INVITED REVIEW
Different modes of NF-
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 |
The eukaryotic transcription
factor nuclear factor-
B (NF-
B)/Rel is activated by a large
variety of stimuli. It has been demonstrated that NF-
B/Rel
is induced during the course of cerulein pancreatitis. Here, we show
that NF-
B/Rel is differentially activated in pancreatic lobules.
Cerulein induces NF-
B/Rel via activation of I
B kinase (IKK),
which causes degradation of I
B
but not I
B
. Tumor necrosis
factor-
-mediated IKK activation leads to I
B
and I
B
degradation. In contrast, oxidative stress induced by
H2O2 activates NF-
B/Rel independent of IKK
activation and I
B
degradation; instead I
B
is phosphorylated
on tyrosine. H2O2 but not cerulein-mediated
NF-
B/Rel activation can be blocked by stabilizing microtubules with
Taxol. Inhibition of tubulin polymerization with nocodazole causes
NF-
B/Rel activation in pancreatic lobules. These results propose
three different pathways of NF-
B/Rel activation in pancreatic acinar
cells. Furthermore, these data demonstrate that microtubules play a key
role in IKK-independent NF-
B/Rel activation following oxidative stress.
acute pancreatitis; inhibitor protein I
B; I
B kinase; inflammation; hydrogen peroxide; tyrosine phosphorylation; cytokines; cerulein
 |
INTRODUCTION |
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-
B (NF-
B)/Rel is activated
during cerulein and acute biliary pancreatitis.
NF-
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-
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-
B/Rel is localized in the
cytoplasm complexed to its endogenous inhibitor proteins I
Bs
(7, 8). After stimulation, I
Bs are phosphorylated at
serine residues by a 700-kDa protein complex containing two related
catalytic subunits, IKK
and IKK
and the regulatory subunit IKK
. Various extracellular signaling pathways converge at the level
of IKK activation (8, 28). After phosphorylation, the inhibitor proteins, I
Bs are ubiquitinated and rapidly degraded by
the nonlysosomal, ATP-dependent 26S proteasome, which releases the
transcription factor NF-
B/Rel into the nucleus, where it binds
to DNA sites containing
B motifs.
The role of NF-
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-
B, such as tumor necrosis factor-
(TNF-
), interleukin
(IL)-1 or -6, or interferon-
. In particular, TNF-
has
been implicated in systemic complications of acute pancreatitis (29). Multiple factors are thought to contribute to
NF-
B/Rel induction in acute pancreatitis. This might include trypsin
activation, TNF-
, oxygen radical intermediates, and disruption of
the cytoskeleton (2, 4, 10, 15, 31). The present study was
undertaken to investigate the mode of NF-
B/Rel activation in
pancreatic lobules exposed to different NF-
B/Rel inducers.
 |
MATERIALS AND METHODS |
Reagents.
Cerulein was purchased from Pharmacia (Erlangen, Germany). TNF-
and
Taxol were from Sigma (Deisenhofen, Germany).
H2O2 was delivered by Merck (Darmstadt,
Germany). Anti-
-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-
, 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
B sequences found in the mouse
-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
[
-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 I
B
or I
B
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 I
B
or I
B
(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
-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 I
B kinase (IKK) assay, an anti-IKK
antibody recognizing
IKK
and IKK
(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
-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)-I
B
(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
-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-IKK
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
-glycerophosphate, and 10 MgCl2. Kinase activity was assayed in 40 µl of kinase
buffer containing 10 µM [
-32P]dATP and 3 µg
GST-I
B
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
-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 |
Kinetics of I
B degradation in pancreatic lobules after
stimulation with cerulein and TNF-
.
A supramaximal concentration of cerulein results in acute pancreatitis,
which is characterized by activation of NF-
B/Rel. NF-
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-
. To analyze the effect of cerulein and TNF-
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-I
B
and anti-I
B
. Phosphorylated I
B
was
detected using an antibody recognizing serine-phosphorylated I
B
.
The cytoplasmic I
B
signal almost completely disappeared after
stimulation with 100 nM cerulein for 15 min. In line with this
observation, we could see serine-phosphorylated I
B
after 15 min
of stimulation lasting 2 h. I
B
was not affected by cerulein
(Fig. 1A).

