From the Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1434
Received for publication, October 19, 2000
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ABSTRACT |
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Inducible nitric-oxide synthase (iNOS) is
an important signaling protein involved in the regulation of biological
processes (e.g. vasodilation, inflammation) and is subject
to transcriptional regulation by cytokines and lipopolysaccharide
(LPS). Full activation of the human iNOS (hiNOS) promoter by cytokines
(i.e., tumor necrosis factor- Nitric oxide (NO)1 is a
signaling molecule produced by a family of heme-containing nitric-oxide
synthases (NOS) concomitant with conversion of L-arginine
to L-citrulline (1). Neuronal NOS (NOS1) and endothelial
NOS (NOS3) are constitutively present in a variety of cell types, with
their activities sensitive to changes in intracellular calcium. Much
larger amounts of NO are produced in response to numerous extracellular
stimuli after induction of the calcium-insensitive iNOS (NOS2) (2). The
transient burst of NO production by iNOS has been implicated in
numerous biological processes (e.g. immune modulation,
control of vascular tone) (3-5).
Clues to the role of iNOS in physiologic and pathophysiologic states
resulted from studies of its cellular and subcellular localization as
well as the stimulatory elements and signaling pathways required for
its induction (6). Unfortunately, many of these studies were performed
in animal models, which clearly do not reflect human physiology. For
instance, NO production by iNOS in macrophages appears to be more
important in mice than humans (7). Differences among species in NO
production and NOS expression in response to a given inflammatory
stimulus may reflect differences in the cell signaling mechanisms that
lead to iNOS gene transcription and/or in promoter structure.
Transcriptional regulation is a crucial checkpoint in the initiation of
cytokine-stimulated NO production by human iNOS (hiNOS) (8). 7.2-kb and
8.3-kb 5'-flanking regions of the human iNOS gene were found to contain
NF- MAPKs are signaling proteins rapidly activated by tyrosine
phosphorylation, and tyrosine kinase inhibitors have been studied as
potential therapeutic targets in inflammatory and proliferative disorders (15-19). Activation of MAPK pathways by LPS and cytokines (20, 21) represents a potential signaling mechanism for NO production
during the inflammatory response. Furthermore, the extracellular
signal-regulated kinases (ERKs), c-jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAPK pathways have been implicated in the activation of AP-1 (22), which is
involved in hiNOS promoter activation by cytokines (10).
LPS, tumor necrosis factor- Cell Culture and Cytokine Induction--
A549 cells (American
Type Culture Collection (ATCC) CCL 185), a human alveolar type II
epithelial cell-like lung adenocarcinoma cell line, were grown at
37 °C at 5% CO2 in Ham's F-12 K medium supplemented
with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (all
from Biofluids). To initiate experiments, cells were washed with
serum-free medium and treated with or without inhibitors for 1 h
before incubation for 6 h with cytokine mixture (CM) containing 100 units/ml IFN- Transient Transfection and Determination of hiNOS Promoter
Activity--
A549 cells were transfected with hiNOS promoter
constructs linked to luciferase cDNA (PGL3-hiNOS) (10) without or
with mammalian expression vectors containing cDNAs for various
forms of MAP/ERK kinase 1 (MEK1), MAPK kinase 3 (MKK3), and MKK6.
