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and TNF-
in Mouse Macrophages1
,¶,§,*,
*
Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206;
Department of Biochemistry and Molecular Genetics,
Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine,
§
Department of Pharmacology, and
¶
Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262; and
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Faculty of Medicine, Division of Critical Care Medicine, University of Calgary, Calgary, Alberta, Canada
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and a number of
stimuli, including TNF-
. Recent work has shown that TNF-
activates members of the mitogen-activated protein kinase family that
subsequently trans-activate transcription factors
implicated in the regulation of iNOS expression. The objective of this
study was to systematically evaluate the role of: 1)
p42mapk/erk2, 2) p46 c-Jun
NH2-terminal kinase/stress-activated protein kinase (p46
JNK/SAPK), and 3) p38mapk in the
induction of iNOS expression during costimulation of mouse macrophages
with IFN- and TNF-. All three kinases were activated during
costimulation with IFN- and TNF-. However, specific antagonism of
the p42mapk/erk2 and
p38mapk with PD98059 and SKF86002,
respectively, had no effect on the induction of iNOS expression. In
contrast, blockade of all three kinases with
N-acetylcysteine completely blocked the induction of iNOS
expression. In addition, specific antagonism of the JNK/SAPK upstream
kinases MEKK (mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase kinase) and MKK4 (mitogen-activated
protein kinase kinase 4) with dominant inhibitory mutants blocked
transcriptional activation of the iNOS promoter in response to
costimulation with IFN- and TNF-. Collectively, these findings
support the involvement of p46 JNK/SAPK and its upstream kinases in
regulating the induction of iNOS following ligation of the TNF-
receptor CD120a (p55) in the presence of IFN-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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iNOS expression is induced in macrophages following costimulation with
IFN- and bacterial LPS (11), IFN- and TNF- (12, 13, 14), IFN-
and phorbol ester (15), and a variety of other stimuli (16, 17, 18, 19, 20) that
initiate transcription of iNOS through the activity of several
critically important signal-transduction mechanisms that result in the
trans-activation of the appropriate transcription factors.
The cloning and subsequent analysis of the promoter of the iNOS gene
have revealed two regions that are required for the synergistic
activation of transcription of iNOS mRNA during costimulation with
IFN- and LPS (3, 21). The response to IFN- has been shown to be
localized between positions -913 and -1029 (22). This region contains
a cluster of motifs characteristic of IFN--responsive genes,
including IFN--activated sequence, IFN-stimulated response element,
and IRF element. In work reported by Martin et al. (23), site-directed
mutagenesis of these sequences and electrophoretic mobility shift
assay (EMSA) analyses of nuclear extracts of IFN--stimulated
macrophages have revealed that an IRF element located between positions
-913 to -923 that binds IRF-1 is necessary for the IFN-
enhancement of iNOS transcription. In addition, macrophages obtained
from mice with targeted disruptions of the genes encoding 1) STAT1 or
2) IRF-1 are incapable of expressing iNOS in response to costimulation
with IFN- and LPS (22, 24, 25). Thus, the mechanism underlying the
induction of iNOS appears to involve the IFN--dependent activation
of STAT1, leading to the transcription and translation of IRF-1, which
then indirectly mediates the enhancement of iNOS transcription. The
region of the iNOS promoter required for the response to LPS (region I)
has been localized between positions -48 and -209 and shown to
encompass a critically important NF-
B motif that binds a
trans-activating complex of p50 and c-rel (19, 26).
