(Received for publication, April 6, 1995; and in revised form, June 20, 1995)
From the
Diglycerides are phospholipid-derived second messengers that
serve as cofactors for protein kinase C activation. We have previously
shown that, in rat glomerular mesangial cells, the cytokine,
interleukin-1, and the vasoactive peptide, endothelin, generate
diglycerides from unique phospholipid precursors. However, neither the
molecular species of these diglycerides nor their biological actions
were determined. It is now hypothesized that interleukin-1- and
endothelin-treated mesangial cells form distinct molecular species of
diglycerides which may serve different roles as intracellular signaling
molecules. Diglyceride molecular species were resolved and quantified
by TLC and high performance liquid chromatography as
diglyceride-[
C]acetate derivatives. Endothelin
stimulates predominantly ester-linked species (diacylglycerols) in
contrast to interleukin-1 which stimulates only ether-linked species
(alkyl,acyl- and alkenyl,acylglycerols). In support of these data,
interleukin-1-treated mesangial cells hydrolyze ethanolamine
plasmalogens, vinyl ether-linked phospholipids. It has been reported
that ether-linked, in contrast to ester-linked, diglyceride species do
not activate protein kinase C activity. Thus, we next assessed membrane
protein kinase C activity in endothelin- or interleukin-1-treated
mesangial cells. Even though interleukin-1 has no effect upon basal
protein kinase C activity, this cytokine, through the formation of
ether-linked diglyceride second messengers, inhibits endothelin,
platelet-activating factor, or arginine vasopressin-stimulated protein
kinase C activity. We further demonstrate that ester-linked
diacylglycerols but not alkyl,acyl- or alkenyl,acylglycerols substitute
for phorbol esters in a cell-free protein kinase C assay. In addition,
alkenyl,acylglycerols inhibit diacylglycerol-stimulated
immunoprecipitated protein kinase C
activity in vitro and total protein kinase C activity in permeabilized mesangial
cells ex vivo. Taken together, these data suggest that
interleukin-1-induced formation of ether-linked diglycerides may
physiologically serve to down-regulate receptor-mediated protein kinase
C activity and that individual molecular species of diglycerides may
serve different roles as intracellular signaling molecules.
Classical transmembrane signaling theory suggests that receptors
are linked to a phosphatidylinositol 4,5-bisphosphate
(PtdIns)()-specific phospholipase C generating inositol
phosphates and diglycerides (DG). However, distinct phospholipase C and
phospholipase D activities that hydrolyze ester- and ether-linked
phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEth) may
also release unique molecular species of DG and phosphatidic acids. We
have previously compared and contrasted the early phospholipid-derived
second messengers generated by the vasoconstrictor, endothelin-1 (ET),
and the cytokine, interleukin-1
(IL-1) in rat glomerular mesangial
cells (MC). ET maintains the myogenic phenotype of these smooth muscle
pericytes inducing MC contraction which leads to a decreased glomerular
filtration rate through a diminished ultrafiltration
coefficient(1) . IL-1 induces a phenotypic change in MC,
switching these cells from a contractile to a secretory,
immune-responsive, state with concomitant changes in matrix formation
and adhesion protein expression, events associated with
sclerosis(2) . ET generates DG initially from PtdIns and
secondarily from PtdCho in MC(3) . Furthermore, ET-stimulated
Ins-1,4,5-P
responses are transient, while elevated DG
responses are sustained(4, 5, 6) . In
contrast, IL-1 generates DG from PtdEth hydrolysis totally independent
of any measurable polyphosphoinositide turnover or elevations in
intracellular free calcium concentration
([Ca
]
) in
MC(7) . Similar results demonstrating IL-1-induced DG derived
from PtdCho hydrolysis (8, 9) or from an augmented
sequential lysophosphatidic acid acyltransferase/phosphatidic acid
phosphohydrolase activity (10) without concomitant
Ins-1,4,5-P
generation have also been reported. This
alternative signal transduction pathway which suggests that DG can be
generated solely from sources besides the polyphosphoinositides has
been demonstrated for diverse growth factors and cytokines including
fibroblast growth factor(11, 12) , insulin (13) , EGF(14) , growth hormone(15) ,
interferon
(16) , D59
(17) ,
Ha-ras(18) , IL-3(19) , and IL-1
(20, 21) . Agonists that stimulate hydrolysis of
phospholipids besides PtdIns may (interferon,
IL-3(16, 19) ) or may not (IL-1, fibroblast growth
factor(11, 20, 21, 22) ) stimulate
PKC. IL-1, in all cell types studied, consistently does not induce PKC
translocation or bioactivity. The role of the unique DG species
generated by these agonists is still controversial as PtdCho-derived
DG has been shown to be an effective (23) or ineffective
activator of PKC(24, 25) . Agonists, such as IL-1,
that generate DG independent of any polyphosphoinositide turnover have
never been characterized in terms of their ester- and ether-linked sn-1 configuration. We hypothesize that ET and IL-1 generate
discrete molecular species of DG that differentially regulate PKC.
