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Am J Physiol Gastrointest Liver Physiol 287: G582-G591, 2004. First published April 29, 2004; doi:10.1152/ajpgi.00087.2004
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INFLAMMATION/IMMUNITY/MEDIATORS

PKC-{delta} and -{epsilon} regulate NF-{kappa}B activation induced by cholecystokinin and TNF-{alpha} in pancreatic acinar cells

Akihiko Satoh,1,2 Anna S. Gukovskaya,1 Jose M. Nieto,1 Jason H. Cheng,1 Ilya Gukovsky,1 Joseph R. Reeve, Jr,1 Tooru Shimosegawa,2 and Stephen J. Pandol1

1Research Center for Alcoholic Liver and Pancreatic Diseases, Veterans Affairs Greater Los Angeles Health Care System and University of California, Los Angeles, California 90073; and 2Division of Gastroenterology, Tohoku University Graduate School of Medicine, Miyagi 980-8574, Japan

Submitted 24 February 2004 ; accepted in final form 24 April 2004


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although NF-{kappa}B plays an important role in pancreatitis, mechanisms underlying its activation remain unclear. We investigated the signaling pathways mediating NF-{kappa}B activation in pancreatic acinar cells induced by high-dose cholecystokinin-8 (CCK-8), which causes pancreatitis in rodent models, and TNF-{alpha}, which contributes to inflammatory responses of pancreatitis, especially the role of PKC isoforms. We determined subcellular distribution and kinase activities of PKC isoforms and NF-{kappa}B activation in dispersed rat pancreatic acini. We applied isoform-specific, cell-permeable peptide inhibitors to assess the role of individual PKC isoforms in NF-{kappa}B activation. Both CCK-8 and TNF-{alpha} activated the novel isoforms PKC-{delta} and -{epsilon} and the atypical isoform PKC-{zeta} but not the conventional isoform PKC-{alpha}. Inhibition of the novel PKC isoforms but not the conventional or the atypical isoform resulted in the prevention of NF-{kappa}B activation induced by CCK-8 and TNF-{alpha}. NF-{kappa}B activation by CCK-8 and TNF-{alpha} required translocation but not tyrosine phosphorylation of PKC-{delta}. Activation of PKC-{delta}, PKC-{epsilon}, and NF-{kappa}B with CCK-8 involved both phosphatidylinositol-specific PLC and phosphatidylcholine (PC)-specific PLC, whereas with TNF-{alpha} they only required PC-specific PLC for activation. Results indicate that CCK-8 and TNF-{alpha} initiate NF-{kappa}B activation by different PLC pathways that converge at the novel PKCs ({delta} and {epsilon}) to mediate NF-{kappa}B activation in pancreatic acinar cells. These findings suggest a key role for the novel PKCs in pancreatitis.

phosphatidylcholine-specific phospholipase C; phosphatidylinositol-specific phospholipase C; Src kinases; translocation; tyrosine phosphorylation

ACUTE PANCREATITIS IS A DISORDER the pathophysiology of which remains obscure (5, 47–49). Although the complete mechanism of pancreatitis has not been established, there is a substantial body of evidence suggesting a critical role for the inflammatory response in this disease (5, 13, 19, 49). Results from our group and others indicate that the initial events in this disorder occur in pancreatic acinar cells (13, 19, 39). More specifically, we have shown that pancreatic acinar cells are capable of responding to noxious stimuli by upregulating signaling systems that mediate the production of proinflammatory mediators such as cytokines, chemokines, and adhesion molecules (17, 19, 32, 56). These pancreas-generated mediators subsequently lead to the severe systemic complications of the disease (5, 13, 19, 49).

A key regulator of the expression of these inflammatory molecules is the transcription factor NF-{kappa}B (42, 51, 58). In experimental pancreatitis, NF-{kappa}B activation in acinar cells is one of the earliest events (19, 50, 56), and the inhibition of NF-{kappa}B activation attenuates inflammatory response and the severity of pancreatitis (13, 19, 20, 41, 56). Furthermore, the direct activation of NF-{kappa}B within the pancreas by adenovirus-mediated gene transfer is sufficient for the initiation of pancreatic and systemic inflammatory responses (7).

Although previous studies demonstrated a key role for NF-{kappa}B activation in the mechanism of pancreatitis, the signaling mechanisms mediating the NF-{kappa}B activation are unclear. Multiple factors are thought to contribute to induction of NF-{kappa}B activation in pancreatitis, and one important factor is TNF-{alpha} (3, 17, 19, 24). In vivo experiments on animal models demonstrated that TNF-{alpha}-induced NF-{kappa}B activation correlated with an increase in gene expression of various inflammatory molecules, and the administration of soluble TNF receptor or anti-TNF antibody prevented NF-{kappa}B activation in pancreatic acini and attenuated the inflammatory response and the severity of pancreatitis (14, 19, 25, 31). Furthermore, the severity and mortality of pancreatitis was attenuated in mice deficient in TNF receptor (9). We (17) previously demonstrated the presence of the TNF-{alpha} receptors in rat pancreatic acinar cells and further determined that TNF-{alpha} activates these receptors, thereby initiating signal transduction cascades including NF-{kappa}B activation. However, the postreceptor events that link to NF-{kappa}B activation in pancreatic acinar cells have not been established.

