INVITED REVIEW
Different modes of NF-kappa B/Rel activation in pancreatic lobules

Hana Algül, Yusuke Tando, Michael Beil, Christoph K. Weber, Claus Von Weyhern, Günter Schneider, Guido Adler, and Roland M. Schmid

Department of Internal Medicine I, University of Ulm, 89081 Ulm, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The eukaryotic transcription factor nuclear factor-kappa B (NF-kappa B)/Rel is activated by a large variety of stimuli. It has been demonstrated that NF-kappa B/Rel is induced during the course of cerulein pancreatitis. Here, we show that NF-kappa B/Rel is differentially activated in pancreatic lobules. Cerulein induces NF-kappa B/Rel via activation of Ikappa B kinase (IKK), which causes degradation of Ikappa Balpha but not Ikappa Bbeta . Tumor necrosis factor-alpha -mediated IKK activation leads to Ikappa Balpha and Ikappa Bbeta degradation. In contrast, oxidative stress induced by H2O2 activates NF-kappa B/Rel independent of IKK activation and Ikappa Balpha degradation; instead Ikappa Balpha is phosphorylated on tyrosine. H2O2 but not cerulein-mediated NF-kappa B/Rel activation can be blocked by stabilizing microtubules with Taxol. Inhibition of tubulin polymerization with nocodazole causes NF-kappa B/Rel activation in pancreatic lobules. These results propose three different pathways of NF-kappa B/Rel activation in pancreatic acinar cells. Furthermore, these data demonstrate that microtubules play a key role in IKK-independent NF-kappa B/Rel activation following oxidative stress.

acute pancreatitis; inhibitor protein Ikappa B; Ikappa B kinase; inflammation; hydrogen peroxide; tyrosine phosphorylation; cytokines; cerulein


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DESPITE HUGE EFFORTS, acute pancreatitis remains a serious clinical problem with significant morbidity and mortality. Although animal models do not reflect the mechanisms initiating acute pancreatitis in humans, they share biochemical, morphological, and pathophysiological similarities with acute pancreatitis in humans (27). Supramaximal concentrations of CCK or CCK analog cerulein result in acute pancreatitis, which is characterized by progressive disassembly of microtubules and microfilaments in acinar cells (2, 23, 35). Normal luminal exocytosis is blocked, and digestive enzymes are activated. Acinar cells display cytoplasmic vacuolization. In addition, focal tissue necrosis and edema formation can be observed. Neutrophilic cells infiltrate the pancreas and release reactive oxygen intermediates. In the last few years, it has been shown that a number of signaling pathways is induced during experimental pancreatitis. Mitogen-activated protein kinase (MAPK) as well as heat shock proteins are activated before necrosis and inflammation (12). We and others (13, 18, 20, 21, 36, 44) have shown that the transcription factor nuclear factor-kappa B (NF-kappa B)/Rel is activated during cerulein and acute biliary pancreatitis.

NF-kappa B/Rel is a pleiotropic transcription factor that binds to DNA as homo- or heterodimers and activates a multitude of cellular stress-related and early-response genes such as genes coding for cytokines, growth factors, adhesion molecules, and acute phase proteins (3, 39). NF-kappa B/Rel is activated by a variety of agents ranging from cell-damaging physical factors and viruses to mitogens and cytokines. In unstimulated cells, NF-kappa B/Rel is localized in the cytoplasm complexed to its endogenous inhibitor proteins Ikappa Bs (7, 8). After stimulation, Ikappa Bs are phosphorylated at serine residues by a 700-kDa protein complex containing two related catalytic subunits, IKKalpha and IKKbeta and the regulatory subunit IKKgamma . Various extracellular signaling pathways converge at the level of IKK activation (8, 28). After phosphorylation, the inhibitor proteins, Ikappa Bs are ubiquitinated and rapidly degraded by the nonlysosomal, ATP-dependent 26S proteasome, which releases the transcription factor NF-kappa B/Rel into the nucleus, where it binds to DNA sites containing kappa B motifs.

The role of NF-kappa B/Rel is evident from its participation in the regulation of the acute-phase reaction following infection, trauma, or immunologically mediated inflammations (39). Most of these responses are mediated by cytokines, which are known to be target genes of NF-kappa B, such as tumor necrosis factor-alpha (TNF-alpha ), interleukin (IL)-1 or -6, or interferon-gamma . In particular, TNF-alpha has been implicated in systemic complications of acute pancreatitis (29). Multiple factors are thought to contribute to NF-kappa B/Rel induction in acute pancreatitis. This might include trypsin activation, TNF-alpha , oxygen radical intermediates, and disruption of the cytoskeleton (2, 4, 10, 15, 31). The present study was undertaken to investigate the mode of NF-kappa B/Rel activation in pancreatic lobules exposed to different NF-kappa B/Rel inducers.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Cerulein was purchased from Pharmacia (Erlangen, Germany). TNF-alpha and Taxol were from Sigma (Deisenhofen, Germany). H2O2 was delivered by Merck (Darmstadt, Germany). Anti-alpha -tubulin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA), the anti-mouse antibody Cy3 was from Dianova Immunotech (Hamburg, Germany). All other chemicals were of the highest purity commercially available and were obtained from Sigma.

