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B
Through Preventing IB Kinase Activation in Respiratory Epithelial Cells1
,
,,,,,
*
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea;
Clinical Research Institute, Seoul National University Hospital, Seoul, Korea; and
Lung Institute, Seoul National University Medical Research Center, Seoul, Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation, we
explored the relationship between the anti-inflammatory effect of
HSP induction and the NF-B/IB
pathway. Both HS and sodium
arsenite treatment increased HSP70 expression time dependently at mRNA
and protein levels. Prior induction of HSP suppressed cytokine-induced
IL-8 and TNF- expression at both mRNA and protein levels. Although
HSP induction did not affect total cellular expression of NF-B,
TNF--induced increase in NF-B-DNA binding activity and nuclear
translocation of the p65 subunit of NF-B were inhibited by prior HSP
induction, suggesting that activation of NF-B was blocked.
Cytokine-induced IB phosphorylation and its degradation were
blocked in HSP-induced cells. Immune complex kinase assays demonstrated
that TNF- induced increase in IB kinase activity was suppressed
by prior HSP induction. These results suggest that the
anti-inflammatory effect of HSP induction in respiratory epithelial
cells is related to stabilization of IB, possibly through the
prevention of IB kinase activation, which thereby inhibits
activation of NF-B. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß have been
extensively documented in the generation of acute lung injury
(ALI)3
(1, 2, 3). Although pro-inflammatory cytokines function in
redundant and overlapping ways through cytokine "cascades" or
"networks," transcription of most pro-inflammatory cytokine genes
is regulated by NF-B activation.
NF-B is a ubiquitous transcription factor that plays an essential
role in the regulation of a variety of genes involved in immune
function, inflammatory response, endothelial cell activation, and the
control of cell growth (4, 5, 6). In most cell types,
NF-B, which is normally sequestered in the cytoplasm in an inactive
form by virtue of its association with a class of inhibitory proteins
called IBs, is rapidly activated in response to various stimuli,
including viral infection, LPS, UV irradiation, and pro-inflammatory
cytokines such as TNF- and IL-1ß (4, 5, 6). TNF-
leads to sequential activation of the downstream NF-B-inducing
kinase (NIK) and recently isolated TNF--inducible IB kinase
complex (IKK and IKKß, also known as IKK-1 and IKK-2,
respectively) (7, 8, 9, 10, 11). Upon cell stimulation by a wide
variety of stimuli, signal-responsive IKK and -ß are activated and
directly phosphorylate Ser32 and
Ser36 in the IB, triggering ubiquitination
at Lys21 and Lys22, and
rapid degradation of IB in 26S proteasome (4, 5, 6).
This process liberates NF-B, allowing it to translocate to the
nucleus. In the nucleus, NF-B binds to its cognate site, B
element (GGGAATTCCC), and transactivates the downstream genes. Because
most genes for inflammatory mediators such as TNF-, IL-2, IL-6,
IL-8, lymphotoxin, GM-CSF, ß-IFN, and adhesion molecules have a B
site in the 5' flanking region, their transcriptions are regulated by
NF-B activation (4, 5, 6).
A diverse array of metabolic insults including the exposure of cells to elevated temperatures, heavy metals various ionophores, amino acid analogues, and metabolic poisons result in the increased expression of genes encoding a group of proteins referred to as the heat shock proteins (HSPs). HSPs are a group of proteins ranging in molecular mass from 8 to 110 kDa. These HSPs seem to confer protection against diverse forms of cellular and tissue injury, including ALI. Villar et al. (12) first demonstrated that pretreatment with heat induces the synthesis of the HSPs in the lungs and attenuates lung damage in a rat model of ALI induced by intratracheal instillation of phospholipase A2. Lung damage was also attenuated by prior HSP induction in an animal model of sepsis-induced ALI (13). Recently, induction of the HSPs even after endotoxin challenge was demonstrated to be protective (14). Considering the fact that in vitro induction of the heat shock (HS) response protected lung cells against endotoxin and oxidants, the in vivo protective effect may be through protecting lung cells (15, 16). However, the mechanisms by which the HSPs exert a cytoprotective effect are not well understood.
Although the molecular chaperone properties of the HSPs are regarded as the main mechanism of the cytoprotective effects of HSPs, there is currently no evidence to directly support this as the protective mechanism in the lung. Another important feature of the stress response is inhibition of gene expression of nonstress protein. During stress, cells undergo a prioritization of gene expression characterized by the rapid expression of stress proteins, whereas the expression of various nonstress proteins is transiently inhibited (17). Because the release of pro-inflammatory mediators is associated with injury to the endothelial and epithelial cells of the lung (18, 19), it can be assumed that the cytoprotective effect of HSPs may be related to the inhibition of pro-inflammatory cytokine gene expression. Actually, it is well documented that HSP induction inhibits pro-inflammatory cytokine gene expression in mononuclear cells (20, 21). Recently, RANTES and inducible NO synthase gene expressions were shown to be inhibited by prior HSP induction in human and murine lung epithelial cells, respectively (22, 23). In addition to these in vitro studies, the protective effect of HSP induction was documented to be related to the attenuation of plasma IL-1ß concentrations in an in vivo model of ALI (14). These findings suggest that one potential mechanism of protection may be the ability of HSPs to inhibit pro-inflammatory responses in lung cells.
In this study, we investigated the mechanism by which the HSPs exert a
cytoprotective effect in respiratory epithelial cells. We found that
prior induction of HSP blocked NF-B activation, and TNF--induced
IL-8 and IL-1ß-induced TNF- expression. This blocking was likely
related to stabilization of IB, possibly through the prevention
of IKK activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BEAS-2B cell line, representing normal human bronchial epithelial cells, and A549 cell line, representing type II alveolar epithelial cells, were used in all experiments. BEAS-2B cells were maintained as a monolayer in KGM medium (Clonetics, Walkersville, MD) and A549 cells in RPMI 1640 medium containing 10% FBS, 60 µg/ml penicillin, and 100 µg/ml streptomycin at 37°C under 5% CO2.
