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Departments of
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Pulmonary and Critical Care Medicine,
Cancer Biology,
Immunology, and
§
Microbiology, Cleveland Clinic Foundation, Lerner Research Institute, Cleveland, OH 44195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, but early in infection gene
expression may be induced by the viral replicative intermediate dsRNA
through the dsRNA-activated protein kinase (PKR). Since PKR activates
signaling pathways important in NOS2 gene induction, we determined
whether PKR is a component in the signal transduction pathway leading
to NOS2 gene expression after viral infection of airway epithelium. We
show that NOS2 gene expression in human airway epithelial cells occurs
in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA
leads to rapid activation of PKR, followed by activation of signaling
components including NF-
B and IFN regulatory factor 1. NOS2
expression is markedly diminished and IFN regulatory factor 1 and
NF-B activation are substantially impaired in PKR null cells.
Strikingly, NOS2 induction in response to LPS is abolished in PKR null
cells, confirming a central role for PKR in the general signaling
pathway to NOS2. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NO produced by NOS2 has potent antiviral activity against a number of
viruses (11, 12, 13, 14, 15). However, NO also contributes to
inflammation and injury through formation of toxic reactive nitrogen
intermediates (6, 10, 16). In this context, development of
pneumonia in a murine model of influenza infection has been linked to
host NOS2 expression (10, 17). Although viral infection
often activates NOS2 in murine models (6, 10), the
signaling pathways and effector mechanisms are not known. Viral
mechanisms regulating NOS2 expression in human airway epithelial cells
have not been studied. IFN-, produced by lymphocytes in the airway
mucosa, is likely involved in NOS2 induction later in the course of
viral infection. However, NOS2 induction by virus early in infection
may be mediated by proteins responsive to the viral replicative
intermediate, dsRNA. Intracellular dsRNA formed during viral
replication (18) binds to and activates a serine/threonine
kinase, dsRNA-activated protein kinase (PKR), which has been implicated
in signal transduction (19, 20). In this context, we
hypothesize that PKR is a component of the signaling pathway to NOS2
gene induction after viral infection of human airway epithelium. In
this study, we show induction of NOS2 gene expression in human airway
epithelial cells by influenza A virus or synthetic dsRNA. Importantly,
dsRNA leads to activation of PKR, followed by activation of signal
transduction proteins including NF-B and IFN regulatory factor 1
(IRF-1) in airway epithelial cells. A role for PKR in the signal
transduction pathway of viral-induced NOS2 expression is conclusively
demonstrated in experiments using cells genetically deficient in PKR
(PKR-/-). Impairment of
NOS2 induction in response to LPS is also found in
PKR-/- cells, confirming
a central role for PKR in the general signaling pathway to
NOS2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Influenza A/Japan/305/57, subtype H2N2 (Advanced Biotechnologies, Columbia, MD) was grown in Madin-Darby canine kidney cells and suspended in serum-free MEM with Earle’s salts. The virus stock contained 106.25 TCID50 (50% of tissue culture infectious dose) per ml.
Sampling of human airway epithelial cells
Human airway epithelial cells were obtained through bronchoscopy with a flexible fiberoptic bronchoscope (Olympus BS-IT10; Olympus Optical, Tokyo, Japan) from normal nonsmoking volunteers with no history of lung disease and no medications. Bronchoscopic brush samplings of airway epithelial cells were taken from second and third order bronchi (21). For some experiments, airway epithelial cells were isolated from surgical specimen of tracheas and mainstem bronchi as previously described (22). Informed consent was obtained under a protocol approved by the Institutional Review Board at the Cleveland Clinic Foundation.
