1 Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, and 2 Program in Cell Biology, National Jewish Medical and Research Center, Denver, Colorado 80206
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ABSTRACT |
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Nitric oxide (NO·) produced by inducible nitric oxide synthase (iNOS)
mediates a number of important physiological and pathophysiological processes. The objective of this investigation was to examine the role
of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS
and NO· by interferon- (IFN-
) + lipopolysaccharide (LPS) in macrophages using specific inhibitors and dominant inhibitory mutant
proteins of the MAPK pathways. The signaling pathway utilized by
IFN-
in iNOS induction is well elucidated. To study signaling pathways that are restricted to the LPS-signaling arm, we used a
subclone of the parental RAW 264.7 cell line that is unresponsive to
IFN-
alone with respect to iNOS induction. In this RAW
264.7
NO(
) subclone, IFN-
and LPS are nevertheless required for
synergistic activation of the iNOS promoter. We found that
extracellular signal-regulated kinase (ERK) augmented and
p38mapk inhibited IFN-
+ LPS induction
of iNOS. Dominant-negative MAPK kinase-4 inhibited iNOS promoter
activation by IFN-
+ LPS, also implicating the c-Jun
NH2-terminal kinase (JNK) pathway in mediating iNOS
induction. Inhibition of the ERK pathway markedly reduced IFN-
+ LPS-induced tumor necrosis factor-
protein expression, providing a
possible mechanism by which ERK augments iNOS expression. The
inhibitory effect of p38mapk appears more
complex and may be due to the ability of p38mapk
to inhibit LPS-induced JNK activation. These results indicate that the
MAPKs are important regulators of iNOS-NO· expression by IFN-
+ LPS.
nitric oxide; monocytes/macrophages; protein kinases; lipopolysaccharide; rodent
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INTRODUCTION |
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NITRIC OXIDE (NO·)
is a free radical produced from the catalytic oxidation of the
guanidino group of L-arginine by nitric oxide synthase
(NOS). NO· has been shown to have a number of important biological
functions, including tumor cell killing, host defense against
intracellular pathogens, vasodilatation, neurotransmission, and
inhibition of platelet aggregation (29). NO· produced
from the constitutive forms of NOS, namely, endothelial and neuronal NOS, functions principally as a vasodilator and neurotransmitter, respectively (41). The third form of NOS, known as
inducible NOS (iNOS or NOS II), is generally not present in resting
cells but is induced by various stimuli, which include bacterial
lipopolysaccharide (LPS), tumor necrosis factor- (TNF-
),
interleukin (IL)-1
, picolinic acid, lipoarabinomannan, phorbol
ester, interferon (IFN)-
, and hypoxia (11, 35, 36, 40).
In contrast to the constitutive forms of NOS, iNOS is regulated
primarily at the level of transcription, because calmodulin is already
tightly bound and, thus, iNOS expression is largely independent of
intracellular calcium (33, 40). Increased expression of
iNOS has been associated with disorders as diverse as septic and
hemorrhagic shock, rheumatoid arthritis, and chronic infections such as tuberculosis.
Since the cloning of the mouse iNOS gene, the transcriptional
regulation of iNOS has been extensively characterized (30, 31,
55, 56). Although many potential cis-regulatory
elements have been identified, six enhancer elements have been shown to be important in IFN- + LPS induction of iNOS (15, 17, 24, 34, 39, 57). In the upstream enhancer element known as region II, located between positions
913 and
1029, two IFN-stimulated response elements and an IFN-
-activated sequence have been
identified to be critical for IFN-
signaling of iNOS transcription
(15, 24, 34). For LPS transcriptional regulation of iNOS,
two nuclear factor-
B (NF-
B) sites, one located in the basal
enhancer region I between positions
48 and
209 and another located
in region II, are required (16, 39, 55, 57). More
recently, another cis-regulatory element, an Oct site, which
binds the basal transcriptional element octomer, was found to be
required for maximal iNOS transcription by LPS (15, 17).
NF-
B has also been shown to be required in the regulation of the
human iNOS gene (49).
