Journal of Molecular Neuroscience Copyright © 1999 Hurnana Press Inc. All rights of any nature whatsoever reserved. ISSN0895-8696 / 99 / 12:89-99 / $12.75

Involvement of Lipid Mediators on Cytokine Signaling and Induction of Secretory Phospholipase A 2 in Immortalized Astrocytes (DITNC) Wei Tong, Darshan Shah, Jianfeng Xu, J. Alan Diehl, Alexandra Hans, Mark Hannink, and Grace Y. Sun* Biochemistry Department, University of Missouri, Columbia, MO 65212 Received July 22, 1998; Revised November 23, 1998; Accepted December 2, 1998

Abstract Our previous studies demonstrated the ability of proinflammatory cytokines, such as tumor necrosis factor-~x (TNF-(z) and interleukin 1[3(IL-113),to stimulate NF~cB/DNA binding and synthesis of secretory phospholipase A 2 (sPLA2) in immortalized astrocytes (DITNC). In this study, we examined possible involvement of lipid medi~itors in the cytokine action. Using [14C]serine to label sphingomyelin and ceramide in these cells, subsequent exposure of cells to cytokines did not result in alteration of sphingomyelin/ceramide ratio. Furthermore, neither exogenous sphingomyelinase nor cell-permeable ceramides could stimulate NF~cB/DNA binding. On the other hand, C-2 ceramide (0.3 gM) as well as other lipid mediators, such as lysophosphatidylcholine and arachidonic acid, were able to elicit a small increase in sPLA2 and potentiate the induction of sPLA 2 by TNF-~x. When DITNC cells were prelabeled with [32p]Pi, an increase in labeled phosphatidic acid (PA) was observed on treatment of cells with IL-113 (200 U/mL). However, despite the ability of phorbol myristate acetate (PMA) to stimulate phospholipase D (PLD) and synthesis of phosphatidylethanol (PEt) in these cells, PLD activity was not affected by IL-1[~. With the [32p]labeled cells, however, PA-phosphohydrolase inhibitors, such as chlorpromazine and propranolol, could elicit large increases in labeled PA, indicating active PA metabolism in these cells. Cytokines also caused an increase in levels of diacylglycerol (DG) in these cells, although the source of this lipid pool is presently not understood. Taken together, these results provide evidence for the participation of PA and DG in cytokine signaling activity. Furthermore, although cytokines did not cause the release of ceramide, lipid mediators, such as lysophospholipids, and AA could modulate cytokine-mediated induction of sPLA2 in astrocytes. Index Entries: Lipid mediators; cytokine signaling; secretary phospholipsase A2; immortalized astrocytes; NF~cB/DNA binding. Abbreviations: Phosphatidic acid, PA; phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylserine, PS; arachidonic acid, AA; diacylglycerol, DG.

*Author to whom all correspondence and reprint requests should be addressed.

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Introduction Astrocytes constitute the major cell type in the central nervous system, and they are known to play many functional roles in support of different types of neuronal activities in brain. In particular, astrocytes are noted for their ability to respond to the inflammatory cytokines, such as tumor necrosis factor c~ (TNF-c~) and interleukin 113(IL-113), which is associated with activation of the pleiotropic transcription factor, NF~cB,and induction of target genes (Benveniste, 1992; Benveniste and Benos, 1995). In nonneural cells, studies to undertand the cytokine signaling pathways have indicated the participation of a large number of lipid mediators (for a review, see Serhan et al., 1996), which are generated from sphingomyelinase hydrolysis (Schutze et al., 1992, 1994; Hannun, 1994; Kolesnick and Golde, 1994) and different types of phospholipases (Murakami et al., 1997). However, studies to examine these pathways in astrocytes have been lacking. Previously, we demonstrated the ability of the immortalized astrocytes (DITNC) to respond to the proinflammatory cytokines, which in turn, led to stimulation of NF~cB/DNA binding (Diehl et al., 1995) and induction of a secretory phospholipase Aa (sPLA2) (Tong et al., 1995). Despite the involvement of several lipid mediators in the cytokine signaling pathway in nonneural cell, it is not clear whether these pathways are also associated with the cytokine action in astrocytes. In this stud?6 immortalized astrocytes were used to investigate whether hydrolysis of sphingomyelin and generation of ceramides participate in the cytokine signaling pathway leading to the stimulation of NF~cB/ DNAbinding and induction of sPLA2. Results show that although exposure of DITNC cells to TNF-ct or IL-lb alone did not stimulate sphingomyelin hydrolysis nor could cellpermeable ceramides activate NFKB/DNAbinding, lipid mediators including ceramides, lysophospholipids, and arachidonic acid could modulate the induction of sPLA2 by TNF-ot. Furthermore, this study provided evidence for participation of phosphatidic acid (PA) and diacylglycerol (DG) in cytokine signaling activity in DITNC cells.

