(Received for publication, April 11, 1997)
From the Department of Physiology and Biophysics and
¶ Department of Medicine, Case Western Reserve University,
Cleveland, Ohio 44106-4949 and the § Institute for
Biological Sciences, National Research Council of Canada, Ottawa,
Ontario, Canada K1A OR6
It is hypothesized that inflammatory cytokines
and vasoactive peptides stimulate distinct species of diglycerides that
differentially regulate protein kinase C isotypes. In published data,
we demonstrated that interleukin-1, in contrast to endothelin,
selectively generates ether-linked diglyceride species (alkyl, acyl-
and alkenyl, acylglycerols) in rat mesangial cells, a smooth
muscle-like pericyte in the glomerulus. We now demonstrate both in
intact cell and in cell-free preparations that these interleukin-1
receptor-generated ether-linked diglycerides inhibit
immunoprecipitated protein kinase C and
but not
activity.
Neither interleukin-1 nor endothelin affect de novo protein
expression of these protein kinase C isotypes. As down-regulation of
calcium-insensitive protein kinase C isotypes has been linked to
antimitogenic activity, we investigated growth arrest as a functional
correlate for IL-1-generated ether-linked diglycerides. Cell-permeable ether-linked diglycerides mimic the effects of interleukin-1 to induce a growth-arrested state in both
G-protein-linked receptor- and tyrosine kinase receptor-stimulated
mesangial cells. This signaling mechanism implicates cytokine
receptor-induced ether-linked diglycerides as second messengers that
inhibit the bioactivity of calcium-insensitive protein kinase C
isotypes resulting in growth arrest.
Interleukin-1
(IL-1)1-induced activation of
rat glomerular mesangial cells (MC) culminates in an inflammatory
phenotype often observed in vivo in models of
glomerulosclerosis. Our laboratory has been investigating the early,
lipid-mediated signal transduction pathways for inflammatory cytokines
in MC with particular emphasis on the distinct molecular species of
diglycerides (DG) generated and their regulation of protein kinase C
(PKC) activity. We have demonstrated previously that the inflammatory
cytokine interleukin-1 and the vasoconstrictor peptide, endothelin-1
(ET-1) generate distinct species of DG from different
membrane-associated phospholipids in MC (1-3). IL-1 receptor
activation selectively generates ether-linked species of DG, namely
alkyl, acyl- and alkenyl, acylglycerols, whereas ET-1 receptor
activation produces predominantly ester-linked diacylglycerols (1). Our
laboratory has also demonstrated previously that these IL-1 generated
ether-linked DG, in contrast to the PKC-activating diacylglycerols,
inhibit total PKC activity as well as inhibit diacylglycerol-stimulated
PKC
activation (1). In fact, ether-linked DG may competitively
inhibit PKC activation induced by diacylglycerol species (4).
Additional studies support this signaling mechanism, as ether-linked DG
fail to activate total PKC activity in vitro or only
activate PKC in the presence of pharmacological concentrations of
calcium (5-8).
In light of these findings, our interest has now turned to regulation
by ether-linked DG of specific PKC isotypes. The PKC family now
includes 12 distinct isotypes subdivided into three categories (9). The
calcium-sensitive conventional PKCs consist of alpha (), beta
(
1 and
11), and gamma (
) isotypes,
while the calcium-insensitive novel isotypes include delta (
),
epsilon (
), nu (
), mu (µ), and theta (
). PKCs zeta (
),
iota (
), and lamda (
) are known as atypical PKCs as they have
only one cysteine-rich zinc finger domain and are not regulated by DG
or phorbol esters. We have chosen to investigate the regulation of the
calcium-insenstitive isotypes
and
by DG, as IL-1
receptor-mediated production of ether-linked DG is not accompanied by
an increase in intracellular free calcium concentration (3, 10)
It is not surprising that in addition to varying in primary structure, these PKC isotypes are differentially regulated in terms of expression, subcellular localization, cofactor, or substrate specificities and ligand activation (9, 11). However, the mechanisms by which distinct receptors target specific PKC isotypes have not been elucidated. Mechanisms have focused on ligand-induced PKC translocation and/or down-regulation and catabolism as well as PKC accessory or binding proteins. We now hypothesize a novel acute mechanism by which inflammatory cytokine receptors form ether-linked DG species that competitively inhibit the bioactivity of calcium-insensitive PKC isotypes. Moreover, it is hypothesized that inhibition of calcium-insensitive PKC isotypes correlates with a growth-arrested, inflamed MC phenotype.