Cholecystokinin-8 (CCK-8) stimulation of isolated rat pancreatic acini can be also used to investigate the mechanism of NF-{kappa}B activation (3, 19–22, 50, 52). CCK is a physiological regulator of pancreatic digestive enzyme secretion; however, supramaximally stimulating doses of CCK-8 cause the inflammatory response that underlies many of the features of human pancreatitis (19, 20, 50, 57). In vivo and in vitro experiments using pancreatic acini demonstrated that supramaximal but not submaximal stimulation with CCK-8 activates NF-{kappa}B through I{kappa}B degradation (3, 19–22, 50, 52). Similar to TNF-{alpha}, the postreceptor events mediating NF-{kappa}B activation by CCK-8 are poorly understood.

One candidate for mediating NF-{kappa}B activation in pancreatic acinar cells is the family of PKCs, because the incubation of pancreatic acinar cells with phorbol esters, a general activator of PKCs, causes NF-{kappa}B activation (18, 22, 52). PKCs are a family of serine/threonine kinases comprising 10 isoforms that differ in their structures and regulations (8, 38). These isoforms are subdivided into three classes on the basis of their molecular structure and mode of activation, namely, conventional PKC isoforms ({alpha}, {beta}I, {beta}II, and {gamma}), novel PKC isoforms ({delta}, {epsilon}, {eta}, and {theta}), and atypical PKC isoforms ({zeta} and {lambda}/{iota}). The conventional PKC isoforms are activated by Ca2+ and by diacylglycerol (DAG) or phorbol esters. Of note, CCK stimulates increases in Ca2+ and DAG in pancreatic acinar cells through the phospholipase C (PLC) pathway (57). The novel PKC isoforms are also activated by DAG and phorbol esters but are Ca2+ independent. The atypical PKC isoforms are unresponsive to Ca2+, DAG, and phorbol esters. In addition to the regulation by Ca2+ and lipid messengers, the activity of PKCs is regulated by phosphorylation (8, 15, 35, 38), and one important mediator of this pathway is the family of Src kinases (8, 15, 35, 38). Each PKC isoform has a different pattern of cell distribution, can be activated independently by specific stimuli, and mediates distinct biological functions. In general, the activation of PKCs is associated with their translocation to distinct intracellular compartments, and specific anchoring proteins target individual PKCs to different intracellular components and confer specificity for different substrates (8, 15, 30, 38).

In pancreatic acinar cells, four PKC isoforms ({alpha}, {delta}, {epsilon}, and {zeta}), have been detected (4, 37). Although the involvement of PKC in NF-{kappa}B activation by CCK-8 has been addressed (18, 22, 52), the roles of individual isoforms for NF-{kappa}B activation have not been determined. Furthermore, participation of PKC in NF-{kappa}B activation by other stimuli (i.e., TNF-{alpha}) in pancreatic acinar cells has not been studied. The aims of the present study were to determine 1) which specific PKC isoforms are involved in NF-{kappa}B activation by CCK-8; 2) whether PKC isoforms contribute to the activation of NF-{kappa}B induced by TNF-{alpha}; and 3) whether the upstream signaling mechanisms, namely, PLC and Src kinases, are involved in the regulation of specific PKC isoforms and NF-{kappa}B. We found that PKC-{delta} and -{epsilon} are responsible for both CCK-8-induced and TNF-{alpha}-induced NF-{kappa}B activation in pancreatic acinar cells. Translocation but not tyrosine phosphorylation of PKC-{delta} is necessary for mediating NF-{kappa}B activation. Pharmacological analysis showed that both phosphatidylinositol (PI)-specific PLC and phosphatidylcholine (PC)-specific PLC are necessary for the activation of PKC-{delta}, PKC-{epsilon}, and NF-{kappa}B by CCK-8. In contrast, these responses occur only through PC-specific PLC in acini stimulated with TNF-{alpha}. Although CCK-8 and TNF-{alpha} initiate NF-{kappa}B activation by different PLC pathways, these pathways converge on the activation of PKC-{delta} and -{epsilon}, leading to NF-{kappa}B activation in pancreatic acinar cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. CCK-8 was from American Peptide (Sunnyvale, CA); recombinant TNF-{alpha} was from BD Biosciences (San Diego, CA); medium 199 was from GIBCO-BRL (Grand Island, NY); [{gamma}-32P]ATP was from ICN Biomedicals (Costa Mesa, CA); GF109203X, Gö6976, PKC-{delta} peptide substrate, and PKC-{epsilon} peptide substrate were from Calbiochem (La Jolla, CA); PKC-{zeta} substrate and PKC-{zeta} pseudosubstrate were from Biosource International (Camarillo, CA); antibodies against inhibitor {kappa}B{alpha} (I{kappa}B{alpha}) and PKC-{alpha}, -{delta}, -{epsilon}, and -{zeta} were from Santa Cruz Biotechnology (Santa Cruz, CA); 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2), D-609, and U-73122 were from Biomol (Plymouth Meeting, PA); conventional PKC substrate and anti-phosphotyrosine antibody were from Upstate Biotechnology (Charlottesville, VA); T4 polynucleotide kinase was from New England Biolabs (Beverly, MA); and poly(dI-dC) was from Boehringer Mannheim (Indianapolis, IN). All other chemicals were from Sigma (St. Louis, MO).

Preparation of dispersed pancreatic acini. Pancreatic acini were prepared from Sprague-Dawley rats (75–100 g) using a collagenase digestion method as we described previously (17–20) and were then incubated in medium 199 supplemented with penicillin (100 U/ml) and streptomycin (0.1 mg/ml) for 3 h at 37°C in a 5% CO2 humidified atmosphere.