Reagents
Recombinant human TNF- and IL-1ß and the ELISA kit for
TNF- and IL-8 were purchased from R&D Systems (Minneapolis, MN).
Stock solutions of the cytokines were prepared in distilled water and
aliquots were stored at -70°C until use. Rabbit polyclonal
anti-IB, anti-p65, anti-p50, anti-IKK Abs,
mouse monoclonal anti-HSP70 Ab, and recombinant GST-IB were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit
polyclonal anti-phosphorylated-IB Ab
(Ser32) was supplied by New England Biolabs
(Beverly, MA). Goat anti-rabbit secondary Ab conjugated with HRP
and T4 polynucleotide kinase were purchased from Promega (Madison, WI).
Rhodamine isothiocyanate-conjugated goat anti-rabbit IgG Ab was
obtained from Jackson ImmunoResearch (West Grove, PA). Protein-G
Sepharose beads and the enhanced chemiluminescence kit were supplied by
Amersham Pharmacia Biotech (Uppsala, Sweden). Protease inhibitors were
obtained from Roche (Mannheim, Germany). Proteasome inhibitor MG132
(Z-Leu-Leu-Leu-H) was purchased from the Peptide Institute (Osaka,
Japan). TRIZOL reagent was obtained from Life Technologies
(Gaithersburg, MD). [-32P]dCTP and
[
-32P]ATP were supplied by ICN
Pharmaceuticals (Costa Mesa, CA). Random priming kit was purchased from
Stratagene (La Jolla, CA).
HS and sodium arsenite (SA) treatment
In all experiments, the HS response was induced by incubating
cells in a water bath at 43°C for 2 h with BEAS-2B cells, and
4 h with A549 cells. For SA treatment, culture dishes were
maintained at 37°C and SA was added at a concentration of 1 mM for
2 h. After this initial treatment with HS or SA, culture medium
was removed and replaced with fresh medium. Cells were then allowed to
recover in 5% CO2 incubator at 37°C for
various times before stimulation with IL-1ß or TNF- at a
concentration of 5 ng/ml.
Northern blot analysis
Total cellular RNA was isolated using TRIZOL reagent. The
precipitated total RNA pellet was washed with 1 ml of 75% ethanol,
air-dried, and resuspended in 20 µl of sterile
diethylpropylcarbonate-treated water. Equal amounts of total RNA (20
µg/lane) from each sample were loaded on to a 1.0% agarose/2%
formaldehyde gel and capillary transferred to a nylon membrane. The RNA
was cross-linked to the nylon membrane by 1500 Joules UV irradiation in
a UV cross-linker (Stratagene). The human cDNA for inducible HSP70,
TNF-, and IL-8 were radiolabeled with
[-32P]dCTP using random priming kit. After
prehybridizing the membranes for 2 h at 45°C in hybridization
buffer, radiolabeled cDNA probe (1 x 106
cpm/ml final concentration) was added and incubated overnight at
45°C. The membranes were then washed at 45°C, 50°C, and then
55°C, sequentially. The membranes were exposed to x-ray film (Eastman
Kodak, Rochester, NY) in a cassette with intensifying screen for up to
5 days at -70°C.
IL-8 and TNF- ELISA
Cells (1 x 104) were grown in
96-well culture plates in equal numbers. The supernatants were
collected and stored at -70°C until being analyzed. IL-8 and TNF-
concentrations were quantitated using ELISA kit according to the
manufacturer’s specifications.
Preparation of cytoplasmic and nuclear extracts
Cells were washed twice with 1x PBS and allowed to equilibrate for 5 min in ice-cold cytoplasmic extraction buffer (CEB) consisting of 10 mM Tris-HCl (pH 7.9), 60 mM KCl, l mM EDTA, 1 mM DTT. Cells were lysed on ice for 5 min in 0.4% Nonidet P-40/CEB/protease inhibitor cocktail (50 µg/ml antipain, 40 µg/ml bestatin, 100 µg/ml chymostatin, 4 µg/ml E-64, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 20 µg/ml phosphoramidon, 0.4 mg/ml pefabloc SC, 0.2 mg/ml EDTA, 2 µg/ml aprotinin, and 0.5 mM PMSF). Cells were gently scraped with rubber policeman. Following centrifugation at 2500 rpm for 5 min, the supernatants (cytoplasmic extracts) were collected and snap frozen on dry ice. The nuclear pellets were washed in detergent-free CEB containing all the protease inhibitors, then suspended in nuclear extraction buffer (NEB) consisting of 20 mM Tris-HCl (pH 7.9), 0.4 M NaCl, 1.5 mM MgCl2, 1.5 mM EDTA, 1 mM DTT, 25% glycerol, and the protease inhibitor cocktails listed above. After vigorous vortex mixing at maximum speed and incubating for 10 min on ice, the solution was clarified by centrifugation at 10,000 rpm for 5 min, and the supernatant (nuclear extract) was collected and snap frozen on dry ice before storage at -70°C. The protein concentration was determined by the Bradford method.
Western Blot Analysis
Cells were lysed in whole lysis buffer (0.1% Nonidet P-40, 5 mM
EDTA, 50 mM Tris (pH 
7.5–8.0), 250 mM NaCl, and 50 mM NaF).
