Department of Medicine I, University of Ulm, 89070 Ulm, Germany
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ABSTRACT |
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The eukaryotic transcription factor NF-B/Rel
is activated by a large variety of stimuli. We have recently shown that
NF-
B/Rel is induced during the course of caerulein
pancreatitis. Here, we show that activation of NF-
B/Rel by
caerulein, a CCK analog, requires increasing intracellular
Ca2+ levels and protein kinase C
activation. Caerulein induces a dose-dependent increase of nuclear
NF-
B/Rel binding activity in pancreatic lobules, which is paralleled
by degradation of I
B
. I
B
was only slightly affected by
caerulein treatment. Consistent with an involvement of
Ca2+, the endoplasmic
reticulum-resident Ca2+-ATPase
inhibitor thapsigargin activated NF-
B/Rel in pancreatic lobules. The
intracellular Ca2+ chelator TMB-8
prevented I
B
degradation and subsequent nuclear translocation of
NF-
B/Rel induced by caerulein. BAPTA-AM was less effective.
Cyclosporin A, a
Ca2+/calmodulin-dependent protein
phosphatase (PP2B) inhibitor, decreased caerulein-induced NF-
B/Rel
activation and I
B
degradation. The inhibitory
effect of bisindolylmaleimide suggests that protein kinase C activity
is also required for caerulein-induced NF-
B/Rel activation. These
data suggest that Ca2+- as well as
protein kinase C-dependent mechanisms are required for
caerulein-induced NF-
B/Rel activation.
pancreas; signaling; acute pancreatitis
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INTRODUCTION |
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IN RODENTS, SUPRAMAXIMAL stimulation of the pancreas with the CCK analog caerulein causes morphological, biochemical, and pathophysiological alterations similar to acute pancreatitis in humans (1, 21). The hormone-receptor mechanism underlying this phenomenon has been well characterized and involves the low-affinity state of the CCK receptor (14). Receptor binding of CCK causes an initial release of Ca2+ from intracellular stores via inositol 1,4,5-trisphosphate, which is maintained by subsequent influx of Ca2+ into the cell leading to an increase in the free ionized cytosolic Ca2+ concentration ([Ca2+]i) (41). Physiological doses of CCK are known to produce oscillations in [Ca2+]i, which are believed to be important in organizing and controlling complex intracellular functions (25). Supramaximal doses of CCK produce a single, large, sustained increase in [Ca2+]i in acinar cells, which is known to be associated with cell damage in several cell types, and may be implicated in the pathogenesis of acute pancreatitis (24, 28). A corresponding increase in 1,2-diacylglycerol is observed with high doses of caerulein in excess of that required to stimulate a maximal secretory response (44).
In addition to these signaling events, supramaximal doses of caerulein produce a high but transient increase of p42map and p44map as well as other upstream components of the mitogen-activated protein kinase signaling cascade, including mitogen-activated protein kinase kinase and Ras (5, 10, 11). Furthermore, supramaximal doses of caerulein induced a dramatic increase in c-Jun NH2-terminal kinase (JNK) activity (5). The pronounced increase in JNK activity may reflect the response to cellular stress.
The transcription factor NF-B is a
trans-acting factor that binds to
enhancer elements involved in the immune and inflammatory response
[2, 36 (reviewed)]. This transcription factor is formed by
different homo- and heterodimers of members of this now called NF-
B/Rel family. In unstimulated cells, NF-
B/Rel complexes are found in the cytoplasm bound to I
B
and I
B
, which prevent
the dimers from entering the nucleus. When cells are activated, I
Bs are phosphorylated within minutes, causing their rapid degradation by
proteasomes (2, 4, 36). The release of NF-
B/Rel from I
Bs results
in the passage of NF-
B/Rel into the nucleus where it binds to
specific sequences in the promoter regions of target genes. Therefore,
I
B proteins control the presence of NF-
B/Rel binding activity in
the nucleus. Many stimuli activate NF-
B/Rel including cytokines,
activators of protein kinase C (PKC), lipopolysaccharides, and
oxidative stress [2, 36 (reviewed)]. We and others have
recently shown that the transcription factor NF-
B/Rel is activated
following caerulein pancreatitis (12, 17, 18, 38).
In an attempt to characterize signaling events involved in
caerulein-induced NF-B/Rel activation, we investigated DNA binding and nuclear translocation of NF-
B/Rel complexes in pancreatic lobules. Caerulein-induced I
B
degradation and subsequent nuclear translocation of NF-
B/Rel activation can be prevented by the intracellular Ca2+ chelator
8-(diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) and to a lesser
extent by
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM as well as by the
Ca2+/calmodulin-dependent protein
phosphatase (PP2B) inhibitor cyclosporin A. The inhibitory effect of
bisindolylmaleimide (BIS) suggests that PKC activity is also required
for caerulein-induced NF-
B/Rel activation. These data suggest that
Ca2+ as well as PKC-dependent
mechanisms are required for caerulein-induced NF-
B/Rel activation in
pancreatic acinar cells.
