Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622
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ABSTRACT |
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In the cholecystokinin (CCK)
hyperstimulation model of acute pancreatitis, two early intracellular
events, activation of trypsinogen and activation of nuclear factor-B
(NF-
B), are thought to be important in the development of the
disease. In this study, the relationship between these two events was
investigated. NF-
B activity was monitored by using a DNA binding
assay and mob-1 chemokine gene expression. Intracellular
trypsin activity was measured by using a fluorogenic substrate.
Protease inhibitors including FUT-175, Pefabloc, and E-64d prevented
CCK stimulation of intracellular trypsinogen and NF-
B activation.
Likewise, the NF-
B inhibitors pyrrolidine dithiocarbamate and
N-acetyl-L-cysteine inhibited CCK stimulation of
NF-
B and intracellular trypsinogen activation. These results
suggested a possible codependency of these two events. However, CCK
stimulated NF-
B activation in Chinese hamster ovary-CCKA
cells, which do not express trypsinogen, indicating that trypsin is not
necessary for CCK activation of NF-
B. Furthermore,
adenovirus-mediated expression in acinar cells of active p65 subunits
to stimulate NF-
B, or of inhibitory
B-
molecules to inhibit
NF-
B, did not affect either basal or CCK-mediated trypsinogen
activation. Thus trypsinogen and NF-
B activation are independent
events stimulated by CCK.
pancreatitis; inflammation; cholecystokinin; nuclear
factor-B
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INTRODUCTION |
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ACUTE PANCREATITIS
is a disease of increasing incidence with significant morbidity and
mortality and one for which few effective therapies exist. The lack of
effective therapies is due primarily to a lack of understanding of the
pathophysiology of this disease. Despite years of study, the cellular
mechanisms that initiate the development of this disease remain obscure
(29). Two mechanisms that are thought to be important are
the activation of trypsinogen to the active protease trypsin and the
activation of the transcription factor nuclear factor-B (NF-
B).
Support for the central role of trypsin in acute pancreatitis comes
from the observation that pancreatitis is ameliorated by cell-permeant
inhibitors that block trypsin activity (17, 36).
Furthermore, hereditary pancreatitis, a rare form of the disease, has
been linked to a mutation in the cationic trypsinogen gene
(35). Similar to the inappropriate activation of
trypsinogen, NF-
B activation has been observed in several models of
acute experimental pancreatitis (3, 5, 7, 30). NF-
B
activation was found to be required for the production of chemokines by
pancreatic acinar cells (8). Chemokines and cytokines are
thought to initiate the inflammatory cascade observed in acute
pancreatitis (14, 19, 20). Supporting the importance of
the role of cytokines and chemokines in acute pancreatitis are
experiments indicating that a reduction in the levels of these
inflammatory mediators reduces the severity of the disease
(21). Thus both trypsinogen and NF-
B activation occur
and are thought to be important during acute pancreatitis. However, the
relationships between these two events are unknown.
To investigate early events in the initiation of acute pancreatitis,
researchers have turned to animal models. The most commonly utilized
animal model for acute pancreatitis consists of treating rats with
high, nonphysiological concentrations of the secretagogue cholecystokinin (CCK) or its analog, caerulein (28). More
recently, to investigate acinar cell mechanisms in the absence of the
confounding influence of other cell types, researchers have
investigated the effects of CCK on trypsinogen activation (23,
24) and NF-B activation (9) in isolated acinar
cells in vitro. These two processes appear remarkably similar in terms
of time course and concentration dependence. Both processes occur
within minutes. Both processes show monophasic concentration dependence
and require supraphysiological concentrations of CCK. Furthermore, both
trypsinogen activation (23) and NF-
B activation
(9) require increased intracellular Ca2+.
These similarities led us to the hypothesis that the two processes may
be mechanistically linked.
The purpose of the present study was to determine whether CCK
activation of these two events is dependent or independent. We found
that treatment of rat pancreatic acinar cells with protease inhibitors
abolished trypsin activation, as expected, and unexpectedly also
significantly inhibited NF-B activation induced by CCK
hyperstimulation. We also found that inhibitors of NF-
B blocked
NF-
B activation, as expected, and unexpectedly also reduced
CCK-induced trypsin activation. Thus there is significant
cross-interference by the various inhibitors with these two processes.