Male Wistar rats (250-300 g body wt) were obtained from the breeding colony of Ulm University Animal Facilities. They were housed in nalgene shoebox cages under a 12:12-h light-dark cycle with free access to standard diet and water. All animal experiments were conducted according to the guidelines of the local Animal Use and Care Committees and executed according to the National Animal Welfare Law.

Preparation of pancreatic lobules. Pancreatic lobules were prepared as previously described (37). In brief, after an overnight fast, rats were killed by exsanguination under light ether anesthesia. The pancreas was removed and incubated in DMEM (GIBCO Life Technologies, Paisley, Scotland). Equal quantities of lobules were incubated in medium for 15 min at 37°C under continuous oxygenation in a shaking water bath. After this adaptation period, lobules were incubated with Taxol (5 µM) as pretreatment or left with medium alone. Thereafter, lobules were stimulated by cerulein, TNF-alpha , or H2O2. After the respective incubation periods, lobules were immediately frozen in liquid nitrogen and stored at -70°C.

Protein extracts. Nuclear protein extracts were prepared essentially as described by Dignam et al. (11), with some modifications as follows. Pancreatic lobules were homogenized in a sucrose buffer containing protease inhibitors. Nuclei were separated by centrifugation, and proteins were eluted using a high-salt buffer as previously described (44).

For cytoplasmic protein extracts, pancreatic lobules were homogenized in 150 mM NaCl, 50 mM Tris · HCl, 50 mM CaCl2, 1.0% Nonidet P-40, 5 mM NaF, 0.1 M phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 35 µg/ml pepstatin, 10 µg/ml aprotinin, and 0.5 mM 1,4-dithiothreitol, pH 7.2, and centrifuged at 15.000 rpm for 20 min at 4°C. Aliquots of the supernatant were stored at -70°C. Protein concentrations were determined by the method of Bradford (Bio-Rad Laboratories, München, Germany).

Electrophoretic mobility shift assays. Electrophoretic mobility shift assays (EMSAs) were performed as previously described (49). The DNA probe used for EMSAs corresponded to the high-affinity kappa B sequences found in the mouse kappa -light-chain enhancer and in the HIV-1 promoter region. Two oligonucleotides were annealed to generate a double-stranded probe: sense 5'-AGCTTGGGGACTTTCCACTAGTACG-3' and antisense 5'-AATTCGTACTAGTGGAAAGTCCCCA-3' (the binding sites are underlined). Labeling was accomplished by treatment with Klenow in the presence of dGTP, dCTP, dTTP, and [alpha -32P]dATP. Labeled oligonucleotides were purified on push columns (Stratagene, Heidelberg, Germany). Labeled double-stranded probe (80,000 counts/min) was added to 10 µg of nuclear protein in the presence of 5 µg poly(dIdC) as nonspecific competitor (Pharmaka Biotech, Freiburg, Germany). Binding reactions were carried out in 10 mM Tris · HCl, pH 7.5, 100 mM NaCl, and 4% glycerol for 30 min at 4°C.

DNA protein complexes were resolved by electrophoresis on a 4% nondenaturating polyacrylamide gel in 1 × Tris-glycine-EDTA buffer. Gels were vacuum dried and exposed to Kodak Bio Max MS-1 film at -70°C with intensifying screens. Competition was performed by adding specific unlabeled double-stranded oligonucleotide to the reaction mixture in 10-, 50-, or 100-fold molar excess.

Western blotting. Cellular protein extracts were analyzed by immunoblotting. Samples were diluted in SDS-PAGE loading buffer in a ratio of 1:5 and heated at 97.5°C for 10 min. Recombinant Ikappa Balpha or Ikappa Bbeta was prepared by transfecting 293 human embryonic kidney cells with respective eukaryotic expression vector and loaded as controls (data not shown). Protein complexes were resolved by electrophoresis on 10% nondenaturating polyacrylamide gels in 1 × Tris-glycine-SDS buffer at room temperature. After SDS-PAGE, gels were transferred to 0.45-µm polyvinylidene difluoride membranes for 25 min at 40 mA at room temperature (Schleicher and Schuell, Dassel, Germany). Nonspecific binding was blocked in 5% (wt/vol) skim milk in Tris-buffered saline (TBS), pH 7.5, at 4°C. Blots were then incubated for 1 h with antibodies directed against Ikappa Balpha or Ikappa Bbeta (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:1,000 in 5% (wt/vol) skim milk powder in TBS, washed three times with 0.05% Tween 20 in TBS (T-TBS), and incubated for 1 h with a secondary antibody, goat anti-rabbit IgG peroxidase (Dianova-Immunotech, Hamburg, Germany), at a dilution of 1:5,000 in 5% (wt/vol) skim milk powder in TBS. Blots were washed three times with T-TBS and developed with enhanced chemiluminescence reagents (Amersham Buchler, Braunschweig, Germany).

