(Received for publication, May 24, 1996, and in revised form, October 30, 1996)
From the Department of Biochemistry (Signal Transduction Laboratories) and the Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
The mechanism of inhibition of phospholipase D
(PLD) by ceramides was determined using granulocytes differentiated
from human promyelocytic leukemic (HL-60) cells. In a cell-free system,
hydrolysis of phosphatidylcholine by membrane-bound PLD depended upon
phosphatidylinositol 4,5-bisphosphate, guanosine
5-3-O-(thio)triphosphate) (GTP
S), and cytosolic factors
including ADP-ribosylating factor (ARF) and RhoA. C2-
(N-acetyl-), C8- (N-octanoyl-), and
long-chain ceramides, but not dihydro-C2-ceramide,
inhibited PLD activity. Apyrase or okadaic acid did not modify the
inhibition of PLD by ceramides, indicating that the effect in the
cell-free system was unlikely to be dependent upon a
ceramide-stimulated kinase or phosphoprotein phosphatases.
C2- and C8-ceramides prevented the
GTP
S-induced translocation of ARF1 and RhoA from the cytosol to the
membrane fraction. In whole cells, C2-ceramide, but not
dihydro-C2-ceramide, inhibited the stimulation of PLD by
N-formylmethionylleucylphenylalanine and decreased the
amounts of ARF1, RhoA, CDC42, Rab4, and protein kinase C-
and
-
1 that were associated with the membrane fraction, but
did not alter the distribution of protein kinase C-
and -
. It is
concluded that one mechanism by which ceramides prevent the activation
of PLD is inhibition of the translocation to membranes of G-proteins
and protein kinase C isoforms that are required for PLD activity.
PLD1 in mammalian cells plays a key
role in signal transduction and its activation occurs in a wide range
of cell types in response to hormones and growth factors. Several
components such as G-proteins, PKC, and Ca2+ are involved
in regulating PLD (for review, see Refs. 1 and 2). PLD catalyzes the
hydrolysis of cell phospholipids, mainly PC, resulting in the formation
of PA, which may also be the precursor of lysoPA. These two lipids are
bioactive, and they have many effects that are similar. PA or lysoPA
been reported to stimulate the respiratory burst in neutrophils (3),
monoacylglycerol acyltransferase (4), phospholipase C (5),
phosphatidylinositol-4-phosphate kinase (6), PKC-
(7), and the
Ras-Raf-mitogen-activated protein kinase pathway (8) and to inhibit
adenylate cyclase (9). PA may also be dephosphorylated by phosphatidate
phosphohydrolase to diacylglycerol (10), a well characterized activator
of PKC (2).
In cell-free assays, the activation of membrane-associated PLD by
GTPS is dependent on the presence of both membranes and cytosol
components. The latter consist of small G-proteins of the Ras
superfamily, such as ARF (11, 12), RhoA (13, 14), and CDC42 (15, 16).
ARF was first identified as a cofactor necessary for the
ADP-ribosylation of the
-subunit of heterotrimeric G-proteins,
i.e. Gs, by cholera toxin (17). ARF has also
been implicated in vesicular transport in the Golgi (18) and in
endocytosis (19). ARF-stimulated PLD has been partially purified from
HL-60 cell membranes, and this stimulation was dependent on the
presence of PIP2 (12). Subsequently, ARF-stimulated PLD was
separated from oleate-stimulated PLD after solubilization from brain
membranes, and its activation by class I, II, and III mammalian ARFs
was demonstrated (20). Rho proteins regulate the assembly of focal adhesion complexes and actin stress fibers in fibroblasts (21), they
inhibit phorbol ester-induced and integrin-dependent
aggregation in lymphocytes (22), and they play a critical role in
coupling of G-protein-linked chemoattractant receptors to
integrin-mediated adhesion in leukocytes (23). The exact mechanisms by
which ARF and RhoA are involved in the PLD activation are still
unclear.
Hammond et al. (24) identified human PLD cDNA, which
defines a new and highly conserved gene family, and described the
critical role for PIP2 in ARF-stimulated-PLD. In cell-free
assays, ARF translocation correlates with potentiation of
GTPS-stimulated PLD activity in membranes fractions of HL-60 cells
(25). Several studies show that ARF acts synergistically with members
of the Rho family (26), and with other cytosolic factors from human neutrophils (27) and HL-60 cells (28). In these studies, the cytosolic
stimulating fraction prepared by gel filtration chromatography was
estimated to be 50 kDa. Singer et al. (29) recently reported that ARF and RhoA functions are synergistic with PKC-
in the activation of PLD that was partially purified from membranes of porcine
brain. This effect of PKC-
is independent of ATP. By contrast,
Ohguchi et al. (30) demonstrated that the synergism between
PKC-
and RhoA in the activation of membrane-bound PLD was absolutely
dependent on ATP in HL-60 cells. Although Rho can activate PLD in
cell-free systems, its physiological involvement has been questioned
and the major stimulation by G-proteins was concluded to be mediated by
ARF (31).