Further investigation with the use of more specific approaches
unambiguously showed that these two events were independent. NF-
B
activation is independent of trypsinogen activation, because CCK
activated NF-
B in the absence of trypsin activation in Chinese
hamster ovary (CHO) cells expressing ectopic CCKA
receptors. Trypsinogen activation is independent of NF-
B activity,
because intracellular trypsin activity was not affected by inhibition
or stimulation of NF-
B with the use of adenovirus-mediated gene
transfer of either inhibitory or active NF-
B subunits to pancreatic
acinar cells. Therefore, trypsinogen activation and NF-
B activation
are independent events that occur early in the development of acute
experimental pancreatitis.
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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), Na3VO4, HEPES,
(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbuthane ethyl ester (E-64d), pyrrolidine dithiocarbamate (PDTC),
N-acetyl-L-cysteine (NAC),
4-(2-aminoethyl)benzenesulfonyl fluoride (Pefabloc), trypsin, and
glutamine were obtained from Sigma Chemical (St. Louis, MO). CCK was
purchased from Research Plus (Bayonne, NJ). Nafamostat mesilate
(FUT-175) was a kind gift from Dr. M. Kurumi (Torii Pharmaceutical, Chiba-shi, Japan). Boc-Gln-Ala-Arg-MCA was purchased from Peptides International (Louisville, KY). Enhanced chemiluminescence (ECL) detection reagents, [
-32P]ATP, and
[
-32P]dCTP were from Amersham (Arlington Heights, IL).
The electrophoretic mobility shift assay systems kit was purchased from
Promega (Madison, WI). The rabbit polyclonal antibodies to inhibitory
B-
(I
B-
) and the NF-
B subunits p65, p50, and c-Rel, as
well as the goat anti-rabbit IgG horseradish peroxidase conjugate, were
from Santa Cruz Biotechnology (Santa Cruz, CA). Eagle's minimum
essential amino acids, guanidine thiocyanate, and agarose were from
GIBCO BRL (Gaithersburg, MD).
Cells and treatments. CHO cells stably expressing CCKA receptors (CHO-CCKA) were described previously (37). Cells were routinely cultured in Dulbecco's modified Eagle's medium supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), amphotericin B (50 µg/ml), and 5% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2.
The preparation of pancreatic acini was performed as previously described. 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 orifices (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% bovine serum albumin (BSA) and were resuspended in HEPES-buffered Ringer solution (HR; pH 7.5) supplemented with 0.2% glucose, Eagle's minimum essential amino acids, 2 mM glutamine, SBTI (0.1 mg/ml), and 0.5% BSA. The dispersed acini were aliquoted 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.Adenovirus construction and infection.
The adenovirus Adp65, with separate cytomegalovirus (CMV) promoters
driving expression of the NF-B p65 subunit and green fluorescent
protein (GFP), was constructed using the AdEasy system according to the
method of He et al. (10). Briefly, the full-length cDNA
encoding p65 was excised from a pBluescript SK+ plasmid as
an XhoI/XbaI restriction fragment (from Dr. G. Nabel, University of Michigan). This was cloned into the
XhoI/XbaI site of the shuttle vector,
pAdTrack-CMV. The pAdTrack-p65 was then linearized with
PmeI and cotransfected along with the pAdEasy-1 adenoviral
backbone plasmid into BJ5183 Escherichia coli. Recombinants were selected for kanamycin resistance and confirmed by restriction endonuclease analysis. The linearized recombinant was transfected into
293 cells, where the recombinant adenovirus was generated and packaged.
The adenovirus AdI
B-
, with a CMV promoter driving expression of a
full-length I
B-
cDNA modified by the addition of a nuclear
translocation sequence, was a kind gift from Dr. J. Anrather (Beth
Israel Deaconess Medical Center, Boston, MA) (26). The
adenovirus AdLacZ, expressing bacterial
-galactosidase and GFP from separate CMV promoters, was a gift from Dr. T. C. He
(Johns Hopkins Oncology Center, Baltimore, MD) and was utilized as a
control. Acini prepared as described above were infected with these
adenoviruses as described previously (18). The adenovirus titer used for infection was 109 plaque-forming units/mg
acinar protein (multiplicity of infection was ~1,000). Fluorescence
microscopy confirmed that adenovirus-transferred genes were expressed
in nearly 100% of acinar cells 4-6 h after infection (data not
shown), which is similar to the efficiency previously reported
(8, 15). Therefore, acini were then incubated for 6 h
before the addition of CCK-8.
