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Am J Physiol Gastrointest Liver Physiol 292: G667-G677, 2007. First published November 9, 2006; doi:10.1152/ajpgi.00433.2006
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HORMONES AND SIGNALING

Induction of early response genes in trypsin inhibitor-induced pancreatic growth

Lili Guo,1 Maria Dolors Sans,1 Grzegorz T. Gurda,1 Sae-Hong Lee,1 Stephen A. Ernst,2 and John A. Williams1,3

Departments of 1Molecular and Integrative Physiology, 2Cellular and Developmental Biology, and 3Internal Medicine, The University of Michigan Medical School, Ann Arbor, Michigan

Submitted 20 September 2006 ; accepted in final form 3 November 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Endogenous CCK release induced by a synthetic trypsin inhibitor, camostat, stimulates pancreatic growth; however, the mechanisms mediating this growth are not well established. Early response genes often couple short-term signals with long-term responses. To study their participation in the pancreatic growth response, mice were fasted for 18 h and refed chow containing 0.1% camostat for 1–24 h. Expression of 18 early response genes were evaluated by quantitative PCR; mRNA for 17 of the 18 increased at 1, 2, 4, or 8 h. Protein expression for c-jun, c-fos, ATF-3, Egr-1, and JunB peaked at 2 h. Nuclear localization was confirmed by immunohistochemistry of c-fos, c-jun, and Egr-1. Refeeding regular chow induced only a small increase of c-jun and none in c-fos expression. JNKs and ERKs were activated 1 h after camostat feeding as was the phosphorylation of c-jun and ATF-2. AP-1 DNA binding evaluated by EMSA showed a significant increase 1–2 h after camostat feeding with participation of c-jun, c-fos, ATF-2, ATF-3, and JunB shown by supershift. The CCK antagonist IQM-95,333 blocked camostat feeding-induced c-jun and c-fos expression by 67 and 84%, respectively, and AP-1 DNA binding was also inhibited. In CCK-deficient mice, the maximal response of c-jun induction and AP-1 DNA binding were reduced by 64 and 70%, respectively. These results indicate that camostat feeding induces a spectrum of early response gene expression and AP-1 DNA binding and that these effects are mainly CCK dependent.

CCK; camostat; c-jun; c-fos; AP-1

PANCREATIC GROWTH OCCURS DURING fetal and early postnatal development after which cell proliferation in the adult pancreas is minimal compared with other organs within the gastrointestinal system. There are circumstances, however, in which proliferation of pancreatic acinar cells occurs, including adaptation to high-protein diets (18, 19, 28), pregnancy, and lactation (25) and with regeneration from injury following pancreatitis (15, 21). One of the main regulators of adult pancreatic growth is the gastrointestinal hormone cholecystokinin (CCK). Administration of exogenous CCK or its analog caerulein induces pancreatic growth (11, 36, 38, 47). Feeding protease inhibitors such as soybean trypsin inhibitor or the synthetic trypsin inhibitor camostat induces endogenous CCK release and leads to pancreatic growth in many species including the mouse (36, 51), rat (17, 35), hamster (12), and chicken (32). The effect involves new DNA synthesis and growth or division of acinar cells (30, 48). Coadministration of a CCK receptor antagonist blocks the effects of camostat on the pancreas (12, 59), and this growth response is absent in CCK- or CCKA-receptor deficient mice (42, 51). Although it is well known that both exogenous and endogenous CCK can induce pancreatic growth, the mechanisms involved are not well understood.

CCK activates a number of intracellular signal transduction pathways in pancreatic acinar cells in addition to the Ca2+ signaling that mediates secretion (57). These pathways include the three mitogen-activated protein kinases (MAPKs) c-jun NH2-terminal kinases (JNKs), extracellular signal-related kinases (ERKs), and P38 MAPK, as well as the PI3K-PKB-mTOR pathway (58). MAPKs are activated in response to a wide variety of stimuli, and they transduce signals from the cell membrane to the nucleus, phosphorylate nuclear transcription factors, and thereby enhance their ability to activate transcription (7). MAPKs regulate cellular activities ranging from cell growth to survival and apoptosis (24). A previous study showed that ERKs and JNKs were activated during camostat feeding (50). Moreover, CCK has been observed to activate ERKs and JNKs in isolated rat pancreatic acini (9, 10, 13).