Preparation of nuclear extracts and EMSA. Preparation of nuclear and cytosolic protein extracts and the EMSA have been described in detail (19, 20). Briefly, pancreatic acinar cells were lysed on ice in a hypotonic buffer A (19) supplemented with 1 mM PMSF, 1 mM DTT, and protease inhibitor cocktail containing 5 µg/ml each of pepstatin, leupeptin, chymostatin, antipain, and aprotinin. Cells were left to swell on ice for a 20- to 25-min period, 0.3% (vol/vol) Igepal CA-630 was then added, and the nuclei were collected by microcentrifugation. The supernatant (cytosolic proteins) was saved for Western blot analysis of I{kappa}B{alpha}, and the nuclear pellet was resuspended in a high-salt buffer C (19) supplemented with 1 mM PMSF, 1 mM DTT, and the protease inhibitor cocktail described above. After incubating at 4°C, membrane debris were pelleted by microcentrifugation for 10 min, and the clear supernatant (nuclear extract) was aliquoted and stored at –80°C. Protein concentration in the extracts was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).

For EMSA, aliquots of nuclear extracts with equal amounts of protein (5–10 µg) were mixed in 20-µl reactions with a buffer containing 10 mM HEPES (pH 7.8), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 10% glycerol, and 3 µg poly(dI-dC). Binding reactions were started by the addition of 32P-labeled DNA probe and incubated at room temperature for 20 min. The oligo probe 5'-GCAGAGGGGACTTTCCGAGA-3' containing {kappa}B binding motif (underlined) was annealed to the complementary oligonucleotide and end-labeled by using T4 polynucleotide kinase. Samples were electrophoresed on a native 4.5% polyacrylamide gel at 200 V in 0.5 x TBE buffer (1 x TBE: 89 mM Tris base, 89 mM boric acid, 2 mM EDTA). Gels were dried and densitometrically quantified in the PhosphorImager (Molecular Dynamics, Sunnyvale, CA). In pancreatic acinar cells, the NF-{kappa}B band has two components: we have previously shown (19, 32) that the upper component corresponds to the p50/p65 heterodimer and the lower component to the p50/p50 homodimer. In the present study, we quantified the total (combined) intensity of the NF-{kappa}B band.

Subcellular fractionation. The dispersed acini were homogenized with 50 strokes in a Dounce homoginizer in an ice-cold homogenization buffer containing (in mM) 130 NaCl, 50 Tris·HCl (pH 7.5), 5 EGTA, 5 EDTA, 1.5 MgCl2, 10 NaF, 1 Na3VO4, 10 Na4P2O7, 1 PMSF, and 10% (vol/vol) glycerol plus 5 µg/ml each of pepstatin, leupeptin, chymostatin, antipain, and aprotinin. Homogenates were centrifuged at 500 g for 10 min at 4°C to remove unbroken cells, nuclei, and other debris. Supernatants were recovered and ultracentrifuged at 150,000 g for 45 min at 4°C to separate the cytosolic fraction (the resulting supernatant) and the pellet for translocation experiments. The pellet was washed five times with homogenization buffer, resuspended in a homogenization buffer containing 2% (vol/vol) Triton X-100, sonicated five times for 10 s on ice, and incubated for 30 min at 4°C. At the end of incubation, the samples were centrifuged at 15,000 g for 15 min, and the resulting supernatant was designated the membrane fraction.

Immunoprecipitation. Pancreatic acini were suspended in 1 ml of ice-cold homogenization buffer, sonicated five times for 10 s on ice, and incubated for 45 min at 4°C. After the centrifugation for 15 min at 15,000 g, specific antibody against individual PKC isoform (1:100 dilution) was added to the lysate, which was rotated overnight at 4°C. Protein A-Sepharose beads (50% slurry) were added and rotated for 2 h at 4°C. The beads were washed twice in the lysis buffer, followed by additional three washes with the kinase buffer [(in mM) 20 MOPS (pH 7.2), 25 {beta}-glycerophosphate, 5 EGTA, 1 Na3VO4, and 1 DTT]. The beads were resuspended in a final 50 µl of kinase buffer.

Isoform-specific PKC kinase assay. The kinase assay was performed by using the PKC assay kit (Upstate Biotechnology) according to the manufacturer's instruction with minor modifications. We used substrates optimized for individual PKC isoforms. We used the kinase buffer described in Immunoprecipitation for the measurement of PKC-{delta}, -{epsilon}, and -{zeta}, and supplemented it with 1 mM CaCl2 for PKC-{alpha}. The assay was started with the addition of a magnesium/ATP mixture (75 mM MgCl2 and 0.5 mM ATP) containing 10 µCi of [{gamma}-32P]ATP to the sample containing 10 µl of the PKC isoform-specific immunoprecipitate, 30 µl of kinase buffer, and 40 µM of substrate, and the reaction was incubated for 10 min at 30°C. Reactions were stopped by the addition of 50 µl of 0.75% phosphoric acid, and the samples were applied onto p81 phosphocellulose paper. The p81 papers were washed three times with 0.75% phosphoric acid and once with acetone. The amount of 32P was determined by liquid scintillation counting. Background measurements of 32P were determined from incubations conducted in the absence of substrate and were subtracted from the 32P values in experimental samples. Measurements were performed in duplicate.

Western blot analysis. Immunoprecipitate of PKC-{delta} for tyrosine phosphorylation analysis, cytosolic extracts for I{kappa}B{alpha} analysis, or subcellular fractions for PKC translocation studies were used as samples for Western blot analysis. After samples were adjusted for protein concentration, equal amounts of protein were fractionated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. The membranes were blocked by overnight incubation in Tris-buffered saline supplemented with 5% nonfat dry milk and probed with an antibody against I{kappa}B{alpha} (1:100 dilution); PKC-{alpha}, -{delta}, -{epsilon}, and -{zeta} (1:200 dilution each); or phosphotyrosine (1:500 dilution) for 2 h at room temperature. The membranes were incubated with secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. Blots were developed by using the enhanced chemiluminescence detection kit (Pierce). When reprobing was necessary, the membrane was stripped of bound antibody by incubating in stripping buffer at room temperature for 20 min.