The mitogen-activated protein kinases (MAPKs) are Ser-Thr kinases that
have been shown to activate a number of transcription factors,
including activator protein-1 (AP-1), activating transcription factor (ATF)-2, cAMP-responsive element binding protein, NF-B, and
certain members of the Ets family (19, 22, 37). MAPKs are
comprised of three principal family members with distinct isoforms
within each member: extracellular signal-regulated kinases (ERKs),
p38mapks, and c-Jun NH2-terminal
kinases (JNKs) or stress-activated protein kinases (SAPKs) (9,
20, 21, 26). MAPKs are activated by a family of dual-specificity
kinases [MAPK kinases (MKKs)] that phosphorylate MAPKs on specific
Thr and Tyr residues: MKK4 and MKK7 [also known as JNKK1 and JNKK2 or
SAPK/ERK kinase (SEK)-1 and -2] activate the JNK/SAPKs, MKK3 and MKK6
activate the p38mapks, and MAPK/ERK kinase
(MEK)-1 and -2 activate the ERKs (10, 28, 45). Further
upstream, the MEK kinases (MEKKs) and other kinases such as germinal
center kinase, mixed-lineage kinase, p21-activated kinase, and tyrosine
phenol-lyase are known to activate the JNK and ERK pathways (8,
13, 42). We and others previously examined the role of the MAPKs
in the induction of iNOS by TNF-
and other stimuli (2, 3, 5,
7, 18, 43, 51). We showed that the MEKK1-MKK4-JNK pathway played
an important role in the transcriptional regulation of iNOS by TNF-
(5). Furthermore, we showed that ERK and
p38mapk were not involved in the regulation of
iNOS by IFN-
+ TNF-
. We undertook this study to determine
the role of the MAPKs in the regulation of iNOS with IFN-
+ LPS
stimulation (Fig. 1). Because the
IFN-
-signaling pathway has been elucidated in regard to iNOS
induction, we restricted our study to the MAPK family of signaling
molecules that is activated by LPS. The parental RAW 264.7 macrophage
strain is known to express iNOS-NO· in response to IFN-
alone,
making it difficult to discriminate the relative contribution of
IFN-
and LPS on costimulation. Moreover, the use of cells that are
capable of producing NO· with IFN-
stimulation alone would be
problematic when pharmacological or dominant-negative (DN) inhibitors
of the LPS-induced MAPK pathways are utilized to sort out the
regulatory signaling pathways. To eliminate this obstacle, we used a
subclone of RAW 264.7 cells that does not respond to IFN-
alone with
respect to iNOS induction (39). Nevertheless, in these RAW
264.7
NO(
) cells, it is important to emphasize that costimulation
with IFN-
and LPS is essential for induction of iNOS-NO·
(39).
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EXPERIMENTAL PROCEDURES |
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Materials.
RAW 264.7NO(
) macrophages, which were used for all the studies,
were generously provided by Dr. William J. Murphy (University of Kansas
Medical Center) (39). Unlike the parent RAW 264.7 line
(TIB-71; American Type Culture Collection, Rockville, MD), the RAW
264.7
NO(
) line does not respond to IFN-
alone by producing NO·. LPS purified from Salmonella typhimurium was
purchased from Sigma Chemical (St. Louis, MO). Fetal bovine serum (FBS)
was purchased from Irvine Scientific (Irvine, CA).
Glutathione-Sepharose beads were purchased from Pharmacia (Piscataway,
NJ). Enhanced chemiluminescence assay kits were obtained from Amersham
Life Sciences (Arlington Heights, IL). Recombinant
c-Jun-(1-79)-glutathione S-transferase (GST) and DN-MEKK1 mutant in a cDNA3 expression vector were kindly provided by Dr. Gary Johnson (University of Colorado Health Sciences Center). The DN-MKK4 mutant (K116R) and DN-c-Jun, which lacks the
transactivating domain but contains the DNA-binding domain (DBD-c-Jun),
both in an LNCX expression vector, were gifts from Dr. Lynn Heasley
(University of Colorado Health Sciences Center). Rabbit polyclonal
anti-p46 JNK, rabbit polyclonal anti-p42 ERK, and mouse monoclonal
phospho-specific p46-p54 JNK antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Mouse IFN-
and neutralizing
anti-TNF-
and anti-IL-1
antibodies were obtained from R & D
Systems (Minneapolis, MN). Rabbit anti-iNOS polyclonal antibody was
purchased from Alexis (San Diego, CA). [
-32P]ATP
(>3,000 Ci/mmol) was purchased from NEN Research Products DuPont
(Wilmington, DE). The MEK1 inhibitor (PD-98059) and phospho-specific p38mapk antibody were purchased from New England
Biolabs (Beverly, MA). SB-203580 (p38mapk
inhibitor) was purchased from Calbiochem (San Diego, CA). The iNOS-luciferase reporter construct was generously provided by Dr.