Cell culture and treatments
Human airway epithelial cells obtained by bronchial brushing were cultured in serum-free Lechner and LaVeck media (LHC8, Biofluids, Rockville, MD) on plates precoated with coating media containing 29 µg/ml collagen (Vitrogen, Collagen Corp., Palo Alto, CA), 10 µg/ml BSA (Biofluids), and 10 µg/ml fibronectin (Calbiochem, La Jolla, CA) for 5 min (21). The cells were passaged at 60–80% confluence by dissociation from plates with 0.02% trypsin (E-PET, Biofluids) which was neutralized with soybean trypsin inhibitor (Biofluids). Primary cultures of passage 0–2 were used in experiments. The epithelial nature of primary and cultured cells was confirmed by immunocytochemical staining as previously described (23). A549 cells, an epithelial cell line derived from lung adenocarcinoma (American Type Culture Collection, Manassas, VA), were cultured in MEM (Life Technologies, Gaithersburg, MD) with 10% FCS, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (23). Confluent monolayers of human airway epithelial cells and A549 in 35-mm-diameter dishes were exposed to virus stock diluted in LHC8 medium and serum-free MEM, respectively.
Mice homozygous for a targeted deletion of PKR were generated as previously described (20). Mouse embryo fibroblasts (MEF) derived from wild-type PKR+/+ or PKR-/- mice (129/SvEv x C57BL/6) were isolated and cultured in DMEM (Life Technologies) with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin (20).
Human IFN- was a gift from Genentech (South San Francisco, CA) or
purchased from R & D Systems (Minneapolis, MN). Recombinant human
IL-1ß and TNF-
were purchased from Genzyme (Cambridge, MA).
Polyinosinic-polycytidylic acid (poly IC) and LPS (Escherichia
coli serotype O55:B5) were from Sigma (St. Louis, MO). Recombinant
murine IFN- was from Genzyme.
Immunofluorescent detection of influenza
Confluent monolayers of human airway epithelial cells and A549 grown on 12-mm-diameter coverslips of shell vial system (BioWhittaker, Walkersville, MD) were infected at a concentration of 5 x 104.25 TCID50/ml with influenza A. At 24 h postinfection, cells were fixed for 30 min at -20°C with cold methanol, washed with PBS, and incubated for 1 h at room temperature with monoclonal anti-influenza A Ab (1:400 dilution) (Chemicon International, Temecula, CA). After being washed with PBS, cells were incubated for 1 h at room temperature with a FITC-labeled goat anti-mouse Ab (1:100 dilution) (Biosource, Camarillo, CA). Samples were analyzed by immunofluorescent microscopy.
RNA extraction and Northern analysis
Total RNA was extracted and evaluated by Northern analysis as
previously described using a 32P-labeled 1.9-kb
NOS2 cDNA probe (pCCF21) or, as a control, a 2-kb -actin cDNA probe
(pHFA-1) and a 1.5-kb 18S cDNA probe (HHCS65, American Type Culture
Collection) (21, 23) and then subjected to
autoradiography. RNA from A549 cells stimulated with 100 U/ml IFN-,
10 ng/ml TNF-, and 10 U/ml IL-1ß for 8 h was used as a
positive control (23). Expression of NOS2 mRNA relative to
-actin or 18S mRNA was accomplished using a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
Western analysis
Cell lysate was prepared by freeze/thaw of A549, human airway
epithelial cells, and MEF; cultured for the indicated times with
IFN-, TNF-, IL-1ß, and/or poly IC; and isolated in lysate
buffer (3 mM DTT, 5 µg/ml aprotinin, 1 µg/ml leupeptin and
pepstatin A, 0.1 mM PMSF, 1% Nonidet P-40, and 40 mM HEPES, pH 7.5).
Total protein was measured by bicinchoninic protein assay (Pierce,
Rockford, IL). Purified recombinant murine NOS2 (24) and
lysate from A549 cells stimulated with 100 U/ml IFN-, 10 ng/ml
TNF-, and 10 U/ml IL-1ß for 24 h were used as a positive
controls for NOS2 expression. Primary Abs used for Western analyses
included a rabbit polyclonal primary Ab directed against the C-terminal
10 amino acids of human NOS2 (NO53, Merck, Rahway, NJ)
(25), and a rabbit affinity-purified polyclonal Ab
directed against peptide 961-1144 of rodent NOS2 (Transduction
Laboratories, Lexington, KY) for detection of murine NOS2, a polyclonal
rabbit antiserum raised against the N-terminal 170 residues of human
PKR, and anti-IRF-1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA).