Expression vectors for dominant negative MEK1 (pcDNA3-MEK1 dn),
MKK6 (pcEFLGST-MKK6 dn), MKK3 (pcDNA3-MKK3 dn), as well as those
for wild-type and constitutively active MKK3 (pcDNA-MKK3 wt,
pcDNA-MKK3 ca) were kind gifts from Dr. J. S. Gutkind, NIDCR,
National Institutes of Health, Bethesda, MD. In separate experiments,
expression of the above proteins in A549 cells was confirmed by Western
blot analysis (data not shown). After 36 h, cells were induced
with cytokines for 6 h before harvest of cells for luciferase
activity determination (Luciferase Assay System Kit, Promega). hiNOS
promoter regional sequence requirements for LPS/IFN-
Luciferase activities of both stimulated (CM or LPS/IFN- Preparation of Nuclear Extracts--
Nuclear extracts were
prepared from cells grown near confluence in 100-mm2
dishes, incubated for 1 h without or with inhibitors, before stimulation with CM or LPS/IFN- Oligonucleotides Used in Electrophoretic Mobility Gel Shift
Assay--
Complementary oligonucleotides were synthesized on an
Applied Biosystems (Foster City, CA) 380B DNA Synthesizer. The positive strands of the double-stranded oligonucleotides corresponding to the
identical regions of the hiNOS promoter are as follows: wild-type
NF- Electrophoretic Mobility Gel Shift Assay--
NF- Measurement of Cell Viability--
Cell viability was assessed
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay (25). Briefly, cells plated in 96-well plates (2.5 × 105 cells/well) were incubated for 1 day before addition of
LPS/IFN- Effects of LPS and IFN- Role of AP-1 and NF-
In cells transfected with the hiNOS promoter construct lacking the
region upstream from the two AP-1 sites ( Role of Tyrosine Kinase Activity in CM-induced hiNOS Promoter
Activation--
The general tyrosine kinase inhibitors genistein and
erbstatin each attenuated CM-induced hiNOS promoter activity in a
concentration-dependent manner (Table
II). The effects of the more specific
tyrphostins, AG126 and AG1288, as well as the JAK2 inhibitor AG490,
were then investigated. All three tyrphostins decreased CM- and
LPS/IFN-
To define better the MAPKs involved in activation of the hiNOS
promoter, we used a molecular approach by coexpressing dominant negative, wild-type, and constitutively active forms of ERK and p38
MAPK kinases. Complementary results were obtained with pharmacological inhibitors of MEK1 (PD98059) and p38 MAPK (SB203580). PD98059 produced
25 and 42% reductions in CM-and LPS/IFN-
SB203580 caused 43 and 45% inhibition of CM- and
LPS/IFN- Effects of p38 MAPK and MEK1 Inhibition on Transcription Factor
Binding to hiNOS Promoter Regulatory Elements--
Using DNA probes
corresponding to the respective NF- The production of NO by iNOS has a variety of important biological
effects including oxidative stress and signaling (3, 23). Production of
iNOS protein is tightly regulated at the transcriptional level, and the
signaling pathways by which human iNOS transcription is controlled in
response to inflammatory stimuli are poorly understood. Here, we report
that LPS, in combination with IFN- The 8.3-kb hiNOS promoter construct has several distinct advantages
over measures of NO production or NOS expression, most importantly, the
ability to study the molecular determinants of transcriptional
activation (i.e. NF- Animal models of iNOS induction do not accurately reflect signaling
mechanisms in human cells. In a region containing critical cytokine
response elements ( A role for tyrosine kinases in iNOS induction has been established
(31-36). The importance of early gene activation was stressed in
animal models of sepsis, in which tyrphostins decreased mortality only
when administered before, or soon after, the initiation of sepsis (16,
17, 19). The JAK2 inhibitor AG490 attenuated CM stimulation of hiNOS
promoter activity. However, none of the contributory interferon
response elements usually responsible for JAK/STAT-activated gene
transcription had been identified in a previous characterization of
hiNOS promoter activation by cytokines (24). One possible explanation
is that IFN- Both p38 and ERK pathways involve upstream protein tyrosine
phosphorylation by specific MAPK kinases and can lead to activation of
AP-1 elements (39-42). Others have reported a role for MAPK-mediated AP-1 activation of gene transcription (43, 44). In our study, the use
of both pharmacological and molecular inhibitors of MAPK pathways was
instrumental in dissecting the mechanisms of hiNOS transcriptional
activation. The p38 inhibitor SB203580 and MEK1 inhibitor PD98059
reduced LPS/IFN- The lack of effect of dominant negative MKK6 on hiNOS activation
suggests selectivity for MKK3 in CM- and LPS/IFN- The decrease in hiNOS promoter activity caused by MAPK inhibitors was
accompanied by reduced CM- and LPS/IFN- Although MAPKs can also activate NF- The p38 and MEK1 inhibitors SB203580 and PD98059 caused additive
inhibition of hiNOS promoter activity and AP-1 binding to downstream,
but not upstream sites, possibly reflecting the different binding site
sequences. Although additive reduction in AP-1 binding likely explains
the additive inhibition of hiNOS promoter activity, transcription
factor binding to promoter regulatory elements is one of several
potential mechanisms for control of gene transactivation (58).
Consistent with AP-1 binding as the critical step in hiNOS promoter
activation, cytokines stimulated de novo JunD and Fra-2 expression, coinciding with increased AP-1 binding (10). However, in
addition to affecting AP-1 binding, MAPKs can also influence post-translational modification of AP-1 subunits (22). In addition, TATA-binding protein phosphorylation plays a role in MAPK-induced gene
transcription. ERK2 directly phosphorylated the TATA-binding protein
amino-terminal domain during phorbol 12-myristate 13-acetate-induced differentiation and G0-G1 transition (59).
