Effect of heat shock preconditioning on NF-kappa B/I-kappa B pathway during I/R injury of the rat liver

Hiroshi Uchinami, Yuzo Yamamoto, Makoto Kume, Kei Yonezawa, Yasuhide Ishikawa, Kojiro Taura, Akio Nakajima, Koichiro Hata, and Yoshio Yamaoka

Department of Gastroenterological Surgery, Graduate School of Medicine, Kyoto University, Kyoto 606 - 8507, Japan


    ABSTRACT
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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-kappa B (NF-kappa B) is an important event in the hepatic I/R-induced inflammatory response, the effect of HS preconditioning on the pathway for NF-kappa B activation was investigated. In the control group, NF-kappa 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-kappa B activation, NF-kappa B inhibitor I-kappa 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-kappa B and the subsequent expression of proinflammatory mediators through the stabilization of I-kappa B proteins.

hepatic ischemia-reperfusion; nuclear factor-kappa B proinflammatory mediators


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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-kappa B (NF-kappa 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-kappa B is retained in cytoplasm by its inhibitor I-kappa B proteins. In response to a variety of stimuli, cytoplasmic NF-kappa B/I-kappa B complex is disassociated, and free NF-kappa 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-kappa B activation. In this study, we evaluated the effect of HS preconditioning on the NF-kappa B/I-kappa 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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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.

The protein concentration of each extract was determined by bicinchoninic acid (BCA) protein assay reagent using bovine serum albumin as a reference standard (Pierce, Rockford, IL).

Electrophoretic mobility shift assay. A modified procedure based on the method of Diaz-Guerra et al. (16) was used. The NF-kappa B binding sequence, derived from the murine inducible nitric oxide synthase (iNOS) promoter and also containing a functional NF-kappa B element (5'-CCAACTGGGGACT-CTCCCTTTGGGAACA-3') was used as a probe. Double-stranded oligonucleotide was end-labeled with [gamma -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-kappa 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 beta -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-alpha (TNF-alpha ), and macrophage inflammatory protein-2 (MIP-2). beta -Actin cDNA was coamplified as an internal standard. Primers used in the PCR were as follows: TNF-alpha 5' primer (5'-CACGCTCTTCTGTCTACTGA-3') and TNF-alpha 3' primer (5'-GGACTCCGTGATGTCTAAGT-3') to give a 541-bp product; beta -actin 5' primer (5'-CTACAATGAGCTGCGTGTGG-3') and beta -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-alpha , 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 beta -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-alpha , and MIP-2 mRNA were normalized to the level of beta -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% beta -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-kappa Balpha antibody (1:2,500 dilution; Santa Cruz), polyclonal rabbit anti-I-kappa Bbeta 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-kappa 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-kappa 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 beta -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-kappa 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 beta -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 [gamma -32P]ATP, 20µM ATP, and 1 µg of glutathione S-transferase-I-kappa Balpha 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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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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Fig. 1.   The expression of heat shock (HS) proteins (HSPs) in livers was analyzed using Western blotting (left) and was evaluated by band intensity on the same film (right). A: HSP-72 was not expressed in the livers of control (C) group (group C; 48 h after 37°C treatment) and no-treatment livers (NT). However, it was strongly expressed in an HS group (group HS; 48 h after 42°C treatment). B: HSP-60 was constitutively expressed, and there was no difference in its expression level between the groups. C: HSP-90 was expressed in both groups. The amount of HSP-90 was slightly increased in group HS. All data were expressed as the means ± SE (n = 5 in each group). #P < 0.01 vs. group C. OD, optical density.

HS preconditioning inhibited NF-kappa B activation during I/R. To assess the effect of HS preconditioning on the DNA binding activity of NF-kappa 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-kappa 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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Fig. 2.   Nuclear factor-kappa B (NF-kappa B) activation during hepatic ischemia-reperfusion. A: the electrophoretic mobility shift assay (EMSA) was performed to determine the NF-kappa B DNA binding activity in the nuclear extracts from livers obtained 60 min after reperfusion. NF-kappa B activation was clearly demonstrated 60 min after reperfusion in group C, but activation was not observed in group HS. EMSA assay was triplicated in both groups using 3 independent samples. B: specificity of the NF-kappa B/DNA complex was confirmed by a supershift assay. Lane 1 shows EMSA 60 min after reperfusion in group C. Lanes 2 and 3 show the supershift assay using antibodies (Ab) of p50 (lane 2) and p65 (lane 3). Lanes 5 and 6 show the competition assay with 100× excess molar of unlabeled NF-kappa B oligonucleotide (lane 4) and activator protein-1 (AP-1) oligonucleotide (lane 5). The open arrow indicates a supershift band of p50, and the closed arrow indicates a supershift band of p65. C: a time course of NF-kappa B activation during hepatic ischemia-reperfusion. Nuclear extracts prepared from liver samples obtained at the preischemic phase (48 h after preconditioning) and at 0, 30, 60, and 120 min postreperfusion were subjected to EMSA. D: the expression level of NF-kappa B subunit, p65, in the nucleus. Nuclear extracts from livers obtained at the preischemic phase and at 60 and 120 min postreperfusion were subjected to Western blotting. D: the expression of p65 in cytoplasmic extract from the liver obtained at the preischemic phase was also evaluated to confirm that p65 was not affected by HS preconditioning. Pre, before ischemia; R0, just after ischemia; R30, 30 min after reperfusion; R60, 60 min after reperfusion; R120, 120 min after reperfusion.WT, wild-type oligonucleotide; MT, mutant oligonucleotide.

