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Department of Medicine, University of Iowa College of Medicine and Veterans Administration Medical Center, Iowa City, IA 52242
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and this activation
was inhibited by D609. LPS-activated PKC phosphorylated MAP kinase
kinase, the kinase directly upstream of the ERK kinases. LPS-induced
cytokine production (RNA and protein) was also inhibited by D609. As an
aggregate, these studies support the hypothesis that one way by which
LPS activates the ERK kinases is via activation of PC-PLC and that
activation of a PC-PLC is an important component of macrophage
activation by LPS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pathways by which the ERK kinases may become activated have
been studied in a number of cell systems. These kinases are known to be
directly activated by mitogen-activated protein kinase kinase, a kinase
directly upstream of the ERK kinases 15 . The mechanism by which MEK
becomes activated varies, depending on the stimulus and the cell type.
The best-described pathway involves the sequential activation of ras,
raf-1, MEK, and the ERK kinases 15, 19, 20 . Ueda et al. 21
described a ras-independent pathway that requires raf-1 and PKC 
.
Berra et al. 22, 23 used a dominant-negative mutant of PKC to
block serum and TNF activation of the ERK kinases. These studies
suggest that there are multiple pathways by which MEK and the ERK
kinases can be activated.
No studies have determined how LPS activates the ERK kinases, but
several studies provided clues that have led to our hypothesis. A study
by Grove et al. 24 showed that LPS activated a
phosphatidylcholine-specific phospholipase C (PC-PLC) in macrophages.
Several studies using other stimuli have linked activation of a PC-PLC
to activation of the ERK kinases in other types of cells 25, 26, 27 .
Potentially, downstream of PC-PLC, ceramide may serve as an activator
of PKC . This has been described in a study by Muller et al. 28 ,
which used TNF as a stimulus. Collectively, these observations led us
to hypothesize that LPS-induced activation of ERK kinases in alveolar
macrophages would be mediated by sequential activation of PC-PLC,
induction of cellular diacylglycerol (DAG) and ceramide, and activation
of PKC . We further hypothesized that activation of a PC-PLC would
play an important role in the production of cytokines by LPS-treated
alveolar macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alveolar macrophages were obtained from bronchoalveolar lavage as previously described 29 . Briefly, normal volunteers with a lifetime nonsmoking history, no acute or chronic illness, and no current medications underwent bronchoalveolar lavage. The lavage procedure used five 25-ml aliquots of sterile, warmed saline in each of three segments of the lung. The lavage fluid was filtered through two layers of gauze and centrifuged at 1500 x g for 5 min. The cell pellet was washed twice in HBSS without Ca2+ and Mg2+ and suspended in complete medium, RPMI tissue culture medium (Life Technologies, Gaithersburg, MD) with 5% FCS (HyClone, Logan, UT), and added gentamicin (80 µg/ml). Differential cell counts were determined using a Wright-Giemsa-stained cytocentrifuge preparation. All cell preparations had between 90 and 100% alveolar macrophages. This study was approved by the Committee for Investigations Involving Human Subjects at the University of Iowa.
Immunoprecipitation of ERK kinase and PKC
Alveolar macrophages were cultured in complete medium with or
without LPS (1 µg/ml, Sigma, St. Louis, MO), and/or
tricyclodecan-9-yl xanthogenate (D609, 100 µM, Calbiochem, San Diego,
CA) a relatively specific inhibitor of PC-PLC, 30, 31, 32 . In some
instances, cells were also exposed to C2 ceramide (16 µM,
Biomol, Plymouth Meeting, MA) or bacterial-derived sphingomyelinase
(0.4 U/ml, Biomol). After culture, cells were lysed on ice for 20 min
in 500 µl of lysis buffer (0.05 M Tris (pH 7.4), 0.15 M NaCl, 1%
Nonidet P-40, 0.5 M PMSF, 50 µg/ml aprotinin, 10 µg/ml leupeptin,
50 µg/ml pepstatin, 0.4 mM sodium o-vanadate, 10 mM sodium
fluoride, and 10 mM sodium pyrophosphate, all from Boehringer Mannheim,
Indianapolis, IN). The lysates were then spun at 15,000 x
g for 10 min, and the supernatant was saved. Protein was
measured, and 600 µg from each sample were removed for
immunoprecipitation. The samples were cleared by incubation for 2
h with 1 µg/sample rabbit IgG and 10 µl/sample GammaBind Sepharose
(Pharmacia, Piscataway, NJ). After centrifugation, the supernatants
were transferred to a tube containing 3 µg/sample rabbit anti-ERK
Ab (sc-154, Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit
anti-PKC b (sc-216, Santa Cruz Biotechnology) bound to
GammaBind Sepharose and rotated at 4°C overnight. The beads were
subsequently washed three times with high salt buffer (0.05 M Tris (pH
7.4), 0.50 M NaCl, and 1% Nonidet P-40) and three times with lysis
buffer without protease inhibitors. The ERK or PKC complexes were
either released with 2x sample buffer for Western analysis or used to
determine kinase activity.