The data presented here highlight several similarities and differences
between LPS- and cytokine-induced signal transduction. Both p38 MAPK
and MEK1 activation were responsible for augmented AP-1 binding in both
CM- and LPS/IFN- Despite these similarities, LPS/IFN-, interleukin-1
,
interferon-
(IFN-
)) required downstream and upstream nuclear
factor-
B (
115,
8283) and activator protein-1 (AP-1) (
5115,
5301) transcription factor binding sites. Human lung epithelial
(A549) cells were transiently transfected with luciferase reporter
plasmids containing an 8.3-kilobase human iNOS promoter to
examine the molecular signaling events necessary for hiNOS
transcriptional activation. The combination of LPS and IFN-
, but
neither alone, increased hiNOS promoter activity 28-fold, in a reaction
requiring two critical AP-1 (JunD·Fra-2) promoter binding
sites. Mitogen-activated protein kinases (MAPKs) were assessed as
potential activators of AP-1 and the hiNOS promoter. Both
pharmacological and molecular inhibitors of the extracellular signal-related kinase (ERK) and p38 pathways reduced cytokine mixture
(CM)- and LPS/IFN-
-induced promoter activation. By gel retardation
analysis, the addition of MAP/ERK kinase-1 and p38 inhibitors
significantly diminished AP-1 binding in both CM- and LPS/IFN-
-stimulated cells. Thus, p38- and ERK-dependent
pathways, through effects on the AP-1 complex, activate the hiNOS
promoter in cells stimulated with CM or LPS/IFN-
.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and activator protein-1 (AP-1) binding regulatory regions (9,
10). JunD and Fra-2 were identified in the heterodimers that bound to
upstream and downstream AP-1 sites in the 8.3-kb hiNOS promoter. The
upstream and downstream NF-
B sites bound RelA/RelA and RelA/p50,
respectively (10). Because both lipopolysaccharide (LPS) and cytokines
can activate AP-1 and NF-
B (11-14), the promoter provides a
molecular model with which to define the signaling determinants of iNOS
activation in response to inflammatory mediators.
, interferon-
(IFN-
), and
interleukin-1
(IL-1
), which are present in human airways during Gram-negative infection, cause inflammation in part by initiating NO
production by iNOS (23). Because AP-1 and NF-
B activation are major
determinants of hiNOS induction in A549 cells, we hypothesized that
MAPK proteins were upstream signaling effectors of hiNOS promoter
activation in response to LPS and cytokines. Using both pharmacological
and molecular approaches, the involvement of p38 MAPK and ERK pathways
in hiNOS promoter activation by cytokines and LPS/IFN-
via binding
of AP-1 to specific promoter sequences was demonstrated.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Roche Molecular Biochemicals), 0.5 ng/ml IL-1
(Genzyme), and 10 ng/ml tumor necrosis factor-
(Roche Molecular Biochemicals), or a mixture of 100 µg/ml LPS (Sigma) and 100 units/ml IFN-
. Erbstatin, genistein, SB203580, and PD98059 (Calbiochem) dissolved in dimethyl sulfoxide (Me2SO) were added
directly to the cells.
-stimulated
activation were assessed using deletion mutants as reported previously
(24). Luminescence units were expressed relative to total lysate
protein, determined using the bicinchoninic acid protein assay (Pierce).
) and
unstimulated cells are the means of values from at least two independent experiments with assays in triplicate. The fold induction was calculated by dividing the stimulated by the unstimulated luciferase activity. For pharmacological inhibition experiments, the
percentage of control is the fold induction in the presence of
inhibitor divided by that in the absence times 100. For MKK cotransfection experiments, the percentage of control is the hiNOS promoter activity of cells cotransfected with a mammalian expression vector containing MKK cDNA divided by that of cells cotransfected with the corresponding empty vector times 100. Me2SO (up to
1% final concentration) did not affect LPS/IFN-
- or CM-stimulated promoter activity (data not shown).
for 3 h, and harvested as
described (10). Protein concentrations were determined using a Bradford assay kit with bovine serum albumin as standard.