Gomez-Muñoz et al. (32, 33) showed that
C2- and C6-ceramides inhibited the stimulation
of PLD activity by PA, lysoPA, sphingosine 1-phosphate, and a variety
of growth factors in intact rat fibroblasts. Furthermore,
C2-ceramide and sphingoid bases partially inhibited
diradylglycerol formation by the PLD pathway in neutrophils (34) and
C6-ceramide inhibited the serum-stimulated DAG accumulation
and PLD activation in senescent cells (35). Sphingomyelin hydrolysis
and the production of ceramide and sphingosine has also been implicated
in a signal transduction (36, 37, 38, 39), and there is "cross-talk"
between the sphingolipid and glycerolipid signaling pathways (40, 41, 42).
Ceramides promote the dephosphorylation of PA, lysoPA, sphingosine
1-phosphate, and ceramide 1-phosphate by the plasma membrane
phosphatidate phosphohydrolase, which acts upon all of these substrates
(32, 33, 40, 41, 42). Ceramides are also competitive inhibitors of DAG
kinase in HL-60 cells (43). Recent studies indicate that pretreatment
of mouse epidermal or human skin fibroblasts with sphingomyelinase or
C2-ceramide specifically blocked the translocation of
PKC- but not that of PKC-
to particulate material (44). An
inhibitory effect of C2-ceramide on PLD activation was also observed in rat basophilic leukemia (RBL-2H3) cells (45). In the same
work, the authors demonstrated that the translocation of PKC-
,
-
1, and
2 isozymes from cytosol to membrane
fraction was specifically prevented during C2-ceramide
inhibition.
The mitogenic effects of PA, lysoPA, sphingosine 1-phosphate, and ceramide 1-phosphate are also blocked by cell-permeable ceramides (32, 33, 40). In addition, C2-ceramide induces cell differentiation potently in HL-60 cells and inhibits cell growth (46). C8-ceramide inhibits protein and DNA synthesis in Medin-Darby canine kidney cells (47). Ceramides can affect signal transduction by stimulating the serine phosphorylation of proteins, e.g. the epidermal growth factor receptor in A431 human epidermoid carcinoma cells (48). Alternatively, they can modify protein phosphorylation by stimulating phosphoprotein phosphatase activities (37).
In the present study we investigated the mechanism of the inhibition of
PLD activation by ceramides using differentiated HL-60 cells. We
demonstrate that short-, medium-, and long-chain ceramides inhibit the
GTPS- and cytosol-dependent stimulation of
membrane-bound PLD in a cell-free assay. C2-ceramide, but
not dihydro-C2-ceramide, inhibited the ARF1- and
RhoA-stimulated PLD activity by preventing the translocation of ARF1
and RhoA from cytosol to the membrane fraction. Furthermore, in intact
HL 60 cells C2-ceramide, but not
dihydro-C2-ceramide, prevented the fMLP-induced stimulation of PLD and it decreased the amount of ARF1, RhoA, CDC42, PKC-
, and
PKC-
1 that was associated with the membrane fraction. These results
provide a novel mechanism for the inhibition of PLD by ceramides, which
decrease the association of PLD with the low molecular weight
G-proteins and PKC isoforms that are required for activity. In
addition, the effects of ceramides in modifying the subcellular
distribution of low molecular weight G-proteins could have a profound
effect on signal transduction by pathways other than those involving
PLD.
RPMI 1640, penicillin, streptomycin, and fetal
bovine serum were obtained from Life Technologies, Inc. GDPS,
GTP
S, PIP2, and okadaic acid were purchased from
Boehringer Mannheim and DAG kinase (from Escherichia coli)
was from Calbiochem. Bovine serum albumin, apyrase, cytochalasin B,
fMLP, ceramides (from bovine brain sphingomyelin), DETAPAC,
cardiolipin, and 1,2-dipalmitoyl-sn-glycerol were from
Sigma. D-erythro-Sphingosine,
D-erythro-C2-ceramide, D-erythro-dihydro-C2-ceramide
(N-acetyldihydrosphingosine),
D-erythro-C8-ceramide, and
D-erythro-sphingosine were from Biomol (Plymouth
Meeting, PA). L-
-PC (from bovine brain),
L-
-PE, sn-1,2-dioleoylglycerol, and
L-
-PEt were from Avanti Polar Lipids, Inc., and
octyl-
-D-glucoside was from ICN. Rabbit polyclonal
anti-RhoA, anti-Rab4, anti-CDC42, and anti-PKC-
, -
1,
-
, and -
antibodies were purchased from Santa Cruz Biotechnology,
Inc. Rabbit antiserum that cross-reacts with ARF1 and ARF4 (25) was a
generous gift from Dr. S. Bourgoin (Université de Laval,
Québec, Canada). Thin layer chromatography plates of Silica Gel
60 without fluorescent indicator were from Merck.