ERK and PKC kinase activity
After immunoprecipitation of ERK or PKC from alveolar
macrophages, the protein-containing pellet was washed twice with kinase
buffer (20 mM MgCl2, 25 mM HEPES, 20 mM
ß-glycerophosphate, 20 mM p-nitrophenylphosphate, 20 mM
sodium o-vanadate, and 2 mM DTT). The pellet was then
suspended in 20 µl of kinase buffer, and the following were added: 20
µM ATP, 5 µCi of [
-32P]ATP (BLU 002Z, DuPont/NEN,
Boston, MA), and 10 µg of myelin basic protein (MBP, Sigma) or in one
instance, 8 µg of MEK-1 (55-kDa polyhistidine-tagged fusion protein,
Santa Cruz Biotechnology). The reaction was continued for 15 min (ERK
kinase) or 30 min (PKC ) at 25°C and then stopped by the addition
of 40 µl/sample of 2x sample buffer. The samples were boiled for 5
min and run on a 10% SDS-PAGE gel. The gel was dried, and
autoradiography was performed to visualize the 32P-labeled
MBP or MEK-1. Densitometry was performed on films, and fold increase
was calculated as experimental sample/control sample.
Western analysis
To evaluate equal loading of the protein in the kinase activity
assay, we performed Western analysis on a portion of the
immunoprecipitated kinase. A 15% portion of the total sample was mixed
1:1 with 2x sample buffer (20% glycerol, 4% SDS, 10% 2-ME, 0.05%
bromophenol blue, and 1.25 M Tris, pH 6.8; all chemicals from Sigma),
loaded onto a 10% SDS-PAGE gel, and run at 30 mA for 3 h. Cell
proteins were transferred to nitrocellulose (ECL, Amersham, Arlington
Heights, IL) overnight at 30 V. The nitrocellulose was then blocked
with 5% milk in Tris-buffered saline with 0.1% Tween 20 (TTBS) for
1 h, washed, and then incubated with the primary Ab (1:1000
dilution, anti-ERK or anti-PKC from Santa Cruz
Biotechnology) for 1 h. The blots were washed four times with TTBS
and incubated for 1 h with horseradish peroxidase-conjugated
anti-rabbit IgG Ab (Amersham, at 1/5000 dilution). Immunoreactive
bands were developed using a chemiluminescent substrate (ECL,
Amersham). Autoradiography was performed, with exposure times of 10
s–2 min, which were adequate for visualization.
Detection of ceramide and DAG
The ceramide and DAG mass were evaluated by a slight
modification of the procedure as described by Preiss et al. 33 .
Lipids were extracted from alveolar macrophages as described, after
exposure to medium alone (control), LPS (1 µg/ml), or LPS and D609
(100 µM); dried under nitrogen gas; and solubilized in an aliquot of
octyl-ß-D-glucoside-cardiolipin solution. The lipid
mixture contained 31 µl of 3.19 µM cardiolipin, 10 µl of a 15%
octy-ß-D-glucoside solution, and 20 µl of 1 mM DETAPAC
(all from Sigma). After drying, 20 µl of DETAPAC were added, and the
solution was sonicated briefly in a bath sonicator. Fifty microliters
of 2x reaction buffer (100 mM imidazole, 100 mM NaCl, 25 mM
MgCl2, 2 mM EGTA, pH 6.6), 10 µl of 20 mM DTT, and 10
µl of diluted diacylglycerol kinase membranes were then added to the
assay mixture. The reaction was initiated by the addition of 10 µl of
a [-32P]ATP solution. The remainder of the procedure
was performed as described 23 . The DAG kinase phosphorylates both
ceramide and DAG, converting the lipids to ceramide phosphate and
phosphatidic acid (PA), respectively. These species were resolved using
TLC (ceramide phosphate (Rf = 0.14) and DAG
(Rf of PA = 0.35)). The films were analyzed
with densitometry, and fold increase was calculated as experimental
sample/control sample.
