Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622
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ABSTRACT |
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Supraphysiological
concentrations of cholecystokinin (CCK) induce chemokine expression in
rat pancreatic acini through the activation of the transcription factor
NF-B. In the current study, the intracellular signals involved in
these pathophysiological effects of CCK were investigated. CCK
induction of mob-1 expression in
isolated rat pancreatic acini was blocked by the protein kinase C (PKC)
inhibitors GF-109203X and Ro-32-0432 and by the intracellular Ca2+ chelator BAPTA. CCK induced
NF-
B nuclear translocation, and DNA binding was also blocked by
GF-109203X and BAPTA. Direct activation of PKC with TPA induced
mob-1 chemokine expression and
activated NF-
B DNA binding to a similar extent as did CCK.
Increasing intracellular Ca2+
using ionomycin had no effect on mob-1
mRNA levels or NF-
B activity. Both CCK and TPA treatments decreased
inhibitory
B-
(I
B-
) levels, whereas ionomycin had no
effect. However, the effects of TPA on I
B-
degradation were less
complete than for CCK. In combination, TPA and ionomycin degraded
I
B-
to a similar extent as CCK. Therefore, activation of NF-
B
and mob-1 expression by supraphysiological CCK is likely mediated by both PKC activation and
elevated intracellular Ca2+.
pancreas; acinar cells; pancreatitis; chemokine; cholecystokinin; nuclear factor-B
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INTRODUCTION |
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CHOLECYSTOKININ (CCK) is both a hormone and a neurotransmitter involved in various aspects of gastrointestinal function, including stimulation of gallbladder contraction, inhibition of gastric emptying, and induction of satiety (26). CCK also influences pancreatic function. Actions of CCK on the rodent pancreas are mediated by CCK-A receptors, which are G protein-coupled receptors. Physiological activation of CCK receptors on pancreatic acinar cells leads to secretion of digestive enzymes and has a general trophic action on the organ. However, supramaximal stimulation of these receptors leads to an acute inflammatory response resembling aspects of the clinically important disease acute pancreatitis (41). This latter observation has led to the widespread use of supramaximal concentrations of the CCK analogue caerulein as an experimental model of acute pancreatitis.
Of particular importance for the development of experimental
pancreatitis may be the induction of chemokines and cytokines in
pancreatic cells. We have previously shown that the induction of
chemokines is due to the ability of supraphysiological concentrations of CCK to activate the transcription factor NF-B (19). NF-
B/Rel transcription factors bind to enhancer elements stimulating the expression of a variety of genes involved in immune and inflammatory responses. NF-
B was originally identified as a nuclear factor that
bound to the enhancer element of the immunoglobulin kappa light chain
gene (39). Functional NF-
B is composed of hetero- or homodimeric
combinations of NF-
B/Rel proteins. The prototypical NF-
B complex
is a heterodimer containing p50 and p65 subunits. Other members of the
NF-
B/Rel family of proteins are c-Rel, NF-
B1 (p50/p105), NF-
B2
(p52/p100), Rel A (p65), Rel B, and the
Drosophila proteins Dorsal, Dif, and
Relish (1). These proteins interact through
NH2-terminal Rel homology domains.
The Rel homology domains also function in DNA binding and in
interaction with inhibitor proteins known as I
Bs.
The mechanism of activation of NF-B has been an intensive area of
study due to its importance in many inflammatory diseases. In most
cells, NF-
B is sequestered in the cytoplasm through interactions with the I
B family of inhibitory proteins. Activation of NF-
B by
a wide variety of stimuli such as mitogens, cytokines, bacterial lipopolysaccharide, viral infection, and ultraviolet light is thought
to involve the dissociation of NF-
B from I
Bs (36). Stimulation of
cells with inducers of NF-
B leads to rapid phosphorylation, ubiquitination, and degradation of I
Bs. NF-
B is then released and
translocates into the nucleus where it activates the expression of
target genes. Therefore, early studies implicated I
B phosphorylation as a crucial step for NF-
B activation. Recently, it was shown that
two highly related serine kinases, IKK-
and IKK-
, are activated in response to the NF-
B inducer tumor necrosis factor-
(TNF-
) and are responsible for the phosphorylation of I
Bs (49). These kinases appear to be downstream of MEKK1, a component of the
mitogen-activated protein kinase signaling cascade. However, other
kinases, including p90rsk (13),
have also been reported to phosphorylate I
B. Furthermore, there
appear to be important cell type-specific differences in the mechanisms
involved in NF-
B signaling (5, 6, 21).