sn-1,2-Dipalmitoylglycerophosphoryl[N-methyl-3H]choline
([3H-methyl]PC), [
-32P]ATP,
[3H] myristate, and enhanced chemiluminescence kit (ECL) were
from Amersham Life Science Inc. Recombinant ARF and RhoA were acquired and purified as described previously (31). All the other reagents were
of the best quality available.
Human promyelocytic leukemic cells (HL-60) were grown in liquid suspension in RPMI 1640 medium, supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. HL-60 cell lines were maintained in a humidified atmosphere of 5% CO2 at 37 °C, and they were passaged under subconfluent conditions. To induce granulocyte differentiation, 5 × 105 cells/ml were incubated in medium containing 1.3% Me2SO (v/v) for 3-7 days. HL-60 granulocytes were labeled with [3H]myristate (1 µCi/ml) for 12 h at 37 °C. The labeled cells were washed twice with PBS and resuspended in buffer A (137 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 2.5 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 1 mM benzamidine, pH 7.5) to be used for experiments with isolated cell fractions. For studies with whole cells (5 × 105 cells/ml), labeled or washed unlabeled HL-60 cells in RPMI 1640 were preincubated in the presence or absence of C2-ceramide for 1 h at 37 °C. Cells were pretreated with 10 µM cytochalasin B for 5 min and stimulated with 100 nM fMLP for 10 min in the presence of 200 mM ethanol. Incubations were stopped either by the addition of methanol/chloroform (2:1, v/v) (after which lipids were extracted for PLD activity) or by diluting the cells with five volumes of ice-cold RPMI 1640.
Unlabeled and labeled cells were washed twice with ice-cold
phosphate-buffered saline, harvested by centrifugation, and resuspended in buffer A. Cells were then sonicated twice for 10 s each with an
Astracon ultrasonic processor W-385, and centrifuged for 5 min at
800 × g. Unbroken cells and nuclei were discarded, and the cytosol was separated from the membranes by centrifuging at 250,000 × g for 60 min using Beckman TL-100
ultracentrifuge. Membranes were washed twice and resuspended in buffer
A and stored at 70 °C before being used for the PLD assay, or for
Western blot analysis.
For the cell-free assay, membranes and
cytosol (25 µg of protein for each) were premixed at 4 °C as
indicated in a volume of 70 µl with 30 µM GTPS
dissolved in a solution of 30 mM MgCl2, 20 mM CaCl2, plus 400 mM NaCl plus any
other specified additions. The substrate, composed of PE,
PIP2, PC (16:1.4:1, molar ratio), and
[3H-methyl]PC (60,000 dpm/assay), was prepared
freshly in the form of phospholipid vesicles (12) and added in 30 µl
to the assay mixture. The final concentration of PIP2 and
PC in the assays was 12 and 8.6 µM, respectively. All
ceramides, sphingosine, and sn-1,2-dipalmitoylglycerol were
co-sonicated with the substrate liposome (PE/PIP2/PC). This
was achieved by drying all lipid components of the liposome substrate
along with the ceramide, sphingosine, or sn-1,2
dipalmitoylglycerol, creating mixed liposomes by probe sonication and
starting the assay by adding this substrate to the HL-60 membranes and
cytosol. Reactions were performed at 37 °C for 60 min and stopped
with methanol/chloroform (2:1, v/v), and lipids were extracted (49).
Water-soluble products were isolated and separation of
[3H]choline from phosphoryl[3H]choline was
achieved by using a column of Dowex cation exchange resin (50). The
column was washed with 10 ml of water, and the bound
[3H]choline was eluted with 10 ml of 1 M KCl
and quantitated by liquid scintillation counting. The majority (>96%)
of the water-soluble product was [3H]choline, indicating
that there was little PC-phospholipase C activity under these
conditions.
PLD activity was also measured in membranes in the presence of 30 µM GTPS and in whole cells by prelabeling PC with
[3H]myristate as described previously (51, 52) and by
measuring the formation of [3H]PEt. The results are
expressed as a percentage of the total radioactive counts recovered in
PEt.
