©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence That Ceramide Selectively Inhibits Protein Kinase C- Translocation and Modulates Bradykinin Activation of Phospholipase D (*)

(Received for publication, September 14, 1994; and in revised form, November 11, 1994)

Meril J. Jones (§) Andrew W. Murray

From the School of Biological Sciences, Faculty of Science and Engineering, Flinders University, GPO Box 2100, Adelaide, South Australia 5001, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Sphingomyelinase (SMase) treatment (0.1 unit/ml for up to 30 min) of mouse epidermal (HEL-37) or human skin fibroblast (SF 3155) cells preincubated with [^3H]serine to label the sphingomyelin pool caused the accumulation of labeled ceramide but not sphingosine or ceramide 1-phosphate. Incubation of HEL-37 cells with dioctanoylglycerol (diC(8)) or SF 3155 cells with bradykinin caused translocation of calcium/phosphatidylserine-dependent protein kinase C (PKC) activity to particulate material. In both cell lines the translocation was blocked by SMase treatment of the cells or by incubation with the cell-permeable ceramide analogue N-acetylsphingosine (C(2)-Cer). Western blot analysis indicated that treatment of HEL-37 cells with diC(8) or SF 3155 cells with bradykinin resulted in the translocation of both PKC-alpha and PKC- to particulate material. Treatment with SMase or C(2)-Cer specifically blocked the translocation of PKC-alpha but not that of PKC-. Pretreatment of cells with SMase or C(2)-Cer also inhibited the activation of phospholipase D activity induced by either diC(8) (HEL-37 cells) or bradykinin (SF 3155 cells). The data provide strong evidence that ceramide can negatively regulate the translocation of PKC-alpha but not PKC- and further suggest that PKC-alpha may be involved in regulating phospholipase D activity.


INTRODUCTION

Sphingolipids have received increasing attention over the past decade as modulators of biological events (for review, see (1) ). Many investigations have implicated sphingomyelin (SM) (^1)hydrolysis products as biologically active molecules (for review, see (2) ). Although ceramide is the immediate product of sphingomyelinase (SMase) hydrolysis of SM, most attention has focused on sphingosine, which has been shown to modulate numerous cellular functions (for review, see (2) ) including inhibition of PKC activity(3) . More recently, ceramide has been demonstrated to activate a protein kinase in A431 cells and HL-60 cells (4) , stimulate phosphorylation of epidermal growth factor receptor in A431 cells(5) , inhibit DAG kinase in HL-60 cells(6) , stimulate a protein phosphatase in T9 glioma cell membranes(7, 8) , induce apoptosis in a leukemic cell line(9) , and activate mitogen-activated protein kinase in HL-60 cells(10) . Interest in ceramide as a second messenger molecule (for review, see (11, 12, 13) ) has been particularly stimulated by the reports that dihydroxyvitamin D(3), tumor necrosis factor-alpha, interleukin 1beta, and interferon- receptors appear to be tightly linked to activation of SMase, a linkage that may involve fatty acids(14) .

Protein kinase C (PKC) is a family of structurally related serine/threonine kinases that plays an important role in signal transduction and numerous cellular functions (for review, see (15) ). Differential regulation of the activation of different forms of PKC is not well understood. Calcium concentrations are probably important, as one group of isozymes (alpha, beta(I), beta, and ) are regulated by phosphatidylserine (PS), Ca, and DAG. However the activities of the novel isozymes (, , /L, and ) are independent of Ca, and is independent of both Ca and DAG. A crucial role for Ca has been reinforced in a recent paper by Ha and Exton(16) . Incubation of IIC9 cells with thrombin caused a transient increase in cytosolic Ca concentration and a more sustained elevation of DAG derived from both inositol and non-inositol lipids. This was associated with a transient translocation of PKC-alpha (Ca-dependent) and a more prolonged translocation of PKC- (Ca-independent). Furthermore, cells stimulated with platelet-derived growth factor, which showed an increase in phosphatidylcholine-derived DAG without change in intracellular Ca levels, resulted in the translocation of PKC- only. The isozyme did not translocate in response to either agonist. From these data the authors concluded that the differential translocation of the isozymes related to different calcium requirements rather than to the source of DAG.

The cellular role of the different PKC isozymes remains unclear, although studies using overexpression of specific PKC isozymes are now associating specific functions with individual isoforms. One study (17) using NIH 3T3 cells transfected with PKC- or PKC- showed that overexpression of PKC- resulted in cells with altered morphology, slower growth rate, and a decrease in cell density at confluence. In contrast, overexpression of PKC- led to increased growth rate and higher cell densities in monolayers without change in morphology. PKC isozyme function can also be studied using antisense technology. In a recent study, Balboa and colleagues (18) selectively reduced the levels of either PKC-alpha or PKC-beta(I) by transfection of Madin-Darby canine kidney D1 cells with corresponding antisense oligonucleotides. This work implicated PKC-alpha but not PKC-beta(I) in the activation of phospholipase D.