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MATERIALS AND METHODS |
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Reagents. Caerulein was purchased from Pharmacia (Erlangen, Germany). Phorbol 12-myristate 13-acetate (PMA), thapsigargin, and cyclosporin A were from Sigma Chemical (Deisenhofen, Germany). BAPTA-AM, TMB-8, and BIS (GF-109203X) were from Calbiochem (La Jolla, CA). 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 by a modified method previously described (33, 43). 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 either incubated with BIS (1, 5, 10, or 15 µM), BAPTA-AM (20 or 40 µM), TMB-8 (250, 500, or 1,000 µM), or cyclosporin A (100 or 500 nM) as pretreatment or left with medium alone. Thereafter, lobules were stimulated with caerulein (0.1, 1, 10, or 100 nM), PMA (100 ng/ml), or thapsigargin (10 µM). After the respective incubation periods, lobules were immediately frozen in liquid nitrogen and stored atProtein extracts. Nuclear protein extracts were prepared essentially as described by Dignam et al. (7), 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 (38).
For cytoplasmic protein extracts, pancreatic lobules were homogenized in 150 mM NaCl, 50 mM Tris · HCl, 50 mM CaCl2, 1.0% NP-40, 5 mM natrium fluoride, 0.1 M phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 35 µg/ml pepstatin, 10 µg/ml aprotinin, and 0.5 mM DTT, pH 7.2, and centrifuged at 15,000 rpm for 20 min at 4°C. Aliquots of the supernatant were stored atElectrophoretic mobility shift assays.
Electrophoretic mobility shift assays (EMSAs) were performed as
previously described (38). 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'-AGC
ACTAGTACG-3' and antisense
5'-AATTCGTACTA
A-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 cpm) was added to 10 µg of nuclear protein in the presence of 5 µg poly(dIdC) as
nonspecific competitor (Pharmacia Biotech Enzyme, 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.
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 IB
or I
B
was prepared by
transfecting 293 human embryonic kidney cells with the respective
eukaryotic expression vector as previously described and loaded as
controls (data not shown) (34). Protein complexes were resolved by
electrophoresis on 10% nondenaturing polyacrylamide gels in 1×
Tris-glycine-SDS buffer at room temperature. After SDS-PAGE, the gels
were transferred to 0.45-µm polyvinylidene difluoride membranes for
25 min at 40 mA at room temperature (Schleicher & 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 primary antibodies 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%
(vol/vol) 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. After blots were washed three times
with T-TBS, they were developed with enhanced chemiluminescence reagents (Amersham Buchler, Braunschweig, Germany).
Immunohistochemistry.
To analyze cell types contributing to NF-B/Rel activity, nuclear
translocation of RelA (p65) was visualized using immunohistochemistry as previously described (42). Pancreatic lobules were pretreated with
or without the inhibitors BAPTA-AM, TMB-8, or BIS followed by
stimulation with caerulein or left with medium as control. After
indicated time points, lobules were shock frozen in liquid nitrogen.
Frozen sections (4 µm) were air dried overnight and fixed with 4%
methanol-free formalin (Polyscience, Warrington, PA) in PBS at room
temperature for 15 min. After a short wash with washing solution (PBS
containing 0.02% Tween 20), samples were sequentially treated with
0.1% Triton X-100 in PBS for 5 min, rinsed with washing solution
twice, treated with 5 mg/ml RNase A in PBS for 30 min, and incubated in
blocking solution (PBS containing 2% BSA and 3% normal goat serum)
for 30 min at room temperature. Primary antibody (murine anti-p65,
Boehringer Mannhein) was diluted 1:200 in blocking solution and
incubated overnight at 4°C in a humidified chamber. Negative
controls were incubated with a nonspecific mouse IgG in blocking
solution. After the sections were washed five times for 5 min in
washing solution, sections were incubated with the secondary antibody
at 1:2,000 (anti-mouse Alexa 568, Molecular Probes, Eugene, OR) for 30 min in a dark, humidified chamber followed by five washes for 5 min. Thereafter, sections were counterstained for 15 min with a solution containing Yo-Pro-1 iodide (Molecular Probes) at 1:2,500 in 0.2× PBS followed by three washes in 0.2× PBS. Sections were mounted and kept in the dark until analysis with a confocal microscope (Leica,
TCS 4D, Heidelberg, Germany).
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RESULTS |
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Dose- and time-dependent induction of NF-B/Rel by
caerulein.
