Caerulein-induced NF-kappa B/Rel activation requires both Ca2+ and protein kinase C as messengers

Yusuke Tando, Hana Algül, Martin Wagner, Hans Weidenbach, Guido Adler, and Roland M. Schmid

Department of Medicine I, University of Ulm, 89070 Ulm, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The eukaryotic transcription factor NF-kappa B/Rel is activated by a large variety of stimuli. We have recently shown that NF-kappa B/Rel is induced during the course of caerulein pancreatitis. Here, we show that activation of NF-kappa B/Rel by caerulein, a CCK analog, requires increasing intracellular Ca2+ levels and protein kinase C activation. Caerulein induces a dose-dependent increase of nuclear NF-kappa B/Rel binding activity in pancreatic lobules, which is paralleled by degradation of Ikappa Balpha . Ikappa Bbeta was only slightly affected by caerulein treatment. Consistent with an involvement of Ca2+, the endoplasmic reticulum-resident Ca2+-ATPase inhibitor thapsigargin activated NF-kappa B/Rel in pancreatic lobules. The intracellular Ca2+ chelator TMB-8 prevented Ikappa Balpha degradation and subsequent nuclear translocation of NF-kappa B/Rel induced by caerulein. BAPTA-AM was less effective. Cyclosporin A, a Ca2+/calmodulin-dependent protein phosphatase (PP2B) inhibitor, decreased caerulein-induced NF-kappa B/Rel activation and Ikappa Balpha degradation. The inhibitory effect of bisindolylmaleimide suggests that protein kinase C activity is also required for caerulein-induced NF-kappa B/Rel activation. These data suggest that Ca2+- as well as protein kinase C-dependent mechanisms are required for caerulein-induced NF-kappa B/Rel activation.

pancreas; signaling; acute pancreatitis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN RODENTS, SUPRAMAXIMAL stimulation of the pancreas with the CCK analog caerulein causes morphological, biochemical, and pathophysiological alterations similar to acute pancreatitis in humans (1, 21). The hormone-receptor mechanism underlying this phenomenon has been well characterized and involves the low-affinity state of the CCK receptor (14). Receptor binding of CCK causes an initial release of Ca2+ from intracellular stores via inositol 1,4,5-trisphosphate, which is maintained by subsequent influx of Ca2+ into the cell leading to an increase in the free ionized cytosolic Ca2+ concentration ([Ca2+]i) (41). Physiological doses of CCK are known to produce oscillations in [Ca2+]i, which are believed to be important in organizing and controlling complex intracellular functions (25). Supramaximal doses of CCK produce a single, large, sustained increase in [Ca2+]i in acinar cells, which is known to be associated with cell damage in several cell types, and may be implicated in the pathogenesis of acute pancreatitis (24, 28). A corresponding increase in 1,2-diacylglycerol is observed with high doses of caerulein in excess of that required to stimulate a maximal secretory response (44).

In addition to these signaling events, supramaximal doses of caerulein produce a high but transient increase of p42map and p44map as well as other upstream components of the mitogen-activated protein kinase signaling cascade, including mitogen-activated protein kinase kinase and Ras (5, 10, 11). Furthermore, supramaximal doses of caerulein induced a dramatic increase in c-Jun NH2-terminal kinase (JNK) activity (5). The pronounced increase in JNK activity may reflect the response to cellular stress.

The transcription factor NF-kappa B is a trans-acting factor that binds to enhancer elements involved in the immune and inflammatory response [2, 36 (reviewed)]. This transcription factor is formed by different homo- and heterodimers of members of this now called NF-kappa B/Rel family. In unstimulated cells, NF-kappa B/Rel complexes are found in the cytoplasm bound to Ikappa Balpha and Ikappa Bbeta , which prevent the dimers from entering the nucleus. When cells are activated, Ikappa Bs are phosphorylated within minutes, causing their rapid degradation by proteasomes (2, 4, 36). The release of NF-kappa B/Rel from Ikappa Bs results in the passage of NF-kappa B/Rel into the nucleus where it binds to specific sequences in the promoter regions of target genes. Therefore, Ikappa B proteins control the presence of NF-kappa B/Rel binding activity in the nucleus. Many stimuli activate NF-kappa B/Rel including cytokines, activators of protein kinase C (PKC), lipopolysaccharides, and oxidative stress [2, 36 (reviewed)]. We and others have recently shown that the transcription factor NF-kappa B/Rel is activated following caerulein pancreatitis (12, 17, 18, 38).