In pancreatic acinar cells, the mechanisms involved in the activation
of NF-B by CCK remain unknown. In the current study, we investigated
the major signaling pathways activated by CCK for their effects on
NF-
B and mob-1 chemokine
expression. We observed that the ability of CCK to stimulate
mob-1 gene expression or to activate
NF-
B was completely blocked by inhibition of protein kinase C
(PKC) or chelation of intracellular
Ca2+. Activation of PKC using the
phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA) also activated NF-
B and induced
mob-1 expression. However, there were
differences between the effects of TPA and CCK in terms of
effects on I
B-
. CCK caused a rapid and nearly complete
decrease in I
B-
levels. In contrast, TPA only caused a small
decrease in I
B-
levels. This difference was at least in part
explained by an observed requirement for increased
Ca2+ in the degradation of
I
B-
. Thus the effects of CCK on NF-
B and chemokine
expression are likely due to the combined actions of PKC and
Ca2+.
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MATERIALS AND METHODS |
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Materials. Chromatographically
purified collagenase was purchased from Worthington Biochemical
(Freehold, NJ). Soybean trypsin inhibitor (SBTI), -mercaptoethanol,
phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, TPA, HEPES,
and glutamine were obtained from Sigma Chemicals (St. Louis, MO). CCK
was purchased from Research Plus (Bayonne, NJ). BAPTA-AM,
Ro-32-0432, and ionomycin were purchased from Calbiochem (La
Jolla, CA). Bisindolylmaleimide I (GF-109203X) was purchased from LC
Laboratories (Woburn, MA). Enhanced chemiluminescence detection
reagents (ECL),
[
-32P]ATP, and
[
-32P]dCTP were
from Amersham (Arlington Heights, IL). An electrophoretic mobility
shift assay systems kit was purchased from Promega (Madison, WI). The
rabbit polyclonal antibodies to I
B-
and the NF-
B subunits p65,
p50, and c-Rel and the goat anti-rabbit IgG horseradish peroxidase conjugate were from Santa Cruz Biotechnology (Santa Cruz, CA). The
phosphospecific I
B-
antibody was from New England Biolabs (Beverly, MA). Eagle's minimal essential amino acids, guanidine thiocyanate, and agarose were from GIBCO-BRL Life Technologies (Gaithersburg, MD).
Pancreatic acini isolation and treatments. The preparation of pancreatic acini was performed as previously described (19). Briefly, pancreata from male Wistar rats were injected with collagenase (100 U/ml) and incubated at 37°C for 45-50 min with shaking (120 cycles/min). Acini were then dispersed by triturating the pancreas through polypropylene pipettes with decreasing orifice (3.0, 2.4, and 1.2 mm) and filtration through a 150-µm nylon mesh. Acini were purified by centrifugation through a solution containing 4% BSA and were resuspended in HEPES-buffered Ringer solution (HR, pH 7.5) supplemented with 0.2% glucose, Eagle's minimal essential amino acids, 2 mM glutamine, 0.1 mg/ml SBTI, and 0.5% BSA. The dispersed acini were separated into aliquots and treated with CCK and pharmacological agents at indicated concentrations in HR for specified times in tissue culture dishes. All treatments and incubations were conducted in a cell culture incubator at 37°C in a humidified atmosphere.
