CCK stimulates mob-1 expression and NF-kappa B activation via protein kinase C and intracellular Ca2+

Bing Han and Craig D. Logsdon

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Supraphysiological concentrations of cholecystokinin (CCK) induce chemokine expression in rat pancreatic acini through the activation of the transcription factor NF-kappa B. In the current study, the intracellular signals involved in these pathophysiological effects of CCK were investigated. CCK induction of mob-1 expression in isolated rat pancreatic acini was blocked by the protein kinase C (PKC) inhibitors GF-109203X and Ro-32-0432 and by the intracellular Ca2+ chelator BAPTA. CCK induced NF-kappa B nuclear translocation, and DNA binding was also blocked by GF-109203X and BAPTA. Direct activation of PKC with TPA induced mob-1 chemokine expression and activated NF-kappa B DNA binding to a similar extent as did CCK. Increasing intracellular Ca2+ using ionomycin had no effect on mob-1 mRNA levels or NF-kappa B activity. Both CCK and TPA treatments decreased inhibitory kappa B-alpha (Ikappa B-alpha ) levels, whereas ionomycin had no effect. However, the effects of TPA on Ikappa B-alpha degradation were less complete than for CCK. In combination, TPA and ionomycin degraded Ikappa B-alpha to a similar extent as CCK. Therefore, activation of NF-kappa B and mob-1 expression by supraphysiological CCK is likely mediated by both PKC activation and elevated intracellular Ca2+.

pancreas; acinar cells; pancreatitis; chemokine; cholecystokinin; nuclear factor-kappa B


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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CHOLECYSTOKININ (CCK) is both a hormone and a neurotransmitter involved in various aspects of gastrointestinal function, including stimulation of gallbladder contraction, inhibition of gastric emptying, and induction of satiety (26). CCK also influences pancreatic function. Actions of CCK on the rodent pancreas are mediated by CCK-A receptors, which are G protein-coupled receptors. Physiological activation of CCK receptors on pancreatic acinar cells leads to secretion of digestive enzymes and has a general trophic action on the organ. However, supramaximal stimulation of these receptors leads to an acute inflammatory response resembling aspects of the clinically important disease acute pancreatitis (41). This latter observation has led to the widespread use of supramaximal concentrations of the CCK analogue caerulein as an experimental model of acute pancreatitis.

Of particular importance for the development of experimental pancreatitis may be the induction of chemokines and cytokines in pancreatic cells. We have previously shown that the induction of chemokines is due to the ability of supraphysiological concentrations of CCK to activate the transcription factor NF-kappa B (19). NF-kappa B/Rel transcription factors bind to enhancer elements stimulating the expression of a variety of genes involved in immune and inflammatory responses. NF-kappa B was originally identified as a nuclear factor that bound to the enhancer element of the immunoglobulin kappa light chain gene (39). Functional NF-kappa B is composed of hetero- or homodimeric combinations of NF-kappa B/Rel proteins. The prototypical NF-kappa B complex is a heterodimer containing p50 and p65 subunits. Other members of the NF-kappa B/Rel family of proteins are c-Rel, NF-kappa B1 (p50/p105), NF-kappa B2 (p52/p100), Rel A (p65), Rel B, and the Drosophila proteins Dorsal, Dif, and Relish (1). These proteins interact through NH2-terminal Rel homology domains. The Rel homology domains also function in DNA binding and in interaction with inhibitor proteins known as Ikappa Bs.

The mechanism of activation of NF-kappa B has been an intensive area of study due to its importance in many inflammatory diseases. In most cells, NF-kappa B is sequestered in the cytoplasm through interactions with the Ikappa B family of inhibitory proteins. Activation of NF-kappa B by a wide variety of stimuli such as mitogens, cytokines, bacterial lipopolysaccharide, viral infection, and ultraviolet light is thought to involve the dissociation of NF-kappa B from Ikappa Bs (36). Stimulation of cells with inducers of NF-kappa B leads to rapid phosphorylation, ubiquitination, and degradation of Ikappa Bs. NF-kappa B is then released and translocates into the nucleus where it activates the expression of target genes. Therefore, early studies implicated Ikappa B phosphorylation as a crucial step for NF-kappa B activation. Recently, it was shown that two highly related serine kinases, IKK-alpha and IKK-beta , are activated in response to the NF-kappa B inducer tumor necrosis factor-alpha (TNF-alpha ) and are responsible for the phosphorylation of Ikappa Bs (49). These kinases appear to be downstream of MEKK1, a component of the mitogen-activated protein kinase signaling cascade. However, other kinases, including p90rsk (13), have also been reported to phosphorylate Ikappa B. Furthermore, there appear to be important cell type-specific differences in the mechanisms involved in NF-kappa B signaling (5, 6, 21).