Trypsin activity assay. Intracellular trypsin activity in pancreatic acinar cells was measured fluorometrically by using Boc-Gln-Ala-Arg-MCA as the substrate according to the method of Kawabata et al. (13). After acini were treated with various agents, the cells were washed twice with HR and then homogenized in ice-cold MOPS buffer containing 250 mM sucrose, 5 mM MOPS, and 1 mM MgSO4 (pH 6.5) with a motorized glass-Teflon homogenizer. After centrifugation (14,000 g for 15 min), 100 µl of supernatant were added to a cuvette containing assay buffer (50 mM Tris, 150 mM NaCl, 1 mM CaCl2, and 0.1% BSA, pH 8.0). The reaction was initiated by adding substrate, and the fluorescence emitted at 440 nm after excitation at 380 nm was monitored. The trypsin activity in the samples was calculated by using a standard curve generated by assaying purified trypsin. The maximal values for trypsin activity after CCK hyperstimulation induced trypsinogen activation in acinar cells were in the range of 20-50 ng trypsin/mg protein. To compare values between different treatment groups, the data were expressed as the percentage of maximal activity obtained when acini were incubated with 100 nM CCK for 20 min in each experiment, unless otherwise noted.
Preparations of nuclear extracts. Nuclear extracts were prepared by using a modified version of the method of Maire et al. (16) as previously described (8). Pancreatic acini and CHO-CCKA cells were collected by brief centrifugation and washed with ice-cold phosphate-buffered saline (PBS) containing 1 mM EDTA. The pellets were then resuspended in 0.5 ml of 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 then homogenized with a motor-driven pestle for 5-10 strokes on ice. Nuclei were collected by centrifugation at 30,000 g for 30 min at 4°C, washed with 1 ml of 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 were added. The nuclear suspension was incubated on ice for 15-30 min with intermittent mixing and then centrifuged at 14,000 g for 5 min at 4°C. Protein concentration in the nuclear extract was determined by using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA).
Electrophoretic mobility shift assay.
Aliquots of nuclear extract with equal amounts of protein (10 µg)
were utilized in 20-µl reactions in a buffer containing 10 mM HEPES
(pH 7.9), 10% glycerol (vol/vol), 1 mM DTT, 1 µg of poly(dI-dC), and
5 µg of nuclease-free BSA, as previously described (8).
The binding reaction was started by adding 10,000 cpm of the 22-base
pair oligonucleotide 5'-AGT TGA GGG GAC TTT CCC AGG C-3'
containing the NF-B consensus sequence (underlined) (Promega,
Madison, WI) 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 the antibody supershift
assays, 2 µg of specific antibodies to the NF-
B protein subunits
p65, p50, and c-Rel were incubated with nuclear extracts for 1 h
at room temperature before the labeled probe was added. 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, visualized with the use of a GS-505 Molecular Imaging System
(Bio-Rad Laboratories, Richmond, CA) and quantitated with the Molecular
Analyst software (Bio-Rad Laboratories, Hercules, CA).
Immunoblot analysis.
After treatments were completed, dispersed acini were washed with
ice-cold PBS containing 1 mM Na3VO4. The
pellets were then lysed by sonication 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, and 0.5 mM PMSF to which a protease
inhibitor cocktail containing 10 µg/ml each of aprotinin, leupeptin,
and pepstatin was added before use. After centrifugation, the
supernatant was removed as whole cell lysate and was assayed for
protein by using the Bio-Rad protein assay. Equal amounts of protein
(25 µg) were resolved by SDS-PAGE and transferred to nitrocellulose
membrane. Immunoblot analysis was performed with anti-IB-
and
NF-
B p65 subunit antibodies as described previously (8)
and was visualized with ECL reagent on film. Film images were scanned
with an Agfa Arcus II scanner (Bayer, Ridgefield Park, NJ) to create a
digital image.
