Research Center for Alcoholic Liver and Pancreatic Injury, Veterans Affairs Greater Los Angeles Healthcare System, and the University of California, Los Angeles, California 90073
Submitted 20 February 2003 ; accepted in final form 28 August 2003
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ABSTRACT |
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alcoholic pancreatitis; inflammatory response; cholecystokinin; calcineurin
Although the complete molecular mechanism of pancreatitis has not been established, recent evidence suggests a critical role for the inflammatory response in this disease (5, 11, 30, 36, 43). Results from our group and others (6, 10, 11, 13, 16, 18-20, 36, 48, 53) indicate that the inflammatory response is initiated by injured pancreatic acinar cells that produce inflammatory mediators, such as cytokines (e.g., tumor necrosis factor-) and adhesion molecules (e.g., intercellular adhesion molecule-1), ultimately leading to systemic complications. Recent data (8, 10, 18-20, 22, 31, 38, 44, 48, 53) suggest that the transcription factor NF-
B is a key regulator of inflammation in pancreatitis. The importance of NF-
B activation in both acute and chronic inflammatory states is that it upregulates a multitude of proinflammatory cytokines, chemokines, and other inflammatory mediators (51).
In our report demonstrating the sensitizing effect of ethanol diet on pancreatitits (31), we found marked NF-B activation as well as cytokine upregulation occurring in the rats that received the ethanol diet and the low-dose CCK-8 but not in the rats receiving control diet and low-dose CCK-8. These results suggested that the sensitizing effect of ethanol on pancreatitis was due, at least in part, to its ability to augment NF-
B activation in the pancreas. Of note, at the same time, we found that the ethanol diet alone attenuated the basal NF-
B activity in the pancreas (31). We also reported (17) that ethanol (as well as its oxidative metabolite acetaldehyde) decreased the basal NF-
B activity in isolated pancreatic acini.
NF-B comprises a family of transcription factors regulating the inflammatory, immune, and cell death responses (12, 51). NF-
B is composed of hetero- or homodimers of the Rel family proteins. In most nonstimulated cells, NF-
B is kept inactive in the cytoplasm by association with the inhibitory (I
B) proteins. On activation, I
Bs are phosphorylated by specific I
B kinases (IKKs) and rapidly degraded via the proteasome-involving pathway. NF-
B then translocates into the nucleus and activates the expression of genes that have
B-binding sites in their promoters/enhancers.
Many characteristics of pancreatitis and, in particular, NF-B activation can be studied ex vivo in isolated pancreatic acini stimulated with supraphysiological doses of CCK-8 or its analog cerulein (6, 15, 18-22, 35, 42, 50, 53). The mechanisms of NF-
B activation in the pancreatic acinar cell remain poorly understood. It has recently been shown (21, 45) that intracellular Ca2+ mobilization and PKC, two major signaling pathways activated by CCK-8 (50), mediate CCK-induced NF-
B activation in isolated pancreatic acini or lobules. However, no in vitro model reproducing the effects of ethanol on NF-
B that we observed in vivo (31) has been developed. The roles of Ca2+ and PKC pathways in ethanol's effects on NF-
B activation in pancreas have not been addressed.
The mechanisms through which Ca2+ and PKC regulate NF-B in other systems are tissue and stimulus specific. For example, Ca2+ can regulate NF-
B through Ca2+-dependent protein phosphatase 2b (calcineurin) (9, 46, 49) or through Ca2+-dependent ("conventional") isoforms of PKC (12). Ca2+-independent ("novel" and "atypical") PKC isoforms were also shown to mediate NF-
B activation in several cell types (7, 25, 34). The PKC isoforms that mediate NF-
B activation in the pancreatic acinar cell have not been determined. Of note, several PKC isoforms, including conventional
and
, novel
and
, and atypical
, were detected in pancreatic acinar cells (4, 33).
In the present study, we applied a model of isolated pancreatic acini to investigate in vitro the effect of ethanol on CCK-induced NF-B activation. We found that the in vitro ethanol treatment had similar effects to those that we observed in ethanol-fed rats, namely, augmentation of the CCK-8-induced activation of NF-
B and attenuation of the basal NF-
B activity in acinar cells. Ethanol prevented NF-
B activation induced by thapsigargin, an agent that directly mobilizes the intracellular Ca2+. Our results indicate that the inhibitory effect of ethanol on NF-
B is likely mediated through the Ca2+/calcineurin pathway and that the augmentation effect is mediated through PKC signaling pathways. Furthermore, the results indicate that the nonconventional Ca2+-independent isoforms of PKC mediate NF-
B activation in pancreatic acinar cells. These divergent effects of ethanol on NF-
B activation may play a role in the ability of ethanol to sensitize pancreas to the inflammatory response and pancreatitis.