In the present report we studied the differential regulation of PKC isozymes by ceramide and established that the lipid can block the translocation of PKC-alpha but not PKC- to particulate material. Furthermore, it was found that ceramide inhibited the diC(8)-mediated (HEL-37 cells) or bradykinin-mediated (SF 3155 cells) activation of phospholipase D, providing additional evidence that PKC-alpha is involved in regulating phospholipase D activity.


EXPERIMENTAL PROCEDURES

Materials

ATP, benzamidine, diC(8), ceramides (Type III, from brain sphingomyelin), histone (Type III-S), phenylmethylsulfonyl fluoride, phosphatidylserine, SM, SMase (from Staphylococcus areus), and sphingosine were from Sigma. L-[^3H]Serine (31.1 Ci/mmol) and [9,10-^3H]palmitic acid (60 Ci/mmol) were from DuPont NEN, and [-P]ATP (4000 Ci/mmol) was from Bresatec, Adelaide, South Australia. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was from P-L Biochemicals, and leupeptin was from Tokyo Kasai Kogyo Co. The cell culture media Eagle's minimal essential medium, RPMI 1640, and fetal calf serum were obtained from the Commonwealth Serum Laboratories, Melbourne, Australia. N-Octanoylsphingosine (C(8)-Cer) was synthesized as described (19) and stored in chloroform/methanol (98.5:1.5) under nitrogen. N-Acetylsphingosine (C(2)-Cer) was from Sapphire Biochemicals, Sydney, Australia. When chromatographed on silica gel thin layer plates in chloroform/methanol (95:5) ceramide, C(8)-Cer and C(2)-Cer had R(F) values of 0.68, 0.58, and 0.32, respectively. Before use, an aliquot was dried under nitrogen, dissolved in ethanol, and sonicated into medium as required. The mouse epidermal cell line (HEL-37) was maintained as described(20) . SF 3155 human fibroblast cells were supplied by Dr A. Poulos, Women's and Children's Hospital, North Adelaide, and were grown and maintained in RPMI 1640 culture medium. The cells were grown in Nunc cell culture flasks and passaged weekly. When required for experimentation, both cell types were grown to confluency in 6- or 10-cm dishes. For experiments involving bradykinin, SF 3155 cells were grown to confluency and depleted of exogenous growth factors and agonists by replacement of the growth medium with fresh RPMI medium without fetal calf serum and incubated for a further 24 h.

Measurement of Particulate PKC Activity

Confluent HEL-37 cells or SF 3155 cells in 10-cm dishes were washed (2 times 5 ml) with warm phosphate-buffered saline and harvested by scraping into 7 ml of buffer containing Tris-HCl (20 mM, pH 7.4), 5 mM EGTA, 2 mM EDTA, 10 mM benzamidine, 0.001% leupeptin, 10 mM 2-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride, and 0.25 M sucrose (buffer A). The cells were sonicated (3 times 10 s), and the sonicate was centrifuged (100,000 times g; 30 min; 4 °C). The pellet was sonicated in buffer A containing 0.2% Triton X-100, incubated on ice for 30 min, and centrifuged as before. The supernatant (particulate fraction) was fractionated on DE-52 columns as described(21) . Alternatively a ``batch elution'' method was used to partially purify PKC from particulate fractions using the method of Leach et al.(22) . Briefly, small columns containing 0.5 ml of DE-52 (packed volume) were equilibrated with column buffer(21) , and the particulate fraction extracts were applied to the columns. After washing with 3 ml of column buffer, PKC was eluted with 1 ml of column buffer containing 0.12 M NaCl and 0.001% leupeptin. Protein was estimated as described (23) using bovine serum albumin as standard. Calcium/phospholipid-dependent PKC activity was measured by the phosphorylation of histone III-S as described(24) .

In some experiments particulate fractions were prepared by sonicating the cells in a buffer containing Tris-HCl (25 mM, pH 7.4), 0.5 mM EGTA, 2 mM EDTA, 10 mM benzamidine, 0.001% leupeptin, 10 mM 2-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride, 0.25 M sucrose, and 0.02% Triton X-100 (buffer B).

Western Blotting and Detection of PKC Isozymes

Aliquots of particulate fraction extracts (described above) were added to well-loading buffer (20 mM Tris-HCl (pH 6.8), 40% sucrose, 6% SDS, and 10 mM 2-mercaptoethanol) (2:1 (v/v)) and heated (5 min; 100 °C). Aliquots (60 µg of protein) were separated by SDS-polyacrylamide gel electrophoresis (25) using 12% gels in a Bio-Rad mini gel system (200 V; 45 min) and then electrophoretically transferred to Schleicher and Schuell nitrocellulose paper (0.2 micron) (100 V; 1.5 h)(26) . Development of the blots and detection of the anti-PKC antibodies by enhanced chemiluminescence (ECL) was that described in the kit obtained from Amersham Corp. Briefly, the blots were exposed to the primary antibody for 2 h at room temperature and the secondary antibody for a further 45 min prior to exposure to ECL reagent. PKC-alpha was detected using peptide-purified rabbit antibodies prepared against the bovine PKC peptide sequence 313-326(27) . Anti-PKC-, from Boehringer Mannheim, was raised in rabbit against the peptide sequence 726-737 of bovine PKC.