Inhibition analysis. For pharmacological analysis, we used a broad-spectrum PKC inhibitor, GF109203X; a specific inhibitor of conventional PKC isoforms, Gö6976; and a specific PKC-{zeta} inhibitor, PKC-{zeta} pseudosubstrate. We synthesized a specific PKC-{delta} translocation inhibitor ({delta}V1–1: S-F-N-S-Y-E-L-G-S-L), PKC-{epsilon} translocation inhibitor ({epsilon}V1–2: E-A-V-S-L-K-P-T), and scrambled peptide (L-S-E-T-K-P-A-V) according to previous studies (6, 11). For each of the PKC isoforms, these peptides correspond to specific sequences in the V1 regions that are responsible for anchoring the individual isoform to its translocation site. Thus the peptides competitively inhibit the binding of a specific isoform of PKC to its anchoring protein. Each of these peptides was conjugated to a Drosophila antennapedia peptide (R-Q-I-K-I-W-F-Q-N-R-R-M-K-W-K-K) to make it cell permeable.

We (18–20) have previously shown that CCK-8 causes a rapid and prolonged NF-{kappa}B activation in isolated rat pancreatic acini in a dose- and time-dependent manner and that the NF-{kappa}B response to 100 nM CCK-8 reaches a maximum at 30 min after the stimulation. We also showed the NF-{kappa}B activation in response to TNF-{alpha} (17). On the basis of these results, we preincubated pancreatic acini with each PKC inhibitor (10 µM) or the same volume of DMSO for 3 h and then stimulated them with CCK-8 (100 nM) or TNF-{alpha} (100 ng/ml) for 30 min. The scrambled peptide (10 µM) was used instead of DMSO as the control for the translocation inhibitors.

Statistical analysis. Values are expressed as means ± SE. The percent changes in NF-{kappa}B activation and PKC activity were calculated as the difference between stimulated and unstimulated (basal) conditions. Statistics were performed by using a paired t-test. A difference with a P value of <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
CCK-8 activates PKC-{delta}, -{epsilon}, and -{zeta}, but not PKC-{alpha}, in rat pancreatic acini. We first examined the presence and translocation of each PKC isoform by Western blot analysis. As reported previously (4, 37), immunoreactivities to four isoforms of PKC ({alpha}, {delta}, {epsilon}, and {zeta}) were detected in untreated rat pancreatic acini, with a large percentage of each isoform residing in the cytosolic fraction (Fig. 1A, Table 1). Treatment with 100 nM CCK-8 decreased the presence of PKC-{delta} and -{epsilon} in the cytosolic fraction and increased them in the membrane fraction, indicating translocation from cytosol to cell membranes. In contrast, no changes in the subcellular localization of PKC-{alpha} or -{zeta} were detected after CCK-8 stimulation (Fig. 1A, Table 1).



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  		Fig. 1. Subcellular distribution and kinase activities of PKC isoforms in pancreatic acini stimulated by cholecystokinin-8 (CCK-8). Dispersed rat pancreatic acini were preincubated for 3 h and then stimulated with 100 nM CCK-8 for 30 min. A: subcellular distribution of PKC isoforms in response to CCK-8 was determined in cytosolic and membrane fractions using isoform-specific PKC antibodies and Western blot analysis. Shown are representative Western blots from 3 independent experiments. B: changes in PKC kinase activities stimulated by CCK-8. Individual PKC isoforms were immunoprecipitated from whole cell lysates, and PKC activities were measured by kinase assay using isoform-optimized substrates. For each PKC isoform, activity values were normalized on its basal activity in unstimulated control acini. Values are means ± SE (n = 5–10). *P < 0.05 compared with each isoform basal activity.

 

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  		Table 1. Effects of CCK-8 and TNF-{alpha} on the distribution of PKC isoforms between membrane and cytosolic fractions in pancreatic acini

 
We then performed kinase assays using PKC isoform-specific immunoprecipitates. The CCK-8 treatment increased kinase activities for PKC-{delta}, -{epsilon}, and -{zeta} (Fig. 1B). At the same time, PKC-{alpha} activity was not significantly altered by CCK-8. The following data show the distinct responses of PKC isoforms: 1) CCK-8 stimulated both kinase activity and translocation of PKC-{delta} and -{epsilon}; 2) it increased PKC-{zeta} activity without affecting its translocation; and 3) it had no effect on kinase activity and translocation of PKC-{alpha}.