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MATERIALS AND METHODS |
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Measurement of free cytosolic Ca concentration. Cytosolic Ca concentration ([Ca2+]i) was monitored by the change in the fluorescence intensity of fura-2-loaded cells, as we described previously (32). Acini were incubated for 3 h at 37°C with or without 100 mM ethanol and for the last 30 min with 2 µM fura-2 AM. Cells were then washed twice by centrifugation and resuspended in a buffer containing (in mM) 20 HEPES (pH 7.4), 120 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, 10 sodium pyruvate, and 10 ascorbic acid with 0.1% BSA and 0.01% soybean trypsin inhibitor, with or without ethanol. [Ca2+]i responses to CCK-8 or thapsigargin were measured at 37°C by monitoring fura-2 fluorescence in a Shimadzu RF-1501 spectrofluorimeter with excitation at 340 and 380 nm and emission at 510 nm.
Preparation of protein extracts. Nuclear and cytosolic protein extracts were prepared as we described previously (6, 16, 18, 19, 31, 48, 53). Briefly, pancreatic acini were lysed on ice in a hypotonic buffer A (16) 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 20-25 min, then 0.3% Igepal CA-630 was added, and the nuclei were collected by microcentrifugation. The supernatant (cytosolic protein) was saved for Western blot analysis of IB, and the nuclear pellet was resuspended and incubated at 4°C for up to 1 h in a high-salt buffer C (16) supplemented with 1 mM PMSF, 1 mM DTT, and the protease inhibitor cocktail described above. Membrane debris were pelleted by microcentrifugation for 10 min, and the clear supernatant (nuclear extract) was aliquoted and stored at -80°C. Protein concentrations in the nuclear and cytosolic extracts were determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).
EMSA. For the EMSA, aliquots of nuclear extracts with equal amounts of protein (10-20 µg) were mixed in 20-µl reactions with a buffer containing (in mM) 10 HEPES (pH 7.8), 50 KCl, 0.1 EDTA, and 1 DTT with 10% glycerol and 3 µg poly(dI-dC). Binding reactions were started by addition of 32P-labeled DNA probe and incubated at room temperature for 20 min. The probe for NF-B was 5'-GCAGAGGGGACTTTCCGAGA, and that for NFATc was 5'-CGCCCAAAGAGGAAAATTTGTTTCATA (binding motifs underlined). They were annealed to corresponding complementary oligonucleotides and end-labeled using T4 polynucleotide kinase.
Samples were electrophoresed at room temperature on a 4.5% native 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).
Western blot analysis. For IB analysis, the cytosolic extracts (see Preparation of protein extracts) were adjusted to equal protein concentration, fractionated by SDS-PAGE, and electrophoretically transferred onto a nitrocellulose membrane. The membranes were blocked by overnight incubation in Tris-buffered saline supplemented with 5% dry milk and probed with antibodies against I
B
, as we described previously (18, 19). Band intensities in the immunoblots were quantified by densitometry using Scion Image software (Scion, Frederick, MD).
Measurement of lactate dehydrogenase release. Acinar cell necrosis was determined by release of lactate dehydrogenase (LDH) into incubation medium. LDH activity was measured spectrofluorimetrically as the production of NAD from pyruvic acid and NADH, as we described previously (15).
Statistical analysis of data. This was done using two-tailed Student's t-test. P values <0.05 were considered statistically significant.
Reagents. CCK-8 was from American Peptide (Sunnyvale, CA); medium 199 was from GIBCO-BRL (Grand Island, NY); [-32P]ATP was from ICN Biomedicals (Costa Mesa, CA); fura-2 AM and thapsigargin were from Molecular Probes (Eugene, OR); cyclosporin A, FK506, GF-109203X, Ro-32-0432, and Go-6976 were from Calbiochem (La Jolla, CA); T4 polynucleotide kinase was from New England BioLaboratories (Beverly, MA); I
B
antibody was from Santa Cruz Biotechnology (Santa Cruz, CA); poly(dI-dC) was from Boehringer Mannheim (Indianapolis, IN); and enhanced chemiluminescence kit was from Pierce (Rockford, IL). All other chemicals were from Sigma (St. Louis, MO).