Early response genes are a class of genes that are rapidly upregulated following a number of extracellular stimuli. They function as mediators coupling short-term signals to long-term cellular responses such as growth and differentiation by acting as transcription factors to regulate the expression of other genes (39). Two of the best characterized early response genes are c-jun and c-fos. In isolated rat pancreatic acinar cells, CCK stimulation induced an increase in mRNA expression for c-jun, c-fos, and c-myc (31). In pancreas-derived AR42J cells, c-jun and c-fos expression were induced by gastrin (53) and carbachol (54). Both c-jun and c-fos belong to the AP-1 (activator protein 1) family, which is one of the first mammalian transcription factors identified. AP-1 is a group of dimeric basic region Leucine zipper (bZIP) proteins including Jun (c-jun, JunB, JunD), Fos (c-fos, FosB, Fra-1, Fra-2), ATF (ATF-2, ATF-3, B-ATF), and Maf subfamilies (45). AP-1 activities are known to be induced by growth factors, cytokines, neurotransmitters, polypeptide hormones, cell matrix interactions, and bacterial and viral infections, as well as a variety of physical and chemical stress. AP-1 regulates a wide range of cellular processes including cell proliferation, death, survival, and differentiation (44).

Although early response genes can be induced in acinar and acini-derived cells, the complete spectrum of response has not been studied in detail in the context of a physiological response. In the present work we used quantitative real-time PCR, Western blots, immunohistochemistry, and gel shift assays to evaluate the expression and nuclear localization of early response genes in response to acute camostat feeding of mice. The results show rapid induction and phosphorylation of a large number of these proteins and direct future studies to potential downstream targets that activate pancreatic acinar cell growth and division.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Camostat was provided by Ono Pharmaceutical (Osaka, Japan). IQM-95,333 was a gift from Dr. M. T. García-López (Consejo Superior de Investigaciones Científicas Instituto de Química Médica, Madrid, Spain) (2). Rabbit polyclonal antibodies to c-jun, JunB, c-fos, ATF-3, Egr-1, lamin A/C, and JNK1 were from Santa Cruz Biotechnology (Santa Cruz, CA). Other rabbit polyclonal antibodies including anti-phospho-ERK1/2 (Thr 202/Tyr 204), anti-phospho-JNK1/2 (Thr 183/ Tyr 185), anti-phospho-c-jun (Serine 63), anti-phospho-ATF-2 (Thr 71), anti-ATF-2, and anti-ERK1/2 were from Cell Signaling (Beverly, MA). 32P-ATP was from Amersham Pharmacia Biotech (Piscataway, NJ), T4 polynucleotide kinase was from New England Biolabs (Ipswich, MA), and the AP-1 oligonucleotide and gel shift binding buffer were from Promega (Madison, WI). TRIzol and all the primers used for quantitative real-time PCR were obtained from Invitrogen (Carlsbad, CA) and RNAlater from Ambion (Austin, TX).

Animals and treatment. Male ICR mice weighing 26–30 g from Harlan Sprague Dawley (Indianapolis, IN) or CCK-deficient and littermate control mice on a 129/SV background weighing 19–22 g (28) were used in all experiments. The mice were housed at 22–24°C on a 12:12-h light-dark cycle changing at 6 AM and 6 PM with free access to water and were fed a standard rodent chow (5001 Rodent Diet, PMI Nutrition International, St. Louis, MO). All studies were approved by the University of Michigan Committee on Use and Care of Animals. Three days before an experiment, mice were switched to the powdered form of the same diet and then fasted for 18 h beginning at 3 PM. The next day at 9 AM mice were refed ad libitum with powder diet containing 0.1% camostat for 1, 2, 4, 8, 12, or 24 h. For the CCK antagonist experiment, mice were injected intraperitoneally with IQM-95,333 in vehicle (saline) at 500 µg/kg or with vehicle alone 1 h before refeeding. The pancreas was removed following CO2 asphyxiation and either directly homogenized, frozen in liquid nitrogen, or placed in RNAlater at 4°C.