ET-1 was obtained from the Peptide Institute (Osaka, Japan),
while IL-1 and platelet-derived growth factor (PDGF) were purchased from Life Technologies, Inc. [-32P]ATP was obtained
from ICN and [3H]thymidine was purchased from NEN Life
Science Products. DG and phospholipid standards were obtained from
Serdary Biochemicals (London, Ontario, Canada) or Deva Biologicals
(Hatboro, PA). Polyclonal anti-PKC antibodies were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). All other materials were purchased
from either Sigma or Calbiochem. All cell culture media and reagents
were purchased from Life Technologies, Inc.
MC were isolated from collagenase-digested glomeruli obtained from 100-g male Harlan Sprague Dawley rats by a sequential sieving technique. MC were grown in RPMI 1640 or Dulbecco's modified Eagle's medium supplemented with 12% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml insulin, 5 µg/ml transferrin, amd 5 ng/ml selenium at 37 °C in 5% CO2. Cells were used in their 3rd to 15th passage. We have verified previously that the MC cultures are devoid of epithelial, endothelial, macrophage, and fibroblast contamination (12).
Western Blot AnalysisWestern immunoblotting using
polyclonal antibodies to PKC ,
,
,
,
, and
was
performed primarily as described previously (1, 13) except that the
lysis buffer now consisted of 20 mM Hepes, pH 7.5, 40 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM benzamidine hydrochloride. The
cell lysate proteins were separated by SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose. The nitrocellulose
blots were incubated for 2 h at 25 °C with one of the
polyclonal anti-PKC antibodies (1:1000 dilution) and then incubated for
1 h at 25 °C with the secondary antibody, goat anti-rabbit IgG,
coupled to horseradish peroxidase at 1:5000 dilution. Enhanced
chemiluminescence substrates were used to reveal positive bands
according to the manufacturer's (Amersham Corp.) instructions.
Confirming the results of another laboratory (14), we observed basal
expression of PKC isotypes
,
,
,
, and
, but not
in
MC as well as in A7r5 vascular smooth muscle cells (data not shown). To
confirm the negative results with PKC
1 and
11 as well as the positive with PKC
in MC, we
utilized polyclonal antibodies from both Santa Cruz Biotechnology and
Transduction Laboratories (Lexington, KY) that were derived from
different immunogens. Specificity of anti-PKC antibodies was determined
on MC immunoprecipitates as described later. Human foreskin fibroblasts
served as positive controls in all experiments.
The immunoprecipitation of PKC isotypes and the subsequent
reconstitution assay were adapted from previous methods (1). The
bioactivity of the immunoprecipitate has been demonstrated with our
polyclonal antibodies (15); and this immunoprecipitation strategy for
PKC activity has been used successfully in MC (16). Briefly, either PKC
,
, or
was immunoprecipitated from cleared lysates using 0.5 µg of polyclonal anti-PKC isotype (Santa Cruz Biotechnology). The
formed immunocomplexes were subsequently collected with goat
anti-rabbit IgG agarose after a 2-h incubation at 4 °C. As described
previously (1), the kinase reaction contained 10 µg of histone
IIIS/reaction as exogenous substrate in the presence or absence of 40 µg/ml phosphatidylserine/reaction and 24 µg/ml phorbol 12-myristate
13-acetate or various concentrations of diradylglycerols with identical
chain lengths and variable sn-1 linkages. The phosphorylated proteins were resolved by 15% SDS-polyacrylamide gel electrophoresis, visualized by autoradiography, and quantified by laser
densitometry.