Sphingolipid Metabolism

Subconfluent HEL-37 cells or SF 3155 cells in 6-cm dishes were radiolabeled by the addition of [^3H]serine (1 µCi/ml) to the culture medium for 24 h. The confluent cells were washed with phosphate-buffered saline (2 times 2 ml) and incubated for 5 h in Eagle's minimal essential medium or RPMI growth medium containing unlabeled serine (5 mM). The cells were washed as before and incubated with SMase (0.1 unit/ml) in Eagle's minimal essential medium medium or RPMI containing bovine serum albumin (1 mg/ml) for the appropriate time. The medium was removed, and the cells were precipitated in cold methanol (1 ml) containing 20 µg/ml each of SM, sphingosine, and ceramide. The sphingolipids were isolated essentially as described(28) . Briefly, the lipids were extracted by incubation with chloroform/methanol (1:2, by volume) for 1 h and then with chloroform/methanol (2:1, by volume) for a further 30 min. The phases were separated by the addition of 1.5 ml of water, and the organic phase was incubated with 0.1 M methanolic KOH (final concentration) for 1 h at 37 °C. The lipids in the chloroform phase were dried, solubilized in chloroform/methanol (2:1 (v/v)), and separated by sequential thin layer chromotography (TLC) in solvent containing chloroform/methanol/ammonia/water (80:20:1:1 (v/v/v/v)) to 9.5 cm, followed by solvent containing diethyl ether/methanol (99:1 (v/v)) to 19.5 cm. Sphingolipids were located by iodine staining, and the silica gel was scraped into 0.5 ml of water and sonicated. Radioactivity was determined by liquid scintillation spectrometry.

Assay of Phospholipase D Activity

Subconfluent HEL-37 cells or SF 3155 cells in 6-cm dishes were incubated with [^3H]palmitic acid (1 µCi/ml) for 24 h prior to use. The cells were washed in warm phosphate-buffered saline (2 times 2 ml) and incubated at 37 °C in Eagle's minimal essential medium or RPMI containing the test substance and either butanol (0.5%) or ethanol (0.5%) as indicated in the figure legends. The lipids were extracted (29) , and phosphatidylethanol (PEth) or phosphatidylbutanol (PBut) were separated using a one-dimensional TLC system (30) consisting of ethyl acetate/trimethyl-pentane/acetic acid/water (39:30:6:9) and (33:15:6:30) for PEth and PBut, respectively. Radioactivity associated with PEth or PBut was determined by liquid scintillation spectroscopy using Triton X-114/xylene scintillation fluid.

Measurement of Inositol Phosphates

Total inositol phosphates in SMase-treated cells stimulated with bradykinin were measured essentially as described(31) . SF 3155 cells were grown in inositol-free RPMI medium in 100-mm dishes and labeled with myo-[^3H]inositol (1 µCi/ml) for 24 h prior to use. The cells were washed in warm phosphate-buffered saline (2 times5 ml) and incubated with SMase (0.1 unit/ml) or vehicle in RPMI (inositol-free) containing bovine serum albumin (1 mg/ml) for 10 min at 37 °C. The cells were washed as before and incubated in RPMI medium containing 20 mM LiCl(2) for 10 min at 37 °C. After incubation with bradykinin (or buffer) (5 min; 37 °C), the cells were precipitated with 1 ml of 10 mM formic acid (cold) and left on ice for 30 min. The precipitate was scraped from the dish, diluted with 3 ml of 5 mM NH(4)OH (final pH, 8-9), and centrifuged (3000 rpm; 5 min; 4 °C). The supernatant was applied to columns containing 0.75 ml of Bio-Rad anion exchange resin (AG, 1 times 8, formate form, 200-400 mesh). Free inositol and glycerophosphoinositol were removed by washing the column with 4 ml of 40 mM ammonium formate, formic acid, pH 5.0. Total inositol phosphates were eluted with 4 ml of 2 M ammonium formate formic acid, pH 5.0. A 500-µl aliquot of total inositol phosphates was counted for radioactivity using a liquid scintillation counter.