In pancreatic acinar cells, the mechanisms involved in the activation of NF-kappa B by CCK remain unknown. In the current study, we investigated the major signaling pathways activated by CCK for their effects on NF-kappa B and mob-1 chemokine expression. We observed that the ability of CCK to stimulate mob-1 gene expression or to activate NF-kappa B was completely blocked by inhibition of protein kinase C (PKC) or chelation of intracellular Ca2+. Activation of PKC using the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) also activated NF-kappa B and induced mob-1 expression. However, there were differences between the effects of TPA and CCK in terms of effects on Ikappa B-alpha . CCK caused a rapid and nearly complete decrease in Ikappa B-alpha levels. In contrast, TPA only caused a small decrease in Ikappa B-alpha levels. This difference was at least in part explained by an observed requirement for increased Ca2+ in the degradation of Ikappa B-alpha . Thus the effects of CCK on NF-kappa B and chemokine expression are likely due to the combined actions of PKC and Ca2+.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Materials. Chromatographically purified collagenase was purchased from Worthington Biochemical (Freehold, NJ). Soybean trypsin inhibitor (SBTI), beta -mercaptoethanol, phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, TPA, HEPES, and glutamine were obtained from Sigma Chemicals (St. Louis, MO). CCK was purchased from Research Plus (Bayonne, NJ). BAPTA-AM, Ro-32-0432, and ionomycin were purchased from Calbiochem (La Jolla, CA). Bisindolylmaleimide I (GF-109203X) was purchased from LC Laboratories (Woburn, MA). Enhanced chemiluminescence detection reagents (ECL), [gamma -32P]ATP, and [alpha -32P]dCTP were from Amersham (Arlington Heights, IL). An electrophoretic mobility shift assay systems kit was purchased from Promega (Madison, WI). The rabbit polyclonal antibodies to Ikappa B-alpha and the NF-kappa B subunits p65, p50, and c-Rel and the goat anti-rabbit IgG horseradish peroxidase conjugate were from Santa Cruz Biotechnology (Santa Cruz, CA). The phosphospecific Ikappa B-alpha antibody was from New England Biolabs (Beverly, MA). Eagle's minimal essential amino acids, guanidine thiocyanate, and agarose were from GIBCO-BRL Life Technologies (Gaithersburg, MD).

Pancreatic acini isolation and treatments. The preparation of pancreatic acini was performed as previously described (19). Briefly, pancreata from male Wistar rats were injected with collagenase (100 U/ml) and incubated at 37°C for 45-50 min with shaking (120 cycles/min). Acini were then dispersed by triturating the pancreas through polypropylene pipettes with decreasing orifice (3.0, 2.4, and 1.2 mm) and filtration through a 150-µm nylon mesh. Acini were purified by centrifugation through a solution containing 4% BSA and were resuspended in HEPES-buffered Ringer solution (HR, pH 7.5) supplemented with 0.2% glucose, Eagle's minimal essential amino acids, 2 mM glutamine, 0.1 mg/ml SBTI, and 0.5% BSA. The dispersed acini were separated into aliquots and treated with CCK and pharmacological agents at indicated concentrations in HR for specified times in tissue culture dishes. All treatments and incubations were conducted in a cell culture incubator at 37°C in a humidified atmosphere.

Isolation of RNA and analysis of mob-1 mRNA expression. Total RNA was isolated by a modified acid guanidinium-thiocyanate-phenol-chloroform extraction as previously described (19). RNA was quantitated spectrophotometrically: 25 µg of each sample were electrophoresed in 1% agarose and 2.2 M formaldehyde gels in MOPS buffer and were transferred to a nylon membrane. mob-1 mRNA was detected using a full-length 1.2-kb cDNA of rat mob-1 (25). Membranes were hybridized at high stringency using QuickHyb solution (Stratagene, La Jolla, CA) with the [alpha -32P]dCTP-labeled mob-1 probe at 68°C for 2 h. After hybridization, the membranes were exposed to a B-1 phosphoimaging screen and were visualized by the use of a GS-505 Molecular Imaging System (Bio-Rad Laboratories, Richmond, CA). Images were imported into Photoshop 4.0 (Adobe Systems Incorporated, San Jose, CA) for preparation of Figs. 1-6.

