Department of Gastroenterological Surgery, Graduate School of Medicine, Kyoto University, Kyoto 606 - 8507, Japan
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ABSTRACT |
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Hepatic ischemia-reperfusion
(I/R) injury continues to be a fatal complication after liver surgery.
Heat shock (HS) preconditioning is an effective strategy for protecting
the liver from I/R injury, but its exact mechanism is still unclear.
Because the activation of nuclear factor-B (NF-
B) is an important
event in the hepatic I/R-induced inflammatory response, the effect of
HS preconditioning on the pathway for NF-
B activation was
investigated. In the control group, NF-
B was activated 60 min after
reperfusion, but this activation was suppressed in the HS group.
Messenger RNA expressions of proinflammatory mediators during
reperfusion were also reduced with HS preconditioning. Concomitant with
NF-
B activation, NF-
B inhibitor I-
B proteins were degraded in
the control group, but this degradation was suppressed in the HS group.
This study shows that HS preconditioning protected the liver from I/R
injury by suppressing the activation of NF-
B and the subsequent
expression of proinflammatory mediators through the stabilization of
I-
B proteins.
hepatic ischemia-reperfusion; nuclear factor-B
proinflammatory mediators
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INTRODUCTION |
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AN ORGANISM HAS ENDOGENOUS ability to respond to environmental stresses for survival (37, 39). This highly conserved response is called the "stress response" and is associated with the expression of a wide spectrum of proteins related to organic defense mechanisms. For example, the induction of oxygen-free radical scavenging enzymes (12, 26) and several members of the heat shock protein (HSP) family (5, 35, 52), particularly HSP-72, have been noted. HSP-72 functions as a molecular chaperone (23, 25) and contributes to the folding, assembling, and stabilization of intracellular proteins. It is believed that HSP-72 enables an organism to survive noxious stresses (10, 38). We have previously reported that heat shock (HS) preconditioning reduces liver damage caused by ischemia-reperfusion (I/R) injury resulting in a remarkable increase in survival rate (31, 44, 59, 60). We have also demonstrated a correlation between HS preconditioning-induced expression levels of HSP-72 in the liver and resulting tolerance against hepatic I/R injury. I/R injury facilitates the depletion of ATP, the deterioration of intracellular Ca2+ homeostasis (4, 21), the activation of cytotoxic enzymes (proteases, phospholipases, arachidonic acid, etc.) (20), and the generation of reactive oxygen species (ROS) (17). However, little is known about the mechanism with which HS preconditioning or HSP-72 provides protection against hepatic I/R injury.
It has recently been shown that several signaling pathways are
activated in response to hepatic I/R, and the activated transcription factors induce a variety of cellular gene expressions. For example, nuclear factor-B (NF-
B) is a ubiquitous, inducible transcription factor that regulates the expression of numerous cellular genes, particularly those involved in the inflammatory response
(2). Recently, its activation during hepatic I/R has been
well documented (64-66). NF-
B is retained in
cytoplasm by its inhibitor I-
B proteins. In response to a variety of
stimuli, cytoplasmic NF-
B/I-
B complex is disassociated, and free
NF-
B is then allowed to migrate into the nucleus where it can bind
to cognate DNA binding sites (2). We hypothesized that the
protective action of HS preconditioning on I/R injury might be mediated
by, at least in part, the suppression of NF-
B activation. In this
study, we evaluated the effect of HS preconditioning on the
NF-
B/I-
B pathway using a hepatic I/R model in rats. We also
sought to determine the role of HS preconditioning on the expression of
proinflammatory mediators during I/R of the liver.
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MATERIALS AND METHODS |
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Experimental model and animals. We have previously reported that HS preconditioning protects the liver from I/R injury by the use of a rat model of 30-min hepatic ischemia with 15-min HS preconditioning 48 h before ischemia. In this model, protective effects were clearly demonstrated with serum liver-related enzyme levels, recovery of hepatic ATP and energy charge levels, and survival rate (31, 44, 59). Therefore, the same animal model was employed in the present study.