As DG can be generated through distinct biochemical pathways and
from different phospholipid precursors, it is very surprising that
little information is currently available correlating agonist-induced
DG structure with physiological function. Significant ether-linked DG
species have been observed in PMA- and fMLP
(formylmethionylleucylphenylalanine)-stimulated neutrophils (26) while only diacyl species have been observed with
thrombin-stimulated I1 Cg cells (27) and IgG-stimulated mast
cells(28) . The characterization of molecular species of DG
from discrete phospholipid pools is especially important as
ether-linked DG (alkyl,acyl- or alkenyl,acyl-DG species), in contrast
to diacyl species, may inhibit PKC, not affect PKC (29, 30, 31, 32) , or only activate
PKC in the presence of elevated
levels(33) . Thus, cytokines such as IL-1 which
generate DG at physiological levels of calcium may still be unable to
activate PKC. In addition, ether-linked DG actually inhibit PKC
activation induced by diacylglycerol species(30) . To date,
there have been no studies that directly link agonist-stimulated
ether-linked DG with an altered PKC activity.
Given the different signaling pathways that generate ET- and IL-1-induced second messengers in MC, our first aim was to characterize the molecular species of DG generated by these agonists as either diacyl-, alkyl,acyl-, or alkenyl,acylglycerol species. Our second aim was to correlate the formation of these individual molecular species of DG with PKC activation utilizing both cell-free and intact-cell methods.
ET-1 was obtained from the Peptide Institute (Osaka, Japan)
while IL-1 was purchased from U.B.I. (New York). Radiolabeled materials
including [H]glycerol and
[
C]acetic anhydride were obtained from Amersham.
DG and phospholipid standards were obtained from Deva Biologicals
(Penn) or Serdary Biochemicals (London, Ontario, Canada). Polyclonal
anti-PKC
antibody was obtained from Santa Cruz Biotechnology. All
other materials were purchased from either Sigma or Calbiochem.
In
selected experiments, diradylglycerols with identical chain lengths and
variable sn-1 linkages were substituted for PMA in the PKC
assays and cell permeabilization experiments. The prepared
diradylglycerols used in these studies all consisted of 16:0 and 18:1
hydrocarbon substituents in the sn-1- and sn-2-position, respectively. The concentration of our
phospholipase C-derived DG species was determined from the initial
concentration of phospholipid precursor. The phospholipase C reaction
goes to completion and showed no preference for ether- or ester-linked
phospholipids. Permeabilization of MC with 20 µg/ml saponin for 5
min was accomplished as described previously(38) . Efficiency
of permeabilization was determined using
[S]GTP
S and
[
H]glycerol-labeled DG. This procedure did not
elevate basal PKC activity or phospholipase C or D
activities(38) . As should be expected, DG species did not
affect membrane protein kinase C activity in nonpermeabilized cells.
