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, -ßI, or -
But Not -
Inhibit Lipopolysaccharide-Induced Nitric Oxide Synthase Expression in RAW 264.7 Macrophages: Involvement of a Nuclear Factor 
B-Dependent Mechanism1
Institutes of
*
Pharmacology and
Medical Technology, College of Medicine, National Taiwan University, Taipei, Taiwan
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, -ßI,
-
, -
, and -
in RAW cells; down-regulation of PKC-, -ßI,
and -, but not -, was seen after long-term TPA treatment,
indicating the possible involvement of one or all of PKC-, -ßI,
and -, but not -, in LPS-mediated effects. Treatment with
antisense oligonucleotides for these isoforms further demonstrated the
involvement of PKC-, -ßI, and , but not -, in LPS responses.
Stimulation of cells with LPS for 1 h caused activation of NF-
B
in the nuclei by detection of NF-B-specific DNA-protein binding;
this was inhibited by genestein, U73122, D609, calphostin C, or
antisense oligonucleotides for PKC-, -ßI, and -, but not -.
These data suggest that LPS activates PI-PLC and PC-PLC via an upstream
tyrosine kinase to induce PKC activation, resulting in the stimulation
of NF-B DNA-protein binding, then initiated the expression of iNOS
and NO release. PKC isoforms , ßI, and were shown to be
involved in the regulation of these LPS-induced
events. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Changes in NO production in iNOS-expressing cells usually correlate
with similar changes in iNOS mRNA abundance, indicating that a major
part of iNOS regulation occurs at the level of transcription. The
promotor region of the iNOS gene contains several consensus sequences
for the binding of transcriptional factors, such as NF-B and
activator protein-1 (AP-1), as well as for various members of the
CCAAT/enhancer-binding protein (C/EBP), activating transcription factor
(ATF)/cAMP response element-binding protein (CREB), and STAT family of
transcription factors (13, 14, 15). Of these, proteins of the NF-B
family appear to be essential components for the enhanced iNOS gene
expression in macrophages exposed to the active components of
endotoxin, LPS (16, 17). In unstimulated cells, NF-B is retained in
the cytoplasm by binding to IB but is released by signal induction
and translocates to the nucleus, where it activates the responsive gene
(18). The macrophage iNOS regulates NO synthesis over a period of
several hours following cell stimulation with LPS (19). The sustained
production of NO endows macrophages with cytotoxic activity against
viruses, bacteria, fungi protozoa, and tumor cells (20). However, the
intracellular signaling pathways by which LPS causes iNOS expression
are largely unresolved; a number have been proposed, including the
activation of tyrosine kinases (21, 22), protein kinase C (PKC) (22, 23), phosphatidylcholine-specific phospholipase C (PC-PLC) (24, 25),
and sphingomyelinase (26). However, the relationships between these
pathways are unknown. Although the role of PKC isoforms in LPS-induced
NO production and iNOS induction has been studied in J774 and RAW 264.7
macrophages, respectively (26, 27), both studies used only the phorbol
ester, TPA, as a research tool, and conclusive results were not
obtained. Furthermore, the signal pathways for LPS-induced PKC
activation are still unknown, and the role of PKC in the mechanism of
LPS-induced NO production and iNOS expression have not been addressed.
In the present study, we explored the intracellular signaling pathway
of LPS-induced PKC activation and its involvement in the LPS-stimulated
NO production in RAW 264.7 macrophages, the role of PKC isoforms in
LPS-induced NO release, iNOS expression and NF-B activation being
further elucidated using isoform-specific antisense oligonucleotides.