Aliquots containing 30 µg of total protein were resolved on 10%
SDS-PAGE, and transferred to nitrocellulose. The membranes were blocked
with 5% skim milk-PBS/0.1% Tween 20 for 1 h before overnight
incubation at room temperature with mouse monoclonal anti-HSP70,
rabbit polyclonal anti-p65, anti-IB Ab, or Ab specific to
phosphorylated IB, and diluted 1:1000 in 5% skim milk-PBS/0.1%
Tween 20. Membranes were washed three times in 1x PBS/0.1% Tween 20,
and incubated with HRP-conjugated secondary Ab, and then diluted 1:2000
in 5% skim milk-PBS/0.1% Tween 20 for 1 h. Following successive
washes, the membranes were developed with an enhanced
chemiluminescence kit.
Immunofluorescent staining for NF-B
Cells grown in 2-well chamber slides were fixed with freshly prepared 3% formaldehyde at room temperature for 5 min and permeabilized with 0.5% Triton X-100 on ice for 5 min. Cells were then rinsed twice with 1x PBS. After 30 min of blocking with 1% BSA, cells were incubated with rabbit polyclonal anti-p65 Ab, and diluted 1:100 in 1% BSA, for 30 min. Cells were washed with 1x PBS and then incubated with rhodamine isothiocyanate-conjugated goat anti-rabbit Ig G Ab, diluted 1:100 in 1% BSA, for 30 min. After mounting with 50% glycerol, slides were analyzed using an MRC-100 confocal microscope (Bio-Rad, Hercules, CA).
EMSA
The NF-B double-stranded oligonucleotide corresponding to the
NF-B consensus sequence in the light chain enhancer in B cells
(5'-AGT TGA GGG GAC TTT CCC AGG C-3') was end-labeled with
[-32P]ATP and T4 polynucleotide kinase and
purified with G-25 columns. Nuclear extracts (10 µg) were added
to radiolabeled NF-B oligonucleotide (50,000–200,000 cpm) in a
binding buffer containing 4% glycerol, 1 mM
MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl,
10 mM Tris-HCl (pH 7.5), and poly(dI-dC)·poly(dI-dC). Reaction
mixtures were incubated for 20 min at room temperature. In competition
experiments, 50-fold molar excess of unlabeled oligonucleotide was
added to the nuclear extracts and binding buffer and the reaction
mixture was incubated for 5 min before the addition of the radiolabeled
probe. In supershift experiments, after the oligonucleotide had reacted
for 20 min with the nuclear extract, 0.4 µg of anti-p65 or
anti-p50 Ab was added and allowed to react for 45 min at room
temperature. DNA-protein complexes were resolved on 4% nondenaturing
polyacrylamide gel (80:1 acrylamide:bisacrylamide). Gels were dried and
autoradiographed at -70°C.
IKK assay
Immune complex kinase assay for endogenous IKK activity was
performed by the methods of DiDonato et al. (7). Whole
cell lysates were prepared by lysing cells in a buffer containing 20 mM
Tris-HCl (pH 7.6), 150 mM NaCl, 25 mM ß-glycerophosphate, 2 mM EDTA,
2 mM pyrophosphate, 1 mM sodium orthovanadate, 10% glycerol, 1%
Triton X-100, 1 mM DTT, 10 µg/ml leupeptin, and 1 mM PMSF. After
centrifugation of the lysate at 16,000 x g for 10 min
at 4°C, the supernatant was incubated with anti-IKK Ab,
diluted 1:100, and with 50 µl of protein-G Sepharose beads with
end-over-end rotation overnight at 4°C. The beads were then washed
twice sequentially in buffer A (1 M NaCl, 20 µM Tris-HCl (pH 7.4),
and 0.1% Nonidet P-40), buffer B (200 µM NaCl, 20 µM Tris-HCl (pH
7.4), 1% Nonidet P-40, 0.1% SDS, and 1 µM EDTA), and buffer C (20
µM Tris-HCl (pH 7.4) and 0.1% Nonidet P-40). Kinase reactions were
initiated by the addition of 10 µl of buffer (20 mM HEPES (pH 7.6),
20 mM ß-glycerophosphate, 0.1 mM sodium orthovanadate, 10 mM
MgCl2, 50 mM NaCl, and 1 mM DTT) containing 0.5
µg GST-IB (containing aa 1–317), and 10 µCi of
[-32P]ATP. The reaction mixture was
incubated at 30°C for 30 min. The kinase reaction was terminated by
adding protein sample buffer. Kinase reaction products were
subjected to SDS/PAGE in 10% gels followed by transfer to a
nitrocellulose membrane and autoradiography. This membrane was later
used for immunoblot with anti-IKK Ab to ensure that equal
amounts of kinase were immunoprecipitated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the optimal condition for HSP induction in
respiratory epithelial cells, we first analyzed the time-dependent
effect of stress on HSP expression. BEAS-2B and A549 cells were
heat-shocked at 43°C for 2 and 4 h, respectively, or treated
with SA for 2 h, and then incubated in 5%
CO2 incubator at 37°C for 1, 2, 4, and 8 h
after changing the medium. HSP70 mRNA levels were determined by
Northern blot analysis. Although HSP70 mRNA was hardly detectable in
control cells, both HS and SA treatment induced HSP70 mRNA expression
1 h after stress, and persisted up to 8 h (Fig. 1
A). To see whether this
increase in HSP70 mRNA expression resulted in an increase in HSP70,
total cellular extracts from control and HS- or SA-treated cells were
subjected to Western blot analysis. Low levels of HSP70 were detectable
in control cells. Inducible HSP70 increased after 4 h of HS or SA
treatment in BEAS-2B cells, and after 1 h in A549 cells. The
increased expression of HSP70 persisted up to 24 h in both cells
(Fig. 1B).