Experiments were performed with male Wistar rats (obtained from Shizuoka Laboratory Animal Center, Shizuoka, Japan) weighing between 280 and 320 g. They were housed in a climatized room with a 12:12-h light-dark cycle and had free access to water and food. They received humane care in compliance with the Animal Protection Guidelines of Kyoto University.Preconditioning. Rats were divided into either an HS group (group HS) or control group (group C). To monitor their rectal temperature, all rats were anesthetized with pentobarbital sodium (40 mg/kg ip) and fitted with a thermocouple probe inserted in the rectum 2-3 cm beyond the anal sphincter. Because rectal temperature during HS treatment shows good parallelism with the directly measured liver temperature, as reported elsewhere (31), we monitored rectal temperature in place of liver temperature in this study. Rats in group HS were bathed in 42.5°C water until their rectal temperature reached 42°C and were then moved to 41.5°C water to maintain their temperatures at 42°C for 15 min. In group C, rats were bathed in a 37°C water bath for 35 min to proximate the total duration of preconditioning in group HS, which was 35-40 min. Rats were allowed to recover from anesthesia and were returned to their cages.
I/R. Rats from both pretreatment groups recovered for 48 h and were anesthetized again. A midline laparotomy was performed, and total hepatic ischemia was induced by clamping the hepatoduodenal ligament for 30 min using an atraumatic microvascular clip (Pringle's maneuver). Reperfusion was accomplished by removing the clip. At several indicated time points, rats were killed by exsanguination. Their liver tissue was taken for analysis and stored in liquid nitrogen until use.
Preparation of whole cell, nuclear, and cytoplasmic extracts.
Liver tissue was rinsed with PBS and then homogenized on ice in 3 ml of
buffer containing (in mM) 50 Tris · HCl (pH 7.5), 150 NaCl, 1 1,4-dithiothreitol (DTT), 1 phenylmethylsulfonyl fluoride (PMSF),
protease inhibitor cocktail (Roche, Mannheim, Germany), 50 NaF, and 0.1 Na3VO4, with 0.05% Triton X-100 using a Potter homogenizer. The homogenates were incubated for 15 min on ice and
centrifuged at 15,000 g for 20 min. The supernatants were stored at 80°C as whole cell extracts. For the isolation of nuclear and cytoplasmic protein extracts, a procedure modified from the method
of Zwacka et al. (65) was used. Briefly, liver tissue was
rinsed with ice-cold PBS and homogenized on ice in 6 ml of ice-cold
buffer A [in mM: 10 mM HEPES (pH 7.9), 1.5 MgCl2, 10 NaCl, 1 DTT, 1 PMSF, 50 NaF, and 0.1 Na3VO4, with protease inhibitor cocktail]
using a Potter homogenizer. After a 10-min incubation on ice, the
homogenate was transferred to a polypropylene centrifuge tube and
centrifuged at 850 g for 10 min at 4°C. The supernatants were stored as cytoplasmic extracts at
80°C. The pellet was
suspended in an ice-cold buffer B (0.1% Triton X-100 in
buffer A), incubated on ice for 10 min, and then centrifuged
as above. The crude nuclear pellet was resuspended in buffer
C (buffer A with 1.7 M sucrose) and overlaid on 1 ml of
cushion buffer (buffer A with 2.2 M sucrose) followed by
centrifugation at 75,000 g for 2 h at 4°C. The
supernatant was removed, and the purified nuclear pellet was
resuspended in 250 µl of Dignam C buffer [20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.42 M NaCl, 25% glycerol, 0.2 mM EDTA, 1 mM
PMSF, 1 mM DTT, and protease inhibitor cocktail]. The suspension was
incubated for 30 min at 4°C with frequent vortexing. After the
supernatants were centrifuged at 15,000 g for 15 min at
4°C, they were transferred to new tubes in aliquotes to freeze in
liquid nitrogen and stored at
80°C until use.
Electrophoretic mobility shift assay.
A modified procedure based on the method of Diaz-Guerra et al.
(16) was used. The NF-B binding sequence, derived from
the murine inducible nitric oxide synthase (iNOS) promoter and also containing a functional NF-
B element
(5'-CCAACTGGGGACT-CTCCCTTTGGGAACA-3') was used as a probe.