Materials and Methods Materials An immortalized astrocyte cell line (DITNC) from rat diencephalon was provided by Deschep-

Journal of Molecular Neuroscience

per (Clinical Research Institute of Montreal, Montreal, Canada). Recombinant murine TNF-o~ and IL-l[3 (SA 1 U/5-10 pg) were obtained from R&D Systems (Minneapolis, MN). [1-14ClArachidonic acid, [1-14C]palmitic acid, and [1-14C]myristic acid (specific r a d i o a c t i v i t y , 55 m C i / m m o l ) w e r e p u r c h a s e d from A m e r i c a n R a d i o l a b e l e d (St. Louis, MO). [7-32p]ATP (3000 C i / m m o l ) a n d [32p]orthophosphate (Pi) (8500-9120 C i / m m o l ) w e r e p u r c h a s e d from N e w E n g l a n d N u c l e a r (Boston, MA). Cell-culture reagents were obtained from Cell and Immunobiology Core Facility (University of Missouri, Columbia, MO). PA, chlorpromazine, propranolol, and sphingomyelinase (from Staphylococcus aureus) were purchased from Sigma Chemical (St. Louis, MO). DG kinase (specific activity, 8.3 u n i t / m g protein) was from Calbiochem (La Jolla, CA). Cell-permeable ceramides (C2 and C6) were purchased from Matreya, Inc. (Pleasant Gap, PA). Phosphatidylethanol was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Whatman HP-K high-performance silica gel plates (10 x 10 cm, 200 ~t) were obtained from Fisher Scientific Co. (St. Louis, MO). BioSafe-NA scintillation fluid was from Research Product International Corp. (Mount Prospect, IL).

Cell Culture and Treatment The immortalized astrocytes (DITNC, passages 66-75) have been shown to exhibit characteristic properties of type i astrocytes (Radany et al., 1992). Cells were cultured in T75 cm 2 flasks containing 10 mL Dulbecco's Modified Eagle's Medium (DMEM) with 5% fetal bovine serum (FBS), 1% penicillin, 1% streptomycin, and 1% fungizone. Cells were maintained at 37°C in a Steri-Cult 200 incubator (Forma Scientific, Marietta, OH) under a humidified atmosphere of 95% air and 5% CO 2, and culture medium was changed twice weekly. After cells r e a c h e d 90% c o n f l u e n c y , t h e y w e r e b r i e f l y trypsinized and subcultured to 35-mm dishes with 1 mL of the same medium. After reaching confluency in 2-3 d, cells were used for experiments.

Probing Sphingomyelin Metabolism in [14ClSerine-LabeledDITNC Cells In order to assess cytokine effect on sphingomyelin hydrolysis, DITNC cells in 35-mm dishes

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Lipid Mediators in Cytokine-lnduced sPLA2 w e r e i n c u b a t e d in D M E M w i t h [14C]serine (5 gCi/ml) for 6 h. After removing the excess label in the c u l t u r e m e d i u m , cells w e r e s t i m u l a t e d with TNF-ct (200 u / m L ) and IL-I[3 (100 u / m L ) in a serum-free m e d i u m for different periods ranging from 5 min to 2 h. After stimulation, the culture m e d i u m was removed, and lipids in the cells were e x t r a c t e d by p a r t i t i o n i n g w i t h c h l o r o f o r m methanol-H20 (4:2:1.5, by vol). The labeled lipids were s e p a r a t e d by a t w o - d i m e n s i o n a l HPTLC system as described by Sun (1988). This system separated individual phospholipids in the cells, includi n g s p h i n g o m y e l i n . The n e u t r a l l i p i d s t h a t appeared in the u p p e r right corner of the HPTLC plate were extracted with chloroform/methanol, 2:1 (v/v), and separated in a one-dimensional solvent system containing chloroform-methanolacetone-acetic a c i d - a m m o n i u m acetate (70:30:27. 5:2.3:5, by vol). This solvent system can separate individual neutral lipids, i n c l u d i n g ceramides. I n d i v i d u a l lipids from the HPTLC plate were removed into scintillation vials for measurement of radioactivity (Beckman LS 3800, Beckman Instrument Co., Sunnyvale, CA). Sphingomyelinase activity was depicted by the decrease in radioactivity of s p h i n g o m y e l i n a n d a c o n c o m i t a n t increase in ceramide.