The concentration of our phospholipid-derived DG species was determined
from the initial concentration of phospholipid precursor as described
previously (1). The phospholipase C reaction goes to completion and
showed no preference for ester- or ether-linked phospholipids. We
utilized either
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine, 1-O-palmityl-2-oleoyl-sn-glycero-3-phosphorylcholine,
or phosphatidylcholine plasmalogen derived from bovine heart as
phospholipid substrate. Thus, the prepared diacyl- and alkyl,
acylglycerol species utilized in these PKC assays all consisted of 16:0
and 18:1 hydrocarbon substituents in the sn-1 and
sn-2 position, respectively. The prepared alkenyl,
acylglycerol species consisted predominantly of 16:0 and 18:1
substituents;
1-O-palmit-1-ene-yl-2-oleoyl-sn-glycerol.2
DG mass was confirmed by acetylation with 1 µCi of
[14C]acetic anhydride and pyridine for 3 h at
37 °C. The DG acetates (diacyl-, RF = 0.465 ± 0.008; alkyl,acyl, RF = 0.585 ± 0.002;
alkenyl,acyl, RF = 0.645 ± 0.005) are well
resolved from underivatized DG (RF = 0.09 ± 0.01) using a hexane/diethyl/methanol/acetic acid, 90:20:3:2 (v/v/v/v) elution system followed by drying the TLC plates and then a toluene (100, v) TLC solvent system run in the identical direction. The DG
acetate molecular species were also well resolved by C18 high performance liquid chromatography using an acetonitrile, 2-propranol, methyl-5-butyl ether, H20 elution system, 83:10:5:2
(v/v/v/v).
Specificity of the immunoprecipitation procedure was determined by
Western blotting. Immunoprecipitated PKC isotypes were resolubilized
and run on 10% acrylamide gels. Blots which were probed for the
immunoprecipitated isotype were subsequently stripped and reprobed with
another anti-PKC antibody, upon which no positive bands were revealed.
For example, Western blots of immunoprecipitated PKC revealed a
78-kDa band when anti-PKC
, but not when anti-PKC
, was used as a
probe (data not shown). Also, the position of the band for each isotype
corresponded to the correct molecular weight as determined by molecular
weight markers. Furthermore, it has been shown that PKC
isotype-specific peptide immunogens can abolish subsequent
visualization of the PKC isotype by Western analysis.3 We determined that
this procedure precipitated equal masses of each isotype by
visualization of the protein bands on the membranes with Ponceau S
(Sigma).
Initial experiments investigated the DG cofactor requirement for the
immunoprecipitated PKC isotypes. As a control, the kinase assay was
performed in the absence of any immunoprecipitated PKC protein, which
resulted in no observable histone phosphorylation. After
immunoprecipitation of either PKC ,
, or
from treated MC,
in vitro reconstitution assays were run with
phosphatidylserine and either 100 nM (16:0, 18:1)
diacylglycerol or vehicle control. Immunoprecipitated PKC
and
from ET (100 nM)-, but not IL-1 (10 ng/ml)-, stimulated MC
could phosphorylate histone substrate in the absence of exogenous DAG
(data not shown). In the presence of DAG, control-, and IL-1-induced
histone phosphorylation was enhanced but was still significantly below
the level observed from ET-stimulated cells. Supermaximal activation of
immunoprecipitated PKC
and
from ET-treated MC was also observed
when OAG or phorbol 12-myristate 13-acetate substituted for DAG in the
kinase assay. Other laboratories have also noted a supermaximal
stimulation of receptor-activated immunoprecipitated PKC isotypes in
the presence of exogenous DAG or phorbol 12-myristate 13-acetate (15,
16). In contrast to PKC
or
, exogenous diacylglycerol had no
effect upon either control or ET-stimulated PKC
bioactivity. This
confirms numerous reports that activation of PKC
is
DAG-independent. These data suggest, but do not prove, that ET, and not
IL-1, treatment may induce a conformational or post-translational
alteration of PKC
,
, and
, which can be maintained throughout
the immunoprecipitation procedure.
Proliferation of quiescent MC was evaluated by modification of our procedure to assess [3H]thymidine uptake into acid-insoluble DNA (17). Cells were subcultured into 12-well dishes and incubated with RPMI 1640 medium supplemented with 12% bovine serum albumin overnight. Twenty-four hours subsequent to the time of subculture, cells were down-regulated by incubation with serum-free medium for an additional 24 h. Cells were then stimulated with the appropriate agonist and further incubated for an additional 18 h, after which cells were pulsed with 1 µCi of [3H]thymidine/ml of medium. Thymidine incorporation was stopped after 6 h by aspiration of medium and washing the cells twice with ice-cold Dulbecco's phosphate-buffered saline. Cells were fixed for 1 h at 4 °C with 1 ml/well of fixing solution (40:50:10, v/v/v, water:methanol:acetic acid). A solution of 1% SDS (w/v) was applied to each well (0.5 ml/well) for 5 min at 4 °C. The SDS solution was then removed from each well and counted in a liquid scintillation counter.