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 (8). RNA was quantitated spectrophotometrically,
and 25 µg of RNA from each sample were electrophoresed in 1% agarose
and 2.2 M formaldehyde gels in MOPS buffer and then transferred to
nylon membrane. mob-1 mRNA was detected by using a
full-length 1.2-kilobase cDNA of rat mob-1
(15). Membranes were hybridized at high stringency by
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
phosphoimaging screens and visualized using the GS-505 Molecular
Imaging System. Images were imported into Photoshop 4.0 (Adobe Systems,
San Jose, CA) for preparation of figures.
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RESULTS |
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Action of inhibitors on CCK activation of intracellular trypsin and
NF-B.
To determine the relationships between the ability of high
concentrations of CCK to activate intracellular trypsinogen and NF-
B, we initially tested the effects of inhibitors of each pathway on the ability of CCK to activate trypsin intracellularly. High concentrations of CCK caused a dramatic activation of trypsin within
pancreatic acinar cells (Fig. 1). As
expected, preincubating pancreatic acinar cells with the specific
trypsin inhibitor FUT-175 (17), the cell-permeant
cathepsin inhibitor E-64d (24), or the serine protease
inhibitor Pefabloc (24) for 30 min completely abolished
the effect of CCK hyperstimulation on intracellular trypsin
activation. Unexpectedly, preincubation of acini with the
NF-
B inhibitors PDTC and NAC also abolished the effect of CCK
hyperstimulation on trypsin activation. These data suggest that either
NF-
B activation is necessary for CCK activation of intracellular
trypsin or that the NF-
B inhibitors have nonspecific effects on
trypsin activation.
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NF-B activation is independent of trypsinogen activation.
One interpretation of the results obtained by testing the effects of
protease inhibitors on CCK stimulation of NF-
B activation is that
trypsinogen activation is required for NF-
B activation. To
investigate the relationship between CCK-stimulated trypsinogen activation and NF-
B activation, we tested the ability of CCK to
activate NF-
B in a non-pancreatic cell model. CHO-CCKA
cells, which do not produce trypsin, were treated with CCK, and NF-
B activity was measured in gel shift assays. CCK treatment caused a
significant increase of NF-
B DNA binding (Fig.
4). CCK activation of NF-
B in CHO
cells was dose dependent and only occurred at concentrations of CCK >1
nM (data not shown). To determine whether the NF-
B activation
observed in CHO cells is similar to that observed in pancreatic acinar
cells, we analyzed the NF-
B subunit composition by using a
supershift assay. Antibodies to the NF-
B subunits p65 and p50, but
not to c-Rel, led to a supershift of the observed band, similar to what
was observed in pancreatic acinar cells (8). Specificity
of binding was also supported by competition with cold oligonucleotides
(data not shown). These data indicate that CCK can activate NF-
B in
the absence of trypsinogen activation in a CHO cell. To determine
whether the inhibitory effects previously noted for the protease
inhibitors are specific to pancreatic acinar cells, we tested their
effects on NF-
B activation in the CHO cell model (Fig.
5). The NF-
B inhibitors (PDTC
and NAC) and the protease inhibitors (FUT, E64D, and Pefabloc)
inhibited the ability of CCK to activate NF-
B DNA binding in CHO
cells. Therefore, the ability of CCK to activate NF-
B is independent of its ability to activate trypsinogen, and the protease inhibitors act
at a site separate from trypsin.
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Trypsin activation is independent of NF-B activation.
