 µM). SMase was measured at various concentrations of MgCl2, MnCl2, CaCl2, ZnCl2, or CuCl2. B, dependence of nSMase2 activity on PS. Activity of nSMase2 on SM was measured at various concentrations of PS and in the presence of 3.3 mol % of SM. C, effects of various phospholipids. The indicated phospholipids were delivered at a final concentration of 6.7 mol % (100 µM), and enzyme was assayed in the absence of PS. PEOH, phosphatidylethanol; PG, phosphatidylglycerol; LPC, lyso-PC; PE, phosphatidylethanolamine; LPAF, lyso-PAF; DAG, diacylglycerol; PI, phosphatidylinositol; PA, phosphatidic acid; LPA, lyso-PA; PC, phosphatidylcholine; CL, cardiolipin. The results are averages of one experiment for duplicate. Similar results were obtained in two different experiments.

Effects of GW4869 on nSMase2 "in Vitro"-- In order to determine whether GW4869, a novel inhibitor of the Mg2+-dependent N-SMase, acts on nSMase2, in vitro experiments were performed using delipidated microsomes from the yeast overexpressors. Fig. 3A shows that preincubation of the enzyme with GW4869 (15 µM) inhibited activity by ~40%. The inhibition was dose-dependent with maximum inhibition achieved at 45 µM (70%) GW4869. In non-delipidated membranes, the inhibition was more complex, and only 30% inhibition was observed at 45 µM GW4869, suggesting effects of membrane lipids on inhibition of the enzyme. As shown in Fig. 3B, the effect of GW4869 was dependent on the concentration of PS present in the assay. When the activity was measured at 0.03 mol % of PS, total inhibition was observed at 30 µM GW4869. Importantly, the effects of PS on GW4869 inhibition of the N-SMase activity were very similar to those observed using partially purified rat brain N-SMase (17). Thus, nSMase2 is a direct in vitro target for GW4869.


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Fig. 3.   Inhibition of nSMase2 by GW4869: effects of varied [PS]. A, microsomal fractions from yeast overexpressors and controls were preincubated at the indicated concentrations of GW4869 for 30 min at 37 °C. The activity was assayed in the presence of 3.3 mol % PS (50 µM) as described under "Experimental Procedures." Results are average ± S.D. of three different experiments. B, delipidated microsomal fraction was assayed for activity as described under "Experimental Procedures" in the absence or presence of GW4869 at different PS concentrations. The results are averages of duplicates. Similar results were obtained in two different experiments.

Studies of nSMase2 in Mammalian Cells

To study the biochemical effects of nSMase2 in mammalian cells, we stably transfected MCF7 breast cancer cells with the cloned enzyme in pRc/CMV vector. SMase activity in the cell lysates of nSMase2 stable transfectants was increased by ~20-fold in the presence of 3.3 mol % PS, compared with vector transfectant cells. The biochemical properties were similar to those observed with the membranes from yeast. The presence of dithiothreitol did not affect significantly the enzymatic activity (Fig. 4A). The Km and Vmax values for SM in the presence of PS (6.7 mol %) were 24 µM (1.6 mol %) and 0.43 nmol/µg/h, respectively (Fig. 4B). Therefore, as seen with the original study (20), nSMase2 expressed SMase activity in mammalian cells.


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Fig. 4.   nSMase2 activity in MCF7 overexpressors. A, N-SMase activity was measured in total lysate of MCF7 stably transfected with the pRc/CMV nSMase2 in the presence or absence of 5 mM dithiothreitol and 3.3 mol % PS (50 µM). B, phospholipase C activity on SM was measured at increasing concentrations of SM and in the presence of 6.7 mol % PS (100 µM) and 5 mM Mg2+. The inset shows a Lineweaver-Burk plot of the data. Results are from one experiment representative of two separate experiments.