RESULTS

The Effect of SMase on Particulate PKC Activity in diC(8)-treated Epidermal Cells

An earlier report by Kolesnick and Clegg (32) provided the first indication that SM hydrolysis products may regulate PKC activity. Incubation of GH(3) pituitary cells with the phorbol ester TPA resulted in the translocation of PKC to a membrane fraction, and subsequent treatment of the cells with SMase caused the release of the membrane-bound PKC into the soluble fraction. In preliminary experiments, we confirmed this result using HEL-37 cells preincubated with TPA (data not shown). However, pretreatment of the cells with SMase had no effect on the subsequent translocation of PKC when cells were exposed to TPA (data not shown). It was reasoned that this latter result may be due to the high affinity of TPA for PKC, which may ``over-ride'' subtle regulatory mechanisms operating with physiological regulators. Consequently, a series of experiments was carried out using the DAG analogue diC(8) at concentrations that resulted in a submaximal translocation of PKC. At concentrations between 0 and 10 µg/ml, diC(8) induced a dose-dependent increase in Ca/PS-dependent PKC activity (data not shown), and a concentration of 5 µg/ml was chosen for routine use. At this concentration, PKC translocation was markedly inhibited by preincubation of the cells with SMase (0.1 unit/ml; 30 min) (Fig. 1). When this experiment was repeated using 10 µg/ml of diC(8), SMase pretreatment had only a small effect on translocation (data not shown). This result reinforces the notion that high levels of either diglyceride or phorbol ester can over-ride the SMase-mediated modulation of PKC translocation.


Figure 1: The effect of SMase on diC(8)-induced translocation of PKC in HEL-37 cells. HEL-37 cells were pretreated with SMase (0.1 unit/ml), or buffer, for 10 min at 37 °C and then incubated with diC(8) (5 µg/ml) for a further 10 min. The cells were collected, and the PKC extracted from the particulate fractions was partially purified by DE52 cellulose chromatography as described under ``Experimental Procedures.'' PKC activity was assayed in the presence of Ca and PS (circle) or with EGTA without PS (bullet) as described under ``Experimental Procedures.'' PanelsA-D show PKC activity after incubation with vehicle, SMase, diC(8), or SMase plus diC(8), respectively. The protein loaded onto the columns was 10.7, 10.5, 11.2, and 12.0 mg in panelsA-D, respectively. The data are representative of three experiments.



Sphingolipid Metabolism in HEL-37 cells

This result provided evidence that a metabolite of SMase inhibited the translocation of PKC to the membrane. Phosphate analysis of the membrane phospholipids showed that incubation of HEL-37 cells with SMase reduced the mass of SM by 77%. SMase treatment had no significant effect on the levels of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol/phosphatidylserine in these cells (data not shown).

To examine the products of SM hydrolysis, HEL-37 cells were incubated for 24 h with [^3H]serine to label the SM pool, followed by a 5-h chase with unlabeled serine. Incubation of the prelabeled cells with SMase resulted in a rapid decrease in SM-associated radioactivity, while that associated with ceramide increased (Fig. 2). Over the time course studied, essentially no radioactivity was associated with sphingosine. A similar result was reported in 3T3 cells(33) . No other labeled products were detected, suggesting that ceramide is the major SMase-induced metabolite in HEL-37 cells. While the phosphorylation of ceramide to ceramide 1-phosphate has been reported(34) , this metabolite was not detected in P(i) (20 µCi/ml)-labeled cells exposed to SMase for up to 30 min (data not shown).


Figure 2: The effect of SMase on sphingolipids in HEL-37 cells. [^3H]Serine-labeled HEL-37 cells were incubated with SMase (0.1 unit/ml), or buffer, for varying times at 37 °C. The sphingolipids were extracted from the cells and separated by TLC as described under ``Experimental Procedures.'' Sphingolipids from untreated cells (SM (box), ceramide (up triangle), and sphingosine (circle)) and from SMase-treated cells (SM (), ceramide ([), and sphingosine (bullet)). The data are means (± S.E.) of triplicate determinations and are representative of two experiments.



Inhibition of Particulate PKC Activity by C(2)-Cer in diC(8)-treated HEL-37 Cells-The above data strongly suggest that the effects of SMase on PKC translocation are mediated through ceramide. However, although the lipid determinations indicated that SM was preferentially attacked by the added SMase, it is difficult to exclude an involvement of small amounts of hydrolysis products produced from other lipids. Experiments were therefore carried out using ceramide analogues. Because natural ceramides are not permeable to cells, the short acyl chain ceramide analogues, C(2)-Cer and C(8)-Cer, were used. These analogues are cell-permeable and have been demonstrated to mimic the effects of exogenously applied SMase(33) . As shown in Fig. 3, preincubation with C(2)-Cer had little effect on the basal activity of membrane-bound PKC in HEL-37 cells but markedly reduced the diC(8)-induced translocation of the enzyme at both concentrations of C(2)-cer tested.


Figure 3: The effect of C(2)-Cer on diC(8)-stimulated translocation of PKC in HEL-37 cells. HEL-37 cells were pretreated with C(2)-Cer (0, 2.5, or 5.0 µg/ml) for 5 min at 37 °C and then incubated with or without diC(8) (5 µg/ml) for a further 10 min. The cells were collected, and the PKC extracted from the particulate fraction was partially purified by DE52 cellulose using ``batch elution'' as described under ``Experimental Procedures.'' The PKC activity shown represents the difference between the Ca/PS-dependent and -independent PKC activities. The data are means (± S.E.) of triplicate determinations and are representative of three experiments. *p < 0.02 when compared with the diC(8)-treated cells without pretreatment with C(2)-Cer.