Isolation of RNA and analysis of mob-1 mRNA
expression. Total RNA was isolated by a modified acid
guanidinium-thiocyanate-phenol-chloroform extraction as previously
described (19). RNA was quantitated spectrophotometrically: 25 µg of each sample were electrophoresed in 1% agarose and
2.2 M formaldehyde gels in MOPS buffer and were transferred
to a nylon membrane. mob-1 mRNA was
detected using a full-length 1.2-kb cDNA of rat
mob-1 (25). Membranes were hybridized
at high stringency using QuickHyb solution (Stratagene, La Jolla, CA)
with the
[-32P]dCTP-labeled
mob-1 probe at 68°C for 2 h. After
hybridization, the membranes were exposed to a B-1 phosphoimaging
screen and were visualized by the use of a GS-505 Molecular Imaging
System (Bio-Rad Laboratories, Richmond, CA). Images were imported into Photoshop 4.0 (Adobe Systems Incorporated, San Jose, CA) for
preparation of Figs. 1-6.
Preparations of nuclear extracts. Nuclear extracts were prepared using a modified version of the method of Maire et al. (27) as previously described (19). Pancreatic acini were collected by brief centrifugation and were washed with ice-cold PBS containing 1 mM EDTA. The pellets were then resuspended in 0.5 ml homogenization buffer containing 10 mM HEPES (pH 7.9), 2 M sucrose, 10% glycerol (vol/vol), 25 mM KCl, 150 mM spermine, 500 mM spermidine, and 2 mM EDTA to which 1 mM dithiothreitol (DTT) and a protease inhibitor cocktail containing 10 µg/ml each of aprotinin, leupeptin, and pepstatin were added before use. Cells were homogenized with a motor-driven pestle for 5-10 strokes on ice. Nuclei were collected by centrifugation at 62,000 g for 30 min at 4°C, washed with 1 ml PBS containing 1 mM EDTA, and then centrifuged at 14,000 g for 5 min at 4°C. The nuclei were resuspended in an appropriate volume (~100 µl) of ice-cold high-salt buffer containing 10 mM HEPES (pH 7.9), 10% glycerol (vol/vol), 0.42 M NaCl, 100 mM KCl, 3 mM MgCl2, and 0.1 mM EDTA, to which DTT and the protease inhibitor cocktail described above were added. The nuclear suspension was incubated on ice for 15-30 min with intermittent mixing and was centrifuged at 14,000 g for 5 min at 4°C. Protein concentration in the nuclear extract was determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA).
Immunoblot analysis. Dispersed acini
were treated as described in the legends for Figs. 1-6. The
treatments were terminated by washing the acini with ice-cold PBS
containing 1 mM
Na3VO4. For analysis of whole cell protein levels, the pellets were lysed by
sonicating for 5 s in a solution containing 50 mM
Tris · HCl (pH 7.5), 150 mM NaCl, 2 mM EGTA, 2 mM
EDTA, 1% Triton X-100, 0.5 mM PMSF, and the protease inhibitor
cocktail described above. The supernatant was removed as whole cell
lysate and was assayed for protein by the Bio-Rad protein assay. For
analysis of nuclear protein levels, nuclear extracts were prepared as
described above. Equal amounts of protein (25 µg) were resolved by
SDS-PAGE and were transferred to a nitrocellulose membrane. IB
immunoblot analysis was performed as described previously (18) and
visualized with ECL reagent on film or a screen. The NF-
B protein
was detected by specific antibodies against subunit p65 of
NF-
B. Film images were scanned with an Agfa Arcus II scanner (Bayer,
Ridgefield Park, NJ) to create a digital image.
Electrophoretic mobility shift assay.
Aliquots of nuclear extract with equal amounts of protein (6-12
µg) were used in 10-µl reactions in a buffer containing 10 mM HEPES
(pH 7.9), 10% glycerol (vol/vol), 1 mM DTT, 1 µg poly(dI-dC), and 5 µg nuclease-free BSA as previously described (19). The binding
reaction was started by addition of 10,000 counts/min of the
22-base pair oligonucleotide 5'-AGT TGA
C AGG C-3' containing the NF-
B consensus sequence (underlined; Promega) that had been labeled with
[
-32P]ATP (10 mCi/mmol) by T4 polynucleotide kinase. The reaction was allowed to
proceed for 30 min at room temperature. For cold competition
experiments, unlabeled NF-
B oligonucleotide as a specific competitor
or OCT1 (40) oligonucleotide (5'-TGT CGA ATG CAA ATC ACT AGA
A-3') as a nonspecific competitor (300×) was added to the
binding reaction 5 min before the addition of the radiolabeled probe.