Inhibition of PKC-{delta} and -{epsilon} prevents CCK-8-induced NF-{kappa}B activation. To determine the PKC isoform(s) that mediate NF-{kappa}B activation by CCK-8, we performed pharmacological analysis with isoform-specific PKC inhibitors. Activation of NF-{kappa}B was determined by NF-{kappa}B binding activity and I{kappa}B{alpha} degradation. The CCK-8-induced NF-{kappa}B activation was inhibited by the broad-spectrum PKC inhibitor GF109203X, the PKC-{delta} translocation inhibitor {delta}V1–1, and the PKC-{epsilon} translocation inhibitor {epsilon}V1–2, by 98, 76, and 80%, respectively (Fig. 2, A and B). The conventional PKC isoform inhibitor Gö6976 did not inhibit but even enhanced the NF-{kappa}B response (Fig. 2, A and B). PKC-{zeta} pseudosubstrate did not affect NF-{kappa}B activation (Fig. 2, A and B) while abolishing the increase in kinase activity of PKC-{zeta} (data not shown). None of the inhibitors alone affected the basal NF-{kappa}B activity (Fig. 2B). The degradation of I{kappa}B{alpha} correlated with the increased NF-{kappa}B binding activity in CCK-8-treated cells, and the blockade of I{kappa}B{alpha} degradation by {delta}V1–1 or {epsilon}V1–2 was consistent with their inhibitory effects on NF-{kappa}B binding activity (Fig. 2, A–C). Gö6976 enhanced CCK-8-induced I{kappa}B{alpha} degradation (Fig. 2C). These results indicate that PKC-{delta} and -{epsilon} are responsible for CCK-8-induced NF-{kappa}B activation in pancreatic acinar cells. In contrast, the data suggest that PKC-{alpha} may exert an inhibitory effect on the NF-{kappa}B activation.



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  		Fig. 2. Effects of PKC inhibitors on CCK-8-induced NF-{kappa}B activation in pancreatic acini. Pancreatic acini were preincubated for 3 h with PKC broad-spectrum inhibitor GF109203X (GF), conventional PKC isoform inhibitor Gö6976 (Gö), PKC-{delta} translocation inhibitor ({delta}V1–1); PKC-{epsilon} translocation inhibitor ({epsilon}V1–2); PKC-{zeta} inhibitor PKC-{zeta} pseudosubstrate ({zeta} pseudo), 10 µM each, or with DMSO and then stimulated with 100 nM CCK-8 for 30 min. A: NF-{kappa}B binding activity was measured in nuclear extracts by EMSA. B: NF-{kappa}B band intensities were quantified in the PhosphorImager and normalized on the band intensity in unstimulated control acini. Values are means ± SE (n = 5). *P < 0.05 compared with unstimulated control. #P < 0.05 compared with CCK-8 alone. C: I{kappa}B{alpha} degradation was measured in cytosolic extracts by Western blot analysis. Representative of 5 independent experiments.

 
To demonstrate the specificity of the translocation inhibitors {delta}V1–1 and {epsilon}V1–2 in pancreatic acini, we examined their effects on PKC-{delta} and -{epsilon}. As shown in Fig. 3A, {delta}V1–1 prevented the translocation of PKC-{delta} but not that of PKC-{epsilon}, whereas {epsilon}V1–2 blocked the translocation of PKC-{epsilon} without affecting PKC-{delta}. Furthermore, each inhibitor abolished the increase in its target isoform kinase activity without affecting the other isoform activity (Fig. 3B). The scrambled peptide did not affect the translocation and kinase activity of either isoform. The effects of both {delta}V1–1 and {epsilon}V1–2 were evident at 0.1 µM and reached maximum at 1 µM (Fig. 3C). We also confirmed that neither {delta}V1–1 nor {epsilon}V1–2 inhibited kinase activity of the PKCs when applied directly into the assay (data not shown).



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  		Fig. 3. The specificity of the isoform-specific translocation inhibitors. Pancreatic acini were preincubated with PKC translocation inhibitors {delta}V1–1 or {epsilon}V1–2 (10 µM each), scrambled peptide (10 µM), or DMSO for 3 h and then stimulated with 100 nM CCK-8 for 30 min. A: cytosolic and membrane fractions were subjected to SDS-PAGE and blotted by using antibodies specific for PKC-{delta} or -{epsilon}. Shown are representative blots from 3 independent experiments. B: effects of PKC translocation inhibitors on kinase activity. For each PKC isoform, activity values were normalized on its basal activity in unstimulated control acini. Values are means ± SE (n = 5–10). *P < 0.05 compared with unstimulated control; #P < 0.05 compared with CCK-8 alone. C: pancreatic acini were preincubated with the indicated concentration of {delta}V1–1 (top) or {epsilon}V1–2 (bottom) for 3 h and then stimulated with 100 nM CCK-8 for 30 min. The kinase activity responding to CCK-8 in the absence of the inhibitors was considered as 100%. Values are means ± SE (n = 3–10). *P < 0.05 compared with CCK-8 alone.

 
TNF-{alpha} activates PKC-{delta}, -{epsilon}, and -{zeta}, but not PKC-{alpha}, in rat pancreatic acini. Inhibition of PKC-{delta} and -{epsilon} prevents TNF-{alpha}-induced NF-{kappa}B activation. In the next set of experiments, pancreatic acini were stimulated with 100 ng/ml TNF-{alpha}. Similar to CCK-8, TNF-{alpha} induced translocation of PKC-{delta} and -{epsilon}, but not -{alpha} or -{zeta} (Fig. 4A, Table 1). In cells stimulated by TNF-{alpha}, increases in kinase activity were observed in PKC-{delta}, -{epsilon}, and -{zeta}, but not in -{alpha} (Fig. 4B). As shown in Fig. 5, TNF-{alpha} caused NF-{kappa}B activation in pancreatic acini. Compared with CCK-8, the responses of both PKC and NF-{kappa}B to TNF-{alpha} were relatively smaller (see Figs. 2B and 5B). When acini were pretreated with GF109203X, the increase in NF-{kappa}B binding activity by TNF-{alpha} was abolished, indicating participation of PKC isoforms in the NF-{kappa}B activation by TNF-{alpha}. The TNF-{alpha}-induced NF-{kappa}B activation was inhibited by GF109203X, {delta}V1–1, and {epsilon}V1–2, by 81, 57, and 58%, respectively (Fig. 5, A and B). The conventional PKC isoform inhibitor Gö6976 did not inhibit but enhanced the NF-{kappa}B response. PKC-{zeta} pseudosubstrate did not affect NF-{kappa}B activation (Fig. 5, A and B). Consistent with the results of NF-{kappa}B binding activity, TNF-{alpha}-induced degradation of I{kappa}B{alpha} was blocked by {delta}V1–1 and {epsilon}V1–2, enhanced by Gö6976, and unaffected by PKC-{zeta} pseudosubstrate (Fig. 5C). These results suggest that PKC-{delta} and -{epsilon} are responsible for TNF-{alpha}-induced NF-{kappa}B activation and that PKC-{alpha} may exert an inhibitory effect on the NF-{kappa}B activation.