SMase Inhibition of diC(8)-induced PKC-alpha Translocation in HEL-37 Cells

Western blotting experiments established that the predominant PKC isozyme expressed in HEL-37 cells was PKC-alpha, with a low level of PKC- and no detectable PKC-beta(I), -beta, or - (data not shown). Immunoreactivity with both PKC-alpha and PKC- was abolished in competition experiments with the peptide used for raising the respective antibody (not shown).

DiC(8) induced only a limited amount of PKC-alpha translocation to the fraction extracted between 0.02 and 0.2% Triton X-100, and this translocation was insensitive to SMase treatment (Fig. 4, lanes2-5). This result is similar to that reported by Ha and Exton(16) , who concluded that elevated Ca levels were required for translocation of PKC-alpha in IIC9 cells. However diC(8) did induce substantial PKC-alpha translocation in HEL-37 cells when the 0.02% Triton extraction was eliminated (Fig. 4, lanes6-9), and this translocation was strongly inhibited by treatment of the cells with SMase (Fig. 4, lane9) or with C(2)-Cer (not shown). This result indicates that the ceramide-sensitive pool of PKC-alpha is only loosely associated with the membrane.


Figure 4: Western blot analysis of the effect of SMase on PKC-alpha translocation in HEL-37 cells harvested in the presence or absence of 0.02% Triton X-100. HEL-37 cells were pretreated with SMase (0.1 unit/ml) or buffer for 10 min at 37 °C and then incubated with diC(8) (5 µg/ml) for a further 10 min. The cells were collected in buffer containing 0.02% Triton X-100 (buffer B; lanes2-5), buffer without added Triton X-100 (buffer A; lanes6-9), and the PKC extracted from the particulate fractions as described under ``Experimental Procedures.'' The extracted proteins were separated by SDS-polyacrylamide gel electrophoresis, and PKC-alpha was detected by Western blot as described under ``Experimental Procedures.'' Lane1 shows PKC-alpha standard; lanes2 and 6, 3 and 7, 4 and 8, and 5 and 9, show extracts after incubation with buffer, diC(8), SMase, or SMase plus diC(8), respectively. Molecular mass markers are as indicated. The data are representative of three experiments.



SMase and C(8)-Cer Inhibition of diC(8)-stimulated Phospholipase D Activation in HEL-37 Cells

We next examined the effect of SMase and ceramide analogues on the diC(8)-induced activation of phospholipase D in HEL-37 cells. In the presence of primary alcohols, this enzyme catalyzes a transphosphatidylation reaction resulting in the transfer of the phosphatidyl moiety of the phospholipid to primary alcohols forming phosphatidylalcohol and free base(35, 36) . In these experiments, the phospholipid pool of subconfluent cultured cells was prelabeled by incubation with [^3H]palmitic acid, and phospholipase D activity was quantified by measuring the accumulation of radioactive PEth or PBut when cells were incubated with diC(8) in the presence of ethanol or butanol, respectively. As shown in Fig. 5A, HEL-37 cells accumulated labeled PBut when treated with increasing concentrations of diC(8). This accumulation was decreased in cells preincubated with SMase (0.1 unit/ml; 30 min). In separate experiments, preincubation of cells with C(2)-Cer also decreased the diC(8)-stimulated phospholipase D activity (Fig. 5B). At a concentration of 1 µg/ml, C(8)-Cer radioactivity associated with PEth was reduced to approximately 20% of the control.


Figure 5: The effect of SMase or C(8)-Cer on diC(8)-stimulated activation of phospholipase D in HEL-37 cells. HEL-37 cells were pretreated with buffer (circle), or SMase (0.1 unit/ml) (bullet) for 10 min at 37 °C (panelA) or C(8)-Cer for 5 min (panelB) and incubated for a further 10 min after the addition of diC(8) (5 µg/ml) in the presence of 0.5% butanol (panelA) or ethanol (panelB). The lipids were extracted, and PBut or PEth were separated by TLC as described under ``Experimental Procedures.'' The data are means of triplicate determinations (± S.E.) and are representative of two experiments.