Pancreatic lobules were used for studying NF-
B/Rel activation in
vitro. To study the effect of different doses of caerulein on
NF-
B/Rel binding activity, EMSAs were performed. Pancreatic lobules
were incubated with increasing doses of caerulein, and nuclear extracts
were prepared and incubated with an end
32P-labeled DNA oligonucleotide
containing the recognition site of NF-
B. Although very little
NF-
B/Rel binding activity was detected in the unstimulated state,
caerulein induced NF-
B/Rel binding in a gradual manner. Maximal
activation of the inducible DNA-binding activity required 100 nM
caerulein. The specificity of NF-
B/Rel DNA binding induced by
caerulein was confirmed in competition experiments (data not shown). In
unstimulated cells, NF-
B/Rel heterodimers are kept as inactive
complexes in the cytoplasm by inhibitory proteins, such as I
B
and
I
B
. After stimulation, the I
Bs are phosphorylated and then
degraded, and free NF-
B/Rel dimers translocate into the nucleus. We
tested whether the degradation of I
B
or I
B
could take place
after treatment of pancreatic lobules with caerulein at different
doses. Cytoplasmic extracts were analyzed by Western blotting using
anti-I
B
and anti-I
B
antibodies. The cytoplasmic I
B
signal almost completely disappeared at 1 and 10 nM caerulein and was
completely absent when caerulein was added at 100 nM. Protein levels of
I
B
were not affected following caerulein treatment for 30 min
(data not shown). To study time-dependent induction of NF-
B/Rel,
pancreatic lobules were stimulated with caerulein at 100 nM over time.
Caerulein-mediated induction of NF-
B/Rel binding was detected after
10 min with a maximum at 30 min and thereafter decreased gradually. To
test the degradation of I
B
and I
B
after treatment with
caerulein, cytoplasmic extracts were analyzed by Western blotting using
anti-I
B
and anti-I
B
antibodies. Thirty minutes after
treatment with 100 nM caerulein, cytoplasmic I
B
levels were
almost completely reduced compared with unstimulated cells. After 60 min, I
B
reappeared and was back to original levels at 120 min.
Cytoplasmic I
B
levels decreased only slightly at 60 and 120 min
after caerulein treatment (data not shown).
Inhibition of endoplasmic reticulum-resident
Ca2+-ATPase
induces NF-B/Rel activation in pancreatic lobules.
The compound thapsigargin inhibits the endoplasmic reticulum
(ER)-resident Ca2+-ATPase, thereby
causing a rapid efflux of Ca2+
from the ER lumen into the cytoplasm. Pancreatic lobules were incubated
with 10 µM thapsigargin for 30 min. Nuclear extracts were prepared
and incubated with an end
32P-labeled DNA oligonucleotide
containing the recognition site of NF-
B. Compared with unstimulated
pancreatic lobules, thapsigargin activated NF-
B/Rel binding activity
after 30 min (Fig.
1A,
compare lanes 1 and
2). This activation can be reduced
by preincubation of pancreatic lobules for 15 min with 20 and 40 µM
BAPTA-AM or with 500 µM TMB-8, effective intracellular
Ca2+ chelators
(lanes 3-6), whereas 250 µM
TMB-8 had only a slight inhibitory effect. To further assess the
potential action of intracellular Ca2+ in pancreatic lobules, the
capacity of BAPTA-AM and TMB-8 to influence thapsigargin degradation of
I
B
and I
B
was assayed. Doses of 20 and 40 µM BAPTA-AM as
well as 250 and 500 µM TMB-8 prevented the I
B
degradation
induced by thapsigargin (Fig. 1B, lanes 3-6), with BAPTA-AM being
more effective.
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Ca2+
chelators interfere with caerulein-induced NF-B/Rel
activation.
To investigate whether intracellular
Ca2+ is required for
caerulein-induced NF-
B/Rel activation, pancreatic lobules were
preincubated with 20 or 40 µM BAPTA-AM or 250 or 500 µM TMB-8 for
15 min and then stimulated with 100 nM caerulein for 30 min (Fig.
2A).
Thereafter, nuclear extracts were prepared and assayed for NF-
B/Rel
activity. NF-
B/Rel activation is inhibited by pretreatment with
Ca2+ chelators, with TMB-8 being
most effective. BAPTA-AM caused only a slight reduction in
caerulein-mediated NF-
B/Rel binding activity (lanes
3 and 4). Neither
BAPTA-AM nor TMB-8 exerted a detectable effect on the basal levels of
NF-
B/Rel activity as judged by EMSA (data not shown). Preincubation
of pancreatic lobules with TMB-8 abrogated caerulein-stimulated
degradation of I
B
at 250 and 500 µM (Fig.
2B, lanes
5 and 6).
Pretreatment with BAPTA-AM also inhibited I
B
degradation but to a
much lesser extent (lanes 3 and
4).
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Cyclosporin A pretreatment decreased caerulein-induced
NF-B/Rel activation.