In an attempt to characterize signaling events involved in caerulein-induced NF-kappa B/Rel activation, we investigated DNA binding and nuclear translocation of NF-kappa B/Rel complexes in pancreatic lobules. Caerulein-induced Ikappa Balpha degradation and subsequent nuclear translocation of NF-kappa B/Rel activation can be prevented by the intracellular Ca2+ chelator 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) and to a lesser extent by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM as well as by the Ca2+/calmodulin-dependent protein phosphatase (PP2B) inhibitor cyclosporin A. The inhibitory effect of bisindolylmaleimide (BIS) suggests that PKC activity is also required for caerulein-induced NF-kappa B/Rel activation. These data suggest that Ca2+ as well as PKC-dependent mechanisms are required for caerulein-induced NF-kappa B/Rel activation in pancreatic acinar cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Caerulein was purchased from Pharmacia (Erlangen, Germany). Phorbol 12-myristate 13-acetate (PMA), thapsigargin, and cyclosporin A were from Sigma Chemical (Deisenhofen, Germany). BAPTA-AM, TMB-8, and BIS (GF-109203X) were from Calbiochem (La Jolla, CA). All other chemicals were of the highest purity commercially available and were obtained from Sigma.

Male Wistar rats (250-300 g body wt) were obtained from the breeding colony of Ulm University Animal Facilities. They were housed in nalgene shoebox cages under a 12:12-h light-dark cycle with free access to standard diet and water. All animal experiments were conducted according to the guidelines of the local Animal Use and Care Committees and executed according to the National Animal Welfare Law.

Preparation of pancreatic lobules. Pancreatic lobules were prepared by a modified method previously described (33, 43). In brief, after an overnight fast, rats were killed by exsanguination under light ether anesthesia. The pancreas was removed and incubated in DMEM (GIBCO Life Technologies, Paisley, Scotland).

Equal quantities of lobules were incubated in medium for 15 min at 37°C under continuous oxygenation in a shaking water bath. After this adaptation period, lobules were either incubated with BIS (1, 5, 10, or 15 µM), BAPTA-AM (20 or 40 µM), TMB-8 (250, 500, or 1,000 µM), or cyclosporin A (100 or 500 nM) as pretreatment or left with medium alone. Thereafter, lobules were stimulated with caerulein (0.1, 1, 10, or 100 nM), PMA (100 ng/ml), or thapsigargin (10 µM). After the respective incubation periods, lobules were immediately frozen in liquid nitrogen and stored at -70°C.

Protein extracts. Nuclear protein extracts were prepared essentially as described by Dignam et al. (7), with some modifications as follows. Pancreatic lobules were homogenized in a sucrose buffer containing protease inhibitors. Nuclei were separated by centrifugation, and proteins were eluted using a high-salt buffer as previously described (38).

For cytoplasmic protein extracts, pancreatic lobules were homogenized in 150 mM NaCl, 50 mM Tris · HCl, 50 mM CaCl2, 1.0% NP-40, 5 mM natrium fluoride, 0.1 M phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 35 µg/ml pepstatin, 10 µg/ml aprotinin, and 0.5 mM DTT, pH 7.2, and centrifuged at 15,000 rpm for 20 min at 4°C. Aliquots of the supernatant were stored at -70°C. Protein concentrations were determined by the method of Bradford (Bio-Rad Laboratories, München, Germany).

Electrophoretic mobility shift assays. Electrophoretic mobility shift assays (EMSAs) were performed as previously described (38). The DNA probe used for EMSAs corresponded to the high-affinity kappa B sequences found in the mouse kappa -light-chain enhancer and in the HIV-1 promoter region. Two oligonucleotides were annealed to generate a double-stranded probe: sense 5'-AGCTT<UNL>GGGGACTTTCC</UNL>ACTAGTACG-3' and antisense 5'-AATTCGTACTAGT<UNL>GGAAAGTCCCC</UNL>A-3' (the binding sites are underlined). Labeling was accomplished by treatment with Klenow in the presence of dGTP, dCTP, dTTP, and [32P]dATP. Labeled oligonucleotides were purified on push columns (Stratagene, Heidelberg, Germany). Labeled double-stranded probe (80,000 cpm) was added to 10 µg of nuclear protein in the presence of 5 µg poly(dIdC) as nonspecific competitor (Pharmacia Biotech Enzyme, Freiburg, Germany). Binding reactions were carried out in 10 mM Tris · HCl, pH 7.5, 100 mM NaCl, and 4% glycerol for 30 min at 4°C.