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expressions are suppressed by HSP
induction
To determine whether HSP induction influenced the expression of
pro-inflammatory cytokines, we incubated BEAS-2B and A549 cells at
37°C or 43°C for 2 and 4 h, respectively, after which cells
were allowed to recover in 5% CO2 incubator at
37°C for 4 h before addition of IL-1ß or TNF-. IL-8 and
TNF- protein levels in culture supernatants were determined by ELISA
18 h after stimulation. In control cells, IL-1ß and TNF-
induced production of TNF- and IL-8, respectively. These inductions
were significantly suppressed by prior stress (Fig. 2A). Decreased productions of
TNF- and IL-8 proteins in HSP-induced cells could be due to changes
in mRNA levels and/or protein translation. To determine whether mRNA
levels were decreased, we measured TNF- and IL-8 mRNA levels 4
h after stimulation with IL-1ß by Northern blot analysis. Prior
induction of HSP completely blocked IL-1ß-induced TNF- and IL-8
mRNA expressions (Fig. 2B), suggesting suppression of gene
expression.
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B activation
Because most of the pro-inflammatory cytokine genes including
TNF- and IL-8 contain B-binding motifs in their promoter regions,
their transcriptions are dependent on NF-B activation. To test the
possibility that the effects of HSP induction on the suppression of
pro-inflammatory cytokine expression are due to blocking of NF-B
activation, we measured TNF--induced NF-B activity. NF-B
activity was assayed by two approaches: one was measuring the
NF-B-DNA binding activity by EMSA and the other was assessing the
nuclear translocation of NF-B. Control and HSP-induced cells were
stimulated with TNF- (5 ng/ml) for 30 min. To evaluate the effect of
prior HSP induction on the NF-B-DNA binding activity, nuclear
extracts were prepared and subjected to EMSA with B site DNA probe.
Nuclear extracts from TNF--stimulated cells had more active NF-B
available to bind to the B probe compared with extracts from
untreated cells. This TNF--induced increase in NF-B-DNA binding
activity was inhibited by prior HSP induction (Fig. 3A). When 50-fold molar excess
of unlabeled double-stranded NF-B oligonucleotide was added to the
binding reaction, the retarded band disappeared, suggesting specificity
of the binding (data not shown). The supershift assay showed the
presence of p50 and p65 subunits (data not shown). To confirm that
nuclear p65 levels were reduced in HSP-induced cells, as well as to
verify the identity of the nuclear Ag detected, we subjected
cytoplasmic and nuclear extracts from control and HSP-induced cells to
Western blot analysis. Although the majority of p65 was located in the
cytoplasmic fraction of control cells in basal states, 30 min of
incubation with TNF- caused an increase in the nuclear expression of
p65. In contrast, nuclear p65 levels were greatly reduced by prior HSP
induction (Fig. 3B). We also investigated the subcellular
localization of p65 by immunofluorescent staining. In control cells,
incubation with TNF- caused nuclear uptake of p65 as demonstrated by
clear nuclear staining, as opposed to the cytoplasmic distribution in
unstimulated cells. In HSP-induced cells, nuclear translocation of p65
was reduced compared with TNF- treated cells (Fig. 3C).
These results indicate that prior HSP induction suppresses
TNF--induced NF-B activation.
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To investigate whether HSP induction inhibits NF-B activation
by decreasing total cellular p65 expression, we measured p65 protein
levels at various times after HSP induction. The total cellular level
of p65 did not change up to 24 h after HS or SA treatment (Fig. 4).
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B by blocking IB
phosphorylation
Because NF-B exists in an inactive form in the cytoplasm bound
to inhibitory protein IB, degradation of IB is a
prerequisite for the activation of NF-B. To test the possibility
that the effects of HSP induction on NF-B activation were due to a
stabilization of IB, we measured IB protein levels from
control and HSP-induced cells after stimulation with IL-1ß and
TNF- by Western blot analysis. In control cells, IB was
markedly degraded after 30 min of incubation with IL-1ß and TNF-.
In contrast, degradation of IB was blocked by prior HSP induction
(Fig. 5A). These observations
suggest that blocking of NF-B activation is likely to be due to the
stabilization of IB by prior HSP induction. Because IB
degradation is preceded by phosphorylation of two serine residues
(Ser32 and Ser36), we
tested IL-1ß- and TNF--induced phosphorylation of IB from
control and HSP-induced cells to evaluate the mechanism involved in the
stabilization of IB by HSP. Control and HSP-induced cells were
pretreated with proteasome inhibitor MG132 for 1 h before addition
of IL-1ß and TNF-. Total cellular extracts were subjected to
Western blot analysis. In control cells, pretreatment of MG132
stabilized the phosphorylated IB in response to IL-1ß and
TNF-, which was detectable as a slower migrating band as previously
reported (24, 25, 26). In contrast, this slowly migrated band
was not observed in HSP-induced cells (Fig. 5B). These
slowly migrated bands were confirmed as phosphorylated IB in
immunoblotting with Ab specific to phosphorylated IB at
Ser32 (Fig. 5C). These observations
suggest that IB stabilization is likely to occur at the level of
IB phosphorylation.
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To evaluate the effect of HSP induction on IKK activity, IKK
activities were measured by immune complex kinase assays after TNF-
stimulation in control and HSP-induced cells. In control cells, IKK
activity was induced by TNF- stimulation. In contrast, IKK activity
was markedly suppressed in HSP-induced cells (Fig. 6). This inhibition of IKK activity by
prior HSP induction was not due to a decrease in IKK protein levels,
as immunoblot analysis demonstrated comparable IKK expression at all
conditions (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As molecular chaperones, HSPs have the capacity to bind to folding intermediates and misfolded or denatured proteins and prevent their irreversible denaturation. Furthermore, they assist in renaturation and correct refolding of misfolded proteins (27, 28, 29). Another important feature of the stress response is the inhibitory effect on the gene expression of the nonstress protein (17). In this study, we investigated the effect of HSP induction on the expression of pro-inflammatory cytokines. We demonstrated that the induction of the HSPs inhibited pro-inflammatory cytokine expressions in vitro. This is consistent with previous findings (23, 30, 31, 32, 33). The suppressive effect of HSP induction on the pro-inflammatory cytokine expression can be generalized to respiratory epithelial cells, because the same effect was observed in both BEAS-2B and A549 cells, which represent bronchial and alveolar epithelial cells, respectively. Cell survival studies indicated that an attenuation of pro-inflammatory cytokine expression caused by HSP induction was not due to cytotoxicity (data not shown). Although we did not directly measure the transcriptional rate, reductions in both steady-state mRNA and protein levels by prior HS or SA treatment can be accounted for at least in part by the transcriptional regulation of HSPs. Because the release of pro-inflammatory mediators is associated with injury to the endothelial and epithelial cells of the lung (18, 19), an attenuation of pro-inflammatory cytokine expression by prior HSP induction means that HSPs may exert a cytoprotective effect to respiratory epithelial cells by blocking the inflammatory response.