One interpretation of the observed ability of NF-
B inhibitors to
block CCK-induced intracellular trypsinogen activation is that NF-
B
activity is required for trypsin activation. To further understand the
relationship between NF-
B and trypsin activation, we utilized
adenovirus-mediated gene transfer to directly stimulate or inhibit
NF-
B in pancreatic acinar cells. Acinar cells were infected with
adenovirus bearing either the NF-
B p65 subunit gene, to directly
activate NF-
B, or the inhibitory protein I
B-
gene, to inhibit
CCK-mediated NF-
B activation. The acinar cells were infected with
adenovirus for 6 h, at which time nearly 100% of the cells
expressed the GFP coded for by the virus (data not shown), and were
then treated with CCK. Western blotting with specific antibodies was
utilized to indicate expression of the ectopically expressed genes
(Fig. 6). NF-
B p65 subunits and
I
B-
were overexpressed in acinar cells within the time period of
the experiments. To determine whether expression of these subunits had
the expected effects on NF-
B activity, we analyzed their effects on
the expression of the chemokine mob-1, a known target of
NF-
B. Overexpression of p65 led to high levels of mob-1
gene expression, verifying its ability to activate the NF-
B pathway. Overexpression of I
B-
completely blocked CCK-induced
mob-1 gene expression, indicating both that the molecule was
effective as an inhibitor and that the adenoviral gene transfer was
highly efficient. Infection with control virus had no effect on either p65 or I
B-
protein levels or mob-1 gene expression in
acinar cells. Despite their abilities to stimulate or inhibit NF-
B
activity, overexpression of neither p65 subunits nor I
B-
had any
effect on basal or CCK-stimulated intracellular trypsin activity (Fig. 7). These results indicate that trypsin
activity in pancreatic acinar cells is independent of NF-
B activity.
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DISCUSSION |
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Acute pancreatitis is a complex disease with both local and
systemic aspects. The cellular mechanisms that initiate this disease are not completely understood, but trypsinogen activation and NF-B
activation each have been suggested to be central to this process. Our
working hypothesis was that these two cellular mechanisms were
independent and played different roles in the development of the
disease. In this model, trypsin is responsible for local damage to the
pancreas, while NF-
B, through its ability to induce the expression
of chemokines and cytokines, is responsible for initiating the systemic
inflammatory response. Alternative hypotheses include the possibilities
that trypsin activation is the primary initiating event and NF-
B
activation is a secondary consequence of trypsin activity or that
NF-
B activation is primary and trypsin is a consequence of the
effects of NF-
B activity. In the current study, we found that the
use of pharmacological inhibitors, which had been a tool in previous
studies of the importance of these cellular mechanisms in the disease
(1, 11, 22, 27, 31, 32, 34), was complicated by a
significant overlap in their effects. These observations suggest that
it may be necessary to reevaluate the conclusions of previous studies.
Utilizing a variety of highly specific experimental approaches, we
found that trypsin activation and NF-
B activation were independent events.
Pretreatment with protease inhibitors previously has been shown to
ameliorate the acute pancreatitis induced in several animal models of
the disease. Unfortunately, clinical trials using protease inhibitors
in patients with acute pancreatitis have been unsuccessful (25). However, pretreatment of patients with protease
inhibitors has been reported to reduce the incidence of pancreatitis
associated with endoscopic retrograde cholangiopancreatography
(2). These results suggest that pretreatment is
more successful than posttreatment and have been interpreted to support
the idea that trypsin activation is a central initiator of the disease
but is not necessarily involved in its maintenance. However, in our
studies we found that protease inhibitors could also inhibit NF-B
activation. Therefore, it is unclear whether the previous successful
results with protease inhibitor pretreatment were due to effects on
trypsinogen activation or NF-
B activation, or both.
The possibility that CCK stimulation could activate NF-B in the
absence of trypsin was shown by the ability of CCK to stimulate NF-
B
activation in the CHO cell line. These cells do not produce digestive
enzymes and do not express trypsinogen, yet activation of ectopically
expressed CCK receptors activated NF-
B in these cells. We
(37) have previously shown that the CCK receptor expressed in the CHO cell couples to similar intracellular signaling mechanisms as it does in pancreatic acinar cells. Also, we (8) and
others (33) have previously reported that activation of
NF-
B within the pancreatic acinar cell involves increases in
intracellular Ca2+ and activation of protein kinase C. Thus
these intracellular pathways are likely responsible for CCK-induced
NF-
B activation in both CHO and acinar cells, and they do not
require trypsin activity. However, while trypsin activity is not
required for CCK activation of NF-
B, whether activation of
intracellular trypsin may lead to the activation of NF-
B remains
unknown. Trypsin activity within the cell cytoplasm would be expected
to cause cell stress. A wide variety of stressful stimuli activate
NF-
B. Therefore, it is likely that intracellular activation of
trypsin leads indirectly to activation of NF-
B. Further
investigation is required to answer this question.