The immunosuppressive drug cyclosporin A acts by blocking a
Ca2+-mediated signaling pathway
contributing to the induction of NF-
B/Rel (13, 37). Pancreatic
lobules were incubated with 100 or 500 nM cyclosporin A for 30 min.
Nuclear extracts were prepared and assayed for
B binding activity.
Cytoplasmic extracts were analyzed using Western analysis. NF-
B/Rel
binding activity was suppressed following pretreatment with cyclosporin
A (Fig.
3A,
lanes 3 and 4). Cyclosporin A prevents the
inducible degradation of I
B
when stimulated with caerulein (Fig.
3B, lanes
3 and 4). These
results indicate that calcineurin, a
Ca2+/calmodulin-dependent protein
phosphatase, is required for caerulein-mediated activation of
NF-
B/Rel.
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PMA activates NF-B/Rel in pancreatic lobules.
Pancreatic lobules were preincubated with or without 5, 10, or 15 µM
BIS for 15 min followed by stimulation with the active phorbol ester
PMA (100 ng/ml) and harvested after 30 min. Nuclear extracts were
prepared and assayed for DNA binding activity of NF-
B. Cytoplasmic
extracts were used for Western analysis. PMA induced NF-
B/Rel
binding activity compared with controls (Fig. 4A,
compare lanes 1 and
2). Pretreatment with BIS blocked
PMA-induced NF-
B/Rel activation completely (lanes
3-5). Degradation of I
B
induced by PMA was
sustained by preincubation with BIS at all doses tested (Fig.
4B).
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NF-B/Rel activation by caerulein requires PKC.
Pancreatic lobules were pretreated with or without 1, 5, 10, or 15 µM
BIS for 15 min followed by stimulation with or without 100 nM caerulein
for 30 min. Nuclear and cytoplasmic extracts were prepared and assayed
in EMSAs using the
-light-chain enhancer high-affinity
B site as
probe and Western analysis with anti-I
B
and anti-I
B
antibodies. BIS caused a slight induction of NF-
B/Rel activity at 1 and 5 µM but had no effect on basal NF-
B/Rel activity at 10 and 15 µM (Fig. 5, lanes
3-6). Caerulein-induced NF-
B/Rel activity was
efficiently blocked by BIS at 5, 10, and 15 µM, with the
highest dose being most effective. Degradation of I
B
by caerulein
was already sustained by BIS at 1 µM and completely blocked by BIS at
5, 10, or 15 µM (Fig. 5,
lanes 8-10).
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Supramaximal doses of caerulein induce NF-B/Rel
activation in acinar cells.
In unstimulated cells, NF-
B/Rel is bound to the inhibitory I
B
subunits. To monitor its activation at a cellular level, a monoclonal
antibody can be used that recognizes an epitope that includes the
nuclear localization signal of the RelA (p65) subunit. This antibody
therefore recognizes RelA (p65) only when I
B was not bound to p65
(20). To determine active RelA (p65) in acinar cells, pancreatic
lobules were prepared and incubated with or without 100 nM caerulein
for 10, 30, or 60 min. Lobules were shock frozen, sectioned, fixed,
stained with the anti-p65 antibody (red fluorescence), and
counterstained with Yo-Pro to stain nuclei (green fluorescence).
Overlaying the red fluorescence with the green counterstain results in
a yellow nuclear staining indicative of nuclear localization of active
RelA (p65). Active nuclear RelA (p65) was observed as early as 30 min
after treatment with 100 nM caerulein as seen by the yellow nuclear
staining (Fig.
6B). No
nuclear staining of RelA (p65) was observed within the first 10 min
(Fig. 6A). The amount of nuclear
immunoreactivity for RelA (p65) further increased after 60 min (Fig.
6C) and 120 min (data not shown).
The saline-treated control showed very weak staining after 60 min (Fig.
6F). No nuclear staining for RelA
(p65) was observed at earlier time points (Fig. 6,
D and
E).
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Pretreatment with BAPTA-AM, TMB-8, or BIS abrogates caerulein
induced NF-B/Rel activation in acinar cells.
Pretreatment of pancreatic lobules with 40 µM BAPTA-AM (Fig.
7A),
1,000 µM TMB-8 (Fig. 7B), or 15 µM BIS (Fig. 7C) markedly reduced
the nuclear staining of RelA (p65) after a 30-min treatment with 100 nM
caerulein compared with the control, which did not receive an inhibitor
(Fig. 7D). The nuclear translocation
of RelA (p65) was not completely abolished in lobules pretreated with BAPTA-AM, indicative of nuclear staining for RelA (p65) in
some cells (Fig 7A).