Charles J. Lowenstein (Johns Hopkins University School of Medicine,
Baltimore, MD) and Dr. Robert Scheinman (University of Colorado Health
Sciences Center). Phospho-specific ERK polyclonal antibody and the
firefly luciferase reporter assay system were purchased from Promega
(Madison, WI). The Lipofectamine reagent used for the transfection
experiments was purchased from GIBCO BRL (Gaithersburg, MD). TNF-
ELISA kit was purchased from Genzyme (Cambridge, MA). The
cytomegalovirus
-galactosidase plasmid used for normalizing
transfection efficiency was kindly provided by Dr. Dwight Klemm
(National Jewish Medical and Research Center). All other reagents were
of the highest purity.
Analysis of nitrite anion accumulation.
Nitrite anion (NO) accumulation in the supernatant
was determined as previously reported (11). Briefly, RAW
264.7
NO(
) cell monolayers were stimulated with LPS (1 ng/ml) and
IFN-
(10 U/ml) or were coincubated with PD-98059 (30 µM) or
SB-203580 (30 µM) for 18 h. One hundred microliters of
supernatant were combined with an equal volume of Greiss reagent, and
the samples were incubated at room temperature for 10 min before the absorbance was quantified at 550 nm. With the use of a standard curve,
the nanomoles of NO
produced were determined and
normalized to total cell number in each sample.
Determination of JNK activity.
For measurement of JNK activity, the RAW 264.7NO(
) cells were
lysed at 4°C with 500 µl of ice-cold lysis buffer [50 mM
Tris · HCl, pH 8.0, containing 137 mM NaCl, 10% (vol/vol)
glycerol, 1% (vol/vol) Nonidet P-40, 1 mM NaF, 10 µg/ml leupeptin,
10 µg/ml aprotinin, 2 mM Na3VO4, and 1 mM
phenylmethylsulfonyl fluoride (21)]. After the protein
content was normalized between samples, JNK in each sample of lysate
was bound to 15 µl of a 1:1 slurry of lysis
buffer-GST-c-Jun-(1-79)-Sepharose beads and incubated at 4°C for 2 h. The beads were then washed twice with 500 µl
of lysis buffer and twice with 500 µl of JNK buffer (20 mM HEPES buffer, pH 7.2, containing 30 mM
-glycerophosphate, 10 mM
p-nitrophenylphosphate, 10 mM MgCl2, 0.5 mM
dithiothreitol, and 50 µM Na3VO4). The
activity of JNK was detected by phosphorylation of c-Jun-GST in an in
vitro kinase assay and was assessed by incorporation of
[
-32P]ATP (10 µCi/sample) in JNK buffer incubated at
30°C for 30 min. The kinase reactions were then stopped with an equal
volume of 2× Laemmli sample buffer containing 20 mM dithiothreitol and
boiled for 3 min. The proteins present in the supernatants were
separated by SDS-PAGE through a 12% polyacrylamide gel and transferred
onto nitrocellulose membranes. 32P-labeled c-Jun-GST was
detected by autoradiography.
Western blot analysis. Samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes as described elsewhere (50). The blots were then washed in Tris-Tween-buffered saline [TTBS, 20 mM Tris · HCl buffer, pH 7.6, containing 137 mM NaCl and 0.05% (vol/vol) Tween 20], blocked overnight with 5% (wt/vol) nonfat dry milk, and probed according to the method described by Towbin et al. (50) with a polyclonal iNOS antibody or with phospho-specific antibodies to p46-p54 JNK, p42/p44 ERK, and p38mapk antibodies in 5% (wt/vol) BSA dissolved in TTBS. With the use of horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody, bound antibodies were detected by enhanced chemiluminescence. To determine equal loading of proteins between samples, the membranes were probed with rabbit polyclonal p46 JNK, p42/p44 ERK, and p38mapk antibodies.
Transient transfection and luciferase assay.
RAW 264.7NO(
) cells were plated at a density of 1 × 106 cells per six-well plate in RPMI containing penicillin
(100 U/ml), streptomycin (100 µg/ml), and 10% (vol/vol)
heat-inactivated FBS. After 24 h of growth to ~30-40%
confluence, the cells were transfected with plasmids using
Lipofectamine as described by the manufacturer's protocol (GIBCO BRL).
Briefly, 0.3 µg of iNOS-luceriferase plasmid was combined with 2 µg
of DN-MEKK, 2 µg of DN-MKK4, or 2 µg of DBD-c-Jun plasmid, 10 µl
of Lipofectamine reagent, and 100 µl of Optimem serum-free medium. To
normalize for the amount of DNA transfected, equivalent amounts of the
pc-DNA3 empty vector (for DN-MEKK1) or LNCX empty vector (for DN-MKK4
or DBD-c-Jun) were cotransfected in the controls. To normalize for
transfection efficiency between samples, 1 µg of cytomegalovirus
-galactosidase plasmid was cotransfected for each sample. The
lipid-DNA mixture was incubated for 30 min at room temperature. Each
well was then washed with 2 ml of Optimem medium and replaced with 1 ml
of the Lipofectamine-DNA mixture. After 5 h of incubation, 1 ml of
RPMI containing 20% (vol/vol) FBS and 1%
penicillin-streptomycin-L-glutamine was added to each well.