Double-stranded oligonucleotide was end-labeled with
[
-32P]ATP (3,000 Ci/mmol at 10 mCi/ml; Amersham
Pharmacia Biotechnology, Tokyo, Japan) using T4 polynucleotide kinase
(Nippon gene, Toyama, Japan) and purified in G-50 sephadex columns
(Roche). Nuclear extracts (10 µg) were incubated with electrophoretic
mobility shift assay (EMSA) buffer [in mM: 10 Tris · HCl (pH
7.5), 50 NaCl, 1 MgCl2, 1 EDTA, and 1 DTT, with 4% glycerol (vol/vol)
and 1µg poly(dI-dC)] in a final volume of 14 µl for 15 min at
4°C, and then incubated with 1µl of radiolabeled oligonucleotide
(35 fmol/µl) for 20 min at room temperature. DNA-protein complexes
were analyzed on a 5% native polyacrylamide gel run in 0.25 × Tris borate/EDTA buffer for 90 min at 150 mV. In a competition assay, a
100-fold molar excess of nonradioactive NF-
B or activator protein-1
(AP-1) oligonucleotides was added to the binding reaction for 1 h
before the addition of radiolabeled probes. In a supershift analysis, the binding reaction, containing 1µg of anti-p50 or -p65 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), was incubated for 1 h
on ice before adding radioactive probes. The dried gel on Whatman No. 3 MM paper (Whatman, Maidstone, UK) was exposed to an imaging plate and
visualized on Fuji BAS 2000 apparatus (Fuji, Tokyo, Japan).
Semiquantitative RT-PCR.
From liver tissue, total RNA was extracted using TRIzol reagent
(GIBCO-BRL, Life Technologies, Rockville, MD) and was treated with
deoxyribonuclease (RT grade; Nippon gene) for 15 min at 37°C to avoid
DNA contamination during the PCR. RNA (5 mg) was reverse transcribed
using a first-strand cDNA kit (Amersham Pharmacia Biotechnology,
Buckinghamshire, UK) in a 15-µl reaction mixture according to the
manufacturer's protocol. Because the performance of PCR was
exclusively higher for -actin cDNA than other cDNAs, the RT products
were amplified in a two-step PCR (32) using Taq
DNA polymerase (GIBCO-BRL) and specific cDNA primers for iNOS, tumor
necrosis factor-
(TNF-
), and macrophage inflammatory protein-2 (MIP-2).
-Actin cDNA was coamplified as an internal standard. Primers used in the PCR were as follows: TNF-
5' primer
(5'-CACGCTCTTCTGTCTACTGA-3') and TNF-
3' primer
(5'-GGACTCCGTGATGTCTAAGT-3') to give a 541-bp product;
-actin 5'
primer (5'-CTACAATGAGCTGCGTGTGG-3') and
-actin 3' primer
(5'-CGCGTAACCCCATAGA-TGG-3') to give a 241-bp product; and diluted iNOS
and MIP-2 primer pairs (Biosource, Camarillo, CA) to give 563- and
219-bp products, respectively. Each PCR cycle consisted of a
heat-denaturation step at 94°C for 30 s, annealing of primers at
60°C for 45 s, and polymerization at 72°C for 45 s. The
PCRs for TNF-
, MIP-2, and iNOS were first initiated for 8, 8, and 10 cycles, respectively, using only their specific primers. After the
first-step PCR, primers for
-actin were added to the reaction
mixture and an additional 20 cycles of amplification were carried out.
These PCR cycle numbers were determined by a kinetic study (22-34
cycles) for each set of primers to ensure that all PCR products remain
proportional to initial gene expression templates. The PCR products
were run on 2.5% agarose gel, stained with ethidium bromide, and then
visualized by ultraviolet illumination. Signal intensities were
evaluated using Bio-Rad's image-analysis systems (Bio-Rad, Hercules,
CA). The levels of iNOS, TNF-
, and MIP-2 mRNA were normalized to the
level of
-actin mRNA. Results were expressed in arbitrary units.
Western blot analysis.