Using [C]acetic anhydride, we have
assessed diacyl-, alkyl,acyl-, and
alkenyl,acylglycerol-[
C]acetate species from
vehicle- and ET- or IL-1-treated MC (Fig. 1). Previous
experiments using [
H]arachidonate (3, 7, 34) established 5 min as an optimal
point to assess DG. These results, expressed as a mass measurement,
indicated that ET generates predominantly ester-linked species
(diacylglycerol) while IL-1 generates only ether-linked (alkyl,acyl-
and alkenyl,acylglycerol) species. ET stimulate a 2.8-fold increase in
diester-linked species in contrast to a nonsignificant 1.6-fold
increase for ether-linked species. IL-1 treatment significantly
increased alkyl,acylglycerol formation 2.2-fold and
alkenyl,acylglycerol formation 2.8-fold. In data not shown, IL-1
concentrations as high as 30 ng/ml did not induce diacylglycerol
formation. MC pretreated with IL-1 4 min before subsequent ET
stimulation displayed an additive 5.0-fold increase in
alkenyl,acylglycerol species formation which was not observed for the
other species. Also, IL-1 treatment did not reduce ET-stimulated
diacylglycerol formation. Expressed as ether/ester-DG percentages,
control values of 54% were increased to 120% with IL-1 and reduced to
34% with ET. IL-1 restored this diminished ET-induced ether/ester-DG
percentage to control levels of 56%. We next analyzed IL-1-stimulated
(10 ng/ml) DG species at several time points that correspond to
previously published data assessing IL-1-induced
[
H]DG formation(7) . As depicted in Fig. 2, IL-1 induced alkyl,acyl- and alkenyl,acyl- but not
diacylglycerol at 1, 5, and 15 min. IL-1-stimulated ether-linked DG
returned to baseline values by 30 min, confirming the earlier
radioactive flux measurements (7) .
Figure 1:
ET-stimulated MC generate predominantly
ester-linked DG while IL-1-stimulated MC form only ether-linked DG
species. MC were treated with ET and/or IL-1 for 5 min. DG mass was
assessed as DG-[C]acetate for TLC-separated
diacyl-, alkyl,acyl-, and alkenyl,acylglycerol species. n = 3 experiments, each experiment replicated in duplicate,
mean ± S.E., *, p <
0.05.
Figure 2:
IL-1-treated MC form ether- but not
ester-linked DG as a function of time. DG mass was assessed as
DG-[C]acetate for TLC-separated diacyl-,
alkyl,acyl-, and alkenyl,acylglycerol species. n = 4
experiments, each experiment replicated in duplicate, mean ±
S.E., p < 0.05 by one-way ANOVA for alkyl and alkenyl
data.
We have utilized a spectrophotometric assay that ascertains the plasmalogen content (a vinyl ether-linked phospholipid) of TLC-separated phospholipids, to investigate if the elevation in IL-1-generated ether-linked DG corresponds to a decrease in ether-linked phospholipid species (Fig. 3). IL-1 (3 ng/ml)-treated MC responded with a decrease in ethanolamine plasmalogen but not choline plasmalogen mass which corresponds to the time course of IL-1-induced DG generation(7) . In addition, these studies confirmed our earlier studies (7) that, in MC, IL-1 receptor signaling is linked to hydrolysis of ethanolamine- but not choline-containing phospholipid species.
Figure 3: IL-1-treated MC hydrolyze ethanolamine but not choline plasmalogens. Plasmalogen (alkenylphospholipid) content was assessed spectrophotometrically by measuring long chain fatty aldehydes released from the sn-1-position of the phospholipid. n = 4, mean ± S.E., p < 0.01 by two-way ANOVA.
As it has been reported that ether-linked diglyceride species, in contrast to diacylglycerol species, do not activate PKC activity, we treated MC with either IL-1 which generates only ether-linked species or ET which generates predominantly diacyl species and assessed PKC activity (Fig. 4). Using this Triton X-100 mixed micelle EGF-receptor binding domain phosphorylation assay system (Amersham) and a partially purified MC membrane preparation, we have previously demonstrated that PMA activates PKC activity in the presence but not the absence of exogenous PtdSer, calcium, or membrane preparation. ET (100 nM) stimulated membrane PKC activity compared to vehicle- or IL-1 (3 ng/ml)-treated MC. The doses selected elicited maximal DG stimulation in MC. In data not shown, IL-1 concentrations as high as 100 ng/ml did not stimulate membrane PKC activity. Moreover, in data not shown, in a complementary manner, ET reduced cytosolic PKC activity. At 5 min, membrane PKC activity was 32, 29, and 71% of total PKC activity for control, IL-1-, and ET-stimulated MC, respectively, suggesting that ET but not IL-1 translocates PKC activity to the membrane. These studies which demonstrate that IL-1 does not stimulate PKC activity or translocation as assessed by phosphorylation of an EGF-receptor binding domain have been confirmed using a PKC pseudosubstrate (Life Technologies, Inc.) or histone as alternative substrates.