RNA isolation, reverse transcription, and quantitative real-time PCR. RNA isolation, cDNA synthesis, and quantitative real-time PCR followed the same procedures as described by Sans et al. (41). Briefly, total RNA was isolated from mouse pancreas by use of TRIzol and RNeasy spin columns. RNA quality and quantity were evaluated by agarose gel electrophoresis and UV spectrometry (OD260/OD280 >1.7). One microgram of total RNA was reverse transcribed by using TaqMan reverse transcription reagents with random hexamers as primers. Quantitative PCR was carried out using a Bio-Rad I-Cycler IQ real-time PCR detection system with 96-well plates. A mixture of 10.3 µl HPLC water, 2 µl 10x PCR buffer, 2.2 µl MgCl2 (50 mM), 1 µl fluorescein, 0.4 µl dNTP (10 mM), 2 µl primer mixtures (consisting of 0.1 nmol/µl concentration of each forward and reverse primer and 1 µl 100x SYBRgreen in 100 µl volume), and 2 µl cDNA were used for each reaction. PCR was performed in triplicate wells for each sample at 95°C for 3 min, 60°C for 1 min (repeated 40 times), and 55°C for 1 min. The fluorescence resulting from the incorporation of SYBRgreen 1 dye into the double-stranded DNA produced during the PCR reaction was quantitated to obtain the threshold cycle (CT) value for each sample. Because CT readings represent measurements on a log scale, a mean CT value was calculated and the final relative amounts of mRNA for each animal were calculated as 2{Delta}CT, where {Delta}CT is the mean control CT minus the individual CT. This converts the log scale CT values to a relative number in linear form that can be used to calculate a mean ± SE for each group.

The primers used for quantitative RT-PCR were designed with Primer Express software from ABI (Foster City, CA) based on gene sequences obtained from the GenBank NCBI Sequence Viewer (http://www.ncbi.nlm.nih.gov) and are given in Table 1.


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  		Table 1. Pancreatic early response gene expression induced by camostat feeding

 
Preparation of pancreatic tissue lysate for electrophoresis. Pancreatic tissue was homogenized with a polytron homogenizer in ice-cold buffer as previously described (51). The supernatant was prepared for SDS-PAGE after which protein was transferred to nitrocellulose membrane and Western blotting was conducted using specific antibodies (20).

Nuclear protein extraction. Pancreas was removed and a portion was homogenized in 2 ml of ice-cold homogenization buffer (2 M sucrose, 10% glycerol, 10 mM HEPES pH 7.9, 25 mM KCl, 150 mM spermine, 500 mM spermidine, 2 mM EDTA, 10 mg/l leupeptin, 10 mg/l aprotinin, 1 mM benzamidine, 1 mM PMSF, 1 mM DTT) with a motor-driven pestle. Nuclei were collected by centrifugation at 30,000 rpm in a Beckman optima TLX ultracentrifuge for 30 min at 4°C. The pellet was washed with 1 ml washing buffer (1 mM EDTA in PBS) and then centrifuged in a refrigerated microcentrifuge for 5 min. The pellet was resuspended in 250 µl of ice-cold nuclear resuspension buffer (10% glycerol, 10 mM HEPES pH 7.9, 0.42 M NaCl, 100 mM KCl, 3 mM MgCl, 0.1 mM EDTA, 1 mM DTT, 10 mg/l leupeptin, 10 mg/l aprotinin, 1 mM benzamidine, 1 mM PMSF) and incubated on ice for 30 min with intermittent mixing, after which the samples were centrifuged for 15 min and supernatant was saved.

EMSA. A double-stranded oligonucleotide of AP-1 with the consensus sequence TGACTCA was labeled with {gamma}-[32P]-ATP and T4 polynucleotide kinase. Pancreatic nuclear extract (2.5 µg) was incubated with the gel shift binding buffer supplied by the manufacturer (Promega). After 20 min of incubation at room temperature, the labeled AP-1 probe was added and incubation continued for another 30 min. Subsequently, the reaction mixture was subjected to a nondenaturing 5% polyacrylamide gel electrophoresis with 0.5x Tris-borate EDTA, dried and exposed to X-ray film. Competitive inhibition was carried out with 50-fold excess of unlabeled cold AP-1 oligonucleotide. Supershift assays were conducted by adding 4 µl of specific antibody to the binding reaction mixture 20 min before addition of the labeled oligonucleotide.

Immunofluorescence. Small pieces of pancreas were fixed for 2 h with 4% formaldehyde, prepared from paraformaldehyde, cryoprotected, and frozen as described previously (37). Immunofluorescence localization was performed following methods described previously in detail (37). Primary antibodies were rabbit anti-c-fos, anti-c-jun, and anti-Egr-1, diluted 1:800 to 1:600; secondary antibody was Cy-3-conjugated donkey anti-IgG diluted 1:200. 4'6-Diamidino-2-phenylindole (DAPI) was added to the mounting medium to counterstain nuclei. Digital images were taken with an Olympus BX-51 microscope and processed using Photoshop 6.0 software (Adobe System, Mountain View, CA).