Western blotting was performed according to a method described
elsewhere (31). Nuclear, cytoplasmic or whole cell,
extract was boiled for 5 min in equal volumes of 2× sample buffer
[250 mM Tris · HCl (pH 6.8), 4% SDS, 10% glycerol, 2%
-mercaptoethanol, and 0.003% blomophenol blue] at 95°C. Protein
samples (20 µg) were separated on denaturing 10% SDS-polyacrylamide
gels and transferred to a polyvinylidene fluoride membrane (Millipore,
Tokyo, Japan) using a semidry transfer system (Bio-Rad). The gels were
stained with Coomassie to ensure that equal amounts of loading proteins were used. The membranes were blocked overnight at 4°C with a blocking buffer (5% nonfat dry milk in PBS with 0.1% Tween 20). Membranes were washed three times for 5 min in PBS containing 0.05%
Tween 20 (TPBS) and then incubated for 2 h at room temperature with polyclonal rabbit anti-p65 antibody (1:2,000 dilution; Santa Cruz), anti-I-
B
antibody (1:2,500 dilution; Santa Cruz),
polyclonal rabbit anti-I-
B
antibody (1:2,000 dilution; Santa
Cruz), monoclonal mouse anti-HSP-72 antibody (1:3,000 dilution;
StressGen, Victoria, Canada), monoclonal mouse anti-HSP-60 antibody
(1:2,000 dilution; StessGen), and monoclonal mouse anti-HSP-90
antibody (1:2,000 dilution; StressGen) in blocking buffers.
Membranes were washed three times in TPBS for 10 min and incubated for
1 h at room temperature with an appropriate secondary antibody
(anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase
(1:1,500 dilution; Santa Cruz) in TPBS. After the membranes were washed
four times in TPBS for 15 min, they were developed with the enhanced
chemiluminescence system (Amersham Pharmacia Biotechnology) according
to the manufacturer's protocol and then exposed to films. Protein
levels were quantified by scanning densitometry using image-analysis
systems (Bio-Rad). Expression levels of I-
Bs were evaluated by their
relative integrated intensity vs. a normal liver and presented as a
percentage of the standard.
Kinase assay.
For measuring I-B kinase (IKK) activity, kinase assay was performed,
with some modifications, using the method of Schwabe et al.
(49). Extracts were prepared by incubating liver tissue in
lysis buffers [20 mM Tris · HCl (pH 7.5), 200 mM NaCl, 10% glycerol, 0.5% Nonidet P-40 (NP-40), 50 mM NaF, 0.1 mM
Na3VO4, 1 mM DTT, protease inhibitor cocktail,
1 mM PMSF, and 20 mM
-glycerophosphate]. Protein concentrations
were determined using the BCA method. Three milligrams of
protein were precleared in a 900-µl pull-down buffer [20 mM
Tris · HCl (pH 7.5), 200 mM NaCl, 10% glycerol, 0.05% NP-40, and 2 mM EDTA] containing protease and phosphatase inhibitor for 30 min at 4°C on a rocking platform by adding 30 µl of protein A-agarose (Roche). After centrifugation, 2 µg of anti-NF-
B
essential modulator (NEMO) antibody (Santa Cruz) was added to the
supernatant and incubated overnight with continuous rocking at 4°C.
Then, 30 µl of protein A-agarose were added to the samples, and they were incubated for another 3 h at 4°C. Precipitates were washed twice in pull-down buffers and twice in kinase buffers [in mM: 20 HEPES (pH 7.5), 20 MgCl2, 1 EDTA, 2 DTT, 20
-glycerophosphate, 1 NaF, and 0.1 Na3VO4]. The kinase reaction was
performed for 30 min at 30°C in a 30-µl kinase buffer containing
3µCi of [
-32P]ATP, 20µM ATP, and 1 µg of
glutathione S-transferase-I-
B
substrate (Santa Cruz). The
reactions were terminated by addition a 2× sample buffer, and the
reaction mixtures were resolved on 10% SDS-acrylamide gels. After
electrophoresis, the gels were dried and exposed at
80°C to Kodak
X-ray films.