Detection of DAG in phosphatidylcholine-labeled cells
To evaluate DAG in labeled cells, the following methods were utilized. Alveolar macrophages were cultured overnight in complete medium with added [1-14C]arachidonic acid (NEC661, Dupont/NEN), 0.1 µCi/107 cells, or L-1-palmitoyl-1-14C]lysopalmitoylphosphatidylcholine (lyso-PC, NEC683, DuPont/NEN), 0.1 µCi/107 cells. The cells were then washed with PBS and resuspended at 107 cells/ml of complete medium and placed at 1 ml/tube in 1.5-ml microtubes. After stimulation with LPS (1 µg/ml) or LPS and D609 (100 µM) for 5 min, lipids were extracted using the Bligh-Dyer method 34 . After extraction, samples were dried under nitrogen gas and resuspended in 50 µl/sample chloroform-methanol (2:1). Samples were then spotted on a TLC plate, (LK 5D, Whatman, Clifton, NJ) and run in a solvent of hexane-diethyl ether-acetic acid (60:40:1). The DAG bands were found by comparison with the position of a known standard (1,2-dioleolyl-sn-glycerol, C18:1, cis-9, Sigma). Quantitation was done, by analyzing the TLC plate on an AMBIS radioisotope scanner (Scanalytics, Billerica, MA). Data are presented as fold increase, experimental sample/control sample. To confirm that the palmitoyl-1-14C lyso-PC label remained in the phosphatidylcholine (PC) fraction during the 24-h labeling incubation, we evaluated the distribution of the label immediately before the LPS stimulation. Alveolar macrophages were labeled overnight with palmitoyl-1-14C lyso-PC, and then lipids extracted using the Bligh-Dyer method. The major phospholipids (PC, sphingomyelinase, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol) were resolved using TLC in chloroform-methanol-acetic acid-water-acetone (40:25:4:2:4). The phospholipids that comigrated with known standards were scraped, and counts were determined using a liquid scintillation counter.
Expression of cytokine mRNA
Whole-cell RNA was isolated using RNA Stat-60 according to the
manufacturer’s instructions (Tel-Test "B," Friendswood, TX). Cells
were lysed in RNA Stat-60 solution, containing phenol and guanidinium
thiocyanate. The mixture was then shaken vigorously, allowed to sit for
2–3 min, and then centrifuged at 12,000 rpm for 15 min at 4°C. The
RNA was removed from the upper aqueous layer, precipitated with
isopropanol for 30 min, centrifuged at 12,000 rpm for 10 min at 4°C,
and washed with 75% ethanol. The isolated RNA was fractionated in a
1.5% denaturing agarose gel containing 2.2 M formaldehyde. An RNA
ladder (0.24–9.5 kb; Life Technologies) was included as a molecular
size standard. RNA loading was confirmed by equivalent ethidium bromide
staining in each lane. The RNAs were transferred to GeneScreen Plus
(DuPont/NEN) as suggested by the manufacturer. IL-6 and TNF cDNA probes
(generated by PCR with primers obtained from Clontech, Palo Alto, CA)
were labeled with [-32P]CTP (DuPont/NEN) by random
primer method. Blots were prehybridized for 3 h at 42°C (10 ml
formamide, 5 mM NaCl, 4 ml 50% dextran, 10% SDS, 1 M Tris (pH 7.0),
and 0.4 ml of 50x Denhardt’s solution) and then hybridized with the
labeled probe overnight at 42°C. The filters were washed twice with
1x SSC at 25°C, twice with 1x SSC plus 1% SDS at 65°C, and then
once with 0.1x SSC at 25°C. The filters were exposed to
autoradiographic film at -70°C.
Release of cytokines
For these studies, alveolar macrophages were cultured in RPMI medium with 5% FCS for 24 h in the presence or absence of LPS and with and without D609, as described above. After the culture period, the supernatants were harvested and stored at -70° until assayed. The amounts of IL-6 and TNF in the supernatant of the cells were measured by ELISA (R&D Systems, Minneapolis, MN).
Statistical analysis
All of the cytokine measurements and densitometry data are shown as means ± SEM. Statistical comparisons were performed using a paired t test with a probability value of p < 0.05 considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although prior studies showed that LPS activates ERK kinases in
murine macrophages and human monocytes 9, 10, 12 , no studies have
shown that LPS activates these kinases in human alveolar macrophages.
Thus, we first showed that LPS activates the ERK 2 MAP kinase in
alveolar macrophages (Fig. 1
).
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A). A Western blot shows equal loading of the kinase in the
activity assay. Fig. 1B shows composite data from three
separate experiments. These data suggest that PC-PLC activation is an
important step in the up-regulation of ERK activity in LPS-treated
alveolar macrophages. To further confirm that PC-PLC was linked to ERK
2 activation, we evaluated whether phospholipase D (PLD) was linked to
ERK 2 activation. To evaluate this, we first determined whether
propranolol, an inhibitor of PA phosphohydrolase, blocked ERK 2
activation 35 . Propranolol did not inhibit LPS-induced ERK 2
activation (Fig. 2). This observation
suggests that PLD-derived DAG is not linked to ERK 2 activation. We
also used 2,3-bis-diphosphoglycerate, an inhibitor of PLD 36 . This
compound also did not inhibit LPS-induced ERK 2 activation (data not
shown). This observation suggests that neither activation of PLD nor
PLD-derived PA is linked to LPS-induced ERK 2 activation.