The media were changed 24 h after transfection, and after an
additional 48 h, the cells were stimulated with IFN-
(10 U/ml)
and LPS (1 ng/ml) for 8 h. The cells were then washed with PBS,
lysed in a luciferase lysis buffer, and assayed for luciferase activity
according to the manufacturer's instructions. The amount of luciferase
activity was normalized to
-galactosidase activity and reported as
magnitude increase in activity.
Statistical analysis. Replicate experiments were independent, and summary results are presented as means ± SE. Differences were considered significant for P < 0.05. Group means were compared by repeated-measures ANOVA using Fisher's least significant difference.
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RESULTS |
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IFN- synergizes with LPS in the expression of
NO
.
We first showed that, unlike the parental RAW 264.7 cell line available
from American Type Culture Collection, these selected RAW
264.7
NO(
) cells do not produce NO· with IFN-
stimulation alone. We considered it paramount to use this macrophage phenotype, because it would allow the detection of any effects of inhibitors of
the LPS-induced MAPK signaling pathways on iNOS-NO· induction. Thus
RAW 264.7
NO(
) macrophages were stimulated with IFN-
(10 U/ml),
LPS (1 ng/ml), or both for 18 h, and NO
(a
stable metabolite of NO·) levels in the culture supernatants were
measured using the Greiss reagent assay. As shown in Fig. 2, cells stimulated with IFN-
or LPS
produced no more NO
than unstimulated cells. In
contrast, there was marked synergy of NO
production
when cells were costimulated with IFN-
+ LPS.
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LPS induces phosphorylation of ERK, p38mapk, and JNK.
Activation of the MAPKs is dependent on the phosphorylation by their
respective upstream MAPK kinases of Thr and Tyr residues on
MAPK-specific tripeptide motifs: TPY for JNK, TEY for ERK, and TGY for
p38mapk. Thus, before the study of the role of
the MAPKs in the regulation of iNOS by LPS, we examined the
phosphorylation of the MAPKs by LPS using phospho-specific antibodies
that recognize the TXY motifs. RAW 264.7NO(
) macrophages were
stimulated with LPS at 100 ng/ml for 0-3 h. The cells were then
lysed with Nonidet P-40 lysis buffer, and the whole cell lysates were
normalized for protein content and then separated by SDS-PAGE. This was
followed by immunoblot of the separated proteins with phospho-specific
antibodies to each of the MAPKs. As shown in Fig.
3, phosphorylation of Thr and Tyr
residues of all three MAPKs occurred after 30 min of LPS stimulation,
peaking at 60 min, and remained sustained above basal level even after
3 h of stimulation. This pattern of phosphorylation is
significantly more prolonged than that induced by TNF-
stimulation in bone marrow-derived macrophages, in which the MAPKs were
phosphorylated and activated after only 10 min of stimulation and
returned to near-basal level by 30 min (4, 53, 54).
IFN-
alone does not activate any of the MAPKs (0-18 h of
stimulation), and costimulation of IFN-
+ LPS does not alter
the kinetics of MAPK activation compared with LPS alone (data not
shown).
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MEK1-ERK inhibition attenuates NO expression by
inhibition of LPS-induced TNF-
production.
To investigate the role of ERK in the modulation of iNOS expression by
LPS, we determined the effect of PD-98059, a specific inhibitor of
MEK1-ERK, on NO
production. Macrophages were
pretreated with PD-98059 (30 µM) for 1 h and then costimulated with IFN-
+ LPS in the presence of the inhibitor for 18 h
before quantification of NO
accumulation in culture supernatants. Compared with IFN-
+ LPS stimulation alone,
treatment with PD-98059 reduced NO
production by
~25-30% (Fig. 4A),
suggesting that ERK plays a modest, although positive, regulatory role
in the induction of iNOS by IFN-
+ LPS. The vehicle DMSO, at a
concentration equivalent to 30 µM PD-98059 (0.075%), did not affect
stimulated NO
expression. To examine whether iNOS
protein level was also influenced by PD-98059, we treated the RAW
264.7
NO(
) cells as described above and, after lysis with a Nonidet
P-40 buffer, performed an immunoblot on nuclear-free whole cell lysate
with a polyclonal iNOS antibody. As shown in Fig. 4B,
PD-98059 also inhibited iNOS protein expression in a fashion that was
similar to that observed for NO
, whereas DMSO had no
effect. A possible toxic or antiproliferative effect of PD-98059 was
assessed by a cell viability assay in which metabolically induced
formazan production was measured (CellTiter 96 Aqueous One Solution
Cell Proliferation Assay; Promega). Compared with unstimulated cells or
cells stimulated with IFN-
+ LPS, addition of 30 µM PD-98059
for 18 h had no effect on cell viability (data not shown).
