CCK independently activates intracellular trypsinogen and NF-kappa B in rat pancreatic acinar cells

Bing Han, Baoan Ji, and Craig D. Logsdon

Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the cholecystokinin (CCK) hyperstimulation model of acute pancreatitis, two early intracellular events, activation of trypsinogen and activation of nuclear factor-kappa B (NF-kappa B), are thought to be important in the development of the disease. In this study, the relationship between these two events was investigated. NF-kappa 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-kappa B activation. Likewise, the NF-kappa B inhibitors pyrrolidine dithiocarbamate and N-acetyl-L-cysteine inhibited CCK stimulation of NF-kappa B and intracellular trypsinogen activation. These results suggested a possible codependency of these two events. However, CCK stimulated NF-kappa B activation in Chinese hamster ovary-CCKA cells, which do not express trypsinogen, indicating that trypsin is not necessary for CCK activation of NF-kappa B. Furthermore, adenovirus-mediated expression in acinar cells of active p65 subunits to stimulate NF-kappa B, or of inhibitory kappa B-alpha molecules to inhibit NF-kappa B, did not affect either basal or CCK-mediated trypsinogen activation. Thus trypsinogen and NF-kappa B activation are independent events stimulated by CCK.

pancreatitis; inflammation; cholecystokinin; nuclear factor-kappa B


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa B (NF-kappa 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-kappa B activation has been observed in several models of acute experimental pancreatitis (3, 5, 7, 30). NF-kappa 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-kappa 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-kappa 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-kappa 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-kappa B activation induced by CCK hyperstimulation. We also found that inhibitors of NF-kappa B blocked NF-kappa 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-kappa B activation is independent of trypsinogen activation, because CCK activated NF-kappa B in the absence of trypsin activation in Chinese hamster ovary (CHO) cells expressing ectopic CCKA receptors. Trypsinogen activation is independent of NF-kappa B activity, because intracellular trypsin activity was not affected by inhibition or stimulation of NF-kappa B with the use of adenovirus-mediated gene transfer of either inhibitory or active NF-kappa B subunits to pancreatic acinar cells. Therefore, trypsinogen activation and NF-kappa B activation are independent events that occur early in the development of acute experimental pancreatitis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Chromatographically purified collagenase was purchased from Worthington Biochemical (Freehold, NJ). Soybean trypsin inhibitor (SBTI), beta -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, [gamma -32P]ATP, and [alpha -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 kappa B-alpha (Ikappa B-alpha ) and the NF-kappa 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-kappa 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 AdIkappa B-alpha , with a CMV promoter driving expression of a full-length Ikappa B-alpha 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 beta -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-kappa B consensus sequence (underlined) (Promega, Madison, WI) that had been labeled with [gamma -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-kappa 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-Ikappa B-alpha and NF-kappa 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 [alpha -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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Action of inhibitors on CCK activation of intracellular trypsin and NF-kappa B. To determine the relationships between the ability of high concentrations of CCK to activate intracellular trypsinogen and NF-kappa 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-kappa B inhibitors PDTC and NAC also abolished the effect of CCK hyperstimulation on trypsin activation. These data suggest that either NF-kappa B activation is necessary for CCK activation of intracellular trypsin or that the NF-kappa B inhibitors have nonspecific effects on trypsin activation.


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Fig. 1.   Effect of inhibitors on cholecystokinin (CCK) activation of intracellular trypsinogen. Rat pancreatic acini were preincubated with HEPES-Ringer (HR) buffer alone or buffer containing either protease inhibitors [1 mM FUT-175 (FUT), 0.5 mM E-64d, or 2 mM Pefabloc] (open bars) or nuclear factor-kappa B (NF-kappa B) inhibitors [10 mM pyrrolidine dithiocarbamate (PDTC) or 10 mM N-acetyl-L-cysteine (NAC)] (filled bars) for 30 min and then further incubated in the absence or presence of 100 nM CCK for 20 min. The acinar cells were collected, and the trypsin activity in the homogenized acini was measured. Data are expressed as the percentage of maximal activity obtained in each experiment (range of maximal activity observed was 20-50 ng trypsin/mg protein). Values are means ± SE obtained from at least 3 independent experiments.

We also investigated the effects of the inhibitors on the ability of CCK to activate the NF-kappa B pathway. NF-kappa B inhibitors completely blocked the CCK induction of NF-kappa B DNA binding, as expected (Fig. 2). The protease inhibitors, however, also markedly inhibited the induction of NF-kappa B DNA binding by CCK. These data suggest that either intracellular trypsinogen activation is required for CCK activation of NF-kappa B or that protease inhibitors have nonspecific effects on the NF-kappa B pathway. Because DNA binding as measured by electrophoretic mobility shift assays (EMSA) is not a direct indication of transcriptional activity, we also examined the CCK-mediated induction of mob-1 mRNA expression (Fig. 3), since the expression of this chemokine has been shown to be a consequence of NF-kappa B induction (8). As expected, pretreatment of acinar cells with inhibitors of NF-kappa B for 30 min completely blocked the CCK-mediated stimulation of mob-1 mRNA levels (Fig. 3). Unexpectedly, preincubation of acini with the protease inhibitors also dramatically inhibited the effects of CCK on mob-1 gene expression.