For the antibody supershift assays, 2 µg of specific antibodies to
NF-
B protein subunits p65, p50, and c-Rel were incubated with
nuclear extracts for 1 h at room temperature before addition of labeled
probe. All reaction mixtures were subjected to PAGE on 4.5% gel in
0.5× TBE buffer (44.5 mM Tris base, 44.5 mM boric
acid, and 1 mM disodium EDTA, pH 8.3) at 200 V. Gels were dried and
directly exposed to a B-1 phosphoimaging screen.
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RESULTS |
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CCK induction of mob-1 gene expression in pancreatic acinar cells is
dependent on PKC activation and elevated intracellular
Ca2+.
Chemokine gene expression is an early event in the initiation of
inflammatory responses during experimental pancreatitis (15). In the
current study, we initially examined the effects of inhibiting the
major intracellular signaling pathways activated by CCK in pancreatic
acinar cells on the induction of a representative chemokine, mob-1. A major action of CCK in acinar cells is the
activation of phospholipase C, leading to the generation of
diacylglycerol (DAG) and inositol trisphosphate
(IP3). DAG activates PKC, and IP3 causes the release of
Ca2+ from intracellular stores. To
study the role of PKC, we used the specific inhibitors
bisindolylmaleimide I (GF-109203X; see Ref. 45) and Ro-32-0432
(47) and the PKC activator TPA. Pretreatment of acini with GF-109203X
(20 µM) or Ro-32-0432 (10 µM) for 30 min completely blocked
mob-1 gene expression induced by CCK
(Fig. 1A).
These results suggest a requirement for PKC activation in the actions
of CCK on the expression of this gene. Direct activation of PKC with
TPA (1 µM) also induced mob-1
chemokine expression (Figs. 1 and 2); this
effect was concentration dependent, with significant effects noted at
10 nM and maximal effects at 100 nM (Fig. 2). The effects of TPA were
blocked by GF-109203X (Fig. 1A). Taken together,
these data suggest that PKC activation is required and is sufficient
for the induction of mob-1 gene
expression in pancreatic acinar cells by CCK.
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TPA stimulates NF-B nuclear translocation and
specific consensus site DNA binding in a
Ca2+-dependent
manner.
Previously, we have shown that CCK induction of
mob-1 gene expression requires
activation of NF-
B (19). PKC is known to activate NF-
B in a
variety of cell types. To confirm that activation of PKC leads to
NF-
B activation in pancreatic acinar cells, we used TPA to directly
activate PKC. To examine whether PKC activity is important for the
effects of CCK, we used GF-109203X to inhibit PKC. NF-
B protein
nuclear translocation was analyzed by Western blotting, and a
B-specific consensus site DNA binding was assessed using
electrophoretic mobility shift assay. Compared with untreated controls
(Fig. 3, lanes
1 and 7), CCK (100 nM) induced a significant increase of p65 nuclear translocation and
B consensus DNA binding activity (Fig. 3, lanes
2 and 8). TPA (1 µM) also induced p65 nuclear translocation and
B binding (Fig. 3,
lanes 4 and
10). These effects of CCK and TPA
were blocked by pretreatment with the PKC inhibitor GF-109203X (Fig. 3,
lanes 3 and
5). GF-109203X itself had no effect
on p65 nuclear translocation or
B binding (Fig. 3,
lane 6). These data support the
hypothesis that PKC activity is required and sufficient for activation
of NF-
B by CCK in pancreatic acinar cells.
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Intracellular
Ca2+ influences
IB-
phosphorylation and degradation by
CCK and TPA.
Previously, it was found that supraphysiological stimulation of
pancreatic acini with CCK caused a rapid degradation of I
B-
. Therefore, we examined I
B-
levels to determine whether the
inhibitors that blocked CCK-mediated induction of
mob-1 gene expression and NF-
B
activation affected I
B-
degradation (Fig.