Figure 4: ET but not IL-1 stimulates membrane PKC activity in MC. For these experiments, ionomycin, IL-1, and/or ET were added to MC monolayers, membranes were prepared and assayed for PKC activity in the presence of all cofactors including calcium. n = 7 to 30 experiments, each experiment replicated in triplicate or quadruplicate, mean ± S.E., *, p < 0.05.
We have shown previously that IL-1 does not stimulate inositol trisphosphate production or induce elevations in intracellular free calcium concentrations(7) . Thus, we used ionomycin (a calcium ionophore) to elevate intracellular calcium concentration to levels observed with 100 nM ET stimulation and then assessed the effects of IL-1 upon membrane PKC activity (Fig. 4). Ionomycin induced a slight increase in PKC activity that was not potentiated with IL-1 suggesting that IL-1 does not activate PKC in the presence or absence of elevation of intracellular calcium. To test if IL-1-generated ether-linked DG have an inhibitory effect upon receptor-activated PKC activity, we pretreated MC with IL-1 or vehicle for 5 min and then assessed ET-stimulated PKC activity for an additional 15 min (Fig. 4). IL-1 had no effect on basal PKC activity but did reduce ET-stimulated PKC activity, suggesting a physiological inhibitory role for IL-1-generated ether-linked DG. In data not shown, this inhibitory effect of IL-1 upon ET-stimulated PKC activity was evident even without preincubation. In addition, IL-1 also inhibited platelet-activating factor- and arginine vasopressin-stimulated PKC activity. ET (100 nM) stimulated membrane PKC activity in a time-dependent manner (Fig. 5), and we chose either a 5-min or 15-min ET stimulation for all subsequent experiments, as these time points correspond to maximal ET-stimulated DG formation and PKC activation. At all time points studied, IL-1 (3 ng/ml) did not stimulate PKC activity above control values.
Figure 5:
ET
(10M) stimulates membrane PKC activity in
a time-dependent manner. Total PKC activity was assessed by
phosphorylation of the EGF-receptor binding domain peptide as an
exogenous substrate. n = 3 experiments, each replicated
in triplicate, mean ± S.E., *, p < 0.05, one-way
ANOVA.
To test
whether ET-induced diacylglycerols but not IL-1-generated ether-linked
diglycerols stimulate PKC, we next substituted diacyl-, alkyl,acyl-,
and alkenyl,acylglycerol species for PMA in the cell-free PKC assay
system (Fig. 6). Substituting diacylglycerol but not alkyl,acyl-
and alkenyl,acylglycerols for PMA in the PKC assay elevated basal
membrane PKC activity. PtdSer stimulated PKC activity in the absence of
PMA and served as baseline control. The mol % of PtdSer and DG have
previously been shown to be optimal for this assay(34) . To
confirm and extend these cell-free studies that assess total PKC
activity, we next specifically evaluated immunoprecipitated PKC
in an in vitro kinase assay (Fig. 7). MC express PKC
which is acutely activated by G-protein-linked receptors (43) . (
)A 4-min preincubation with 10 nM alkenyl,acylglycerol (AAG) diminished 100 nM diacylglycerol (DAG)-stimulated, immunoprecipitated, PKC
activity as assessed by histone phosphorylation. Actual counts/min/lane
values for the excised histone bands were, respectively, 3507, 200,
and 173 for diacylglycerol-, alkenyl,acylglycerol-, and
alkenyl,acylglycerol/diacylglycerol-treated immunoprecipitates. In
addition, alkenyl,acylglycerol inhibited diacylglycerol-induced
autophosphorylation of PKC
. These data suggest that IL-1- and
ET-receptors increase the content of distinct species of DG that
differentially activate PKC in MC.