Bu (
8287 to
8270), 5'-CCCTGGGGAACTCCTGCA-3'; mutated NF-
Bu, 5'-CCCTATAGAACTAATGCA-3'; wild-type
NF-
Bd (
102 to
119), 5'-GCTGGGGACACTCCCTTT-3'; mutated NF-
Bd,
5'-GCTGATAACACTAACTTT-3'; wild-type AP-1u (
5307
to
5290), 5'-CCAGCTTGAGTCACACTC-3'; mutated AP-1u,
5'-CCAGCTTAATTAACACTC-3'; wild-type AP-1d (
5121 to
5104), 5'-TTTGTGTGACTCACGCCC-3'; mutated AP-1d, 5'-TTTGTGTAATTAACGCCC-3'. After
purification on G-25 spin columns, (Amersham Pharmacia Biotech),
complementary strands in equal concentrations were mixed and annealed
by slowly cooling to room temperature after heating to 95 °C for 5 min. Double-stranded oligonucleotides were stored at
20 °C at a
concentration of 1.8 pmol/µl in 50 mM NaCl. For
electrophoretic mobility gel shift assay, oligonucleotide probes were
labeled with [
-32P]ATP using a T4 polynucleotide
kinase kit and purified on G-25 spin columns (all from Amersham
Pharmacia Biotech).
B and AP-1
binding activity was determined by electrophoretic mobility gel shift
assay using the Promega Gel Shift Assay System. Samples (5 µg) of
nuclear proteins were incubated with the indicated radiolabeled
oligonucleotide for 20 min at room temperature (~21 °C).
Specificities of the binding reactions were tested in competition
assays in which a 100-fold excess of unlabeled wild-type or mutated
oligonucleotide was added to extracts from stimulated cells 15 min
before the labeled probe. In assays without nuclear protein, none of
the probes caused any discernible band shift (data not shown).
Protein-nucleotide complexes were separated by electrophoresis in a 6%
DNA-retardation gel (Novex) with Tris borate/EDTA (50 mM
Tris-HCl, 50 mM boric acid, 1 mM EDTA, pH 8.3) at constant current (30 mA) at room temperature. Photographic film was
exposed to dried gels at
70 °C and scanned using an Epson
Expression 636 scanner. Band densities were measured using Scion Image
beta 3b software. Because of their variable nature, band density
results are mean gray value for a given area minus a measured gray
background value for each gel. Me2SO alone (up to 1% final
concentration) did not affect LPS/IFN-
- or CM-stimulated transcription factor binding (data not shown).
or CM with and without inhibitors or Me2SO
alone (eight wells/group), and 6 h later, MTT (500 µg/ml). After
4 h in the dark, plates were centrifuged (450 × g
for 10 min); the precipitate was dissolved in Me2SO and
shaken for 10 min. Absorbance at 540 nm was directly proportional to
the number of cells plated (data not shown). Viability was assessed as
the ratio of absorbances for inhibitor-treated and untreated cells. No
differences in viability among differently treated cells were found
(data not shown).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
on hiNOS Promoter Activity in A549
Cells--
After incubation of A549 cells with different
concentrations of LPS without or with IFN-
for 6 h, neither
IFN-
nor LPS alone significantly activated the hiNOS promoter. In
the presence of IFN-
, however, LPS increased promoter activity in a
concentration-dependent manner to a maximum of 28-fold the
control value (Table I). Results were
similar for 12-h incubations, with a 45-fold increase in hiNOS promoter
activity attributable to LPS/IFN-
(data not shown).
Activation of the hiNOS promoter by the combination of LPS and
IFN-
, as indicated, for 6 h. Data are the means
of values from two experiments with assays in triplicate (± half the
range), with luciferase activity expressed relative to that of
unstimulated cells without additions = 1.0.
B Binding Sites in hiNOS Promoter Activation
by LPS/IFN-
--
It had been shown that removal of the upstream
NF-
B binding site reduced the promoter response to the cytokine
mixture by 82% (24); removal of promoter regions upstream of AP-1
binding sites (deletions
6796,
6534, and
5774 bp), had no further
effect (Fig. 1A). For the
response to LPS/IFN-
, the
7277-bp deletion mutant retained 50% of
the native promoter activity (Fig. 1B), suggesting that
upstream NF-
B binding (RelA/RelA) was more important for CM
activation than for that by LPS/IFN-
. The region between
5774 bp
and
7277 bp accounted for a 4.5-fold increase in
LPS/IFN-
-stimulated promoter activity. In an attempt to identify a
discrete binding site responsible for enhancement of hiNOS activation
by LPS/IFN-
, we constructed promoter fragments containing serial
truncations at short intervals (
6796-bp,
6534-bp,
5774-bp).