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RESULTS |
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In acini treated with ethanol, 0.1 nM CCK-8 induced NF-B binding activity, whereas it did not activate NF-
B in acini not treated with ethanol (Fig. 1, A and B). Furthermore, in ethanol-treated acini, 100 nM CCK-8 produced greater NF-
B activation than without ethanol (Fig. 1, B and C). Thus ethanol potentiates NF-
B activation caused by both maximal and supramaximal doses of CCK-8. The effect of ethanol on CCK-induced NF-
B activation was dose and time dependent. At 3 h incubation, the sensitization was only observed with 100 mM but not with 50 mM ethanol. However, the sensitizing effect of 50 mM ethanol was detected at 5 h incubation with ethanol (Fig. 1C).
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Figure 1, A and B, also shows that ethanol treatment decreased basal NF-B binding activity by
30%. Thus ethanol had two effects on NF-
B in pancreatic acinar cells: it decreased the basal NF-
B activity and potentiated the CCK-8-induced NF-
B activation. As a result, in ethanol-treated cells, the NF-
B response to 100 nM CCK-8 was two times greater than in cells not treated with ethanol (Fig. 1C).
The effects of ethanol on NF-B in isolated pancreatic acini are similar to those we found in vivo in an experimental model of ethanol + CCK pancreatitis (31). Although the conditions of the in vivo and in vitro applications of ethanol are different, in both models ethanol potentiates CCK-8-induced NF-
B activation while having an inhibitory effect on NF-
B basal activity. Of note, in our in vivo model, alcohol concentration in the blood of rats continuously fed ethanol is in the range of 20-120 mM (1, 3, 31, 47).
Ethanol was reported to affect osmotic balance of the incubation medium (2, 27). To test whether the observed effects of ethanol were caused by changes in osmolarity, we measured the effects of hyperosmolarity on the CCK-8-induced NF-B activation. Pancreatic acini were incubated for 3 h in the presence or absence of 100 mM mannitol and then stimulated for 30 min with 100 nM CCK-8. In the presence of mannitol, CCK-8 increased NF-
B binding activity 1.5 ± 0.2-fold, and in the absence of mannitol increased it 1.7 ± 0.2-fold (n = 3), the responses being statistically not different. These results suggest that changes in osmolarity are not a major factor in the effect of ethanol on NF-
B activation in pancreatic acinar cells.
We and others (18, 20, 45) reported that CCK-8-induced NF-B activation in the pancreatic acinar cell is associated with degradation of the inhibitory I
B proteins. Figure 1D shows that incubation with ethanol potentiated the CCK-8-induced degradation of I
B
. In particular, 0.1 nM CCK-8 stimulated I
B
degradation in ethanol-treated but not in control acini, and 100 nM CCK-8 produced a greater degradation of I
B
in acini treated with ethanol than in control acini. The greater degradation of I
B
in cells treated with CCK-8 in the presence of ethanol (Fig. 1D) correlates with the increased NF-
B binding activity in these preparations (Fig. 1, A-C). We also observed increased basal levels of I
B
(Fig. 1D) in acini treated with ethanol, correlating with the diminished basal NF-
B binding activity (Fig. 1A).
Effects of ethanol on NF-B activation induced by Ca2+ mobilization. As stated in the introduction, previous studies (21, 45) have demonstrated that Ca2+ mobilization mediates, in part, the effect of CCK-8 on NF-
B activation. To determine whether ethanol regulates NF-
B binding activity through effects on the Ca2+-mobilization pathway, we measured the effects of ethanol on NF-
B activation induced by thapsigargin, an agent that directly causes intracellular Ca2+ mobilization bypassing the receptor. Figure 2 shows that thapsigargin stimulated NF-
B activation in acini incubated for 3 h in the absence of ethanol. The stimulation of NF-
B binding activity by thapsigargin was
1.5-fold compared with a twofold increase with 100 nM CCK-8. Previously, thapsigargin has been reported to induce NF-
B activation in freshly isolated rat pancreatic acini (21) or lobules (45).
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Pretreatment with ethanol greatly inhibited both the thapsigargin-induced NF-B activation (Fig. 2, A and B) and I
B
degradation (Fig. 2C). Thus ethanol produced opposite effects on the CCK-8- and thapsigargin-induced NF-
B activation in pancreatic acini: ethanol potentiated the former but inhibited the latter response (Figs. 1 and 2). These results show that the augmenting effect of ethanol on CCK-8-induced NF-
B activation cannot be through ethanol's effect on the Ca2+ mobilization pathway alone.