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DISCUSSION |
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We have previously shown that high doses of caerulein lead to the
induction of the transcription factor NF-B/Rel (38). Because active
NF-
B/Rel translocates directly to the nucleus and induces gene
transcription, this establishes a novel signal transduction pathway
between the CCK receptor and the cell nucleus. NF-
B/Rel is
detectable within minutes after stimulation of pancreatic lobules with
high doses of caerulein. Both I
B
and I
B
contribute to
nuclear NF-
B/Rel binding activity. Whereas I
B
is degraded in
the initial phase and correlates with rapid NF-
B/Rel induction, decreased I
B
levels might be responsible for prolonged
NF-
B/Rel activation.
The present study was undertaken to determine the second messenger
molecules that mediate this activation. Caerulein, similar to CCK on
binding to its receptors on acinar cells, activates phospholipase C,
generates inositol trisphosphate, and induces free intracellular
Ca2+. The observation that
inhibition of the ER-resident
Ca2+-ATPase, which causes rapid
release of Ca2+ from the
organelle, activates NF-B/Rel in pancreatic lobules suggested a role
for caerulein-mediated Ca2+
release in NF-
B activation in acinar cells. We show here that two
structurally unrelated Ca2+
chelators inhibit NF-
B/Rel activation in response to caerulein. Whereas one inhibitor, TMB-8, inhibited NF-
B/Rel activation in response to caerulein efficiently, a second inhibitor, BAPTA-AM, was
less effective. This may be explained by the different mechanisms of
action of the two inhibitors. Moreover, cyclosporin A, an inhibitor of
the Ca2+/calmodulin-dependent
protein phosphatase (PP2B), decreased caerulein-mediated NF-
B/Rel. These data indicate that free
intracellular Ca2+ is necessary at
a postreceptor signaling step for caerulein/CCK-mediated activation of
NF-
B/Rel, as measured by DNA binding activity and enhancement of
I
B
degradation.
Interestingly, NF-B/Rel is strongly activated only at supramaximal
doses of caerulein known to cause a single, large sustained increase in
[Ca2+]i
in acinar cells. In contrast, physiological doses of CCK known to
produce oscillations in
[Ca2+]i
do not activate NF-
B/Rel, suggesting that the NF-
B/Rel activation is mediated by the low-affinity state of the CCK-A receptor (24, 25,
28, 44). In agreement with these data, JMV-180, known to activate the
high-affinity state of the CCK-A receptor and to induce
[Ca2+]i
oscillations, did not activate NF-
B/Rel in the pancreas (18). Furthermore, a recent report suggested that
[Ca2+]i
activation of different transcription systems in B cells can differ
greatly with regard to the amount of free intracellular Ca2+ required (39). In that
system, NF-
B is selectively activated by a large transient
[Ca2+]i
rise and nuclear factor for activation of transcription (NFAT) is
already turned on by a low, sustained
Ca2+ plateau, whereas neither
factor is induced by
[Ca2+]i
oscillations (8, 9). The activation of NF-
B/Rel by
Ca2+ ionophores on their own has
been only observed in lymphocytes. This cell type-specific regulation
is characterized by a synergistic PKC and
Ca2+-dependent phosphorylation and
degradation of I
B
(37). Among the numerous agents that induce
NF-
B/Rel activity in epithelial cells, only stimulation with
epidermal growth factor or ER stress-inducing conditions may require
input from free intracellular Ca2+
(26, 39). In U-937 monocytic cells, sphingosine-1-phosphate activates
NF-
B/Rel in a Ca2+-dependent
manner (32). Sphingosine-1-phosphate-induced NF-
B/Rel activation was
inhibited by cyclosporin A, suggesting a role for the
Ca2+/calmodulin-dependent
phosphatase calcineurin. Furthermore,
Ca2+-dependent pathways that
induce the phosphatase calcineurin synergize with PKC to degrade
I
B
and result in the activation of NF-
B/Rel (13, 37).
The activation of phospholipase C generates at least two second
messengers, one is Ca2+ and the
other is diacylglycerol, which stimulate PKC. An increase in
intracellular Ca2+ may activate
Ca2+/phospholipid-dependent PKC
isozymes. Moreover, the Ca2+
chelator TMB-8 has been reported to have effects on phospholipid metabolism and PKC (27). We therefore assessed whether activation of
PKC played a role in caerulein-mediated NF-B/Rel activation. Caerulein-induced NF-
B/Rel activation can be abrogated by
pretreatment with a PKC-inhibitor that is more specific than the ones
currently available (40). Although the mechanism of blocking NF-
B
translocation by PKC-inhibitors is unclear at this time, our current
results indicate that PKC is upstream of I
B
phosphorylation and
its inhibition selectively abrogates caerulein-induced I
B
degradation in pancreatic lobules. Previous studies indicated that in
vitro phosphorylation of I
B
by different kinases, including PKC,
was sufficient to dissociate NF-
B from I
B
in cytosolic
extracts and that, in vivo, stimuli such as PMA activate both PKC and
NF-
B (16, 35).