Total proteins were separated by 6, 8, and 10% SDS-PAGE under
denaturing and reducing conditions for NOS2, PKR, and IRF-1,
respectively. Signal detection was accomplished with a
peroxidase-linked species-specific donkey anti-rabbit secondary Ab
(Amersham, Arlington Heights, IL) and enhanced chemiluminescence
(Amersham). Blots were also hybridized with a mouse monoclonal primary
anti-ß-actin Ab (Sigma) followed by a sheep secondary
anti-mouse Ig (Amersham). The images of signals were electronically
digitalized by scanning, and the intensity of images relative
to ß-actin was quantitated by the software, ImageQuant version 1.2
(Molecular Dynamics).
Kinase assay for detection of PKR activity
After stimulation with poly IC, human airway epithelial cells
were resuspended in immunoprecipitation lysis buffer (50 mM Tris-HCl
(pH 7.6), 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 5 mM EDTA, 1 mM
DTT, 100 mM NaF, 2 mM sodium pyrophosphate, 2 mM sodium orthovanadate,
1 mM PMSF, and 10 µg/ml each of aprotinin and leupeptin)
and stored on ice for 20 min before clarification by centrifugation
(18,000 x g, 20 min, 4°C). PKR was
immunoprecipitated from 30–50 µg lysate using mAb specific for human
PKR (26). After immunoprecipitation at 4°C with
mAb-protein G-Sepharose, the bound kinase was washed with lysis buffer,
then with DBGA (10 mM Tris-HCl (pH 7.6), 50 mM KCl, 2 mM magnesium
acetate, 20% glycerol, 7 mM mercaptoethanol) before incubation in DBGA
containing 0.83 mM manganese chloride, 0.8 µM ATP, 500 ng histone
(Sigma), and 9 µCi [-32P]ATP for 20 min at
30°C. The reaction was stopped by the addition of 2 volumes of
SDS-PAGE sample buffer. Phosphoproteins were resolved on 10% SDS-PAGE
gels and quantitated by PhosphorImager.
Electrophoretic mobility shift assay
Whole cell extracts were prepared by a modification of a previously described method (27). In brief, adherent cells were harvested by a cell lifter, and the cell suspensions were centrifuged, washed with PBS, and resuspended in ice-cold low salt buffer (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM PMSF, 0.5 mM DTT). After a 5-min incubation on ice, cells were washed in the same buffer and then pelleted. A volume of high salt extraction buffer equal to the volume of cell pellet was added (20 mM HEPES (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, 5 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 µg/ml pepstatin), and the mixture was placed on ice for 30 min. Whole cell extracts were clarified by centrifugation at 12,000 x g for 20 min at 4°C. The protein concentration was measured by bicinchoninic protein assay (Pierce).
Oligonucleotides used for EMSA include the B sequence motif
(5'-gatcTACTCCGGGAATTTCCCTGGCC-3') (28), and
the multimerized hexamer probe, (AAGTGA)4,
originally used to clone IRF-1 (29). These synthetic
oligonucleotides were either end-labeled with
[-32P]ATP by polynucleotide kinase or
fill-in labeled with [-32P]dCTP by Klenow.
For binding reactions, whole cell extracts (5 µg total protein) were
incubated in 24 µl total reaction volume containing 20 mM HEPES (pH
7.9), 10% glycerol, 60 mM NaCl, 5 mM MgCl2, 4 mM
Tris-HCl, 1 mM DTT, 0.6 mM EDTA, 200 µg/ml BSA, and 2 µg
polydeoxyinosinic:polydeoxycytidylic acid (Amersham) for 15 min at
4°C. The 32P-labeled oligonucleotide (0.2 ng,
2 x 105 cpm) was added to the reaction
mixture and incubated for 20 min at room temperature.