Reductions in promoter activity due to successive small deletions were
incremental but small, suggesting heterogeneity in the unknown enhancer
elements responsible for full hiNOS activation by LPS/IFN-
and the
absence of a discrete site (Fig. 1, B and C).
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Fig. 1.
Deletions of hiNOS promoter regions influence
LPS/IFN- -mediated hiNOS induction. A549
cells transfected with the indicated construct of the hiNOS promoter
were incubated for 12 h without or with CM (A) or 10 µg/ml LPS and 100 units/ml IFN-
(B) before assay of
luciferase activity. Data are the means of values from two
experiments (± half the range) with assays in triplicate, expressed
relative to luciferase activity of unstimulated cells = 1.0. C, positions of upstream and downstream NF-
B (NF-
Bu,
NF-
Bd) and AP-1 (AP-1u, AP-1d) in truncation mutants are depicted,
followed by data for induction by LPS/IFN-
(a) or CM
(b), and the ratio of LPS/IFN-
-stimulated promoter
activity to CM-stimulated promoter activity
(a/b).
5774 bp), CM caused a
7.6-fold stimulation, twice that caused by LPS-IFN-
. Removal of the
two AP-1 sites (
3665 bp) further decreased LPS/IFN-
stimulation
3.5-fold. Because this region accounted for the largest discrete drop
in LPS/IFN-
-stimulated hiNOS promoter activation, and the presence
of intact AP-1 binding sites was critical for CM stimulation, we
examined the signaling pathways that might lead to hiNOS promoter
activation via AP-1.
-induced activation in concentration-dependent
manner (Table III), suggesting that MAPK
and JAK pathways are involved in hiNOS promoter induction.
Tyrosine kinase activity is required for activation of the hiNOS
promoter
Effect of tyrphostins on CM-mediated hiNOS activation
-stimulated hiNOS
induction, respectively (Fig.
2A). As was the case for
PD98059, coexpression of dominant negative MEK1 lead to a significant
reduction in basal as well as CM- and LPS/IFN-
-stimulated hiNOS
activation (Fig. 2B), indicating a role for ERK activation
in hiNOS transcriptional activation.
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Fig. 2.
Effects of ERK pathway inhibition on CM- and
LPS/IFN- -stimulated hiNOS promoter
activity. A, A549 cells transiently transfected with
the hiNOS promoter were incubated for 1 h with the indicated
concentration of the MEK1 inhibitor PD98059 before continued incubation
for 6 h without or with CM (black bars) or LPS/IFN-
(white bars). The fold induction in the absence of inhibitor
(100% control) was 55 ± 15 and 31 ± 0.4 for CM and
LPS/IFN-
, respectively. Data are the means of values from three
experiments (± S.E.) with assays in triplicate. B, A549
cells were transfected with 2 µg/ml empty vector (pcDNA3) or
vector containing MEK1 dn cDNA and 1 µg/ml pGL3 hiNOS promoter.
In separate experiments, after 36 h, cells were incubated for
6 h without and with CM (i) or LPS/IFN-
(ii) before cell lysis. Luciferase activity/µg of total
protein in cells transfected with MEK1 dn cDNA was normalized to
that measured in cells transfected with empty vector (% control). Data
are the means of values from four experiments (± S.E.) with assays in
triplicate.
-stimulated hiNOS promoter activity, respectively (Fig.
3A). Cotransfection of
dominant negative forms of MKK3, but not of MKK6, with the hiNOS
promoter led to a significant reduction in CM- or
LPS/IFN-
-stimulated hiNOS promoter activity (Fig. 3B).
Coexpression of wild-type or constitutively active MKK3 enhanced basal
hiNOS promoter activity, confirming the involvement of the p38 MAPK
pathway (Fig. 4). hiNOS promoter
activation by CM and LPS/IFN-
was not enhanced further by the
overexpression of wild-type and constitutively active MKK3. Administration of SB203580 reversed the hiNOS promoter activation attributed to expression of constitutively active MKK3, demonstrating specificity of SB203580 for p38 MAPK in the context of
cytokine-stimulated hiNOS activation. Together the two pharmacological
inhibitors reduced CM- and LPS/IFN-
-stimulated hiNOS promoter
activity 86 and 89%, respectively (Fig.
5). Apparently additive effects of the
p38 and MEK1 inhibitors suggest that the two kinases contribute independently to the activation of the hiNOS promoter.
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Fig. 3.