The Levels of SM and Ceramide in Stable Transfectant Cells-- Studies with nSMase1 showed that whereas the enzyme utilized SM as a substrate in vitro, it did not modulate SM/ceramide metabolism in cells where it appeared to act primarily as a lyso-PAF PLC (21). Therefore, to determine whether the overexpressed nSMase2 functions as a N-SMase in cells, the mass of SM and ceramide in the vector or nSMase2 transfectant MCF7 cells was measured. The mass of SM was 30-40% lower in nSMase2 transfectant cells compared with the vector transfectants (Fig. 5A). The observed decreased in SM corresponded to an increase in the levels of ceramide (40-70%) (Fig. 5B). In contrast, PC and diacylglycerol levels were unchanged (Fig. 5, B and C). These changes in the levels of SM and ceramide in overexpressing cells suggest a role for this enzyme in the metabolism of SM.


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Fig. 5.   SM, PC, and ceramide levels in vector and pRc/CMV nSMase2 transfectant cells. The levels of SM (A) and PC (C) were determined by mass measurement as described under "Experimental Procedures." B, the levels of ceramide and diacylglycerol (DAG) were determined by diacylglycerol kinase assay as described under "Experimental Procedures." Results are average ± S.D. of three different experiments. WT, wild type.

Metabolism of SM and PC in Transfectant Cells-- To determine the effects of nSMase2 on SM metabolism in cells, we performed radiolabeling experiments with [3H]choline. When vector or pRc/CMV nSMase2 transfectant cells were continuously labeled with [3H]choline, the time course of the increase in labeled PC or SM was similar in both transfectant cells (Fig. 6, A and B). There was a slight decrease in the level of labeled SM in nSMase2 transfectant cells compared with that in vector transfectant cells until the cells reached steady state. When vector or nSMase2 transfectant cells were then chased, after replacement of medium, labeled SM was metabolized significantly faster in the nSMase2 overexpressors. There was a 20-30% decrease at 6-20 h of chase (Fig. 6D). PC, on the other hand, was cleared slower in the overexpressors compared with the vector control cells (Fig. 6C). These results suggest a significant effect of the enzyme on the catabolism of SM in the overexpressors. Similar results were observed with transient overexpression of nSMase2, demonstrating that these effects were not due to "adaptive" changes in the stable transfectants (data not shown).


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Fig. 6.   The metabolism of SM and PC in vector and pRc/CMV nSMase2 transfectant cells. Cells were labeled with 0.5 µCi of [3H]choline/ml for the indicated times. Lipids were extracted by the method of Bligh and Dyer (31) and separated by TLC in solvent system A. PC (A) and SM (B) levels were determined as described under "Experimental Procedures." C and D, cells were labeled with 0.5 µCi of [3H]choline/ml for 3 days and then chased for the indicated times as described under "Experimental Procedures." Results are average ± S.D. of three different experiments.

To investigate whether the levels of lipids other than SM, PC, or ceramide might be changed in nSMase2 transfectant compared with vector transfectant cells, both transfectants were labeled with [3H]palmitic acid, and the levels of labeled lipids were compared between vector and nSMase2 transfectant cells. The labeled lipids (other than SM or ceramide) showed no apparent difference in various TLC solvent systems (data not shown).

Effects on Lyso-PAF in Vitro and in Cells

It has been shown that nSMase1 expresses lyso-PAF phospholipase C activity in vitro and acts preferentially to modulate lyso-PAF metabolism in cells (21); therefore, we studied whether lyso-PAF serves as a substrate for nSMase2. In vitro assays were conducted using 1-alkyl-3H-lyso-PAF as a substrate in the JK9-3d yeast strain. In the overexpressors, PLC activity on lyso-PAF was substantially lower (0.37 nmol/µg/h) than that on SM (14.5 nmol/µg/h) (Table I). In contrast to N-SMase activity, which requires detergents such as Triton X-100, lyso-PAF-PLC activity could only be detected in the absence of Triton X-100. In fact, addition of Triton X-100 in the assay inhibited lyso-PAF-PLC activity (data not shown). No PAF-PLC activity was detected in microsomes isolated from either vector or nSMase2 transfectant yeast (Table I). Thus unlike nSMase1, nSMase2 does not display significant lyso-PAF-PLC activity in vitro.