To specifically identify NF-B and IRF-1 proteins in binding
complexes, 2–4 µg of rabbit anti-P65 polyclonal Ab, rabbit
anti-IRF-1 or anti-IRF-2 Ab (Santa Cruz Biotechnology) were
added to the binding reaction mix and incubated for 30 min at 4°C
before adding the 32P-labeled oligonucleotide.
The reaction products were analyzed by electrophoresis on a 4%
polyacrylamide gel with 0.25x 22.3 mM Tris, 22.2 mM borate, 0.5 mM
EDTA buffer for NF-B or a 4% polyacrylamide gel containing 50 mM
Tris-HCl (pH 7.5), 0.38 M glycine, and 2 mM EDTA for IRF-1. The gels
were dried and analyzed by autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NOS2 mRNA is present in A549 cells infected with influenza A as
early as 8 h after infection (Fig. 1
A). Because intracellular
dsRNA is produced during the viral replication cycle, and appears to be
the active component in viral infection that stimulates gene expression
in infected cells (30, 31), poly IC, a synthetic dsRNA,
was used to simulate the viral-infected state and to study dsRNA-viral
mechanisms regulating cellular gene expression. Accordingly, we tested
whether dsRNA would induce NOS2 expression in human airway epithelial
cells. Cells exposed to poly IC express NOS2 mRNA maximally between 8
and 24 h, returning to near baseline at 48 h (Fig. 1, A and B). NOS2 protein mirrors mRNA expression
and is present between 8 and 24 h, but is lost by 48 h (Fig. 1C). In contrast to poly IC induction, IFN--induced NOS2
mRNA is maintained for up to 6 days (Fig. 1A).
Immunofluorescence using influenza A Ab 24 h after exposure to
virus confirms effective infection and replication by influenza A virus
in human airway epithelial cells in vitro (Fig. 2, A and B).
Immunostaining of influenza-infected cells in culture is similar in
intensity and pattern to cells obtained from a nasopharyngeal swab of a
child with influenza pneumonia (Fig. 2C), with cells showing
nuclear and/or cytoplasmic staining. Together, these results suggest
that the early induction of NOS2 involves effector proteins responsive
to viral replicative intermediate dsRNA. Because dsRNA activates PKR
which may play a role in cell signaling events for gene expression
(20, 32), we investigated the involvement of PKR in the
mechanisms of dsRNA virus-induced NOS2 expression in human airway
epithelial cells.
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The effects of IFN- and/or poly IC on PKR protein induction in
human airway epithelial cells were examined by Western analysis.
Unstimulated cells express PKR protein constitutively. PKR expression
increases 1.8-fold with IFN- and 2.5-fold with poly IC (Fig. 3A). IFN- does not
potentiate poly IC-induced PKR protein expression. To examine the
effect of poly IC on PKR activation, human airway epithelial cells were
stimulated with poly IC and harvested at 0, 30, 60, and 120 min to
evaluate PKR kinase activity by quantitation of autophosphorylation of
PKR or phosphorylation of histone protein. PKR kinase activity
increases at 30 min, peaks at 60 min, and declines thereafter (Fig. 3B). These results show that dsRNA induces and activates
PKR, which may subsequently influence many cellular responses/signaling
pathways in human airway epithelium.
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To directly test PKR involvement in the signaling pathways leading
to NOS2 expression, we studied NOS2 induction in MEF derived from mice
with homozygous targeted deletion of the PKR gene. Rationale for the
use of MEF to investigate dsRNA viral-induced NOS2 in human airway
epithelial cells include the fact that poly IC induction of NOS2 is
similar in both cell types. PKR wild-type
(PKR+/+) and PKR knockout
(PKR-/-) MEF were
stimulated with LPS, poly IC, and/or IFN- for 8 h, and NOS2
mRNA was evaluated by Northern analysis. In wild-type cells, poly IC
and LPS induce NOS2 mRNA. In contrast, NOS2 induction is strikingly
diminished in knockout cells exposed to poly IC or LPS (Fig. 4A). IFN- alone does not
induce NOS2 mRNA in MEF. However, IFN- potentiates poly IC-induced
and LPS-induced NOS2 expression, although no potentiation occurs with
the combination of poly IC and LPS (data not shown).
