Molecular species of sphingomyelin in sphingomyelinase-sensitive and sphingomyelinase-resistant...

Molecular Species of Sphingomyelin in Sphingomyelinase-Sensitive

and Sphingomyelinase-Resistant Pools of HL-60 Cells Veronica Fitzgerald, Merle L. Blank and Fred Snyder*

Medical Sciences Division, Oak Ridge Associated Universities, Oak Ridge, Tennessee 37831-0117

ABSTRACT: This study of sphingomyelin molecular species in undifferentiated and differentiated (granulocytic form) HL-60 cells demonstrated only minor differences in the distribution of species between the sphingomyelinase-sensitive and sphingomyelinase-resistant pools of sphingomyelin in these cells. The two most prominent species of sphingosine present in both the undifferentiated and differentiated cells were those containing 16:0 (slightly higher in the sphingomyelinase-resistant membranes) and 24:1 N-acyl moieties. Cell differentiation exerted little effect on the distribution of molecular species of sphingomyelin between the sphingomyelinase-sensitive and sphingomyelinase-resistant pools in HL-60 cells, although the levels of N-palmitoyl sphinganine were significantly lower and the N- nervonoyl sphingosine higher in both pools from the differentiated cells. Our results indicate the same species of sphingomyelin, available at both the outer layer of the plasma membrane and inner layer of the plasma membrane (plus intracellular membranes) of HL-60 cells, serve as precursors for generation of the ceramides that participate in signal transduction processes initiated by cell activation. tipids 30, 805--809 (1995).

Recent research has indicated that sphingomyelin, in addition to being a structural component of cellular membranes, is involved in signal transduction processes through the generation of ceramides by the action of a sphingomyelinase.(l-10). Several investigators have utilized HL-60 cells as the biological system for their studies ( I -4 ,9 , t0) . Yet as far as we are aware, the molecular species of sphingomyelin in HL-60 cells have not been analyzed. Determinatioa of molecuiar species of sphingomyelin would seem to take on greater importance after a recent report suggesting that a specific "signall'ing" pool of sphingomyelin is resistant to treatment with sl~in- gomyelinase ( I i ) in HL-60 cells. The question then arises as to whether this precursor pool of sphingomyelin may consist of different types of molecular species than present in the

*To whom correspondence should be addressed at Medical Sciences Divi- sion, Oak Ridge Associated Universities, P.O. Box 117, Oak Ridge, "YN 37831-0117. Abbreviations: GLC, gas-liquid chromatography; HPLC, high-performance liquid chromatography; PBS, phosphate buffeted saline; TLC, thin-layer chromatography; UV, ultraviolet.

more inert pool of sphingomyelin associated with the outer portion of the plasma membrane. We therefore investigated the possibility of whether a specific distribution of sphingomyelin molecular species in the membranes of HL-60 cells could contribute to the difference in biological activity associated with the sphingomyelinase-sensitive and sphingomyelinase-resistant pools of sphingomyelin in these cells.

MATERIALS AND METHODS

Cell culture. Human leukemic cells (HL-60; purchased from the American Type Culture Collection, Rockville, MD) were grown in RPMI 1640 media containing 10% heat inactivated fetal calf serum, 100 units of penicillin per mL, 100 I~g o f streptomycin per mL, 2 mM L-glutamine, and 20 mM HEPES buffer. Cells were maintained in a humidified, 5% CO 2 at- mosphere at 37~ Stock cultures were usually diluted 1:4 with fresh media when they reached a cell density.of 1.5 x 106 cells/mL. Cells to be treated with sphingomyelinase were isolated from stock cultures by centrifugation at.200 • g for ! 5 rain and washed twice with Dulbecco's phosphate.buffered saline (PBS) with the same centrifugation between washes. The washed cells were resuspended in the same media (0.4 • 106 cellstmL) used for stock cultures except the fetal calf

