(Received for publication, September 8, 1994; and in revised form, December 7, 1994)
From the
The effect of alkylglycerol supplementation on protein kinase C
(PKC)-mediated signaling events has been studied in fibroblasts from
Zellweger patients (SF 3271 cells). Western blotting analysis
established that Zellweger fibroblasts express PKC ,
, and
. Incubation with bradykinin induced a rapid transient
translocation of PKC
and a more sustained translocation of PKC
to the particulate fraction; translocation of PKC
was
unaffected. Bradykinin-induced translocation and activation of PKC
, but not translocation of PKC
, was blocked in SF 3271 cells
which had been incubated with 1-O-hexadecylglycerol
(1-O-HDG; 20 µg/ml) for 24 h and then incubated in the
absence of 1-O-HDG and serum for a further 24 h.
Supplementation with 1-O-HDG increased the mass of
ether-linked phospholipid. Bradykinin initiated a transient increase in
cytosolic Ca
concentration in both control and
1-O-HDG supplemented cells, indicating that the initial
receptor linked events were not affected by 1-O-HDG
supplementation. Bradykinin also caused a rapid activation of
phospholipase D (PLD), measured by phosphatidylbutanol accumulation,
and mitogen-activated protein kinase (MAPK) determined by myelin basic
protein phosphorylation of Mono Q fractions. Both events were blocked
by preincubation of the cells with
12-O-tetradecanoylphorbol-13-acetate for 24 h to deplete PKC
protein. 1-O-HDG supplementation prevented the
bradykinin-induced activation of PLD, but had no effect on the
stimulation of MAPK activity. These results establish that modulation
of the ether lipid composition of membranes can alter PKC isozyme
translocation and indicate that a PKC isozyme other than PKC
,
most likely PKC
, is involved in MAPK activation.
Protein kinase C (PKC) ()is a family of structurally
related isozymes that has been shown to participate in the transduction
of signals generated by hormones, neurotransmitters, and growth
factors(1) . The most widely studied of these signals are
diglyceride (DG) and Ca
generated as a consequence of
phosphatidylinositol 4,5-bisphosphate hydrolysis and DG derived from
phosphatidylcholine (PC)(2) .
Molecular cloning has
described multiple closely related PKC isozymes which have been divided
into groups on the basis of biochemical and structural
properties(3) . The classical PKC isozymes ,
I,
II, and
are regulated by phosphatidylserine, DG, and
Ca
. The novel isozymes such as PKC
,
,
, and
lack the Ca
binding domain C2 and
are Ca
-independent, and atypical forms such as PKC
are independent of both Ca
and DG. PKC isozymes
exhibit distinct tissue distribution patterns, with multiple isozymes
often found in a single cell type. Together with the different cofactor
dependence and activator specificity, this suggests that PKC isotypes
participate in distinct signal transduction pathways within the cell,
and this has been supported by a number of recent studies.
For the
most part these studies have demonstrated changes in signaling pathways
following the overexpression of specific PKC
isozymes(4, 5, 6, 7) or have used
antisense technology to deplete the cells of individual PKC
subspecies(8, 9, 10) . However there is very
little information on the factors which may be important in regulating
individual forms of PKC. The recent report by Ha and
Exton(11) , which demonstrated differential translocation of
PKC isozymes in response to thrombin in IIC9 fibroblasts, was the first
to demonstrate the effect of endogenous factors on PKC translocation.
The authors presented evidence that the rapid transient translocation
of PKC required both an increase of DG and cytosolic
Ca
as a result of phosphatidylinositol
4,5-bisphosphate hydrolysis. PKC
translocation was rapid and
sustained in response to thrombin but could be induced by increases in
DG alone and was therefore independent of an increase in cytosolic
Ca
.
As part of a program to study the role of ether phospholipids in transmembrane signaling, we have used skin fibroblast cells isolated from patients with Zellweger syndrome. This syndrome is characterized by an absence of peroxisomes, organelles that contain the enzymes which catalyze the two initial steps in ether lipid biosynthesis. As a consequence tissues and cells from Zellweger patients have low levels of ether lipids(12) . However incubation of cells with alkylglycerol by-passes the enzyme deficiencies (13) and enables the accumulation of high levels of ether lipids. In the present study we have examined the effect of alkylglycerol supplementation on the translocation of PKC isozymes in bradykinin-stimulated skin fibroblasts and on two responses which can be regulated by PKC, activation of PLD and stimulation of the MAPK cascade. We show differential effects of supplementation on PKC translocation and on the two PKC-mediated responses.