Inhibition of Particulate PKC Activity by SMase and C(2)-Cer in Bradykinin-treated Fibroblasts

The effects of SMase, C(2)-Cer, and C(8)-Cer on PKC translocation and phospholipase D activation were also determined in a human skin fibroblast cell line (SF 3155) incubated with bradykinin. Bradykinin is a nonapeptide that stimulates B(2) receptors resulting in a cascade of events leading to inositol lipid hydrolysis, activation of PKC(37) , and subsequent choline lipid metabolism(38, 39) . Incubation of SF 3155 cells with 25 nM bradykinin resulted in a transient translocation of Ca/PS-dependent PKC activity to the particulate fraction after a 0.75-min incubation that had returned to near control values after 2 min (data not shown). A similar transient bradykinin-stimulated translocation of PKC has been demonstrated in Madin-Darby canine kidney cells(40) . Pre-exposure of SF 3155 cells to SMase for 10 min caused a marked inhibition of bradykinin-stimulated translocation of PKC (Fig. 6), providing further evidence that a metabolite of SM inhibits the association of PKC with the membrane. However, to ensure that the effect of SMase on bradykinin-induced PKC translocation was not due to an effect on receptor binding or subsequent inositol lipid hydrolysis, the production of inositol phosphates in SMase-treated or untreated cells was determined. As shown in Table 1, the bradykinin-stimulated accumulation of total inositol phosphates was unaffected by preincubation of SF 3155 cells with SMase. It was therefore concluded that SMase does not interfere with the initial receptor-mediated responses to bradykinin.


Figure 6: The effect of SMase on the bradykinin-stimulated translocation of PKC in SF 3155 cells. SF 3155 cells were pretreated with SMase (0.1 unit/ml), or buffer, for 10 min at 37 °C before incubation with 25 nM bradykinin for 0.5 min. The cells were collected, and the PKC extracted from the particulate material was partially purified by DE52 cellulose chromatography as described under ``Experimental Procedures.'' PKC activity was assayed in the presence of Ca and PS (circle) or with EGTA without added PS (bullet) as described under ``Experimental Procedures.'' PanelsA-D show PKC activity from cells incubated with vehicle, SMase, bradykinin, or SMase plus bradykinin, respectively. The protein loaded onto the column was 3.1, 3.5, 3.1, and 3.0 mg in panelsA-D, respectively. The data are representative of two experiments.





Ceramide Accumulation in SMase-treated SF 3155 Cells

To confirm that ceramide was the major sphingolipid metabolite produced following SMase treatment, confluent SF 3155 cells were prelabeled with [^3H]serine for 24 h, and the sphingolipid metabolites were isolated as described for HEL-37 cells. Again, ceramide was the only radiolabeled sphingolipid metabolite detected (Fig. 7).


Figure 7: The effect of SMase on sphingolipids in SF 3155 cells. [^3H] serine-labeled SF 3155 cells were incubated with SMase (0.1 unit/ml) or buffer for varying times at 37 °C. The sphingolipids were extracted from the cells and separated by TLC as described under ``Experimental Procedures.'' Sphingolipids from untreated cells (SM (box), ceramide (up triangle), and sphingosine (circle)) and from SMase-treated cells (SM (), ceramide (), and sphingosine (bullet)). The data are means (± S.E.) of triplicate determinations and are representative of two experiments.



SMase and C(2)-Cer Induce Selective Inhibition of PKC-alpha Translocation in Bradykinin-treated SF 3155 Cells

The bradykinin-stimulated translocation of PKC in SF 3155 cells was inhibited by C(2)-Cer (Fig. 8). This result contrasts with a report by Younes et al.(6) who found that C(8)-Cer was a competitive inhibitor of DAG kinase in HL-60 cells and resulted in the accumulation of DAG, accompanied by increased translocation of PKC. In the present studies, C(2)-Cer did not induce any increase in basal PKC activity in either HEL-37 or SF 3155 cells. The small apparent increases seen in SF 3155 cells (Fig. 8) were not significantly different from the control when tested using Student's t test.


Figure 8: The effect of C(2)-Cer on bradykinin-stimulated translocation of PKC in SF 3155 cells. SF 3155 cells were pretreated with C(2)-Cer (0, 2.5, or 5 µg/ml) for 10 min at 37 °C and then incubated with or without 25 nM bradykinin for a further 0.5 min. The cells were collected, and the PKC was extracted and activity assayed as for Fig. 3. *p < 0.02 when compared with the bradykinin-stimulated cells without pretreatment of C(2)-Cer.**p > 0.05 when compared with the untreated bradykinin-stimulated cells.



The predominant PKC isoforms expressed in SF 3155 cells were PKC-alpha, PKC-, and PKC-; PKC-beta or PKC- could not be detected. Analysis of the 0-0.2% Triton extract of SF 3155 cells by Western blotting demonstrated that bradykinin induced a rapid translocation of PKC-alpha, which was strongly inhibited by treatment with SMase (Fig. 9) or C(2)-Cer (not shown). As observed in HEL-37 cells incubated with diC(8), the bradykinin-induced translocation of PKC-alpha was markedly decreased when the 0.02-0.2% Triton X-100 extract was analyzed (not shown). By contrast, bradykinin induced a strong translocation of PKC- to the fraction extracted with 0.02-0.2% Triton X-100, and this translocation was not inhibited by SMase treatment (Fig. 10) or C(2)-Cer (not shown). In fact, SMase treatment promoted the translocation of PKC- (Fig. 10, lane3), and this was enhanced by the addition of bradykinin. Incubation of cells with C(2)-Cer also resulted in translocation of PKC- (data not shown). Although the enhancement of PKC- translocation by SMase treatment or C(2)-Cer was reproducible (2 separate experiments in each case), it was not examined further in the present studies. Samples prepared without a preliminary extraction with 0.02% Triton contained high basal levels of PKC-, which tended to mask the bradykinin-induced translocation of this subspecies. The differing extraction conditions used to isolate PKC-alpha and PKC- suggests that PKC- is more tightly associated with the membrane than is the former subspecies. Furthermore, these results highlight the importance of the extraction conditions used when following the translocation of PKC ioszymes.