DNA protein complexes were resolved by electrophoresis on a 4% nondenaturing polyacrylamide gel in 1× Tris-glycine-EDTA buffer. Gels were vacuum dried and exposed to Kodak Bio Max MS-1 film at -70°C with intensifying screens. Competition was performed by adding specific unlabeled double-stranded oligonucleotide to the reaction mixture in 10-, 50-, or 100-fold molar excess.

Western blotting. Cellular protein extracts were analyzed by immunoblotting. Samples were diluted in SDS-PAGE loading buffer in a ratio of 1:5 and heated at 97.5°C for 10 min. Recombinant Ikappa Balpha or Ikappa Bbeta was prepared by transfecting 293 human embryonic kidney cells with the respective eukaryotic expression vector as previously described and loaded as controls (data not shown) (34). Protein complexes were resolved by electrophoresis on 10% nondenaturing polyacrylamide gels in 1× Tris-glycine-SDS buffer at room temperature. After SDS-PAGE, the gels were transferred to 0.45-µm polyvinylidene difluoride membranes for 25 min at 40 mA at room temperature (Schleicher & Schuell, Dassel, Germany). Nonspecific binding was blocked in 5% (wt/vol) skim milk in Tris-buffered saline (TBS), pH 7.5, at 4°C. Blots were then incubated for 1 h with primary antibodies Ikappa Balpha or Ikappa Bbeta (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:1,000 in 5% (wt/vol) skim milk powder in TBS, washed three times with 0.05% (vol/vol) Tween 20 in TBS (T-TBS), and incubated for 1 h with a secondary antibody, goat anti-rabbit IgG peroxidase (Dianova-Immunotech, Hamburg, Germany) at a dilution of 1:5,000 in 5% (wt/vol) skim milk powder in TBS. After blots were washed three times with T-TBS, they were developed with enhanced chemiluminescence reagents (Amersham Buchler, Braunschweig, Germany).

Immunohistochemistry. To analyze cell types contributing to NF-kappa B/Rel activity, nuclear translocation of RelA (p65) was visualized using immunohistochemistry as previously described (42). Pancreatic lobules were pretreated with or without the inhibitors BAPTA-AM, TMB-8, or BIS followed by stimulation with caerulein or left with medium as control. After indicated time points, lobules were shock frozen in liquid nitrogen. Frozen sections (4 µm) were air dried overnight and fixed with 4% methanol-free formalin (Polyscience, Warrington, PA) in PBS at room temperature for 15 min. After a short wash with washing solution (PBS containing 0.02% Tween 20), samples were sequentially treated with 0.1% Triton X-100 in PBS for 5 min, rinsed with washing solution twice, treated with 5 mg/ml RNase A in PBS for 30 min, and incubated in blocking solution (PBS containing 2% BSA and 3% normal goat serum) for 30 min at room temperature. Primary antibody (murine anti-p65, Boehringer Mannhein) was diluted 1:200 in blocking solution and incubated overnight at 4°C in a humidified chamber. Negative controls were incubated with a nonspecific mouse IgG in blocking solution. After the sections were washed five times for 5 min in washing solution, sections were incubated with the secondary antibody at 1:2,000 (anti-mouse Alexa 568, Molecular Probes, Eugene, OR) for 30 min in a dark, humidified chamber followed by five washes for 5 min. Thereafter, sections were counterstained for 15 min with a solution containing Yo-Pro-1 iodide (Molecular Probes) at 1:2,500 in 0.2× PBS followed by three washes in 0.2× PBS. Sections were mounted and kept in the dark until analysis with a confocal microscope (Leica, TCS 4D, Heidelberg, Germany).