In contrast to the comprehensive understanding of the
mechanisms underlying the IFN- and LPS induction of iNOS,
comparatively little is known about how TNF- stimulates iNOS
expression in the presence of IFN- and, in particular, the
signal-transduction mechanisms that couple ligation of the TNF-receptor
CD120a (p55) to the activation of iNOS expression. Work conducted in
this laboratory has focused on understanding the early signaling events
that are activated in macrophages in response to ligation of CD120a
(p55). This work has revealed the rapid, transient, concomitant, and
preferential activation of specific members of the MAP kinase family,
namely p42mapk/erk2 (27), p46 JNK/SAPK
(28), and p38mapk (29). Furthermore, we
have shown that the upstream MAPK kinases MEK1 (30), MKK4, and MKK3
(29) are all activated in response to stimulation with TNF-, as is
the MAPK kinase, MEKK1 (31). However, while these studies have revealed
much about signaling heterogeneity in macrophages, they raise the
obvious question as to its role, if any, in the initiation of
downstream macrophage responses, including the induction of iNOS
expression. Therefore, in the present study, we have investigated the
potential involvement of the ERK, JNK/SAPK, and
p38mapk subfamilies of MAPKs in the
induction of iNOS expression at both the mRNA and catalytic level. Our
data support the conclusion that while activation of all three
subfamilies of MAPKs occurs in macrophages costimulated with IFN-
and TNF-, the activation of the p46 JNK/SAPK subfamily and its
upstream kinases is necessary for the activation of iNOS expression and
NO2- production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C3H/HeJ mice were used throughout the study and were bred at the
National Jewish Biological Resource Center. FBS was purchased from
Irvine Scientific (Santa Ana, CA). Glutathione-Sepharose beads
were purchased from Pharmacia (Piscataway, NJ). Protein A-Sepharose
beads and N-acetylcysteine (NAC) were purchased from Sigma
(St. Louis, MO). Enhanced chemiluminescence assay kits were obtained
from Amersham Life Sciences (Arlington Heights, IL). Rabbit polyclonal
p38mapk Ab, recombinant
c-Jun1–79 -GST, and dominant-negative MEKK1 mutant in
a pcDNA3 expression vector were generously provided by Dr. Gary Johnson
(National Jewish Medical and Research Center, Denver, CO). The
dominant-negative MKK4 (JNKK1) mutant (K116R) in an LNCx expression
vector was a kind gift from Dr. Lynn Heasley (University of Colorado
School of Medicine, Denver, CO). Rabbit polyclonal anti-p46 JNK and
rabbit polyclonal anti-p42mapk/erk2 Abs
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Recombinant mouse TNF- and IFN- were generously provided by
Genentech (San Francisco, CA). [-32P]ATP (>3000
Ci/mmol) was purchased from NEN Research Products DuPont (Wilmington,
DE). p38mapk inhibitor (SKF86002-A2) was a
generous gift from Smith Kline Beecham (King of Prussia, PA), and the
MEK1 inhibitor (PD98059) was purchased from New England Biolabs
(Beverly, MA). SB203580 (also a p38mapk
inhibitor) was purchased from Calbiochem (San Diego, CA). The iNOS cDNA
probe and iNOS-luciferase reporter construct were generously provided
by Dr. Charles Lowenstein (The Johns Hopkins School of Medicine,
Baltimore, MD). IRF-1 and GAPDH cDNA probes were generous gifts of Drs.
Jack Routes and John Shannon, National Jewish Medical and Research
Center, respectively. The firefly luciferase reporter assay system was
purchased from Promega (Madison, WI). The CAT ELISA kit was obtained
from Boehringer Mannheim (Indianapolis, IN). The LipofectAMINE reagent
was purchased from Life Technologies (Gaithersburg, MD). All other
reagents were of the highest purity.
Macrophage isolation and culture
Monolayers of mouse bone marrow-derived macrophages were
prepared as previously described (32). Briefly, the cells were cultured
in DMEM containing penicillin (100 U/ml), streptomycin (100 µg/ml),
10% (v/v) heat-inactivated FBS, and 10% (v/v) L929 cell-conditioned
medium as a source of CSF-1 at a density of 2.4 x 105
cells/cm2 at 37°C in a 10% (v/v) CO2
atmosphere for 5–6 days. For determination of MAPK activities, the
CSF-1-containing medium was replaced with CSF-1-free medium alone on
day 5 of cell growth (DMEM with penicillin (100 U/ml), streptomycin
(100 µg/ml), and 0.1% (v/v) heat-inactivated FBS) or with IFN-
(10 U/ml) for 18 h before stimulation with 10 ng/ml of TNF-.
Treatment of cells with NAC was as previously described (33). Cells
were treated with NAC for 4 h, followed by stimulation with
TNF- at 10 ng/ml for 10 min.