In some experiments apyrase or okadaic acid was added
to the cell-free system that was used to determine PLD activity, in order to destroy ATP or inhibit phosphoprotein phosphatase activities, respectively. The action of apyrase was tested by adding
[-32P]ATP (44,000 cpm) to 70 µl of the buffer and
GTP
S used in the PLD assay and incubating for 10 min on ice in the
absence of the lipid substrate but in the presence or absence of
membranes plus cytosol. This solution was then incubated for 10 min at
37 °C in the presence or absence of 5 units of apyrase/ml. The
reaction was stopped by adding HClO4, and 32P
was separated from [
-32P]ATP by extracting with
iso-butanol/benzene (53), except that the concentration of
ammonium molybdate was increased by 5-fold to ensure that all of the
32P was recovered in the organic phase. Greater than 92%
of the [
-32P]ATP was hydrolyzed in incubations
containing apyrase independently of whether membranes and cytosol were
present. In incubations that contained membranes and cytosol but no
apyrase, the degradation of [
-32P]ATP was about 35%
because of endogenous ATPases.
In other experiments 2 µM okadaic acid was substituted
for the apyrase in the incubations, and the combined cytosol plus
membrane fractions that were used for the assay of PLD were also
assayed for phosphoprotein phosphatase activities by determining the
release of 32P from 32P-labeled phosphorylase
a (54). The cytosol and membranes were routinely stored at
70 °C before use, and these preparations contained no detectable
phosphoprotein phosphatase 1 and 2A activities as expected; these
enzymes are unstable to freezing and thawing under these
conditions.2 Fresh unfrozen membranes were
therefore used, and 10 µl of the PLD reaction mixture released 16%
of the 32P from phosphorylase a after incubating
for 10 min at 30 °C. In samples that contained 2 µM
okadaic acid, this rate was inhibited by 94%.
Lipids were extracted from the
cells by the method of Bligh and Dyer (49) and the dried lipid extracts
were stored at 70 °C under N2 until assayed. The
lipids were dissolved in chloroform and samples corresponding to 25-50
nmol of phospholipid were dried in Pyrex glass tubes under nitrogen.
Standards of ceramides (50-2,000 pmol) were prepared in the same way
for each series of assays. The mass of ceramide was then determined
using the method of Preiss et al. (55, 56) with the
following modifications; the reaction mixture containing 50 mM imidazole/HCl, pH 6.6, 1 mM DETAPAC, pH 6.6, 50 mM NaCl, 12.5 mM MgCl2, 1 mM EGTA, 10 mM dithiothreitol, 1 mM
ATP, DAG kinase (approximately 15.5 milliunits/assay), 2.5% octyl-
-D-glucoside, and 1 mM cardiolipin was
incubated at 37 °C for 20 min before 1 µCi/assay of
[
-32P]ATP was added. Samples of this mixture (100 µl) were added to each tube, which was then vortexed and warmed to
37 °C for 5 min. The tubes were then sonicated in a Branson
sonicator for 5 min and then incubated at 37 °C for another 5 min.
The samples were resonicated for 5 min, after which they were incubated
at 37 °C for 20 min. Using this method, the exogenous long-chain
ceramide standards were quantitatively converted to ceramide
1-phosphate, either in the absence, or the presence of up to 50 nmol of
phospholipid from the sample. This shows that the quantity of sample
used did not affect the activity of the kinase (56, 57).
C2-ceramide was phosphorylated with about 30% of the
efficiency of the long-chain ceramides. Lipids were extracted (49) by
stopping the reaction with 470 µl of chloroform/methanol, 10 mM HCl (15:30:2, v/v/v). The phases were separated by
adding 150 µl of chloroform and 1 ml of water. After centrifuging,
the aqueous phase was aspirated and the chloroform phase washed twice
with 1 ml portions of water, before drying the lipid under vacuum
overnight. The products were separated on plastic-backed thin layer
chromatography plates of Silica Gel 60 (Merck). The plates were
developed in chloroform/methanol/NH4OH (65:35:7.5, v/v/v)
for 90% of their lengths, dried, and then developed in
chloroform/methanol/acetic acid/acetone/water (10:2:3:4:1, v/v/v/v/v)
for 80% of their lengths. The locations of the ceramide 1-phosphates
were confirmed with authentic standards (33) and a Bioscan Radioimager,
or by autoradiography. C2-ceramide 1-phosphate migrated
with a combined RF value of about 0.35 compared to
about 0.61 for the long-chain derivatives. Radioactivity was determined
by scintillation counting, and the quantity of lipids was calculated by
reference to the appropriate standard curves.