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Fig. 1.
Kinetics of I B degradation in pancreatic lobules
following cerulein or tumor necrosis factor- (TNF- ) 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- (B,
lanes 2-5) for different time points as indicated.
Cytoplasmic extracts were prepared and subjected to Western analysis
with anti-I B , anti-I B , and phosphoserine specific I B
(P-Ser-I B ) antibodies.
|
|
Next, we analyzed TNF-
-induced degradation of I
Bs in pancreatic
lobules. Maximal activation of NF
B/Rel activity required 10 U/ml
TNF-
in pancreatic lobules (data not shown). Thirty minutes after
treatment with 10 U/ml TNF-
, cytoplasmic I
B
levels were almost
completely reduced compared with unstimulated lobules. After 60 min,
I
B
reappeared and was back to original levels at 120 min.
Cytoplasmic I
B
completely disappeared after 30 min but reappeared
after 60 min. With the use of serine-specific anti-I
B
antibody,
we could detect serine-phosphorylated I
B
after 15 and 30 min
(Fig. 1B).
These data show that TNF-
induces a transient degradation of
I
B
and I
B
, whereas cerulein treatment causes a prolonged degradation of I
B
but does not affect I
B
in vitro.
Time-dependent activation of IKK by cerulein and TNF-
.
To analyze whether degradation of I
B
correlates with IKK
activity, endogenous IKK activity was immunoprecipitated with an anti-IKK antibody. Kinase activity was visualized by incubating the
immunocomplexes with GST-I
B
(1-54) in the
presence of [
-32P]dATP.
Treatment with cerulein for 15 min leads to phosphorylation of
GST-I
B
(1-54), with a peak of IKK activity
after 30 min of stimulation. IKK-activity lasted up to 120 min. The
amounts of precipitated IKK
and IKK
were verified on the same
membrane (Fig. 2A). The
activation of IKK correlates with I
B
degradation, and the
appearance of NF-
B/Rel binding activity in the nucleus (data not
shown).

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Fig. 2.
Time-dependent activation of I B kinase (IKK) by
cerulein and TNF- . 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- (B, lanes 2-5) for time periods
indicated. Cytoplasmic protein extracts were immunoprecipitated with
anti-IKK specific antibody and incubated with glutathione
S-transferase (GST)-I B (1-54) as
substrate in the presence of [ -32P]dATP. The amounts
of immunoprecipated IKK and IKK were verified (A). KA,
kinase assay.
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|
After stimulation with TNF-
, IKK activity peaks at 30 min and lasts
for 2 h later (Fig. 2B). IKK activity correlates with degradation of I
B
and I
B
(Fig. 1B). These data
show that cerulein and TNF-
activate IKK with different kinetics.
Dose- and time-dependent induction of NF-
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-
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-
B/Rel.
H2O2 induced a dose-dependent increase of NF-
B/Rel binding activity with a maximal stimulatory effect of 150 µM. Increasing H2O2 to 500 µM reduced
NF-
B/Rel binding activity compared with 150 µM (Fig.
3A). The specificity of
NF-
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 B
(NF- 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 B motif of the mouse -light-chain
enhancer as probe.
|
|
To study time-dependent induction of NF-
B/Rel, pancreatic
lobules were incubated with H2O2 (150 µM)
over time. H2O2-mediated induction of
NF-
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-
B/Rel as fast as cerulein or
TNF-
.
H2O2 induces tryrosine phosphorylation of
I
B
, which is not mediated by IKK.
In contrast to the classic activators of NF-
B/Rel,
H2O2-mediated induction of NF-
B/Rel has been
shown to be associated with tyrosine phosphorylation (22).
To test how NF-
B/Rel is activated following
H2O2 treatment in pancreatic lobules,
cytoplasmic extracts were analyzed by Western blotting using
anti-I
B
and anti-I
B
antibodies. Both inhibitory proteins
were not degraded after stimulation with H2O2
over time. In contrast, I
B
slightly increased after 15 min. To
test whether I
B
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-I
B
. Figure
4A shows that tyrosine-phosphorylated I
B
can be detected after 15, 30, and even
60 min after stimulation with H2O2. The
detection of tyrosine-phosphorylated I
B
correlates with
NF-
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-I
B
(1-54) in the presence of
[
-32P]dATP. Very little IKK activity was detected with
a slight increase after 30 min (long exposure). The amounts of
precipitated IKK
and IKK
were verified on the same membrane (Fig.
3B). The IKK activity does not precede and correlate
with NF-
B/Rel binding. Therefore, it seems unlikely that IKK is
responsible for tyrosine phosphorylation of I
B
.