Electrophoresis Mobility Shift Assay (EMSA) for NFKB/DNA Binding EMSA w a s c a r r i e d o u t a c c o r d i n g to t h a t described by Diehl et al. (1995). Briefly, the DNA binding probe was synthesized by primer extension with using [c~-32p]dCTP and [ot-B2p]dGTP and 10 m M each of unlabeled dATP and dTTP. The sense strand of the palindromic KB probe was 5'CAACGG C A G G G G A A T T C C C C T C T C C T T - 3 ' , and the sequence of the primer was 5'-AAGGAGAGGG3'. The primer (100 pmol) and the template (1 pmol) were annealed at 80°C and cooled to room temperature. The primer extension reaction was carried out with DNA polymerase I large fragment (Klenow) at 37°C for 1 h in restriction buffer (10 m M Tris, p H 7.5, 10 m M MgCI2, 10 m M dithiothreitol [DTT]). Unlabeled dNTP (100 mM) was added, and the reaction was allowed to continue for 30 min. U n i n c o r p o r a t e d n u c l e o t i d e s were r e m o v e d by p h e n o l extraction a n d t h r o u g h a Sephadex G25 spin column. The probe was pre-

Journal of Molecular Neuroscience

91 cipitated with NH4OAc, 20 gg tRNA, and ethanol at -80°C. Cell nuclear extracts (3 gL) from various treatments were incubated with I gg of poly (dI-dC) in the HDKE buffer containing 20 m M HEPES, 50 m M KC1, 10 m M ETDA, and 2% glycerol for 15 min at room temperature. The probe (10,000 cpm) was a d d e d to the reaction mixture (20 gL) and was incubated for an additional 15 min. The reaction mixture was subjected to electrophoresis using a 5% polyacrylamide gel and a buffer system containing 1X TBE (89 m M Tris-HC1, 89 m M boric acid, 2 m M EDTA, pH 8.0). RelA-DNA complexes were visualized by autoradiography.

Assay of sPLA2 in the Culture Medium Induction of sPLA 2 by cytokines, C2-ceramides, and other lipid mediators was assessed in cellc u l t u r e m e d i u m 24 h after a d d i t i o n of t h e test a g e n t s . The p r o t o c o l for m e a s u r e m e n t of sPLA2 a c t i v i t y u s i n g [ 1 4 C ] a r a c h i d o n o y l phosphatidylethanotamine (PE) as substrate was same as that described by Tong et al. (1995).

Effect of Cytokines on PA Metabolism in [32p]Pi-Labeled Cells In this experimental protocol, DITNC ceils in DMEM containing 1% FBS were incubated with [32p]Pi (5 ~tCi/mL) for 4 h. After labeling, cells were washed twice with 2 mL of DMEM, and replacement with 1 ml of DMEM containing 1% bovine serum albumin (BSA). After equilibration at 37°C for 2 h, ceils were stimulated with IL-11~(100 U / m L ) or TNF-o~ (200 U / m L ) for various time periods ranging from 5 to 60 min. The reaction was terminated by removal of the culture m e d i u m into a scintillation vial for counting of radioactivity and immediately followed by addition of methanol (1 mL) to the cells. Lipids extracted from the cells (together with addition of nonradioactive PA standard) were separated either by the two-dimensional system d e s c r i b e d by S u n a n d Lin (1989) or t h e oned i m e n s i o n a l s y s t e m c o n t a i n i n g ethyl acetateisooctane-acetic acid (90:50:20, by vot) (Gustavsson et al., 1993). After solvent development, the HPTLC plates were exposed to Kodak OMAT-AR films and subsequently to iodine vapors. Individual lipid spots were scraped to scintillation vials for counting of radioactivity.

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Assay of Phospholipase D (PLD) Activity Several labeling protocols were used to examine the PLD activity in DITNC cells and to test whether cytokine-induced increase in labeled PA is associated with activation of this enzyme. Phospholipids in cells were labeled with [14C]palmitic acid or [14C]myristic acid (1 gCi/mL) for 24 h, or with [33p]ATP for 6 h. After labeling, cells were washed twice with 1% BSA in DMEM and were incubated in the same medium together with 0.5% ethanol for 15 min at 37°C prior to a 30-rain stimulation with different agents, i n c l u d i n g phorbol m y r i s t a t e acetate (PMA), TNF-~x, and IL-113. The reaction was t e r m i n a t e d by a d d i n g m e t h a n o l together with nonradioactive PA and phosphatidylethanol (PEt) standards. Lipid extracts were separated by a two-dimensional HPTLC system as described by Sun (1988) with the exception that the HC1 step was omitted. Labeled PA and PEt as well as other phosp h o l i p i d s w e r e r e m o v e d for m e a s u r e m e n t of radioactivity.