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B). NOS2
induction is decreased in knockout cells stimulated with IFN- + poly
IC or IFN- + LPS as compared with wild-type (Fig. 4C).
Similar to mRNA induction, poly IC induction of NOS2 is potentiated by
IFN- (Fig. 4C). IFN- also potentiates LPS induction
of NOS2.
NF-B activation and IRF-1 expression in human airway epithelial
cells
NF-B increases the expression of the genes for many cytokines,
enzymes, and adhesion molecules in the course of inflammation,
including NOS2 (33). On the other hand, IRF-1 is essential
for NOS2 activation in murine macrophages and mediates transcriptional
activation via specific cis-acting elements resident in the
promoters of IFN-stimulated genes (34, 35). To determine
the involvement of NF-B and IRF-1 in dsRNA viral induction of NOS2,
whole cell extracts for EMSA were prepared from human airway epithelial
cells stimulated for 1 h with IFN-, poly IC, or TNF-. Poly
IC and TNF- induce NF-B activation (Fig. 5A), whereas IFN- has
little effect on basal NF-B activity. To detect IRF-1 induction and
activation, human airway epithelial cells were stimulated with IFN-,
TNF-, poly IC, or IL-1ß for 4 h and evaluated by Western
analysis and EMSA. IRF-1 protein is induced by IFN-, and to a lesser
degree by poly IC (Fig. 5B). Neither TNF- nor IL-1ß
induce IRF-1 protein. Both IFN- or poly IC result in the activation
of IRF-1 DNA binding (Fig. 5C). In contrast to IRF-1, IRF-2
activation is not detected in human airway epithelial cells by any of
the stimuli in our experimental systems. Although IFN- activates
STAT-1 in human airway epithelial cells (23), poly IC
does not (data not shown). These data suggest that NOS2 expression in
human airway epithelial cells by dsRNA or IFN- is likely mediated
through the activation of NF-B and/or IRF-1. Loss of NOS2 expression
by poly IC in PKR-/-
cells indicates a crucial role for PKR in the signal transduction
pathway of dsRNA viral-induced NOS2 expression. Moreover, loss of NOS2
induction by LPS, and decreased NOS2 induction by IFN- + poly IC or
IFN- + LPS in PKR-/-
cells suggests an important role for PKR in the signaling pathway to
NOS2 expression by a wide variety of microbes. On the basis of these
findings, we reasoned that PKR signaling to NOS2 gene expression might
be through activation of NF-B and/or IRF-1 pathways.
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B and IRF-1 activation in PKR knockout cells
To confirm the involvement of NF-B and/or IRF-1 in PKR-mediated
NOS2 expression, EMSA were performed on whole cell extracts of
PKR+/+ and
PKR-/- MEF stimulated
with IFN-, LPS, or poly IC for 4 h. NF-B is prominently
activated by poly IC but is reduced in
PKR-/- cells in contrast
to wild-type cells (Fig. 6A).
However, IRF-1 activation by poly IC or LPS also was impaired in
PKR-/- cells (Fig. 6B), whereas comparable levels of constitutive IRF-2 binding
activity are similar in both PKR+/+ and
PKR-/- cells. IRF-1 and
IRF-2 were verified in complexes by using Abs that specifically
supershift the DNA protein band (Fig. 6B). These results
suggest that PKR plays an essential role in the signaling events
leading to NF-B and IRF-1 activation and subsequently NOS2
induction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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also induces NOS2 expression;
however, the kinetics of NOS2 induction by dsRNA and IFN- are
different, suggesting distinct mechanisms of gene regulation.