To further elucidate the contribution of Ca2+ mobilization to the NF-B responses, we applied BAPTA-AM, an intracellular Ca2+ chelator. In separate experiments, we tested that 40 µM BAPTA-AM prevented both the CCK-8- and thapsigargin-induced [Ca2+]i responses in pancreatic acini incubated for 3 h (not illustrated). The results in Fig. 3 show that BAPTA partially inhibited NF-
B activation induced by CCK-8 and almost completely inhibited NF-
B activation induced by thapsigargin. The extent of the inhibitory effect of BAPTA on the CCK-8-induced NF-
B activation was the same in cells treated and not treated with ethanol (not illustrated). These results indicate that Ca2+ mobilization contributes to both the CCK- and thapsigargin-induced NF-
B activation. However, as distinct from thapsigargin, CCK-8-induced NF-
B activation is also mediated through Ca2+-independent mechanisms.
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To test the possibility that ethanol could affect Ca2+-dependent pathways of NF-B activation by directly affecting Ca2+ signals, we measured the effects of ethanol treatment on [Ca2+]i changes induced by CCK-8 and thapsigargin in pancreatic acini. The [Ca2+]i responses to both 0.1 and 100 nM CCK-8 are composed of the initial peak, resulting from intracellular Ca2+ mobilization, followed by an elevated [Ca2+]i plateau, resulting from Ca2+ influx (32, 50). The [Ca2+]i response to thapsigargin also shows the peak-and-plateau pattern (26, 52), but the peak is less abrupt and smaller in magnitude than in the CCK-induced [Ca2+]i response. The data in Table 1 show that ethanol had no significant effects on either the peak or plateau of the CCK-8-induced [Ca2+]i signal. The [Ca2+]i response to thapsigargin was also not significantly affected by ethanol. Ethanol did not affect the basal [Ca2+]i level in rat pancreatic acini (Table 1).
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These data indicate that ethanol affects Ca2+-dependent pathways of NF-B activation by targeting signals downstream of Ca2+ mobilization. Indeed, under conditions used, ethanol had no effect on the thapsigargin- or CCK-8-induced [Ca2+]i signals, whereas it affected the NF-
B responses.
One mediator that can link NF-B activation and intracellular Ca2+ mobilization is the Ca2+-dependent phosphatase calcineurin (9, 21, 45, 46, 49). Calcineurin inhibitors, cyclosporin A and FK506, have been shown to inhibit amylase secretion by both CCK-8 and thapsigargin (14). We found that cyclosporin A and FK506 markedly inhibited NF-
B response induced by thapsigargin in pancreatic acini (Fig. 4A), the extent of their effect being similar to that of BAPTA (Fig. 3). These results suggest that thapsigargin-induced NF-
B activation is predominantly mediated by calcineurin. To further demonstrate that thapsigargin activates calcineurin in pancreatic acinar cells, we measured the effect of thapsigargin on the transcription factor nuclear factor of activated T cells (NFAT), a downstream target of calcineurin (9). Thapsigargin stimulated NFAT binding activity, which was prevented by both cyclosporin A and FK506 (Fig. 4B). The specificity of our binding assay (which used the distal NFATc binding site from murine IL-2 promoter) was shown in a "cold competition" experiment (Fig. 4C).
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In contrast to what we observed for thapsigargin, the calcineurin inhibitors did not affect the CCK-8-induced NF-B activation in cells treated both with and without ethanol (Fig. 5). These data indicate that the Ca2+/calcineurin pathway is not engaged in the CCK-8-induced NF-
B activation in rat pancreatic acini.
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The combined results of Figs. 1, 2, 3, 4, 5 and Table 1 suggest that Ca2+ mobilization mediates NF-B activation induced by thapsigargin and CCK-8 through different mechanisms: calcineurin dependent for thapsigargin and calcineurin independent for CCK-8. The results indicate that the Ca2+/calcineurin pathway does not mediate the observed (Fig. 1) potentiation of CCK-8-induced NF-
B activation by ethanol.
Effects of ethanol on NF-B activation mediated through the PKC pathway. It has been shown previously (21, 45) by using general PKC inhibitors that PKC is involved in the CCK-8-induced NF-
B activation. The experiments presented in Figs. 6, 7, 8, 9 were performed to determine the role of the PKC pathway in the effects of ethanol and CCK-8 on NF-
B activation in pancreatic acini. For this purpose, we applied both broad-spectrum (GF-109203X and Ro-32-0432) PKC inhibitors and an inhibitor of the conventional Ca2+-dependent PKC isoforms, Go-6976 (28, 29). We tested that under conditions used in our experiments, these inhibitors prevented activation of their target PKC isoforms in rat pancreatic acini (37).