The antisera against
PKC ,
, and
were raised in this laboratory against the
amino acid sequences 313-326, 313-329, and 306-318,
respectively (15) and purified against the peptide. Anti-PKC
,
, and
were purchased from Boehringer Mannheim. The
MAPK antibody, raised against residues 333-367 of the C terminus
of rat 43-kDa MAPK (erk I) which recognizes p42, p43, and p44 MAPK, was
a gift from Dr. S. L. Pelech.
Zellweger fibroblasts are deficient in the ability to
synthesize ether lipids. The level of ether lipids can be increased by
supplementation with alkylglycerol which bypasses the enzyme deficiency (13) . To confirm that supplementation increased ether lipid
levels, total phospholipid was separated into diacyl and alkenyl and
alkyl subclasses and the mass determined by phosphate analysis. The
mass of alkenyl and alkyl phospholipid increased from 45 ± 0.5
nmol/10 cells in unsupplemented cells to 65 ± 3
nmol/10
cells in cells incubated with 1-O-HDG (20
µg/ml) for 24 h followed by incubation for a further 24 h in the
absence of 1-O-HDG. In separate experiments cells were labeled
for 24 h with [
H]choline and the PC separated
into diacyl, alkenyl, and alkyl subclasses. The results (data not
shown) show that incubation with 1-O-HDG (20 µg/ml) for 24
h followed by incubation for a further 24 h in the absence of
1-O-HDG, increased the radioactivity associated with
alkyl-linked PC by approximately 10-fold. Similar results were obtained
for alkyl-linked phosphatidylethanolamine when the cells were labeled
with [
H]ethanolamine (data not shown). However,
in these experiments it is not known if equilibrium labeling has been
reached in each of the separate phospholipid subclasses, and this may
be reflected in the difference between the increase in mass and fold
increase observed with the labeling data. These results confirm
previous findings that supplementation of the tissue culture medium of
fibroblasts with alkylglycerol allows its uptake and subsequent
incorporation into ether lipids(23, 24) .
Western
blotting of whole cell extracts established that Zellweger fibroblasts
contained three PKC isozymes (,
, and
) and that PKC
,
, or
were not present in detectable amounts. The
immunoreactivity could be blocked by incubation with the peptide
against which the antibody was raised (data not shown). PKC
and
were located mainly in the cytosol fraction of unstimulated
cells, whereas PKC
appeared to be predominantly associated with
the particulate fraction (data not shown).
Treatment of Zellweger
fibroblasts with bradykinin induced a rapid translocation of PKC as
detected by measurement of enzyme activity and Western blotting (Fig. 1). The enzyme assay conditions used were specific for
Ca-dependent isozymes of PKC. Given that PKC
is
the only Ca
-dependent isozyme expressed by SF 3271,
this form is therefore likely to be responsible for the enzyme activity
shown in Fig. 1. Analysis of 0.2% Triton X-100 extracts of
particulate material by Western blotting with a PKC
-specific
antibody showed that PKC
associated with the membrane 30 s after
bradykinin addition. In separate experiments PKC
associated with
the membrane was shown to have returned to control levels within 2 min
(data not shown). In Zellweger fibroblasts supplemented with
1-O-HDG, the translocation of PKC
and
Ca
/PS-dependent histone phosphorylation was
dramatically reduced (Fig. 1). The reduction of PKC
translocation was not due to a loss of PKC
protein in
supplemented cells, as total cell extracts of control and supplemented
SF 3271 cells contained similar levels of PKC
protein determined
by Western blotting (data not shown).
Figure 1:
Translocation of PKC induced by
bradykinin. A, cells were incubated with either carrier for 24
h and then in the absence of serum for 24 h (
) or with 20
µg/ml 1-O-HDG for 24 h and then in the absence of
1-O-HDG and serum for a further 24 h (
). Cells were then
incubated with bradykinin (10 nM) for the times indicated.
Particulate fractions (extracted with 0.2% Triton X-100) were prepared;
and PKC partially purified by batch elution from DEAE-Sephacel and
assayed for the Ca
/PS-dependent phosphorylation of
histone IIIS. B, cells were incubated either with carrier
(designated by a -) or 1-O-HDG (designated by a +) as for A. Cells were then activated with
bradykinin (10 nM) for the times indicated (times shown are in
seconds) and particulate fractions analyzed by Western blotting with
PKC
antibody. Migration of molecular mass markers (108.5 and 76
kDa) are indicated on the right side of the
figure.
Translocation of PKC was
also observed in response to the synthetic DG analogue DiC
(5 µg/ml) (Fig. 2). Translocation was observed at 5
min and maintained at 20 min, and at both time points 1-O-HDG
supplementation inhibited translocation.