Figure 6: Substituting diacylglycerols but not alkyl,acylglycerols or alkenyl,acylglycerols for PMA in the cell-free PKC assay elevates PKC activity. Membranes prepared from control MC monolayers were assayed for PKC activity in the presence of PtdSer, calcium, and selected DG or PMA. n = 7 experiments, each experiment replicated in quadruplicate, mean ± S.E., *, p < 0.05.
Figure 7:
Alkenyl,acylglycerol inhibits
diacylglycerol activation of PKC as assessed by
[
P]PO
incorporation into histone. Immunoprecipitated PKC
activity
was measured in an in vitro kinase reaction that contained
PtdSer, calcium, and either diacyl (DAG)- or
alkenyl,acylglycerol (AAG) species. For visual clarity, the
autoradiogram exposure time was 6 h for the diacylglycerol lane and 72
h for the AAG and AAG/diacylglycerol lanes. A representative
autoradiogram of two such experiments.
To definitively assess the effects of DG molecular species upon PKC activity, we incubated permeabilized MC monolayers (38) for 10 min with 100 nM diacyl-, alkyl,acyl-, or alkenyl,acylglycerol species or vehicle and then assessed membrane PKC activity (Fig. 8). These intact cell experiments are the in vivo equivalents of the cell-free experiments that substitute DG species for PMA in the assay itself. Using the permeabilized cells, diacylglycerol but not ether-linked glyceride species stimulated PKC activity. Ether-linked DG concentrations as high as 1 µM did not stimulate PKC activity (data not shown). Also, in data not shown, maximal concentration of ET (100 nM) and diacylglycerols (100 nM) did not have a synergistic effect upon PKC activity, suggesting activation through a common pathway.
Figure 8: Exogenous application of diacylglycerol species but not alkyl,acylglycerol or alkenyl,acylglycerol species to permeabilized MC stimulate membrane PKC activity. Saponin-treated MC were incubated with 100 nM concentrations of the individual DG species for 10 min and then homogenized for PKC analysis. In nonpermeabilized cells, 100 nM diacylglycerol-induced PKC activity was only 6.0% above corresponding control. n = 6 experiments, each experiment replicated in quadruplicate, mean ± S.E., *, p < 0.05.
We next tested if alkenyl,acylglycerol could inhibit diacylglycerol-induced PKC activation in this intact cell model. Alkenyl,acylglycerol species (1 nM through 1 µM) were added 4 min before diacylglycerol species (100 nM) and then membrane PKC was assessed after 10 min. (Fig. 9). Similar results were obtained if the alkenyl,acylglycerol species were added simultaneously with the diacylglycerol species (data not shown). Alkenyl,acylglycerol inhibited diacylglycerol-induced PKC activity in a dose-dependent manner. Specifically, 1 nM alkenyl,acylglycerol reduced 62% of membrane PKC activity induced by 0.1 µM diacylglycerol. These data suggest, but do not prove, that ether-linked DG species compete for the diacylglycerol cofactor site on PKC without activating the enzyme.
Figure 9: Exogenous application of alkenyl,acylglycerols inhibit stimulation of membrane PKC activity induced by diacylglycerols in permeabilized MC. Saponin-treated MC were incubated with various doses of alkenyl,acylglycerol for 4 min before addition of 100 nM diacylglycerol for another 6 min. MC membranes were then prepared and assayed for PKC activity. n = 6 experiments, each experiment replicated in quadruplicate, mean ± S.E., *, p < 0.05.