Statistical AnalysisIndependent t tests were used to establish significant differences between groups. The p value of the individual components was adjusted for multiple comparisons by the Bonferroni method. All data points on the concentration-response curves are expressed as arithmetic means ± S.E. and analyzed by nonlinear curve fitting to logistical equations using the PRISM program (18). The goodness-of-fit for competitive and noncompetitive models were compared by an F test on the ratio of residual variance.
To extend our previous studies, which demonstrate that
IL-1-induced ether-linked DG inhibit total PKC activity (1), we now
investigate if individual PKC isotypes are regulated via DG cofactor
specificity. Utilizing intact cells, we asked if there were any effects
of IL-1 pretreatment on either G-protein-coupled receptor (ET) or
tyrosine kinase receptor-linked (PDGF) activation of novel and atypical
calcium-insensitive immunoprecipitated PKC isotypes. IL-1- pretreatment
diminished both ET- and PDGF-stimulated PKC and
, but not
bioactivity (Fig. 1). In these
experiments, IL-1 was added 5 min before a subsequent 5 min stimulation
with either ET, PDGF, or vehicle. IL-1 had no significant effect, by itself, on any of these PKC isotypes. IL-1 pretreatment abrogated approximately 50% of either the ET- or PDGF-induced activation of PKC
and
. In additional data not shown, coincubation of IL-1 and ET
for 10 min or IL-1 pretreatment for 5 min followed by a 30-min ET
stimulation also significantly reduced PKC
and
activity.
However, a 5- or 30-min incubation with IL-1 subsequent to a 5-min
pretreatment with ET failed to significantly diminish PKC
or
activity (data not shown). At minimum, these studies suggest that a
physiological function of IL-1 is, in part, to inhibit specific PKC
isotypes and that IL-1, by itself, is incapable of stimulating MC PKC
isotypes. At maximum, this inhibition may reflect the generation of
second messengers that bind to, but do not activate, selected PKC
isotypes.
We next asked if an IL-1-induced second messenger, ether-linked DG, can
mimic the effect of IL-1 to inhibit PKC isotypes in a cell-free system.
Immunoprecipitated PKC isotypes from control MC were treated with
exogenous alkenyl, acylglycerol (AAG, consisting primarily of
1-O-palmit-1-ene-yl-2-oleoyl-sn-glycerol))
and/or DAG (1-palmitoyl-2-oleoyl-sn-glycerol) in the kinase
assay buffer (Fig. 2). A dose-response
relationship was determined with concentrations of AAG ranging from
10
6 M to 10
10 M,
which were added to the kinase assay buffer already containing a fixed
concentration of DAG (10
7 M or
10
6 M). Increasing concentrations of AAG
dose-dependently inhibited PKC
and
activated with
0.1 µM DAG. Curve fitting analysis showed that inhibition
of PKC
by AAG was both highly potent (IC50 = 13 ± 2 nM) and effective (Emax = 87 ± 3% reduction in activity). AAG was even more potent against PKC
(IC50 = 1.9 ± 0.6 nM,
Emax = 55 ± 1%). Neither AAG nor DAG
stimulated PKC
activity (data not shown). Also in data not shown,
alkyl, acylglycerol (1-O-palmityl-2-oleoylglycerol)
inhibited activated PKC
and
with a similar dose-response
profile. This suggests that it is most likely the sn-1 ether
or vinyl ether linkage and not the chain length or degree of saturation
that leads to inhibition of PKC isotypes.