Because transcription of most pro-inflammatory cytokine genes is
dependent on NF-B activation, it is very likely that the suppression
of pro-inflammatory cytokine expression by prior HSP induction is
closely related to the blocking of NF-B activation. Our EMSA data
clearly demonstrated that TNF--induced NF-B DNA binding activity
was blocked by HSP induction. However, transcriptional activation,
based sorely upon EMSA, may be complicated by the fact that
transcriptionally inactive NF-B complexes lacking p65, for example
p50 homodimers, can also bind to B sites and result in decreased
electrophoretic mobility. We could identify the main retarded bands in
EMSA that were transcriptionally active p65/p50 heterodimers in a
supershift assay (data not shown). To confirm this further, we tested
whether HSP induction did block nuclear translocation of the p65
subunit of NF-B by immunoblot and immunofluorescent staining. The
nuclear translocation of the p65 subunit was reduced by HSP induction.
These results support the possibility that HSP induction reduces
transcriptional activation of pro-inflammatory cytokines by blocking
the activation of NF-B. Because total cellular expression of p65 was
not changed in either HS or SA treatment for up to 24 h, this
blocking effect of HSP induction on the cytokine-induced activation of
NF-B was not due to the decrease in total cellular amount of
NF-B. In vertebrates, NF-B consists of homo- or heterodimers of
p65 (RelA), RelB, c-Rel, p50 (NFKB1), and p52 (NFKB2). Because the
prototype NF-B transcription factor consists of p50 and p65 (RelA),
we focused the effect of HSP induction on p65 and p50 and did not
examine the other Rel family in this study.
One possible mechanism by which HS can interfere with the activation of
NF-B is that HSP70, which also translocates to the nucleus
(34), impedes NF-B nuclear translocation by competing
for access to nuclear pore complexes through which NF-B is
transported (35). Considering the fact that activation of
NF-B needs degradation of IB, a second possibility is that
HSP70 exerts its effect on NF-B through IB. We found that
blocking of NF-B activation by prior HSP induction was secondary to
the stabilization of IB. These observations, in which induction
of the HSPs blocked nuclear translocation of NF-B by inhibiting
IB degradation, are in accord with previous studies (22, 23).
The first step of IB degradation is phosphorylation of IB.
Whereas our results suggest that cytokine-induced IB
phosphorylation was inhibited by prior HSP induction, others have
failed to detect the effects of HS on IB phosphorylation
(22). One factor, which may contribute to this
discrepancy, is the duration of HS and recovery. In the present study,
we induced HS response for 4 h in A549 cells, and then allowed the
cells to recover for 4 h before treatment with TNF-. In
contrast, Ayad et al. (22) heat-shocked cells for 1
h, and then incubated the cells at 37°C for 1 h before
stimulation with TNF-. Our results were not specific to TNF-
stimulation or to A549 cells, because the same inhibition of IB
phosphorylation was also observed in BEAS-2B cells and in cells
stimulated with IL-1ß. A recent report suggested that the HS response
in BEAS-2B cells increased the expression of IB mRNA in a
time-dependent manner (36). It is therefore possible that
an up-regulation of IB might be another potential mechanism by
which HSPs block NF-B activation.
The mechanisms by which HSPs interfere with the cytokine-induced
phosphorylation of IB are not yet known. As phosphorylated
IB is in equilibrium of phosphorylation by IKK and
dephosphorylation by phosphatase, the decrease in phosphorylated
IB by HSP induction could be due to either inhibition of IKK
activity or activation of phosphatase. To differentiate these two
possibilities, we examined the effect of HSP induction on IKK activity
by immune complex in vitro kinase assays. Prior HSP induction reduced
the TNF--induced activation of IKK. These results indicate that
inhibition of IB phosphorylation by prior HSP induction is more
likely related to the inhibition of IKK activation rather than the
activation of phosphatase. The "molecular chaperone" properties of
HSPs led us to speculate that HSP70 may bind IKK to inhibit its
activity. However, we did not observe any binding between HSP70 and IKK
in immunoprecipitation studies (data not shown). Although inhibition of
radiation-induced IKK activation was reported in HeLa cells recently
(37), this is the first report to demonstrate that HSP
induction suppresses cytokine-induced activation of IKK in respiratory
epithelial cells.
The TNF-- and IL-1ß-induced NF-B/IB signaling pathway
involves distinct pathways. TNF- stimulation recruits TNF
receptor-associated factor 2 (TRAF-2) and the receptor-interacting
protein (RIP) (38, 39), whereas IL-1ß uses the IL-1R
accessory protein and the IL-1R-associated kinase (IRAK) to transmit
signals to TRAF-6 (40, 41). The TNF- and IL-1ß
pathways converge on NIK to activate the IKK complex. Thus, the target
to block cytokine-induced degradation of IB could be RIP, TRAF-2,
TRAF-6, NIK, or IKK. However, because prior HSP induction in this study
blocked both TNF-- and IL-1ß-induced phosphorylation of IB
by inhibiting the activation of IKK, it seems likely that HSP induction
interferes with a common signal upstream or parallel to IKK. How HSP
prevents IKK activation was not directly addressed in the present
study. As inhibition of NF-B activation through inhibition of IKK
was attributed to RelB in fibroblasts in a recent report
(42), it cannot be excluded that RelB suppresses cytokine
expression by modulating the stability of IB.