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We next determined whether LPS stimulates PC-PLC activity and
whether this effect is inhibited by D609. We first determined whether
LPS increased DAG mass and whether this increase could be blocked by
D609. Alveolar macrophages were cultured with or without LPS (1
µg/ml) for 1–5 min and with and without D609 (100 µM) which was
added to the cells 30 min before LPS. The cells were harvested, and DAG
mass was determined. LPS increased DAG by 1 min after LPS exposure,
with a significantly greater increase at 5 min (Fig. 3). The LPS-induced increase in DAG was
inhibited by D609. These data show that LPS triggers release of DAG in
alveolar macrophages and that DAG might be derived from PC.
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). LPS increased amounts of DAG,
and the DAG was derived, at least in part, from PC because increases in
labeled DAG were found after using the lyso-PC label. The amount of DAG
detected using this label was similar to that detected using
[14C]arachidonic acid, as a label. We next determined the
distribution of [14C]lyso-PC into major phospholipids. We
observed that the majority of the label incorporated into PC and, to a
lesser extent, sphingomyelinase; only a small percentage of the label
was found in the other phospholipids (Fig. 5). The amount of the label in the
phospholipids other than PC is unlikely to account for the increases
seen in DAG, especially considering that PC is the major phospholipid
found in eukaryotic cells. Thus, these results, taken together, suggest
that the majority of DAG is derived from PC.
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Activation of PC-PLC can be linked to activation of
sphingomyelinase activity 37, 38 . To evaluate this, alveolar
macrophages were cultured with medium alone (control) or with LPS (1
µg/ml) for 30 s–30 min. The cells were then harvested, and the
amount of ceramide in cell lysates was determined. LPS increased
amounts of ceramide in alveolar macrophages as early as 30 s after
stimulation with a return to baseline by 30 min after stimulation (Fig. 6). We next determined whether the LPS
induction of ceramide was linked to activation of PC-PLC. Alveolar
macrophages were incubated with and without D609 (100 µM, for 30 min)
and then stimulated with LPS (1 µg/ml) for 1 or 5 min. The cells were
then harvested, and amounts of ceramide were evaluated in cell lysates.
D609 blocked the LPS-induced increase in ceramide to below baseline
levels (Fig. 7). These data suggest that
LPS activates a sphingomyelinase pathway in alveolar macrophages in a
PC-PLC-dependent manner.
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To determine whether products of the sphingomyelin/ceramide
pathway could induce ERK kinase activity in alveolar macrophages, the
cells were cultured with medium alone (control), or with C2
ceramide (16 µM), or with bacterial-derived sphingomyelinase (0.4
U/ml) for 5 min or for 1 h. Both C2 ceramide and
bacterial-derived sphingomyelinase triggered activation of the ERK
kinase by 5 min (Fig. 8). This kinase
activity was decreased by 1 h. Shown for comparison are the
effects of LPS, which has a more prolonged time course. These studies
show that components of the sphingomyelinase pathway can activate ERK
kinases in alveolar macrophages.
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). This observation suggests that
activation of PC-PLC is a proximal event to generation of ceramide.
These data also show that D609 does not have a nonspecific effect on
ERK 2 activation.
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in alveolar macrophages, and this activation
is blocked by D609
One means by which ceramide might activate the ERK kinases is via
activation of an atypical PKC isoform ( or 
) 39 . In previous
studies, we were able to detect PKC but not PKC in alveolar
macrophages 40 . To determine activation of PKC , we cultured
alveolar macrophages with and without LPS (1 µg/ml) and with and
without D609 (100 µM). After 15 min, the cells were harvested, and
PKC was immunoprecipitated from the cell lysates. Fig. 10A shows two different
kinase activity assays, one using MBP as a substrate and the other
using MEK as a substrate. We also looked at PKC autophosphorylation
with similar results (data not shown). Fig. 10B shows the
composite data from three separate experiments, which use MBP as a
substrate. These studies show that LPS causes activation of PKC and
that this activation is blocked by D609. These experiments also show
that LPS-activated PKC can phosphorylate the cellular substrate
MEK, a kinase directly upstream of the ERK kinases.
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To determine whether D609 would interfere with a macrophage
function that can be tied to ERK kinase activation 41 , we evaluated
the effect of D609 on cytokine release and mRNA production in
LPS-treated alveolar macrophages. IL-6 and TNF are both proinflammatory
cytokines that are released from LPS-activated alveolar macrophages. We
evaluated the effect of D609 on IL-6 and TNF mRNA and protein release.
LPS increased both IL-6 and TNF mRNAs and protein release, and these
were blocked by D609 (Figs. 11 and
12). These studies show that activation
of a PC-PLC can be linked to a physiologically relevant macrophage
function (cytokine production after LPS exposure).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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.