Statistical analysis. The significance of differences was determined by ANOVA. A P value < 0.05 was considered to be significant.
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RESULTS |
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HS preconditioning induced production of HSP-72.
To examine the stress response after HS preconditioning, we
investigated protein expressions of three major HSPs (HSP-60, -72, and
-90) in the liver tissue 48 h after preconditioning. As shown in
Fig. 1, HSP-60 and -90 were
constitutively expressed in the liver tissue. Compared with normal rat
liver tissue, the expression of HSP-60 was not significantly enhanced
in either group C or HS. The expression of HSP-90 was
slightly increased in group HS. In livers of no-treatment
and group C rats, HSP-72 was hardly detectable but was
induced strongly in the liver of group HS.
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HS preconditioning inhibited NF-B activation during I/R.
To assess the effect of HS preconditioning on the DNA binding activity
of NF-
B during hepatic I/R, EMSA was performed on nuclear extracts
obtained from livers undergoing a 30-min ischemia and a 60-min
reperfusion. Two NF-
B/DNA complexes, presented as upper- and
lower-band complexes appeared in group C. In contrast, only
a weak lower-band complex was detected in group HS (Fig. 2A).
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HS preconditioning suppressed expression of iNOS, TNF-, and
MIP-2 mRNA at 120 min after reperfusion.
Because activation of NF-
B is required for the transcription of
proinflammatory mediators, we examined whether production of mRNA for
these mediators was suppressed after hepatic I/R by HS preconditioning.
mRNA levels of iNOS, TNF-
, and MIP-2 were assessed by RT-PCR using
RNA extracts from liver tissue before ischemia and at 120 min
after reperfusion. Before ischemia, the mRNA expression of
these mediators was not detected in either group. At 120 min after
reperfusion, levels of these mRNA expressions were increased in
group C, but the increase was significantly abrogated in
group HS (Fig. 3). These data
showed that HS preconditioning inhibited the mRNA expression of NF-
B
responsible genes.
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HS preconditioning increased I-B family proteins and prevented
their degradation during I/R.
Activation of NF-
B is largely dependent on the degradation of I-
B
family proteins. To delineate the temporal profile of I-
Bs during
I/R, protein levels of I-
B
and I-
B
were assessed by Western
blot analysis using cytoplasmic extracts (Fig.
4). In the cytoplasm of group
C, expression levels of both I-
Bs began to decrease at the end
of ischemia and showed minimal levels between 30 and 60 min
after reperfusion. These levels increased gradually toward 120 min
after reperfusion. On the other hand, preischemic levels of
I-
B
and I-
B
were significantly higher in group
HS than in group C. In addition, HS preconditioning
prevented I/R-induced decrease in I-
B family protein. It was
therefore demonstrated that HS preconditioning induced an increase in
cytoplasmic I-
B proteins and prevented their degradation during
hepatic I/R.
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IKK complex was not activated during hepatic I/R.
A number of studies has demonstrated that the degradation of I-B
requires serine phosphorylation mediated by IKK. IKK is a complex
composed of three subunits: IKK
, IKK
, and NEMO (36, 61). Because hepatic I/R caused the degradation of I-
B
proteins, we hypothesized that the activation of the IKK complex during I/R and the suppression of this activation by HS preconditioning resulted in maintaining the quantity of I-
B protein. To investigate this possibility, activities of IKK complex during hepatic I/R were
examined using an immunocomplex kinase assay (Fig.
5). However, no increase in IKK activity
was detected during the I/R period in group C or in
group HS.