Analysis of NO2- accumulation
Nitrite anion (NO2-) accumulation by macrophage
monolayers was determined as previously reported (13). Briefly,
macrophage monolayers were stimulated with TNF- (10 ng/ml) and
IFN- (10 U/ml) for 18 h. A total of 100 µl of supernatant was
combined with an equal volume of Greiss reagent, and the samples were
incubated at room temperature for 10 min before quantifying the
absorbance at 550 nm. Using a standard curve, the nmol of
NO2- produced was determined and normalized to total
cell number in each sample.
Northern blot analysis
The expression of iNOS mRNA was determined by Northern blot analysis. The extraction, purification, electrophoresis, and transfer of the RNA to nitrocellulose membranes were conducted as described (32). Briefly, macrophage monolayers were extracted with 4 M guanidine isothiocyanate, and the RNA was purified by centrifugation through 5.7 M cesium chloride at 100,000 x g for 18 h. A total of 15 µg of total RNA was electrophoresed under denaturing conditions through a 1% (w/v) agarose-formaldehyde gel, and then transferred to Nytran membranes. The blots were hybridized with 5 x 106 dpm of 32P-labeled iNOS, IRF-1, or GAPDH cDNA probes, and autoradiograms were prepared by exposure to Kodak XAR-5 film at -70°C.
Determination of ERK and p38mapk activities
To determine ERK or p38mapk
activity, the cells were lysed in 500 µl of RIPA lysis buffer (50 mM
Tris/HCl buffer, pH 7.2, containing 0.1% (w/v) SDS, 150 mM NaCl, 0.5%
(w/v) deoxycholate, 1% (v/v) Triton X-100, 10 mM sodium pyrophosphate,
25 mM ß-glycerophosphate, 2 mM Na3VO4, and
2.1 µg/ml aprotinin). The cell lysates were precleared with 15 µl
of protein A-Sepharose beads, and the protein contents were normalized
between samples by the BCA protein assay.
p42mapk/erk2 was immunoprecipitated from
precleared lysates with 0.9 µg of
anti-p42mapk/erk2 Ab and 15 µl of
protein A-Sepharose beads at 4°C for 2 h. Similarly,
p38mapk was immunoprecipitated with 2 µl
of anti-p38mapk antiserum. The beads
were washed twice with RIPA lysis buffer and twice with PAN buffer (10
mM PIPES buffer, pH 7, containing 100 mM NaCl and 21 µg/ml
aprotinin). Both ERK and p38mapk activities
were assessed by resuspending the beads in 50 µl of kinase buffer (20
mM HEPES buffer, pH 7.6, containing 20 mM MgCl2, 20 µM
ATP, 100 µM Na3VO4, 2 mM DTT, 25 mM
ß-glycerophosphate), and 16 µCi [-32P]ATP and 500
ng ATF-2 as substrate. The reactions were conducted for 30 min at
30°C and terminated with an equal volume of 2x Laemlli sample buffer
containing 20 mM DTT and boiled for 5 min. The reaction mixtures were
then separated by SDS-PAGE through a 12% polyacrylamide gel, and
proteins were transferred to nitrocellulose. 32P-labeled
ATF-2 was detected by autoradiography.
Determination of JNK/SAPK activity
For measurement of JNK/SAPK activity, the macrophage monolayers
were lysed at 4°C with 500 µl of ice-cold lysis buffer (50 mM
Tris/HCl, pH 8, containing 137 mM NaCl, 10% (v/v) glycerol, 1% (v/v)
Nonidet P-40, 1 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 2
mM Na3VO4, and 1 mM PMSF (34)). After the
protein content was normalized between samples, JNK/SAPK in each sample
of lysate was bound to 15 µl of a 1:1 slurry of lysis
buffer:GST-c-Jun1–79-Sepharose beads and incubated at
4°C for 2 h. The beads were then washed twice with 500 µl
lysis buffer and twice with 500 µl JNK/SAPK buffer (20 mM HEPES
buffer, pH 7.2, containing 30 mM ß-glycerophosphate, 10 mM
p-nitrophenylphosphate, 10 mM MgCl2, 0.5 mM DTT,
and 50 µM Na3VO4). The activity of JNK/SAPK
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/SAPK buffer incubated at 30°C for 30 min.