The results show that LPS can activate PI-PLC and PC-PLC via tyrosine
phosphorylation, resulting in PKC activation, NF-B activation, iNOS
expression, and, finally, NO production. Of the PKC isoforms , ßI,
, , and expressed in RAW cells and PKC-, -ßI, and -
are involved in the regulation of LPS-induced NF-B activation, iNOS
expression, and NO release.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Affinity-purified rabbit polyclonal anti-iNOS Ab was
obtained from Transduction Laboratories (Lexington, KY). Rabbit
polyclonal Abs specific for PKC-
, -
, or -, DMEM, FCS,
penicillin, and streptomycin were purchased from Life Technologies,
(Gaithersburg, MD). Rabbit polyclonal Abs specific for PKC-, -ßI,
-ßII, -, -
, or - or for the p65, p50, or p52 subunit of
NF-B and the NF-B probe were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). TPA was from L.C. Services (Waburn,
MA). LPS (from E. coli serotype 0127:B8), staurosporine, pyrrolidine
dithiocarbamate (PDTC), sulfanilamide,
N-(1-naphthyl)-ethylenediamine, and histone III-S were from
Sigma (St. Louis, MO). Genestein, calphostin C, Go 6976, Ro 31-8220,
pertussis toxin (PTX), and 1,2-didecanoyl-rac-glycerol were from
Calbiochem (San Diego, CA). D609, U73122, and U73343 were from Research
Biochemicals (Natick, MA). T4 polynucleotide kinase was from New
England Biolab (Beverly, MA). Poly (dI/dc) was from Pharmacia Biotech
(Piscataway, NJ). Phosphatidylserine (PS) and 1,2-dioleoylglycerol (DG)
were from Avanti Polar Lipids (Birmingham, AL). Reagents for SDS-PAGE
were from Bio-Rad (Hercules, CA). Myo-[3H]inositol (23.5
Ci/mmol) and [
-32P]ATP (3000 Ci/mmol) were from
Dupont-New England Nuclear (Boston, MA). Horseradish peroxidase-labeled
donkey anti-rabbit second Ab and the ECL detecting reagent were
purchased from Amersham International (Arlington Heights, IL).
Cell culture
RAW 264.7 cells, a murine macrophage cell line, were obtained
from American Type Culture Collection (ATCC, Manassas, VA) and cultured
in DMEM supplemented with 10% FCS, 100 U/ml penicillin, and 100
µg/ml streptomycin; they were grown in 12-well plates (nitrite
assay); 6-well plates (phosphatidylinositol (PI) hydrolysis assay); or
10-cm dishes (PKC isoform Western blot, PKC activity measurement, iNOS
expression, and NF-B gel shift assay).
Determination of NO concentration
NO production in culture supernatant was assayed by measuring nitrite, its stable degradation product, using the Griess reagent. The DMEM was changed to phenol red-free medium before the cells were stimulated with LPS (1 µg/ml) for 24 h; then isolated supernatant was centrifuged and mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride, 2% phosphoric acid) and incubated at room temperature for 10 min. The absorbance was measured at 550 nm in a microplate reader. Sodium nitrite (NaNO2) was used as a standard. In pretreatment experiments, cells were incubated with genestein (tyrosine kinase inhibitor), U73122 (PI-PLC inhibitor), D609 (PC-PLC inhibitor), propanolol (phosphatidate phosphohyrolase inhibitor), staurosporine, calphostin C, Go 6976, or Ro 31-8220 (PKC inhibitors) for 30 min or with TPA for 24 h before addition of LPS.
Preparation of cell extracts and Western blot analysis of iNOS and PKC isoforms
Following treatment with LPS, or pretreatment with inhibitors, TPA or antisense oligonucleotides (see below) followed by LPS, or treatment with TPA for 10 min or 24 h, the cells were harvested and collected. Cell homogenates for iNOS or PKC isoform (antisense oligonucleotides treatment) expression or cytosolic and membrane fractions for PKC isoform expression (TPA treatment) were prepared as described previously (28) and subjected to SDS-PAGE using a 7.5% (iNOS) or 10% (PKC isoform) running gel. The proteins were transferred to nitrocellulose paper, and immunoblot analyses were performed as described previously (29). Briefly, the membrane was incubated successively with 0.1% milk in TTBS at room temperature for 1 h, with rabbit Abs specific for iNOS or PKC isoforms for 1 h, and with horseradish peroxidase-labeled anti-rabbit Ab for 30 min. After each incubation, the membrane was washed extensively with TTBS. The immunoreactive band was finally detected with ECL detecting reagents and visualized using Hyperfilm-ECL.
Measurement of phosphatidylinositol (PI) hydrolysis
PI hydrolysis was assessed by measuring the accumulation of [3H]inositol phosphates (IP) in cells labeled by a 24-h incubation in growth medium containing myo-[3H]inositol (2.5 µCi/ml) as previously described (29). After incubation, the cells were washed with physiologic salt solution (PSS; 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 1.2 mM KH2PO4, 11 mM glucose, and 20 mM HEPES, pH 7.4) containing 10 mM LiCl and incubated at 37°C for 20 min. In PTX pretreatment experiments, PTX (100 ng/ml) was incubated in the growth medium for 24 h. Pretreatment with U73122 or U73343 (10 µM), genestein (30 µM), or D609 (50 µM) was performed by adding the reagent to the PSS 30 min before stimulation with LPS (1 µg/ml) for 15 min in the presence of 10% FCS.