Protein concentrations were determined routinely using the Bradford procedure (58) with Bio-Rad Dye Reagent and bovine serum albumin as a standard. After incubation for the PLD assay, the reaction mixture was centrifuged at 250,000 × g for 60 min using a Beckman TL-100 ultracentrifuge. Proteins from membranes and cytosol were separated by SDS-12% polyacrylamide gel electrophoresis (70 µg of protein/lane) as described by Laemmli (59) and transferred to nitrocellulose membranes. Nonspecific protein binding sites were blocked by incubating membranes for 1 h with 5% (w/v) nonfat dried milk in PBS. Membranes were then incubated for 2 h with a 1:5000 dilutions of anti-ARF1 serum, or 0.1 µg/ml of anti-RhoA, anti-CDC42, anti-Rab4, or anti-PKC-isoform antibodies. Unbound primary antibodies were removed by three washes (10 min each) with PBS containing 0.05% (v/v) Tween 20. The membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (Amersham) (diluted 1:10,000 with PBS-Tween) for 1 h, followed by three washes (10 min each) in PBS-Tween, and developed with enhanced chemiluminescence Western blotting solutions (Amersham), following the manufacturer's recommendations. The blots were quantitated by densitometry at different exposures to ensure proportionality of the response.
Cytosol, membranes, and the combination of the two were
assayed for PLD activity using a mixed phospholipid vesicle containing [3H-methyl]PC, PIP2, and PE. About
a 5-fold activation of PLD was observed when cytosol and membranes were
mixed together and 30 µM GTPS was added. There was
little activity when membranes or cytosol were incubated alone in the
presence or absence of GTP
S (results not shown). GTP
S renders
G-proteins constitutively active. GDP
S, a non-hydrolyzable and
non-phosphorylatable form of GDP, resulted in more than a 60%
reduction in the GTP
S-stimulated PLD activity (results not shown).
The requirement for the cytosolic fraction and GTP
S confirms the
expected involvement of G-protein(s) in stimulating the membrane-bound
PLD (11, 12, 13, 14, 31, 60).
We showed previously that C2- and
C6-ceramides inhibited both agonist-stimulated and
GTPS-dependent PLD activity in intact and permeabilized
rat fibroblasts, respectively (32). This inhibition in intact cells was
time- and concentration-dependent, with a maximum
inhibition at 50 µM C2- or
C6-ceramide in the presence of 0.5% albumin. The effects
of ceramides on the reconstituted PLD assay were investigated to study
their mechanisms of action. C2-, C8-, and
long-chain ceramides inhibited the GTP
S-dependent PLD
activity in dose-dependent manner (Fig. 1).
By contrast, dihydro-C2-ceramide had no significant effect
and sn-1,2 dipalmitoylglycerol stimulated PLD activity
slightly. D-erythro-Sphingosine did not alter
the activity of PLD significantly until its concentration was increased to about 60 µM. At this point the lipid mixture used as a
substrate for PLD tended to become cloudy, and we think that the
inhibitory action of 60 and 100 µM sphingosine is an
artifact of the cell-free assay.
To determine the mechanism of the ceramide effect further, membranes
and cytosol were preincubated for 10 min at 37 °C with apyrase (5 units/ml) to destroy endogenous ATP and thus inhibit kinase activity
(61), or with 2 µM okadaic acid to inhibit protein phosphatases 1 and 2A (for evidence of efficacy, see "Experimental Procedures"). These treatments did not affect the
GTPS-dependent PLD activity significantly. Furthermore,
the average inhibition of PLD in two independent experiments in the
presence of 50 µM C2-ceramide was 42%. The
average inhibitions in the presence of apyrase, or okadaic acid in
these two experiments were 48 and 47%, respectively.
The effects of ceramides on PLD activity were also determined using
membranes labeled in PC with [3H]myristate rather than by
using the PE/PIP2/PC substrate. The GTPS-dependent PLD activity was measured by the
formation of [3H]PEt. C2- and
C8-ceramides inhibited the GTP
S-stimulated PLD activity
in a dose-dependent manner (Fig. 2). By
contrast, dihydro-C2-ceramide had no significant
effect.
Several studies indicated that PLD is regulated by small GTP-binding
proteins such as ARF and RhoA (11, 12, 13, 14, 31). ARF is myristoylated on the
amino-terminal glycine residue, and this myristoylation is necessary
for activity (17). We, therefore, determined if the G-proteins could
replace the need for the cytosol in the assay and whether ceramides
would inhibit the ARF- and RhoA-dependent PLD activity.