The kinase reactions were then stopped with an equal volume of 2x
Laemlli sample buffer containing 20 mM DTT 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 (35). 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% (v/v) Tween 20), blocked overnight with 5% (w/v) BSA, and probed according to the method described by Towbin et al. (35) with rabbit polyclonal p46 JNK/SAPK, p42/p44mapk/erk2/1, and p38mapk Abs in 5% (w/v) BSA dissolved in TTBS. Using horseradish peroxidase-conjugated secondary anti-rabbit Ab, bound Abs were detected by enhanced chemoluminescence.
Transient transfection and luciferase assay
NIH3T3 fibroblasts were plated to a density of 1 x
106 cells per 6-well plate in DMEM containing penicillin
(100 U/ml), streptomycin (100 µg/ml), and 10% (v/v) heat-inactivated
FBS. After 24 h of growth to 
30–40% confluence, the cells
were transfected with plasmids, as described in the manufacturer’s
(Life Technologies) protocol. Briefly, 0.3 µg of iNOS-luciferase
plasmid was combined with 2 µg DN-MEKK or DN-MKK4 plasmid, 1 µg CAT
reporter plasmid that contained an SV40 basic enhancer, 10 µl
LipofectAMINE reagent, and 100 µl Optimem serum-free medium.
The lipid-DNA mixture was incubated for 30 min at room temperature.
Each well was then washed with 2 ml Optimem medium and replaced with 1
ml LipofectAMINE-DNA mixture. After 5 h of incubation, 1 ml DMEM
containing 20% (v/v) 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 TNF-
(1 ng/ml) and IFN- (1 U/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.
Expression of CAT, used to normalize for transfection efficiency
between samples, was quantified using a commercially available kit.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To accurately reproduce the conditions that result in iNOS
expression, we investigated the effects of costimulation with IFN-
and TNF- on the activation of
p42mapk/erk2, p46 JNK/SAPK, and
p38mapk in mouse macrophages. As can be
seen in Fig. 1
, exposure to TNF- in
the presence of IFN- resulted in a modest, though consistent,
increase in the level of activation of both
p42mapk/erk2 and p46 JNK/SAPK compared with
cells exposed to TNF- in the absence of IFN-. In contrast, there
was no additional effect by IFN- plus TNF- on the activation of
p38mapk compared with that seen in the
absence of IFN-. Costimulation with IFN- and TNF- without
pretreating with IFN- for 18 h resulted in a pattern of
activation of the three MAPKs that was indistinguishable from that seen
with TNF- alone (data not shown). Unlike the effects of TNF-,
IFN- alone failed to stimulate MAPK activity. Thus, costimulation
with IFN- and TNF- initiates a qualitatively similar pattern of
activation of p42mapk/erk2, p46 JNK/SAPK,
and p38mapk to that seen in the absence of
IFN-.
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To begin to discriminate the roles of
p42mapk/erk2, p46 JNK/SAPK, and
p38mapk in the induction of iNOS expression
following costimulation with IFN- and TNF-, we investigated the
effects of the specific MAP kinase inhibitors PD98059, a specific
inhibitor of MEK1 (36), and SKF86002, a competitive inhibitor of
p38mapk (37). Specific pharmacologic
antagonists of p46 JNK/SAPK or its activators, MKK4, are currently not
available. Monolayers of mouse macrophages were pretreated with PD98059
or SKF86002 (30 µM each for 1 h) and stimulated with IFN- (10
U/ml), TNF- (10 ng/ml), and each of the inhibitors for 18 h
before quantifying NO2- levels in the culture
supernatants. As can be seen in Fig. 2,
neither PD98059 nor SKF86002 exhibited any significant inhibitory
effect on the induction of NO2- accumulation in
response to costimulation with IFN- and TNF-. In addition,
pretreatment of macrophages with the combination of both PD98059 and
SKF86002 (each at 30 µM), nor pretreatment with the alternative
p38mapk inhibitor SB203580, likewise, did
not affect the accumulation of NO2- in response to
costimulation with IFN- and TNF- (data not shown).