5). CCK caused a rapid and complete
degradation of I
B-
(92 ± 3% degraded,
n = 5). However, TPA caused only a
relatively small decrease in the cellular level of I
B-
(30 ± 8% degraded, n = 5). Inhibition of
PKC with GF-109203X reduced, but did not completely block, the ability
of CCK to stimulate I
B-
degradation. In the presence of
GF-109203X, CCK treatment caused the formation of additional bands in
the Western blot that may represent partial degradation products. These
data suggested that PKC activity was not sufficient, but is important,
for the effects of CCK on I
B-
degradation. In contrast, the
degradation of I
B-
was blocked by the
Ca2+ chelator BAPTA (Figs. 5 and
6). The relatively minor effect of TPA on
I
B-
was also blocked by BAPTA-AM pretreatment (Figs. 5 and 6).
Increasing intracellular Ca2+ with
ionomycin did not itself cause a decrease in I
B-
levels (Fig. 5).
However, in combination with TPA, ionomycin caused a decrease in
I
B-
comparable to that observed with maximal concentrations of
CCK. These data supported the hypothesis that increased intracellular Ca2+ is required, but not
sufficient, for I
B-
degradation.
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DISCUSSION |
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NF-B is activated by a large number of factors and treatments
ranging from proinflammtory cytokines, such as TNF-
, to cell stresses, such as ultraviolet irradiation (40). The cellular mechanisms
mediating the effects of TNF-
have been particularly well
elucidated, and details concerning the signaling components continue to
emerge. However, the pathways whereby G protein-coupled receptors such
as CCK activate NF-
B have been much less studied. CCK is a
gastrointestinal hormone with a variety of physiological actions in the
gut. CCK signaling has been widely studied in the context of its
physiological roles in secretion and pancreatic protein synthesis.
Supraphysiological concentrations of CCK induce pancreatic and systemic
manifestations of pancreatitis, such that CCK hyperstimulation is the
most widely used model of experimental pancreatitis. Supraphysiological
concentrations of CCK activate NF-
B, which has been suggested to be
important for the development of acute pancreatitis (15, 17, 19, 43).
Therefore, activation of NF-
B constitutes an additional action of
CCK that is likely relevant for our understanding of pancreatic
inflammatory responses.
Supraphysiological concentrations of CCK trigger rapid degradation of
IB-
, the nuclear translocation of NF-
B, the binding to
DNA
B consensus sites of p65/p50 complexes, and expression of
cytokines and chemokines, including
mob-1 (19). High- and low-affinity CCK
receptors are known to generate different patterns of second messengers
and cellular response (29). High-affinity CCK receptors are responsible
for low levels of phosphatidylinositol hydrolysis and an oscillatory
pattern of intracellular Ca2+
release (28) and are responsible for activation of extracellular signal-regulated kinases (10),
p90rsk-1 (7), and p38/Hog
(35). Thus these signals induced by high-affinity CCK
receptors, which are primarily involved in physiological actions of
CCK, are neither sufficient nor necessary for activating NF-
B.
Low-affinity CCK receptors cause a prolonged increase in DAG (28),
strongly activate PKC, cause a peak and plateau pattern of
Ca2+ release, and activate Jun
kinases (8). Of these signaling mechanisms, activation of PKC was a
strong candidate for coupling to NF-B activation and chemokine
expression based on known effects in other cell models. CCK activates
PKC by increasing cellular levels of the PKC activator DAG. CCK
receptors activate a biphasic increase in cellular DAG levels (30).
This is thought to be due to a rapid but transient increase in
hydrolysis of phosphatidylinositol, followed by a more prolonged
increase due to hydrolysis of phosphatidylcholine. The strong
activation of PKC observed with high concentrations of CCK is likely
responsible for the ability of CCK to activate NF-
B in pancreatic
acinar cells. Interestingly, the lower level of PKC activation observed
with physiological concentrations of CCK is obviously not sufficient
for activation of NF-
B.