Preparations of nuclear extracts. Nuclear extracts were prepared using a modified version of the method of Maire et al. (27) as previously described (19). Pancreatic acini were collected by brief centrifugation and were washed with ice-cold PBS containing 1 mM EDTA. The pellets were then resuspended in 0.5 ml homogenization buffer containing 10 mM HEPES (pH 7.9), 2 M sucrose, 10% glycerol (vol/vol), 25 mM KCl, 150 mM spermine, 500 mM spermidine, and 2 mM EDTA to which 1 mM dithiothreitol (DTT) and a protease inhibitor cocktail containing 10 µg/ml each of aprotinin, leupeptin, and pepstatin were added before use. Cells were homogenized with a motor-driven pestle for 5-10 strokes on ice. Nuclei were collected by centrifugation at 62,000 g for 30 min at 4°C, washed with 1 ml PBS containing 1 mM EDTA, and then centrifuged at 14,000 g for 5 min at 4°C. The nuclei were resuspended in an appropriate volume (~100 µl) of ice-cold high-salt buffer containing 10 mM HEPES (pH 7.9), 10% glycerol (vol/vol), 0.42 M NaCl, 100 mM KCl, 3 mM MgCl2, and 0.1 mM EDTA, to which DTT and the protease inhibitor cocktail described above were added. The nuclear suspension was incubated on ice for 15-30 min with intermittent mixing and was centrifuged at 14,000 g for 5 min at 4°C. Protein concentration in the nuclear extract was determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA).

Immunoblot analysis. Dispersed acini were treated as described in the legends for Figs. 1-6. The treatments were terminated by washing the acini with ice-cold PBS containing 1 mM Na3VO4. For analysis of whole cell protein levels, the pellets were lysed by sonicating for 5 s in a solution containing 50 mM Tris · HCl (pH 7.5), 150 mM NaCl, 2 mM EGTA, 2 mM EDTA, 1% Triton X-100, 0.5 mM PMSF, and the protease inhibitor cocktail described above. The supernatant was removed as whole cell lysate and was assayed for protein by the Bio-Rad protein assay. For analysis of nuclear protein levels, nuclear extracts were prepared as described above. Equal amounts of protein (25 µg) were resolved by SDS-PAGE and were transferred to a nitrocellulose membrane. Ikappa B immunoblot analysis was performed as described previously (18) and visualized with ECL reagent on film or a screen. The NF-kappa B protein was detected by specific antibodies against subunit p65 of NF-kappa B. Film images were scanned with an Agfa Arcus II scanner (Bayer, Ridgefield Park, NJ) to create a digital image.

Electrophoretic mobility shift assay. Aliquots of nuclear extract with equal amounts of protein (6-12 µg) were used in 10-µl reactions in a buffer containing 10 mM HEPES (pH 7.9), 10% glycerol (vol/vol), 1 mM DTT, 1 µg poly(dI-dC), and 5 µg nuclease-free BSA as previously described (19). The binding reaction was started by addition of 10,000 counts/min of the 22-base pair oligonucleotide 5'-AGT TGA G<UNL>GG GAC TTT CC</UNL>C AGG C-3' containing the NF-kappa B consensus sequence (underlined; Promega) that had been labeled with [gamma -32P]ATP (10 mCi/mmol) by T4 polynucleotide kinase. The reaction was allowed to proceed for 30 min at room temperature. For cold competition experiments, unlabeled NF-kappa B oligonucleotide as a specific competitor or OCT1 (40) oligonucleotide (5'-TGT CGA ATG CAA ATC ACT AGA A-3') as a nonspecific competitor (300×) was added to the binding reaction 5 min before the addition of the radiolabeled probe. For the antibody supershift assays, 2 µg of specific antibodies to NF-kappa B protein subunits p65, p50, and c-Rel were incubated with nuclear extracts for 1 h at room temperature before addition of labeled probe. All reaction mixtures were subjected to PAGE on 4.5% gel in 0.5× TBE buffer (44.5 mM Tris base, 44.5 mM boric acid, and 1 mM disodium EDTA, pH 8.3) at 200 V. Gels were dried and directly exposed to a B-1 phosphoimaging screen.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CCK induction of mob-1 gene expression in pancreatic acinar cells is dependent on PKC activation and elevated intracellular Ca2+. Chemokine gene expression is an early event in the initiation of inflammatory responses during experimental pancreatitis (15). In the current study, we initially examined the effects of inhibiting the major intracellular signaling pathways activated by CCK in pancreatic acinar cells on the induction of a representative chemokine, mob-1. A major action of CCK in acinar cells is the activation of phospholipase C, leading to the generation of diacylglycerol (DAG) and inositol trisphosphate (IP3). DAG activates PKC, and IP3 causes the release of Ca2+ from intracellular stores. To study the role of PKC, we used the specific inhibitors bisindolylmaleimide I (GF-109203X; see Ref. 45) and Ro-32-0432 (47) and the PKC activator TPA. Pretreatment of acini with GF-109203X (20 µM) or Ro-32-0432 (10 µM) for 30 min completely blocked mob-1 gene expression induced by CCK (Fig. 1A). These results suggest a requirement for PKC activation in the actions of CCK on the expression of this gene. Direct activation of PKC with TPA (1 µM) also induced mob-1 chemokine expression (Figs. 1 and 2); this effect was concentration dependent, with significant effects noted at 10 nM and maximal effects at 100 nM (Fig. 2). The effects of TPA were blocked by GF-109203X (Fig. 1A). Taken together, these data suggest that PKC activation is required and is sufficient for the induction of mob-1 gene expression in pancreatic acinar cells by CCK.