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Fig. 4.
H2O2-induced tyrosine
phosphorylation of I B (P-Tyr-I B ), 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-I B and
anti-I B antibodies. Cytoplasmic lysates were immunoprecipitated
with anti-phosphotyrosine followed by Western blot analysis with
anti-I B antiserum. B: cell lysates were analyzed for
IKK activity. Endogenous IKK complexes were immunprecipitated with an
IKK specific antibody and incubated with GST-I B
(1-54) as substrate in the presence of
[ 32P]dATP. The amount of IKK and IKK was
verified on the same membrane by immunoblotting.
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These data show that H2O2 induces
phosphorylation of I
B
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
-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. -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.
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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-
and Taxol do not affect the microtubule system
by themselves (data not shown).
Taxol does not induce NF-
B/Rel activation in pancreatic lobules.
Taxol has been shown to activate NF-
B/Rel in murine macrophages but
has no effect on Hela cells (30, 33). To test whether Taxol induces NF-
B/Rel in the pancreas, we have incubated pancreatic lobules with 5 µM Taxol for 30 min. Taxol does not stimulate
NF-
B/Rel (Fig. 6) (background
activity). Consistently, the I
B
and
-levels do not display
significant differences.

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Fig. 6.
Taxol does not induce NF- 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 B specific probe. Cytoplasmic protein extracts of
same samples were separated in SDS-PAGE and immunblotted with
antibodies specific for I B and I B .
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Taxol treatment does not interfere with cerulein-induced NF-
B
activation.
It has been previously shown (33) that the cytoskeleton
controls gene expression and depolymerization of microtubules activates NF-
B/Rel. In an attempt to analyze the association of NF-
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-
B/Rel (Fig.
7A). Cytoplasmic extracts were
used for Western analysis (Fig. 7B). Pretreatment with Taxol
does not block cerulein-induced NF-
B/Rel activation (lanes
3-7). Consistent with these results, I
B
degradation
mediated by cerulein cannot be blocked. I
B-protein levels remained
unaffected as expected (Fig. 7B).

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Fig. 7.
Taxol treatment does not interfere with cerulein-induced
NF- 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 B specific
probe. B: cytoplasmic protein extracts of same samples were
separated in SDS-PAGE and immunblotted with antibodies specific for
I B and I B .
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These data suggest that disorganization of the microtubule system is
not required for cerulein-mediated NF-
B/Rel activation.
H2O2 induced NF-
B activation requires
microtubule depolymerization.
To test whether H2O2-induced NF-
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-
B/Rel.
Increasing doses of Taxol block
H2O2-induced NF-
B/Rel activation most
efficiently at a doses of 5 µM (Fig. 8).

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Fig. 8.
H2O2-induced NF- 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 B specific probe. B: cytoplasmic
protein extracts of same samples were separated in SDS-PAGE and
immunoblotting with antibodies specific for I B and I B .
Cytoplasmic lysates were also immunoprecipitated with
anti-phosphotyrosine followed by a Western blot analysis with
anti-I B .
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To test whether Taxol treatment acts upstream of I
B
, we prepared
cytoplasmic extracts and performed immunoprecipitations with
anti-phosphotyrosine antibody. Pretreatment of 0.5 µM Taxol already
suppressed kinase activity. Protein levels of I
B
and I
B
remained unchanged (Fig. 8B).
These data suggest that changes of the cytoskeleton mediate
H2O2-induced NF-
B/Rel activation.
Activation of NF-
B by nocodazole in pancreatic lobules.
Because the microtubule system is involved in
H2O2-mediated NF-
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-
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-
B/Rel binding activity. As shown in Fig.
9A, nocodazole induces
NF-
B/Rel binding with the most prominent effect after 30 min.