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p38mapk inhibitor (SB-203580) augments
NO independent of IFN-
+
LPS-induced TNF-
expression.
To determine the role of p38mapk on
NO
and iNOS protein expression induced by
IFN-
+ LPS, we pretreated RAW 264.7
NO(
) cells with
SB-203580 (30 µM) for 1 h, costimulated the cells with
IFN-
+ LPS in the presence of the inhibitor for 18 h, and
then quantified NO
accumulation in culture
supernatants and lysis of cells for immunoblotting for iNOS protein. In
contrast to that observed with MEK1 inhibition, inhibition of
p38mapk augmented NO
production, indicating that p38mapk plays an
inhibitory role in NO· induction (Fig. 4A). There was a
small increase in the amount of iNOS protein expression in the presence
of SB-203580 (Fig. 4B).
Potential paracrine and autocrine role of TNF- and IL-1
in
IFN-
+ LPS induction of iNOS-NO·.
LPS is known to induce the expression of a number of cytokines such as
TNF-
, which also has the capacity, in conjunction with IFN-
, to
induce iNOS expression. Thus a possible mechanism by which ERK may
enhance IFN-
+ LPS-induced expression of NO· is by signaling
the expression of cytokines such as TNF-
with IFN-
+ LPS
stimulation. We therefore measured the amount of TNF-
protein levels
in macrophages stimulated with IFN-
+ LPS in the presence of
MEK1-ERK inhibition by PD-98059. As shown in Fig. 5A, stimulation of cells with
IFN-
+ LPS resulted in a significant increase in the amount of
TNF-
measured in the supernatant. Coincubation with PD-98059
substantially inhibited TNF-
protein expression in the supernatant
of RAW 264.7
NO(
) cells. Interestingly, despite the fact that
SB-203580 augmented NO
expression by IFN-
+ LPS, it also inhibited TNF-
protein expression, although the level
of inhibition was not as marked as that seen with MEK1-ERK inhibition
(Fig. 5A). The vehicle DMSO at a concentration equivalent to
30 µM PD-98059 (0.075%) had no effect on stimulated TNF-
expression (Fig. 5A). To determine whether induction of
NO· by IFN-
+ LPS is due to an autocrine or paracrine effect
of TNF-
and/or IL-1
, we neutralized TNF-
, IL-1
, or both
with neutralizing antibodies in the presence of IFN-
+ LPS
stimulation. RAW 264.7 cells were coincubated with IFN-
+ LPS
along with neutralizing concentrations of anti-TNF-
antibody
(2.5-5 µg/ml), anti-IL-1
antibody (2.5-5 µg/ml),
anti-TNF-
antibody + anti-IL-1
antibody (5 µg/ml each), or
nonimmune IgG isotype antibody (2.5-10 µg/ml). After 18 h
of incubation, the supernatants were assayed for NO
. As shown in Fig. 5B, incubation with neutralizing
anti-TNF-
antibody resulted in a partial inhibition of
NO
, with ~33% inhibition at 5 µg/ml
anti-TNF-
IgG. In contrast, neither anti-IL-1
antibody nor
nonimmune IgG isotype had any inhibitory effect. Neutralization of
TNF-
and IL-1
was not significantly different from neutralization
of TNF-
alone.
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DN-MKK4, but not DN-MEKK1, inhibits iNOS promoter activation by
IFN- + LPS.