PKC activity assay
Cells treated with LPS for 10 min, 30 min, 1 h, 12 h,
or 24 h were scraped and collected, and cytosolic and membrane
fractions were prepared and assayed for PKC activity as previously
described (30); the assay was performed at 30°C for 5 min in 25 µl
of 30 mM Tris-HCl buffer, pH 7.5, containing 6 mM magnesium acetate,
0.12 mM [-32P]ATP, 0.4 mM CaCl2, 40 µg/ml
phosphatidylserine (PS), 8 µg/ml 1,2-dioleoylglycerol (DG), 1 mg/ml
histone III-S, and the enzyme preparation (2.5–5.0 µg protein) from
cytosolic or membrane fractions. The Ca2+ and
phospholipid-independent activity was measured under the same
conditions in the absence of Ca2+ and phospholipid, and in
the presence of 2 mM EGTA.
Synthesis of antisense oligonucleotides and treatment of cells with oligonucleotides
Phosphorothioate oligodeoxynucleotides were synthesized in
trityl-on mode using an Applied Biosystem Model 391 DNA synthesizer as
described previously (31). The A,G,C, and T phosphoramidites,
controlled pore glass supports, and sulfuring reagent were purchased
from Glen Research (Sterling, VA). The oligodeoxynucleotides were
deblocked and cleaved from the solid support using concentrated ammonia
water by a standard procedure. After evaporation of the ammonia, the
deprotected oligodeoxynucleotides were purified using Sep-Pak C18
cartridges (Millipore, Milford, MA) as reported previously (32).
Control sequences (reversed polarity or scrambled versions of the
antisense oligonucleotides) were also synthesized, with each antisense
oligonucleotide and its control having contained the same base
composition. The sequences of antisense oligonucleotides for the
PKC-, -ßI, -, -, and - and controls used in this study
are listed in Table I
.
|
, --control, -ßI,
-ßI-control, -, --control, -, or - was added, and
incubation was continued for 5 days before challenge with 1 µg/ml of
LPS for 24 h. The protein levels of each PKC isoform after
treatment with antisense oligonucleotides were determined by
immunoblotting using PKC isoform , ßI, , or -specific
Ab. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
Control cells, or cells pretreated with genestein, calphostin C, or antisense oligonucleotides of PKC isoform were treated with 1 µg/ml of LPS for 1 h. Nuclear extracts were then isolated as described previously (33). Briefly, cells were washed with ice-cold PBS and pelleted. The cell pellet was resuspended in hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 1 mM NaF, and 1 mM Na3VO4) and incubated for 15 min on ice; then the cells were lysed by addition of 0.5% Nonidet P-40, followed by vigorous vortexing for 10 s. The nuclei were pelleted and resuspended in extraction buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 mM NaF, and 1 mM Na3VO4), and the tube was vigorously shaken at 4°C for 15 min on a shaking platform. The nuclear extracts were then centrifuged and the supernatants aliquoted and stored at -80°C.
A double-stranded oligonucleotide probe containing NF-B sequences
(5'-AGTTGAGGGGACTTTCCCAGGGC-3') was purchased (Santa Cruz)
and end labeled with [-32P]ATP using T4 polynucleotide
kinase. The nuclear extract (6–10 µg) was incubated with 1 ng of
32P-labeled NF-B probe (40,000–60,000 cpm) in 10 µl
of binding buffer containing 1 µg poly(dI-dc), 15 mM HEPES, pH 7.6,
80 mM NaCl, 1 mM EGTA, 1 mM DTT, and 10% glycerol at 30°C for 20
min. DNA/nuclear protein complexes were separated from the DNA probe by
electrophoresis on a native 6% polyacrylamide gel; then the gels were
vacuum dried and subjected to autoradiography with an intensifying
screen at -80°C. When supershift assays were performed, polyclonal
Abs against p65, p50, or p52 were added to the nuclear extracts 30 min
before the binding reaction, and the DNA/nuclear protein complexes were
separated on a 4.5% polyacrylamide gel.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Exposure of RAW 264.7 macrophages to LPS stimulates nitrite production and the expression of 130-kDa iNOS in a concentration- and time-dependent manner (54), with the maximum being seen using 1 µg/ml of LPS for 24 h; this condition was therefore used in the following NO production experiments.