To determine whether AAG inhibition was competitive with respect to
DAG, we tested the effect of AAG on PKC activity stimulated with a
10-fold higher concentration of DAG (Fig. 2). The inhibitory effect of
AAG was markedly blunted in the presence of 1.0 µM DAG. These data were analyzed according to either a competitive model (increase in IC50 with no concomitant change in
Emax) or a noncompetitive model (no change in
IC50 with a decrease in Emax). The
competitive model gave the best fit for both PKC and
(F(3,3) = 40 and 39, respectively; p < 0.01). Only the curves obtained in the competitive fit are shown in
Fig. 2. These results show that DAG and AAG interact competitively and
probably act at the same site on PKC. It may be inferred from these
experiments that IL-1-induced ether-linked DG may function as second
messengers to diminish the activity of calcium-insensitive PKC isotypes
by binding to the DAG cofactor domain without leading to activation of
PKC.
We next investigated if the protein expression of PKC isotypes in MC
can be differentially regulated by inflammatory cytokines or vasoactive
peptides. Western blots depicting a time course of PKC ,
, and
expression after ET (10
7 M) and IL-1 (10 ng/ml) treatment are shown in Fig. 3.
There were no significant differences observed in the level of protein
expression at any of the time points for either of the treatments.
PDGF-
(10 ng/ml) treatment also did not significantly alter PKC
,
, or
expression over time (data not shown). In addition,
IL-1 cotreatment with either ET or PDGF did not affect PKC isotype expression (data not shown). The constitutive expression of these PKC
isotypes suggests that regulation of bioactivity by IL-1 is most likely
a post-translational event and not a consequence of translational
regulation or down-regulation by catabolism.
We next investigated growth arrest as a physiological correlate for
IL-1-induced ether-linked DG inhibition of selected PKC isotypes. Since
physiological DG cannot penetrate the plasma membrane of intact cells,
we utilized cell-permeable, sn-2 short chain, DG analogues.
We initially asked if an ether-linked, cell-permeable, DG analogue
could mimic the effect of AAG to reduce PKC activation. In cell-free
systems, 1-palmityl-2-acetylglycerol (PAG) preincubation (5 min)
reduced DAG (1-palmitoyl-2-oleoylglycerol)-stimulated immunoprecipitated PKC and
activity. Specifically, DAG
(10
8 M) activated PKC
and
58 and
56%, respectively. PAG pretreatment reduced DAG stimulated activity to
6 and 4% of vehicle control for PKC
and
, respectively. Thus,
we are confident in using PAG as an ether-linked DG analogue to
potentially demonstrate inhibition of ET- or PDGF-induced
proliferation.
We next asked if either IL-1 or the cell-permeable, ether-linked, DG
analogue can induce growth arrest. Quiescent MC, in the absence of
serum, proliferated in response to both ET and PDGF, but not IL-1,
stimulation (Fig. 4A).
However, IL-1 pretreatment reduced both ET (56%)- and PDGF
(42%)-induced MC proliferation to a level consistent with the
reduction of PKC and
bioactivity by IL-1 pretreatment. We next
asked if the ether-linked DG analogue, PAG, could mimic the effect of
IL-1 to induce growth-arrest. PAG reduced ET-induced proliferation to
basal levels (Fig. 4B). In contrast, the ester-linked DG
analogue, OAG, elevated ET-induced proliferation. Both PAG and OAG did
not by themselves significantly demonstrate any proliferative effect.
As PAG can be metabolized into platelet-activating factor
(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), we utilized a platelet-activating factor receptor blocker (BN52021) to
show that this autacoid does not mediate the inhibitory actions of PAG
upon proliferation (data not shown). These data suggest that
IL-1-generated ether-linked DG can induce a growth-arrested state in
both G-protein-linked receptor and tyrosine kinase receptor-stimulated MC. We conclude that the cell-permeable ether-linked DG analogue, PAG,
not only mimics the actions of physiological (IL-1 receptor-generated) ether-linked DG to inhibit the activation of PKC isotypes, but also
reduces ET- or PDGF-induced proliferation.
We have demonstrated a signaling mechanism by which receptor generation
of unique molecular species of diglycerides differentially regulate the
bioactivity of individual PKC isotypes. Recent reports have also begun
to establish a role for DG cofactor specificity for individual PKC
isotypes. 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
(19). Phosphatidylinositol-derived DG
preferentially containing arachidonate at the sn-2 position are a better cofactor for PKC
compared with PKC
or
, in
contrast to, phosphatidylcholine-derived DG species, which are
equipotent in stimulating PKC isoenzymes (20). Interferon, which like
IL-1, does not elevate [Ca2+]i and forms
phosphatidylcholine-derived DG, translocates PKC
but not
in
Daudi cells (21). Our studies may begin to clarify apparent paradoxes
by demonstrating an acute receptor-mediated mechanism to diminish
activation of selected PKC isotypes.