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Fig. 1.   Protein kinase C (PKC) activity and intracellular Ca2+ are required for cholecystokinin (CCK) induction of mob-1 gene expression. A: freshly isolated pancreatic acini were preincubated for 30 min in the absence or presence of GF-109203X (GFX; 20 µM) or Ro-32-0423 (RO; 10 µM) to inhibit PKC activity and then were treated with CCK (100 nM) for 2 h. To examine the effects of directly activating PKC, acini were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA; 1 µM) for 2 h after preincubation for 30 min in the presence or absence of GF-109203X (20 µM). B: acini were preincubated for 30 min in the absence or presence of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (20 µM) to chelate intracellular Ca2+ and then were treated with CCK (100 nM) or TPA (1 µM) for 2 h. Acini were treated with ionomycin (2 µM) for 2 h to directly increase intracellular Ca2. Acini were also treated with the combination of TPA and ionomycin. Data are representative of 3-4 independent experiments.



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Fig. 2.   Concentration dependence of TPA effects on inhibitory kappa B (Ikappa B)-alpha degradation and mob-1 gene expression in pancreatic acinar cells. Freshly isolated acini were treated with TPA at different concentrations as indicated. Ikappa B-alpha protein levels were determined after 30 min of treatment by Western blotting using an anti-Ikappa B-alpha antibody. mob-1 mRNA levels were determined after 2 h of treatment by Northern blotting (25 µg total RNA) using a full length of mob-1 cDNA. Data are representative of 4 independent experiments.

Next, we investigated the role of intracellular Ca2+ in the effects of CCK on mob-1 gene expression. To examine the effects of blocking the CCK-induced increase in intracellular Ca2+, we utilized the intracellular Ca2+ chelator BAPTA. BAPTA has previously been shown to block the increase in intracellular Ca2+ normally observed in response to secretagogues (9). Pretreatment of the cells for 30 min with BAPTA-AM completely blocked the ability of CCK to induce an increase in mob-1 mRNA levels (Fig. 1B). BAPTA also blocked the effects of TPA on mob-1 gene expression. These data indicated that intracellular Ca2+ is required for the induction of mob-1 gene expression. However, treatment of acini with the Ca2+ ionophore ionomycin, which directly increases intracellular Ca2+ (14), did not affect mob-1 levels itself, nor did ionomycin alter the effects of TPA (Fig. 1B) or CCK (data not shown). These data suggest that increased intracellular Ca2+ is not sufficient for mob-1 gene expression.

TPA stimulates NF-kappa B nuclear translocation and specific consensus site DNA binding in a Ca2+-dependent manner. Previously, we have shown that CCK induction of mob-1 gene expression requires activation of NF-kappa B (19). PKC is known to activate NF-kappa B in a variety of cell types. To confirm that activation of PKC leads to NF-kappa B activation in pancreatic acinar cells, we used TPA to directly activate PKC. To examine whether PKC activity is important for the effects of CCK, we used GF-109203X to inhibit PKC. NF-kappa B protein nuclear translocation was analyzed by Western blotting, and a kappa B-specific consensus site DNA binding was assessed using electrophoretic mobility shift assay. Compared with untreated controls (Fig. 3, lanes 1 and 7), CCK (100 nM) induced a significant increase of p65 nuclear translocation and kappa B consensus DNA binding activity (Fig. 3, lanes 2 and 8). TPA (1 µM) also induced p65 nuclear translocation and kappa B binding (Fig. 3, lanes 4 and 10). These effects of CCK and TPA were blocked by pretreatment with the PKC inhibitor GF-109203X (Fig. 3, lanes 3 and 5). GF-109203X itself had no effect on p65 nuclear translocation or kappa B binding (Fig. 3, lane 6). These data support the hypothesis that PKC activity is required and sufficient for activation of NF-kappa B by CCK in pancreatic acinar cells.