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The results in Figs. 6 and 7 show that the broad-spectrum PKC inhibitors prevented the NF-B activation induced by maximal (0.1 nM) and supramaximal (100 nM) CCK-8, both with and without the ethanol treatment. GF-109203X (Fig. 7) and Ro-32-0432 (not illustrated) caused inhibition of the CCK-8-induced I
B
degradation. The blockade of I
B
degradation by PKC inhibitors concomitantly with their inhibitory effect on the CCK-induced NF-
B binding activity (Fig. 6) indicates that PKC regulates the NF-
B response to CCK-8 at the level of I
B degradation or upstream.
In contrast to GF-109203X and Ro-32-0432, Go-6976, the inhibitor of conventional PKC isoform, did not block the CCK-8-induced NF-B activation in cells treated both with and without ethanol (Fig. 6). Also, Go-6976 did not inhibit the CCK-8-induced I
B
degradation (Fig. 7). These results indicate that the conventional Ca2+-dependent PKC isoforms do not mediate the CCK-8-induced NF-
B activation in rat pancreatic acinar cells.
To further explore the role of PKC in NF-B activation in ethanol-treated pancreatic acinar cells, we used PMA, a general activator of PKC. PMA activates both Ca2+-dependent and -independent PKC isoforms in rat pancreatic acini (33). Figure 8 shows that cell treatment with ethanol potentiated PMA-induced NF-
B activation in pancreatic acini, providing further evidence that PKC mediates ethanol's effects on NF-
B. We next examined the effects of PKC inhibitors on PMA-induced NF-
B activation. PMA-induced NF-
B activation in ethanol-treated acini was prevented by GF-109203X and Ro-32-0432 but was unaffected by Go-6976 (Fig. 9). Similar results were obtained in cells not treated with ethanol (not illustrated). The results with Go-6976 further indicate that activation of Ca2+-dependent PKC isoforms (which occurs with PMA treatment) does not contribute to NF-
B activation in pancreatic acinar cells.
The data presented in Figs. 6, 7, 8, 9 indicate that it is Ca2+-independent (novel and/or atypical) but not the Ca2+-dependent (conventional) PKC isoforms that mediate NF-B activation induced by both CCK-8 and PMA in pancreatic acinar cells and the augmentation of NF-
B responses by ethanol.
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DISCUSSION |
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Our results show that ethanol has diverse effects on the signaling pathways mediating NF-B activation in pancreatic acinar cells. Ethanol can cause both stimulation and inhibition of NF-
B activation, and it affects both Ca2+- and PKC-mediated pathways of NF-
B activation.
As was recently shown (21, 45) and corroborated by the results of the present study, CCK-8 causes NF-B activation through both Ca2+- and PKC-mediated pathways. Our data indicate that there are at least two different mechanisms through which Ca2+ mobilization causes activation of NF-
B in pancreatic acinar cells, one of which is employed by thapsigargin and the other by CCK-8. Our results with cyclosporin A and FK506 indicate that thapsigargin-induced NF-
B activation is mediated by the Ca2+-dependent protein phosphatase 2b (calcineurin). With the calcineurin inhibitors, we also found an inhibition of thapsigargin-induced activation of NFAT, a downstream target of calcineurin. Of note, calcineurin has recently been shown to mediate Ca2+-dependent NF-
B activation in other cell types (46, 49).
By contrast, the inhibitory analysis indicated that calcineurin is not engaged in the CCK-8-induced pathway of NF-B. The reason for these differences in the involvement of calcineurin is unknown. One possibility is that CCK-8 but not thapsigargin elicits a signal blocking the Ca2+/calcineurin pathway of NF-
B activation. The mechanisms through which Ca2+ mediates the CCK-8-induced NF-
B activation in pancreatic acinar cells remain to be determined.
Ethanol produced opposite effects on NF-B activation induced by thapsigargin and CCK-8. Ethanol inhibited the thapsigargin-induced while potentiating the CCK-8-induced NF-
B activation. Ethanol's effects were not due to its direct action on [Ca2+]i signals elicited by thapsigargin or CCK-8. One mechanism of the inhibitory effect of ethanol on thapsigargin-induced NF-
B activation could be through inhibition of the Ca2+/calcineurin pathway by ethanol. Indeed, both ethanol and the calcineurin inhibitors markedly inhibited thapsigargin-induced NF-
B activation (as did the intracellular Ca2+ chelator BAPTA).