Effects of p38 MAPK pathway inhibition on CM-
and LPS/IFN- -stimulated hiNOS promoter
activity. A, A549 cells transiently transfected with
the hiNOS promoter were incubated for 1 h with the indicated
concentration of the p38 inhibitor SB203580 before continued incubation
for 6 h without or with CM (black bars) or LPS/IFN-
(white bars). The fold induction in the absence of inhibitor
(100% control) was 36 ± 0.4 and 31 ± 0.4 for CM and
LPS/IFN-
, respectively. Data are the means of values from three
experiments (± S.E.) with assays in triplicate. B, A549
cells were transfected with 1 µg/ml pGL3-hiNOS and 1 µg/ml empty
vector (pcDNA3 or pcEFL-GST), pcEFL-GST-MKK6 dn, or pcDNA3-MKK3
dn. In separate experiments, after 36 h, cells were incubated for
6 h without and with CM or LPS/IFN-
before cell lysis.
Luciferase activity/µg of total protein in cells cotransfected with
MKK6 dn or MKK3 dn cDNA was normalized to that in cells
cotransfected with the corresponding empty vector (% control). Data
are means of values from three to five experiments (± S.E.) with
assays in triplicate.
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Fig. 4.
Effect of wild-type (wt) and
constitutively active (ca) MKK3 overexpression on CM-
and LPS/IFN- -stimulated hiNOS promoter
activity. A549 cells were transfected with 1 µg/ml pGL3-hiNOS
and 1 µg/ml empty vector (pcDNA3), pcDNA3-MKK3 wt, or
pcDNA-MKK3 ca. After 36 h, cells were incubated as indicated
for 1 h without or with 40 µM SB203580 and then for
6 h without and with CM (A) or LPS/IFN-
(B) before cell lysis. Luciferase activity/µg of total
protein in cells transfected with MKK3 constructs was normalized to
that in cells transfected with empty vector (% control). Data are the
means of values from five experiments with assays in triplicate (± S.E.).
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Fig. 5.
Effect of MEK1 inhibition, p38 MAPK
inhibition, and both on CM- and
LPS/IFN- -stimulated hiNOS promoter
activity. A549 cells were incubated without or with CM
(black bars) or LPS/IFN-
(white bars) and
without (C) or with 40 µM SB203580
(SB), 50 µM PD98059 (PD), or both
for 6 h before cell lysis. The fold induction in the absence of
inhibitor (100% control) was 60 ± 6.2 and 37 ± 4.2 for CM
and LPS/IFN-
, respectively. Data are the means of values from two
experiments with assays in triplicate (± half the range).
Bu, NF-
Bd, AP-1u, and AP-1d
binding sites, we assessed the effects of ERK and p38 MAPK pathway
inhibition on transcription factor binding by gel retardation analysis.
Exposure to CM or LPS/IFN-
for 3 h increased to similar extents
the binding of NF-
B and AP-1 complexes (Fig.
6, A and B). The
gel migration distance of the DNA probe-transcription factor complex
was the same for CM- and LPS/IFN-
-stimulated cells, consistent with
the binding of identical dimer complexes to the oligonucleotide probes
regardless of the agonist used (data not shown). In unstimulated cells,
SB203580 and PD98059 did not affect binding to NF-
B sites, whereas
the combination of SB203580 and PD98059 increased binding to AP-1
sites. SB203580 and PD98059 inhibited LPS/IFN-
- but not
CM-stimulated NF-
B downstream (
115) binding. SB203580 and PD98059
augmented both LPS/IFN-
- and CM-stimulated NF-
B upstream (
8283)
binding. SB203580 or PD98059, singly or together, decreased CM- and
LPS/IFN-
- stimulated AP-1 binding. Of note, the inhibitory effects
of PD98059 and SB203580 on the CM- and LPS/IFN-
-induced increases in
AP-1 downstream binding were additive. These data point to a crucial
role for MAPKs in AP-1-dependent human iNOS induction.
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Fig. 6.
Effect of MAPK inhibitors on CM- and
LPS/IFN- -stimulated transcription factor
binding to NF-
B and AP-1 regulatory sites in
the hiNOS promoter. A549 cells were incubated for 1 h without
or with 40 µM SB203580, 50 µM PD98059, or
both before the addition of CM (A) or LPS/IFN-
(B) and continued incubation for 3 h before cells were
lysed and nuclear protein was extracted. From each sample, 5 µg of
nuclear protein were incubated with 32P-labeled
oligonucleotide probes encoding specific upstream and downstream
NF-
B (NF-
Bu, NF-
Bd) and AP-1 (AP-1u, AP-1d) binding sequences
in the hiNOS promoter. The specificity of binding reactions was
assessed by the addition of a 100-fold excess of unlabeled wild-type
(cold WT) or mutant (cold MT) oligonucleotide 15 min before the labeled probe. The autoradiographs are representative of
four or five experiments. The mean band density (± S.E.) is recorded
below each gel lane.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, activated specific NF-
B- and
AP-1-binding elements in an 8.3-kb hiNOS promoter. The ERK and p38 MAPK
pathways played a crucial role in human iNOS transcriptional activation
via modulation of AP-1 binding to specific promoter sequences.