Figure 9: Western blot analysis of the effect of SMase on PKC-alpha translocation in bradykinin-stimulated SF 3155 cells. SF 3155 cells were pretreated with SMase (0.1 unit/ml), or buffer for 10 min at 37 °C and then incubated with 25 nM bradykinin for 0 (lanes2 and 3), 0.5 (lanes4 and 5), or 1.0 min (lanes6 and 7). The cells were collected in buffer A, and the PKC was extracted from the particulate fractions as described under ``Experimental Procedures.'' The extracted proteins were separated by SDS-polyacrylamide gel electrophoresis, and PKC-alpha was detected by Western blot as described under ``Experimental Procedures.'' Lane1 shows PKC-alpha standard; lanes2, 4, and 6 show pretreated with buffer; and lanes3, 5, and 7 show SMase-pretreated. Molecular mass markers are as indicated. The data are representative of three experiments.




Figure 10: Western blot analysis of the effect of SMase on PKC- translocation in bradykinin-stimulated SF 3155 cells. SF 3155 cells were pretreated with SMase (0.1 unit/ml) or buffer for 10 min at 37 °C and then incubated with 25 nM bradykinin or buffer for a further 0.5 min. The cells were collected in buffer B, and the PKC was extracted from the particulate fractions as described under ``Experimental Procedures.'' The extracted proteins were separated by SDS-polyacrylamide gel electrophoresis and the PKC- detected by Western blot as described under ``Experimental Procedures.'' Lanes1-4 show PKC- from cells treated with vehicle, bradykinin, SMase or SMase plus bradykinin. Lane5 shows PKC- standard, and the molecular mass markers are as indicated. The data are representative of three experiments.



SMase and C(8)-Cer Inhibition of Phospholipase D Activity in Bradykinin-treated SF 3155 Cells

The effect of SMase and C(8)-cer on the activation of phospholipase D was also studied in SF 3155 cells stimulated with bradykinin. As shown in Fig. 11, bradykinin stimulated the formation of PBut, and SMase reduced the radioactivity associated with PBut by greater than 50% at all bradykinin concentrations tested (panelA). Inhibition of phospholipase D activity in bradykinin-stimulated cells by C(8)-Cer mimicked that of SMase resulting in a marked decrease in radioactivity associated with PEth formation (panelB).


Figure 11: The effect of SMase or C(8)-Cer on bradykinin-stimulated activation of phospholipase D in SF 3155 cells. SF 3155 cells were pretreated with buffer (circle), or SMase (0.1 unit/ml) (bullet) for 10 min at 37 °C (panelA) or C(8)-Cer for 5 min (panelB) and incubated for a further 0.5 min after the addition of 25 nM bradykinin, or buffer, in the presence of 0.5% butanol (panelA) or ethanol (panelB). The lipids were extracted, and PBut or PEth were separated by TLC as described under ``Experimental Procedures.'' The data are means of triplicate determinations (± S.E.) and are representative of two experiments.



To exclude the possibility that the ceramide analogues were deacylated to sphingosine, lipid extracts from C(2)-Cer-treated HEL-37 cells and SF 3155 cells were chromatographed in the solvent system used to separate PEth. In this system PEth (R(F) 0.52) is well separated from C(2)-Cer (R(F) 0.69) and sphingosine (R 0.23). Although a clear iodine-stained spot was apparent corresponding to the C(2)-cer marker, no sphingosine was detectable (data not shown). Furthermore, as reported by Lavie and Liscovitch, sphingosine activates phospholipase D(41) . To confirm this, HEL-37 cells and SF 3155 cells incubated with sphingosine (25 µM) resulted in the stimulation of PEth formation, which was enhanced by the addition of diC(8) or bradykinin (data not shown). These data show conclusively that the results obtained using SMase or ceramide analogues cannot be attributed to the accumulation of sphingosine.