Immunoprecipitation. Pancreatic lobules were homogenized in lysis buffer containing (in mM) 150 NaCl, 25 Tris · HCl, 2 EGTA, 50 NaF, 25 Na pyrophosphate, and 50 beta -glycerophosphate, pH 8.0, with 1.0% Triton X-100, 10% glycerin, and complete protease inhibitor mixture (Boehringer-Mannheim, Mannheim, Germany) and centrifuged at 14,000 rpm for 20 min at 4°C.

For Ikappa B kinase (IKK) assay, an anti-IKKalpha antibody recognizing IKKalpha and IKKbeta (Santa Cruz Biotechnology) and 35 µM protein A agarose were added to the supernatant (3 mg protein) and mixed at 4°C for 2 h. Immunoprecipitated material was washed three times with lysate buffer and once with kinase buffer containing (in mM) 25 HEPES, 150 NaCl, 25 beta -glycerophosphate, and 10 MgCl2.

Cytoplasmic extracts were used for the immunoprecipitation of tyrosine-phosphorylated proteins. Seven microliters of phosphotyrosine antibodies and 50 µl of washed anti-mouse-IgG-agarose beads (Santa Cruz Biotechnology) were added to the supernatant and mixed at 4°C for 3 h. Immunoprecipated material was washed with lysate buffer. Samples were diluted in SDS-PAGE loading buffer in a ratio of 1:5 and heated at 97.5°C for 10 min. The immunoprecipitated material was then analyzed using Western blot analysis.

IKK activity assay. IKK activity was detected by immunoprecipitation of IKK followed by a kinase assay using glutathione S-transferase (GST)-Ikappa Balpha (1-54) substrate as previously described, with some modifications (50). Pancreatic lobules were homogenized in lysis buffer containing (in mM) 150 NaCl, 25 Tris · HCl, 2 EGTA, 2 EDTA, 50 NaF, 25 Na pyrophosphate, and 50 beta -glycerophosphate, pH 8.0, with 1.0% Triton X-100, 10% glycerin and complete protease inhibitor mixture (Boehringer-Mannheim) and centrifugated at 14,000 rpm for 20 min at 4°C. Anti-IKKalpha antibody (Santa Cruz Biotechnology) and 35 µM washed protein A agarose (Boehringer-Mannheim) were added to the supernatant (3 mg protein) and mixed at 4°C for 2 h. Immunoprecipitated material was washed three times with lysate buffer and once with kinase buffer containing (in mM) 25 HEPES, 150 NaCl, 25 beta -glycerophosphate, and 10 MgCl2. Kinase activity was assayed in 40 µl of kinase buffer containing 10 µM [gamma -32P]dATP and 3 µg GST-Ikappa Balpha for 20 min at room temperature. The reaction was stopped by the addition of SDS gel sample buffer and analyzed by SDS-PAGE and autoradiography.

Immunoflourescence. Pancreatic lobules were prepared as described and incubated for different times and stimuli as indicated. Samples were fixed in 4% formaline for 1 h and washed three times in cold PBS. Next, tissue samples were embedded in agarose. Fifty-micrometer sections were prepared and incubated with anti-mouse alpha -tubulin antibody (1:100 dilution in PBS/0.4% Triton X-100) overnight at 4°C and washed three times with cold PBS. Next samples were incubated with an anti-mouse Cy3-conjugated antibody (1:1,000 in PBS) for 1 h at room temperature. After being incubated with the second antibody, samples were washed three times again with cold PBS for 30 min. The stained microtubule system was visualized with the confocal microscope (Leica, TCS 4D, Heidelberg, Germany).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Kinetics of Ikappa B degradation in pancreatic lobules after stimulation with cerulein and TNF-alpha . A supramaximal concentration of cerulein results in acute pancreatitis, which is characterized by activation of NF-kappa B/Rel. NF-kappa B activation might be a result of direct signaling via CCK-receptor activation or induction of cytokines acting in a pivotal fashion, such as TNF-alpha . To analyze the effect of cerulein and TNF-alpha separately, we used isolated pancreatic lobules. Pancreatic lobules were prepared and incubated with cerulein for 15, 30, 60, or 120 min, respectively. Cytoplasmic extracts were analyzed by Western blotting using anti-Ikappa Balpha and anti-Ikappa Bbeta . Phosphorylated Ikappa Balpha was detected using an antibody recognizing serine-phosphorylated Ikappa Balpha . The cytoplasmic Ikappa Balpha signal almost completely disappeared after stimulation with 100 nM cerulein for 15 min. In line with this observation, we could see serine-phosphorylated Ikappa Balpha after 15 min of stimulation lasting 2 h. Ikappa Bbeta was not affected by cerulein (Fig. 1A).


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Fig. 1.   Kinetics of Ikappa B degradation in pancreatic lobules following cerulein or tumor necrosis factor-alpha (TNF-alpha ) stimulation. Pancreatic lobules were left untreated (A and B, lane 1) or treated with 100 nM cerulein (A, lanes 2-5) or 10 U/ml TNF-alpha (B, lanes 2-5) for different time points as indicated. Cytoplasmic extracts were prepared and subjected to Western analysis with anti-Ikappa Balpha , anti-Ikappa Bbeta , and phosphoserine specific Ikappa Balpha (P-Ser-Ikappa Balpha ) antibodies.