Some additional action other than IKK inhibition could be considered as
the possible mechanism of blocking IB phosphorylation. The
association of IB with NF-B occurs via interaction of IB
ankyrin domains with nuclear localization sites. Mutational analysis
has confirmed the presence of a nuclear localization site region in
human HSP70 (34), which raises the possibility that HSP70
can specifically interact with ankyrin domains present in IB.
Such an interaction could conceivably hinder IB
phosphorylation.
In this study, we have shown that HSP induction inhibits
pro-inflammatory cytokine production and blocks NF-B activation in
respiratory epithelial cells. This inhibitory effect may be related to
stabilization of IB, possibly through the prevention of IKK
activation. Taken together with protective effects of HSP induction in
an in vivo animal model of ALI (13, 14), the findings
presented here suggest that HSP induction may be utilized a novel
therapeutic modality in ALI.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Chul-Gyu Yoo, Department of Internal Medicine, Lung Institute, and Clinical Research Institute, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea.
3 Abbreviations used in this paper: ALI, acute lung injury; IKK, IB kinase; HS, heat shock; SA, sodium arsenite; HSP, HS protein; MG132, Z-Leu-Leu-Leu-H (aldehyde); NIK, NF-B-inducing kinase.
Received for publication December 21, 1999. Accepted for publication March 6, 2000.
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References |
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B and IB proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[Medline]
B: a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med. 336:1066.B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16:225.[Medline]
B kinase that activates the transcription factor NF-B. Nature 388:548.[Medline]
B kinases essential for NF-B activation. Science 278:860.B kinase complex (IKK) contains two kinase subunits, IKK and IKKß, necessary for IB phosphorylation and NF-B activation. Cell 91:243.[Medline]
B kinase. Cell 90:373.[Medline]
B kinase-ß: NF-B activation and complex formation with IB kinase and NIK. Science 278:866.-B degradation and NF-B nuclear translocation. Biochem. Biophys. Res. Commun. 231:257.[Medline]
B1 precursor protein and the activation of NF-B. Cell 78:773.[Medline]
B to the ubiquitin-proteasome pathway. Genes Dev. 9:1586.B nuclear translocation and increases I-B expression in A549 cells. J. Clin. Invest. 99:2423.[Medline]
B promoter and increases I-B mRNA expression. Cell Stress Chaperones 4:1.[Medline]
B via inhibition of I-B kinase. J. Biol. Chem. 274:23061.B stability as a mechanism of transcription suppression of interleukin-1 (IL-1), IL-1ß, and tumor necrosis factor in fibroblasts. Mol. Cell. Biol. 19:7688.This article has been cited by other articles:
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  M. Liang, X. Wang, Y. Yuan, Q. Zhou, C. Tong, and W. Jiang Different effect of glutamine on macrophage tumor necrosis factor-alpha release and heat shock protein 72 expression in vitro and in vivo Acta Biochim Biophys Sin, February 1, 2009; 41(2): 171 - 177. [Abstract] [Full Text] [PDF]
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 G. C. Bowick, H. M. Spratt, A. E. Hogg, J. J. Endsley, J. E. Wiktorowicz, A. Kurosky, B. A. Luxon, D. G. Gorenstein, and N. K. Herzog Analysis of the Differential Host Cell Nuclear Proteome Induced by Attenuated and Virulent Hemorrhagic Arenavirus Infection J. Virol., January 15, 2009; 83(2): 687 - 700. [Abstract] [Full Text] [PDF]
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 E Rodriguez, J Guevara, A Paez, E Zapata, M. Collados, T. Fortoul, R Lopez-Marure, F Masso, and L. Montano The altered expression of inflammation-related molecules and secretion of IL-6 and IL-8 by HUVEC from newborns with maternal inactive systemic lupus erythematosus is modified by estrogens Lupus, December 1, 2008; 17(12): 1086 - 1095. [Abstract] [PDF]
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 I. S. Singh, A. Gupta, A. Nagarsekar, Z. Cooper, C. Manka, L. Hester, I. J. Benjamin, J.-r. He, and J. D. Hasday Heat Shock Co-Activates Interleukin-8 Transcription Am. J. Respir. Cell Mol. Biol., August 1, 2008; 39(2): 235 - 242. [Abstract] [Full Text] [PDF]
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 J. A. Poff, C. T. Allen, B. Traughber, A. Colunga, J. Xie, Z. Chen, B. J. Wood, C. Van Waes, K. C. P. Li, and V. Frenkel Pulsed High-Intensity Focused Ultrasound Enhances Apoptosis and Growth Inhibition of Squamous Cell Carcinoma Xenografts with Proteasome Inhibitor Bortezomib Radiology, August 1, 2008; 248(2): 485 - 491. [Abstract] [Full Text] [PDF]
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 M. Choi, B. Salanova, S. Rolle, M. Wellner, W. Schneider, F. C. Luft, and R. Kettritz Short-Term Heat Exposure Inhibits Inflammation by Abrogating Recruitment of and Nuclear Factor-{kappa}B Activation in Neutrophils Exposed to Chemotactic Cytokines Am. J. Pathol., February 1, 2008; 172(2): 367 - 777. [Abstract] [Full Text] [PDF]