To study the intracellular signaling pathway involved in the
LPS-induced NO production and iNOS expression, the tyrosine kinase
inhibitor genestein was used. When cells were pretreated for 30 min
with 10 µM or 30 µM genestein, LPS-induced nitrite production was
inhibited by 45.2% or 55.4%, respectively (Fig. 1A). This effect was
accompanied by the decreased iNOS expression (Fig. 1C). When
cells were pretreated for 30 min with 10 µM U73122 (PI-PLC
inhibitor), 10 µM U73343 (an inactive analogue of U73122), 50 µM
D609 (PC-PLC inhibitor), or 100 µM propranolol (phosphatidate
phosphohydrolase inhibitor), LPS-induced nitrite production was
inhibited 61.3% or 76.1% by U73122 or D609, respectively, while
U73343 and propanolol had no effect (Fig. 1B); iNOS
expression was also inhibited by U73122 or D609, but not by U73343 or
propranolol (Fig. 1, D and E).
|
), indicating that, in RAW 264.7 cells,
LPS stimulates the PI-PLC pathway by activating tyrosine kinase.
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A, PKC activity was decreased
in the cytosol and increased in the membrane after treatment with LPS
for 10 min; this effect was seen up to 12 h of treatment with LPS,
then lost at 24 h. The LPS-induced increase in membrane PKC
activity was inhibited by 30 min pretreatment with 30 µM genestein or
100 nM staurosporine or by overnight pretreatment with 1 µM TPA, the
respective inhibition was 73.7%, 79.3%, or 65% (Fig. 3B).
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To determine whether activation of PKC by LPS was involved in the
regulation of LPS-induced NO production, PKC inhibitors were used.
Pretreatment of cells for 30 min with staurosporine, calphostin C, Ro
31-8220, or Go 6976 inhibited LPS-induced nitrite production in a
dose-dependent manner (Fig. 4,
A and B). LPS-induced iNOS expression was also
inhibited by these four inhibitors (Fig. 4C). Long-term
(24-h) pretreatment of cells with 1 µM TPA also inhibited both
LPS-induced nitrite production (70% inhibition) and iNOS expression
(Fig. 5).
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, ßI, ßII, , , , , , or
), only PKC-, -ßI, -, -, and - were detected in RAW
cells, while all nine isoforms were detected in cell homogenates of rat
brain (Fig. 6A). Exposure of
the cells to 1 µM TPA for 10 min resulted in marked translocation of
PKC-, -ßI, -, and - from the cytosol to the membrane. After
long-term (24-h) treatment, complete down-regulation of PKC-, -ßI,
and - was seen, while translocated PKC- was not down-regulated
(Fig. 6B; 33), suggesting the possible involvement of one or
all of PKC-, -ßI, and -, but not -, in the LPS-induced NO
production.
|
, -ßI, and/or - is involved in the LPS-mediated NO
production and iNOS expression, the effect of direct TPA-mediated
activation of PKC on NO release and iNOS expression was examined. TPA
alone did not induce nitrite release (data not shown). The effect of
PKC activation on the LPS response could not be tested in combination
with TPA pretreatment, since the combination resulted in
down-regulation of PKC-, -ßI, and - due to following 24 h
treatment. Instead, a cell-permeable PKC activator,
1,2-didecanoyl-rac-glycerol, which activates PKC but does not result in
its down-regulation, when tested at 10 µM, had no effect on
LPS-induced NO production (data not shown). Inhibitory effect of PKC isoform antisense oligonucleotides on LPS-induced NO production and iNOS expression
To further study the involvement of PKC-, -ßI, and -, but
not -, antisense oligonucleotides for these isoforms and their
corresponding scrambled or reversed controls were used. Ten percent
confluent cells were treated for 5 days with the respective antisense
oligonucleotides, and the level of each isoform protein was determined
by Western blot. As shown in Fig. 7A, 5 µM of PKC-, -ßI,
-, or - antisense oligonucleotides caused a specific reduction in
the level of their corresponding immunoreactive isoform protein; e.g.
the antisense oligonucleotides of PKC- specifically inhibited 85%
of the expression of PKC- protein but had no effect on the
expression of PKC-ßI, -, or -.