serum was replaced withinsulin (10 pg/mL), transferrin (10 ~ug/mL), sodium selenite (100 tag/mL), and fatty acid free bovine serum albumin (0.1% wt/vol). Cells were then incubated in the serum-free media for 48'h, pelleted, washed once with ~PBS, .resuspended i.n serum-free media (1.6 x 106 cells/mL), and incubated 2 h before adding 0.1 unit of sphingomyelinase ,(Staphlococcus aureus) per mL of media (11 ). An identicid 'volume of the sphingomyelinase vehicle (0.25 M phosphate ~btrffer pH 7.4 in glycerol/water, 1 : 1) was added to control cultures. After 20 min in the presence of the sphingomyelinase at 37~ the cells were ,pelleted by centrifuga- tioa at 600 x g for l0 rain.and the cellular lipids extracted (12). These procedures are nearly identical to those used previously by others to hydrolyze,the sphingomyelin in the outer layer of the plasma merrlbr~me of,HL-60 cells (9,11). A simi- lax approach also has been ,.used to study the asymmetric dis- tribulitm of stahingomyelin ra~lecarlar species in human ery-

Copyright �9 1995 by AOCS Press 805 ~Lipids, Vol. 30, no. 9 (1995)

806 V. FITZGERALD ETAL.

throcyte membranes (13). The validity of using sphingomyelinase to determine the external and internal amounts of sphingomyelin also has been demonstrated recently on he- patocytes (14).

Some HL-60 cells were also differentiated into granulocytic type cells by adding dimethyi sulfoxide to the media (15) six days prior to the sphingomyelinase treatment. The average extent of cellular differentiation was 77.7 _ 5.5% (mean • SE) as measured by the intracellular reduction of nitro blue tetrazolium (16).

Determination of sphingomyelin hydrolysis by sphingomyelinase. Phospholipids from sphingomyelinase-treated cells and vehicle-treated control cells were separated by thin- layer chromatography (TLC) on plates coated with silica gel H that were developed in chloroform/methanol/glacial acetic acid/water (50:25:8:4, by vol). The developed plates were sprayed with sulfuric acid, charred by heating, and the amount of phosphorus in each phospholipid class was measured (17). The percentage decrease in sphingomyelin between the cdn- trois and sphingomyelinase-treated cells was taken as the percent of sphingomyelin that had been hydrolyzed.

Preparation of benzoate derivatives. Cellular lipid extracts were divided in hal l and one portion was benzoylated using benzoic anhydride and dimethylaminopyridine as previously described for diradylglycerols (18). Ceramide dibenzoates, representing the sphingomyelin that was originally associated with the outer leaflet of the plasma membrane (1 I), were isolated from these reaction products by preparative TLC on silica gel G plates developed in hexane/diethyl ether ( l : l , vol/vol). The developed plates were sprayed with a solution of 0.05% 2,7-dichlorofluorescein in 50% ethanol and viewed under ultraviolet (UV) light to locate the ceramide dibenzoate band (Rf- 0.3) which was extracted (12). Sphingomyelinase- resistant sphingomyelin was isolated from the second half of the total lipid extract by TLC on silica gel H plates developed in chloroform/methanol/glacial acetic acid/water (25:15:4:3, by vol). Developed plates were exposed to ammonia fumes for - 3 min and then sprayed with 0.05% 2,7-dichlorofluorescein in absolute ethanol to locate the sphingomyelin band under UV light. The sphingomyelin was extracted from the silica gel by the method of Bligh and Dyer (12) and subjected to phospholipase C hydrolysis (19,20) to remove the phosphocholine group. Total products from this phospholipase C hydrolysis were benzoylated and the ceramide dibenzoates isolated by TLC as described earlier.

Analysis of ceramide dibenzoates by high-performance liquid chromatography (HPLC). The identical dual pump HPLC system (Beckman Instruments, Berkeley, CA) previously used for separation of diradylglycerolbenzoate molecular species (20) was used to separate the molecular species of the dibenzoate derivatives of ceramides. A mobile phase of acetonitrile/isopropanol (3:2, vol/vol) at a flow rate of I mL per min and a detector set at 230 nm was used for the cc- ramide dibenzoate analyses.