Figure 2:
Translocation of PKC induced by
DiC
. Cells were incubated with either carrier for 24 h and
then in the absence of serum for 24 h (designated by a -) or with 1-O-HDG (20 µg/ml) for 24 h and
then in the absence of 1-O-HDG and serum for a further 24 h
(designated by a +). Cells were then incubated with
DiC
(5 µg/ml) for the times indicated (times shown are
in minutes). Particulate fractions (extracted with 0.2% Triton X-100)
were prepared and analyzed by Western blotting with PKC
antibody.
Migration of molecular mass markers (108.5 and 76 kDa) are indicated on
the right side of the figure.
Western blotting analysis
was also used to examine the effect of bradykinin on the translocation
of PKC and
. In these experiments the extraction conditions
were altered from those used to detect PKC
as only low levels of
PKC
was solubilized with 0.2% Triton X-100. However when the
Triton X-100 concentration was increased to 1%, translocation of PKC
was clearly visible (Fig. 3). PKC
translocation was
still observed under these extraction conditions. In the time course
shown in Fig. 3, PKC
translocation had returned to basal
levels within 1 min, whereas PKC
translocation, which was also
rapid, was sustained for at least 5 min. Time points beyond 5 min were
not examined. Supplementation had no effect on the translocation of PKC
in response to bradykinin. PKC
was associated with the
particulate fraction at high levels in unstimulated cells, and no
change was observed when cells were incubated with either bradykinin or
TPA. Similarly, there was no change in the membrane association of PKC
in cells supplemented with 1-O-HDG. The higher molecular
weight band seen in the TPA-treated tracks corresponds to PKC
as
the anti-PKC
antibody used in these studies cross-reacts with PKC
, as has been reported earlier(11) .
Figure 3:
Translocation of PKC isozymes ,
, and
induced by bradykinin. Cells were incubated with
either carrier for 24 h and then in the absence of serum for 24 h
(designated by a -) or with 1-O-HDG (20
µg/ml) for 24 h and then in the absence of 1-O-HDG and
serum for a further 24 h (designated by a +). Cells were
then incubated with bradykinin (10 nM) or TPA (100
nM) as indicated for the times shown (time is in minutes).
Particulate fractions (extracted with 1% Triton X-100) were prepared
and analyzed by Western blotting with antibodies specific for the PKC
isozyme indicated. Migration of molecular mass markers (108.5 and 76
kDa) are indicated on the right side of the
figure.
It is clear from
the above results that supplementation with 1-O-HDG
selectively blocked the bradykinin-induced translocation of PKC ,
a Ca
-dependent form of PKC. The effect of
supplementation on the release of intracellular Ca
in
response to bradykinin was therefore measured (Fig. 4).
Bradykinin caused a very rapid increase in intracellular Ca
that returned to control levels within 1 min consistent with
previous reports(25, 26) . A second addition of
agonist 3 min after the first did not result in a further mobilization
of Ca
, indicating that the cells became refractory to
the agonist. The bradykinin-induced increase in Ca
concentration was unaffected by supplementation of cells with
1-O-HDG.
Figure 4:
Changes of cytosolic Ca
with bradykinin. Cells were incubated with either carrier for 24 h and
then in the absence of serum for 24 h (A) or with
1-O-HDG (20 µg/ml) for 24 h and then in the absence of
1-O-HDG and serum for a further 24 h (B). Cells were
then trypsinized, loaded with Fura-2AM, followed by the addition of
solvent (1 min) or bradykinin (10 nM; 2 and 5 min). The
additions are indicated by arrows. Cytosolic Ca
measurements were carried out as described under
``Experimental Procedures.''
Phospholipid metabolism in bradykinin-stimulated
human fibroblasts has been extensively
studied(27, 28, 29) . We have confirmed some
of these findings in Zellweger fibroblasts. Bradykinin caused a rapid
activation of PLD as measured by PBut accumulation in the presence of
butanol. This activation was not observed in cells pretreated with TPA
for 24 h (Table 1), consistent with an involvement of PKC. The
accumulation of PBut induced by bradykinin was maximal at 5 min (Table 2). In cells supplemented with 1-O-HDG
bradykinin-stimulated PLD activation was strongly inhibited, and no
PBut accumulation was observed for up to 15 min after bradykinin
addition (Table 2). Increased concentrations (up to 1
µM) of bradykinin did not activate PLD in supplemented
cells (data not shown). The inhibition observed was not due to a total
loss of PLD activity in supplemented cells, as TPA was still able to
activate PBut accumulation, although to a lesser extent than in control
cells (Table 2). In these experiments, PLD activity was measured
by prelabeling the phospholipid pool with
[H]palmitic acid. It was therefore important to
establish that the absence of bradykinin-stimulated
[
H]PBut in supplemented cells was not due to
changes in the initial labeling pattern of cellular lipids.