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Fig. 3.   Roles of PKC and intracellular Ca2+ in the activation of nuclear factor (NF)-kappa B translocation and DNA binding in pancreatic acinar cells. Pancreatic acini were preincubated for 30 min in the absence or presence of GF-109203X (20 µM) or BAPTA-AM (20 µM) and then were treated with or without CCK (100 nM) or TPA (1 µM) for 1 h. Nuclei were isolated, and nuclear protein was extracted. To determine the extent of NF-kappa B nuclear translocation, aliquots of 20 µg nuclear protein were separated on a 10% SDS-polyacrylamide gel and were probed with anti-NF-kappa B protein subunit p65 antibody. To determine DNA binding, aliquots of 6 µg nuclear protein were used to form protein-DNA complex with the 32P-labeled oligonucleotide-containing NF-kappa B consensus and then were subjected to electrophoretic mobility shift assay. Data are representative of 3 independent experiments.

The effects on NF-kappa B translocation and DNA binding of both CCK and TPA were also blocked by the Ca2+ chelator BAPTA (Fig. 3, lanes 9 and 11). BAPTA itself had no effect on NF-kappa B nuclear translocation or DNA binding (Fig. 3, lane 12). Similar to what was observed concerning mob-1 gene expression, ionomycin treatment had no effect on NF-kappa B activation (data not shown). These data indicate that increased intracellular Ca2+ is required, but not sufficient, for the activation of NF-kappa B by CCK and TPA in pancreatic acinar cells.

To confirm that the NF-kappa B activated by TPA was composed of similar subunits as that induced by CCK, supershift analysis was performed. After treatment with TPA, NF-kappa B subunits p65 and p50 but not c-Rel, were involved in the nuclear DNA binding (Fig. 4, lanes 3-5). The binding specificity was confirmed by the blockade of the appearance of the NF-kappa B consensus binding band by preincubation with excess cold kappa B, but not OCT1, oligonucleotide (Fig. 4, lanes 6 and 7). These data were entirely similar to what was previously observed with CCK (19).


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Fig. 4.   TPA-induced NF-kappa B DNA binding. Pancreatic acini were treated with TPA (1 µM) for 1 h, and aliquots of 12 µg nuclear protein were subjected to electrophoretic mobility shift assay. For gel supershift assay, antibodies against NF-kappa B protein subunits p65, p50, and c-Rel (2 µg/each) were incubated with the nuclear extracts for 1 h at room temperature before the addition of radiolabeled oligonucleotide probe. Open arrowheads indicate supershift complexes. The original locations of gel-shifted complexes and nonspecific bands are indicated by filled arrowheads. To confirm the specificity of the complexes, cold competition assays were conducted by adding excess of unlabeled NF-kappa B oligonucleotide or OCT1 oligonucleotide, as a nonspecific competitor, before the addition of the labeled probe. Results are representative of 3 independent experiments.

Intracellular Ca2+ influences Ikappa B-alpha phosphorylation and degradation by CCK and TPA. Previously, it was found that supraphysiological stimulation of pancreatic acini with CCK caused a rapid degradation of Ikappa B-alpha . Therefore, we examined Ikappa B-alpha levels to determine whether the inhibitors that blocked CCK-mediated induction of mob-1 gene expression and NF-kappa B activation affected Ikappa B-alpha degradation (Fig. 5). CCK caused a rapid and complete degradation of Ikappa B-alpha (92 ± 3% degraded, n = 5). However, TPA caused only a relatively small decrease in the cellular level of Ikappa B-alpha (30 ± 8% degraded, n = 5). Inhibition of PKC with GF-109203X reduced, but did not completely block, the ability of CCK to stimulate Ikappa B-alpha degradation. In the presence of GF-109203X, CCK treatment caused the formation of additional bands in the Western blot that may represent partial degradation products. These data suggested that PKC activity was not sufficient, but is important, for the effects of CCK on Ikappa B-alpha degradation. In contrast, the degradation of Ikappa B-alpha was blocked by the Ca2+ chelator BAPTA (Figs. 5 and 6). The relatively minor effect of TPA on Ikappa B-alpha was also blocked by BAPTA-AM pretreatment (Figs. 5 and 6). Increasing intracellular Ca2+ with ionomycin did not itself cause a decrease in Ikappa B-alpha levels (Fig. 5). However, in combination with TPA, ionomycin caused a decrease in Ikappa B-alpha comparable to that observed with maximal concentrations of CCK. These data supported the hypothesis that increased intracellular Ca2+ is required, but not sufficient, for Ikappa B-alpha degradation.