To investigate the metabolism of lyso-PAF in mammalian cells, both overexpressors and vector control cells were labeled with 1-alkyl-3H-lyso-PAF. In both cases, 90% of lyso-PAF was metabolized into diradyl-PC (80%), phosphatidylethanolamine (5%), and diacylglycerol (2.5%). There was no difference in the levels of alkyl-acylglycerols, which represented 2.5-3% of the total radioactivity (Fig. 7). Therefore, unlike nSMase1, nSMase2 did not affect lyso-PAF metabolism; thus, nSMase2 has much more restricted substrate specificity than nSMase1 and appears to act preferentially on SM as a substrate in cells.


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Fig. 7.   The metabolism of lyso-PAF in transfectant cells. A vector (lane V) and nSMase2 (lane N) transfectant cells were labeled with 1 µCi/ml of 1-O-octadecyl-3H-lyso-PAF for 1 h. Lipids were extracted by the method of Bligh and Dyer (31) and separated by TLC in solvent system described under "Experimental Procedures." Equal radioactivity was loaded on each lane. AG, 1-alkyl- or 1-acyl-glycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; DAG, diacylglycerol. The results are from one experiment representative of three separated experiments.

Effects of nSMase2 on Growth of MCF7 Cells

Increased ceramide levels have been shown to induce cell cycle arrest, apoptosis, and/or cell differentiation (1-3). To assess whether the effects of changes in cellular sphingolipid content with increased activity of nSMase2 could modulate changes in growth, the growth rates of MCF7 nSMase2 overexpressors were compared with the control cells. It was observed that, over a typical growth experiment, nSMase2 overexpressors grew only to 70% the level of control cells as evaluated by the MTT assay which monitors the conversion of the MTT tetrazolium salt to a colored formazan and which requires the mitochondrial respiratory chain (Fig. 8A). Therefore, the MTT assay detects viable cells, and decreased MTT-positive cells can be due to either death or inhibition of growth. To evaluate if nSMase2 accelerated cell death, trypan blue exclusion assays were conducted as described under "Experimental Procedures." Trypan blue is taken up by cells with damaged plasma membranes due to apoptosis or necrosis, and therefore its exclusion reflects viability. As shown in Fig. 8B, the overexpression of nSMase2 reduced the number of viable cells; however, the number of cells that incorporated the trypan blue (dead cells) was very low and similar in nSMase2 overexpressors and control cells (data not shown). These results suggest that the overexpression of nSMase2 causes growth inhibition but not cell death. The growth inhibition was further evaluated by examining the incorporation of [3H]thymidine into trichloroacetic acid-precipitable macromolecules (Fig. 8C). It was observed that [3H]thymidine incorporation also decreased by ~30%. These results support a role of this enzyme in mediating growth arrest through alteration of SM/ceramide metabolism.


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Fig. 8.   Growth rates of vector and pRc/CMV nSMase2 transfectant cells. A, the effects of nSMase2 overexpression on cell growth were determined by the MTT assay. Triplicate samples were used in MTT assays in at least four independent experiments. B, cell viability was assayed by trypan blue exclusion as described under "Experimental Procedures." The figure presented is representative of three independent experiments. C, thymidine incorporation was examined after 98 h of growth by detection of [3H]thymidine incorporation into trichloroacetic acid-precipitable macromolecules as described under "Experimental Procedures." Results are average ± S.D. of three different experiments in A and C, and one experiment in B is representative of two separate experiments.