Addition of ARF (25 nM) to HL-60 membranes in the presence
of GTPS increased PLD activity by about 1.8-fold (Fig.
3). When the ARF was separated by gel filtration into
fractions containing mainly myristoylated versus
non-myristoylated-ARF, the former fraction was about 4 times more
potent at activating PLD (results not shown). Addition of RhoA (1 nM) to HL-60 membranes resulted in a 1.5-fold activation (Fig. 3). ARF and RhoA, when added together, activated PLD in an
additive manner, resulting in a 2.3-fold activation. Addition of
C2-ceramide (100 µM) inhibited PLD activity
in the presence of ARF, RhoA, or ARF and RhoA by 52-62%. There was
also a ceramide-induced inhibition of the basal PLD activity in the
absence of exogenous G-proteins. This PLD activity may depend partly on
the endogenous ARF and Rho that was present in the membranes (Figs.
4 and 5).
In light of these results, we tested the hypothesis that ceramides
inhibit PLD by preventing its activation by cytosolic factors such as
ARF and possibly Rho. We therefore examined the distribution of ARF1
and RhoA between the membrane fraction and cytosol in order to
correlate this to PLD activity. A membrane fraction and cytosol were
separated after the incubations and subjected to Western blot analysis.
GTPS induced translocation of ARF1 from the cytosol to the membrane
fraction by about 66%, and there was also about a 148% increase in
membrane-bound RhoA (Figs. 4 and 5, respectively).
C2-ceramide (50 µM and 100 µM)
and C8-ceramide (100 µM) decreased the amount
of ARF1 and RhoA in the membrane fraction. In the case of
C2-ceramide, the decrease in membrane-bound ARF1 and RhoA
was accompanied by an increase in the cytosol. This situation also
applied for RhoA with C8-ceramide (Fig. 5), but in the case
of ARF1 there was not a proportional increase in the cytosol;
therefore, there was an incomplete recovery. By contrast to
C2- and C8-ceramides, 100 µM
dihydro-C2-ceramide did not modify the distribution of ARF1
and RhoA between the membrane fraction and the cytosol.
The results in Figs. 4 and 5 indicate that the changes in the
distribution of ARF1 after treatment with the ceramides correlate better with the activity of PLD (Fig. 1) than do those for RhoA. In
preliminary experiments we were not able to detect a significant change
in the amount of PKC- that was associated with the total membrane
fraction when treated with either C2-ceramide or
dihydro-C2-ceramide (results not shown).
The relationship of these effects observed on PLD activity in a cell-free system were examined further in intact HL 60 cells. fMLP-stimulated PLD activity in the cells by about 3.5-fold as expected (Table I), and this increase was blocked by about 90% with C2-ceramide (p < 0.015). There was also an apparent decrease in PLD activity when 50 µM dihydro-C2-ceramide was added, but this was only about 34% and the decrease was not statistically significant (p > 0.07). Furthermore, neither C2-ceramide nor dihydro-C2-ceramide altered significantly the low activity of PLD that was observed in the absence of fMLP. This lack of effect was observed whether or not the cells were primed with cytochalasin B.
|
We therefore tested whether this inhibition of PLD was also accompanied
by a decrease in the membrane association of those proteins that are
involved in the stimulation of PLD. Treatment of the cells with fMLP
increased the amount of ARF1, RhoA, CDC42, PKC-, and
PKC-
1 in the membrane fraction by about 146, 181, 195, 192, and 89%, respectively (Fig. 6).
C2-ceramide, but not dihydro-C2-ceramide,
decreased the amount of ARF1, RhoA, CDC42, PKC-
, and
PKC-
1 on the membranes in the presence of fMLP by about
47, 36, 82, 46, and 57% respectively. By contrast, there was no
significant effect of C2-ceramide or
dihydro-C2-ceramide in the presence of fMLP on the amount
of PKC-
and -
in the membrane fraction. The effects of fMLP and
ceramides were also determined for comparison on the distribution of
Rab4, which belongs to a G-protein family that participates in
regulating vesicle movement involving endocytotic and exocytotic
pathways (62). Rab4 is often found in endosomes (62), and it also
appears to be involved in the insulin-dependent
translocation of vesicles that contain the glucotransporter, GLUT4 (63,
64). Rab4 has relatively little effect in activating PLD (15).
Stimulation of the cells with fMLP in the presence of cytochalasin B
produces a very marked movement of Rab4 to the membranes, and the
presence of C2-ceramide prevented this movement (Fig. 6).
Dihydro-C2-ceramide did decrease the amount of Rab4 on the
membranes by about 52% but it was much less effective than
C2-ceramide.