A ceramide dibenzoate standard was prepared from beef brain ceramides (Sigma Chemical Co., St. Louis, MO) and

purified by TLC as described in a previous section. The purified dibenzoate derivative had a molar absorptivity of 2.62 x 104 at 230 nm, which would be the expected value for a dibenzoate derivative (20). According to earlier work, treatment of ceramides with benzoic anhydride leads to O-benzoylation but not to N-benzoylation (21). The components of 13 different HPLC peaks from the beef brain ceramide dibenzoates were collected and treated with HCI-methanol at 70~ for 18 h to produce the free sphingoid bases and methyl esters (22). These products were benzoylated (! 8,20), and the resulting mixture of methyl esters and benzoate derivatives of the sphingoid bases was separated by TLC on silica gel G plates developed in hexane/diethyl ether (I : I, vol/vol). The developed plates were sprayed with 0.05% 2,7-dichiorofluo- rescein in 50% ethanol, viewed under UV light to locate the benzoylated sphingoid bases (R r - 0.3) and methyl esters (Rf

0.7), which were then extracted (12) from the silica gel. Ad- dition of 2-3 drops of ammonium hydroxide during this extraction results in almost no contamination of dichlorofluorescein in the lower chloroform layer. The methyl esters were then analyzed by gas-liquid chromatography (GLC), and the sphingoid benzoates were separated by HPLC. An elution solvent of acetonitrile:isopropanol (4:1, vol/vol) at a flow rate of 1 mL per min through a 250 mm x 4.6 mm column packed with 5 pM Ultrasphere-ODS (Beckman Instruments) caused the sphingosine tribenzoate to be eluted at 7.9 min and sphinganine tribenzoate at 9.4 min. On the basis of graphical plots of log retention time vs. chain length the sphingosine (purchased from Sigma Chemicals) contained small amounts of 16:1 and 20:1 (<10%) bases in addition to the expected 18:1 sphingosine base. These analyses permitted identification of ! 3 major peaks in the beef brain ceramide benzoate standard (tentative identification of other minor peaks were extrapo- lated through the linear relationship that exists between log retention time and chain lengths). The composition of the beef brain ceramide benzoates was similar to that reported previously using slightly different methodology (23).

Identification of sphingomyelin molecular species in samples from HL-60 cells was based primarily on comparison of HPLC retention times of the ceramide dibenzoate derivatives between the samples and the standard prepared from beef brain. In addition, four of the larger separated ceramide dibenzoate fractions from undifferentiated HL-60 cells were also collected from the HPLC separation and subjected to the analysis of N-acyl and sphingoid base groups as described for the beef brain ceramides.

RESULTS AND DISCUSSION

Although HL-60 cells do contain low amounts of ceramide (1,2), it was calculated from information on the amounts of sphingomyelin in HL-60 cells (24) that sphingomyelin levels are about five times higher than the ceramide contents. There- fore, the small amount of free ceramides present in HL-60 cells would not likely cause a problem in interpreting our results. Also, based on the fact that no sphingosine and very lit-

Lipids, Vol. 30, no. 9 (1995)

SPHINGOMYELIN MOLECULAR SPECIES IN HL-60 CELLS 807

tie sphingomyelin (2.8% in 12 h) was produced by HL-60 cells labeled with [3H]acetylsphingosine (2), any changes in the distribution of ceramide species caused by further metabolism of ceramides during the sphingomyelinase treatment would appear to be minimal.

The extent of sphingomyelin hydrolysis by treatment of HL-60 cells with sphingomyelinase for 20 min was 56%; this accessible pool is considered to be on the outside surface of the cells. The nanomoles of sphingomyelin per 107 cells were 10.32 and 4.52 before and after treatment, respectively. Our results agree well with the previously reported amount of sphingomyelinase-accessible sphingomyelin (60%) present in the outer leaflet of the plasma membranes from HL-60 cells (11) using a slightly different analytical technique. TLC analysis of lipids extracted from the media of HL-60 cells after treatment with sphingomyelinase revealed only barely detectable amounts of ceramides, indicating that the ceramide portion of the sphingomyelin molecule remained associated with the outer plasma membrane after sphingomyelinase treatment. The distribution of molecular species in ceramides obtained by either sphingomyelinase or phospholipase C treatment of total sphingomyelin isolated from undifferentiated HL-60 cells was identical (data not shown). Thus, there was no change in the molecular species distribution that could be ascribed to a difference in selectivity between sphingomyelinase and phospholipase C treatments.