Statistical analysis. Results are expressed as means ± SE, which were obtained from two to five different experiments yielding a total of 4–10 animals in each group. One-way ANOVA with the Dunnett’s test was carried out using Graphpad Prism software. P < 0.05 represents statistical significance.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Induction of early response gene expression shortly after camostat feeding in mice. To evaluate the early time course of the initial growth response (51), we fasted mice for 18 h and then refed them with chow containing camostat, which mice eat vigorously. At the earliest time point evaluated, all mice had food in their stomachs. Based in part on an increase in expression in an unpublished microarray study of mice fed camostat and in part on relevance to growth in other cell types, we studied 18 early response genes and characterized five of them in detail including c-jun (Fig. 1A), c-fos (Fig. 1B), JunB (Fig. 1C), ATF-3 (Fig. 1D), and Egr-1 (Fig. 1G), which are known to be related to cell proliferation (1, 6, 16, 22, 23, 34, 46). Camostat feeding rapidly upregulated the expression of 17 of 18 early response genes at the mRNA level by 2.4- to 600-fold (Table 1). Their expression rapidly increased, peaked at 1, 2, 4, or 8 h, and then decreased (Fig. 1). The expression of c-myc was the only one not affected by camostat feeding. In contrast to the early response genes, the expression of housekeeping genes 18sRNA (Fig. 1E), GAPDH (Fig. 1F), and cyclophilin A (not shown) was not affected by camostat administration.


Figure 1
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  		Fig. 1. Effect of camostat feeding on expression of the early response genes c-jun (A), c-fos (B), JunB (C), and ATF-3 (D) in the pancreas. Mice were adapted to powder diet for 3 days, fasted for 18 h, and refed powdered chow containing 0.1% camostat at 9 AM for the indicated period of time. Pancreatic RNA was isolated and samples were analyzed by quantitative real-time PCR as described in METHODS. GAPDH (E) and 18s RNA (F) were included as controls. G: results for mRNA expression of the Egr family members and their corepressor Nab2. Mice were refed powdered chow containing 0.1% camostat for the indicated period of time from 1 to 24 h. All results shown are means ± SE of 6–10 mice. **P < 0.01; *P < 0.05 compared with fasted control.

 
The expression of mRNA for Egr family members Egr-1, Egr-2, and Egr-3 was also induced by camostat (Fig. 1G). Expression peaked at 1 h (Egr-2 and Egr-3) or 2 h (Egr-1) followed by the decrease of mRNA level; the increase in expression of Egr-1 and Egr-2 was much greater than for Egr-3. Nab2, a delayed early response gene, is a nuclear corepressor protein to Egr-1 and Egr-2 (49), and its expression was induced by camostat as well (Fig. 1G). It peaked at 8 h with a 13-fold increase and then decreased but was still elevated at 24 h of camostat feeding.

We were also able to detect the increased expression of the five early response genes c-jun, JunB, c-fos, ATF-3, and Egr-1 at the protein level by using Western blotting. Results were clearer with use of a nuclear fraction although c-jun could be easily detected in a total pancreatic lysate. The increased protein expression was observed 1 h after camostat feeding and showed a similar pattern as mRNA, but with all peaking at 2 h after camostat feeding (Fig. 2). The nuclear protein lamin A/C was used as a loading control, and its expression was not affected by camostat feeding. Since the basal protein levels of these early response genes were very low, we calculated the protein level at each time point as percentage of the 2-h value. However, as a fold increase c-jun protein increased 27-fold, JunB 294-fold, c-fos 7.7-fold, ATF-3 18-fold, and Egr-1 36-fold.


Figure 2
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  		Fig. 2. Effect of camostat feeding on pancreatic early response gene expression of c-jun (A), c-fos (B), ATF-3 (C), Egr-1 (D), and JunB (E) at the protein level; Lamin A/C (F) is included as a control. Nuclear protein was extracted and samples were analyzed by Western blotting and quantitative densitometry. For each panel a representative blot is shown along with quantitative data expressed as a percentage of the 2-h value. Quantitative results shown are means ± SE of 6–9 mice. **P < 0.01; *P < 0.05 compared with the fasted control.

 
Because the pancreas does not grow in response to feeding regular chow, we predicted that the early response gene expression increase would be much greater in mice fed diet containing camostat than those fed regular chow. At the mRNA level, fasting decreased c-jun and c-fos, but this was not significant. Refeeding regular chow increased c-jun mRNA by 1.9-fold (Fig. 3A) but had no effect on c-fos (Fig. 3B). At the protein level, there was a small but insignificant increase in c-jun (Fig. 3C) and this was less than 10% of the response to refeeding chow with camostat. The c-fos protein did not change with refeeding regular chow (Fig. 3D).