A pharmacological inhibitor of the JNK pathway is not available
commercially. To investigate the role of the MEKK1-MKK4-JNK pathway in
iNOS expression by LPS, we used a reporter gene assay in which RAW
264.7
NO(
) cells were cotransfected with 0.3 µg of an iNOS
promoter-luciferase plasmid (iNOS-luc) and DN-MEKK1 or DN-MKK4
(10, 28, 45, 58). After transfection and growth for
72 h, the cells were stimulated with IFN-
(10 U/ml) + LPS (l ng/ml) for 8 h, and luciferase activity in cell lysates was measured. As shown in Fig. 6A,
transfection of the iNOS-luc alone followed by IFN-
+ LPS
stimulation resulted in an ~20-fold induction of luciferase activity
compared with unstimulated cells. However, in cells cotransfected with
the DN-MKK4, there was a significant decrease in luciferase activity
(Fig. 6A). We next determined the effects of DN-MEKK1 on
iNOS promoter regulation by IFN-
+ LPS. After transfection of
iNOS-luc with or without DN-MEKK1, RAW 264.7
NO(
) cells were
stimulated with IFN-
(10 U/ml) + LPS (l ng/ml) for 8 h. In
contrast to the inhibitory effects observed with the DN-MKK4 construct,
DN-MEKK1 did not inhibit iNOS promoter activity after stimulation with
IFN-
+ LPS (Fig. 6B).
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DN-c-Jun does not inhibit iNOS promoter activity by IFN-
+ LPS.
Preliminary work by others with truncated forms of the iNOS-luc
constructs suggested that AP-1 was not involved in its regulation by
IFN-
+ LPS (30, 55). However, the significant
inhibition of iNOS promoter activity by DN-MKK4 in IFN-
+ LPS-treated cells raises the question of how the JNK pathway may be
regulating iNOS expression by LPS. One possibility is that c-Jun
participates as a trans-activating factor, via AP-1 or in
association with another transcription factor. Thus we tested the
effects of a DBD-c-Jun, which contains the DNA-binding domain but lacks
the transactivating domain, in the iNOS-luc reporter assay. With
IFN-
+ LPS stimulation, there was no significant difference in
iNOS promoter activity in the presence or absence of DBD-c-Jun (Fig. 7). Therefore, although the MKK4-JNK
pathway plays a positive regulatory role in the promoter activity of
iNOS stimulated with IFN-
+ LPS, c-Jun does not appear to be
involved.
|
SB-203580 augments JNK phosphorylation and activation by
IFN- + LPS.
p38mapk has been shown to inhibit ERK
activation (23). Thus the dichotomy we observed in
IFN-
+ LPS-stimulated iNOS induction showing the inhibitory
effect of p38mapk vs. the enhancing effect of
JNK and ERK raises the possibility that MAPK interaction may also be
occurring. Therefore, we examined the effects of SB-203580 on
IFN-
+ LPS-induced JNK and ERK phosphorylation and the effects
of PD-98059 on the phosphorylation of JNK and p38mapk. RAW 264.7
NO(
) cells were
pretreated with SB-203580 (15 and 30 µM) or PD-98059 (15 and 30 µM)
for 1 h and then costimulated with LPS (100 ng/ml) + IFN-
(10 U/ml) for an additional 1 h. After the cells were lysed and
the lysates were normalized for protein content, a portion was
separated by SDS-PAGE and immunoblotted with phospho-specific JNK, ERK,
or p38mapk antibody. As expected, PD-98059
inhibited phosphorylation of ERK (Fig.
8A). PD-98059 also did not
affect TXY phosphorylation of p38mapk or JNK by
LPS + IFN-
(Fig. 8, B and C). As shown in
Fig. 8B, SB-203580 is not expected to inhibit the
phosphorylation of p38mapk in a dose-responsive
fashion, although we have shown that it does inhibit
p38mapk activity (5). Inhibition of
p38mapk with SB-203580 also did not affect the
phosphorylation of ERK by LPS + IFN-
(Fig. 8A).
However, SB-203580 substantially and consistently increased the
phosphorylation of Thr-183 and Tyr-185 residues of both JNK isoforms
(Fig. 8C). To determine whether this augmentation in
phosphorylation of JNK by SB-203580 is also accompanied by an increase
in JNK activity, another portion of the whole cell lysate was subjected
to a solid-phase in vitro kinase assay with c-Jun-GST beads as the
substrate. As shown in Fig. 9
(top), IFN-
+ LPS-induced JNK activity was
significantly augmented by SB-203580. Because JNK activation also
correlates with the binding of JNK to c-Jun (4), we
immunoblotted the nitrocellulose membrane with p46 JNK1 antibody. As
shown in Fig. 9, bottom, the amount of p46 JNK1 bound to
c-Jun correlated with JNK activity, although the amount was modest.