Furthermore, we show that PKR is essential for dsRNA induction of NOS2
using cells genetically deficient in PKR.
PKR is important for host antiviral mechanisms, as evidenced by
impaired antiviral responses in mice with a homozygous targeted
deletion in the PKR gene (20). First identified as a
component of IFN-inducible cellular antiviral defenses, PKR exhibits
two distinct kinase activities after activation by dsRNA,
autophosphorylation/activation and phosphorylation of substrates
(38). One antiviral effect mediated by PKR is the
phosphorylation of eukaryotic initiation factor-2, effectively
restricting viral protein translation and subsequent replication
(38). In addition to effects on translation, PKR regulates
transcriptional events by phosphorylation of proteins related to signal
transduction pathways. For example, PKR is required for the activation
of NF-B in immortal cell lines in response to different stimuli
(19). In this study, dsRNA-induced PKR autophosphorylation
and substrate phosphorylation in human airway epithelial cells provide
conclusive evidence of a functional PKR pathway in these cells. These
findings provide strong support that NF-B activation by dsRNA in
human airway epithelial cells is most likely due to PKR activation and
phosphorylation of the inhibitor of NF-B (IB). Thus, PKR appears
to mediate signal transduction in human airway epithelial cells in part
through NF-B. In addition, PKR may have impact on the signaling
pathways through transcriptional and/or posttranslational effects on
IRF-1 (32). For example, expression of IRF-1 protein in
cells does not manifest functional DNA-binding activity unless a
phosphorylation signal is provided (39), potentially by
PKR (40, 41). In support of this concept, IRF-1 protein is
induced and activated by dsRNA in human airway epithelial cells.
NOS2 is subject to predominantly transcriptional regulation
(42). The molecular basis for induction of the human NOS2
gene is only partially understood (33, 43). In contrast,
regions in the murine macrophage NOS2 promotor essential for conferring
inducibility of NOS2 to LPS and IFN- have been well defined
(44, 45). An NF-B element at positions -76 to -85 bp
relative to the transcription start point binds members of the
NF-B/Rel family of proteins in response to LPS (46),
and further upstream an IFN-stimulated response element site binds
IRF-1 on stimulation of RAW 264.7 cells with IFN-
(34).
Originally identified as a transcriptional activator of IFN-ß
as well as IFN-inducible genes (39), IRF-1 is essential
for NOS2 activation in murine macrophages (34, 35).
However, the role of IRF-1 in human NOS2 gene expression is unknown.
This study provides the first clear evidence of IRF-1 expression and
activation in human airway epithelial cells by IFN- or poly IC. The
promoter of the IRF-1 gene contains an NF-B binding site and an
IFN--activated sequence (47). Thus, IFN- induction
of IRF-1 is likely mediated through STAT-1 activation and binding to
promoter elements. Interestingly, TNF- or IL-1ß activate NF-B,
but do not induce IRF-1 expression. Thus, NF-B activation alone is
not sufficient for IRF-1 induction in human airway epithelial cells. In
the context that dsRNA does not activate STAT-1, the mechanism of
IRF-1 induction by dsRNA is unclear but may be mediated through a
specific dsRNA virus-inducible element in coordination with NF-B.
Importantly, these studies demonstrate that NOS2 expression parallels
IRF-1 expression, with IRF-1 expression preceding NOS2 mRNA
accumulation. Collectively, the characteristics of these responses
strongly support that the expression of NOS2 in human airway epithelial
cells may depend, as it does in other cell types, on induction of
IRF-1. Although NF-B activation is not sufficient for NOS2
expression in human airway epithelial cells, studies of the 5'-flanking
region of the human NOS2 gene reveal that NF-B binding sites
are critical for cytokine inducibility of the gene in human epithelial
cell lines (33). Thus, we cannot exclude the possibility
that dsRNA induction of NOS2 may be mediated in part through PKR
activation of NF-B (19).