Relative differences in their abilities to activate PKC may explain
differences observed in the abilities of CCK and another pancreatic
secretagogue, bombesin, to initiate an acute inflammatory response in
the pancreas. Bombesin receptor activation on pancreatic acinar cells
has been reported to generate either little (30) or no (33) DAG.
Therefore, bombesin likely does not activate PKC to the same extent as
CCK. In the current study, PKC activity was required for NF-B
activation by CCK. Bombesin treatment also does not provoke
pancreatitis (34) nor does it activate NF-
B in pancreatic acinar
cells (15, 19). Thus it is likely that differences in the abilities of
CCK and bombesin receptors to cause a strong activation of PKC explains
the observation that CCK, but not bombesin, is able to initiate an
acute inflammatory response in the pancreas.
It has long been recognized that TPA acts as an activator of NF-B.
However, the mechanisms involved in this process remain to be resolved.
TPA activates the conventional isoforms of PKC, which are activated by
Ca2+ and DAG, and the novel PKCs,
which are activated by DAG but do not respond to
Ca2+. Activation of atypical
isoforms of PKC, which do not require DAG or
Ca2+, has been shown to be
required for NF-
B activation by TNF-
(11). However, neither
conventional, novel, nor atypical forms of PKC directly phosphorylate
I
B (24). Recently, it was shown that both the atypical isoform
PKC
and the conventional PKC
activate IKK
(24).
The IKKs phosphorylate I
B-
on serines 32 and 36; this
phosphorylation is thought to trigger the subsequent ubiquitination and
proteasomal degradation of I
Bs. However, in many cell lines, PKC is
an excellent activator of NF-
B DNA binding and gene transcription
but is not efficient at causing I
B degradation (12, 42). In the
current study, inhibition of PKC blocked the ability of CCK to activate
NF-
B nuclear localization, DNA binding, and
mob-1 gene expression. However,
inhibition of PKC only partially blocked the ability of CCK to cause
the degradation of I
B-
. The presence of multiple protein bands
after CCK treatment in the presence of the PKC inhibitor suggests that
unidentified PKC-independent kinases may be involved in the effects of
CCK on I
B-
. Moreover, TPA activated NF-
B but caused only a
minor amount of I
B-
degradation in the pancreatic acinar cells.
Taken together, the data support a model in which PKC is capable of activating NF-
B in the absence of profound I
B degradation.
Recently, it was shown that TPA activation of PKC in enterocytes also
induces NF-
B without leading to profound I
B degradation (48).
Thus there appear to be mechanisms other than I
B-
degradation
involved in the effects of PKC on NF-
B activation in some cell types.
Recent studies have suggested that IB degradation and NF-
B
nuclear translocation are insufficient for transcription from NF-
B-regulated promoters and have proposed the existence of
important parallel signaling mechanisms (3). These parallel signaling mechanisms may involve phosphorylation of NF-
B p65 subunits (4, 32,
46). TPA activation of PKC has previously been shown in vitro to lead
to phosphorylation of a defined region within a transactivation domain
of the p65 subunit of NF-
B (37). This phosphorylation was found to
stimulate transcriptional activity. However, no evidence is available
as to whether or not this phosphorylation event interrupts the
interaction between I
B-
and NF-
B and thus might account for a
dissociation of NF-
B in the absence of I
B-
degradation. A
mechanism allowing the dissociation of NF-
B from I
B-
without
I
B-
degradation would explain the increased p65 nuclear
accumulation stimulated by TPA in pancreatic acini observed in the
current study or in the case of enterocytes (48). One such mechanism
might include the tyrosine phosphorylation of I
B-
. NF-
B
activation has previously been reported to occur by the tyrosine
phosphorylation of I
B-
and in the absence of I
B-
degradation (2, 20, 23). Further details of the NF-
B activation machinery will be required to determine the mechanisms involved in PKC
activation of NF-
B in pancreatic acinar cells.