As stated above, our results indicate that the Ca2+/calcineurin pathway of NF-B activation does not contribute significantly to the NF-
B response to CCK-8. This may be one reason why ethanol does not inhibit (but instead potentiates) the NF-
B response to CCK-8. Another factor underlying the differential effects of ethanol on thapsigargin- and CCK-8-induced NF-
B activation, as discussed below, is the involvement of the PKC pathway in the latter but not the former response.
The mechanisms through which ethanol inhibits the basal NF-B activity remain to be determined. As we have shown (6), this activity is caused by stresses of the process of acinar cell isolation from the pancreas. We have also reported that ethanol and its oxidative metabolite, acetaldehyde, decreased basal NF-
B activity in isolated acini (17). One explanation for the decreased basal NF-
B activity in ethanol-treated cells, compared with cells not treated with ethanol, could be an inhibitory effect of ethanol on the Ca2+/calcineurin pathway of NF-
B activation. However, this mechanism remains to be determined.
Our data suggest that the potentiation of the CCK-8-induced NF-B activation by ethanol is mediated by ethanol's effect on the PKC pathway. We found that the CCK-8-induced NF-
B activation in cells both treated and not treated with ethanol was blocked by general PKC inhibitors. In contrast, a specific inhibitor of conventional Ca2+-dependent PKC isoforms did not inhibit the CCK-8-induced NF-
B activation. We also observed similar effects of the PKC inhibitors on NF-
B activation induced by PMA. Thus our results indicate that the conventional isoforms of PKC are not involved in either CCK-8-induced or PMA-induced NF-
B activation. It is the Ca2+-independent (novel and/or atypical) PKC isoforms that regulate NF-
B activation in pancreatic acinar cells. Of note, both the conventional and the novel and atypical isoforms of PKC are expressed in pancreatic acinar cells (4, 33, 37). In other cell types, both Ca2+-dependent and -independent isoforms of PKC have been shown to mediate NF-
B activation (7, 12, 25, 34). However, the mechanisms through which PKC isoforms regulate NF-
B are not well understood.
Our findings suggest that the effect of ethanol to augment CCK-8-induced NF-B activation results from ethanol's actions on the PKC pathway. First, we found that ethanol treatment augmented the effect of PMA on NF-
B activation. Second, PKC inhibitors prevented the augmented NF-
B activation by CCK-8 in ethanol-treated acini. The mechanisms through which ethanol affects PKC-mediated pathways of NF-
B activation in pancreatic acinar cells remain to be determined. Ethanol could directly affect PKC activity or regulate PKC-mediated signaling. The finding that ethanol augmented the CCK-8-induced degradation of I
B
suggests that ethanol's effects on PKC are upstream of I
B degradation, possibly at the level of IKK activation. Recent evidence indicates that IKKs are regulated through phosphorylation or protein-protein interaction by a variety of kinases, including PKC isoforms (12). Our results do not exclude the possibility that pathways other than those that are PKC mediated contribute to the potentiation of CCK-8-induced NF-
B activation by ethanol. In particular, ethanol may directly affect IKKs or upstream kinases unrelated to PKC.
In conclusion, the results of the present study demonstrate that ethanol augments the NF-B response caused by CCK-8 in pancreatic acinar cells while inhibiting the basal NF-
B activity and NF-
B activation induced by thapsigargin. These effects on isolated acini are similar to ethanol's effects that we observed in vivo with ethanol feeding (31). The inhibition of NF-
B by ethanol is likely mediated through the Ca2+/calcineurin pathway, and the augmentation effect is likely mediated through signaling pathways involving novel or atypical PKC isoforms. These divergent effects of ethanol on NF-
B may play a role in the ability of ethanol to sensitize pancreas to the inflammatory response and pancreatitis.
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ACKNOWLEDGMENTS |
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GRANTS
The study was supported by the Department of Veterans Affairs, the National Institute of Diabetes and Digestive and Kidney Diseases (Grant DK-59508), and the Research Center for Alcoholic Liver and Pancreatic Diseases [National Institute on Alcohol Abuse and Alcoholism (NIAAA) Grant P50-A11999]. S. Hosseini was supported by the Training Program in Alcoholic Liver and Pancreatic Diseases (T32-AA-07578) funded by the NIAAA.
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FOOTNOTES |
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REFERENCES |
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