Next, we analyzed TNF-alpha -induced degradation of Ikappa Bs in pancreatic lobules. Maximal activation of NFkappa B/Rel activity required 10 U/ml TNF-alpha in pancreatic lobules (data not shown). Thirty minutes after treatment with 10 U/ml TNF-alpha , cytoplasmic Ikappa Balpha levels were almost completely reduced compared with unstimulated lobules. After 60 min, Ikappa Balpha reappeared and was back to original levels at 120 min. Cytoplasmic Ikappa Bbeta completely disappeared after 30 min but reappeared after 60 min. With the use of serine-specific anti-Ikappa Balpha antibody, we could detect serine-phosphorylated Ikappa Balpha after 15 and 30 min (Fig. 1B).

These data show that TNF-alpha induces a transient degradation of Ikappa Balpha and Ikappa Bbeta , whereas cerulein treatment causes a prolonged degradation of Ikappa Balpha but does not affect Ikappa Bbeta in vitro.

Time-dependent activation of IKK by cerulein and TNF-alpha . To analyze whether degradation of Ikappa Balpha correlates with IKK activity, endogenous IKK activity was immunoprecipitated with an anti-IKK antibody. Kinase activity was visualized by incubating the immunocomplexes with GST-Ikappa Balpha (1-54) in the presence of [gamma -32P]dATP.

Treatment with cerulein for 15 min leads to phosphorylation of GST-Ikappa Balpha (1-54), with a peak of IKK activity after 30 min of stimulation. IKK-activity lasted up to 120 min. The amounts of precipitated IKKalpha and IKKbeta were verified on the same membrane (Fig. 2A). The activation of IKK correlates with Ikappa Balpha degradation, and the appearance of NF-kappa B/Rel binding activity in the nucleus (data not shown).


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Fig. 2.   Time-dependent activation of Ikappa B kinase (IKK) by cerulein and TNF-alpha . Pancreatic lobules were left untreated (A and B, lane 1) or incubated with 100 nM cerulein (A, lanes 2-5) or 10 U/ml TNF-alpha (B, lanes 2-5) for time periods indicated. Cytoplasmic protein extracts were immunoprecipitated with anti-IKKalpha specific antibody and incubated with glutathione S-transferase (GST)-Ikappa Balpha (1-54) as substrate in the presence of [gamma -32P]dATP. The amounts of immunoprecipated IKKalpha and IKKbeta were verified (A). KA, kinase assay.

After stimulation with TNF-alpha , IKK activity peaks at 30 min and lasts for 2 h later (Fig. 2B). IKK activity correlates with degradation of Ikappa Balpha and Ikappa Bbeta (Fig. 1B). These data show that cerulein and TNF-alpha activate IKK with different kinetics.

Dose- and time-dependent induction of NF-kappa B/Rel by H2O2 in pancreatic lobules. It has been suggested that oxidative stress plays an important role in the early stages of acute pancreatitis. More recently, the enhanced formation of oxygen radicals and their adducts has been detected early during induction of acute pancreatitis in mice (15).

To assess the role of oxidative stress on NF-kappa B/Rel binding activity, rat pancreatic lobules were incubated with H2O2 at different doses for 30 min. Nuclear extracts were prepared and incubated with 32P-labeled DNA oligonucleotide containing a high-affinity recognition site for NF-kappa B/Rel. H2O2 induced a dose-dependent increase of NF-kappa B/Rel binding activity with a maximal stimulatory effect of 150 µM. Increasing H2O2 to 500 µM reduced NF-kappa B/Rel binding activity compared with 150 µM (Fig. 3A). The specificity of NF-kappa B/Rel binding activity was confirmed by competitions with unlabeled probes (data not shown).


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Fig. 3.   Dose- and time-dependent induction of nuclear factor kappa B (NF-kappa B)/Rel by H2O2 in pancreatic lobules. A: pancreatic lobules were left untreated (lane 1) or incubated with 50, 150, or 500 µM H2O2 for 30 min (lanes 2-4). B: pancreatic lobules were left untreated (lane 1) or incubated with 150 µM H202 for time periods as indicated (lanes 2-5). Extracts were prepared, and equal amounts of protein were used in electromobility shift analysis with the kappa B motif of the mouse kappa -light-chain enhancer as probe.

To study time-dependent induction of NF-kappa B/Rel, pancreatic lobules were incubated with H2O2 (150 µM) over time. H2O2-mediated induction of NF-kappa B/Rel binding was detected after 15 min up to 60 min and disappeared after 2 h (Fig. 3B). These data suggest that oxidative stress activates NF-kappa B/Rel as fast as cerulein or TNF-alpha .