In the absence of fMLP, C2-ceramide appeared to increase
the amount of PKC-, and CDC42 that was isolated with the membranes by about 73% and 84%, respectively. Dihydro-C2-ceramide
had a similar effect. The amount of Rab4 in the membranes was
undetectable when no C2-ceramide or
dihydro-C2-ceramide was present, and these lipids appeared
to increase membrane-bound Rab4 slightly. There was no significant
effect of C2-ceramide and dihydro-C2-ceramide on the binding of ARF1, RhoA, and PKC-
, -
1, and -
to membranes in the absence of fMLP (Fig. 6).
We also measured the concentrations of long-chain ceramides and C2-ceramide in the cells to evaluate the magnitude of the interaction with the cell-permeable ceramide. Cell treated with 50 µM C2-ceramide for 1 h contained 32 ± 6 (S.D. from three experiments) pmol of this lipid/nmol of phospholipid, and this was responsible for an almost complete (90%) inhibition of the stimulation of PLD by fMLP (Table I). It is therefore concluded that more modest increases in C2-ceramide concentrations would also inhibit PLD activity. The equivalent concentrations of long-chain ceramide were about 2.5 pmol/nmol of phospholipid. The concentrations of C2- and long-chain ceramides were not changed significantly by the presence of fMLP (results not shown).
Work from our laboratory with intact cultured cells (31, 39) and
that of others (34, 43) showed that ceramides block the activation of
PLD by a variety of agonists. This work establishes PLD to be a target
for ceramide action, but the mechanism for this effect was not
elucidated completely. The action of ceramides might be mediated at the
level of the transmission of the signal from an activated receptor to
the stimulation of low molecular weight G-proteins or the PKC isoforms
that are required for PLD activity. The present work first examined the
mechanism for the ceramide inhibition of PLD activity in a cell-free
system. This involved a reconstituted assay that required
PIP2, GTPS and the presence of cytosolic proteins. In
agreement with previous work (11, 12, 13, 14, 26, 31), the cytosolic factors
could be replaced by purified ARF and Rho.
Short-, medium-, and long-chain ceramides inhibited the PLD activity
that was dependent on GTPS and the presence of the cytosolic fraction, or recombinant ARF and Rho. This effect was seen when using
exogenous [3H]PC and measuring the formation of
[3H]choline, or with [3H]PC incorporated
into the membranes that contained the PLD activity and determining the
formation of [3H]PEt. Dihydro-C2-ceramide was
inactive in this respect, and dipalmitoylglycerol stimulated the
reaction slightly rather than causing inhibition. In other work,
stearoylarachidonoylglycerol also failed to modify PLD activity to a
significant extent in a reconstituted assay (31). We also tested the
effects of D-erythro-sphingosine, which had no
significant effect on the PLD activity until its concentration reached
about 60 µM, whereas the ceramides were inhibitory at 25 µM. These inhibitory effects of higher sphingosine
concentrations are probably an artifact, since there was a change in
the physical appearance of the mixed lipid substrate. These inhibitory
effects of sphingosine are not likely to have physiological relevance, since sphingosine stimulates rather than inhibits PLD activity in whole
cell systems (34, 40, 42).
The combined results from Figs. 1 and 2 indicate that the inhibitory action of ceramides compared to the dihydro-analogue is specific and is an effect on the ARF- and Rho-dependent PLD activities (Fig. 3). The action of the low molecular weight G-proteins in activating PLD involves their translocation from the cytosol and interaction with the membrane-bound PLD. Our results demonstrate that ceramides prevent the interaction of ARF and Rho with the membrane compartment, thus indicating that this is a site of action in this cell-free experimental system. This system could produce results that are not found in intact cells, and it was therefore vital to determine whether the effects of ceramides could be seen in a more physiological situation.
Cell-permeable ceramides (50 µM) also prevented the fMLP
stimulation of PLD in intact cells, and this was accompanied by a decrease in the amount of membrane-associated ARF1, RhoA, and CDC42.