Effect of TNF on nSMase2 Activity

Since it has been reported (17) that N-SMase contributes, at least in part to the generation of ceramide in response to TNF in MCF7, we investigated if nSMase2 may be regulated by TNF. As shown in Fig. 9, the stimulation of MCF7 with 3 nM TNF for 12 h produced a 43 ± 11% increase in the N-SMase activity of nSMase2 overexpressors when compared with the control cells. Similar activation was observed in the vector transfectant cells in the presence of 3 nM TNF (60 ± 22%); however, the absolute increase in activity was significantly higher in the overexpressors. These results suggest that nSMase2 is responsive to TNF action and that it is a regulated enzyme.


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Fig. 9.   Effects of TNFalpha on cellular activity of nSMase2 transfectant cells. Cells transiently transfected with either vector (control) or nSMase2 were treated with 3 nM TNF for 12 h. The cells were lysed, and enzyme activity was determined as described under "Experimental Procedures." The results are averages ± S.D. of four different experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although the existence of N-SMase has been known for 3 decades, lack of information on its molecular properties and direct mechanisms of cellular regulation has significantly handicapped efforts to delineate pathways involving this enzyme (38-40). In this study we demonstrate that the cloned nSMase2 functions as a neutral Mg2+-dependent sphingomyelinase in cells and that the protein exerts SMase activity both in vitro and in cells. We provide evidence of the biological effect of the overexpression of nSMase2 on cell growth, and finally we report that nSMase2 is a target for the new N-SMase inhibitor, GW4869.

In the past few years, increased attention has focused on the N-SMase for its suggested roles in mediating a variety of cellular processes including differentiation, cell cycle arrest, and programmed cell death (apoptosis) through the generation of ceramide (1, 2). We provide in this report several criteria that demonstrate that nSMase2 is indeed a N-SMase and that it is involved in the regulation of SM metabolism. First in vitro experiments showed that the overexpressed nSMase2 has a strict substrate specificity toward SM. More importantly, nSMase2 does not exhibit significant activity toward PC, lyso-PAF, or PAF. Second, nSMase2 is able to regulate the metabolism of SM and ceramide in cells with a decrease in SM and corresponding increase in ceramide levels. Indeed, [H3]choline labeling experiments showed that the nSMase2 overexpressors display accelerated catabolism of SM. These properties are different from the ones reported for nSMase1, which displays similar activity toward SM and lyso-PAF as substrates in vitro and which also does not influence the metabolism of SM in cells (21). The lack of a role of nSMase2 in PAF and lyso-PAF metabolism was further demonstrated with cellular experiments that failed to demonstrate differences in the metabolism of lyso-PAF in response to nSMase2 overexpression.

Biochemically, the partially purified rat brain N-SMase and the yeast Isc1p showed dependence on anionic phospholipids such as PS for in vitro activity (27, 37). Interestingly, site-directed mutagenesis on Isc1p revealed the importance of positively charged amino acid residues in the carboxyl-terminal region for protein-anionic phospholipid interactions (41). The results of this study demonstrate that nSMase2 also is activated by PS and other anionic phospholipids, especially cardiolipin and phosphatidylglycerol, suggesting the presence of an anionic phospholipid-selective binding domain in nSMase2. The dependence on anionic phospholipids may have implication for cellular activation of the enzyme. PS is highly enriched in the plasma membrane; however, nSMase2 was suggested to localize in a subcompartment of the Golgi apparatus (20). On the other hand, mitochondria contain large amounts of cardiolipin that is not found in other subcellular compartments, and this may be another candidate compartment for activation of nSMase2. Obviously, further studies on nSMase2 localization and regulation are required.

Two main routes have been defined for the generation of ceramide: de novo biosynthesis and the hydrolysis of sphingomyelin by the action of sphingomyelinases that operate at different pH optima (acid, neutral, or alkaline) (2). The study of the novo partway has benefited tremendously from the availability of highly specific inhibitors and from the identification of the key enzymes on this pathway (42, 43). On the other hand, very few tools have been available to dissect the neutral sphingomyelinase pathway. One molecule, scyphostatin, has been described to exert inhibitory activity versus N-SMase (44, 45). Recently, a high throughput assay for N-SMase identified another molecule, GW4869, that exhibited significant and specific inhibitory activity on N-SMase, whereas it showed no significant inhibition of lyso-PAF PLC or acid SMase (17). Interestingly, the current results show that GW4869 is able to inhibit nSMase2 in vitro and in a dose-dependent manner, and with kinetics similar to those observed for the partially purified N-SMase. Thus, nSMase2 becomes the first molecularly defined target for GW4869 which may emerge as a helpful tool to investigate the roles of nSMase2 in stress-induced ceramide generation.