Determination of DG and Ceramide Levels by the DG-Kinase Method DITNC cells in DMEM containing 1% BSA were stimulated with IL-113and TNF-o~ for 0, 15, 30, and 60 rain. The m e d i u m was removed, and lipids in the cells were extracted by addition of I mL of icecold methanol. DG and ceramide in the lipid extract were assayed using the DG kinase p r o c e d u r e according to Preiss et al. (1986) with minor modifications. Briefly, solvents in the lipid extract were evaporated u n d e r nitrogen and the lipids were solubilized in 40 gL of a solution containing 7.5% octyl-[3-glucoside and 5 mM cardiolipin in 1 mM diethylenetriaminepenta-acetic acid (DETAPAC) with vigorous mixing. The incubation system contained 100 btL of reaction buffer (100 mM imidazole HC1, 100 mM NaC1, 25 mM MgC12,2 mM EGTA, pH 6.6), 20 gL freshly prepared 20 mM DTT, 20 gL recombinant DG kinase (5 gg/sample) and 1 ~tCi 7-[32p]ATP in 20 gLbuffer (1 mM DETAPAC, 10 mM ATP, and 100 mM imidazole, pH 6.6). The reaction mixture was vortexed and incubated at 25°C for 30 min. The reaction was terminated by adding 4 vol of chloroform-methanol (2:1, v / v ) together with nonlabeled PA. After phase separation, solvents in the organic phase were e v a p o r a t e d and lipids spotted on HPTLC plate. In each experiment, a

Journal of Molecular Neuroscience

Tong et al. standard curve was constructed using DG and ceramide as standards. Plates were developed in an acidic solvent system containing chloroformmethanol-acetone-acetic acid-ammonium acetate (70:30:27.5:2.3:5, by vol). The lipid bands were identified by exposing the plate to iodide vapor and were transferred to scintillation vials for counting of radioactivity.

Results Cytokines Did Not Elicit an Increase in Ceramide in [14C]Serine-Labeled DITNC Cells Since serine is an essential component for de novo biosynthesis of sphingosine, this amino acid was used to label the sphingolipids in the cells. When DITNC cells were incubated with [14C]serine for 6 h, labeling of s p h i n g o m y e l i n and c e r a m i d e accounted for 18.8 +_2.4% and 21.9 + 1.3% (n = 3) of the total phospholipids, respectively. In addition, l a b e l e d s e r i n e w a s i n c o r p o r a t e d into p h o s phatidylserine (PS) (28.8%), PE (13.1%), as well as PC (10.0%), suggesting active metabolism of the serine-containing phospholipid head groups in these cells. When cells prelabeled with [14C]serine for 6 h were exposed to sphingomyetinase from S. aureus (1 U/mL) for 30 min, radioactivity in sphingomyelin decreased from 18.8 to 15.5% and that in ceramide increased from 21.9 to 25.1%. Since exogenous sphingomyelinase cannot penetrate the cell surface, only sphingomyelins present in the outer monolayer of the cells are hydrolyzed. Thus, the change in labeling represents the proportion o f sphingomyelin present in the outer monlayer of the plasma membrane. Using this labeling protocol, we failed to observe a consistent increase in ceramide/sphingomyelin ratio on exposing cells to IL-113 (100 U / m L ) or TNF-0~ (200 U / m L ) at different time-points ranging from 5 to 60 min (data not shown).

Cell Permeable Ceramides and Exogenous Sphingomyelinase Did Not Activate NF~B/DNA Binding EMSA assay was used to examine whether cellp e r m e a b l e c e r a m i d e s (C2 and C6) as well as ceramides generated by exogenous sphingomyeli-

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Lipid Mediators in Cytokine-lnduced sPLA2

A

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Fig. 1. Effect of exogenous sphingomyelinase from S. aureus (1 U/mL) (A) and C-6 ceramide (B) on NFKB/DNA binding in DITNC cells.

nase could activate NFKB / DNAbinding. Although exposing cells to TNF-ct could result in a timedependent increase in NF~cB/DNA binding, treatment of cells with sphingomyelinase (Fig. 1A) or C-6 ceramides (Fig. IB) failed to elicit this event.

Effects of Ceramide and Other Lipid Mediators on Cytokine Induction of sPLA2 Release In this experiment, sPLA 2 activity in culture m e d i u m was assessed in cells exposed to C2ceramide (0.3 ~tM), TNF-cz (200 U/mL), IL-113 (100 U/mL), and their combinations for 24 h. As discussed previously (Tong et al., 1995), DITNC cells responded to IL-I~ at 100 U / m L b e t t e r than to TNFcz at 200 U / m L in their ability to induce sPLA2. Exposing cells to C2-ceramide alone resulted in a small increase in sPLA2 release (Fig. 2). The presence of ceramide also potentiated the induction of sPLA2 release by TNF-ot. On the other hand, C-2 ceramide did not further enhance the sPLA2 release owing to induction by IL-I~, probably because IL1[~ already maximally induced sPLA2 in these cells (Fig. 2). In a study to examine the dose-effect of the