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 K. D. Singleton and P. E. Wischmeyer Glutamine's protection against sepsis and lung injury is dependent on heat shock protein 70 expression Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2007; 292(5): R1839 - R1845. [Abstract] [Full Text] [PDF]
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 X. Feng, S. Bonni, and K. Riabowol HSP70 Induction by ING Proteins Sensitizes Cells to Tumor Necrosis Factor Alpha Receptor-Mediated Apoptosis Mol. Cell. Biol., December 15, 2006; 26(24): 9244 - 9255. [Abstract] [Full Text] [PDF]
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 S.-K. Jo, G. J. Ko, C. S. Boo, W. Y. Cho, and H. K. Kim Heat Preconditioning Attenuates Renal Injury in Ischemic ARF in Rats: Role of Heat-Shock Protein 70 on NF-{kappa}B-Mediated Inflammation and on Tubular Cell Injury J. Am. Soc. Nephrol., November 1, 2006; 17(11): 3082 - 3092. [Abstract] [Full Text] [PDF]
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 S. J. Kammanadiminti and K. Chadee Suppression of NF-{kappa}B Activation by Entamoeba histolytica in Intestinal Epithelial Cells Is Mediated by Heat Shock Protein 27 J. Biol. Chem., September 8, 2006; 281(36): 26112 - 26120. [Abstract] [Full Text] [PDF]
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 I. Kuwahara, E. P. Lillehoj, W. Lu, I. S. Singh, Y. Isohama, T. Miyata, and K. C. Kim Neutrophil elastase induces IL-8 gene transcription and protein release through p38/NF-{kappa}B activation via EGFR transactivation in a lung epithelial cell line Am J Physiol Lung Cell Mol Physiol, September 1, 2006; 291(3): L407 - L416. [Abstract] [Full Text] [PDF]
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J. K. L. Walker, A. Ahumada, B. Frank, R. Gaspard, K. Berman, J. Quackenbush, and D. A. Schwartz Multistrain Genetic Comparisons Reveal CCR5 as a Receptor Involved in Airway Hyperresponsiveness Am. J. Respir. Cell Mol. Biol., June 1, 2006; 34(6): 711 - 718. [Abstract] [Full Text] [PDF]
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 J. D. Johnson and M. Fleshner Releasing signals, secretory pathways, and immune function of endogenous extracellular heat shock protein 72 J. Leukoc. Biol., March 1, 2006; 79(3): 425 - 434. [Abstract] [Full Text] [PDF]
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 Y. Chen and R. W. Currie Small interfering RNA knocks down heat shock factor-1 (HSF-1) and exacerbates pro-inflammatory activation of NF-{kappa}B and AP-1 in vascular smooth muscle cells Cardiovasc Res, January 1, 2006; 69(1): 66 - 75. [Abstract] [Full Text] [PDF]
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 H. R. Wong, K. E. Dunsmore, K. Page, and T. P. Shanley Heat shock-mediated regulation of MKP-1 Am J Physiol Cell Physiol, November 1, 2005; 289(5): C1152 - C1158. [Abstract] [Full Text] [PDF]
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K.-H. Lee, C.-T. Lee, Y. W. Kim, S. K. Han, Y.-S. Shim, and C.-G. Yoo Preheating Accelerates Mitogen-activated Protein (MAP) Kinase Inactivation Post-heat Shock via a Heat Shock Protein 70-mediated Increase in Phosphorylated MAP Kinase Phosphatase-1 J. Biol. Chem., April 1, 2005; 280(13): 13179 - 13186. [Abstract] [Full Text] [PDF]
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 P. Rice, E. Martin, J.-R. He, M. Frank, L. DeTolla, L. Hester, T. O'Neill, C. Manka, I. Benjamin, A. Nagarsekar, et al. Febrile-Range Hyperthermia Augments Neutrophil Accumulation and Enhances Lung Injury in Experimental Gram-Negative Bacterial Pneumonia J. Immunol., March 15, 2005; 174(6): 3676 - 3685. [Abstract] [Full Text] [PDF]
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H. Cheng, C. Cenciarelli, G. Nelkin, R. Tsan, D. Fan, C. Cheng-Mayer, and I. J. Fidler Molecular Mechanism of hTid-1, the Human Homolog of Drosophila Tumor Suppressor l(2)Tid, in the Regulation of NF-{kappa}B Activity and Suppression of Tumor Growth Mol. Cell. Biol., January 1, 2005; 25(1): 44 - 59. [Abstract] [Full Text] [PDF]
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J.-F. Pittet, H. Lee, M. Pespeni, A. O'Mahony, J. Roux, and W. J. Welch Stress-Induced Inhibition of the NF-{kappa}B Signaling Pathway Results from the Insolubilization of the I{kappa}B Kinase Complex following Its Dissociation from Heat Shock Protein 90 J. Immunol., January 1, 2005; 174(1): 384 - 394. [Abstract] [Full Text] [PDF]
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 J. Y.H. Chan, C.-C. Ou, L.-L. Wang, and S. H.H. Chan Heat Shock Protein 70 Confers Cardiovascular Protection During Endotoxemia via Inhibition of Nuclear Factor-{kappa}B Activation and Inducible Nitric Oxide Synthase Expression in the Rostral Ventrolateral Medulla Circulation, December 7, 2004; 110(23): 3560 - 3566. [Abstract] [Full Text] [PDF]
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Y. Wu, S. Adam, L. Hamann, H. Heine, A. J. Ulmer, U. Buwitt-Beckmann, and C. Stamme Accumulation of Inhibitory {kappa}B-{alpha} as a Mechanism Contributing to the Anti-Inflammatory Effects of Surfactant Protein-A Am. J. Respir. Cell Mol. Biol., December 1, 2004; 31(6): 587 - 594. [Abstract] [Full Text] [PDF]