An example of the HPLC separation of ceramide dibenzoates, prepared by phospholipase C hydrolysis and benzoylation of the sphingomyelin that remained after treatment of HL-60 cells with sphingomyelinase, is shown in Figure 1. The largest component (peak A), identified as the dibenzoate

E t -

O

v

f . ) C

0 .Q <

A B C D E F G

I I I I I I I I I 5 10 15 20 25 30 35 40 45 50

Minutes

FIG. 1. High-performance liquid chromatography tracing of ceramide dibenzoate derivatives prepared as described in the Materials and Meth- ods section from the sphingomyelin located on the outer membrane surface (sphingomyelinase-sensitive) of undifferentiated HL-60 cells. Full- scale absorbance at 230 nm for this chromatogram was 0.013 O.D. units. See Tables 1 and 2 for identification of the molecular species associated with letters located below the peaks.

of N-palmitoyl sphingosine, makes up about 50 mole% of the total species. The molecular species eluting with peak E con- tains nervonic acid and was the next most abundant (-16 mole %) component of the sphingomyelin in HL-60 cells. Identification of peaks A, B, E, and G were verified to contain at least 90% of the indicated molecular species by GLC analysis of their N-acyl groups (as methyl esters) and HPLC separation of the benzoate derivatives of the sphingoid bases as described in the Materials and Methods section. Identifica- tion of other peaks is based solely on their retention times from HPLC.

Table 1 shows the distribution of molecular species of sphingomyelin that were sensitive (outside) or resistant (inside) to sphingomyelinase treatment of undifferentiated HL- 60 cells. Small but statistically significant differences in per- centages between the sphingomyelinase-sensitive (outside) and sphingomyelinase-insensitive (inside) sphingomyelin of undifferentiated HL-60 cells were seen in molecular species eluting with benzoate derivatives of N-palmitoyl (higher on the inside) and N-lignoceroyl sphingosine (higher on the outside, Table 1). However, we did not observe the extensive asymmetric distribution of sphingomyelin molecular species in HL-60 cells that is present in human erythrocyte membranes (13). Except for a small difference in the amount of the N-palmitoyl sphinganine species, the differentiated HL- 60 cells did not show a pronounced difference in distribution of sphingomyelin molecular species between the sphingomyelinase-sensitive and sphingomyelinase-resistant membrane pools (Table 2). However, the amount of N-palmitoyl sphinganine was significantly lower (P < 0.02), and the level of N-nervonoyl sphingosine was significantly higher (P < 0.02) in differentiated cells than was found in the sphingomyelin from both sphingomyelinase-sensitive and sphingomyelinase-resistant pools of sphingomyelin from undifferentiated cells. These data indicate that, although differentia-

TABLE 1 Distribution of Sphingomyelin Species Between the Sphingornyelinase- Sensitive and Sphingomyelinase-Resistant Pools in Membranes of Undifferentiated HL-60 Cells a

N-acyl Inside Outside Peak group (mole percent)

A 16:0 54.0 _+ 1.4 47.5 • 1.6 b B 16:0 c 11.4 • 2.3 9.4 • 1.3 C 18:0 3.2 • 0.2 3.2 • 0.5 D 22:1 2.7 • 0.2 2.6 • 0.2 E 24:1 16.1 • 1.4 16.5 • F 22:0 3.8 + 0.3 4.2 + 0.2 G 24:0 6.0 • 0.7 9.5 • 0.7 d

"Values are the average percent (_+ SE from three separate experiments, each with duplicate samples) of either the molecular species hydrolyzed by sphingomyelinase treatment of the intact HL-60 cells (outside) or those that were resistant to the sphingomyelinase (inside). Other species were also present but none at levels above 2.5 tool%. Determinations of statistically significant differences in the distribution of species between the inside and outside surfaces were based on Student's t-test for paired variables (bp < 0.05 and dp< 0.01 ). High-performance liquid chromatography retention times correspond to those of ceramide dibenzoate species with the indicated N-acyl groups connected to a sphingosine base, except for (c), in which the 16:0 N- acyl group is linked to a sphinganine base.

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808 V. FITZGERALD ETAL.