These G-proteins are able to activate PLD (11, 12, 13, 14, 15, 16, 17, 18). In our experiments
with whole cells, there was also a ceramide-induced decrease in
membrane-bound PKC- and -
1 (Fig. 6), which also
stimulate PLD activity (65). These results for PKC-
and
-
1 agree with other work (45). By contrast, there was no
significant effect of ceramides on the amount of membrane-bound PKC-
and PKC-
(Fig. 6), and these isoforms have little effect on PLD
activity (65). Venable et al. (66) also described an
inhibition by ceramide of PLD stimulation by PKC, but ascribed this to
an upstream effect on PKC rather than a decrease in the translocation
to membranes. The interaction of PKC-
with PLD produces an
ATP-independent activation (61) that is synergistic with the
stimulation by ARF and Rho (29). The lack of inhibitory effect of
dihydro-C2-ceramide on PLD activity in our experiments
confirms the specificity of the ceramide action. This specificity is
also reflected in the relative lack of effect of
dihydro-C2-ceramide in decreasing the amounts of ARF1, Rho, CDC42, PKC-
, and PKC-
1 in the membranes. It is also
evident that C2-ceramide is not simply displacing
peripheral proteins from the membranes, since there was no significant
effect on the distribution of PKC-
and -
in the presence of fMLP
(Fig. 6). In addition, in the absence of fMLP there was an increase in
membrane-bound RhoA, CDC42, Rab4, and PKC-
with
C2-ceramide. This effect was relatively nonspecific since a
similar action was observed with dihydro-C2-ceramide.
The fact that C2-ceramide also had a very marked effect in preventing the fMLP-stimulated movement of Rab4 to the membrane fraction indicates that ceramide may modify signal transduction through G-proteins other than those that are directly involved in PLD activation. The Rab family of G-proteins are involved in endocytosis and exocytosis (62, 63, 64), and the prevention of association of Rab proteins with membranes (Fig. 6) may contribute to the observed effect of ceramides in inhibiting vesicle movement (67). Similarly, ARF participates in vesicle transport from the Golgi (18, 19) and Rho is involved in the organization of the cytoskeleton (68). Again, our results indicate that the effects of ceramides on the subcellular distribution of these G-proteins could modify their effects on signal transduction and cell morphology.
The inhibition of total PLD activity in the presence of fMLP by 50 µM C2-ceramide was about 63% (Table I). Our results suggest that the action of ceramides on PLD activity may be competitive with PIP2, since the inhibition at 100 µM C2-ceramide in Fig. 2 was about 70% compared to about 45% when the cell-free system was fortified with 12 µM PIP2 (Fig. 1). Furthermore, when the PIP2 concentration in the latter system was decreased to 6 µM, the ceramide inhibition increased to about 99% (results not shown). This competition with limiting PIP2 concentrations probably explains why the inhibition of PLD activity by C2-ceramide was relatively complete in the intact cell system compared to that shown in Fig. 1.
Ceramides produce some of their signaling effects by modifying the
phosphorylation of target proteins by activating a
ceramide-dependent kinase (38) or by stimulating of
phosphoprotein phosphatases (37). When ceramides were added to the
cell-free system in the absence of ATP and in the presence of apyrase,
there was no significant change in the PLD activity or in the
inhibition by ceramide. Therefore, the ceramide-dependent
inhibition observed in this system is unlikely to be mediated via
stimulation of a ceramide-dependent kinase, since the
effect was not decreased by ATP depletion. Similarly, the ceramide
inhibition of GTPS-dependent PLD activity was also observed in the presence of a high concentration of okadaic acid, which
showed that the effect was unlikely to be dependent on the activities
of protein phosphatases 1 and 2A. These results do not preclude the
possibility that, in intact cells, there may be effects of ceramides on
PLD activity that are mediated via kinases or phosphatases.
Our results therefore demonstrate that PLD can be a target of ceramide
action. This was demonstrated in the cell-free assay system and was
accompanied by a decrease in the association of RhoA and ARF with the
membranes in the presence of GTPS. This inhibition of PLD by
ceramides was also observed in HL-60 cells that were stimulated with
fMLP. C2-ceramide also decreased interaction of ARF1, RhoA,
CDC42, PKC-
, and PKC-
1 with the membrane fraction. These proteins are known to be able to stimulate PLD activity. The
present results establish for the first time the potential of ceramides
to modify the subcellular distribution of low molecular weight
G-proteins including ARF, Rho, CDC42, and thus prevent PLD activation.
Furthermore, ceramides also prevented the association of Rab4 with the
membranes in the presence of cytochalasin B and fMLP. Such an effect
might have more generalized implications in regulating signal
transduction and vesicle movement in addition to any direct effects on
the production of phosphatidate and diacylglycerol via the activation
of PLD.
We thank Dr. S. Bourgoin for the generous gift of ARF1 antiserum and for advice. The work illustrated in Fig. 3 was performed in Birmingham, UK under the supervision of Drs. A. Martin and M. J. O. Wakelam. We thank them and acknowledge the generous use of their facilities, for helping us with the reconstituted PLD assay, for making ARF and Rho available to us and for their valuable advice. We also thank Hue Anh Luu and Dr. C. F. B. Holmes for performing the assays of phosphoprotein phosphatase activities and for advice.