    RESULTS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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Dose- and time-dependent induction of NF-kappa B/Rel by caerulein. Pancreatic lobules were used for studying NF-kappa B/Rel activation in vitro. To study the effect of different doses of caerulein on NF-kappa B/Rel binding activity, EMSAs were performed. Pancreatic lobules were incubated with increasing doses of caerulein, and nuclear extracts were prepared and incubated with an end 32P-labeled DNA oligonucleotide containing the recognition site of NF-kappa B. Although very little NF-kappa B/Rel binding activity was detected in the unstimulated state, caerulein induced NF-kappa B/Rel binding in a gradual manner. Maximal activation of the inducible DNA-binding activity required 100 nM caerulein. The specificity of NF-kappa B/Rel DNA binding induced by caerulein was confirmed in competition experiments (data not shown). In unstimulated cells, NF-kappa B/Rel heterodimers are kept as inactive complexes in the cytoplasm by inhibitory proteins, such as Ikappa Balpha and Ikappa Bbeta . After stimulation, the Ikappa Bs are phosphorylated and then degraded, and free NF-kappa B/Rel dimers translocate into the nucleus. We tested whether the degradation of Ikappa Balpha or Ikappa Bbeta could take place after treatment of pancreatic lobules with caerulein at different doses. Cytoplasmic extracts were analyzed by Western blotting using anti-Ikappa Balpha and anti-Ikappa Bbeta antibodies. The cytoplasmic Ikappa Balpha signal almost completely disappeared at 1 and 10 nM caerulein and was completely absent when caerulein was added at 100 nM. Protein levels of Ikappa Bbeta were not affected following caerulein treatment for 30 min (data not shown). To study time-dependent induction of NF-kappa B/Rel, pancreatic lobules were stimulated with caerulein at 100 nM over time. Caerulein-mediated induction of NF-kappa B/Rel binding was detected after 10 min with a maximum at 30 min and thereafter decreased gradually. To test the degradation of Ikappa Balpha and Ikappa Bbeta after treatment with caerulein, cytoplasmic extracts were analyzed by Western blotting using anti-Ikappa Balpha and anti-Ikappa Bbeta antibodies. Thirty minutes after treatment with 100 nM caerulein, cytoplasmic Ikappa Balpha levels were almost completely reduced compared with unstimulated cells. After 60 min, Ikappa Balpha reappeared and was back to original levels at 120 min. Cytoplasmic Ikappa Bbeta levels decreased only slightly at 60 and 120 min after caerulein treatment (data not shown).

Inhibition of endoplasmic reticulum-resident Ca2+-ATPase induces NF-kappa B/Rel activation in pancreatic lobules. The compound thapsigargin inhibits the endoplasmic reticulum (ER)-resident Ca2+-ATPase, thereby causing a rapid efflux of Ca2+ from the ER lumen into the cytoplasm. Pancreatic lobules were incubated with 10 µM thapsigargin for 30 min. Nuclear extracts were prepared and incubated with an end 32P-labeled DNA oligonucleotide containing the recognition site of NF-kappa B. Compared with unstimulated pancreatic lobules, thapsigargin activated NF-kappa B/Rel binding activity after 30 min (Fig. 1A, compare lanes 1 and 2). This activation can be reduced by preincubation of pancreatic lobules for 15 min with 20 and 40 µM BAPTA-AM or with 500 µM TMB-8, effective intracellular Ca2+ chelators (lanes 3-6), whereas 250 µM TMB-8 had only a slight inhibitory effect. To further assess the potential action of intracellular Ca2+ in pancreatic lobules, the capacity of BAPTA-AM and TMB-8 to influence thapsigargin degradation of Ikappa Balpha and Ikappa Bbeta was assayed. Doses of 20 and 40 µM BAPTA-AM as well as 250 and 500 µM TMB-8 prevented the Ikappa Balpha degradation induced by thapsigargin (Fig. 1B, lanes 3-6), with BAPTA-AM being more effective.



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Fig. 1.   Inhibition of endoplasmic reticulum-resident Ca2+-ATPase induces NF-kappa B/Rel activation in pancreatic lobules. A: pancreatic lobules were prepared and pretreated with or without 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) followed by stimulation with 10 µM thapsigargin for 30 min. Nuclear cell extracts were prepared, and equal amounts of protein (10 µg) were used in electromobility shift analysis with the kappa B motif of the mouse kappa -light-chain enhancer as probe. B: Western blot analysis of cytoplasmic extracts treated as in A. Antibodies directed against Ikappa Balpha and Ikappa Bbeta were used as described in MATERIALS AND METHODS.