H2O2 induces tryrosine phosphorylation of Ikappa Balpha , which is not mediated by IKK. In contrast to the classic activators of NF-kappa B/Rel, H2O2-mediated induction of NF-kappa B/Rel has been shown to be associated with tyrosine phosphorylation (22). To test how NF-kappa B/Rel is activated following H2O2 treatment in pancreatic lobules, cytoplasmic extracts were analyzed by Western blotting using anti-Ikappa Balpha and anti-Ikappa Balpha antibodies. Both inhibitory proteins were not degraded after stimulation with H2O2 over time. In contrast, Ikappa Balpha slightly increased after 15 min. To test whether Ikappa Balpha is phosphorylated at tyrosine, cytoplasmic extracts were immunoprecipitated with phosphotyrosine antibody. In a next step, we separated the precipitated complexes on SDS-PAGE and incubated the membrane with anti-Ikappa Balpha . Figure 4A shows that tyrosine-phosphorylated Ikappa Balpha can be detected after 15, 30, and even 60 min after stimulation with H2O2. The detection of tyrosine-phosphorylated Ikappa Balpha correlates with NF-kappa B/Rel binding activity (Fig. 3B). To analyze whether tyrosine phosphorylation is accompanied by IKK activity, we analyzed IKK activity after stimulation with H2O2 over time. Cytoplasmic extracts were used to immunoprecipitate endogenous IKK activity with anti-IKK antibody. Immunocomplexes were assayed for kinase activity by incubation with GST-Ikappa Balpha (1-54) in the presence of [gamma -32P]dATP. Very little IKK activity was detected with a slight increase after 30 min (long exposure). The amounts of precipitated IKKalpha and IKKbeta were verified on the same membrane (Fig. 3B). The IKK activity does not precede and correlate with NF-kappa B/Rel binding. Therefore, it seems unlikely that IKK is responsible for tyrosine phosphorylation of Ikappa Balpha .


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Fig. 4.   H2O2-induced tyrosine phosphorylation of Ikappa Balpha (P-Tyr-Ikappa Balpha ), which is not mediated by IKK. A: pancreatic lobules were left untreated (lane 1) or incubated with H2O2 for different time points (lanes 2-5). Cytoplasmic extracts were prepared and used either for Western blot analysis or immunoprecipitations. Equal amounts of protein were separated by SDS-PAGE and subsequently analyzed using anti-Ikappa Balpha and anti-Ikappa Bbeta antibodies. Cytoplasmic lysates were immunoprecipitated with anti-phosphotyrosine followed by Western blot analysis with anti-Ikappa Balpha antiserum. B: cell lysates were analyzed for IKK activity. Endogenous IKK complexes were immunprecipitated with an IKKalpha specific antibody and incubated with GST-Ikappa Balpha (1-54) as substrate in the presence of [gamma 32P]dATP. The amount of IKKalpha and IKKbeta was verified on the same membrane by immunoblotting.

These data show that H2O2 induces phosphorylation of Ikappa Balpha at a tyrosine residue, which does not result in its degradation.

Effect of H2O2 and cerulein on the microtubule system. Destruction of the cytoskeleton is another hallmark during the onset of the acute pancreatitis (23). It has been shown that administration of the substance Taxol prevents the block of pancreatic digestive enzyme secretion induced by supramaximal cerulein stimulation and attenuates the development of cerulein-induced pancreatitis (47).

To analyze the effect of cerulein or H2O2 on the integrity of the microtubule system of the pancreas, we incubated pancreatic lobules with cerulein or H2O2 for 15 or 30 min or those left untreated. To assess the effect of Taxol on the microtubule system, pancreatic lobules were preincubated with 5 µM Taxol and stimulated thereafter with cerulein or H2O2. Lobules were fixed in formalin and embedded in agarose. Vibratone sections (50 µm) were incubated with alpha -tubulin antibodies to visualize the integrity of the cytoskeleton. In both controls, the microtubule system seems to be unaffected after incubation in buffer for 15 and 45 min (Fig. 5, A and E). The pancreatic lobules exhibited long, curving microtubules extending to the cell margins and dense bundles of microtubules coursing around the nucleus and out into the cytoplasm. Incubation with cerulein 100 nM leads to a complete destruction of the microtubule system (Fig. 5B). With increasing treatment time, further microtubule disruption occured until the microtubule network virtually collapsed (Fig. 5C). Preincubation with Taxol prevents the cerulein-mediated disruption of the microtubule system completely (Fig. 5D).


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Fig. 5.   Effect of H2O2 and cerulein on the microtubule system. alpha -Tubulin was detected by direct immunoflourescence using an Cy3-conjugated antibody. Pancreatic lobules were treated with either cerulein or H2O2 for 15 (B and F) or 30 min (C and G). D and H: pancreatic lobules pretreated with 5 µM Taxol and subsequently stimulated with cerulein or H2O2 for 30 min. Untreated pancreatic lobules are shown in A and E.

Pancreatic lobules treated with H2O2 also show a complete destruction of the microtubule system (Fig. 5, F and G). However, the microtubule disruption induced by H2O2 was mild and modest compared with cerulein. Preincubation with Taxol prevented the H2O2-induced microtuble destruction (Fig. 5H). TNF-alpha and Taxol do not affect the microtubule system by themselves (data not shown).