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|
Fig. 9.
Activation of NF- 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 B motif of the mouse
-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-I B and
anti-I B antibodies. Cytoplasmic lysates were immunoprecipitated
with anti-phosphotyrosine, separated by SDS-PAGE, and transferred to
Western blot analysis using anti-I B antibody.
|
|
NF-
B/Rel activation is not associated with subsequent degradation of
the inhibitory proteins I
B
and I
B
, as depicted in Fig.
9B. To analyze whether the inhibitory protein I
B
is
tyrosine phosphorylated, we prepared cytoplasmic extracts and performed an immunoprecipitation with anti-phosphotyrosine followed by Western blot analysis anti-I
B
antiserum. As shown in Fig. 9B,
we could detect tyrosine-phosphorylated I
B
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-
B/Rel in pancreatic lobules via
a proteasome-independent pathway.
Nocodazole-induced NF-
B activation is microtubule dependent.
To test whether nocodazole-mediated NF-
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-
B/Rel. As shown in Fig.
10, Taxol blocks the
nocodazole-mediated NF-
B/Rel over a dose range from 0.5 to 5 µM.

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|
Fig. 10.
Nocodazole-induced NF- 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 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. -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-
B/Rel activation in pancreatic lobules.
 |
DISCUSSION |
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-
B/Rel activation in pancreatic lobules. This activation is not
accompanied by serine phosphorylation and subsequent degradation of
I
B
; instead, we could detect tyrosine-phosphorylated I
B
. Unlike serine phosphorylation of I
B
, phosphorylation of tyrosine residue 42 does not lead to its degradation but rather causes the
dissociation of intact I
B
from NF-
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
-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 I
B
is mediated by at least
one of these tyrosine kinases.
We demonstrate that in pancreatic lobules,
H2O2-induced NF-
B/Rel activation can be
blocked by Taxol, a substance that stabilizes microtubules against
depolymerization. Although Taxol has been shown to activate NF-
B/Rel
in murine macrophages, others have reported that phorbol 12-myristate
13-acetate- or nocodazole-induced NF-
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-
B/Rel via tyrosine phosphorylation of I
B
. These data would
suggest that the IKK-independent NF-
B/Rel activation via tyrosine
phosphorylation of I
B
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 I
B
might
be induced by ROS and associated with the cytoskeleton
(33). This is consistent with Crepieux et al.
(9), who were able to colocalize I
B
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-
B/Rel activation
remained unaffected. These results suggest that IKK-mediated activation
of NF-
B/Rel by cerulein does not require cytoskeleton disorganization. However, it is possible that cerulein-mediated disruption of the cytoskeleton contributes to the NF-
B/Rel activation.
Several lines of evidence suggest an important role of cytokines, such
as TNF-
and IL-6, in human and experimental pancreatitis, which are
known to be target genes of NF-
B/Rel (29). In animal models, IL-1, IL-6, and TNF-
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 I
B
and I
B
undergo degradation during the course of cerulein pancreatitis. Because
cerulein does not affect I
B
in vitro, our results suggest that
the I
B
degradation might be mediated by endogenous TNF-
(19, 46). The TNF-
-mediated NF-
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-
during the processes of pancreatitis
(19). It is also interesting that very low concentrations of TNF-
are sufficient to induce NF-
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-
B/Rel activation in pancreatic lobules (Fig.
12). Cerulein and TNF-
induce
NF-
B/Rel via IKK activation, whereas H2O2
leads to tyrosine phosphorylation of I
B
. Although Taxol might
induce different cellular responses, it selectively blocks
H2O2-mediated NF-
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-
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).
There is still controversy about the inhibition of NF-
B/Rel during
experimental pancreatitis. In light of the different modes of
NF-
B/Rel activation in the pancreas, experimental use of nonspecific inhibitors of NF-
B/Rel has to be reevaluated. Specific inhibition of
different signaling pathways of NF-
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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