Ca2+ chelators interfere with caerulein-induced NF-kappa B/Rel activation. To investigate whether intracellular Ca2+ is required for caerulein-induced NF-kappa B/Rel activation, pancreatic lobules were preincubated with 20 or 40 µM BAPTA-AM or 250 or 500 µM TMB-8 for 15 min and then stimulated with 100 nM caerulein for 30 min (Fig. 2A). Thereafter, nuclear extracts were prepared and assayed for NF-kappa B/Rel activity. NF-kappa B/Rel activation is inhibited by pretreatment with Ca2+ chelators, with TMB-8 being most effective. BAPTA-AM caused only a slight reduction in caerulein-mediated NF-kappa B/Rel binding activity (lanes 3 and 4). Neither BAPTA-AM nor TMB-8 exerted a detectable effect on the basal levels of NF-kappa B/Rel activity as judged by EMSA (data not shown). Preincubation of pancreatic lobules with TMB-8 abrogated caerulein-stimulated degradation of Ikappa Balpha at 250 and 500 µM (Fig. 2B, lanes 5 and 6). Pretreatment with BAPTA-AM also inhibited Ikappa Balpha degradation but to a much lesser extent (lanes 3 and 4).



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Fig. 2.   Ca2+ chelators interfere with caerulein-induced NF-kappa B/Rel activation. A: pancreatic lobules were preincubated with BAPTA-AM for 15 min (lanes 3 and 4), TMB-8 (lanes 5 and 6), or left untreated (lanes 1 and 2), after which they were stimulated with 100 nM caerulein for 30 min (lanes 2-6). Nuclear extracts were prepared and assayed in an electrophoretic mobility shift assay (EMSA) using the kappa -light-chain kappa B site as probe. B: cytoplasmic extracts from samples used in A were analyzed in Western blots using antibodies directed against Ikappa Balpha and Ikappa Bbeta .

Cyclosporin A pretreatment decreased caerulein-induced NF-kappa B/Rel activation. The immunosuppressive drug cyclosporin A acts by blocking a Ca2+-mediated signaling pathway contributing to the induction of NF-kappa B/Rel (13, 37). Pancreatic lobules were incubated with 100 or 500 nM cyclosporin A for 30 min. Nuclear extracts were prepared and assayed for kappa B binding activity. Cytoplasmic extracts were analyzed using Western analysis. NF-kappa B/Rel binding activity was suppressed following pretreatment with cyclosporin A (Fig. 3A, lanes 3 and 4). Cyclosporin A prevents the inducible degradation of Ikappa Balpha when stimulated with caerulein (Fig. 3B, lanes 3 and 4). These results indicate that calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is required for caerulein-mediated activation of NF-kappa B/Rel.



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Fig. 3.   Cyclosporin A (CsA) inhibits caerulein-induced NF-kappa B/Rel activation. A: pancreatic lobules were pretreated with 100 or 500 nM cyclosporin A for 15 min (lanes 3 and 4) followed by stimulation with 100 nM caerulein for 30 min (lanes 2 to 4). Lane 1, control. Nuclear extracts were assayed for kappa B binding activity in an EMSA. B: cytoplasmic extracts were analyzed in Western blots using antibodies against Ikappa Balpha and Ikappa Bbeta .

PMA activates NF-kappa B/Rel in pancreatic lobules. Pancreatic lobules were preincubated with or without 5, 10, or 15 µM BIS for 15 min followed by stimulation with the active phorbol ester PMA (100 ng/ml) and harvested after 30 min. Nuclear extracts were prepared and assayed for DNA binding activity of NF-kappa B. Cytoplasmic extracts were used for Western analysis. PMA induced NF-kappa B/Rel binding activity compared with controls (Fig. 4A, compare lanes 1 and 2). Pretreatment with BIS blocked PMA-induced NF-kappa B/Rel activation completely (lanes 3-5). Degradation of Ikappa Balpha induced by PMA was sustained by preincubation with BIS at all doses tested (Fig. 4B).



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Fig. 4.   Phorbol 12-myristate 13-acetate (PMA) induces NF-kappa B/Rel binding activity in pancreatic lobules. A: pancreatic lobules were pretreated with or without 5, 10, or 15 µM bisindolylmaleimide (BIS) for 15 min followed by stimulation with 100 ng/ml PMA for 30 min. Extracts were prepared, and equal amounts of protein (10 µg) were used in electromobility shift analysis with the kappa B motif of the mouse kappa -light-chain enhancer as probe. B: Western blot analysis of cytoplasmic extracts treated as in A. Antibodies directed against Ikappa Balpha and Ikappa Bbeta were used as described in MATERIALS AND METHODS.