Journal of Molecular Neuroscience

C-2 ceramide (0.3-5 ~tM/mL) on sPLA 2 induction, results indicated that only the 0.3-pM concentration was effective (data not shown). We also tested the effects of other lipid mediators, such as lysophosphatidic acid (LPA), lyso-phosphatidylcholine (LPC), and arachidonic acid (AA), for their ability to induce sPLA2 and to enhance sPLA2 induction by TNF-ot. As shown in Fig. 3, addition of LPA, LPC, o r A A (at 10 ~tM each) to the cells gave rise to a small, but significant increase in sPLA2 release. F u r t h e r m o r e , LPC (but not LPA) and AA also enhanced the sPLA 2 release induced by TNF-ot. When LPC was added together with AA, an additive effect on sPLA 2 release was observed regardless of the presence or absence of TNF-cz (Fig. 3).

Cytokine-lncreased Labeling of PA in pzPi]Cells After incubation of cells with [32p]Pi for 4 h, a substantial amount of the label was incorporated into the membrane phospholipids. Under this incubation condition, labeled PA contributed only 0.36 % of the total labeled phospholipids in the cells. When this labeling protocol was used to probe the effect

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Tong et al. 20-

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Fig. 2. Effects of C2-ceramide (0.3 ~M), TNF-~ (200 U/mL), IL-113(100 U/mL), and their combinations on induction of sPLA2 in DITNC cells. Cells were treated with the test agents for 24 h, and culture medium was taken for assay of sPLA2 activity as described in text. Data represent the % FFA released from U4C]arachi donoy[-PE (10,000 dpm) and are means + SD from 3 dishes in each incubation condition.

of TNF-c~ and IL-I[3 on PA metabolism, we initially observed that m a n y factors could alter labeling of PA, including shaking the culture dish and removing cells from the incubator. Nevertheless, addition of TNF-cx or IL-l[3 to the cells consistently showed a transient increase in labeled PA during the initial 30 min (data not shown). Results in Fig. 4 show an increase in labeled PA on stimulating cells with IL-113 in a typical experiement. Active PA metabolism in DITNC cells can be demonstrated by treating cells with propranolol or chlorpromazine, agents known to inhibit PAphosphohydrolase. Data in Fig. 5 show a dosedependent increase in labeled PA on incubating cells with these inhibitors at 37 ° for 30 min. Furthermore, w h e n [32p]-cells were pretreated with chloropromazine (500 gM) for 15 min and then followed by stimulation with TNF-c~ (200 U/mL), a further increase in labeled PA was observed (Fig. 6). We also tested whether chlorpromazine-induced increase in PA could cause the induction of sPLA2 and whether this condition could enhance TNF-a mediated induction of sPLA 2. This experiment was not successful because of apparent toxic effect of this drug on the cells.

Journal of Molecular Neuroscience

Cont LPA LPC AA LPC ÷

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Fig. 3. Effects of lipid mediators on induction of sPLA2 and their ability to potentiate sPLA2 induction byTN F-~z.Cells were exposed to lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), and arachidonic acid (AA) (10 ~tM each) in the presence and absence of TNF-o~ (200 U/mL) for 24 h. After incubation, cub ture medium was taken for assay of sPLA2activity. Data represent the % FFA released from [~4C]arachidonoy[PE (10,000 dpm) and are means _+SD from 3 dishes in each incubation condition.

Cytokine-lnduced Increase in PA Is Not Owing to PLD We tested whether PLD is present in DITNC cells, because activation of this e n z y m e can be a source of PA. In this experiment, [33p]ATP was used to label the cells. In this experiment, labeled ATP was found incorporated into most phospholipids similar to the labeling pattern after incubating cells with [32p]Pi. The autoradiographic pictures in Fig. 7 show that this two-dimensional HPTLC clearly separated PA and PEt from other phospholipids. Furthermore, an increase in labeling of PEt was observed on incubating cells with PMA (100 nM) in the presence of ethanol (0.5%) (Table 1). We also tested PEt formation using the conventional labeling protocol with [14C]palmitic and [I4C]myristic acids. Although PEt was formed in the presence of ethanol, addition of TNF-c~ or IL-1[~ to the cells did not increase its synthesis further (data not shown).

