(Received for publication, September 14, 1994; and in revised form, November 11, 1994)
From the
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 [H]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
) 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
-Cer). Western blot analysis
indicated that treatment of HEL-37 cells with diC
or SF
3155 cells with bradykinin resulted in the translocation of both
PKC-
and PKC-
to particulate material. Treatment with SMase
or C
-Cer specifically blocked the translocation of
PKC-
but not that of PKC-
. Pretreatment of cells with SMase
or C
-Cer also inhibited the activation of phospholipase D
activity induced by either diC
(HEL-37 cells) or bradykinin
(SF 3155 cells). The data provide strong evidence that ceramide can
negatively regulate the translocation of PKC-
but not PKC-
and further suggest that PKC-
may be involved in regulating
phospholipase D activity.
Sphingolipids have received increasing attention over the past
decade as modulators of biological events (for review, see (1) ). Many investigations have implicated sphingomyelin (SM) ()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
, tumor necrosis factor-
, interleukin 1
, 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 (,
,
,
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-
(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-
or
PKC-
by transfection of Madin-Darby canine kidney D1
cells with corresponding antisense oligonucleotides. This work
implicated PKC-
but not PKC-
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- but not PKC-
to
particulate material. Furthermore, it was found that ceramide inhibited
the diC
-mediated (HEL-37 cells) or bradykinin-mediated (SF
3155 cells) activation of phospholipase D, providing additional
evidence that PKC-
is involved in regulating phospholipase D
activity.
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).
Figure 1:
The effect of SMase on
diC-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
(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 (
) or with EGTA without PS (
) as
described under ``Experimental Procedures.'' PanelsA-D show PKC activity after incubation with
vehicle, SMase, diC
, or SMase plus diC
,
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.
To examine the products of SM hydrolysis, HEL-37 cells were
incubated for 24 h with [H]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
(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. [H]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 (
), ceramide (
),
and sphingosine (
)) and from SMase-treated cells (SM (
),
ceramide ([
), and sphingosine (
)). The data are means
(± S.E.) of triplicate determinations and are representative of
two experiments.
Inhibition of Particulate PKC Activity by
C-Cer in diC
-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
-Cer and C
-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
-Cer had little
effect on the basal activity of membrane-bound PKC in HEL-37 cells but
markedly reduced the diC
-induced translocation of the
enzyme at both concentrations of C
-cer tested.
Figure 3:
The effect of C-Cer on
diC
-stimulated translocation of PKC in HEL-37 cells. HEL-37
cells were pretreated with C
-Cer (0, 2.5, or 5.0 µg/ml)
for 5 min at 37 °C and then incubated with or without diC
(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
-treated
cells without pretreatment with
C
-Cer.
DiC induced only a
limited amount of PKC-
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-
in IIC9
cells. However diC
did induce substantial PKC-
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
-Cer (not shown). This result indicates that the
ceramide-sensitive pool of PKC-
is only loosely associated with
the membrane.
Figure 4:
Western blot analysis of the effect of
SMase on PKC- 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
(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-
was detected by
Western blot as described under ``Experimental Procedures.'' Lane1 shows PKC-
standard; lanes2 and 6, 3 and 7, 4 and 8, and 5 and 9, show extracts after
incubation with buffer, diC
, SMase, or SMase plus
diC
, respectively. Molecular mass markers are as indicated.
The data are representative of three
experiments.
Figure 5:
The effect of SMase or C-Cer
on diC
-stimulated activation of phospholipase D in HEL-37
cells. HEL-37 cells were pretreated with buffer (
), or SMase (0.1
unit/ml) (
) for 10 min at 37 °C (panelA)
or C
-Cer for 5 min (panelB) and
incubated for a further 10 min after the addition of diC
(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.
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 (
) or with EGTA without added PS
(
) 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.
Figure 7:
The effect of SMase on sphingolipids in SF
3155 cells. [H] 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 (
), ceramide (
),
and sphingosine (
)) and from SMase-treated cells (SM (
),
ceramide (
), and sphingosine (
)). The data are means
(± S.E.) of triplicate determinations and are representative of
two experiments.
Figure 8:
The effect of C-Cer on
bradykinin-stimulated translocation of PKC in SF 3155 cells. SF 3155
cells were pretreated with C
-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
-Cer.**p > 0.05 when
compared with the untreated bradykinin-stimulated
cells.
The
predominant PKC isoforms expressed in SF 3155 cells were PKC-,
PKC-
, and PKC-
; PKC-
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-
, which was strongly inhibited by treatment with SMase (Fig. 9) or C
-Cer (not shown). As observed in HEL-37
cells incubated with diC
, the bradykinin-induced
translocation of PKC-
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
-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
-Cer also resulted in translocation of
PKC-
(data not shown). Although the enhancement of PKC-
translocation by SMase treatment or C
-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-
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- 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-
was detected by Western blot as described under ``Experimental
Procedures.'' Lane1 shows PKC-
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.
Figure 11:
The effect of SMase or C-Cer
on bradykinin-stimulated activation of phospholipase D in SF 3155
cells. SF 3155 cells were pretreated with buffer (
), or SMase (0.1
unit/ml) (
) for 10 min at 37 °C (panelA)
or C
-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-Cer-treated HEL-37 cells and SF 3155 cells were
chromatographed in the solvent system used to separate PEth. In this
system PEth (R
0.52) is well separated
from C
-Cer (R
0.69) and sphingosine (R 0.23). Although a clear iodine-stained spot was apparent
corresponding to the C
-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
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.
The major finding of this study is that ceramide selectively
inhibits the translocation of PKC- but not that of PKC-
in
cells incubated either with diC
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
or bradykinin-induced stimulation of phospholipase D
strongly implicates PKC-
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-
and -
has, however, demonstrated
that PKC-
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-
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- 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
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- 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
-Cer also inhibited PKC-
translocation induced by
diC
, 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- and interleukin-1
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-
and interleukin-1
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-
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.