NF-kappa B/Rel activation by caerulein requires PKC. Pancreatic lobules were pretreated with or without 1, 5, 10, or 15 µM BIS for 15 min followed by stimulation with or without 100 nM caerulein for 30 min. Nuclear and cytoplasmic extracts were prepared and assayed in EMSAs using the kappa -light-chain enhancer high-affinity kappa B site as probe and Western analysis with anti-Ikappa Balpha and anti-Ikappa Bbeta antibodies. BIS caused a slight induction of NF-kappa B/Rel activity at 1 and 5 µM but had no effect on basal NF-kappa B/Rel activity at 10 and 15 µM (Fig. 5, lanes 3-6). Caerulein-induced NF-kappa B/Rel activity was efficiently blocked by BIS at 5, 10, and 15 µM, with the highest dose being most effective. Degradation of Ikappa Balpha by caerulein was already sustained by BIS at 1 µM and completely blocked by BIS at 5, 10, or 15 µM (Fig. 5, lanes 8-10).


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Fig. 5.   Caerulein induction of NF-kappa B/Rel activation is dependent on protein kinase C. Top: pancreatic lobules were pretreated with or without 1, 5, 10, or 15 µM BIS for 15 min followed by stimulation with or without 100 nM caerulein for 30 min. Nuclear extracts were prepared and assayed in EMSAs using the kappa -light-chain enhancer high-affinity kappa B site as probe. Bottom: cytoplasmic extracts were prepared from samples as in top and used for Western analysis with anti-Ikappa Balpha and anti-Ikappa Bbeta .

Supramaximal doses of caerulein induce NF-kappa B/Rel activation in acinar cells. In unstimulated cells, NF-kappa B/Rel is bound to the inhibitory Ikappa B subunits. To monitor its activation at a cellular level, a monoclonal antibody can be used that recognizes an epitope that includes the nuclear localization signal of the RelA (p65) subunit. This antibody therefore recognizes RelA (p65) only when Ikappa B was not bound to p65 (20). To determine active RelA (p65) in acinar cells, pancreatic lobules were prepared and incubated with or without 100 nM caerulein for 10, 30, or 60 min. Lobules were shock frozen, sectioned, fixed, stained with the anti-p65 antibody (red fluorescence), and counterstained with Yo-Pro to stain nuclei (green fluorescence). Overlaying the red fluorescence with the green counterstain results in a yellow nuclear staining indicative of nuclear localization of active RelA (p65). Active nuclear RelA (p65) was observed as early as 30 min after treatment with 100 nM caerulein as seen by the yellow nuclear staining (Fig. 6B). No nuclear staining of RelA (p65) was observed within the first 10 min (Fig. 6A). The amount of nuclear immunoreactivity for RelA (p65) further increased after 60 min (Fig. 6C) and 120 min (data not shown). The saline-treated control showed very weak staining after 60 min (Fig. 6F). No nuclear staining for RelA (p65) was observed at earlier time points (Fig. 6, D and E).


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Fig. 6.   Nuclear localization of RelA (p65) in acinar cells in pancreatic lobules after treatment with caerulein. Frozen section of pancreatic lobules were stained with an anti-p65 antibody recognizing only the active Ikappa B-dissociated RelA (p65). RelA (p65)-specific immunoreactivity is indicated by red fluorescence. Nuclei were stained with Yo-Pro and appear as green fluorescence. Overlaying the green fluorescence of the nuclear counterstaining with red fluorescence results in a yellow nuclear staining indicative of nuclear localization of active RelA (p65). Nuclear RelA (p65) is observed 30 min after treatment with caerulein (B) but not after 10 min (A) nor in saline-treated controls (D-F). Nuclear staining further increases after 60 min of treatment in the caerulein group (C). Original magnification = ×150.