To assess the role of PKR in signaling pathways essential for
activation of NOS2, MEF derived from mice with homozygous deletions for
PKR were studied. Although NOS2 expression is regulated in a cell- and
species-specific manner, investigation of mechanisms of PKR signaling
to NOS2 in MEF are justified by similarity of dsRNA induction of NOS2
in airway epithelial cells and MEF and by the definitive model system.
These studies provide conclusive evidence that PKR is essential in the
signaling pathway for dsRNA induction of NOS2. Similar to previous
studies, we show that PKR contributes significantly to activation of
signaling pathways including NF-B and IRF-1, which are important for
NOS2 gene expression (31, 32). Interestingly, PKR is also
essential for LPS induction of NOS2 in murine cells, confirming a
central role for PKR in microbial-induced signaling pathway to NOS2. On
the other hand, continued NOS2 expression in
PKR-/- cells exposed to a
combination of IFN- and LPS or poly IC, albeit at lower levels than
in PKR+/+ cells, points out the possibility of
inducible alternative signaling pathways to NOS2, which are independent
of PKR.
On the basis of these data, we suggest a model for regulation of
NOS2 in the airway early in the course of viral infection (Fig. 7). Upon infection of human airway
epithelial cells, the viral replicative intermediate dsRNA binds to and
activates PKR, which leads to activation of NF-B. dsRNA also induces
de novo synthesis and activation of IRF-1 through PKR activation. IRF-1
in cooperation with other factor(s), such as NF-B, leads to NOS2
gene induction. Soon, release of IFN- by activated lymphocytes in
the virus-infected airway induces prolonged NOS2 gene expression in
human airway epithelium. Increases in airway NOS2 expression early in
viral infection may be antimicrobial and limit spread of virus.
However, IFN--mediated high level NOS2 expression that persists for
days to weeks most likely contributes to airway inflammation and injury
and associated clinical respiratory symptoms.
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Acknowledgments |
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A-1, Genentech for
human IFN-, R. A. Dweik for assistance in bronchoscopic
sampling of airways, and W. N. Sholtis for technical
support. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Serpil C. Erzurum, Departments of Pulmonary and Critical Care Medicine, and Cancer Biology, Cleveland Clinic Foundation, 9500 Euclid Avenue/A90, Cleveland, OH 44195.
3 Abbreviations used in this paper: NOS2, NO synthase 2; PKR, dsRNA-activated protein kinase; IRF-1, IFN regulatory factor 1; TCID50, 50% of tissue infectious dose; MEF, mouse embryo fibroblasts; poly IC, polyinosinic-polycytidylic acid.
Received for publication March 6, 2000. Accepted for publication May 1, 2000.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B-dependent transcriptional activation. J. Clin. Invest. 97:421.[Medline]
B-mediated transcription. J. Biol. Chem. 274:9707.-induced nitric oxide synthase. Science 261:1445.-dependent clearance of influenza A virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice. J. Exp. Med. 188:1541.B by phosphorylating IB. Proc. Natl. Acad. Sci. USA 91:6288. and interleukin 4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators. J. Clin. Invest. 100:829.[Medline]
B sequence motifs cooperatively control LPS-induced KC gene transcription in mouse macrophages. J. Immunol. 155:3593.[Abstract]
B. EMBO J. 16:406.[Medline]
B-binding sites. J. Biol. Chem. 273:22201. and lipopolysaccharide. Proc. Natl. Acad. Sci. USA 90:9730. and bacterial lipopolysaccharide. J. Exp. Med. 177:1779.B/Rel in induction of nitric oxide synthase. J. Biol. Chem. 269:4705.This article has been cited by other articles:
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 M. Cabanski, M. Steinmuller, L. M. Marsh, E. Surdziel, W. Seeger, and J. Lohmeyer PKR Regulates TLR2/TLR4-Dependent Signaling in Murine Alveolar Macrophages Am. J. Respir. Cell Mol. Biol., January 1, 2008; 38(1): 26 - 31. [Abstract] [Full Text] [PDF]
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|
 D. Proud, S. P. Sanders, and S. Wiehler Human Rhinovirus Infection Induces Airway Epithelial Cell Production of Human {beta}-Defensin 2 Both In Vitro and In Vivo J. Immunol., April 1, 2004; 172(7): 4637 - 4645. [Abstract] [Full Text] [PDF]
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 W. Xu, S. A. A. Comhair, S. Zheng, S. C. Chu, J. Marks-Konczalik, J. Moss, S. J. Haque, and S. C. Erzurum STAT-1 and c-Fos interaction in nitric oxide synthase-2 gene activation Am J Physiol Lung Cell Mol Physiol, July 1, 2003; 285(1): L137 - L148. [Abstract] [Full Text] [PDF]
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J. E. Gern, D. A. French, K. A. Grindle, R. A. Brockman-Schneider, S.-I. Konno, and W. W. Busse Double-Stranded RNA Induces the Synthesis of Specific Chemokines by Bronchial Epithelial Cells Am. J. Respir. Cell Mol. Biol., June 1, 2003; 28(6): 731 - 737. [Abstract] [Full Text] [PDF]
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T. R. Meusel, K. E. Kehoe, and F. Imani Protein Kinase R Regulates Double-Stranded RNA Induction of TNF-{alpha} But Not IL-1{beta} mRNA in Human Epithelial Cells J. Immunol., June 15, 2002; 168(12): 6429 - 6435. [Abstract] [Full Text] [PDF]
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 D. Liu, A. K. Cardozo, M. I. Darville, and D. L. Eizirik Double-Stranded RNA Cooperates with Interferon-{gamma} and IL-1{beta} to Induce Both Chemokine Expression and Nuclear Factor-{kappa}B-Dependent Apoptosis in Pancreatic {beta}-Cells: Potential Mechanisms for Viral-Induced Insulitis and {beta}-Cell Death in Type 1 Diabetes Mellitus Endocrinology, April 1, 2002; 143(4): 1225 - 1234. [Abstract] [Full Text] [PDF]
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T. Kawai, O. Takeuchi, T. Fujita, J.-i. Inoue, P. F. Muhlradt, S. Sato, K. Hoshino, and S. Akira Lipopolysaccharide Stimulates the MyD88-Independent Pathway and Results in Activation of IFN-Regulatory Factor 3 and the Expression of a Subset of Lipopolysaccharide-Inducible Genes J. Immunol., November 15, 2001; 167(10): 5887 - 5894. [Abstract] [Full Text] [PDF]
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 I. Davis and S. Matalon Reactive Species in Viral Pneumonitis: Lessons From Animal Models Physiology, August 1, 2001; 16(4): 185 - 190. [Abstract] [Full Text] [PDF]
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 M. J. de Veer, M. Holko, M. Frevel, E. Walker, S. Der, J. M. Paranjape, R. H. Silverman, and B. R. G. Williams Functional classification of interferon-stimulated genes identified using microarrays J. Leukoc. Biol., June 1, 2001; 69(6): 912 - 920. [Abstract] [Full Text] [PDF]
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D. Liu, M. Darville, and D. L. Eizirik Double-Stranded Ribonucleic Acid (RNA) Induces {beta}-Cell Fas Messenger RNA Expression and Increases Cytokine-Induced {beta}-Cell Apoptosis Endocrinology, June 1, 2001; 142(6): 2593 - 2599. [Abstract] [Full Text] [PDF]
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R. A. Dweik, S. A. A. Comhair, B. Gaston, F. B. J. M. Thunnissen, C. Farver, M. J. Thomassen, M. Kavuru, J. Hammel, H. M. Abu-Soud, and S. C. Erzurum NO chemical events in the human airway during the immediate and late antigen-induced asthmatic response PNAS, February 27, 2001; 98(5): 2622 - 2627. [Abstract] [Full Text] [PDF]
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