Vasoconstrictors, cytokines, and growth factors stimulate the
formation of DG second messengers, yet each agonist leads to separate
physiological end points. To further define the biological actions of
ET-1 (vasoconstriction and proliferation) and IL-1 (inducibility of
genes associated with matrix formation and synthesis of other
proinflammatory mediators), we investigated the early signal
transduction pathways for these agonists with particular emphasis on
the distinct molecular species of DG generated and their potential
regulation of PKC activity. The biochemical information contained in
the structure of these unique DG species may determine, in part, the
phenotypic changes induced by each of these agonist groups. Solely on
the basis of the sn-1 bond linkage of the DG, we have
demonstrated that ET and IL-1 generate distinct DG species that
differentially regulate PKC. IL-1-treated MC generated only
ether-linked DG species in contrast to ET-1-stimulated MC which formed
predominantly ester-linked (diacyl) DG species. Furthermore, ET but not
IL-1 stimulated basal membrane PKC activity, while IL-1 decreased
receptor-stimulated PKC activity. Our results further suggest that it
is the elevation of vinyl ether-linked DG species and not a
corresponding decrease in diacylglycerols that leads to IL-1
receptor-linked reduction of ET-activated PKC. The action of
IL-1-induced ether-linked DG to inhibit ET-stimulated ester-linked DG
was shown with both immunoprecipitated PKC in a cell-free system
and with total PKC in a permeabilized cell protocol. Thus, the
physiological role of IL-1-induced formation of ether-linked DG may be
to down-regulate receptor-mediated PKC activation.
We demonstrate that IL-1 inhibits receptor-stimulated PKC activity by generating ether-linked DG which have been suggested to competitively bind to PKC without activating PKC(44) . Alternatively, IL-1 pretreatment of ET-stimulated MC maintains ether/ester-DG percentages at levels that do not favor PKC activation. It has been suggested that the carboxyl groups of the ester bond at the sn-1-position as well as the resulting bond angle are essential to PKC activation (45, 46) . It follows that it may be the characteristic kink in the sn-1 ether bond of alkyl- or alkenylphospholipids which allows IL-1 receptors to activate a phospholipase that selectively hydrolyzes these substrates. It is now widely believed that a physical property of DG to lower the binding energy for membrane/PKC interactions favors translocation of the kinase to the membrane(44) . Thus, as an alternative mechanism of action, ether-linked DG may not increase the apparent membrane binding constant for PKC. Recent studies have suggested that it is not PKC activation or translocation that is ultimately responsible for an altered phenotype, but, in fact, it is down-regulation and/or proteolysis of the activated PKC isotypes(47) . This theory might explain the observation that chronic but not acute phorbol ester treatment is a cell proliferation signal(48) . If down-regulation of a specific isotype is critical for activation of a specific cellular phenotype, then a more direct mechanism, competitive inhibition of selected PKC isotypes with ether-linked DG, might also lead to an altered cellular phenotype.
In terms of mass, there is a
discrepancy between IL-1-induced hydrolysis of ethanolamine plasmalogen
and formation of alkenyl,acylglycerol. Nearly 20 pmol of plasmalogen
are hydrolyzed while only 1 pmol of alkenyl,acylglycerol is formed.
This observation may reflect the fact that IL-1 might activate
alkenylphospholipid-selective phospholipases, including A and D which would degrade plasmalogen without directly forming
alkenyl-DG species(49, 50) . Vinyl ether-selective
phospholipase A
(plasmalogenase) and lysophospholipase
A
(lysoplasmalogenase) activities have also been described
which may be regulated by IL-1-receptors(51, 52) .
Supporting our data, this phospholipase A
activity is
specific for ethanolamine but not choline plasmalogens(51) .
In MC, PKC isoenzymes ,
,
, and
are expressed,
and
,
,
but not
can be down-regulated by chronic
PMA treatment (43) . The role of IL-1-induced ether-linked DG
to inhibit vasoconstrictor-stimulated PKC
is supported by the
following observations: 1) IL-1 inhibits ET-, arginine vasopressin-,
and platelet-activating factor-stimulated PKC activity; 2) all of these
G protein-linked vasoconstrictors activate PKC
; and 3)
ether-linked DG inhibit immunoprecipitate PKC
in an in vitro assay. The putative actions of IL-1-stimulated ether-linked DG to
negatively regulate calcium-insensitive PKC isotypes awaits further
investigation. Even though IL does not stimulate PKC translocation or
activation, it could be that IL-1 induces activation of PKC
which
is not regulated by DG cofactors. It is for this reason we have used
the EGF-receptor binding domain to assess PKC activation as this
substrate, and not histones, is phosphorylated in vitro by PKC
(53, 54) . Moreover, we confirmed our findings by
utilizing the alanine to serine mutated pseudosubstrate peptide
sequence as a PKC substrate(55) .