PKC activates MEK, which subsequently leads to ERK kinase
activation. To test this hypothesis, we showed that LPS activates the
ERK 2 kinase in alveolar macrophages and that this activation is
inhibited by D609, a relatively specific inhibitor of PC-PLC. We next
showed that LPS increases amounts of DAG and ceramide and that both of
these effects of LPS are inhibited by D609. Our present studies
indicate that LPS induction of DAG in alveolar macrophages is derived,
at least in part, from a PC-containing phospholipid. Thus, we conclude
from these experiments that the LPS-induced DAG is derived from
hydrolysis of PC via activation of PC-PLC. In separate studies, the
addition of exogenous C2 ceramide or bacterial-derived
sphingomyelinase activated ERK 2 kinase in alveolar macrophages, and
this activation was not inhibited by D609. Finally, we showed that LPS
activates PKC , and that PKC from LPS-treated cells can activate
MEK. The LPS-induced activation of PKC also was inhibited by D609.
Collectively, these studies strongly suggest that LPS activates the ERK
kinases in alveolar macrophages via sequential activation of a PC-PLC,
sphingomyelin hydrolysis, and PKC . We have not proved that this is
the only pathway by which LPS activates ERK kinases, but our studies
strongly support the hypothesis that this is an important effector
mechanism by which LPS activates ERK kinases in alveolar macrophages.
We further show that LPS activation of a PC-PLC is linked to important
macrophage functions, because inhibition of PC-PLC interferes with
cytokine production in LPS-treated alveolar macrophages. Several studies support the idea that hydrolysis of PC is coupled to activation of ERK kinases. Cai et al., using NIH 3T3 cells, have shown that the addition of exogenous PC-PLC induces phosphorylation of MEK, which subsequently activates ERK kinases 25 . In fibroblasts (Rat-1), van Dijk et al. 26 have shown that stimulation of PC-PLC activity results in activation of the ERK kinases. Work by Sands et al. 42 suggests that PC hydrolysis might be a relevant signaling pathway in endotoxin-exposed macrophages. Using murine macrophages, LPS treatment increased DAG, which was due to activation of PC-PLC. These studies are consistent with our finding that LPS activates ERK kinases in alveolar macrophages via an activation of PC-PLC.
Our observation that LPS activation of a sphingomyelinase pathway by
PC-PLC-generated DAG is supported by studies evaluating TNF and IL-1
signaling in a variety of cell types (reviewed by Kolesnick and Golde
43). Several studies have linked generation of DAG to activation of a
sphingomyelinase activity and subsequent ceramide generation. Of
special relevance to our studies are the observations of Lozano et al.
39 and Muller et al. 28 which reported that the addition of
exogenous sphingomyelinase to cells activates PKC (Lozano), and
that PKC is activated by ceramide (Muller). These studies are
consistent with our findings for both ceramide generation and PKC
activation in LPS-treated alveolar macrophages.
These results are also in agreement with prior studies, which link
PC-PLC to PKC and ERK kinase activation. Bjorkoy et al. 44 have
shown in NIH 3T3 cells that PKC activation is a necessary
downstream component of PC-PLC cell transformation. A study by van Dijk
et al. 26 used an inhibitor of PKC (Ro31-8220) to block
PC-PLC-induced MAP kinase activation. They showed that expression of a
dominant negative mutant of PKC , unlike down-regulation of other
PKC isoforms (
, , and 
), inhibited the MAP kinase activation
by PC-PLC. Berra et al. 23 also showed that a dominant negative
mutant of PKC dramatically impaired the activation of both MEK and
MAP kinase in serum- and TNF-treated COS cells. These studies are
consistent with our finding that PKC plays a central role in ERK
kinase activation by LPS in alveolar macrophages. DAG, phosphatidic
acid, or ceramide have all been shown to stimulate PKC in vitro
26, 28, 45 . The observation that neither propranolol nor
2,3-bis-diphosphoglycerate inhibits LPS-induced ERK 2 activation argues
that PA is of little importance in this system. In addition, the
observation that ceramide inhibits DAG kinase suggests that
PC-PLC-derived PA might be limiting and therefore of lesser importance
in LPS induction of PKC activity 46 .
Aside from LPS activation of MEK via PKC , a number of studies
suggest a role for Raf-1 in ERK kinase activation. One possible
parallel pathway could include LPS
PC-PLCDAGnovel PKC
isoformraf-1MEKERK. This pathway is suggested in studies where
dominant-negative mutants of raf-1 inhibited signaling through PC-PLC
25 . Other studies demonstrate that LPS causes phosphorylation and
activation of Raf-1, which was temporally associated with activation of
MEK and ERK 12 . These data suggest that LPS also could activate ERK
kinases through a raf-1-dependent pathway.