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Fig. 5.   Increased intracellular Ca2+ and PKC activity have a synergistic effect on Ikappa B-alpha degradation in pancreatic acinar cells. Pancreatic acini were pretreated for 30 min without or with BAPTA-AM (20 µM) or GF-109203X (20 µM) and then were treated with CCK (100 nM) or TPA (1 µM) for 30 min. Aliquots of 25 µg protein from whole cell lysates were subjected to Western blotting and were probed with an anti-Ikappa B-alpha antibody. Data shown are representative of 3-5 independent experiments.



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Fig. 6.   Time course of the effects of CCK and TPA on Ikappa B-alpha phosphorylation and degradation in pancreatic acinar cells. Freshly isolated pancreatic acini were preincubated in the presence or absence of 20 µM BAPTA-AM at 37°C for 30 min then were treated with 100 nM CCK or 1 µM TPA. Whole cell lysates were extracted at indicated times, and aliquots of 25 µg protein were subjected to electrophoresis on a 10% SDS-polyacrylamide gel. Western blotting was performed using an anti-phospho-Ikappa B-alpha antibody, and then aliquots were stripped and reprobed with an anti-Ikappa B-alpha antibody. Data shown are representative of 3 independent experiments.

To further explore the differences between the effects of CCK, which activates PKC and increases intracellular Ca2+, and those of TPA, which activates PKC without increasing intracellular Ca2+, we determined the time course of Ikappa B-alpha degradation and phosphorylation (Fig. 6). CCK led to a significant decrease in Ikappa B-alpha levels within 10 min, which was maximal within 30 min. Pretreatment with BAPTA-AM blocked this effect for at least 30 min. The effects of TPA on Ikappa B-alpha degradation were both less pronounced and slower in onset. Pretreatment with BAPTA-AM also blocked the effects of TPA on Ikappa B-alpha degradation. Ikappa B-alpha phosphorylation was monitored using an anti-phospho-Ikappa B-alpha antibody specific for phosphorylation of serine 32. CCK treatment increased the level of phosphorylated Ikappa B-alpha within 10 min (Fig. 6). The levels of phosphorylated Ikappa B-alpha then declined, likely due to degradation of the phosphorylated protein. Pretreatment with BAPTA-AM blocked CCK-induced Ikappa B-alpha phosphorylation. TPA also induced the phosphorylation of Ikappa B-alpha . However, the effects were slower, being maximal after 30 min. This effect of TPA was also blocked by pretreatment with BAPTA-AM. Thus, although TPA stimulates Ikappa B-alpha phosphorylation, the time course is delayed compared with the effects of CCK, and this phosphorylation is not sufficient to cause full degradation of Ikappa B-alpha .


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NF-kappa B is activated by a large number of factors and treatments ranging from proinflammtory cytokines, such as TNF-alpha , to cell stresses, such as ultraviolet irradiation (40). The cellular mechanisms mediating the effects of TNF-alpha have been particularly well elucidated, and details concerning the signaling components continue to emerge. However, the pathways whereby G protein-coupled receptors such as CCK activate NF-kappa B have been much less studied. CCK is a gastrointestinal hormone with a variety of physiological actions in the gut. CCK signaling has been widely studied in the context of its physiological roles in secretion and pancreatic protein synthesis. Supraphysiological concentrations of CCK induce pancreatic and systemic manifestations of pancreatitis, such that CCK hyperstimulation is the most widely used model of experimental pancreatitis. Supraphysiological concentrations of CCK activate NF-kappa B, which has been suggested to be important for the development of acute pancreatitis (15, 17, 19, 43). Therefore, activation of NF-kappa B constitutes an additional action of CCK that is likely relevant for our understanding of pancreatic inflammatory responses.

Supraphysiological concentrations of CCK trigger rapid degradation of Ikappa B-alpha , the nuclear translocation of NF-kappa B, the binding to DNA kappa B consensus sites of p65/p50 complexes, and expression of cytokines and chemokines, including mob-1 (19). High- and low-affinity CCK receptors are known to generate different patterns of second messengers and cellular response (29). High-affinity CCK receptors are responsible for low levels of phosphatidylinositol hydrolysis and an oscillatory pattern of intracellular Ca2+ release (28) and are responsible for activation of extracellular signal-regulated kinases (10), p90rsk-1 (7), and p38/Hog (35). Thus these signals induced by high-affinity CCK receptors, which are primarily involved in physiological actions of CCK, are neither sufficient nor necessary for activating NF-kappa B.