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DISCUSSION |
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In the liver, I/R induces proinflammatory mediators such as
TNF-, adhesion molecules, chemokines, and iNOS. These locally overexpressed hepatic proinflammatory mediators play a critical role in
the progression of hepatic I/R damage (6-8, 18, 29, 34). Expression of these mediators initiates the activation of
the inflammatory cascade, leading to hepatic neutrophil recruitment, microcirculatory disturbance, and hepatic dysfunction. At the transcriptional level, each of these mediators is commonly controlled, at least in part, by NF-
B (9, 51, 53, 58). Accordingly, many investigators have studied the role of NF-
B activation during I/R in various organs and have suggested that the activation of NF-
B
seems to be an early step in the pathogenesis of I/R injury. The
beneficial effect of NF-
B inhibition on attenuating I/R damage is
supported by the studies. One demonstrated that cerebral I/R injury was
attenuated in p50 knockout mice (47), and a second showed
that in vivo transfection of NF-
B decoy oligodeoxynuleotides could
prevent myocardial infarction (40). A third study tested several therapeutic interventions for hepatic I/R injury (e.g., redox
gene therapy, administration of interleukin-10 or atrial natriuretic
peptide) that resulted in the inhibition of NF-
B activation
(22, 41, 64, 66), and another study illustrated how the
inhibition of NF-
B activation by I-
B superrepressor decreased
cell death from oxidative stress in a hepatocyte cell line
(30).
We have previously reported that HS preconditioning protects the liver
from I/R injury in rats (31, 44, 59, 60), but the
mechanism of that protection remains unclear. In the present study, we
demonstrated that induction of the stress response with HS
preconditioning increases HSP-72 levels and suppresses I/R-induced NF-B activation in the liver. These results are consistent with other in vivo and in vitro experiments in which the stress response was
able to suppress NF-
B activation (14, 15, 19, 45, 54,
55). We confirmed that at 120 min after reperfusion, mRNA levels
of NF-
B target genes (TNF-
, MIP-2, and iNOS) in the liver were
also reduced by HS preconditioning. Although we assessed the expression
of these mediators only at an mRNA level, a recent study demonstrated
that HS preconditioning suppressed elevation of plasma concentration of
TNF-
during I/R (62). These findings suggest that
modulation of NF-
B activation during hepatic I/R by HS
preconditioning may be one of the molecular mechanisms responsible for
its hepatoprotective properties.
The major pathway for NF-B activation depends on activation of the
IKK complex, which leads to the phosphorylation of serine residue of
I-
B and the degradation of I-
B via the ubiquitin-proteasome system (36, 61). Under specific circumstances, other
systems for NF-
B activation have also been implicated. According to
Zwacka et al. (65), during hepatic I/R, NF-
B activation
was initiated not by serine phosphorylation, but by tyrosine
phosphorylation of I-
B and was not followed by the
degradation of either I-
B
or I-
B
. Neither could we, in the
present study, detect serine phosphorylation of I-
B with Western
blot analysis using anti-phospho-I-
B antibody (data not shown).
However, degradation of I-
B
and I-
B
occurred during the
reperfusion phase despite the loss of serine phosphrylation. In
addition, this degradation was not dependent on IKK activation.
Although at this time, we cannot explain the discrepancies between our
results and theirs, these results suggest the complexity of the
activation pathways of NF-
B during I/R of the liver. It might be
different from the generally accepted major pathways demonstrated in
studies using proinflammatory mediators (36, 61). Many
intracellular events (production of ROS, ATP depletion, intracellular
Ca2+ accumulation, and activation of proteases) occur
during hepatic I/R (20, 21, 33, 41, 66), and all of them
can correlate with NF-
B activation through different mechanism. In
particular, the activation of proteases such as calpain, caspase, and
lysosomal enzymes has been demonstrated to degrade I-
B without
requiring IKK activation (11, 48). The COOH-terminal
region of I-
B has signal sequences for protein instability (PEST
sequences) (3, 50), which might be sensitive to
degradation by these proteases. Therefore, it is likely that in our
model, some proteolitic pathways may be involved in this
IKK-independent I-
B degradation. However, further studies are
necessary to evaluate this possibility.
With regard to the inhibition of NF-B activation with HS
preconditioning, our data revealed two possible mechanisms. The first
mechanism is the increase in cytoplasmic I-
B proteins 48 h
after HS preconditioning. Because the amount of cytoplasmic p65 was not
increased by HS preconditioning, the increased I-
Bs after HS
preconditioning is probably free I-
B, which can interfere with the
translocation of activated NF-
B. Many reports have demonstrated that
increased I-
B expression decreases NF-
B activation (1, 46,
57). In addition, Pritts et al. (42) and Wong et
al. (55, 56) demonstrated that the induction of the stress
response increased I-
B
gene expression in vitro and in vivo. That
is, I-
B
may be regarded as a stress protein. Further
investigation is needed to determine whether an increased I-
B
expression level with HS preconditioning is due to increased gene
transcription, RNA stability, translation rate, or protein stability.