The possibility that the effects of protease inhibitors on NF-B are
independent of their ability to block trypsin activity was indicated by
the observation that these inhibitors blocked NF-
B activation in CHO
cells. The mechanisms involved in this unexpected effect of the
protease inhibitors are unknown. It was not the purpose of this study
to investigate the mechanisms responsible for the nonspecific actions
of these inhibitors. However, interestingly, the effects of the
protease inhibitors appear to be specific for CCK-mediated NF-
B
activation and must occur at a point between receptor occupation and
I
B-
phosphorylation, because the effects of tumor necrosis
factor-
on acinar cell NF-
B activation were not inhibited by
these protease inhibitors (data not shown). A variety of
protease inhibitors previously were found to inhibit NF-
B activation
in other models and, in some cases, were also shown to block activation
of I
B-
kinases (4). These results suggest the
presence of an as yet unidentified upstream protease in some pathways
leading to NF-
B activation. Further experiments are necessary to
discover the specific target of these inhibitors. However, it is clear
that these protease inhibitors have effects on cellular mechanisms
other than trypsinogen and that their ability to ameliorate
pancreatitis should not be interpreted as being specifically due to
their ability to block trypsinogen activation.
Inhibitors of NF-B activation have been reported to ameliorate
(3, 5, 7) or exacerbate (30) pancreatitis.
One potential explanation for this discrepancy was that the protective effects were noted early in the course of experimental pancreatitis, whereas the exacerbation was noted after longer times. It has been
speculated that the late exacerbation may be due to the
antiapoptotic actions of NF-
B (6). However, these
experiments are difficult to interpret fully because the specificity of
these inhibitors is unclear. PDTC and NAC inhibit NF-
B activation at
the level of I
B-
degradation, but the exact mechanisms are
unknown. They are both antioxidants, and this is thought to be an
important characteristic for their function as NF-
B inhibitors.
However, these agents also have other activities; for example, PDTC is an iron chelator. In the current study, we found that both PDTC and NAC
could inhibit the ability of CCK to activate cellular trypsinogen. This
finding illustrates the lack of specificity of these agents. Thus the
role of NF-
B activation in acute pancreatitis remains unclear and
requires more specific approaches.
In the current study, stimulation or inhibition of acinar cell NF-B
activity by overexpression of stimulatory or inhibitory subunits did
not affect trypsinogen activation. These studies were conducted by
using adenovirus-mediated gene transfer to the acini in vitro, which we
have shown previously to be highly efficient with infection rates of
nearly 100% (12, 18). Expression of the respective
NF-
B subunits was confirmed by Western blotting. Moreover, their
effects on a known NF-
B-mediated event, mob-1 gene
expression, demonstrated that these manipulations were successful. Expression of the inhibitory subunit completely prevented CCK stimulation of mob-1 expression, while the expression of the
active subunit stimulated mob-1 expression to a high level.
However, neither inhibition, with the use of an inhibitory subunit, nor stimulation, with the use of an active subunit, resulted in any alteration of basal or CCK-stimulated trypsin activity within the
acini. Therefore, it is clear that trypsinogen activation is
independent of NF-
B activity at a cellular level. However, it is
possible that activation of NF-
B in pancreatic acinar cells in vivo
would lead to acinar cell damage and trypsinogen activation due to the
influence of activated inflammatory cells. In the course of advanced
pancreatitis, there is the opportunity for the activation of numerous
and parallel damaging events and pathways. Therefore, these two
pathways may well be linked by indirect events during the course of the
disease. More specific in vivo strategies need to be developed to fully
answer this question.
Thus we have shown that CCK can activate trypsinogen and NF-B
independently. The relative importance and specific roles of trypsinogen and NF-
B activation in acute pancreatitis remain unclear. The use of pharmacological inhibitors is not capable of
differentiating between these two cellular mechanisms. More specific molecular approaches are needed to define the roles of these
cellular events in the disease.
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ACKNOWLEDGEMENTS |
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We thank Drs. J. Williams and D. Simeone for critically reviewing this manuscript.
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FOOTNOTES |
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This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-52067 and the University of Michigan Gastrointestinal Peptide Center/NIDDK Grant DK-34933.
Address for reprint requests and other correspondence: C. D. 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).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 July 2000; accepted in final form 13 October 2000.
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