As increases in ceramide levels have been shown to be associated with apoptosis and cell cycle arrest, the growth rates of the overexpressors were studied. MCF7 overexpressing nSMase2 showed significant decrease in growth (30%) after 48 h as the cells reached the plateau phase. Interestingly, nSMase2 was previously isolated as the rat Cca1 protein (46), which had been identified in a screen for genes involved in contact inhibition of rat 3Y1 fibroblasts. Thus, nSMase2 may play a role in contact inhibition. The biological significance of a putative role of nSMase2 in cell cycle arrest remains to be established, and further experiments are needed to address this interesting possibility.

Various studies (17, 47-49) have suggested a role of N-SMase in the TNF-induced ceramide generation. However, the N-SMase involved in this response has not been identified conclusively. In cells overexpressing nSMase1, treatment with TNF (19) or H2O2 (21) did not elevate ceramide levels or induce apoptosis. In T-cell hybridoma 3DO undergoing apoptosis induced by T-cell receptor engagement, treatment with nSMase1 antisense probe reduced ceramide production (50). However, the specificity and the effect of the antisense on nSMase1 activity were not evaluated. On the other hand, in the Jurkat T-cell line, the overexpression of wild-type or catalytically inactive nSMase1 showed that nSMase1 is not the N-SMase involved on CD95-induced ceramide production (51). In accordance with this, Fensome et al. (23) observed similar results in the DT40 B cell line where an nSMase1-like enzyme seemed not to play a role in the apoptotic response to endoplasmic reticulum stress. Those results argued against a role of nSMase1 in apoptosis. Interestingly, in the current study the cellular activation of nSMase2 by TNF demonstrates that this enzyme responds to TNF and that it is a regulated sphingomyelinase.

In conclusion, these results suggest that nSMase2 is a sphingomyelinase that plays an important role in the regulation of SM metabolism and levels. Further studies are needed to address the role of this enzyme in stress-induced ceramide generation and to identify the mechanisms of its activation in response to agonists. Moreover, the characterization of the molecular properties of nSMase2 described here will facilitate studies of its regulation; and finally the molecular characterization of nSMase2 will contribute to our understanding of signaling pathways mediated by sphingolipid metabolites.

    ACKNOWLEDGEMENTS

We thank Drs. K. Hofmann and S. Tomiuk for generously providing the mouse and human nSMase2 clones, Patrick Roddy for technical assistance, Dr. Yasuo Okamoto for helpful advice on yeast studies, and Dr. Alicja Bielawska for providing [choline-methyl-14C]SM.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM43825 (to Y. A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 173 Ashley Ave., Charleston, SC 29425. Tel.: 843-792-4321; Fax: 843-792-4322; E-mail: hannun@musc.edu.

Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M212262200

2 S. Vaena de Avalos and Y. A. Hannun, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: SMases, sphingomyelinases; SM, sphingomyelin; A-SMase, acid sphingomyelinase; N-SMase, neutral sphingomyelinase; PC, phosphatidylcholine; PAF, platelet-activating factor; lyso-PAF, lyso-platelet-activating factor; 1-alkyl-glycerol, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine; PS, phosphatidylserine; PLC, phospholipase C; Me2SO, dimethyl sulfoxide; PBS, phosphate-buffered saline; TNF, tumor necrosis factor alpha ; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CMV, cytomegalovirus.

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ABSTRACT
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RESULTS
DISCUSSION
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