Specificity of these complexes for NF-kappa B was examined. Unlabeled NF-kappa B oligonuleotide of 100-fold excess inhibited formation of both NF-kappa B/DNA complexes (Fig. 2B, lane 4). However, the same excess of unrelated oligonucleotide (AP-1) had no effect (Fig. 2B, lane 5). These data indicated that both complexes were specific to NF-kappa B. Supershift assay using p50 antibody shifted both bands (Fig. 2B, lane 2), whereas only the upper-band complex was supershifted using p65 antibody (Fig. 2B, lane 3). These results illustrate that the complexes detected by EMSA corresponded to p50/p65 heterodimer and p50/p50 homodimer for the upper and lower bands, respectively.

Figure 2C illustrates the DNA binding activity of NF-kappa B during the time course of hepatic I/R. NF-kappa B binding activity increased by 60 min after reperfusion and remained elevated after 120 min in group C. But in group HS, this activation was not observed at any time. This suggests that HS preconditioning inhibits the increase in DNA binding activity of NF-kappa B during I/R in the liver.

To investigate whether this inhibitory effect on the DNA binding activity of NF-kappa B was associated with its translocation into the nucleus, Western blot analysis was performed with nuclear extract using p65 antibody (Fig. 2D). With results similar to the results of EMSA, the presence of a p65 subunit was demonstrated at 60 and 120 min after reperfusion in the nuclear extracts in group C, but it was not detected in group HS. To investigate whether HS preconditioning suppresses NF-kappa B activation by decreasing the cytoplasmic p65 expression, we also measured p65 protein levels at 48 h after HS. But p65 level in the cytoplasmic fraction did not change after HS preconditioning. This suggests that HS preconditioning-mediated inhibition of DNA binding activity of NF-kappa B was not due to the decrease in the amount of p65 but to the inhibition of some signaling pathway upstream of the translocation of NF-kappa B into the nucleus.

HS preconditioning suppressed expression of iNOS, TNF-alpha , and MIP-2 mRNA at 120 min after reperfusion. Because activation of NF-kappa 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-alpha , 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-kappa B responsible genes.


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Fig. 3.   Messenger RNA expression of inducible nitric oxide synthase (iNOS; A), tumor necrosis factor-alpha (TNF-alpha ; B), and macrophage inflammatory protein-2 (MIP-2; C) in the liver. Total RNA from livers obtained at the preischemic phase and 120 min after reperfusion was subjected to RT-PCR using iNOS, TNF-alpha , and MIP-2 specific primers. beta -actin was coamplified as a reference for quantitation of mRNA. A 2-step PCR method was employed for amplifying RT products. The PCRs for TNF-alpha , 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 beta -actin were added to reaction mixture and an additional 20 cycles of amplification were carried out. PCR products were stained by ethidium bromide and photographed (left). RT-PCR products from the liver obtained 120 min after reperfusion were digitized using an image-analysis system, and the band intensity was normalized to beta -actin (right). Results were expressed as the means ± SE (n = 5 in each group). #P < 0.05 and ##P < 0.01 vs. group C. PC, positive control [mRNA of the liver harvested 2 h after lipopolysaccharide injection (2 mg/kg)]; MWM, molecular weight marker.

HS preconditioning increased I-kappa B family proteins and prevented their degradation during I/R. Activation of NF-kappa B is largely dependent on the degradation of I-kappa B family proteins. To delineate the temporal profile of I-kappa Bs during I/R, protein levels of I-kappa Balpha and I-kappa Bbeta were assessed by Western blot analysis using cytoplasmic extracts (Fig. 4). In the cytoplasm of group C, expression levels of both I-kappa 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-kappa Balpha and I-kappa Bbeta were significantly higher in group HS than in group C. In addition, HS preconditioning prevented I/R-induced decrease in I-kappa B family protein. It was therefore demonstrated that HS preconditioning induced an increase in cytoplasmic I-kappa B proteins and prevented their degradation during hepatic I/R.


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Fig. 4.   The NF-kappa B inhibitor I-kappa B family protein levels in cytoplasm were analyzed using Western blotting analysis. The levels of I-kappa Balpha (A) and I-kappa Bbeta expression (B) during ischemia-reperfusion were examined in each group. Cytoplasmic extracts were prepared from liver samples obtained at the preischemic phase (48 h after preconditioning) and at 0, 30, 60, and 120 min postreperfusion. The standard sample was a cytoplasmic extract from a normal rat liver. The expression levels were evaluated on the same film as the ratio of their integrated intensity to that of the normal liver and presented as a percentage. Results are expressed as the means ± SE (n = 5 for each time point in each group). #P < 0.05 and ##P < 0.01 vs. Pre-C. *P < 0.05 and **P < 0.01 vs. group C. C, group C; HS, group HS; Pre-C, before ischemia in group C.