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  		Fig. 4. Subcellular distribution and kinase activities of PKC isoforms in pancreatic acini stimulated by TNF-{alpha}. Pancreatic acini were preincubated for 3 h and then stimulated with 100 ng/ml TNF-{alpha} for 30 min. A: subcellular distribution of PKC isoforms in response to TNF-{alpha} was determined in cytosolic and membrane fractions using isoform-specific PKC antibodies and Western blot analysis. Shown are representative blots from 3 independent experiments. B: changes in PKC kinase activities stimulated by TNF-{alpha}. Individual PKC isoforms were immunoprecipitated from whole cell lysates, and PKC activities were measured by kinase assay using isoform-optimized substrates. For each PKC isoform, activity values were normalized on its basal activity in unstimulated control acini. Values are means ± SE (n = 3–5). *P < 0.05 compared with each isoform basal activity.

 


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  		Fig. 5. Effects of PKC inhibitors on TNF-{alpha}-induced NF-{kappa}B activation in pancreatic acini. Pancreatic acini were preincubated for 3 h with PKC broad-spectrum inhibitor GF109203X, conventional PKC isoform inhibitor Gö6976, PKC-{delta} translocation inhibitor {delta}V1–1, PKC-{epsilon} translocation inhibitor {epsilon}V1–2, PKC-{zeta} inhibitor PKC-{zeta} pseudosubstrate (10 µM each), or with DMSO and then stimulated with 100 ng/ml TNF-{alpha} for 30 min. A: NF-{kappa}B binding activity was measured in nuclear extracts by EMSA. B: NF-{kappa}B band intensities were quantified in the PhosphorImager and normalized on the band intensity in unstimulated control acini. Values are means ± SE (n = 4). *P < 0.05 compared with unstimulated control. #P < 0.05 compared with TNF-{alpha} alone. C: I{kappa}B{alpha} degradation was measured in cytosolic extracts by Western blot analysis. Representative of 4 independent experiments.

 
Src kinase inhibitor does not prevent CCK-8- or TNF-{alpha}-induced NF-{kappa}B activation. In pancreatic acinar cells, Src kinases have been implicated as upstream modulators of PKC in response to CCK-8 (12, 54, 55). Src kinases have also been linked to NF-{kappa}B activation in a number of cell types (1, 10, 27). To investigate whether Src kinases are involved in the activation of NF-{kappa}B in pancreatic acinar cells, we applied PP2, a specific inhibitor of Src kinases. Pretreatment of pancreatic acini with PP2 inhibited neither NF-{kappa}B binding activity nor I{kappa}B{alpha} degradation induced by CCK-8 (Fig. 6). Similarly, PP2 had no effect on NF-{kappa}B activation induced by TNF-{alpha} (Fig. 6). On the other hand, PP2 almost completely inhibited tyrosine phosphorylation of PKC-{delta} induced by CCK-8 and TNF-{alpha} (Fig. 7). PP1, another Src kinase inhibitor, also prevented CCK-8-induced and TNF-{alpha}-induced tyrosine phosphorylation of PKC-{delta}, whereas it had no effect on the NF-{kappa}B activation. The inactive analog PP3 was ineffective on the tyrosine phosphorylation of PKC-{delta} (data not shown). These results indicate that Src kinases mediate tyrosine phosphorylation of PKC-{delta} but are not involved in NF-{kappa}B activation induced by CCK-8 and TNF-{alpha} in pancreatic acini.



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  		Fig. 6. Effects of Src kinase inhibitor on NF-{kappa}B activation induced by CCK-8 and TNF-{alpha}. Pancreatic acini were preincubated with Src kinase inhibitor, PP2 (20 µM), or DMSO for 3 h and then stimulated with 100 nM CCK-8 or 100 ng/ml of TNF-{alpha} for 30 min. A: NF-{kappa}B binding activity was measured in nuclear extracts by EMSA. B: I{kappa}B{alpha} degradation was measured in cytosolic extracts by Western blot analysis. Representative of 4 independent experiments.

 


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  		Fig. 7. Effects of Src kinase inhibitor on tyrosine phosphorylation of PKC-{delta}. Pancreatic acini were preincubated with Src kinase inhibitor PP2 (20 µM) or DMSO for 3 h and then stimulated with 100 nM CCK-8 or 100 ng/ml of TNF-{alpha} for 30 min. Whole cell lysates were immunoprecipitated with anti-PKC-{delta} antibody and then subjected to SDS-PAGE and blotted by using anti-phosphotyrosine antibody (top). Equal protein loading was verified by using PKC-{delta} antibody after stripping the membranes (bottom). Representative of 4 independent experiments.