Figure 3
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  		Fig. 3. Effect of regular chow feeding on pancreatic early response gene expression. A and B: mice were refed regular powdered chow for 2 h after 18 h fasting and RNA was isolated. Pooled data are means ± SE of 5–6 mice/group and are expressed as fold increase relative to normally fed (unfasted) mice killed at the same time. **P < 0.01 compared with fed control. C and D: mice were refed with either regular powdered chow or powdered chow containing camostat for 2 h. Nuclear protein was extracted and analyzed by Western blot. Data for each group are means ± SE of 5–6 mice. **P < 0.01 compared with fasted control.

 
Cellular localization of early response gene expression. To confirm the acinar cell localization of early response gene expression after camostat feeding, and to resolve the spatial localization within acinar cells, we visualized expression by immunofluorescence in cryostat sections counterstained with the nuclear fluorescent marker DAPI. As shown for c-fos and c-jun expression, pancreatic acinar cells in fasted control mice demonstrated little or no immunostaining (Fig. 4, A and C). In response to camostat feeding (2 h), a large fraction of the pancreatic acinar cells became c-fos and c-jun positive (Fig. 4, B and D), with the fluorescence localized to acinar nuclei, as indicated by colocalization with DAPI (insets in Figs. 4, B and D). Similar results were seen after 1 h camostat feeding (not shown). The increased expression of c-fos and c-jun was absent in the nuclei of periacinar cells (arrows in insets in Figs. 4, B and D), as well as in islet cells (not shown). Similar results were seen in acinar cells with Egr-1. Although sections from fasted control mice showed cytoplasmic staining for Egr-1, this was not accompanied by any nuclear signal (Fig. 4E and inset). In response to camostat feeding, nuclear staining became apparent in acinar cells (Fig. 4F), but not in periacinar cells (inset in Fig. 4F). In contrast to lack of c-fos and c-jun staining in islet cells, islet cell cytoplasm, but not nuclei, were stained for Egr-1 in sections from both fasted and camostat-treated mice (data not shown).


Figure 4
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  		Fig. 4. Effect of camostat feeding on immunofluorescence localization of c-fos, c-jun, and EGR-1 protein in cryostat sections of mouse pancreas. Localization of c-fos, c-jun, and EGR-1 (red) in fasted controls (A, C, E, respectively) and in corresponding sections from mice treated for 2 h with camostat (B, D, F, respectively) are shown, together with colocalizations (insets) with the nuclear marker DAPI (pseudocolored green). In fasted controls, there was little or no nuclear or cytoplasmic staining with c-fos and c-jun (A and C, respectively, and insets); with EGR-1, cytoplasmic staining was present, but nuclei were negative (E, and inset). In response to camostat feeding, acinar nuclei were stained for all 3 proteins (B, D, F); insets show colocalization with DAPI staining for acinar nuclei (orange and yellow), but not periacinar cell nuclei (green) indicated by arrows.

 
Camostat feeding increases MAPK activity. MAPKs are upstream regulators of the expression of c-jun and c-fos and participate in regulation of cell proliferation. To determine the time course of their activation following camostat refeeding after fasting, we used phospho-specific antibodies, which are activity state dependent, to JNKs, ERKs, and P38 MAPK. The phosphorylation of JNKs (p46 and p54) increased 21.4 ± 3.3-fold at 1 h (Fig. 5A) and ERKs (p42 and p44) 5.7 ± 0.45-fold at 2 h after feeding camostat, and then dropped to basal level at 8 h. Of note, the total JNK and ERK protein did not change in response to camostat feeding. Compared with JNKs and ERKs, phospho P38 MAPK did not show a significant difference between the control and camostat-treated group (data not shown). These results indicated that camostat feeding rapidly increased the activity of JNKs and ERKs in mouse pancreas.


Figure 5
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  		Fig. 5. Effect of camostat feeding on JNK (A) and ERK (B) activation. Activity of JNK and ERK was visualized by Western blotting using activity status-dependent phospho-specific antibodies. Data were expressed as fold increase relative to the fasted control. For each panel a representative blot is shown along with quantitative data expressed as the means ± SE from 6–9 mice. **P < 0.01 compared with fasted control.