Consequently, in separate experiments, the incorporation of
radioactivity into diacyl, alkenyl, and alkyl phospholipid subclasses
after a 24-h incubation with [
H]palmitic acid was
determined. The counts incorporated into total phospholipid in
unsupplemented cells (4296 ± 160 dpm/10
cells) were
slightly higher than in supplemented cells (3708 ± 125
dpm/10
cells), and radioactivity associated with the
alkenyl plus alkyl phospholipid pool increased from 5% of that
incorporated into total phospholipid in unsupplemented cells to 8.4% in
supplemented cells (data not shown). It is therefore clear that a large
proportion of [
H]palmitic acid is incorporated
into diacyl phospholipid in both supplemented and unsupplemented cells.
It was independently established that the lipids in supplemented cells
are substrates for PLD as indicated by the accumulation of labeled PBut
in the presence of TPA (Table 2). Consequently we do not believe
that the inhibition of bradykinin-induced PBut formation after
supplementation can be explained by alterations in phospholipid
labeling by [
H]palmitic acid.
We next examined the effects of supplementation on the bradykinin-induced activation of MAPK. In initial experiments it was shown that bradykinin-induced MAPK activity was blocked by TPA pretreatment for 24 h (Table 3), suggesting an involvement of PKC, whereas EGF-induced activation was unaffected. In these experiments MAPK activity (phosphorylation of myelin basic protein) was measured in soluble extracts, partially purified by phenyl-Sepharose chromatography. A time course of bradykinin-induced MAPK activity established a peak of activity at 1-2 min which returned to control levels by 15 min (data not shown). The isoforms of MAPK activated after 2 min incubation with bradykinin were examined using partial purification by Mono Q chromatography and immunoblotting (Fig. 5). Two peaks of activity were partially resolved on Mono Q. Using Western blotting it was established that the earlier eluting peak corresponded predominantly to p42 and the later peak to p44 protein (see inset in Fig. 5). Both isoforms were activated by bradykinin treatment. Bradykinin-induced activation of both isoforms was unaffected by 1-O-HDG supplementation. The time course of bradykinin induction of MAPK activity was also similar in control and supplemented cells (data not shown).
Figure 5:
Activation of mitogen-activated protein
kinase isoforms by bradykinin. Cells were incubated with either carrier
for 24 h and then in the absence of serum for 24 h () or with 20
µg/ml 1-O-HDG for 24 h and then in the absence of
1-O-HDG and serum for a further 24 h (
). Cells were then
incubated with bradykinin (10 nM;
and
) or solvent (+) for 2 min. Soluble fractions were prepared and
subjected to Mono Q chromatography as described under
``Experimental Procedures.'' The column was eluted with a
linear NaCl gradient and fractions analyzed for the ability to
phosphorylate myelin basic protein. The inset shows a Western
blot of the peak fractions of unsupplemented cells (designated by a -) and 1-O-HDG supplemented cells (designated
by a +). The migration of molecular mass markers (49.5
and 32.5 kDa) is indicated on the right side of the
figure.
This is the first report of PKC signaling related events in
fibroblasts isolated from Zellweger patients. Three PKC isozymes
(,
, and
) were identified in SF 3271 fibroblasts by
Western blotting. Bradykinin invoked a rapid transient translocation of
PKC
, which in timing and duration was closely correlated with an
increase in intracellular Ca
. PKC
translocation
was also rapid but sustained for at least 5 min after agonist addition
and therefore does not correlate with the increase in intracellular
Ca
. The membrane association of PKC
was not
affected by either bradykinin or TPA. These results are consistent with
the observations of Ha and Exton (11) who reported similar
effects of
-thrombin on PKC translocation in IIC9 fibroblasts. The
authors also reported that DiC
did not induce the
translocation of PKC
unless cytosolic Ca
was
increased with ionomycin, whereas in our experiments, DiC
alone caused a clear translocation of PKC
(Fig. 2).
The reason for the different results is likely to be the extraction
conditions used. In the earlier work (11) the fraction
extracting between 0.02 and 1% Triton X-100 was analyzed, whereas we
extracted between 0 and 0.2 or 1% Triton X-100. In preliminary
experiments we have shown that a 0.02% Triton X-100 extraction removed
a substantial proportion of either DiC
- or
bradykinin-induced translocation of PKC
, indicating that this
subspecies is relatively loosely bound to the membrane (data not
shown). The translocation of PKC
was unaffected by 0.02% Triton
X-100, suggesting a tighter membrane association.