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Lipid Mediators in Cytokine-lnduced sPLA2

0.8-] 0 /

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Fig. 4. Changes in labeled PA after stimulating DITNC cells with IL-1I]. Cells were first labeled with [32p]Pi (5 luCi/mL/dish) for 4 h. After incubation, cells were washed with 2 mL of 1% BSA/DMEM two times and then replenished with l mL of the same medium. Cells were then stablized for 2 h prior to addition of IL-1 [3 (100 u/mL). The cell lipid extract was subjected to the one-dimensional HPTLC system to separate PA from other phospholipids. Data depict radioactivity of PA as % of total phospholipids and are average from two dishes. Similar results were observed in two other experiments. 3.0-

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Fig. 5. Effect of chlorpromazine and propranolol on labeled PA in [32p]Pi-labe[ed cells. Cells were incubated with [32p]Pi (5 ~tCi/mL/dish) for 4 h and then followed by washing. After equilibration for 2 h, different concentrations of these compounds were added, and cells were incubated for 30 min. Cell lipid extract was subjected to separation of lipids by the two-dimension HPTLC procedure as described in text.Values are %PA in total lipids.

Journal of Molecular Neuroscience

Fig. 6. TNF-0~ increased [32p] PA in chlorpromazinetreated cells. [32p] Pi-labeled cells were preteated with chlorpromazine (500 luM) for 15 rain prior to exposure toTNF-c~ (200 U/mL) for different time periods. Results depict the amount of PA as a % of total phospholipids in cells.

Cytokine Effect on D G and Ceramide in DITNC Cells In this experiment, cells were exposed to either IL-1 ~3or TNF-c~ for different time periods, and the cell lipid extracts were used to determine DG and ceramide levels using the radioenzyme assay protocol as described by Preiss et al. (1986). Data in Table 2 show a small, but significant increase in levels of DG after treating cells with both TNF-c~ and IL-I~. However, a l t h o u g h these same cell extracts contained endogenous ceramides, no significant changes in this lipid were observed owing to exposing cells with cytokines.

Discussion Although proinflammatory cytokines can transcriptionally activate target genes, such as sPLA 2 in astrocytes (Oka and Arita, 1991; Tong et al., 1995), whether the induction of this enzyme is subjected to modification by lipid mediators has not been examined in detail. In some nonneural cell lines, this type of cytokine action has been linked to the s t i m u l a t i o n of s p h i n g o m y e l i n h y d r o l y s i s a n d ceramide production (see reviews by Hannun, 1994;

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-]~ Ill Fig. 7. Autoradiographs depicting two-dimensional separation of phospholipids in DITNC cells after incubation with [3~P]ATP for 4 h. Notice that this system gave a complete separation of PA and PEt from other phospholipids. (A) A typical labeling pattern in control and (B) after incubation with PMA (100 nM) in the presence of ethanol (0.5%).

Kolesnick and Golde, 1994; Schutze et al., 1994). Apparently, the ability of cytokines to stimulate sphingomyelin hydrolysis is cell-specific and several studies failed to demonstrate the ability of TNFor ceramide to activate NFtcB (Dbaido et al., 1993; Betts et al., 1994; Johns et al., 1994). Recent studies further reveal a complex role of ceramide in modulating cell signaling pathways (Gomez-Munoz, 1998) a n d in m e d i a t i n g a p o p t o t i c cell d e a t h

Journal of Molecular Neuroscience

processes (Aggarwal and Higuchi, 1997; Mangoura and Dawson, 1998;Yoshimura et al., 1998). In the present study, we used [~4C]serine to label the sphingolipids as well as other serine-containing phospholipids in the cells. This labeling protocol seems to have successfully labeled all the sphingomyelin pools in the cells, because exogenous sphingomyelinase was able to hydrolyze a portion (~25%) of the labeled sphingomyelin, presumably the pool present in the outer m e m b r a n e of these cells. Using this labeling procedure, however, we were not able to observe consistent changes in sphingomyelin / ceramide ratios on treating cells with TNF-cz and IL- 113.The lack of an effect of exogenous sphingomyelinase and cell-permeable ceramide was further shown in the EMSA study, which failed to elicit an increase in NF~cB/DNA b i n d i n g u n d e r similar t r e a t m e n t conditions. A recent study by Pahan et al. (1998) also failed to d e m o n s t r a t e s p h i n g o m y e l i n a s e or ceramide to induce nitric oxide (NO) directly in primary astrocytes, although increasing ceramide levels in these cells could enhance NO p r o d u c t i o n i n d u c e d by lipopolysaccharides. Our study here also shows that ceramide as well as other lipid mediators, including LPC and AA, could individually increase s P L A 2 in DITNC cells and that these c o m p o u n d s could potentiate the induction of sPLA 2by TNF-0~. These results are in agreement with the notion that cytokine induction pathways are subjected to regulation by these intracellular mediators (Serhan et al., 1996). Although not well understood, a n u m b e r of factors, e.g., the increase intracellular PLA2, m a y lead to the generation of these lipid m e d i a t o r s (Murakami et al., 1997), which in turn, are directly l i n k e d to p r o t e i n k i n a s e C (PKC) a c t i v a t i o n (Nishizuka, 1992). An earlier study by Oka and Arita (1991) had demonstrated the role of PKC in the cytokine signaling p a t h w a y in astrocytes. When DITNC cells were labeled with [32p]Pi, TNF-c~ and IL-I~ could elicit a transient increase in labeled PA. The large increase in PA on treating cells with PA-phosphohydrolase inhibitors, i.e., chlorpromazine and propranolol, revealed active metabolism of PA in these cells. Apparently, a sufficient a m o u n t of [32p]ATP as well as DG, its substrate, must be present within the cells to mediate this rapid phosphorylation event. The active metabolism of PA in these cells is consistent with other studies demonstrating the important role of this