To our knowledge, prior studies have not demonstrated that LPS
induces ceramide, which was observed in our studies to be PC-PLC
dependent and capable of activating the ERK kinase pathway. Ceramide
can also directly activate raf-1 47 . Thus, our data suggest a third
possible pathway: LPSPC-PLCDAGceramideraf-1MEKERK. A
recent paper by Luberto et al. 48 suggests a related possibility for
the sequence of events involved in LPS signaling. This study suggests
that PC-PLC might also function as a sphingomyelin synthase and/or that
D609 cannot distinguish between the enzymatic activities of
sphingomyelin synthase and PC-PLC. Thus, it is possible that activation
of PKC and ERK 2 could result from activation of a sphingomyelin
synthase rather than a PC-PLC. Thus, further studies are necessary to
determine whether LPS-induced activation of ERK 2 is linked to a PC-PLC
or a sphingomyelin synthase. A novel conclusion of these studies is
that activation of a PC-PLC or a sphingomyelin synthase by LPS is
critical for activation of the ERK kinases. The importance of this
observation is strengthened by the recent observations of Machleidt et
al. 49 , who showed that inhibition of PC-PLC by D609 markedly
diminished the manifestations of sepsis (endotoxin) in an animal model
of sepsis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Martha M. Monick, Room 100 EMRB, The University of Iowa Hospitals and Clinics, Iowa City, IA 52242. E-mail address:
3 Abbreviations used in this paper: ARDS, adult respiratory distress syndrome; MAP, mitogen-activated protein; ERK kinases, p42/44 kinases; MEK, mitogen-activated protein kinase kinase; PKC protein kinase C; PC-PLC, phosphatidylcholine-specific phospholipase C; D609, tricyclodecan-9-yl xanthogenate; DAG, diacylglycerol; MBP, myelin basic protein; PA, phosphatidic acid; lyso-PC, L-1-palmitoyl-1-14C]lysopalmitoylphosphatidylcholine; PC, phosphatidylcholine; PLD, phospholipase D.
Received for publication June 8, 1998. Accepted for publication December 4, 1998.
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References |
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is a molecular switch in signal transduction of TNF-, bifunctionally regulated by ceramide and arachidonic acid. EMBO J. 14:1961.[Medline]
B by phosphatidylcholine-specific phospholipase C induced "acidic" sphingomyelin breakdown. Cell 71:765.[Medline]
isoform is critical for B-dependent promoter activation by sphingomyelinase. J. Biol. Chem. 269:19200. and LPS. Biochem. Biophys. Res. Commun. 199:461.[Medline]
overexpressed in COS cells: comparison with other protein kinase C isotypes and other acidic lipids. Biochem. J. 304:1001.
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 J. Cuschieri, J. Billgren, and R. V. Maier Phosphatidylcholine-specific phospholipase C (PC-PLC) is required for LPS-mediated macrophage activation through CD14 J. Leukoc. Biol., August 1, 2006; 80(2): 407 - 414. [Abstract] [Full Text] [PDF]
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M. Yadav, L. Clark, and J. S. Schorey Macrophage's Proinflammatory Response to a Mycobacterial Infection Is Dependent on Sphingosine Kinase-Mediated Activation of Phosphatidylinositol Phospholipase C, Protein Kinase C, ERK1/2, and Phosphatidylinositol 3-Kinase J. Immunol., May 1, 2006; 176(9): 5494 - 5503. [Abstract] [Full Text] [PDF]
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 N. V. Giltiay, A. A. Karakashian, A. P. Alimov, S. Ligthle, and M. N. Nikolova-Karakashian Ceramide- and ERK-dependent pathway for the activation of CCAAT/enhancer binding protein by interleukin-1{beta} in hepatocytes J. Lipid Res., November 1, 2005; 46(11): 2497 - 2505. [Abstract] [Full Text] [PDF]
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 A. P Albert, A. S Piper, and W. A Large Role of phospholipase D and diacylglycerol in activating constitutive TRPC-like cation channels in rabbit ear artery myocytes J. Physiol., August 1, 2005; 566(3): 769 - 780. [Abstract] [Full Text] [PDF]
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S. Falcone, C. Perrotta, C. De Palma, A. Pisconti, C. Sciorati, A. Capobianco, P. Rovere-Querini, A. A. Manfredi, and E. Clementi Activation of Acid Sphingomyelinase and Its Inhibition by the Nitric Oxide/Cyclic Guanosine 3',5'-Monophosphate Pathway: Key Events in Escherichia coli-Elicited Apoptosis of Dendritic Cells J. Immunol., October 1, 2004; 173(7): 4452 - 4463. [Abstract] [Full Text] [PDF]
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J. F. Alcorn and J. R. Wright Surfactant protein A inhibits alveolar macrophage cytokine production by CD14-independent pathway Am J Physiol Lung Cell Mol Physiol, January 1, 2004; 286(1): L129 - L136. [Abstract] [Full Text] [PDF]