Pretreatment with BAPTA-AM, TMB-8, or BIS abrogates caerulein induced NF-kappa B/Rel activation in acinar cells. Pretreatment of pancreatic lobules with 40 µM BAPTA-AM (Fig. 7A), 1,000 µM TMB-8 (Fig. 7B), or 15 µM BIS (Fig. 7C) markedly reduced the nuclear staining of RelA (p65) after a 30-min treatment with 100 nM caerulein compared with the control, which did not receive an inhibitor (Fig. 7D). The nuclear translocation of RelA (p65) was not completely abolished in lobules pretreated with BAPTA-AM, indicative of nuclear staining for RelA (p65) in some cells (Fig 7A).


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Fig. 7.   BAPTA, TMB-8, and BIS prevent nuclear translocation of RelA (p65) in acinar cells. RelA (p65) was visualized by immunohistochemistry using an anti-p65 antibody recognizing only active RelA (p65). Pancreas lobules were pretreated with 40 µM BAPTA-AM (A), 1,000 µM TMB-8 (B), or 15 µM BIS (C) before incubation with 100 nM caerulein for 30 min. Control lobules were stimulated with caerulein only (D). Nuclear localization of RelA (p65) is markedly reduced after preincubation with either BAPTA-AM, TMB-8, or BIS. Original magnification = ×150.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously shown that high doses of caerulein lead to the induction of the transcription factor NF-kappa B/Rel (38). Because active NF-kappa B/Rel translocates directly to the nucleus and induces gene transcription, this establishes a novel signal transduction pathway between the CCK receptor and the cell nucleus. NF-kappa B/Rel is detectable within minutes after stimulation of pancreatic lobules with high doses of caerulein. Both Ikappa Balpha and Ikappa Bbeta contribute to nuclear NF-kappa B/Rel binding activity. Whereas Ikappa Balpha is degraded in the initial phase and correlates with rapid NF-kappa B/Rel induction, decreased Ikappa Bbeta levels might be responsible for prolonged NF-kappa B/Rel activation.

The present study was undertaken to determine the second messenger molecules that mediate this activation. Caerulein, similar to CCK on binding to its receptors on acinar cells, activates phospholipase C, generates inositol trisphosphate, and induces free intracellular Ca2+. The observation that inhibition of the ER-resident Ca2+-ATPase, which causes rapid release of Ca2+ from the organelle, activates NF-kappa B/Rel in pancreatic lobules suggested a role for caerulein-mediated Ca2+ release in NF-kappa B activation in acinar cells. We show here that two structurally unrelated Ca2+ chelators inhibit NF-kappa B/Rel activation in response to caerulein. Whereas one inhibitor, TMB-8, inhibited NF-kappa B/Rel activation in response to caerulein efficiently, a second inhibitor, BAPTA-AM, was less effective. This may be explained by the different mechanisms of action of the two inhibitors. Moreover, cyclosporin A, an inhibitor of the Ca2+/calmodulin-dependent protein phosphatase (PP2B), decreased caerulein-mediated NF-kappa B/Rel. These data indicate that free intracellular Ca2+ is necessary at a postreceptor signaling step for caerulein/CCK-mediated activation of NF-kappa B/Rel, as measured by DNA binding activity and enhancement of Ikappa Balpha degradation.

Interestingly, NF-kappa B/Rel is strongly activated only at supramaximal doses of caerulein known to cause a single, large sustained increase in [Ca2+]i in acinar cells. In contrast, physiological doses of CCK known to produce oscillations in [Ca2+]i do not activate NF-kappa B/Rel, suggesting that the NF-kappa B/Rel activation is mediated by the low-affinity state of the CCK-A receptor (24, 25, 28, 44). In agreement with these data, JMV-180, known to activate the high-affinity state of the CCK-A receptor and to induce [Ca2+]i oscillations, did not activate NF-kappa B/Rel in the pancreas (18). Furthermore, a recent report suggested that [Ca2+]i activation of different transcription systems in B cells can differ greatly with regard to the amount of free intracellular Ca2+ required (39). In that system, NF-kappa B is selectively activated by a large transient [Ca2+]i rise and nuclear factor for activation of transcription (NFAT) is already turned on by a low, sustained Ca2+ plateau, whereas neither factor is induced by [Ca2+]i oscillations (8, 9). The activation of NF-kappa B/Rel by Ca2+ ionophores on their own has been only observed in lymphocytes. This cell type-specific regulation is characterized by a synergistic PKC and Ca2+-dependent phosphorylation and degradation of Ikappa Balpha (37). Among the numerous agents that induce NF-kappa B/Rel activity in epithelial cells, only stimulation with epidermal growth factor or ER stress-inducing conditions may require input from free intracellular Ca2+ (26, 39). In U-937 monocytic cells, sphingosine-1-phosphate activates NF-kappa B/Rel in a Ca2+-dependent manner (32). Sphingosine-1-phosphate-induced NF-kappa B/Rel activation was inhibited by cyclosporin A, suggesting a role for the Ca2+/calmodulin-dependent phosphatase calcineurin. Furthermore, Ca2+-dependent pathways that induce the phosphatase calcineurin synergize with PKC to degrade Ikappa Balpha and result in the activation of NF-kappa B/Rel (13, 37).