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Lipid Mediators in Cytokine-lnduced sPLA2

97

Table 1 PMA-Induced Increase in PA and Phosphatidylethanol (PEt) Synthesis in DITNC Cellsa

PEt PA

Control

PMA

EtOH

PMA + EtOH

0.74 + 0.39 2.65 + 0.23

0.84 + 0.10 3.11 + 0.07 b

0.73 + 0.33 2.52 _ 0.12

1.38 + 0.15 b 3.21 + 0.49

~DITNC cells were prelabeled with [33p]ATP and subsequently treated with PMA a n d / o r ethanol (EtOH) as described in Fig. 7. Results are percentage of labeled PEt or PA of total phospholipids and are means + SD from 3 d i s h e s / p treatment group. bDepicts values that are significantly different from controls based on unpaired Student's t-test, p < 0.05. Table 2 DG and Ceramide Levels in DITNC Cells Stimulated with TNF-c~ and IL-113a Time DG Zero time 15 rain 30 min 60 rain Ceramides Zero time 15 rain 30 min 60 min

Control

TNF-o~

IL-l~3

3.1 _+0.17 2.9 + 0.31 3.0 + 0.23 3.2 + 0.41

-3.2 + 0.23 3.4 + 0.14 b 3.7 + 0.07 b

-3.7 + 0.14 b 3.5 _+0.18 b 3.4 + 0.23

3.4 + 0.03 3.2 + 0.49 3.1 + 0.10 3.2 ___0.18

-3.2 + 0.36 3.7 + 0.20 4.4 + 0.70

-3.6 + 0.09 3.7 + 0.13 4.1 + 0.66

aDITNC ceils in DMEM containing 1% BSA were treated with TNF-c~ and IL-1~3 for the time indicated. Lipids were extracted from each dish of cells and the lipid extract was used for assay of DG and ceramide levels using the DG kinase protocol with [32p]ATP as described by Priess, et al. (1986). Values depict nmol (mean + SD, n = 4 / p treatment condition) of DG or ceramides/p 35-min dish (1 x 106 cells). bDepicts values that are significantly different from controls based on unpaired Student's t-test, p < 0.05. phospholipid in signaling mechanisms (Abraham et al., 1995). Several studies have linked PA to receptor p a t h w a y s t i m u l a t e d by IL-11 (Siddiqui a n d Yang, 1995), platelet-derived growth factor (Fukami and Tekenawa, 1992), angiotensin II (Lassegue et al., 1993), and endothelin (Desagher et al., 1997). A l t h o u g h the physiological significance of PA in these signaling events has not been fully explored, it has been considered a potential signal transducer for cardiac h y p e r t r o p h y (Dhalla et al., 1997), and modulator of the mitogenic and inflammatory pathw a y s (Fukami and Takenawa, 1992;Abraham et al., 1995). ]ntracellular PA biosynthesis can be mediated by multiple biochemical p a t h w a y s , e.g., acti-

Journal of Molecular Neuroscience

vation of PLD acting on PC, DG-kinase (Lester et al., 1989; Dhalla et al., 1997), a n d acyltransferase specific for lyso-PA (Bursten et al.; 1991 A b r a h a m et al., 1995). Similar to p r i m a r y astrocytes (Gust a v s s o n et al., 1993), PLD activity is p r e s e n t in DITNC cells, and PMA can mediate synthesis of PEt in the presence of ethanol. However, PLD pathw a y is probably not the source for the increase in PA, b e c a u s e PLD a c t i v i t y w a s n o t a l t e r e d b y cytokines. Since cytokine treatment resulted in an increase in DG levels in these cells, it is reasonable to attribute the increase in PA labeling to stimulation of the D G - k i n a s e / P A - p h o s p h o h y d r o l a s e pathway. A l t h o u g h the source of DG is not k n o w n a n d

Volume 12, 1999

98 needs to be further investigated, recent cloning studies have identified several DG kinases in the nervous system (Goto et al., 1992), and some with a consensus sequence suggesting a translocation m e c h a n i s m (Kelleher and Sun, 1989; Sakane et al., 1991). It is possible that either PA alone or its increased turnover to DG is associated with activation of specific isoforms of PKC (Limatola et al., 1994; Lang et al., 1995), which in turn, upregulate the cytokine signaling cascade in these cells.