Even though physiological ether-linked DG species have not been studied as growth-arresting lipids, ether-linked phospholipids and alkyl phosphocholine have recently been identified as antineoplastic, antiproliferative, and apoptosis-inducing agents (22, 23). 1-O-Octadecyl-2-O-methyl-rac-glycerophosphocholine, an alkyllysophospholipid analogue, reduced cell proliferation by decreasing serum- or PDGF-activated MAP kinases (24). Both alkyllysophospholipid and hexadecylphosphocholine reduced membrane-associated and cytosolic total PKC activity (25). 1-O-Dodecyl-sn-glycerol, a monoalkylglycerol, reduced membrane-associated total PKC activity, which correlated with cell culture contact inhibition and resulting growth arrest (26). Also, in agreement with our studies, a cell-permeable ether-linked DG analogue (1-O-hexadecyl-2-acetylglycerol), but not an ester-linked DG analogue (1-oleoyl-2-acetyl-rac-glycerol), inhibited cell growth and induced the differentiation of HL60 human promyelocytic leukemic cells to mononuclear phagocytes (27). Thus, ether-linked DG inhibition of selected PKC isotypes may, in part, be the signaling mechanism by which inflammatory cytokine receptor-generated ether-linked DG induce a growth-arrested cellular phenotype.
Our studies suggest that one of the major physiological outcomes of
IL-1-mediated inhibition of calcium-insensitive PKC activity may be the
induction of a growth-arrested phenotype. Previous studies have also
suggested that inhibition and/or down-regulation of calcium-insensitive
isotypes lead to changes in the cellular phenotype and/or growth
arrest. Down-regulation of PKC or
inhibits G1/S
transition (growth arrest) in vascular smooth muscle cells, an event
consistent with cytokine-induced inflammation (28, 29). Furthermore,
overexpression of PKC
induces tumorgenicity in fibroblasts (30,
31).
Our data indicate that one component of IL-1 signaling is ether-linked
DG inhibition of PKC and
activity. However, other signaling
pathways have been suggested, including the nuclear targeting of a
16-kDa N-terminal IL-1 cleavage product that may function as a
trans-activating factor (32). Alternatively, the carboxyl end of IL-1
bound to the IL-1 receptor also localizes to the nucleus (33). IL-1 has
also been shown to activate upstream kinases for heat shock proteins,
-casein, NF-
B, and cap-binding protein (34-36). Our laboratory
has demonstrated recently that IL-1 activates the stress-activated
protein kinase cascade (also known as jun kinase) via ceramide
formation (17). As IL-1 does not activate ceramidase to form the
putative PKC inhibitor sphingosine (37), we can rule out a role for
IL-1-induced sphingosine to augment ether-linked DG-induced inhibition
of PKC. On the other hand, ceramide inhibits PKC
translocation (38)
and autophosphorylation (39) while inducing PKC
autophosphorylation
(40).
In contrast to PKC and
, PKC
was unaffected by DG, perhaps,
reflecting the lack of a integral cysteine-rich zinc finger domain (9).
Regardless of mechanism, our data suggest that inhibition of activated
PKC
is probably not essential for growth arrest. Even though IL-1
may not affect PKC
bioactivity directly, this inflammatory cytokine
has been shown to induce translocation of PKC
from cytosol to a
presumed membrane compartment in MC in the presence of serum (41) or
after long term pretreatment (42).
In summary, we have demonstrated that IL-1-induced ether-linked DG
competitively inhibit activated calcium-insensitive PKC isotypes, an
event consistent with a proinflammatory, growth-arrested phenotype. DG
cofactor specificity for individual PKC isotypes may offer a more
sensitive and specific mechanism for overall PKC regulation.
Down-regulation of PKC and
by IL-1-generated ether-linked DG
may illustrate one mechanism by which a growth-arrested phenotype in MC
can be induced and/or maintained in models of nonproliferative
immunological renal disease (43).
We thank Michael Simonson for critically reviewing this manuscript.