PKC was originally identified as a
Ca2+- and phospholipid-dependent
protein kinase and subsequently shown to be activated by diacylglycerol
and phorbol esters such as PMA. Five PKC isoforms have been detected in
pancreatic acini from rats, including ,
,
,
, and
(3,
29, 30). Whereas PKC-
belongs to the conventional PKC family, being
Ca2+ dependent, this property is
not shared by the
-,
-, and
-isoforms, which are part of the
novel PKC subfamily. PKC-
, an atypical PKC, shows
phospholipid-dependent kinase activity. So far only the isoforms
PKC-
and PKC-
have been reported to activate
B motifs and
therefore are likely candidates to mediate the stimulatory effects on
NF-
B/Rel activation in pancreatic acinar cells (15, 19). Treatment
of acini with CCK caused translocation of PKC-
and enhanced the
immunostaining pattern of PKC-
in the apical region of acinar cells
(3). Interestingly, PMA has been shown to cause translocation of
PKC-
in acinar cells, although PKC-
lacks a phorbol ester binding
site and is not activated by diacylglycerol (3). This suggests that
other isoforms of PKC can mediate the phosphorylation of PKC-
and
thereby induce its translocation. Recently, two central I
B kinases
have been identified (6, 31). These kinases trigger the phosphorylation
and subsequent proteolytic degradation of I
B
. Two other upstream
kinases, the NF-
B-inducing kinase and the mitogen-activated protein
kinase/ERK kinase, as well as other signaling components including
c-Raf and p90rsk1 have been shown
to be involved in the NF-
B activation pathway (22, 23). It remains
to be seen whether these steps are activated and required for
caerulein-induced NF-
B/Rel activation.
In summary, our data suggest that
Ca2+- as well as PKC-dependent
mechanisms are required for caerulein-induced NF-B/Rel activation in
pancreatic lobules.
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ACKNOWLEDGEMENTS |
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We thank Sonja Aigner for assistance with manuscript preparation and Günther Schneider for helpful discussion.
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FOOTNOTES |
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This work was in part supported by grants from the Bundesministerium für Bildung und Forschung to R. M. Schmid and from the Deutsche Forschungsgemeinschaft to G. Adler.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. M. Schmid, Dept. of Medicine I, Univ. of Ulm, Robert-Koch-Str. 8, D-89070 Ulm, Germany (E-mail: roland.schmid{at}medizin.uni-ulm.de).
Received 22 February 1999; accepted in final form 9 June 1999.
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REFERENCES |
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---|
1.
Adler, G.,
T. Hupp,
and
H. F. Kern.
Course and spontaneous regression of acute pancreatitis in the rat.
Virchows Arch.
382:
31-47,
1979.
2.
Baldwin, A. S.
The NF-B and I
B proteins: new discoveries and insights.
Annu. Rev. Immunol.
14:
649-683,
1996[Medline].
3.
Bastani, B.,
L. Yang,
J. J. Baldassare,
D. A. Pollo,
and
J. D. Gardner.
Cellular distribution of isoforms of protein kinase C (PKC) in pancreatic acini.
Biochim. Biophys. Acta
1269:
307-315,
1995[Medline].
4.
Brown, K.,
S. Gerstberger,
L. Carlson,
G. Franzoso,
and
U. Siebenlist.
Control of IB-
proteolysis by site-specific, signal induced phosphorylation.
Science
267:
1485-1488,
1995[Medline].
5.
Dabrowski, A.,
T. Grady,
C. D. Logsdon,
and
J. A. Williams.
Jun kinases are rapidly activated by cholecystokinin in rat pancreas both in vitro and in vivo.
J. Biol. Chem.
271:
5686-5690,
1996
6.
DiDonato, J. A.,
M. Hayakawa,
D. M. Rothwarf,
E. Zandi,
and
M. Karin.
A cytokine-responsive IB kinase that activates the transcription factor NF-
B.
Nature
388:
548-554,
1997[Medline].
7.
Dignam, J. D.,
R. M. Lebowitz,
and
R. G. Roeder.
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nucleii.
Nucleic Acids Res.
11:
1475-1489,
1983[Abstract].
8.
Dolmetsch, R. E.,
R. S. Lewis,
C. C. Goodnow,
and
J. I. Healy.
Differential activation of transcription factors induced by Ca2+ response amplitude and duration.
Nature
386:
855-858,
1997[Medline].
9.
Dolmetsch, R. E.,
K. Xu,
and
R. S. Lewis.
Calcium oscillations increase the efficiency and specificity of gene expression.
Nature
392:
933-936,
1998[Medline].
10.
Duan, R. D.,
and
J. A. Williams.
Cholecystokinin rapidly activates mitogen-activated protein kinase in rat pancreatic acini.
Am. J. Physiol.
267 (Gastrointest. Liver Physiol. 30):
G401-G408,
1994
11.