Acknowledgment This Work was s u p p o r t e d in part by research grants NS 30178 and AA-06661 from NIH and a Research Board grant 95-056 from University of Missouri-Columbia.

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Lipid Mediators in Cytokine-lnduced sPLA2 Limatola C., Schaap D., Moolenaar W. H., and van Blitterswijk W. J. (1994) Phosphatidic acid activation of protein kinase C-zeta overexpressed in COS cells: comparison with other protein kinase C isotypes and other acidic lipids. Biochem. J. 304, 1001-1008. Ma ngoura D. and Dawson G. (1998) Programmed cell death in cortical chick embryo astrocytes is associated with activation of protein kinase PK60 and ceramide formation. J. Neurochem. 70, 130-138. Murakami M., Nakatani Y., Atsumi G., Inoue K., and Kudo I. (1997) Regulatory functions of phospholipase A2. Crit. Rev. hnmunol. 17, 225-283. Nishizuka Y. (1992) Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258, 607-614. Oka S. and Arita H. (1991) Inflammatory factors stimulate expression of group II phospholipase A 2 in rat cultured astrocytes. Two distinct pathways of the gene expression. ]. Biol. Chem. 266, 9956-9960. Pahan K., Sheikh E G., Khan M., Namboodiri A. M. S., and Singh I. (1998) Sphingomyelinase and ceramide stimulate the expression of inducible nitric oxide synthase in rat primary astrocytes. J. Biol. Chem. 273, 2591-2600. Preiss J., Loomis C. R., Bishop W. R., Stein R., Niedel J. E., and Bell R. M. (1986) Quantitative measurement of sn-l,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. J. Biol. Chem. 261, 8597-8600. Radany E. H., Brenner M., Besnard ]7., Bigornia V., Bishop J. M., and Deschepper C. F. (1992) Directed establishment of rat brain cell lines with the phenotypic characteristics of type 1 astrocytes. Proc. Natl. Acad. Sci. USA 89, 6467-6471. Sakane F., Yamada K., Imai S-I., and Kanoh H. (1991) Porcine 80 kDa diacylglycerol kinase is a calciumbinding and c a l c i u m / p h o s p h o l i p i d - d e p e n d e n t enzyme and undergoes calcium-dependent translocation. J. Biol. Chem. 266, 7096-7100.

Journal of Molecular Neuroscience

99 Schutze S., Potthoff K., Machleidt T., Berkovic D., Wiegmann K., and Kronke M. (1992) TNF activates NF-KB by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell 71, 765-776. Schutze S., Machleidt T., and Kronke M. (1994) The role of diacylglycerol and ceramide in tumor necrosis factor and interleukin-1 signal transduction. J. Leukocyte Biol. 56, 533-541. Serhan C. N., Haeggstrom J. Z., and Leslie C. C. (1996) Lipid mediator networks in cell signaling: u p d a t e and impact of cytokines. FASEB J. 10, 1147-1158. Siddiqui R. A. and Yang Y. C. (1995) Interleukin-11 induces phosphatic acid formation and activates MAP kinase in mouse 3T3-L1 cells. Cell. Signalling 7, 247-259. Sun G. Y. (1988) Preparation and analysis of acyl and alkenyl groups of glycerophospholipids from brain subcellular membranes, in Neuromethods : Lipids and Related Compounds, vol. 7 (Boulton, A. A., Baker, G. B., and Horrocks, L. A., eds.), Humana Press, Clifton, New Jersey, pp. 63-82. Sun G. Y. and Lin T. N. (1989) Time course for labeling of brain m e m b r a n e phosphoinositides and other phospholipids after intracerebral injection of [32p]ATP. Evaluation by an improved HPTLC procedure. Life Sci. 44, 689-696. Tong W., Hu Z. Y., and Sun G. Y. (1995) Stimulation of group II p h o s p h o l i p a s e A 2 m R N A expression and release in an immortalized astrocyte cell line (DITNC) by LPS, TNFo~ a n d IL-I~. Interactive effects. Mol. Chem. Neuropathol. 25, 1-17, 1995. Yoshimura S., Banno Y., Nakashima S., Takenaka K., Sakai H., Nishimura Y., et. al. (1998) Ceramide formation leads to caspase-3 activation during hypoxic PC12 cell death. Inhibitory effects of Bcl-2 on ceramide formation and caspase-3 activation. J. Biol. Chem. 273, 6912-6917.

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