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(10 ng/ml, 10 min) and detergent lysis. The
activities of p42mapk/erk2 and
p38mapk were then quantified by
immunoprecipitation of the appropriate kinase, followed by in vitro
kinase assay using rATF-2 as substrate in the presence of
[-32P]ATP. As shown in Fig. 3, PD98059 and SKF86002 predictably and
substantially inhibited p42mapk/erk2 and
p38mapk activities, respectively. Given the
expected pattern and extent of inhibition of
p42mapk/erk2 and
p38mapk by PD98059 and SKF86002 and their
lack of inhibitory effect on the accumulation of
NO2-, it is reasonable to conclude that neither kinase
is involved in the induction of iNOS during costimulation with IFN-
and TNF-.
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The data presented above, while not directly addressing the role
of p46 JNK/SAPK, do not exclude a potential role for this kinase in the
induction of iNOS during costimulation with IFN- and TNF-.
Recently reported studies by Natoli et al. (33) have shown that the
antioxidant, NAC, dramatically inhibits p46 JNK/SAPK activation by
TNF- by interfering with signal transmission between TNF-associated
factor 2 and MEKK, both of which function as upstream regulators
of p46 JNK/SAPK activation. These observations thus afforded us the
opportunity to directly investigate the potential involvement of p46
JNK/SAPK in iNOS induction in response to costimulation with IFN-
and TNF-. First, we investigated the effects of NAC on
NO2- accumulation in response to costimulation with
IFN- and TNF-. Monolayers of mouse bone marrow-derived
macrophages were pretreated with increasing concentrations of NAC
(1–40 mM for 4 h) before stimulation with IFN- (10 U/ml) and
TNF- (10 ng/ml) in the continued presence of NAC for 18 h. As
can be seen in Fig. 4, increasing
concentrations of NAC resulted in a concentration-dependent inhibition
of NO2- accumulation that was detected at
concentrations greater than 10 mM and was essentially complete at 40
mM. To confirm that the level of inhibition of NO2-
accumulation was also reflected by a concomitant reduction in the level
of iNOS, macrophage monolayers were exposed to IFN- and TNF-, as
described above, in the presence and absence of a fixed concentration
of NAC (40 mM), and iNOS expression was determined by Northern blot
analysis. As can be seen in Fig. 5,
costimulation with IFN- and TNF- in the presence of NAC led to a
substantial inhibition of iNOS mRNA expression. To verify that the
inhibition of iNOS expression by NAC was not due to any interference of
IFN- signal transduction, we also determined the effect of the
antioxidant on IRF-1 expression. As shown in Fig. 5, NAC had no effect
on the induction of IRF-1 mRNA expression by IFN- or on basal
expression of GAPDH mRNA. Similarly, NAC was found to have no effect on
the induction of MHC class II expression by IFN-, as determined by
cytofluorography (data not shown). Furthermore, NAC exhibited no toxic
effects on mouse macrophages, as detected by light microscopy (Fig. 6) and trypan blue dye exclusion (data
not shown). Interestingly, however, NAC did block the shape change that
is characteristically found to accompany the induction of iNOS
expression by macrophages (Fig. 6).
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(10 ng/ml) for 10 min. The cells
were then lysed and assayed for
p42mapk/erk2, p46 JNK/SAPK, and
p38mapk activities by either
immunoprecipitation and in vitro kinase assay
(p42mapk/erk2 and
p38mapk) or solid-phase binding to
GST-c-Jun1–79 in an in vitro kinase assay (p46 JNK/SAPK).
As shown in Fig. 7, pretreatment with NAC
almost completely inhibited the activation of
p42mapk/erk2, p46 JNK/SAPK, and
p38mapk in response to stimulation with
TNF-. Thus, while individual inhibition of
p42mapk/erk2 and
p38mapk failed to block iNOS expression,
inhibition of all three kinase cascades was associated with a complete
inhibition of iNOS expression and NO2- accumulation.