DISCUSSION

The major finding of this study is that ceramide selectively inhibits the translocation of PKC-alpha but not that of PKC- in cells incubated either with diC(8) or bradykinin. Clearly this provides a potential mechanism for the differential regulation of PKC isozyme activity. In fact the observation that ceramide inhibited the diC(8) or bradykinin-induced stimulation of phospholipase D strongly implicates PKC-alpha in regulating phospholipase D activity. Some controversy exists about the relationship of PKC to phospholipase D activation. Numerous studies have implicated PKC in the activation of this enzyme, although some inhibitor studies do not support this (for review, see (42) ). A recent report using antisense cDNA against PKC-alpha and -beta(I) has, however, demonstrated that PKC-alpha mediates the phorbol ester activation of phospholipase D in Madin-Darby canine kidney D1 cells(18) . This result is consistent with our observations. The mechanism by which PKC activates phospholipase D is not understood, but enhanced phospholipase D activation was observed in streptolysin O-permeabilized HL-60 cells when both PKC- and G-protein-mediated pathways were stimulated(43) . Although the effects of ceramide on PKC-alpha translocation provide an obvious explanation for the inhibition of phospholipase D activation, we cannot formally exclude the possibility that ceramide acts by inhibiting G-protein or phospholipase D activity directly. As yet we have been unable to prepare cell-free phospholipase D preparations from fibroblasts that would enable us to test the possibility that ceramide acts directly on phospholipase D.

The cell lines used in this study express a limited number of PKC isozymes. Cell lines that express a wider range of PKC isozymes are currently being tested to determine if ceramide inhibits the translocation of other PKC subspecies. It will be of particular interest to determine whether ceramide inhibition of PKC translocation is specific for the Ca-dependent isozymes.

The mechanism for the inhibition of PKC-alpha translocation by ceramide is not known. Ceramide and DAG have similar molecular structures: a neutral lipid backbone with a fatty acid group at the 2 position, hydrogen at the 1-carbon or head-group position, and both lipids are neutral in charge. It is therefore possible that ceramide competes with DAG at the DAG binding site on the PKC protein resulting in the displacement of DAG. This notion is supported by the observation that higher concentrations of diC(8) decreased the inhibitory effect of SMase on PKC translocation. However, preliminary in vitro experiments have indicated that ceramide did not inhibit the activity of purified rat brain PKC (data not shown), and it has been reported that ceramide did not inhibit the activity of PKC in a mixed micelle assay(3) . Consequently the way in which ceramide is presented to the membrane may be crucial. We are currently carrying out experiments using plasma membranes isolated from cells pretreated with ceramide analogues or SMase to study its effect on exogenous PKC association with membranes.

It was also possible that ceramide inhibited bradykinin-induced PKC-alpha translocation by blocking the increase in cytosolic Ca concentration. However SMase treatment had no effect on the bradykinin-stimulated accumulation of inositol phosphates (Table 1) or increase in cytosolic Ca, measured using fura 2/AM (data not shown). In addition, SMase and C(2)-Cer also inhibited PKC-alpha translocation induced by diC(8), which is independent of changes in cytosolic Ca concentration (data not shown).

An interesting recent development has been the proposal that agonists such as tumor necrosis factor-alpha and interleukin-1beta initiate apoptosis via ceramide(9) . Both compounds have been shown to rapidly activate a membrane-bound neutral SMase activity, which results in the prolonged accumulation of ceramide(12) . The effects of tumor necrosis factor-alpha and interleukin-1beta on induction of apoptosis can be mimicked by treating cells with exogenous SMase or with cell-permeable ceramide analogues(9) . A number of potential targets for ceramide have been identified, which include a novel membrane-associated protein kinase(4) , protein phosphatase(7, 8) , and the mitogen-activated protein kinase cascade(10) . Specific effects of ceramide on the translocation of PKC subspecies can now be added to this list. It should be noted that the role of PKC in apoptosis is unclear. In many cell systems, activation of PKC inhibits apoptosis(9) , although stimulatory effects have also been reported (for review, see (44) ). In addition, a recent report (45) showed that glucocorticord-stimulation of apoptosis in immature thymocytes resulted in the selective activation of PKC-. The present observation that ceramide can differentially effect the activity of individual PKC subspecies provides a possible explanation of these apparently conflicting results. Thus the sensitivity of cells to ceramide initiated apoptosis may depend on the relative proportions of PKC-alpha and PKC- and possibly other subspecies of PKC expressed. Clearly more studies are required using cell types expressing a wide range of PKC subspecies and which contain an agonist-regulated SMase.


FOOTNOTES

*
This work was supported by grants from Anticancer Foundation of the Universities of South Australia and the National Health and Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 061-8-201-3836; Fax: 061-8-201-3015.

(^1)
The abbreviations used are: SM, sphingomyelin; PKC, protein kinase C; PS, phosphatidylserine; SMase, sphingomyelinase; diC(8), dioctanoylglycerol; DAG, diacylglycerol; C(2)-Cer, C(2)-ceramide; C(8)-cer, C(8)-ceramide; PBut, phosphatidylbutanol; PEth, phosphatidylethanol; TLC, thin layer chromatography; TPA, 12-O-tetradecanoylphorbol-13-acetate.


ACKNOWLEDGEMENTS

We thank Dr A. Poulos of the Women's and Children's Hospital, North Adelaide, South Australia, Australia for kindly supplying the human fibroblast cell line, SF 3155.


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