 
Phosphorylation increase of c-jun and ATF-2 by camostat feeding. It is well known that activated JNK can phosphorylate c-jun and ATF-2 (activating transcription factor-2) to enhance their transcriptional activity (14, 24). We therefore examined the phosphorylation status of pancreatic c-jun and ATF-2 by Western blot assay with the corresponding phospho-specific antibody. Figure 6 shows that in fasted mice the phosphorylation level of ATF-2 (Fig. 6A) was low and for c-jun was barely detectable (Fig. 6B). The phosphorylation of c-jun and ATF-2 increased 1 h after camostat feeding and reached a maximal level of 7.6-fold increase for phospho-ATF-2 and ~100-fold increase for phospho-c-jun, followed by a decrease in their phosphorylation toward fasted levels. As shown earlier, the total protein level of c-jun increased in response to camostat feeding, whereas the total protein of ATF-2 was not affected. These results showed that in addition to the induction of early response genes by de novo synthesis, other early transcription regulators are activated by posttranslational modification, such as phosphorylation. In addition to ATF-2, we also found that the phosphorylation of CREB was increased following camostat feeding (data not shown).


Figure 6
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  		Fig. 6. Effect of camostat feeding on phosphorylation of ATF-2 (A) and c-jun (B). The phosphorylation of ATF-2 and c-jun was analyzed by Western blot using phospho-specific antibody. Phospho-ATF-2 value was expressed as fold increase vs. fasted control, and the value for phospho-c-jun was expressed as the percentage of 2 h. Quantitative data are expressed as the means ± SE from 6–9 mice. **P < 0.01 compared with fasted control.

 
AP-1 DNA binding was enhanced by camostat feeding. ATF-3, c-jun, and c-fos are members of the AP-1 family. They can form heterodimers or homodimers and bind AP-1 sites to elicit expression of other genes. EMSA was carried out to determine whether camostat feeding can affect AP-1 DNA binding. As shown in Fig. 7A, AP-1 DNA binding activity increased at 1 h after camostat feeding, peaked at 2 h, and then decreased. The DNA protein complex was competed away by the cold probe as shown in Fig. 7B. Supershift assay with specific antibodies was carried out to determine the major components of AP-1 induced by camostat feeding. As shown in Fig. 7B, the presence of c-jun, c-fos, ATF-3, and phospho-ATF-2 specific antibody in EMSA reaction resulted in the formation of supershifted DNA-protein complexes (arrows indicate the position of antibody retarded AP-1 complexes). The shifted band was also detected in the presence of JunB antibody (data not shown). This indicates that c-jun, JunB, c-fos, ATF-2, and ATF-3 are involved in AP-1 DNA binding induced by camostat feeding in mouse pancreas. As expected, the antibody to Egr-1 had no effect on AP-1 DNA binding.


Figure 7
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  		Fig. 7. Effect of camostat feeding on AP-1 DNA binding and the components of AP-1. The binding activity of AP-1 was analyzed by EMSA. A: time course of AP-1 binding activity. B: specificity of AP-1 DNA binding was determined by addition of excess of 50-fold of unlabeled AP-1 consensus sequence (competitor). Supershift EMSA was performed to determine the components of AP-1 by using antibody to Egr-1, c-jun, c-fos, ATF-3, and phospho-ATF-2. The results are representative from at least 3 independent experiments.

 
Camostat-induced early response gene expression is dependent on CCK. To determine whether CCK is required for camostat feeding-induced early response gene expression, we first utilized a highly selective CCKA receptor antagonist IQM-95,333. As shown in Figs. 2 and 7, c-jun and c-fos induction and AP-1 DNA binding reached their maximum after 2 h of camostat feeding. Accordingly, we choose 2 h as the time point to study the effect of camostat feeding with IQM-95,333 or vehicle injection. As shown in Fig. 8, IQM-95,333 at a dose of 500 µg/kg partially blocked camostat feeding induced c-jun (Fig. 8A) and c-fos (Fig. 8B) expression by 67 and 84%, respectively. AP-1 DNA binding activity was also inhibited by IQM-95,333 as shown in Fig. 8C. By contrast, injection with vehicle did not affect c-jun, c-fos induction, and AP-1 DNA binding stimulated by camostat feeding.


Figure 8
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  		Fig. 8. Camostat-induced early response gene expression is dependent on CCK. Mice were fasted overnight and injected with IQM-95,333 (IQM) at 500 µg/mg or vehicle 1 h before refeeding with camostat containing powdered chow. Expression of c-jun (A) and c-fos (B) was analyzed by Western blotting with nuclear protein extract. Pooled data for each group are means ± SE from 6–9 mice. C: EMSA was performed to detect AP-1 DNA binding. In A and B pooled quantitative data are from 6–9 mice. The results in C are representative of 3 independent experiments. **P < 0.01 compared with fasted control.