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and IFN-
To further explore the concept that the MEKK
JNKKJNK/SAPK
pathway is a necessary component in signaling iNOS induction by
TNF-, we investigated the ability of dominant-negative mutants of
MEKK1 and MKK4 that are catalytically inactive, but are still able to
bind to their respective substrates, to influence iNOS promoter
activity in a luciferase reporter assay system in NIH3T3 cells. A
number of macrophage cell lines were initially screened for suitability
in terms of both transfection and responses to IFN- and TNF-.
However, most were found to have very low transfection efficiencies.
The RAW 264.7 cell line was reasonably transfectable, but exhibited a
significant increase in iNOS promoter activity with IFN- alone and,
due to the inherently low TNF-receptor number expressed on the cell
surface, these cells were poorly responsive to TNF- stimulation.
Therefore, we examined the response of NI H3T3 cells to TNF-,
IFN-, or both cytokines in both the presence and absence of the MAPK
inhibitors, and found that these cells behaved in a fashion that was
similar to mouse bone marrow-derived macrophages with respect to the
synergy between IFN- and TNF-, and to the pattern of inhibition
by PD98059, SFK86002, and NAC (Fig. 8,
A and B). Thus, these cells were used in the
reporter assay with the iNOS-luciferase construct. After determining
that the expression of a CAT reporter plasmid driven by an SV40
enhancer element was not altered by cytokine stimulation (data not
shown), the CAT reporter plasmid was cotransfected to normalize for
transfection efficiency. As shown in Fig. 9, both DN-MEKK1 and DN-MKK4 mutants
markedly inhibit iNOS promoter activity upon stimulation with TNF-
and IFN- when compared with their respective empty expression
vector.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and LPS have been substantially defined, the
mechanisms that mediate induction of iNOS during costimulation with
IFN- and TNF- have not previously been systematically
investigated. The goal of this study was to investigate the involvement
of members of the MAP kinase family of Ser-Thr protein kinases in iNOS
induction by IFN- and TNF-. The major findings are that: 1) while
selective blockade of p42mapk/erk2 and
p38mapk activation, either alone or
together, had no effect on the induction of NO2-
production, blockade of the activation of all three enzymes almost
completely inhibited the induction of iNOS expression and
NO2- production, and 2) dominant-negative mutants of
MEKK1 and MKK4 blocked luciferase reporter gene expression driven by
the iNOS promoter. Collectively, these data suggest that: 1)
coordination of signals arising from both the IFN- receptor and the
TNF receptor, CD120a (p55), is necessary for activation of iNOS
transcription, and 2) during costimulation with IFN-, the
TNF--dependent activation of kinases leading to the activation p46
JNK/SAPK, including the upstream kinases MEKK and MKK4, is necessary
for the induction of iNOS expression.
To probe the role of each MAPK in the initiation of iNOS and
NO2- expression, we utilized specific pharmacologic
antagonists that inhibit the phosphorylation of MEK1 and
p38mapk. When macrophages were pretreated
with each antagonist, either alone or together, and then costimulated
with IFN- and TNF-, NO2- production was detected
at levels that were indistinguishable from cells stimulated in the
absence of inhibitors. However, specific catalytic assays of
p42mapk/erk2 and
p38mapk revealed that, as expected, PD98059
and SKF86002 fully inhibited the activity of these kinases,
respectively. These findings thus indicate that while both
p42mapk/erk2 and
p38mapk are activated in response to
costimulation with IFN- and TNF-, neither kinase, either alone or
in combination, is necessary for the activation of iNOS expression.