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DISCUSSION |
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A greater understanding of the signaling pathways involved in the
regulation of iNOS by LPS may have potential therapeutic implications
in a number of disorders in which LPS and NO· play important
deleterious or beneficial roles, such as septic shock and host defense
against microbes. Although others have also investigated the role of
the MAPKs in iNOS regulation by various stimuli, the role of the JNK
pathway has not been systematically examined with LPS stimulation. A
broad conclusion that can be drawn from these works is that, depending
on the stimulus and cell type, MAPKs may play a positive, negative, or
neutral regulatory role in iNOS expression (2, 7, 14, 18, 47,
51). Bhat and coworkers (2) examined the role of
ERK and p38mapk in the expression of NO· by
LPS in primary glial cells. Similar to our findings, they showed that
inhibition of MEK1-ERK suppressed NO· and iNOS expression. However,
in contrast to the present study, they found that
p38mapk inhibition also inhibited NO·
expression by LPS. Chen and Wang (6) recently reported
that p38mapk augmented iNOS induction by LPS but
that ERK was not involved. Although this finding is opposite to what we
observed, they used an RAW 264.7 cell lineage distinct from that used
in this study. In addition, these investigators stimulated their
macrophages with an amount of LPS that was 1 × 103-1 × 106 greater than the amount
we used. Further evidence that the experimental conditions were
different is that they noted maximal activation of the MAPKs after 10 min of stimulation; in contrast, we did not see any MAPK activation
until after ~30 min of stimulation, which peaked only after 60 min of
stimulation. Ajizian et al. (1) noted that inhibition of
p38mapk or ERK blocked iNOS and TNF-
accumulation in macrophages stimulated with IFN-
+ LPS.
Although their data are consistent with ours in regard to TNF-
production, their data are opposite to our observation of iNOS-NO·
augmentation with inhibition of p38mapk.
Although this discrepancy may also be due to the fact that different lineages of RAW 264.7 cells were used (39), these
investigators measured only iNOS mRNA, and
p38mapk has been shown to also have
posttranscriptional effects (27). Others have also noted
that the role of p38mapk is cell and/or stimulus
specific. For example, Da Silva and coworkers (7)
demonstrated that p38mapk is necessary, but not
sufficient, for iNOS induction by TNF-
+ IL-1
stimulation in
mouse astrocytes. In contrast, Guan et al. (18) found that
p38mapk inhibited NO· expression by IL-1
in
rodent mesangial cells, an effect similar to our observation with
IFN-
+ LPS stimulation.
Here we have demonstrated that the MEK1-ERK and MKK4-JNK pathways
augmented and p38mapk inhibited IFN- + LPS induction of iNOS/NO·. These modulatory effects of the MAPKs
raise the question of how they ultimately affect the transcription of
iNOS with IFN-
+ LPS stimulation. We previously showed that ERK
mediated TNF-
expression on engagement of the Fc
receptor
(44). Thus we reasoned that a possible mechanism by which
ERK augments iNOS induction is through its ability to signal LPS
induction of TNF-
or IL-1
, cytokines that are capable of inducing
NO· expression. Our demonstration that TNF-
expression with
IFN-
+ LPS stimulation is ERK dependent would lend credence to
this hypothesis. The fact that there was only modest inhibition of
NO
expression by PD-90859 is perhaps due to the low
copy number of p55 TNF receptor on the surface of RAW 264.7 cells
(12). This finding was corroborated by experiments in
which neutralization of TNF-
with anti-TNF-
antibody resulted in
a reduction of IFN-
+ LPS-induced NO
expression by ~33% (Fig. 5B), a level of inhibition
similar to that seen with PD-98059 (Fig. 4A). Neutralization
of IL-1
with anti-IL-1
antibody had no effect on IFN-
+ LPS-induced NO
expression, consistent with the fact
that RAW 264.7 cells respond very poorly to IL-1
(25).
Previous works have shown that the three nonconsensus AP-1 sites in the
5'-flanking region of the iNOS promoter were not involved in enhancing
iNOS transcription by LPS (30, 56). The lack of inhibition
of iNOS promoter activity by DBD-c-Jun would substantiate this finding.