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M. M. Monick, T. O. Yarovinsky, L. S. Powers, N. S. Butler, A. B. Carter, G. Gudmundsson, and G. W. Hunninghake Respiratory Syncytial Virus Up-regulates TLR4 and Sensitizes Airway Epithelial Cells to Endotoxin J. Biol. Chem., December 26, 2003; 278(52): 53035 - 53044. [Abstract] [Full Text] [PDF]
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 S. D. Savkovic, A. Koutsouris, and G. Hecht PKC{zeta} participates in activation of inflammatory response induced by enteropathogenic E. coli Am J Physiol Cell Physiol, September 1, 2003; 285(3): C512 - C521. [Abstract] [Full Text] [PDF]
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M. M. Monick, L. S. Powers, N. S. Butler, and G. W. Hunninghake Inhibition of Rho Family GTPases Results in Increased TNF-{alpha} Production After Lipopolysaccharide Exposure J. Immunol., September 1, 2003; 171(5): 2625 - 2630. [Abstract] [Full Text] [PDF]
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 J Berkes, V K Viswanathan, S D Savkovic, and G Hecht Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation Gut, March 1, 2003; 52(3): 439 - 451. [Abstract] [Full Text] [PDF]
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 A. Castrillo, P. G. Traves, P. Martin-Sanz, S. Parkinson, P. J. Parker, and L. Bosca Potentiation of Protein Kinase C {zeta} Activity by 15-Deoxy-{Delta}12,14-Prostaglandin J2 Induces an Imbalance between Mitogen-Activated Protein Kinases and NF-{kappa}B That Promotes Apoptosis in Macrophages Mol. Cell. Biol., February 15, 2003; 23(4): 1196 - 1208. [Abstract] [Full Text] [PDF]
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M. M. Monick, L. Powers, N. Butler, T. Yarovinsky, and G. W. Hunninghake Interaction of matrix with integrin receptors is required for optimal LPS-induced MAP kinase activation Am J Physiol Lung Cell Mol Physiol, August 1, 2002; 283(2): L390 - L402. [Abstract] [Full Text] [PDF]
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 M.M. Monick and G.W. Hunninghake Activation of second messenger pathways in alveolar macrophages by endotoxin Eur. Respir. J., July 1, 2002; 20(1): 210 - 222. [Abstract] [Full Text] [PDF]
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 M. Takeda, A. Takamiya, A. Yoshida, and H. Kiyama Extracellular Signal-Regulated Kinase Activation Predominantly in Muller Cells of Retina with Endotoxin-Induced Uveitis Invest. Ophthalmol. Vis. Sci., April 1, 2002; 43(4): 907 - 911. [Abstract] [Full Text] [PDF]
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 J. Giron-Calle, K. Srivatsa, and H. J. Forman Priming of Alveolar Macrophage Respiratory Burst by H2O2 Is Prevented by Phosphatidylcholine-Specific Phospholipase C Inhibitor Tricyclodecan-9-yl-xanthate (D609) J. Pharmacol. Exp. Ther., April 1, 2002; 301(1): 87 - 94. [Abstract] [Full Text] [PDF]
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Y. Ouadrhiri, C. Pilette, R. C. Monteiro, J.-P. Vaerman, and Y. Sibille Effect of IgA on Respiratory Burst and Cytokine Release by Human Alveolar Macrophages . Role of ERK1/2 Mitogen-Activated Protein Kinases and NF-kappa B Am. J. Respir. Cell Mol. Biol., March 1, 2002; 26(3): 315 - 332. [Abstract] [Full Text] [PDF]
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M. M. Monick, R. K. Mallampalli, A. B. Carter, D. M. Flaherty, D. McCoy, P. K. Robeff, M. W. Peterson, and G. W. Hunninghake Ceramide Regulates Lipopolysaccharide-Induced Phosphatidylinositol 3-Kinase and Akt Activity in Human Alveolar Macrophages J. Immunol., November 15, 2001; 167(10): 5977 - 5985. [Abstract] [Full Text] [PDF]
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A. Castrillo, D. J. Pennington, F. Otto, P. J. Parker, M. J. Owen, and L. Bosca Protein Kinase C{epsilon} Is Required for Macrophage Activation and Defense Against Bacterial Infection J. Exp. Med., October 29, 2001; 194(9): 1231 - 1242. [Abstract] [Full Text] [PDF]
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D. Zhou, C. M. Lauderback, T. Yu, S. A. Brown, D. A. Butterfield, and J. S. Thompson D609 Inhibits Ionizing Radiation-Induced Oxidative Damage by Acting as a Potent Antioxidant J. Pharmacol. Exp. Ther., July 1, 2001; 298(1): 103 - 109. [Abstract] [Full Text]
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B. W. Jones, T. K. Means, K. A. Heldwein, M. A. Keen, P. J. Hill, J. T. Belisle, and M. J. Fenton Different Toll-like receptor agonists induce distinct macrophage responses J. Leukoc. Biol., June 1, 2001; 69(6): 1036 - 1044. [Abstract] [Full Text] [PDF]