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, -ßI, -, or -, LPS-induced nitrite production was
inhibited 54.7, 55.7 or 55.3% by the PKC-, -ßI, and -
antisense oligonucleotides, respectively, whereas the antisense
oligonucleotides of PKC-, although inhibiting the expression of
PKC-, had no effect (Fig. 7
B). The control sequences for
PKC-, -ßI, or -, which did not affect the expression of these
three isoforms, had no effect on the LPS response. Similar results were
seen with LPS-induced iNOS expression (Fig. 7B). The
experiment using antisense oligonucleotides of PKC- is illustrated
in the Discussion.
Since PKC-, -ßI, or - plays a role in regulating the iNOS
expression, the relative contribution of each isoform was assayed using
combinations of oligonucleotides. As shown in Fig. 8, combination of any two of the PKC-,
-ßI, and - antisense oligonuleotides caused a similar increase in
inhibition of LPS-induced nitrite production (
75% inhibition) and
iNOS expression. When all three were added, the inhibition was similar
to that seen with the paired combinations. The combination of all
control sequences for these three isoforms, however, had no effect on
the LPS response (Fig. 8).
|
B in the nuclei of LPS-stimulated RAW 264.7
cells and the inhibitory effect of PKC isoform antisense
oligonucleotides
The NF-B p50/p65 heterodimer is present in the cytosol of
resting cells (18, 34); after stimulation of the cells with various
agents, the cytosolic NF-B/IB complex dissociates, and free
NF-B translocates to the nuclei. We performed an EMSA using
oligonucleotides containing NF-B recognition site-like sequences in
the macrophage iNOS gene (13) and nuclear extracts prepared from
LPS-stimulated cells. In nuclear extracts of unstimulated macrophages,
two faint NF-B-specific DNA-protein complexes were identified. The
intensity of which markedly increased following exposure of the cells
to 1 µg/ml of LPS for 10 min or 1 h (Fig. 9A). After treatment with LPS
for 24 h, the intensity of these DNA-protein complexes decreased,
but was still stronger than in resting cells. For the EMSA, cells were
treated with LPS for 1 h. To identify the specific subunits
involved in the formation of these two banding patterns of the NF-B
dimer after LPS stimulation. Supershift assays were performed in the
presence of Abs against the p65, p50, or p52 subunit. As shown in Fig. 9B, incubation with anti-p65 or anti-p50 Abs induced
a supershift (arrow a and b, respectively). However, there was no shift
in the presence of anti-p52 Ab. Thus, our data agree with those of
Xie et al. (16) in demonstrating that the upper complex is the p65/p50
heterodimer and the lower complex is the p50/p50 homodimer.
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B-specific DNA-protein complex formation was
inhibited (Fig. 9C); as shown in Fig. 10, similar results were obtained when
cells were treated for 5 days with 5 µM of antisense oligonucleotides
of PKC-, -ßI, or -, but not with the contol sequences of
anti- C, anti-ßI C, or anti- C. The
oligonucleotides of anti-, which inhibited the expression of
PKC- (Fig. 7A), had no effect on LPS-induced NF-B
activation in nuclear extracts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6,
and IL-8, was mediated by the interaction of LPS with mCD14 (38, 39, 40).
Recently, Paul et al. found that LPS-induced NO release in RAW cells is
also mediated by mCD14 and is serum dependent (22). Thus, formation of
a LPS/LBP complex might allow binding to, and activation of, mCD14,
then trigger signal transduction to initiate the expression of iNOS and
NO release in RAW cells.
Although many reports have shown the involvement of PKC in LPS-induced
iNOS expression and NO production (22, 23), the signaling pathway
involved in PKC activation is still unknown. In the present study, four
PKC inhibitors, calphostin C, Go 6976, Ro 31-8220, and staurosporine,
dose-dependently inhibited LPS-stimulated iNOS expression and NO
release, indicating that PKC activation is indeed an obligatory event
in the LPS-mediated regulation of NO release and iNOS expression in RAW
cells. LPS induced PKC activation, this phenomenon occurring after 10
min treatment and being sustained for 12 h (Fig. 3). PKC is
activated by the physiologic activator DAG, which can be generated
directly by the action of PLC or indirectly by a pathway involving the
production of phosphatidic acid by phospholipase D (PLD), followed by a
dephosphorylation reaction catalyzed by phosphatidate phosphohydrolase.