IKK complex was not activated during hepatic I/R. A number of studies has demonstrated that the degradation of I-kappa B requires serine phosphorylation mediated by IKK. IKK is a complex composed of three subunits: IKKalpha , IKKbeta , and NEMO (36, 61). Because hepatic I/R caused the degradation of I-kappa 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-kappa 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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Fig. 5.   The I-kappa B kinase (IKK) complex was immunoprecipitated using anti-NF-kappa B essential modulator antibody from liver tissue, and its activity was measured with an immunocomplex kinase assay using glutathione S-transferase-I-kappa Balpha (GST-I-kappa B). A: IKK activity in the liver tissue at 0, 30, 45, and 60 min after lipopolysaccharide (LPS) injection (2 mg/kg) was examined for the control of assay quality. IKK activity was very low in normal liver but dramatically increased after LPS injection in a time-dependent manner. B: activity of the IKK complex during hepatic ischemia-reperfusion. There were no increases in IKK activity in the liver during ischemia-reperfusion in either group. The figure shown is representative of 3 independent experiments.


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In the liver, I/R induces proinflammatory mediators such as TNF-alpha , 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-kappa B (9, 51, 53, 58). Accordingly, many investigators have studied the role of NF-kappa B activation during I/R in various organs and have suggested that the activation of NF-kappa B seems to be an early step in the pathogenesis of I/R injury. The beneficial effect of NF-kappa 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-kappa 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-kappa B activation (22, 41, 64, 66), and another study illustrated how the inhibition of NF-kappa B activation by I-kappa 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-kappa 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-kappa B activation (14, 15, 19, 45, 54, 55). We confirmed that at 120 min after reperfusion, mRNA levels of NF-kappa B target genes (TNF-alpha , 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-alpha during I/R (62). These findings suggest that modulation of NF-kappa 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-kappa B activation depends on activation of the IKK complex, which leads to the phosphorylation of serine residue of I-kappa B and the degradation of I-kappa B via the ubiquitin-proteasome system (36, 61). Under specific circumstances, other systems for NF-kappa B activation have also been implicated. According to Zwacka et al. (65), during hepatic I/R, NF-kappa B activation was initiated not by serine phosphorylation, but by tyrosine phosphorylation of I-kappa B and was not followed by the degradation of either I-kappa Balpha or I-kappa Bbeta . Neither could we, in the present study, detect serine phosphorylation of I-kappa B with Western blot analysis using anti-phospho-I-kappa B antibody (data not shown). However, degradation of I-kappa Balpha and I-kappa Bbeta 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-kappa 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-kappa B activation through different mechanism. In particular, the activation of proteases such as calpain, caspase, and lysosomal enzymes has been demonstrated to degrade I-kappa B without requiring IKK activation (11, 48). The COOH-terminal region of I-kappa 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-kappa B degradation. However, further studies are necessary to evaluate this possibility.

With regard to the inhibition of NF-kappa B activation with HS preconditioning, our data revealed two possible mechanisms. The first mechanism is the increase in cytoplasmic I-kappa B proteins 48 h after HS preconditioning. Because the amount of cytoplasmic p65 was not increased by HS preconditioning, the increased I-kappa Bs after HS preconditioning is probably free I-kappa B, which can interfere with the translocation of activated NF-kappa B. Many reports have demonstrated that increased I-kappa B expression decreases NF-kappa 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-kappa Balpha gene expression in vitro and in vivo. That is, I-kappa Balpha may be regarded as a stress protein. Further investigation is needed to determine whether an increased I-kappa 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-kappa B protein levels similar to those of the control levels during hepatic I/R. Although HS preconditioning did not completely prevent decreases in I-kappa B levels, it blunted the consistent loss of I-kappa 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-kappa 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-kappa 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-kappa 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-kappa B proteins in its role as a molecular chaperone and during hepatic I/R interacts with modified I-kappa B to prevent the subsequent degradation or disassociation from NF-kappa B. This possibility is supported by studies showing that HSP-70 has the ability to bind with I-kappa B proteins using its chaperoning function (11, 24). However, a trial to demonstrate the specific binding of HSP-72 with NF-kappa B/I-kappa B complex using the immunoprecipitation method was unsuccessful. Inclusion of I-kappa 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-kappa B/I-kappa 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-kappa B/I-kappa B complex. We also intended to examine immunoprecipitation using polyclonal anti-I-kappa 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-kappa B protein levels.

A recent report illustrated that the overexpression of Bcl-2 inhibited NF-kappa B activation by forming a stable complex with NF-kappa B components (27). Similar to HSP-72, Bcl-2 is known to be upregulated by HS (28). I-kappa 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-kappa B/I-kappa B complex and HSP-72.

In summary, we have shown that HS preconditioning inhibits hepatic I/R-induced NF-kappa B activation in vivo by affecting I-kappa 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.


    ACKNOWLEDGEMENTS

The authors thank D. A. Brenner, (Dept. of Medicine, Univ. of North Carolina at Chapel Hill) for valuable advice during this study.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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