B, AP-1 binding). Introduction of an
exogenous iNOS promoter makes it possible to focus directly on
transcriptional activation in intact cells, avoiding confounding factors that affect measures of iNOS production. Furthermore, in intact
cells, promoter activation is amenable to molecular characterization,
and iNOS transcription is the rate-limiting step in cytokine-induced NO
production. However, the validity of conclusions regarding signaling
pathways involved in iNOS induction clearly depends on the structural
characteristics of the putative promoter sequence. Taylor et
al. (9) cloned a 7.2-kb hiNOS promoter whose activity was
increased 4-fold by cytokines in A549 cells. The upstream region
contained in the 8.3-kb promoter construct appears also to be important
for full hiNOS transcriptional activation. In addition,
MAPK-dependent hiNOS promoter activation was related to the
enhancement of AP-1 binding activity at the
5115 and
5301 positions
of the 8.3-kb hiNOS 5'-flanking region.
5774 to
3665), two identical AP-1 sites are
present in the hiNOS promoter which are absent in its murine
counterpart (24). The mouse iNOS promoter contains an upstream
regulatory region with a cluster of four IFN-
-response enhancer
elements that account for the potentiation of LPS-stimulated promoter
induction by IFN-
(26-28). In the human promoter, the upstream
putative IFN-
-response elements (
8296 to
7227) apparently do not
contribute to cytokine-stimulated promoter activation (24). Other
transcription factors (e.g. octomer motif, NF-IL6) enhance cytokine-induced murine iNOS expression (29, 30) but are unlikely to
play a direct role in human iNOS induction (24).
-activated factors modified hiNOS transactivation by
NF-
B or AP-1 without binding directly to the promoter.
Alternatively, JAK2 may be responsible for the direct activation of the
MAPK pathway (37). In agreement, AG490 attenuated IL-2-induced T cell
proliferation by inhibiting JAK3-dependent MAPK activation
and AP-1 binding (38). The additive stimulation via these pathways may
explain the requirement for full activation of the hiNOS promoter by
tumor necrosis factor-
/IL-1
to observe an IFN-
effect. In
contrast to CM activation, LPS/IFN-
stimulation of hiNOS promoter
activity was reduced by the deletion of promoter regions containing
putative interferon response elements (
5774 bp to
7727 bp). AG490
may have reduced LPS/IFN-
stimulation of promoter activity by
inhibiting STAT binding to one of these elements.
- and CM-mediated hiNOS promoter activation in
additive fashion, suggesting a potential role for MAPKs in
AP-1-dependent hiNOS induction. Similarly, coexpression of
dominant negative MEK1 led to a 50% attenuation of both basal and CM-
or LPS/IFN-
-stimulated hiNOS promoter activity. Because the pattern
of inhibition by dominant negative MEK1 expression was similar to that
by PD98059, the latter is likely specific for the ERK pathway, insofar
as hiNOS promoter activation is concerned. MKK6 and MKK3 are upstream
kinases with demonstrated specificity for p38 MAPK (45, 46). Consistent
with the action of SB203580, overexpression of dominant negative MKK3
reduced CM- and LPS/IFN-
-stimulated, but not basal, hiNOS promoter
activity. Overexpression of wild-type and constitutively active MKK3
enhanced basal hiNOS promoter activity, confirming the involvement of
the p38 MAPK pathway. The increase in CM- and LPS/IFN-
-stimulated
hiNOS promoter activity observed upon expression of constitutively
active MKK3 was reversed by SB203580, demonstrating that the inhibition
of hiNOS activation by SB203580 was via p38 MAPK.
-stimulated hiNOS
transcriptional activation via p38 MAPK. Similar signaling specificity
was demonstrated when overexpression of PYK2 activated p38 MAPK via
MKK3 and not MKK6 (47). Although both MKK3 and MKK6 can activate p38
MAPK, each exhibited p38 MAPK isoform selectivity (45), and each was
activated differentially by specific extracellular stimuli (48,
49).