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Fig. 2.   Effect of inhibitors on CCK activation of NF-kappa B DNA binding in rat pancreatic acinar cells. After a 30-min preincubation with HR buffer alone or buffer containing either protease inhibitors (1 mM FUT-175, 0.5 mM E-64d, or 2 mM Pefabloc) (B, open bars) or NF-kappa B inhibitors (10 mM PDTC or 10 mM NAC) (B, filled bars), rat pancreatic acini were further incubated in the absence or presence of 100 nM CCK for 2 h. Nuclear extracts were prepared, and aliquots of 10 µg of nuclear protein from each sample were subjected to a gel shift assay. A: representative electrophoretic mobility shift assay (EMSA). Positions of the NF-kappa B band and a nonspecific (NS) band are indicated. B: levels of the NF-kappa B DNA binding were quantitated using the MultiAnalyst System. Data are expressed as the percentage of maximal CCK-stimulated activation in each experiment. Values are means ± SE from 3 independent experiments.



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Fig. 3.   Effect of inhibitors on CCK induction of mob-1 gene expression in rat pancreatic acinar cells. After a 30-min preincubation with HR buffer alone or buffer containing either protease inhibitors (1 mM FUT-175, 0.5 mM E-64d, or 2 mM Pefabloc) (open bars) or NF-kappa B inhibitors (10 mM PDTC or 10 mM NAC) (filled bars), rat pancreatic acini were further incubated in the absence or presence of 100 nM CCK for 2 h. After the various treatments were completed, total RNA was extracted and aliquots of 25 µg of RNA from each sample were subjected to Northern blotting using full-length mob-1 cDNA. A: representative mob-1 mRNA Northern blot. B: quantitated levels of mob-1 mRNA expressed as the percentage of maximal induction by CCK in each experiment. Values are means ± SE from at least 4 independent experiments.

NF-kappa 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-kappa B activation is that trypsinogen activation is required for NF-kappa B activation. To investigate the relationship between CCK-stimulated trypsinogen activation and NF-kappa B activation, we tested the ability of CCK to activate NF-kappa B in a non-pancreatic cell model. CHO-CCKA cells, which do not produce trypsin, were treated with CCK, and NF-kappa B activity was measured in gel shift assays. CCK treatment caused a significant increase of NF-kappa B DNA binding (Fig. 4). CCK activation of NF-kappa B in CHO cells was dose dependent and only occurred at concentrations of CCK >1 nM (data not shown). To determine whether the NF-kappa B activation observed in CHO cells is similar to that observed in pancreatic acinar cells, we analyzed the NF-kappa B subunit composition by using a supershift assay. Antibodies to the NF-kappa 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-kappa 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-kappa B activation in the CHO cell model (Fig. 5). The NF-kappa B inhibitors (PDTC and NAC) and the protease inhibitors (FUT, E64D, and Pefabloc) inhibited the ability of CCK to activate NF-kappa B DNA binding in CHO cells. Therefore, the ability of CCK to activate NF-kappa B is independent of its ability to activate trypsinogen, and the protease inhibitors act at a site separate from trypsin.


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Fig. 4.   Effect of CCK on NF-kappa B DNA binding activity in CHO-CCKA cells. CHO-CCKA cells were treated with or without 100 nM CCK for 2 h. After the medium was removed, CHO-CCKA cells were washed with ice-cold PBS and harvested for nuclear extract preparation. Aliquots of 10 µg of nuclear protein from each sample were subjected to EMSA. To examine the specific NF-kappa B subunits, 2 µg of specific antibodies against the NF-kappa B subunits p65, p50, and c-Rel were added, respectively, to the reaction before the addition of the labeled oligonucleotide. Positions of the NF-kappa B band and a nonspecific (NS) band are indicated by arrows. Data are representative of 5 independent experiments.



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Fig. 5.   Effect of inhibitors on CCK-induced NF-kappa B DNA binding in CHO-CCKA cells. Cells were preincubated with or without either protease inhibitors (1 mM FUT-175, 0.5 mM E-64d, or 2 mM Pefabloc) (B, open bars) or NF-kappa B inhibitors (10 mM PDTC or 10 mM NAC) (B, filled bars) for 30 min, followed by treatment with 100 nM CCK for 1 h. After the medium was removed, CHO-CCKA cells were washed with ice-cold PBS and harvested for nuclear extract preparation. Aliquots of 10 µg of nuclear protein from each sample were subjected to EMSA. A: representative EMSA. Positions of the NF-kappa B band and a nonspecific band are indicated by arrows. B: levels of NF-kappa B DNA binding were quantitated by using the MultiAnalyst System. Data are expressed as the percentage of maximal CCK-stimulated activation in each experiment. Values are means ± SE from 3 independent experiments.