Ca2+-dependent pathways also play
critical roles in NF-B activation in pancreatic acinar cells, as
chelation of intracellular Ca2+
completely blocked CCK stimulation of I
B phosphorylation and degradation, as well as NF-
B nuclear translocation,
B consensus site DNA binding, and mob-1 gene
expression. BAPTA treatment has previously been shown to block the
induction of NF-
B activation by epidermal growth factor (44) and
lysophosphatic acid (38) but not by
H2O2
(22) in fibroblast cell models. In the current study, BAPTA treatment
also inhibited the effects of TPA on
mob-1 mRNA levels and on I
B-
phosphorylation and degradation. It is possible that BAPTA maintained
intracellular free Ca2+ at levels
below those required for maximal activity of conventional PKCs. Thus
one Ca2+ requirement could be PKC
activity itself. However, TPA did not lead to profound degradation of
I
B-
, as was observed with CCK, unless
Ca2+ was artificially elevated.
Thus the response to CCK treatment must involve both PKC activity and
increased intracellular Ca2+.
Similar to what was observed in fibroblasts, in the current study,
increasing intracellular Ca2+ with
an ionophore did not activate NF-
B in pancreatic acinar cells. Thus
increased cytoplasmic Ca2+ levels
are required for some NF-
B inducers, including high
concentrations of CCK, but are not sufficient for NF-
B activation.
The mechanisms involved in the
Ca2+ requirement for NF-B
activation are not completely clear. In the current study, CCK, which causes increases in both Ca2+ and
PKC activity, activated NF-
B and caused a near complete loss of
I
B-
. In contrast, TPA, which increases PKC activity but does not
increase intracellular Ca2+,
activated NF-
B but had only a minor effect on I
B-
degradation. The combination of TPA and a Ca2+
ionophore caused a synergistic reduction in I
B-
. Thus I
B-
degradation appears to require increased intracellular
Ca2+. It has previously been
observed that increased intracellular Ca2+ works in synergy with PKC to
cause I
B degradation in fibroblasts (12, 42). This effect was
suggested to be mediated by calcineurin, a
Ca2+/calmodulin-activated
phosphatase, based upon the observation that calcineurin inhibitors
such as cyclosporin A and FK-506 block the synergistic effect of
Ca2+ ionophores with TPA. However,
more recently, cyclosporin A has been shown to act as an uncompetitive
inhibitor of proteasome activity (31). Proteasomal activity is required
for I
B degradation. Therefore, it is unclear whether the effects of
the calcineurin inhibitors are specific or reflect nonspecific effects
on proteasomes. We found that cyclosporin A inhibited CCK-mediated
I
B-
degradation only at high concentrations [25 µM and
above (data not shown)], whereas 1 µM is sufficient
to inhibit acinar cell calcineurin (16).
In summary, we found that in pancreatic acinar cells the ability of
supraphysiological concentrations of CCK to stimulate NF-B
activation and mob-1 chemokine
expression requires activation of PKC and increased intracellular
Ca2+. PKC activation by TPA
strongly induced NF-
B and caused
mob-1 expression but had only a weak
effect on I
B-
degradation. Increased cytoplasmic
Ca2+ was required for activation
of NF-
B and mob-1 expression by either CCK or TPA but was itself unable to activate these pathways. Increased cytoplasmic Ca2+ acted
in synergy with TPA to degrade I
B-
. The specific cellular targets
of PKC and Ca2+ involved in these
effects remain to be determined. Further understanding of these
signaling mechanisms may be useful in determining intracellular events
important in the development of acute pancreatitis and in the design of
future therapies for this disease.
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ACKNOWLEDGEMENTS |
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We thank Drs. J. A. Williams, B. Nicke, and D. Simeone for critical reviewing of this manuscript.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52067 and the University of Michigan Gastrointestinal Peptide Center Grant DK-34933.
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: C. Logsdon, Dept. of Physiology, Box 0622, Univ. of Michigan, 7710 Medical Sciences Bldg. II, Ann Arbor, MI 48109-0622 (E-mail: clogsdon{at}umich.edu).
Received 24 June 1999; accepted in final form 27 September 1999.
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