Normally, the PLC involved in the production of DAG is PI-PLC, but
PC-PLC may also be involved (41, 42). DAG generated from PI-PLC causes
transient PKC activation while that generated from PC-PLC causes
sustained activation (42). The PI-PLC inhibitor U73122 inhibited
LPS-induced iNOS expression and NO production, while the inactive
analogue U73343 had no effect. LPS also stimulated PI hydrolysis, this
effect being inhibited by U73122, but not by U73343. Genestein also
attenuated LPS-induced PI hydrolysis, indicating that the PI-PLC
involved might be PLC, since PLC is a SH2 domain-containing
protein that utilizes this module to link phosphotyrosine-containing
sequences in a receptor protein or cytoplasmic protein tyrosine kinase
to PI hydrolysis (43). The PC-PLC inhibitor D609 also inhibited
LPS-induced iNOS expression and NO production, but not LPS-stimulated
PI hydrolysis, while the phosphatidate phosphohydrolase inhibitor
propranolol had no effect. Thus, LPS may act through the PI-PLC and
PC-PLC pathway but not the PC-PLD pathway to induce PKC activation in
RAW 264.7 cells. The PC-PLC pathway may contribute to the long-lasting
activation (12 h) of PKC by LPS (Fig. 3A) (41, 42). In
another J774 macrophages, NO synthesis induced by the combination of
IFN- and LPS also involved the activation of PC-PLC (24), and LPS
increased DAG formation via the PC-PLC pathway to activate NF-B
(44). In the present study, genestein inhibited LPS-induced PKC
activation, indicating the requirement for an initial protein tyrosine
phosphorylation event in this activation process. The PI-PLC- indeed
required an upstream activation of protein tyrosine kinase (Fig. 2).
However, the mechanism involved in activation of PC-PLC is still
unknown but might also involve tyrosine phosphorylation (45). The
tyrosine kinase involved might be p53/p56lyn, since in
monocytes LPS activates this kinase, which was associated with CD14
(46, 47). Thus, in RAW 264.7 cells, the LPS/LBP complex binds to mCD14,
then activates PI-PLC- and PC-PLC via an upstream protein tyrosine
phosphorylation to elicit PKC activation and, finally, iNOS expression
and NO production.
To determine which PKC isoform was involved in the regulation of
LPS-induced NO release, down-regulation of PKC by overnight treatment
with TPA was performed, resulting in inhibition of LPS-stimulated iNOS
expression and NO release. Western blot analysis showed the expression
of PKC-, -ßI, -, -, and among nine isoforms (, ßI,
ßII, , , , , , and ). Apart from PKC-, all these
isoforms were translocated by 10 min treatment with TPA, and
down-regulation of PKC-, -ßI, and -, but not -, was seen
after 24 h treatment (Fig. 6; 33). These results suggest that the
activation of one or all of PKC-, -ßI, and -, but not -, was
involved in LPS-elicited iNOS expression and NO release in RAW cells.
To confirm the involvement of these isoforms, antisense
oligonucleotides for PKC-, -ßI, -, -, and their respective
controls were used. The specificity of these antisense oligonucleotides
was demonstrated (Fig. 7A), and the results showed
inhibition of LPS-stimulated iNOS expression and NO production by the
antisense oligonucleotides of PKC-, -ßI, or - but not by the
antisense oligonucleotides of PKC- or the control oligonucleotides
of PKC-, -ßI, or -. Thus, a crucial role for all three
isoforms, PKC-, -ßI, and -, but not -, in the LPS-induced
stimulation of NO production and iNOS expression was demonstrated. This
is further evidence to show that different members of the PKC family
within a single cell elicit specific physiologic responses (41).