Because previous studies have shown that NF-B is a critical
regulator of iNOS transcription by LPS, potential mechanisms for the
role of MKK4-JNK and their feasibility are as follows: 1)
JNK itself has been shown to bind the c-Rel component of NF-
B and to
enhance NF-
B activation (37). Moreover, in an
overexpression system, transfected JNK was able to activate a CAT
reporter gene driven by two
B elements (37). In a
recent report, Janssen-Heininger and coworkers (22) showed
that the JNK pathway was important in the activation of NF-
B by
oxidants and TNF-
in lung epithelial cells. 2) Another
possibility, although not mutually exclusive, is that the bZIP region
of c-Jun may bind directly to the p65 subunit of NF-
B to enhance the
binding of NF-
B to its
B element (48). Although
DBD-c-Jun would be expected to inhibit AP-1 activity, it is not known
whether it would also be capable of inhibiting any NF-
B-c-Jun
interaction, because the mutated c-Jun still contains the bZIP region
that is capable of binding the p65 subunit (48). 3) Although JNK is best known for transactivating c-Jun, it
may also directly or indirectly activate a number of other
transcription factors, including ATF-2, Elk-1, cAMP-responsive element
binding protein, and NF-
B (19, 22, 37, 52). Thus JNK
may activate other uncharacterized transcription factors that may be
directly or indirectly involved in transcriptional control of
LPS-induced iNOS. For example, Welsh (51) showed that JNK1
enhanced IL-1
induction of iNOS by activating ATF-2, which then
physically cooperated with NF-
B to facilitate binding of NF-
B to
its
B sites on the iNOS promoter. More recently, Sawada and
colleagues (46) suggested that, with IL-6 stimulation of
iNOS expression, an octomer-binding protein may physically associate
with and synergize NF-
B activity.
We previously found that, in NIH/3T3 fibroblasts, DN-MEKK1 inhibited
iNOS promoter activity with IFN- + TNF-
stimulation (5). However, we did not find such an effect in RAW
264.7
NO(
) cells stimulated with IFN-
+ LPS. Although MEKK1
has been implicated in the activation of JNK, more recent studies have
shown that other MAPKKK such as germinal center kinases, mixed-lineage
kinases, p21-activated kinases, tyrosine phenol-lyases, SPRK, and other isoforms of MEKK may also participate in the activation of the JNK
pathway, depending on the cell types, stimuli, and culture conditions
(13, 42).
We found an inhibitory role for p38mapk in the
induction of iNOS-NO· by IFN- + LPS. This inhibition by
p38mapk is clearly not due to an inhibition of
IFN
+ LPS induction of TNF-
, because
p38mapk also enhanced IFN-
+ LPS-induced
TNF-
protein expression. Because the MKK4-JNK and MEK1-ERK pathways
augmented iNOS induction by IFN-
+ LPS, we reasoned that a
plausible mechanism of how p38mapk inhibited
IFN-
+ LPS-induced NO
expression is by
inhibiting JNK and/or ERK activation. There is precedence for this
notion that one MAPK may affect the activity of another, because
p38mapk has been shown to inhibit ERK activation
by platelet-derived growth factor or tryptase in airway smooth muscle
cells (23). We therefore explored this possibility and
demonstrated that inhibition of p38mapk
substantially augmented IFN-
+ LPS-induced JNK phosphorylation and activation, providing further insight into the realm of MAPK interactions. Recently, p38mapk was found to
inhibit ERK1- and ERK2-mediated growth of Kaposi's sarcoma cells
(38). Marinissen and colleagues (32) showed an even more complex interaction between JNK,
p38mapk, and ERK5 in the activation of the
c-jun promoter after engagement of muscarinic M1 receptors.
In summary, we have shown that ERK and JNK signal transduction pathways
augmented and p38mapk inhibited iNOS-induction
by IFN- + LPS. Cotransfection studies with the iNOS-luc
reporter gene and dominant-inhibitory mutant proteins of the
MEKK1-MKK4-JNK-c-Jun pathway suggest that although MKK4-JNK appears to
augment IFN-
+ LPS-induced iNOS promoter activity, MEKK1 and
c-Jun do not. These findings imply that, in RAW 264.7
NO(
) cells,
another MAPKKK activates MKK4 with IFN-
+ LPS stimulation and
that the role of MKK4-JNK is due to an AP-1-independent event. Future
studies in the manipulation of these signaling pathways in which LPS
and NO· play critical roles may shed new light on various disease
processes such as endotoxin-mediated septic shock.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Kristin R. Morris, Linda K. Remigio, and Cheryl Leu for expert technical assistance. We are also grateful to Boyd Jacobson, Barry Silverstein, and Nadia de Steckelberg for illustrative assistance.
![]() |
FOOTNOTES |
---|
E. D. Chan is supported by National Heart, Lung, and Blood Institute Clinical Investigator Development Award 1K08 HL-036250-01, the Bettina Garthwaite Lowerre Foundation for Mycobacteriology Research, and the Parke-Davis Atorvastatin Research Grant. D. W. H. Riches is supported by National Heart, Lung, and Blood Institute Grant HL-55549 and Specialized Center of Research Grant HL-56556.
Address for reprint requests and other correspondence: E. D. Chan, K613e, Goodman Bldg., National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206 (E-mail: chane{at}njc.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 July 2000; accepted in final form 25 September 2000.
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