The increase in
ether phospholipid content caused by 1-O-HDG had a dramatic
and unexpected effect on PKC isozyme translocation. 1-O-HDG
supplementation inhibited PKC translocation and PLD activation
without altering Ca
mobilization, PKC
or
translocation, or MAPK activation. It is therefore clear that
modulation of the ether phospholipid content differentially affected
PKC signaling events. Several mechanisms could account for the observed
results. One possibility is the accumulation of a metabolite of
1-O-HDG which specifically inhibits the translocation of PKC
. It has, for example, been reported that alkyl-linked
diglycerides inhibit PKC activity in vitro(30) ,
although possible differential effects on PKC subspecies has not
explored. Alternatively, the incorporation of alkylglycerol into the
phospholipid pool could cause a physical change in the membrane
structure responsible for modulating its interaction with PKC
(31) . It will be of considerable interest to extend these
studies to cells which express a wider range of PKC subspecies, to
determine whether ether lipid supplementation modulates the activity of
only the Ca
-dependent forms of PKC and whether each
of these forms is equally sensitive to supplementation.
Of
particular interest is the observation that the differential effect of
PKC isozyme translocation is also reflected in PKC-dependent downstream
events. It is likely that the bradykinin-induced activation of PLD is
mediated by PKC in SF 3271 cells as activation was lost in cells
preincubated with TPA for 24 h. Western blotting experiments
established that the incubation with TPA resulted in down-regulation of
PKC and PKC
(data not shown). Although a possible effect of
TPA pretreatment on constitutive PLD activity (32) has not been
eliminated in these cells, a number of independent studies have
implicated PKC in the activation of PLD(33, 34) . The
differential effect of 1-O-HDG supplementation on
bradykinin-induced PLD activation and translocation of PKC
therefore suggests a role for this isozyme in stimulating PLD activity.
The results further indicate that PKC
is not involved in PLD
activation in SF 3271 cells. A number of other studies have linked PKC
and activation of PLD. For example, in Madin-Darby canine kidney
cells depleted of PKC
using antisense technology, activation of
PLD by TPA was inhibited(8) . Similarly studies of the
regulation of PLD in membranes prepared from CCL39 fibroblasts
demonstrated that addition of PKC
and PKC
could activate
PLD(35) , although these studies were carried out in the
absence of exogenous ATP and assume activation via a phosphorylation
independent mechanism. In separate studies we have shown that ceramide
specifically inhibits bradykinin-induced activation of PKC
and
PLD activity in cultured fibroblasts. (
)There is therefore
strong evidence from a number of approaches that PKC
is an
important regulator of PLD activity. However there are some reports
which provide conflicting data. For example in mesangial cells there is
evidence that PKC
and not PKC
can activate PLD. In addition
in Swiss 3T3 fibroblasts it has been concluded that overexpression of
PKC
did not influence the acute stimulation of PLD by TPA but
increased the expression of PLD protein. Although we cannot explain the
divergent results, it is possible that cell type differences exist
resulting, for example, from the different expression patterns of PLD
isozymes as suggested earlier(35) .
In contrast to its
effect on PLD activation 1-O-HDG supplementation had no effect
on the bradykinin-induced activation of MAPK. The MAPK cascade is
rapidly activated in response to growth factors which bind tyrosine
kinase receptors and agonists which lead to the activation of
PKC(36) . The events leading to MAPK activation from tyrosine
kinase receptors have been extensively studied and involve the
activation of Ras and Raf proteins. A sequential activation of kinases
leads to the activation of MAPK by phosphorylation on both tyrosine and
threonine residues. Agonists which activate PKC appear to activate the
MAPK cascade at the level of either Raf or Ras(37) . In SF
3271, pretreatment with TPA completely blocked bradykinin-induced MAPK
activation but did not affect activation by EGF, indicating that the
MAPK pathway was functional but that bradykinin requires the action of
a PKC isozyme that is down-regulated by TPA. Given the lack of effect
of 1-O-HDG supplementation on the translocation of PKC
and MAPK activation, it is likely that PKC
, and not PKC
, is
responsible for MAPK activation in SF 3271 cells. However not all known
forms of PKC were examined in this study, and we are currently using an
antisense approach to deplete cells of the specific subspecies.
The assignment of specific PKC-dependent responses to particular isozymes is an exciting area of research. The possibility that endogenous membrane components control individual PKC isozyme translocation is intriguing. However in this study we have experimentally manipulated the levels of ether-linked phospholipids in a cell line which usually has a low level. It would be of interest to determine if similar effects on PKC signaling are observed in other cells, such as neutrophils(38) , which express naturally high levels of ether lipids.