 
CCK-8 activates the novel PKC isoforms and NF-{kappa}B through both PI-specific PLC and PC-specific PLC, whereas TNF-{alpha} activates them through only PC-specific PLC. Previous studies (23, 29, 33, 34, 40) demonstrated that CCK-8 activates PKC through activation of PLC, which results in the hydrolysis of PI as well as PC, resulting in the production of DAG. To investigate whether the activation of PLC is involved in the NF-{kappa}B activation in pancreatic acini, we applied U-73122, an inhibitor of PI-specific PLC inhibitor, and D-609, a PC-specific PLC inhibitor. In D-609-treated cells, NF-{kappa}B activation after stimulation with CCK-8 or TNF-{alpha} was significantly attenuated (Fig. 8). U-73122 prevented the NF-{kappa}B activation induced by CCK-8 but not that by TNF-{alpha} (Fig. 8). U-73122 did not affect the TNF-{alpha}-induced NF-{kappa}B activation at concentrations up to 30 µM (data not shown). As shown in Fig. 9, the CCK-8-induced translocation of PKC-{delta} and -{epsilon} was prevented by both U-73122 and D-609. In contrast, only D-609 inhibited the translocation of PKC-{delta} and -{epsilon} induced by TNF-{alpha}. These results indicate that the activation of NF-{kappa}B and the novel PKCs by CCK-8 is regulated through both PI-specific PLC and PC-specific PLC, whereas TNF-{alpha}-induced responses only involve PC-specific PLC pathway.



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  		Fig. 8. Effects of PLC inhibitors on NF-{kappa}B activation induced by CCK-8 and TNF-{alpha}. Pancreatic acini were preincubated for 3 h with phosphatidylinositol (PI)-specific PLC inhibitor U-73122 (10 µM) or for 30 min with phosphatidylcholine (PC)-specific PLC inhibitor D-609 (50 µM) and then stimulated for 30 min with 100 nM CCK-8 or 100 ng/ml TNF-{alpha}. D-609 was added to the culture medium 30 min before the stimulation, because longer incubation with this inhibitor was toxic for pancreatic acini. A: NF-{kappa}B binding activity was measured in nuclear extracts by EMSA. B: I{kappa}B{alpha} degradation in cytosolic extracts was measured by Western blot analysis. Representative of 3 independent experiments.

 


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  		Fig. 9. Effects of PLC inhibitors on PKC translocation induced by CCK-8 and TNF-{alpha}. Pancreatic acini were preincubated for 3 h with PI-specific PLC inhibitor U-73122 (10 µM) or for 30 min with PC-specific PLC inhibitor D-609 (50 µM) and then stimulated for 30 min with 100 nM CCK-8 (A) or 100 ng/ml TNF-{alpha} (B). Cytosolic and membrane fractions were subjected to SDS-PAGE and blotted by using antibody specific for PKC-{delta} or -{epsilon}. Shown are representative blots from 3 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we investigated the intracellular signaling pathways that mediate NF-{kappa}B activation in rat pancreatic acini. The results indicate that translocation of novel PKC isoforms PKC-{delta} and -{epsilon} is necessary for both CCK-8-induced and TNF-{alpha}-induced NF-{kappa}B activation. Our results show that Src kinases regulate tyrosine phosphorylation of PKC-{delta}, but they do not mediate NF-{kappa}B activation induced by CCK-8 or TNF-{alpha}. Thus tyrosine phosphorylation of PKC-{delta} is not involved in the NF-{kappa}B activation. The activation of PKC-{delta}, PKC-{epsilon}, and NF-{kappa}B with CCK-8 requires both PI-specific PLC and PC-specific PLC pathways, whereas with TNF-{alpha} they require only PC-specific PLC for activation.

To date, four PKC isoforms ({alpha}, {delta}, {epsilon}, and {zeta}) have been identified in the pancreatic acinar cell, each with unique modes of activation (4, 37). However, stimuli-induced changes in kinase activity of each isoform have not been quantitatively characterized. Our results demonstrated the distinct responses of the PKC isoforms. CCK-8 and TNF-{alpha} increased both the kinase activity of PKC-{delta} and -{epsilon} and their translocation from the cytosolic to membrane fractions. Both stimuli increased the kinase activity of PKC-{zeta}, whereas the effect on its translocation was not apparent. It is possible that translocation of PKC-{zeta} at a very weak level is involved in the responses or that CCK-8 and TNF-{alpha} activate PKC-{zeta} without its translocation. On the other hand, we found no increases in either the kinase activity or the translocation of PKC-{alpha} with both CCK-8 and TNF-{alpha}. This result is consistent with the previous study (37) in which CCK-8 did not cause translocation of PKC-{alpha}. Further studies are necessary to understand why CCK-8 does not activate PKC-{alpha} when it causes increases in both DAG and intracellular Ca2+ (57). Similar unresponsiveness of PKC-{alpha} to DAG and Ca2+ has been reported in T84 human intestinal epithelial cells (46).

In general, activation of PKCs is associated with their translocation to distinct intracellular compartments in which each isoform performs its specific function (8, 16, 38). In the present study, the translocation inhibitor peptides {delta}V1–1 and {epsilon}V1–2, designed to competitively inhibit the binding of PKC-{delta} and -{epsilon} to specific anchoring proteins (6, 11), prevented the increases in kinase activity and translocation of their target PKC isoforms. These results indicate that translocation of PKC-{delta} or -{epsilon} is the necessary step for their activation by both CCK-8 and TNF-{alpha} in pancreatic acini. Of note, {delta}V1–1 and {epsilon}V1–2 did not cross inhibit the novel PKC isoforms, indicating the high specificity of these peptide inhibitors. To our knowledge, they have not been previously applied to study the role of PKC in pancreatic acinar cells.