Trypsin activation is independent of NF-kappa B activation. One interpretation of the observed ability of NF-kappa B inhibitors to block CCK-induced intracellular trypsinogen activation is that NF-kappa B activity is required for trypsin activation. To further understand the relationship between NF-kappa B and trypsin activation, we utilized adenovirus-mediated gene transfer to directly stimulate or inhibit NF-kappa B in pancreatic acinar cells. Acinar cells were infected with adenovirus bearing either the NF-kappa B p65 subunit gene, to directly activate NF-kappa B, or the inhibitory protein Ikappa B-alpha gene, to inhibit CCK-mediated NF-kappa 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-kappa B p65 subunits and Ikappa B-alpha 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-kappa B activity, we analyzed their effects on the expression of the chemokine mob-1, a known target of NF-kappa B. Overexpression of p65 led to high levels of mob-1 gene expression, verifying its ability to activate the NF-kappa B pathway. Overexpression of Ikappa B-alpha 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 Ikappa B-alpha protein levels or mob-1 gene expression in acinar cells. Despite their abilities to stimulate or inhibit NF-kappa B activity, overexpression of neither p65 subunits nor Ikappa B-alpha 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-kappa B activity.


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Fig. 6.   Effect of adenovirus-mediated gene transfer on activation of NF-kappa B and mob-1 gene expression in rat pancreatic acinar cells. Isolated rat pancreatic acini were infected without or with adenovirus bearing the gene of NF-kappa B subunit p65, inhibitory kappa B protein Ikappa B-alpha , or bacterial beta -galactosidase (LacZ), respectively, and incubated in HR buffer for 6 h and were then incubated in the absence or presence of 100 nM CCK. Ninety minutes after CCK was added, aliquots were removed and whole cell lysates were prepared, and 25 µg of protein from each sample were subjected to Western blotting by using antibodies against the NF-kappa B p65 subunit (top blot) and Ikappa B-alpha (second blot). Two hours after CCK was added, total RNA was extracted, and 25 µg of RNA from each sample were subjected to Northern blotting by using a full-length mob-1 cDNA (third blot). Ethidium bromide staining was utilized as a loading control for the Northern blotting (bottom blot). Data are representative of 3 independent experiments.



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Fig. 7.   Effect of activation and inhibition of NF-kappa B by adenovirus-mediated gene transfer on trypsin activity in rat pancreatic acinar cells. Isolated rat pancreatic acini were infected with or without adenovirus bearing the gene of NF-kappa B subunit p65, Ikappa B-alpha , or bacterial beta -galactosidase, respectively, incubated in HR buffer for 6 h, and then incubated in the absence or presence of 100 nM CCK for 20 min. The acinar cells were collected by brief centrifugation, and the intracellular trypsin activity was determined. Data are expressed as the percentage of basal trypsin activity obtained in noninfected acinar cells in each experiment. Values are means ± SE obtained from 5 independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa 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-kappa 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-kappa B activation is a secondary consequence of trypsin activity or that NF-kappa B activation is primary and trypsin is a consequence of the effects of NF-kappa 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-kappa 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-kappa B activation. Therefore, it is unclear whether the previous successful results with protease inhibitor pretreatment were due to effects on trypsinogen activation or NF-kappa B activation, or both.

The possibility that CCK stimulation could activate NF-kappa B in the absence of trypsin was shown by the ability of CCK to stimulate NF-kappa 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-kappa 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-kappa 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-kappa 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-kappa B, whether activation of intracellular trypsin may lead to the activation of NF-kappa B remains unknown. Trypsin activity within the cell cytoplasm would be expected to cause cell stress. A wide variety of stressful stimuli activate NF-kappa B. Therefore, it is likely that intracellular activation of trypsin leads indirectly to activation of NF-kappa B. Further investigation is required to answer this question.

The possibility that the effects of protease inhibitors on NF-kappa B are independent of their ability to block trypsin activity was indicated by the observation that these inhibitors blocked NF-kappa 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-kappa B activation and must occur at a point between receptor occupation and Ikappa B-alpha phosphorylation, because the effects of tumor necrosis factor-alpha on acinar cell NF-kappa B activation were not inhibited by these protease inhibitors (data not shown). A variety of protease inhibitors previously were found to inhibit NF-kappa B activation in other models and, in some cases, were also shown to block activation of Ikappa B-alpha kinases (4). These results suggest the presence of an as yet unidentified upstream protease in some pathways leading to NF-kappa 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-kappa 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-kappa B (6). However, these experiments are difficult to interpret fully because the specificity of these inhibitors is unclear. PDTC and NAC inhibit NF-kappa B activation at the level of Ikappa B-alpha 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-kappa 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-kappa B activation in acute pancreatitis remains unclear and requires more specific approaches.

In the current study, stimulation or inhibition of acinar cell NF-kappa 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-kappa B subunits was confirmed by Western blotting. Moreover, their effects on a known NF-kappa 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-kappa B activity at a cellular level. However, it is possible that activation of NF-kappa 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-kappa B independently. The relative importance and specific roles of trypsinogen and NF-kappa 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.


    ACKNOWLEDGEMENTS

We thank Drs. J. Williams and D. Simeone for critically reviewing this manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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