In macrophages, the transcriptional factor NF-B is critical in the
induction of iNOS by LPS (13, 16). In the present study, LPS indeed
increased the levels of the NF-B-specific DNA-protein complex in
nuclear extracts (Fig. 9A), and the NF-B blocker
pyrrolidine dithiocarbamate (PDTC) inhibited LPS-induced NO production
and iNOS expression (54). Translocation of NF-B from the cytosol to
the nucleus and degradation of IB- and IB-ß in the cytosol
were seen (54). The LPS-induced NF-B activation was inhibited by
genestein, U73122, D609, or calphostin C. Furthermore, antisense
oligonucleotides of PKC-, -ßI, or -, but not -, attenuated
this activation, indicating the involvement of PKC-, -ßI, and
-, but not -, in the LPS-stimulated up-regulation of iNOS in RAW
cells. Although involvement of PKC isoforms in the regulation of
LPS-induced NO production has been reported in J774 and RAW 264.7
macrophages (26, 27), the isoform involved was not conclusively
identified and the signaling pathway for PKC activation and the role of
NF-B were not elucidated. Here, our results show that LPS activates
PI-PLC and PC-PLC via protein tyrosine phosphorylation to elicit PKC
activation, NF-B activation, and, finally, iNOS expression and NO
release in RAW 264.7 macrophages. Of the PKC isoforms , ßI, ,
, and present in these cells, PKC-, -ßI, and - were
shown to be involved in the regulation of the LPS effect. To further
elucidate the role of these three isoforms, the effect of combinations
of either two or all three of the antisense oligonucleotides on
LPS-induced response was tested and it was found that the combination
of any two resulted in maximal inhibition (75% inhibition) (Fig. 8). Thus, any combination of two of PKC-, -ßI, and - is
sufficient to mediate the LPS response. Although PKC was shown to be
involved in the LPS effect, direct activation of PKC by TPA did not
induce NF-B activation and NO production in RAW 264.7 cells (data
not shown); similar findings have been reported by other laboratories
(48). This phenomenon contrasts with the situation in peritoneal
macrophages, hepatocytes, HUVEC, and astrocytes in which TPA alone
induces NF-B activation and iNOS expression (49, 50, 51, 52). However, after
ectopic expression of PKC- in RAW 264.7 cells, TPA was sufficient to
induce iNOS synthesis by activation of NF-B (48). The complete
absence of PKC- in RAW cells (Ref. 48; present study) might explain
the lack of response of NO synthesis after incubation of these cells
with TPA (Ref. 48; present study). Thus, the expression of iNOS in
response to LPS in RAW cells does not involve PKC- engagement. This
was confirmed by using antisense oligonucleotides of PKC-, which had
no effect on LPS-induced NO production and iNOS expression (Fig. 7B). Direct activation of PKC not only did not induce NO
release, but it also failed to modulate the LPS response (data not
shown). Thus, PKC-, -ßI, and - are required, but are not
sufficient, for inducing the full response. PKC- was reported to be
activated by ceramide generated from sphingomyelin hydrolysis and
involved in NF-B activation in NIH3T3 cells (53). Whether PKC- is
activated by ceramide and is involved in LPS-induced NO production in
RAW cells requires further investigated. However, in addition to the
inability of the combination of antisense oligonucleotides to
completely inhibit LPS-induced NO production (Fig. 8), four different
PKC inhibitors were also unable to completely block the LPS response
(Fig. 4), indicating that other LPS-activated components might also be
involved in NO production. Actually, we have recently demonstrated that
LPS-induced p38 MAPK activation is also involved and that this pathway
is not dependent on PKC activation (54).
In summary, the signaling pathway for the LPS-induced activation of PKC
was explored and the PKC isoforms , ßI, and , but not , were
found to be involved in the regulation of LPS-induced NF-B
activation, iNOS expression, and NO release in RAW 264.7 macrophages.
This is the first study showing these two mechanisms in the
LPS-stimulated NO release.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ching-Chow Chen, Institute of Pharmacology, College of Medicine, National Taiwan University, No.1, Jen-Ai Road, 1st Section, Taipei 10018, Taiwan. E-mail address:
3 Abbreviations used in this paper: NO, nitric oxide; iNOS, inducible nitric oxide synthase; PC-PLC, phosphatidylcholine-specific phospholipase C; PI-PLC, phosphoinositide-specific phospholipase C; EMSA electrophoretic mobility shift assay; LBP, LPS-binding protein; mCD14, membrane-bound CD14; DAG, diacylglycerol; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol 13-acetate; PI, phosphatidylinositol; PTX, pertussis toxin; IP, inositol phosphates.
4 A. A. Author, and A. A. Author. Title. Submitted for publication.
Received for publication January 23, 1998. Accepted for publication July 30, 1998.
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