The activation of phospholipase C generates at least two second messengers, one is Ca2+ and the other is diacylglycerol, which stimulate PKC. An increase in intracellular Ca2+ may activate Ca2+/phospholipid-dependent PKC isozymes. Moreover, the Ca2+ chelator TMB-8 has been reported to have effects on phospholipid metabolism and PKC (27). We therefore assessed whether activation of PKC played a role in caerulein-mediated NF-kappa B/Rel activation. Caerulein-induced NF-kappa B/Rel activation can be abrogated by pretreatment with a PKC-inhibitor that is more specific than the ones currently available (40). Although the mechanism of blocking NF-kappa B translocation by PKC-inhibitors is unclear at this time, our current results indicate that PKC is upstream of Ikappa Balpha phosphorylation and its inhibition selectively abrogates caerulein-induced Ikappa Balpha degradation in pancreatic lobules. Previous studies indicated that in vitro phosphorylation of Ikappa Balpha by different kinases, including PKC, was sufficient to dissociate NF-kappa B from Ikappa Balpha in cytosolic extracts and that, in vivo, stimuli such as PMA activate both PKC and NF-kappa B (16, 35).

PKC was originally identified as a Ca2+- and phospholipid-dependent protein kinase and subsequently shown to be activated by diacylglycerol and phorbol esters such as PMA. Five PKC isoforms have been detected in pancreatic acini from rats, including alpha , delta , epsilon , theta , and zeta  (3, 29, 30). Whereas PKC-alpha belongs to the conventional PKC family, being Ca2+ dependent, this property is not shared by the delta -, epsilon -, and theta -isoforms, which are part of the novel PKC subfamily. PKC-zeta , an atypical PKC, shows phospholipid-dependent kinase activity. So far only the isoforms PKC-epsilon and PKC-zeta have been reported to activate kappa B motifs and therefore are likely candidates to mediate the stimulatory effects on NF-kappa B/Rel activation in pancreatic acinar cells (15, 19). Treatment of acini with CCK caused translocation of PKC-epsilon and enhanced the immunostaining pattern of PKC-epsilon in the apical region of acinar cells (3). Interestingly, PMA has been shown to cause translocation of PKC-zeta in acinar cells, although PKC-zeta lacks a phorbol ester binding site and is not activated by diacylglycerol (3). This suggests that other isoforms of PKC can mediate the phosphorylation of PKC-zeta and thereby induce its translocation. Recently, two central Ikappa B kinases have been identified (6, 31). These kinases trigger the phosphorylation and subsequent proteolytic degradation of Ikappa Balpha . Two other upstream kinases, the NF-kappa B-inducing kinase and the mitogen-activated protein kinase/ERK kinase, as well as other signaling components including c-Raf and p90rsk1 have been shown to be involved in the NF-kappa B activation pathway (22, 23). It remains to be seen whether these steps are activated and required for caerulein-induced NF-kappa B/Rel activation.

In summary, our data suggest that Ca2+- as well as PKC-dependent mechanisms are required for caerulein-induced NF-kappa B/Rel activation in pancreatic lobules.


    ACKNOWLEDGEMENTS

We thank Sonja Aigner for assistance with manuscript preparation and Günther Schneider for helpful discussion.


    FOOTNOTES

This work was in part supported by grants from the Bundesministerium für Bildung und Forschung to R. M. Schmid and from the Deutsche Forschungsgemeinschaft to G. Adler.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. M. Schmid, Dept. of Medicine I, Univ. of Ulm, Robert-Koch-Str. 8, D-89070 Ulm, Germany (E-mail: roland.schmid{at}medizin.uni-ulm.de).

Received 22 February 1999; accepted in final form 9 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastroint Liver Physiol 277(3):G678-G686
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