Taxol does not induce NF-kappa B/Rel activation in pancreatic lobules. Taxol has been shown to activate NF-kappa B/Rel in murine macrophages but has no effect on Hela cells (30, 33). To test whether Taxol induces NF-kappa B/Rel in the pancreas, we have incubated pancreatic lobules with 5 µM Taxol for 30 min. Taxol does not stimulate NF-kappa B/Rel (Fig. 6) (background activity). Consistently, the Ikappa Balpha and beta -levels do not display significant differences.


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Fig. 6.   Taxol does not induce NF-kappa B/Rel in pancreatic lobules. Pancreatic lobules were left untreated (lane 1) or incubated (lane 2) with 5 µM Taxol for 30 min. Nuclear proteins were prepared, and electrophoretic mobility shift assays (EMSA) were performed using a kappa B specific probe. Cytoplasmic protein extracts of same samples were separated in SDS-PAGE and immunblotted with antibodies specific for Ikappa Balpha and Ikappa Bbeta .

Taxol treatment does not interfere with cerulein-induced NF-kappa B activation. It has been previously shown (33) that the cytoskeleton controls gene expression and depolymerization of microtubules activates NF-kappa B/Rel. In an attempt to analyze the association of NF-kappa B/Rel with the microtubule system, we have prepared pancreatic lobules that were preincubated with or without 0.1, 0.5, 1.0, 2.5, 5 µM Taxol for 15 min followed by stimulation with 100 nM cerulein and harvested after 30 min. Nuclear extracts were prepared and assayed for DNA binding activity for NF-kappa B/Rel (Fig. 7A). Cytoplasmic extracts were used for Western analysis (Fig. 7B). Pretreatment with Taxol does not block cerulein-induced NF-kappa B/Rel activation (lanes 3-7). Consistent with these results, Ikappa Balpha degradation mediated by cerulein cannot be blocked. Ikappa B-protein levels remained unaffected as expected (Fig. 7B).


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Fig. 7.   Taxol treatment does not interfere with cerulein-induced NF-kappa B activation. A: pancreatic lobules were left untreated (lane 1) or pretreated without (lane 2) or with different doses of Taxol for 15 min followed by incubation with cerulein 100 nM for 30 min (lanes 3-7). Nuclear proteins were prepared, and EMSA was performed using a kappa B specific probe. B: cytoplasmic protein extracts of same samples were separated in SDS-PAGE and immunblotted with antibodies specific for Ikappa Balpha and Ikappa Bbeta .

These data suggest that disorganization of the microtubule system is not required for cerulein-mediated NF-kappa B/Rel activation.

H2O2 induced NF-kappa B activation requires microtubule depolymerization. To test whether H2O2-induced NF-kappa B/Rel activation depends on changes of the microtubule system, pancreatic lobules were prepared and preincubated with or without 0.1, 0.5, 1.0, 2.5, or 5 µM Taxol for 15 min followed by stimulation with 150 µM H2O2. After 30 min, nuclear extracts were prepared and assayed for DNA binding activity for NF-kappa B/Rel. Increasing doses of Taxol block H2O2-induced NF-kappa B/Rel activation most efficiently at a doses of 5 µM (Fig. 8).


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Fig. 8.   H2O2-induced NF-kappa B activation requires microtubule depolymerization. A: pancreatic lobules were left untreated (lane 1) or pretreated without (lane 2) or with different doses of Taxol for 15 min followed by incubation with H2O2 for 30 min (lane 3-7). Nuclear proteins were extracted, and EMSA were performed using a kappa B specific probe. B: cytoplasmic protein extracts of same samples were separated in SDS-PAGE and immunoblotting with antibodies specific for Ikappa Balpha and Ikappa Bbeta . Cytoplasmic lysates were also immunoprecipitated with anti-phosphotyrosine followed by a Western blot analysis with anti-Ikappa Balpha .

To test whether Taxol treatment acts upstream of Ikappa Balpha , we prepared cytoplasmic extracts and performed immunoprecipitations with anti-phosphotyrosine antibody. Pretreatment of 0.5 µM Taxol already suppressed kinase activity. Protein levels of Ikappa Balpha and Ikappa Bbeta remained unchanged (Fig. 8B).

These data suggest that changes of the cytoskeleton mediate H2O2-induced NF-kappa B/Rel activation.

Activation of NF-kappa B by nocodazole in pancreatic lobules. Because the microtubule system is involved in H2O2-mediated NF-kappa B/Rel activation and can be prevented by Taxol treatment, it is possible that the induction of the depolymerization of the microtubule system is sufficient to activate NF-kappa B/Rel binding in pancreatic acinar cells. Nocodazole is known to be a reversible inhibitor of tubulin polymerization. Pancreatic lobules were incubated for different time points (15 and 30 min) with 10-5 M nocodazole. Nuclear extracts were examined for NF-kappa B/Rel binding activity. As shown in Fig. 9A, nocodazole induces NF-kappa B/Rel binding with the most prominent effect after 30 min.