Although a specific pharmacologic antagonist of p46 JNK/SAPK is
not available, NAC, an antioxidant whose mechanism of action involves
elevating intracellular glutathione levels, has recently been shown to
block the activation of JNK/SAPKs in response to TNF- (33) by
interfering with signal transmission between the CD120a (p55)
receptor-associated protein TNFR-associated factor 2 and the
downstream kinase MEKK. Previous work from this and other laboratories
has shown MEKK to be rapidly activated by TNF- in mouse macrophages
and to subsequently phosphorylate and activate MKK4/JNKK/SEK1, the
upstream regulator of p46 JNK/SAPK (33, 38, 39, 40). Pretreatment of mouse
macrophages with NAC resulted in an inhibition of iNOS expression and
NO2- production in response to costimulation with
IFN- and TNF-. While others have shown that NAC does not
interfere with TNF--receptor binding to TNF- (41), specific
catalytic assay of p42mapk/erk2,
p38mapk, and p46 JNK/SAPK revealed that NAC
blocked the activation of all three kinases, an effect that is novel
and consistent with data suggesting that its level of action is
proximal in the signaling cascade. Since specific antagonism of the
p42mapk/erk2 and
p38mapk cascades was without effect on iNOS
expression, these findings suggest that p46 JNK/SAPK and/or its
specific upstream kinases and regulators may play a role in mediating
the TNF--induced activation of iNOS expression in the presence of a
functional IFN- signal-transduction pathway. Importantly, we also
showed that NAC had no effect on IFN- signal transduction, as shown
by its lack of effect on IRF-1 expression in response to IFN-.
To obtain more specific indications that p46 JNK/SAPK and its upstream
kinases are involved in iNOS induction, we investigated the effects of
dominant-negative mutants of MEKK and MKK4 on iNOS promoter activity in
a transient luciferase reporter gene assay. Our data showed that both
mutants of this pathway, DN-MEKK1 and DN-MKK4, substantially inhibited
iNOS promoter activity, establishing the importance of this kinase
cascade in iNOS expression by TNF-. These findings also reveal a
clearer understanding of the potential transcription enhancer
element(s) that may be utilized by TNF- in iNOS expression.
The findings of the present study raise the important question of
how activation of p46 JNK/SAPK and its upstream regulators may serve to
initiate iNOS expression during costimulation with IFN- and TNF-.
Although the cis elements involved in the activation of iNOS
expression by TNF- have not been rigorously defined, TNF- has
been shown to activate two important trans-acting factors
represented by cognate cis elements in the 5'-flanking
region of the iNOS gene, namely NF-B and AP-1. The NF-B
cis element beginning 75 bases proximal to the
transcriptional start site has been shown to be necessary for iNOS
expression in response to costimulation with IFN- and LPS (11). In
contrast, putative AP-1 sites beginning at bases -481 and -1063
relative to the transcription start site are not required for LPS
induction of iNOS expression, although they may have the potential to
contribute to TNF--induced expression of this gene. Furthermore, the
potential utilization of NF-B and AP-1 is not mutually exclusive, as
Stein et al. (42) have shown that the p65 subunit of NF-B and
components of the AP-1 complex may form a larger complex that
synergizes to further enhance transcription of the HIV-long terminal
repeat promoter. Similarly, a cooperative interaction between
NF-B and Sp1 enhancers on the HIV long terminal repeat
promoter was also demonstrated (43).