Duan, R. D.,
C. F. Zheng,
K. L. Guan,
and
J. A. Williams.
Activation of MAP kinase kinase (MEK) and Ras by cholecystokinin in rat pancreatic acini.
Am. J. Physiol.
268 (Gastrointest. Liver Physiol. 31):
G1060-G1065,
1995
12.
Dunn, J. A.,
L. Chuanfu,
H. A. Tuanzhu,
L. K. Race,
and
W. Browder.
Therapeutic modification of nuclear factor B binding activity and tumor necrosis factor-
gene expression during acute biliary pancreatitis.
Am. Surg.
63:
1036-1044,
1997[Medline].
13.
Frantz, B.,
E. C. Nordby,
G. Bren,
N. Steffan,
C. V. Paya,
R. Kincaid,
M. J. Tocci,
S. J. O'Keefe,
and
E. A. O'Neil.
Calcineurin acts in synergy with PMA to inactivate IB/MAD3, an inhibitor of NF-
B.
EMBO J.
13:
861-870,
1994[Abstract].
14.
Gardner, J. D.,
and
T. Jensen.
The Pancreas: Biology, Pathobiology, and Disease. (2nd ed.), edited by V.L.W. Go,
E.P. Dimagno,
J.D. Gardner,
E. Lebenthal,
H.A. Reber,
and G.A. Scheele. New York: Raven, 1993, p. 51-166.
15.
Genot, E. M.,
P. J. Parker,
and
D. A. Cantrell.
Analysis of the role of protein kinase C-, -
, and -
in T cell activation.
J. Biol. Chem.
270:
9833-9839,
1995
16.
Ghosh, S.,
and
D. Baltimore.
Activation in vitro of NF-B by phosphorylation of its inhibitor I
B.
Nature
344:
678-682,
1990[Medline].
17.
Grady, T.,
P. Liang,
S. A. Ernst,
and
C. D. Logsdon.
Chemokine gene expression in rat pancreatic acinar cells is an early event associated with acute pancreatitis.
Gastroenterology
113:
1966-1975,
1997[Medline].
18.
Gukovsky, I.,
A. S. Gukovskaya,
T. A. Blinman,
V. Zaninovic,
and
S. J. Pandol.
Early NF-B activation is associated with hormone-induced pancreatitis.
Am. J. Physiol.
275 (Gastrointest. Liver Physiol. 38):
G1402-G1414,
1998
19.
Hirano, M.,
S. Hirai,
K. Mizuno,
S. Osada,
M. Hosaka,
and
S. Ohno.
A protein kinase C isozyme, nPKC , is involved in the activation of NF-
B by 12-O-tetradecanoylphorbol-13-acetate (TPA) in rat 3Y1 fibroblasts.
Biochem. Biophys. Res. Commun.
206:
429-436,
1995[Medline].
20.
Kaltschmidt, C.,
B. Kaltschmidt,
T. Henkel,
H. Stockinger,
and
P. A. Baeuerle.
Selective recognition of the activated form of transcription factor NF-B by a monoclonal antibody.
Biol. Chem.
376:
9-16,
1995.
21.
Lampel, M.,
and
H. Kern.
Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue.
Virchows Arch.
373:
97-117,
1977.
22.
Lee, F. S.,
J. Hagler,
Z. J. Chen,
and
T. Maniatis.
Activation of the IB
kinase complex by MEKK1, a kinase of the JNK pathway.
Cell
88:
213-222,
1997[Medline].
23.
Malinin, N. L.,
M. P. Boldin,
A. V. Kovalenko,
and
D. Wallach.
MAP3K-related kinase involved in NF-B induction of TNF, CD95 and IL-1.
Nature
385:
540-544,
1997[Medline].
24.
Matozaki, T.,
B. Göke,
Y. Tsunoda,
M. Rodriguez,
J. Martinez,
and
J. A. Williams.
Two functionally distinct cholecystokinin receptors show different modes of action on Ca2+ mobilization and phospholipid hydrolisis in isolated rat pancreatic acini. Studies using a new cholecystokinin analog, JMV-180.
J. Biol. Chem.
265:
6247-6247,
1990
25.
Osipchuk, Y. V.,
M. Wakui,
D. I. Yule,
D. V. Gallacher,
and
O. H. Peterson.
Cytoplasmic Ca2+ osciallations evoked by receptor stimulation, G-protein activation, internal application of inositol trisphosphate or Ca2+: simultaneous microfluorimetry and Ca2+ dependent Cl current recording in single pancreatic acinar cells.
EMBO J.
9:
697-704,
1990[Abstract].
26.
Pahl, H. L.,
and
P. A. Baeuerle.
A novel signal transduction pathway from the endoplasmatic reticulum to the nucleus is mediated by transcription factor NF-B.
EMBO J.
14:
2580-2588,
1995[Abstract].
27.