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D. Wirth, F. Bureau, D. Melotte, E. Christians, and P. Gustin Evidence for a role of heat shock factor 1 in inhibition of NF-{kappa}B pathway during heat shock response-mediated lung protection Am J Physiol Lung Cell Mol Physiol, November 1, 2004; 287(5): L953 - L961. [Abstract] [Full Text] [PDF]
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T. Nakagomi, O. Kitada, K. Kuribayashi, H. Yoshikawa, K. Ozawa, S. Ogawa, and T. Matsuyama The 150-Kilodalton Oxygen-Regulated Protein Ameliorates Lipopolysaccharide-Induced Acute Lung Injury in Mice Am. J. Pathol., October 1, 2004; 165(4): 1279 - 1288. [Abstract] [Full Text] [PDF]
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 H. M. Beere `The stress of dying': the role of heat shock proteins in the regulation of apoptosis J. Cell Sci., June 1, 2004; 117(13): 2641 - 2651. [Abstract] [Full Text] [PDF]
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C. H. Kim, K.-H. Lee, C.-T. Lee, Y. W. Kim, S. K. Han, Y.-S. Shim, and C.-G. Yoo Aggregation of {beta}2 integrins activates human neutrophils through the I{kappa}B/NF-{kappa}B pathway J. Leukoc. Biol., February 1, 2004; 75(2): 286 - 292. [Abstract] [Full Text] [PDF]
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R. Schirmbeck, N. Fissolo, P. Chaplin, and J. Reimann Enhanced Priming of Multispecific, Murine CD8+ T Cell Responses by DNA Vaccines Expressing Stress Protein-Binding Polytope Peptides J. Immunol., August 1, 2003; 171(3): 1240 - 1246. [Abstract] [Full Text] [PDF]
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K. M. Park, J.-Y. Byun, C. Kramers, J. I. Kim, P. L. Huang, and J. V. Bonventre Inducible Nitric-oxide Synthase Is an Important Contributor to Prolonged Protective Effects of Ischemic Preconditioning in the Mouse Kidney J. Biol. Chem., July 11, 2003; 278(29): 27256 - 27266. [Abstract] [Full Text] [PDF]
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 M. Qing, J. F. Vazquez-Jimenez, K. Schumacher, R. S. Bhardwaj, B. Klosterhalfen, R. Minkenberg, B. J. Messmer, G. von Bernuth, and M.-C. Seghaye Moderate hypothermia during cardiopulmonary bypass increases intramyocardial synthesis of heat shock protein 72 J. Thorac. Cardiovasc. Surg., October 1, 2002; 124(4): 724 - 731. [Abstract] [Full Text] [PDF]
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 H. Uchinami, Y. Yamamoto, M. Kume, K. Yonezawa, Y. Ishikawa, K. Taura, A. Nakajima, K. Hata, and Y. Yamaoka Effect of heat shock preconditioning on NF-kappa B/I-kappa B pathway during I/R injury of the rat liver Am J Physiol Gastrointest Liver Physiol, June 1, 2002; 282(6): G962 - G971. [Abstract] [Full Text] [PDF]
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H. Cheng, C. Cenciarelli, M. Tao, W. P. Parks, and C. Cheng-Mayer HTLV-1 Tax-associated hTid-1, a Human DnaJ Protein, Is a Repressor of Ikappa B Kinase beta Subunit J. Biol. Chem., May 31, 2002; 277(23): 20605 - 20610. [Abstract] [Full Text] [PDF]
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 L. A. Sonna, J. Fujita, S. L. Gaffin, and C. M. Lilly Molecular Biology of Thermoregulation: Invited Review: Effects of heat and cold stress on mammalian gene expression J Appl Physiol, April 1, 2002; 92(4): 1725 - 1742. [Abstract] [Full Text] [PDF]
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 G.Y. Park, S. Lee, K.H. Park, C.T. Lee, Y.W. Kim, S.K. Han, Y.S. Shim, and C.G. Yoo Anti-inflammatory effect of adenovirus-mediated I{kappa}B{alpha} overexpression in respiratory epithelial cells Eur. Respir. J., November 1, 2001; 18(5): 801 - 809. [Abstract] [Full Text] [PDF]
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P. E. Wischmeyer, M. Kahana, R. Wolfson, H. Ren, M. M. Musch, and E. B. Chang Glutamine induces heat shock protein and protects against endotoxin shock in the rat J Appl Physiol, June 1, 2001; 90(6): 2403 - 2410. [Abstract] [Full Text] [PDF]
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 J. B. Wijeweera, A. J. Gandolfi, A. Parrish, and R. C. Lantz Sodium Arsenite Enhances AP-1 and NF{{kappa}} B DNA Binding and Induces Stress Protein Expression in Precision-Cut Rat Lung Slices Toxicol. Sci., June 1, 2001; 61(2): 283 - 294. [Abstract] [Full Text] [PDF]
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S. Tsuyuki, J. A. Horvath-Arcidiacono, and E. T. Bloom Effect of Redox Modulation on Xenogeneic Target Cells: The Combination of Nitric Oxide and Thiol Deprivation Protects Porcine Endothelial Cells from Lysis by IL-2-Activated Human NK Cells J. Immunol., March 15, 2001; 166(6): 4106 - 4114. [Abstract] [Full Text] [PDF]
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C.-G. Yoo, S. Lee, C.-T. Lee, Y. W. Kim, S. K. Han, and Y.-S. Shim Effect of acetylsalicylic acid on endogenous I{kappa}B kinase activity in lung epithelial cells Am J Physiol Lung Cell Mol Physiol, January 1, 2001; 280(1): L3 - L9. [Abstract] [Full Text] [PDF]
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K. M. Park, C. Kramers, M. Vayssier-Taussat, A. Chen, and J. V. Bonventre Prevention of Kidney Ischemia/Reperfusion-induced Functional Injury, MAPK and MAPK Kinase Activation, and Inflammation by Remote Transient Ureteral Obstruction J. Biol. Chem., January 11, 2002; 277(3): 2040 - 2049. [Abstract] [Full Text] [PDF]
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