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F. Zhang, G. Zhao, and Z. Dong Phosphatidylcholine-specific phospholipase C regulates activation of RAW264.7 macrophage-like cells by lipopeptide JBT3002 J. Leukoc. Biol., June 1, 2001; 69(6): 1060 - 1066. [Abstract] [Full Text] [PDF]
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Y.-W. Hsu, K.-H. Chi, W.-C. Huang, and W.-W. Lin Ceramide Inhibits Lipopolysaccharide-Mediated Nitric Oxide Synthase and Cyclooxygenase-2 Induction in Macrophages: Effects on Protein Kinases and Transcription Factors J. Immunol., May 1, 2001; 166(9): 5388 - 5397. [Abstract] [Full Text] [PDF]
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 A. Gomez, A. Mve-Obiang, B. Vray, W. Rudnicka, I. C. Shamputa, F. Portaels, W. M. Meyers, P.-A. Fonteyne, and L. Realini Detection of Phospholipase C in Nontuberculous Mycobacteria and Its Possible Role in Hemolytic Activity J. Clin. Microbiol., April 1, 2001; 39(4): 1396 - 1401. [Abstract] [Full Text]
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 T. Krakauer Suppression of Endotoxin- and Staphylococcal Exotoxin-Induced Cytokines and Chemokines by a Phospholipase C Inhibitor in Human Peripheral Blood Mononuclear Cells Clin. Vaccine Immunol., March 1, 2001; 8(2): 449 - 453. [Abstract] [Full Text] [PDF]
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M. T. Diaz-Meco and J. Moscat MEK5, a New Target of the Atypical Protein Kinase C Isoforms in Mitogenic Signaling Mol. Cell. Biol., February 15, 2001; 21(4): 1218 - 1227. [Abstract] [Full Text]
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M. M. Monick, J. M. Staber, K. W. Thomas, and G. W. Hunninghake Respiratory Syncytial Virus Infection Results in Activation of Multiple Protein Kinase C Isoforms Leading to Activation of Mitogen-Activated Protein Kinase J. Immunol., February 15, 2001; 166(4): 2681 - 2687. [Abstract] [Full Text] [PDF]
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K. M. Krishna Rao MAP kinase activation in macrophages J. Leukoc. Biol., January 1, 2001; 69(1): 3 - 10. [Abstract] [Full Text]
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M. M. Monick, A. B. Carter, D. M. Flaherty, M. W. Peterson, and G. W. Hunninghake Protein Kinase C {zeta} Plays a Central Role in Activation of the p42/44 Mitogen-Activated Protein Kinase by Endotoxin in Alveolar Macrophages J. Immunol., October 15, 2000; 165(8): 4632 - 4639. [Abstract] [Full Text] [PDF]
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 K. J. Procyk, M. R. Rippo, R. Testi, F. Hofmann, P. J. Parker, and M. Baccarini Lipopolysaccharide induces Jun N-terminal kinase activation in macrophages by a novel Cdc42/Rac-independent pathway involving sequential activation of protein kinase C zeta and phosphatidylcholine-dependent phospholipase C Blood, October 1, 2000; 96(7): 2592 - 2598. [Abstract] [Full Text] [PDF]
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K. C. Corbit, J.-W. Soh, K. Yoshida, E. M. Eves, I. B. Weinstein, and M. R. Rosner Different Protein Kinase C Isoforms Determine Growth Factor Specificity in Neuronal Cells Mol. Cell. Biol., August 1, 2000; 20(15): 5392 - 5403. [Abstract] [Full Text]
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 A. Gomez, A. Mve-Obiang, B. Vray, J. Remacle, K. Chemlal, W. M. Meyers, F. Portaels, and P.-A. Fonteyne Biochemical and Genetic Evidence for Phospholipase C Activity in Mycobacterium ulcerans Infect. Immun., May 1, 2000; 68(5): 2995 - 2997. [Abstract] [Full Text] [PDF]
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A. B. Carter, K. L. Knudtson, M. M. Monick, and G. W. Hunninghake The p38 Mitogen-activated Protein Kinase Is Required for NF-kappa B-dependent Gene Expression. THE ROLE OF TATA-BINDING PROTEIN (TBP) J. Biol. Chem., October 22, 1999; 274(43): 30858 - 30863. [Abstract] [Full Text] [PDF]
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A. B. Carter and G. W. Hunninghake A Constitutive Active MEK right-arrow ERK Pathway Negatively Regulates NF-kappa B-dependent Gene Expression by Modulating TATA-binding Protein Phosphorylation J. Biol. Chem., September 1, 2000; 275(36): 27858 - 27864. [Abstract] [Full Text] [PDF]
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A. B. Carter, L. A. Tephly, and G. W. Hunninghake The Absence of Activator Protein 1-dependent Gene Expression in THP-1 Macrophages Stimulated with Phorbol Esters Is Due to Lack of p38 Mitogen-activated Protein Kinase Activation J. Biol. Chem., August 31, 2001; 276(36): 33826 - 33832. [Abstract] [Full Text] [PDF]
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