Palmer, F. B.,
D. M. Byers,
M. W. Spence,
and
H. W. Cook.
Calcium-independent effects of TMB-8. Modification of phospholipid metabolism in neuroblastoma cells by inhibition of choline uptake.
Biochem. J.
286:
505-512,
1992[Medline].
28.
Petersen, O. H.
Stimulus-secretion coupling: cytoplasmic calcium signals and the control of ion channels in exocrine acinar cells.
J. Physiol. (Lond.)
448:
1-51,
1992[Medline].
29.
Pollo, D. A.,
J. J. Baldassare,
T. Honda,
P. A. Henderson,
V. D. Talkad,
and
J. D. Gardner.
Effects of cholecystokinin (CCK) and other secretagogues on isoforms of protein kinase C (PKC).
Biochim. Biophys. Acta
1224:
127-138,
1994[Medline].
30.
Raffaniello, R. D.,
J. Nam,
I. Cho,
J. Lin,
L. Y. Bao,
J. Michl,
and
J.-P. Raufman.
Protein kinase C isoform expression and function in transformed and non-transformed pancreatic acinar cells.
Biochem. Biophys. Res. Commun.
8:
166-171,
1998.
31.
Regnier, C. H.,
H. Yeong Song,
X. Gao,
D. V. Goeddel,
Z. Cao,
and
M. Rothe.
Identification and characterization of an IB kinase.
Cell
90:
373-383,
1997[Medline].
32.
Shatrov, V. A.,
V. Lehmann,
and
S. Chouaib.
Sphingosine-1-phosphate mobilizes intracellular calcium and activates transcription factor NF-B in U937 cells.
Biochem. Biophys. Res. Commun.
234:
121-124,
1997[Medline].
33.
Scheele, G. A.,
and
G. E. Palade.
Studies on guinea pig pancreas.
J. Biol. Chem.
262:
333-339,
1975
34.
Schmid, R. M.,
S. Liptay,
J. C. Betts,
and
G. J. Nabel.
Structural and functional analysis of NF-B: determinants of DNA binding specificity and protein interaction.
J. Biol. Chem.
269:
32162-32167,
1994
35.
Shirakawa, F.,
and
S. B. Mizel.
In vitro activation and nuclear translocation of NF-B catalyzed by cyclic AMP-dependent protein kinase and protein kinase C.
Mol. Cell. Biol.
9:
2424-2430,
1989[Medline].
36.
Siebenlist, U.,
G. Franzoso,
and
K. Brown.
Structure, regulation and function of NF-B.
Annu. Rev. Cell Biol.
10:
405-455,
1994.
37.
Steffan, N. M.,
G. D. Bren,
B. Frantz,
M. J. Tocci,
E. A. O'Neil,
and
C. V. Paya.
Regulation of IB
phosphorylation by PKC- and Ca2+-dependent signal transduction pathways.
J. Immunol.
155:
4685-4691,
1995[Abstract].
38.
Steinle, A. U.,
H. Weidenbach,
M. Wagner,
G. Adler,
and
R. M. Schmid.
NF-B/Rel activation in cerulein pancreatitis.
Gastroenterology
116:
420-430,
1999[Medline].
39.
Sun, L.,
and
G. Carpenter.
Epidermal growth factor activation of NF-B is mediated through I
B
degradation and intracellular free calcium.
Oncogene
16:
2095-2102,
1998[Medline].
40.
Toullec, D.,
P. Pianetti,
H. Coste,
P. Bellevergue,
T. Grand-Perret,
M. Ajakane,
V. Baudet,
P. Boissin,
E. Boursier,
F. Loriolle,
L. Duhamel,
D. Charon,
and
J. Kirilovsky.
The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C.
J. Biol. Chem.
266:
15771-15781,
1991
41.
Trimble, E. R.,
R. Bruzzone,
C. J. Meehan,
and
T. J. Biden.
Rapid increases in inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and cytosolic free Ca2+ in agonist-stimulated pancreatic acini of the rat.
Biochem. J.
242:
289-292,
1987[Medline].
42.
Wahl, C.,
S. Liptay,
G. Adler,
and
R. M. Schmid.
Sulfasalazine: a potent and specific inhibitor of nuclear factor B.
J. Clin. Invest.
101:
1163-1174,
1998
43.
Weber, C. K.,
T. Gress,
F. Müller-Pillasch,
M. M. Lerch,
H. Weidenbach,
and
G. Adler.
Supramaximal secretagogue stimulation enhances heat shock protein expression in the rat pancreas.
Pancreas
10:
360-367,
1995[Medline].
44.
Williams, J. A.,
and
D. I. Yule.
The Pancreas: Biology, Pathobiology, and Disease (2nd ed.), edited by V. L. W. Go,
E. P. Dimango,
J. D. Gardner,
E. Lebenthal,
H. A. Reber,
and G. A. Scheele. New York: Raven, 1993, p. 167-189.