TABLE 2 Distribution of Sphingomyelin Species Between the Sphingomyetinase- Sensitive and Sphingomyelinase-Resistant Pools in Membranes of Differenliated HL-60 Cells a

N-acyL Inside Outside Peak group (mole percent)

A 1@0 53.8 • 0.7 51.8 • 2.4 B 16:0 c 6.5 • 0.5 4.1 + 0.3 b C 18:0 3.0 + 1.2 2.4 • 0.2 D 22:1 3.3 + 0.7 4.0 • 0.2 E 24:1 22.1 • 0.5 23.6 • 0.6 F 22:0 3.9 + 0.4 4.0 • 0.2 G 24:0 5.2 • 0.7 6.8 • 0.9

aValues are the average percent (+ SE from three separate experiments) of either the molecular species hydrolyzed by sphingomyelinase (outside) or those that were resistant to the sphingomyelinase treatment (inside) in DMSO-induced differeotiated cells. Other footnotes are the same as those described in Table 1.

gomyel in species could be involved in the sphingomyel in cycle associated with the signalling events described recently (I l). Our experiments , while not precluding this possibili ty, do demonstrate that if specific molecular species of intracellular sphingomyel in are involved in cell s ignal l ing events, then essential ly the same species are avai lable for selection on the metabolical ly active internal cel lular membranes (1 !) as are present on the outer surface of the membrane leaflet.

We bel ieve the techniques for analyses of molecular species of sphingomyelin described in this report will be very useful in future studies des igned to establ ish the functional role of sph ingomyel in in cells. Moreover , our data provide basel ine information about the various molecular species of sphingomyel in in HL-60 cells, a cell line that has proven to be a useful model system in a variety of studies dealing with

cell signalling phenomena.

TABLE 3 Distribution of Total Sphingomyelin Species in Undifferentialed and Differentiated HL-60 Cdis a

N-acyl Undifferentiated Differentiated Peak group (mole percent)

A 16:0 55.4 + 0.8 56.4 • 1.3 B b t 6:0 c 11.2 • 1.2 6.0 • 0.2 C 6 18:0 2.9 + 0.1 1.8 • 0.1 D 22:1 2.3 + 0.3 2.8 • 0.5 E b 24:1 15.7 • 1.0 19.4 • 0.7 F 22:0 3.4 • 0.2 3.6 • 0.2 G 24:0 6.0 • 0.7 4.8 • 0.4

aValues are the average mole percent • standard error. Other species also were present but none at levels above 2.5 tool%. Determinations of statistically significant differences in the distribution of species between the undifferentiated and differentiated cells were based on Student's t-test for paired variables (bp< 0.05 and do< 0.001). High-performance liquid chromatography retention times correspond to those of ceramide dibenzoate species for the indicated N-acyl groups connected to a sphingosine base. CThe 16:0 N-acyL group ~or this fraction is linked to a sphinganine base.

tion of HL-60 cells to their granulocyt ic form does not alter the amount of cel lular sphingomyel in (25), the molecular species are indeed modif ied by treatment with dimethyl sulfoxide.

Statist ically significant differences also were observed in some molecular species of the total sphingomyel in between undifferentiated and differentiated HL-60 cells (Table 3) that served as controls (cells not treated with sphingomyelinase). In these controls, the sphingomyel in species containing the N-pa lmi toyl sphinganine base a n d N-s tearoyl sphingosine base were s ignif icant ly h igher in the undifferent iated cells, and the sphingomyel in containing the N-nervonyl sphingosine base was significantly lower; no significant differences were seen in any of the other molecular species of sphingomyelin anatyzed in these cells (Table 3).

There is not enough difference in the distribution of sphingomyel in molecular species between the outer leaflet of the HL-@) plasma membrane and the sphingomyelinase-resistant pool of sph ingomyel in to suggest that a par t icular sphin-

A C K N O W L E D G M E N T S

The authors express their sincere appreciation to Shirley L. Poston for her excellent assistance in the preparation of this manuscript. This work was supported by the Office of Energy Research, U.S. Department of Energy (Contract No. DE-AC05-760R00033).

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[Received April 3, 1995, and in final revised form June 19, 1995; Revision accepted July 24, 1995]

Lipids, VoL 30, no. 9 (1995)

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