Low-affinity CCK receptors cause a prolonged increase in DAG (28), strongly activate PKC, cause a peak and plateau pattern of Ca2+ release, and activate Jun kinases (8). Of these signaling mechanisms, activation of PKC was a strong candidate for coupling to NF-kappa B activation and chemokine expression based on known effects in other cell models. CCK activates PKC by increasing cellular levels of the PKC activator DAG. CCK receptors activate a biphasic increase in cellular DAG levels (30). This is thought to be due to a rapid but transient increase in hydrolysis of phosphatidylinositol, followed by a more prolonged increase due to hydrolysis of phosphatidylcholine. The strong activation of PKC observed with high concentrations of CCK is likely responsible for the ability of CCK to activate NF-kappa B in pancreatic acinar cells. Interestingly, the lower level of PKC activation observed with physiological concentrations of CCK is obviously not sufficient for activation of NF-kappa B.

Relative differences in their abilities to activate PKC may explain differences observed in the abilities of CCK and another pancreatic secretagogue, bombesin, to initiate an acute inflammatory response in the pancreas. Bombesin receptor activation on pancreatic acinar cells has been reported to generate either little (30) or no (33) DAG. Therefore, bombesin likely does not activate PKC to the same extent as CCK. In the current study, PKC activity was required for NF-kappa B activation by CCK. Bombesin treatment also does not provoke pancreatitis (34) nor does it activate NF-kappa B in pancreatic acinar cells (15, 19). Thus it is likely that differences in the abilities of CCK and bombesin receptors to cause a strong activation of PKC explains the observation that CCK, but not bombesin, is able to initiate an acute inflammatory response in the pancreas.

It has long been recognized that TPA acts as an activator of NF-kappa B. However, the mechanisms involved in this process remain to be resolved. TPA activates the conventional isoforms of PKC, which are activated by Ca2+ and DAG, and the novel PKCs, which are activated by DAG but do not respond to Ca2+. Activation of atypical isoforms of PKC, which do not require DAG or Ca2+, has been shown to be required for NF-kappa B activation by TNF-alpha (11). However, neither conventional, novel, nor atypical forms of PKC directly phosphorylate Ikappa B (24). Recently, it was shown that both the atypical isoform PKCzeta and the conventional PKCalpha activate IKKbeta (24). The IKKs phosphorylate Ikappa B-alpha on serines 32 and 36; this phosphorylation is thought to trigger the subsequent ubiquitination and proteasomal degradation of Ikappa Bs. However, in many cell lines, PKC is an excellent activator of NF-kappa B DNA binding and gene transcription but is not efficient at causing Ikappa B degradation (12, 42). In the current study, inhibition of PKC blocked the ability of CCK to activate NF-kappa B nuclear localization, DNA binding, and mob-1 gene expression. However, inhibition of PKC only partially blocked the ability of CCK to cause the degradation of Ikappa B-alpha . The presence of multiple protein bands after CCK treatment in the presence of the PKC inhibitor suggests that unidentified PKC-independent kinases may be involved in the effects of CCK on Ikappa B-alpha . Moreover, TPA activated NF-kappa B but caused only a minor amount of Ikappa B-alpha degradation in the pancreatic acinar cells. Taken together, the data support a model in which PKC is capable of activating NF-kappa B in the absence of profound Ikappa B degradation. Recently, it was shown that TPA activation of PKC in enterocytes also induces NF-kappa B without leading to profound Ikappa B degradation (48). Thus there appear to be mechanisms other than Ikappa B-alpha degradation involved in the effects of PKC on NF-kappa B activation in some cell types.

Recent studies have suggested that Ikappa B degradation and NF-kappa B nuclear translocation are insufficient for transcription from NF-kappa B-regulated promoters and have proposed the existence of important parallel signaling mechanisms (3). These parallel signaling mechanisms may involve phosphorylation of NF-kappa B p65 subunits (4, 32, 46). TPA activation of PKC has previously been shown in vitro to lead to phosphorylation of a defined region within a transactivation domain of the p65 subunit of NF-kappa B (37). This phosphorylation was found to stimulate transcriptional activity. However, no evidence is available as to whether or not this phosphorylation event interrupts the interaction between Ikappa B-alpha and NF-kappa B and thus might account for a dissociation of NF-kappa B in the absence of Ikappa B-alpha degradation. A mechanism allowing the dissociation of NF-kappa B from Ikappa B-alpha without Ikappa B-alpha degradation would explain the increased p65 nuclear accumulation stimulated by TPA in pancreatic acini observed in the current study or in the case of enterocytes (48). One such mechanism might include the tyrosine phosphorylation of Ikappa B-alpha . NF-kappa B activation has previously been reported to occur by the tyrosine phosphorylation of Ikappa B-alpha and in the absence of Ikappa B-alpha degradation (2, 20, 23). Further details of the NF-kappa B activation machinery will be required to determine the mechanisms involved in PKC activation of NF-kappa B in pancreatic acinar cells.