The other important mechanism showed how HS preconditioning maintained
cytoplasmic I-B protein levels similar to those of the control
levels during hepatic I/R. Although HS preconditioning did not
completely prevent decreases in I-
B levels, it blunted the
consistent loss of I-
B proteins in the cytoplasm. These results are
in agreement with the findings of several studies that have demonstrated that the stress response can inhibit I-
B degradation caused by various stimuli (13, 19, 43, 55, 63). Induction of a stress response in our study has been monitored in the liver by
determining the maximum expression of HSP-72. It has been reported in
some studies that this inhibition of I-
B degradation is provided by
blocking IKK activation (13, 43, 63). But IKK was not activated in our model. As another inhibitory mechanism of I-
B degradation, the chaperoning function of HSP-72 may provide a clue. It
is very likely that HSP-72 senses some conformational changes of I-
B
proteins in its role as a molecular chaperone and during hepatic I/R
interacts with modified I-
B to prevent the subsequent degradation or
disassociation from NF-
B. This possibility is supported by studies
showing that HSP-70 has the ability to bind with I-
B proteins using
its chaperoning function (11, 24). However, a trial to
demonstrate the specific binding of HSP-72 with NF-
B/I-
B complex
using the immunoprecipitation method was unsuccessful. Inclusion of
I-
Bs or p65 was not detected in the immunoprecipitates from mouse
monoclonal anti-HSP-72 antibodies (SPA-810; StressGen). Of course,
these results do not preclude the binding of these molecules. It is
possible that the site where HSP-72 binds to NF-
B/I-
B complex is
located very close to the site where SPA-810 recognizes HSP-72,
therefore preventing SPA-810 from recognizing the HSP-72 associated
with the NF-
B/I-
B complex. We also intended to examine
immunoprecipitation using polyclonal anti-I-
B or anti-p65 antibody
from rabbits; but normal rabbit IgG (negative control)
coimmunoprecipitated HSP-72 by itself. Further studies are needed to
fully investigate potential mechanisms with which the stress response
maintains cytoplasmic I-
B protein levels.
A recent report illustrated that the overexpression of Bcl-2 inhibited
NF-B activation by forming a stable complex with NF-
B components
(27). Similar to HSP-72, Bcl-2 is known to be upregulated by HS (28). I-
B may also be inducible by stresses as
described above. It is possible that these stress-inducible molecules
might interact with and stabilize each other to achieve their
cytoprotective functions. This hypothesis leads us to speculate on the
existence of other molecules induced by HS preconditioning. These
molecules might also participate in an interaction between
NF-
B/I-
B complex and HSP-72.
In summary, we have shown that HS preconditioning inhibits hepatic
I/R-induced NF-B activation in vivo by affecting I-
B protein
expression. This can be one of the mechanisms by which HS
preconditioning reduces subsequent I/R damage to the liver. A better
understanding of the role of the stress response in modifying the
proinflammatory mediator cascade will contribute to the clinical application of HS preconditioning in reducing liver damage during I/R
as well as for treating other diseases. It may also provide an
important benchmark for exploring other chemicals or drugs that may
supplant the effects of the stress response.
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ACKNOWLEDGEMENTS |
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The authors thank D. A. Brenner, (Dept. of Medicine, Univ. of North Carolina at Chapel Hill) for valuable advice during this study.
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FOOTNOTES |
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This work was partly supported by a Grant-in-Aid of the Japan Society for the Promotion of Science, Tokyo Japan (Nos. 12470258 and 13557105).
Address for reprint requests and other correspondence: Y. Yamamoto, Dept. of Gastroenterological Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan (E-mail: mai{at}kuhp.kyoto-u.ac.jp).
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.
First published January 30, 2002;10.1152/ajpgi.00466.2001
Received 1 November 2001; accepted in final form 29 January 2002.
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