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Fig. 9.   Activation of NF-kappa B by nocodazole in pancreatic lobules. A: pancreatic lobules were left untreated (lane 1) or incubated for different time points (lanes 2 and 3). Extracts were prepared, and equal amounts of protein (10 µg) were used in EMSA with the kappa B motif of the mouse kappa -light-chain enhancer as probe. B: cytoplasmic extracts of the same samples were prepared and used either for Western blot analysis or immunoprecipitation. Cytoplasmic extracts were separated by SDS-PAGE and analyzed by Western blot using anti-Ikappa Balpha and anti-Ikappa Bbeta antibodies. Cytoplasmic lysates were immunoprecipitated with anti-phosphotyrosine, separated by SDS-PAGE, and transferred to Western blot analysis using anti-Ikappa Balpha antibody.

NF-kappa B/Rel activation is not associated with subsequent degradation of the inhibitory proteins Ikappa Balpha and Ikappa Bbeta , as depicted in Fig. 9B. To analyze whether the inhibitory protein Ikappa Balpha is tyrosine phosphorylated, we prepared cytoplasmic extracts and performed an immunoprecipitation with anti-phosphotyrosine followed by Western blot analysis anti-Ikappa Balpha antiserum. As shown in Fig. 9B, we could detect tyrosine-phosphorylated Ikappa Balpha after 15 min and to a lower extent after 30 min.

These data suggest that depolymerization of the cytoskeleton alone or per se is sufficient to activate NF-kappa B/Rel in pancreatic lobules via a proteasome-independent pathway.

Nocodazole-induced NF-kappa B activation is microtubule dependent. To test whether nocodazole-mediated NF-kappa B/Rel activation is due to microtubule depolymerization, we tested the ability of Taxol to reverse this effect. Pancreatic lobules were prepared and preincubated with or without 0.1, 0.5, 1.0, 2.5, or 5 µM Taxol for 15 min followed by stimulation with 10-5 M nocodazole. The samples were harvested after 45 min. Nuclear extracts were used for the analysis of DNA binding activity of NF-kappa B/Rel. As shown in Fig. 10, Taxol blocks the nocodazole-mediated NF-kappa B/Rel over a dose range from 0.5 to 5 µM.


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Fig. 10.   Nocodazole-induced NF-kappa B activation is microtubule dependent. Pancreatic lobules were left untreated (lane 1) or pretreated without (lane 2) or with different doses of Taxol for 15 min followed by incubation with nocodazole for 30 min (lanes 3-7). Nuclear proteins were extracted, and EMSA were performed using a kappa B specific probe.

Accordingly, we examined the effect of nocodazole on the microtubule system in pancreatic lobules (Fig. 11). Treatment with 10-5 M nocodazole leads to disruption of the normal microtubule cytoskeleton. Substantial shortening and collapse of the cytoskeleton is obvious after 15 and, in particular, after 30 min. Pretreatment with 5 µM Taxol prevented destruction of the microtubulus system.


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Fig. 11.   Effect of nocodazole on the microtubule system. alpha -Tubulin was detected by direct immunoflourescence using a Cy3-conjugated antibody. Pancreatic lobules were treated with 10-5 M nocodazole for 15 (B) and 30 min (C). D: pancreatic lobules preincubated with Taxol 5 µM and subsequently activated with 10-5 M nocodazole for 30 min. Untreated pancreatic lobules are shown in A.

These data show that microtubule depolymerization per se leads to NF-kappa B/Rel activation in pancreatic lobules.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reactive oxygen species (ROS) are generated at sites of inflammation and injury. At low levels, ROS can function as signaling molecules participating as intermediates in the regulation of fundamental cell activities such as cell growth and cell adaptation responses. At higher concentrations, ROS can cause cellular injury and death (25, 41). Several lines of evidence suggest that oxidative stress may play a role in acute pancreatitis (10, 15, 31, 40, 43). The enhanced formation of oxygen radicals and their adducts have been detected early during induction of acute pancreatitis in mice. Here, we showed that H2O2 mediates NF-kappa B/Rel activation in pancreatic lobules. This activation is not accompanied by serine phosphorylation and subsequent degradation of Ikappa Balpha ; instead, we could detect tyrosine-phosphorylated Ikappa Balpha . Unlike serine phosphorylation of Ikappa Balpha , phosphorylation of tyrosine residue 42 does not lead to its degradation but rather causes the dissociation of intact Ikappa Balpha from NF-kappa B/Rel complexes (22, 26). The IKK complex is not involved in this signaling pathway; the responsible kinase has not yet been found. The tyrosine kinase ICK, phosphoinosite-3-kinase, and the tyrosine phosphatase CD 45 have been suggested as potential candidates (5, 22). Increasing evidence indicates that endothelial cells stimulated with H2O2 display increased tyrosine phosphorylation of proteins at focal contacts including beta -catenin, p120, and p130cas (17). Furthermore, tyrosine residues of paxillin and focal adhesion kinase have been phosphorylated in bovine coronary venular endothelial cells (48, 51). In response to oxidant stress, endothelial cells show increased protein kinase C activity via direct oxidative modification of the regulatory domain, secondary to induction of phospholipases (PLC, PLD, PLA2) or through phosphorylation of distinct residues of different PKC isoforms (16). Finally, ROS are known to activate several members of the Src family of protein tyrosine kinases in a wide range of cell types (1, 32, 38). In light of these signaling pathways, it is conceivable that the tyrosine phosphorylation of Ikappa Balpha is mediated by at least one of these tyrosine kinases.