Ca2+-dependent pathways also play critical roles in NF-kappa B activation in pancreatic acinar cells, as chelation of intracellular Ca2+ completely blocked CCK stimulation of Ikappa B phosphorylation and degradation, as well as NF-kappa B nuclear translocation, kappa B consensus site DNA binding, and mob-1 gene expression. BAPTA treatment has previously been shown to block the induction of NF-kappa B activation by epidermal growth factor (44) and lysophosphatic acid (38) but not by H2O2 (22) in fibroblast cell models. In the current study, BAPTA treatment also inhibited the effects of TPA on mob-1 mRNA levels and on Ikappa B-alpha phosphorylation and degradation. It is possible that BAPTA maintained intracellular free Ca2+ at levels below those required for maximal activity of conventional PKCs. Thus one Ca2+ requirement could be PKC activity itself. However, TPA did not lead to profound degradation of Ikappa B-alpha , as was observed with CCK, unless Ca2+ was artificially elevated. Thus the response to CCK treatment must involve both PKC activity and increased intracellular Ca2+. Similar to what was observed in fibroblasts, in the current study, increasing intracellular Ca2+ with an ionophore did not activate NF-kappa B in pancreatic acinar cells. Thus increased cytoplasmic Ca2+ levels are required for some NF-kappa B inducers, including high concentrations of CCK, but are not sufficient for NF-kappa B activation.

The mechanisms involved in the Ca2+ requirement for NF-kappa B activation are not completely clear. In the current study, CCK, which causes increases in both Ca2+ and PKC activity, activated NF-kappa B and caused a near complete loss of Ikappa B-alpha . In contrast, TPA, which increases PKC activity but does not increase intracellular Ca2+, activated NF-kappa B but had only a minor effect on Ikappa B-alpha degradation. The combination of TPA and a Ca2+ ionophore caused a synergistic reduction in Ikappa B-alpha . Thus Ikappa B-alpha degradation appears to require increased intracellular Ca2+. It has previously been observed that increased intracellular Ca2+ works in synergy with PKC to cause Ikappa B degradation in fibroblasts (12, 42). This effect was suggested to be mediated by calcineurin, a Ca2+/calmodulin-activated phosphatase, based upon the observation that calcineurin inhibitors such as cyclosporin A and FK-506 block the synergistic effect of Ca2+ ionophores with TPA. However, more recently, cyclosporin A has been shown to act as an uncompetitive inhibitor of proteasome activity (31). Proteasomal activity is required for Ikappa B degradation. Therefore, it is unclear whether the effects of the calcineurin inhibitors are specific or reflect nonspecific effects on proteasomes. We found that cyclosporin A inhibited CCK-mediated Ikappa B-alpha degradation only at high concentrations [25 µM and above (data not shown)], whereas 1 µM is sufficient to inhibit acinar cell calcineurin (16).

In summary, we found that in pancreatic acinar cells the ability of supraphysiological concentrations of CCK to stimulate NF-kappa B activation and mob-1 chemokine expression requires activation of PKC and increased intracellular Ca2+. PKC activation by TPA strongly induced NF-kappa B and caused mob-1 expression but had only a weak effect on Ikappa B-alpha degradation. Increased cytoplasmic Ca2+ was required for activation of NF-kappa B and mob-1 expression by either CCK or TPA but was itself unable to activate these pathways. Increased cytoplasmic Ca2+ acted in synergy with TPA to degrade Ikappa B-alpha . The specific cellular targets of PKC and Ca2+ involved in these effects remain to be determined. Further understanding of these signaling mechanisms may be useful in determining intracellular events important in the development of acute pancreatitis and in the design of future therapies for this disease.


    ACKNOWLEDGEMENTS

We thank Drs. J. A. Williams, B. Nicke, and D. Simeone for critical reviewing of this manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52067 and the University of Michigan Gastrointestinal Peptide Center Grant DK-34933.

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: C. Logsdon, Dept. of Physiology, Box 0622, Univ. of Michigan, 7710 Medical Sciences Bldg. II, Ann Arbor, MI 48109-0622 (E-mail: clogsdon{at}umich.edu).

Received 24 June 1999; accepted in final form 27 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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