-stimulated AP-1 binding,
whereas binding to the upstream NF-
B (RelA/RelA) sequence was
increased. These data are consistent with an essential role for MAPKs
in AP-1- but not NF-
B-dependent hiNOS activation. The
mechanism by which binding to the NF-
B upstream site was augmented
by SB203580 and PD98059 is unclear, although others have observed
reduction of NF-
B activation by activated MAPK (50). The
oligonucleotide sequence-specific differences in agonist-induced NF-
B binding may reflect the different dimers that bind to upstream (RelA/RelA) and downstream (RelA/p50) sites.
B (51-53), the p38 and MEK1
inhibitors did not reduce CM-induced NF-
B binding. NF-
B binding
to the hiNOS promoter, which is required for full hiNOS promoter
activation, can, therefore, be stimulated via pathways other than those
involving MAPKs. Similarly, others demonstrated that
MAPK-dependent AP-1 activation was partially responsible for human immunodeficiency virus type 1 long terminal repeat activation and that MAPK-independent NF-
B induction was required for full promoter activation (54). A potential MAPK-independent NF-
B inducer,
the recently characterized Toll-like receptor-2, or TLR2 (55) has an
intracytoplasmic IL-1 receptor-like domain and is activated by LPS. In
fact, LPS activation of NF-
B was inhibited by transfection with
dominant negative mutant components of the IL-1 signaling pathway (56),
and a TLR2 homolog, TLR4, plays a role in LPS-induced NF-
B
activation (57). TLR- and IL-1
receptor-mediated activation of
NF-
B in A549 cells may be responsible for the increased NF-
B
binding seen in response to LPS/IFN-
and cytokines.
-stimulated cells. CM- and LPS/IFN-
-stimulated
hiNOS promoter activation required intact ERK and p38 MAPK pathways.
Deletion analysis of the hiNOS promoter confirmed that the two AP-1
sites, as well as the upstream NF-
B site, are important for CM (10)
and LPS/IFN-
stimulation of hiNOS induction. Finally, as shown for
CM (10), LPS/IFN-
increased NF-
B and AP-1 binding to the four key
regulatory sequences in the hiNOS promoter.
alone did not activate all of
the signaling pathways required for full activation of the hiNOS
promoter, as demonstrated by the much greater response to CM. Future
studies can look for the coactivators, or additional signaling
pathways, required for optimal gene transcription. Furthermore, in
contrast to their effects in cells stimulated with CM, inhibitors of
MEK1 and p38 MAPK reduced binding to the downstream NF-
B site. Other
investigators have suggested potential cross-talk between the MAPK and
NF-
B pathways (50, 60, 61). Although
MAPK-dependent NF-
B activation might be important for
the induction of other genes, AP-1 binding is apparently the
rate-limiting step in hiNOS promoter activation. The AP-1 complex
JunD·Fra-2, or its binding site, serves as a molecular target for the
control of diverse biological processes.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Martha Vaughan for useful discussions and critical review of the manuscript. We also thank Dr. J. S. Gutkind for informative and stimulating discussion as well as for providing the mammalian expression vectors instrumental in completion of this study.
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FOOTNOTES |
---|
* 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.
To whom correspondence should be addressed: Bldg. 10, Rm. 5N307,
MSC 1434, National Institutes of Health, 9000 Rockville Pike, Bethesda,
MD 20892-1434. Tel.: 301-496-6980; Fax: 301-402-1610; E-mail:
kristofa@nih.gov.
§ Present address: Metabolism Branch, Bldg. 10, Rm. 4B47, NCI, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892.
Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M009563200
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ABBREVIATIONS |
---|
The abbreviations used are:
NO, nitric oxide;
hiNOS, human inducible nitric-oxide synthase;
kb, kilobase;
NF-B, nuclear factor-
B;
AP-1, activator protein-1;
Fra, Fos-related
antigen;
LPS, lipopolysaccharide;
MAP, mitogen-activated protein;
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated
kinase;
MEK1, MAP/ERK kinase 1;
MKK, MAPK kinase;
IFN-
, interferon-
;
IL, interleukin;
CM, cytokine mixture;
Me2SO, dimethyl sulfoxide;
dn, dominant negative;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
bp, base pair(s);
JAK, Janus-activated kinase;
STAT, signal transducers and
activators of transcription;
TLR, Toll-like receptor;
GST, glutathione
S-transferase.
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