 
To further demonstrate the requirement of CCK in early response gene induction, we used CCK knockout mice to confirm the results obtained with IQM-95,333. As previously shown in ICR mice, c-jun expression (Fig. 9A) and AP-1 DNA binding (Fig. 9B) increased in response to camostat feeding in the pancreas of control 129/sv mice. By contrast, in CCK-deficient mice, the maximal responses of c-jun induction and AP-1 DNA binding were reduced by 64 and 70%, respectively (Fig. 9). Thus inhibition was similar with both a chemical antagonist and gene deletion, indicating that early response gene induction is mainly dependent on CCK.


Figure 9
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  		Fig. 9. Effect of camostat-induced early response gene expression in mice lacking CCK. CCK-deficient (CCK–/–) or wild type (CCK+/+) mice were used. Quantitative results in A are means ± SE from 6–9 mice. The results in B are representative of 3 independent experiments. **P < 0.01 compared with fasted control.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study was designed to evaluate the early events in mouse pancreatic growth shortly after oral administration of camostat. Camostat feeding induces pancreatic acinar cell proliferation in mouse (51), but how the cell cycle machinery is turned on and the molecular basis for cells to exit Go phase and enter the cell cycle is not well understood. A wide spectrum of early response genes act as transcription factors to control expression of other genes and to regulate cell proliferation. Our results showed that the expression of early response genes was rapidly induced by camostat feeding in a time-dependent manner. The decreased expression of these early response genes after 4–8 h of camostat feeding is in keeping with the nature of the early response gene expression but may also be due to decreased food intake and decreased plasma CCK level after initial eating. The increased expression of these genes is related to cell proliferation in other cell types. For example, all five of these early response genes that we studied in detail were induced rapidly during liver regeneration after partial hepatectomy (52), and liver regeneration is impaired in the absence of c-jun (3). By stimulating the transcription of cyclin D1, ATF-3 facilitates the liver cells to exit their Go state (1). Deficiency in c-jun or inhibition of c-fos in mouse fibroblasts results in defect of proliferation (4, 27). The induction of Egr-1 has been demonstrated to occur in a variety of proliferating cells such as the glomerular mesangial cells and beta cells (16, 22). All these results from a variety of cell types demonstrate that c-jun, JunB, c-fos, ATF-3, and Egr-1 play an important role in regulating cell proliferation. The increased expression of a number of early response genes in response to camostat indicates that some of these may also be required for pancreatic acinar cell proliferation. Feeding camostat-containing chow results in acinar cell proliferation that increases at 24 h and peaks at 3 days (8). Pancreas-specific knockout of particular genes will provide an informative tool to further evaluate their involvement in pancreatic growth response.

Both c-jun and c-fos are key members of AP-1 family of transcription factors. One of the main functions of AP-1 is to aid the cells to exit their normally quiescent states and enter the cell cycle (44). AP-1 protein complex promotes cell proliferation through their ability to regulate the expression and function of cell cycle regulatory genes such as cyclin D1, p53, p21cip/waf1, p19ARF, and p16 (44). Camostat-induced mouse pancreatic growth is mainly due to pancreatic acinar cell proliferation. A previous study showed that pancreatic DNA content increased in response to camostat (51) and bromodeoxyuridine incorporation was maximal after 2 days (8). In our study, AP-1 DNA binding activity was increased by camostat feeding; this correlated with increased amount of general AP-1 components such as c-jun, c-fos, and ATF-3. These data suggest that AP-1 proteins may be involved in initiating the cell cycle in pancreatic acinar cells. They may directly control the cell cycle regulatory protein abundance or their activity. It is also possible that they increase the expression of another class of early response genes called late early response genes, which will act as transcription factors to activate expression of other genes that are important for cell cycle progression. Nab2 is an example of a late early response gene that may serve as a feedback inhibitor of Egr-1 action (49), and in our studies (Fig. 1G) Nab2 mRNA showed a delayed and sustained increase compared with Egr-1 and Egr-2. The transcription activity of AP-1 is controlled both by increasing the abundance of AP-1 components and by posttranslational modification of these proteins. The phosphorylation of c-jun and ATF-2 was elevated by camostat administration, and this may potentiate the transcription activity of c-jun by recruitment of other transcription coactivators such as cAMP response element (CRE) binding protein or by increasing its stability (56). ATF-2 and c-jun can form a heterodimer to bind the CRE site of other genes (26). It is known that a CRE site is present in the cyclin A (5) and cyclin D1 (40) promoters; therefore the enhanced expression and activity of c-jun and ATF-2 may act on this site and promote the expression of cyclin A and cyclin D1. Preliminary studies have shown increases in cyclin D, A, and E mRNA that begin 12–24 h after camostat feeding (L. Guo and J. A. Williams, unpublished data).