We demonstrate that in pancreatic lobules, H2O2-induced NF-kappa B/Rel activation can be blocked by Taxol, a substance that stabilizes microtubules against depolymerization. Although Taxol has been shown to activate NF-kappa B/Rel in murine macrophages, others have reported that phorbol 12-myristate 13-acetate- or nocodazole-induced NF-kappa B/Rel activation is blocked by Taxol, suggesting specific effects of Taxol in different cells (30, 33). In line with this observation, nocodazole, which induces depolymerization of the microtubule system, activates NF-kappa B/Rel via tyrosine phosphorylation of Ikappa Balpha . These data would suggest that the IKK-independent NF-kappa B/Rel activation via tyrosine phosphorylation of Ikappa Balpha seems to be a general pathway that is also activated following oxygenation, reoxygenation, and reperfusion in the liver (6, 14, 26, 42, 45, 52). Our data would favor that the kinase responsible for tyrosine phosphorylation of Ikappa Balpha might be induced by ROS and associated with the cytoskeleton (33). This is consistent with Crepieux et al. (9), who were able to colocalize Ikappa Balpha with dynein, a microtubulus protein.

Microtubules, the cytoskeletal structures formed by the polymerization of tubulin heterodimers, play a crucial role in many biological processes including mitosis, cell-cell communication, intracellular transport, cell growth, and programmed cell death or apoptosis (24). It has been suggested that intracellular activation of digestive enzymes resulting from their colocalization with lysosomal enzymes leads to pancreatic autodigestion during the processes of pancreatitis (34). It has been shown that inhibition of digestive enzyme secretion in cerulein-induced pancreatitis occurs due to an impairment of intracellular vesicular transport and that microtubules are probably involved in this phenomenon. Stabilizing microtubules by using Taxol prevents cerulein-induced pancreatitis (47). We confirm that cerulein-mediated destruction of the cytoskeleton can be prevented by Taxol; however, NF-kappa B/Rel activation remained unaffected. These results suggest that IKK-mediated activation of NF-kappa B/Rel by cerulein does not require cytoskeleton disorganization. However, it is possible that cerulein-mediated disruption of the cytoskeleton contributes to the NF-kappa B/Rel activation.

Several lines of evidence suggest an important role of cytokines, such as TNF-alpha and IL-6, in human and experimental pancreatitis, which are known to be target genes of NF-kappa B/Rel (29). In animal models, IL-1, IL-6, and TNF-alpha are induced within the pancreatic parenchyma within 30 min of acute pancreatitis induction and before appreciable changes in pancreatic histology (44). Steinle et al. (44) previously showed that Ikappa Balpha and Ikappa Bbeta undergo degradation during the course of cerulein pancreatitis. Because cerulein does not affect Ikappa Bbeta in vitro, our results suggest that the Ikappa Bbeta degradation might be mediated by endogenous TNF-alpha (19, 46). The TNF-alpha -mediated NF-kappa B/Rel activation leads to degradation of either proteins in vitro. In line with our observations, it has been reported that pancreatic acinar cells produce and release TNF-alpha during the processes of pancreatitis (19). It is also interesting that very low concentrations of TNF-alpha are sufficient to induce NF-kappa B/Rel in pancreatic lobules, whereas much higher doses are required in other cells.

The present study provides evidence for three different pathways of NF-kappa B/Rel activation in pancreatic lobules (Fig. 12). Cerulein and TNF-alpha induce NF-kappa B/Rel via IKK activation, whereas H2O2 leads to tyrosine phosphorylation of Ikappa Balpha . Although Taxol might induce different cellular responses, it selectively blocks H2O2-mediated NF-kappa B/Rel induction in our cell system, suggesting that the microtubulus system plays a key role in the activation of this so-far-unidentified IKK in pancreatic acinar cells. The specific inhibiton of ROS-induced NF-kappa B/Rel activation mediated by Taxol might be an aspect for the better outcome of the experimental pancreatitis as shown by Ueda et al. (47).


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Fig. 12.   Different activation pathways of NF-kappa B/Rel.

There is still controversy about the inhibition of NF-kappa B/Rel during experimental pancreatitis. In light of the different modes of NF-kappa B/Rel activation in the pancreas, experimental use of nonspecific inhibitors of NF-kappa B/Rel has to be reevaluated. Specific inhibition of different signaling pathways of NF-kappa B/Rel might help us to estimate the role of the transcription factor on the course of this inflammatory disease.


    ACKNOWLEDGEMENTS

We are grateful to I. Rueß for assistance with manuscript preparation. We thank members of the laboratory for helpful comments and numerous reagents.


    FOOTNOTES

Address for reprint requests and other correspondence: R. M. Schmid, Dept. of Internal Medicine I, Univ. of Ulm, Robert-Koch-Str. 8, 89081 Ulm, Germany (E-mail: roland.schmid{at}medizin.uni-ulm.de).

10.1152/ajpgi.00407.2001


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