Another regulatory pathway for PKC activation is tyrosine phosphorylation (8, 26, 35, 38). Among the PKC family, PKC-{delta} is the most efficiently tyrosine phosphorylated isoform (15, 26). Recent studies have demonstrated that CCK-8 causes rapid tyrosine phosphorylation of PKC-{delta} in rat pancreatic acini (53, 54) and that Src kinases play a key role in this process (54). We found that TNF-{alpha} also induced tyrosine phosphorylation of PKC-{delta}. Furthermore, pretreatment of pancreatic acini with PP2, a specific inhibitor of Src kinases, abolished the tyrosine phosphorylation of PKC-{delta} induced by CCK-8 and TNF-{alpha}.

PKC-{delta} and -{epsilon} translocation inhibitors prevented both the CCK-8-induced and TNF-{alpha}-induced NF-{kappa}B activation, determined by NF-{kappa}B binding activity and I{kappa}B{alpha} degradation. These results indicate that PKC-{delta} and -{epsilon} are key mediators of the NF-{kappa}B activation in pancreatic acinar cells. Because each isoform-specific inhibitor prevented NF-{kappa}B activation to about the same degree without affecting the kinase activity and localization of the other PKC isoforms, PKC-{delta} and -{epsilon} regulate NF-{kappa}B activation independently at the level of I{kappa}B{alpha} degradation or upstream. In contrast, neither the inhibitor of conventional PKC isoforms nor the PKC-{zeta} inhibitor prevented the NF-{kappa}B activation. Of note, the conventional PKC isoform inhibitor Gö6976 augmented the NF-{kappa}B activation in response to both CCK-8 and TNF-{alpha}. These results indicate that constitutive activity of PKC-{alpha} may have an inhibitory effect on NF-{kappa}B activation.

The Src kinase inhibitor PP2 did not prevent the NF-{kappa}B activation by CCK-8 and TNF-{alpha}, whereas it completely inhibited the tyrosine phosphorylation of PKC-{delta}. Thus it seems likely that neither the activation of Src kinases nor tyrosine phosphorylation of PKC-{delta} is required for NF-{kappa}B activation. It has recently been shown (54) that tyrosine kinase inhibitors, including PP2, blocked the increase in kinase activity of PKC-{delta} stimulated by CCK-8 in pancreatic acini, whereas it had no or little effect on translocation of PKC-{delta}. In conjunction with these data, our results suggest that the NF-{kappa}B activation depends primarily on the translocation of PKC-{delta} but not on kinase activity of PKC-{delta}.

Our results show that the activation of the novel PKCs and NF-{kappa}B by CCK-8 involves both PI-specific PLC and PC-specific PLC. It has been shown that both PI and PC are the main precursors of DAG generation in pancreatic acinar cells after CCK stimulation (23, 29, 33, 34, 57). On the other hand, TNF-{alpha} activated the novel PKCs and NF-{kappa}B in pancreatic acini through only PC-specific PLC. The activation of PC-specific PLC by TNF-{alpha} (28, 43, 45) and the subsequent activation of NF-{kappa}B have been shown in a number of cell types (2, 28, 36, 43–45), and the TNF receptor 1 is associated with the process (2, 28). Importantly, the TNF receptor 1 has been shown to mediate the inflammatory response in pancreatitis (9) and is functional on pancreatic acinar cells (17). Considering these findings, we hypothesize that TNF-{alpha} accelerates the inflammatory response by producing DAG through PC-specific PLC. In turn, DAG mediates NF-{kappa}B activation by promoting translocation of PKC-{delta} and -{epsilon}.

In conclusion, the results of the present study demonstrate several key elements leading to NF-{kappa}B activation in pancreatic acinar cells (Fig. 10). CCK-8 and TNF-{alpha} initiate NF-{kappa}B activation by different PLC pathways; nevertheless, the PLC pathways converge to promote translocation of PKC-{delta} and -{epsilon}, which in turn mediates NF-{kappa}B activation. Because of the key role of NF-{kappa}B activation in the mechanism of pancreatitis, the results implicate an important role for the novel PKC isoforms in pancreatitis.



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  		Fig. 10. Signaling pathways involved in NF-{kappa}B activation induced by CCK-8 and TNF-{alpha} in pancreatic acinar cells. Binding of CCK-8 to its receptor activates both PI-specific and PC-specific PLC, whereas TNF-{alpha} only activates PC-specific PLC. Activation of PLC leads to diacylclycerol (DAG) generation, promotes translocation of PKC-{delta} and -{epsilon}, which in turn mediates I{kappa}B{alpha} degradation and NF-{kappa}B activation. CCK-8 and TNF-{alpha} also induce PKC-{zeta} activation, but it is not involved in NF-{kappa}B activation. Constitutive activity of PKC-{alpha} exerts an inhibitory effect on NF-{kappa}B activation. Although tyrosine phosphorylation of PKC-{delta} is induced by Src, this event is not involved in NF-{kappa}B activation induced by CCK-8 and TNF-{alpha}.

 

   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The study was supported by the Department of Veterans Affairs Merit Grants, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-59508, and National Insititue on Alcohol Abuse and Alcoholism Grant P50-AA-11999 (to the Research Center for Alcoholic Liver and Pancreatic Diseases).


   ACKNOWLEDGMENTS
 
We thank Yoon Jung for help in preparing the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. S. Gukovskaya, Veterans Affairs Greater Los Angeles Healthcare System, West Los Angeles Healthcare Center, Bldg. 258, Rm. 340, 11301 Wilshire Blvd., Los Angeles, CA 90073 (E-mail: agukovsk{at}ucla.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.


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