The DG cofactor substrate
specificity for individual PKC isotypes has only recently been
investigated as a potential signaling mechanism. In a recent report,
supplementation of 1-O-hexadecylglycerol into fibroblasts of
Zellweger patients leads to accumulation of ether-linked DG and a
concomitant inhibition of bradykinin-induced translocation of PKC
but not
or
(56) . Our studies extend these findings
by demonstrating that IL-1-induced ether-linked DG inhibit
immunoprecipitated PKC
activity in an in vitro assay. In
other studies, PtdIns-derived DG preferentially containing arachidonate
at the sn-2-position are a better cofactor for PKC
compared with PKC
or
, in contrast to PtdCho-derived DG
species which are equipotent in stimulating PKC
isoenzymes(57) . Also, the mitogenic effects of insulin have
been linked to PtdCho-dependent phospholipase C activity that generates
undefined species of DG that specifically activates PKC
(58) . In a scenario similar to IL-1-receptor signaling in
MC, interferon which does not elevate
[Ca
]
and forms
PtdCho-derived DG, translocates PKC
but not
in Daudi
cells(59) . Alternatively, a single agonist may have
differential effects on multiple PKC isotypes as retinoic acid-induced
differentiation is associated with PKC
inhibition (60) and PKC
stimulation(61) . Thus, signaling
mechanisms will be defined in the future by the type of PKC isotypes
mobilized as well as the DG cofactor specificity for that PKC isotype.
Our studies are the first report of IL-1 inhibiting
receptor-stimulated PKC activity. However, this is not the first
instance of IL-1 negatively modulating the actions of vasoconstrictors.
IL-1 pretreatment inhibited phenylephrine-induced contraction in rat
aortic rings (62) and inhibited cerebral spinal
fluid-contracted pial arteries (63) . Our data suggest that one
component of IL-1 signaling is ether-linked DG inhibition of PKC
activity. Other signaling pathways that have been postulated include
nuclear targeting of a 16-kDa N-terminal IL-1
cleavage product
that may function as a trans-activating factor(64) .
Alternatively, the carboxyl end of IL-1
bound to the IL-1 receptor
also localizes to the nucleus (65) . The role of JAK/STAT
kinases to amplify early IL-1 receptor signals is an area of active
investigation. In addition to inhibiting PKC activity with subsequent
effects upon contraction and/or differentiation, ether-linked DG may
exert other effects independent of PKC. Ether-linked phospholipids are
preferentially esterified at the sn-2-position with
arachidonate (66) and, thus, these DG may be a potential
substrate for arachidonate release via a DG-lipase. IL-1 has been shown
to induce prostanoids in various cell types(67) , and the
contribution of the DG-lipase mechanism to arachidonate release has not
been investigated. Other PKC-independent actions of DG include
leukocyte chemoattraction(68) , adipocyte glucose
transport(69) , neuronal calcium current
regulation(70) , K
-induced calcium influx
regulation(71) , and translocation of phosphorylcholine
cytidyltransferase activity(72) . DG may serve as a cofactor
for other kinases besides PKC including c-Raf and PAK-2(73) .
Finally, other signaling cascades may augment ether-linked DG-induced
inhibition of PKC. For example, IL-1 and PDGF have recently been shown
to stimulate sphingomyelin metabolism(74, 75) .
However, IL-1, in contrast to PDGF, does not stimulate ceramidase
activity to form the endogenous PKC inhibitor,
sphingosine(76) .
Our studies suggest that IL-1 and ET stimulate distinct species of DG that differentially regulate PKC in MC. This is the first study to conclusively link agonist-stimulated ether-linked DG formation with a physiologically relevant function; i.e. inhibition of PKC activity. An alternative signal transduction pathway is envisioned by which agonists, such as IL-1, that hydrolyze PtdCho and PtdEth but not PtdIns predominantly form ether- but not ester-linked DG species. Our conclusions lead to the provocative speculation that individual molecular species of DG may serve as key metabolic branch points, coupling discrete receptor populations to specific PKC isotypes.