MAPKs are direct upstream regulators of AP-1 transcriptional activity (56). MAPKs are involved in regulation of cell proliferation through their regulation of AP-1 activity (7). JNK drives cyclin D1 expression and hepatocyte proliferation during liver regeneration (43). In a pancreatic regeneration model, ERK activity significantly increased early after pancreatectomy (33). In our previous long-term camostat feeding model, activity of JNK and ERK was increased by camostat and reached their maximum at 2 days and 12 h, respectively (50). In the present study we fasted mice before refeeding to synchronize their eating to evaluate the early events induced by camostat feeding. JNK and ERK activity increased quickly after refeeding, and this is consistent with the phosphorylation of their downstream targets c-jun and ATF-2. In the earlier work, however, mice were not fasted before being switched to camostat-containing chow, and they did not start eating right away. This can explain the different time course of ERK and JNK activation in the two studies.

Pancreatic acinar cells make up ~90% of the pancreas, and therefore we assumed that the increased expression of early response gene occurred mainly in acinar cells. The results from the immunohistochemistry confirmed our assumption. Strong c-jun, c-fos, and Egr-1 nuclear expression was restricted to acinar cells. A previous work showed that CCK directly induced c-jun and c-fos mRNA expression in isolated pancreatic acinar cells (31). Interestingly, in that work CCK also increased c-myc expression by Northern blot whereas we found no increase by quantitative real-time PCR (Table 1). All of these data suggest that c-jun, c-fos, and Egr-1 function only in acinar cells to trigger their proliferation.

Regular food intake increases plasma CCK level from around 1 pM to 3–5 pM and it does not induce pancreatic growth, while camostat increases CCK to 10 pM (51). In the present study, we found that feeding with regular chow induces a small increase of c-jun expression, whereas c-fos expression was not affected. This indicates that it is mainly the presence of camostat in the food that accounts for the robust increase of c-jun and c-fos expression at both mRNA and protein level. The small increase of c-jun expression by feeding with regular chow can be due to the small change of plasma CCK, and this is not enough to induce a growth response.

Camostat-induced pancreatic growth is CCK dependent and can be blocked by CCKA receptor antagonists (59). The CCKA receptor antagonist IQM-95,333, which blocked CCK-induced amylase release in rat pancreatic acini (2), inhibited 70% of c-jun induction and AP-1 DNA binding and completely blocked c-fos induction. These results were further confirmed by using CCK-deficient mice. A previous study showed that camostat failed to stimulate pancreatic growth in mouse with CCK deficiency (51). In our work, camostat feeding induced a small increase of c-jun expression and AP-1 DNA binding in the CCK-deficient mice. This indicates that some other effects besides CCK may also participate in the regulation of c-jun expression. Camostat feeding has also been reported to induce endogenous secretin release (55). In addition, CCK is known to increase neural activity, and part of the CCK effect may be mediated by the vagus (29).

In summary, camostat feeding induces early response gene expression in mouse pancreas. Since it is known that these early response genes are involved in the regulation of proliferation in other cell types, it is likely that all these genes or some of them play a role in the stimulation of pancreatic acinar cell proliferation. Further work is necessary to identify the down stream targets regulated by these early response genes.


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-59578 (J. A. Williams) and P30 DK-34933 (Michigan Gastrointestinal Peptide Center).


   ACKNOWLEDGMENTS
 
We thank Linda Samuelson for CCK-deficient mice, M. T. García-López from Consejo Superior de Investigaciones Científicas Instituto de Química Médica, Madrid, Spain for a gift of the CCK antagonist, and Brad Nelson for assistance with immunohistochemistry.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Williams, Dept. of Molecular and Integrative Physiology, Univ. of Michigan Medical School, 7744 Medical Sciences II, 1301 E. Catherine St., Ann Arbor, MI 48109-0622 (e-mail: jawillms{at}umich.edu)

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


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G. T. Gurda, L. Guo, S.-H. Lee, J. D. Molkentin, and J. A. Williams
Cholecystokinin Activates Pancreatic Calcineurin-NFAT Signaling In Vitro and In Vivo
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