Recently, reported studies have provided strong direct evidence in
support of an involvement of p46 JNK/SAPK and the upstream kinase MEKK
in the regulation of IB phosphorylation and the susbsequent
activation of NF-B. In studies aimed at determining the involvement
of JNK1 in NF-B activation, Meyer and colleagues (44) showed that
JNK1 interacted with c-Rel both in coimmunoprecipitation and yeast
two-hybrid assays. While JNK1 did not directly phosphorylate c-Rel,
NF-B, or IB, it was suggested that JNK1 may phosphorylate other
c-Rel-associated proteins that may be important in the activation of
NF-B. In addition, transient transfection of Jurkat cells with
expression vectors containing constitutively active MEKK or JNK1 in
combination with an NF-B-CAT reporter construct revealed that either
MEKK or JNK1 was capable of inducing transcription through NF-B
activation and that there was synergy when both MEKK and JNK1 were
cotransfected (44). Hirano et al. (45) also showed in NIH3T3 cells that
a constitutively active mutant of MEKK activates NF-B in a fashion
that was blocked by coexpression of IB. Of direct significance to
TNF--induced activation of NF-B, transfection with a
dominant-negative kinase-deficient mutant of MEKK blocked the ability
of TNF- to activate an NF-B reporter gene in NIH3T3 cells. More
recently, Lee et al. (46) also demonstrated that MEKK1 was a critical
component in NF-B activation by TNF- through its ability to
directly activate IB kinase. Thus, these studies lend credence to
the notion that both p46 JNK/SAPK and its upstream kinase MEKK play an
important role in the activation of NF-B by TNF-. Although AP-1
is not considered to contribute much, if at all, in iNOS regulation by
LPS or phorbol esters (19, 21, 47), its role in TNF- regulation of
iNOS has not been established. However, activation of p46 JNK/SAPK is
well known to promote transactivation and homodimerization of c-Jun by
catalyzing the phosphorylation of Ser63 and
Ser73 of c-Jun (48, 49, 50). Furthermore, NF-B may
synergize with other transcription factors, including AP-1 (42), Sp1
(43), or ATF-2 (51, 52, 53), to enhance transcription. Shapiro et al. (54)
have also shown there to be interplay between various transcription
factors in that the c-Rel regulation of IL-2 gene expression was
mediated indirectly through AP-1 activation. To add further
complexities to the potential role played by p46 JNK/SAPK in the
activation of iNOS expression, studies recently reported by Swantek et
al. (55) have shown that stimulation of RAW 264.7 cells with LPS
activates p42mapk/erk, p46 JNK/SAPK, and
p38mapk in an analogous pattern to that
observed in the present study with TNF-. However, only the
activation of p46 JNK/SAPK appeared to be involved in the regulation of
TNF- expression by LPS, through an effect on the translation of
TNF- transcripts and not on the transcription of the TNF- gene.
The specific role of the MAPKs in iNOS regulation has also been
investigated by others. In an insulin-producing cell line, JNK1 was
shown to be involved in IL-1ß induction of iNOS (53). Although
NF-B is necessary in the induction of iNOS by IL-1ß, JNK1 was not
considered to be involved in the signal transduction that resulted in
NF-B activation. Instead, it was hypothesized that activation of
ATF-2 by JNK1 resulted in this transcription factor synergizing with
NF-B to enhance iNOS transcription (53). Da Silva and collegues (52)
demonstrated that p38mapk is necessary, but
not sufficient, for iNOS induction by TNF- and IL-1 stimulation.
In contrast, p38mapk was shown to inhibit
iNOS induction by IL-1ß (56). Similarly, ERK 1 and 2 were shown to be
necessary in iNOS regulation by IL-1ß (57) or by IL-1ß and IFN-
(58), and yet were found not to be involved with TNF-/IL-1
stimulation (52) or LPS/IFN- induction of iNOS (59). In contrast, in
primary culture of glial cells, the induction of iNOS protein and
NO• expression by IFN- and LPS was shown to be
partially blocked by inhibiting
p42/44mapk/erk2/1 and
p38mapk with PD98059 or SB203580,
respectively, and almost completely blocked in the presence of both
inhibitors (60). This diversity in the role of the MAPKs in iNOS
regulation is most likely due to the complex regulation of iNOS
involving interactions between various transcription factors, as
determined by cell type and cytokine specificity. Thus, our present
findings that activation of MEKK and p46 JNK/SAPK by TNF- in the
context of a functional IFN- signal-transduction pathway results in
the regulation of iNOS expression may occur at more than one level.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. D. W. H. Riches, Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Neustadt Room D405, 1400 Jackson Street, Denver, CO 80206. E-mail address:
3 Abbreviations used in this paper: iNOS, inducible nitric oxide synthase; AP-1, activating protein-1; ATF, activating-transcription factor; CAT, chloramphenicol acetyltransferase; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione-S-transferase; IRF, interferon regulatory factor; JNK, c-Jun amino-terminal kinase; JNKK, c-Jun amino-terminal kinase kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase; MKK, mitogen-activated protein kinase kinase; NAC, N-acetylcysteine; NF-B, nuclear factor-B; NO2-, nitrite; SAPK, stress-activated protein kinase.
Received for publication June 25, 1998. Accepted for publication September 1, 1998.
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