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Physiol. Genomics 14: 59-72, 2003. First published April 22, 2003; doi:10.1152/physiolgenomics.00174.2002
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Received 9 December 2002; accepted in final form 11 April 2003.
Physiological Genomics 14:59-72 (2003)
1094-8341/03 $5.00 © 2003 American Physiological Society

Pancreatic gene expression during the initiation of acute pancreatitis: identification of EGR-1 as a key regulator

Baoan Ji 1,*, Xue-qing Chen 1,*, David E. Misek 2, Rork Kuick 2, Samir Hanash 2, Steve Ernst 3, Rebecca Najarian 1 and Craig D. Logsdon 1

1 Departments of Physiology, University of Michigan, Ann Arbor, Michigan 48109
2 Pediatric Oncology, University of Michigan, Ann Arbor, Michigan 48109
3 Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
We hypothesized that genes expressed in pancreatic acinar cells during the initiation of acute pancreatitis determine the severity of the disease. Therefore, we utilized microarrays to identify those genes commonly induced in rat pancreatic acinar cells within 1–4 h in two in vivo models, caerulein and taurocholate administration. This strategy yielded 51 known genes representing a complex array of molecules, including those that are likely to either reduce or increase the severity of the disease. Novel genes identified in the current study included ATF3, BRF1, C/EBPß, CGRP, EGR-1, ephrinA1, villin2, ferredoxin, latexin, lipocalin, MKP-1, NGFI-B, RhoA, tissue factor (TF), and syndecan. To validate these microarray results, the role of EGR-1 was further investigated using quantitative RT-PCR, Western blotting, and immunocytochemistry. EGR-1 expression occurred within acinar cells and correlated with the development of caerulein-induced acute pancreatitis in rats. Furthermore, the levels of the inflammation-related genes MCP-1, PAI, TF, IL-6, and ICAM-1 and the extent of lung inflammation were reduced during the initiation of caerulein-induced acute pancreatitis in EGR-1-deficient mice. Thus this study identified EGR-1 and several other novel genes likely to be important in the development and severity of acute pancreatitis.

real-time quantitative reverse transcription-polymerase chain reaction; acute respiratory distress syndrome; inflammation; pancreas; pancreatic acinar cell


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
ACUTE PANCREATITIS is an inflammatory condition that strikes about 185,000 persons a year in the US, with the incidence of this disease reported to be increasing (29). The majority of cases of acute pancreatitis are mild and self-limiting, but in about 25% of cases the attack is severe, and nearly 50% of these patients will die due to complications of the disease. Specific and effective therapies are lacking, and advances in treatment have been difficult due to a lack of understanding of the early cellular events important in the pathophysiology of this disease. Acute pancreatitis involves a complex cascade of local and systemic events. The initial insult results in injury and destruction of pancreatic epithelial cells accompanied by infiltration of leukocytes and an inflammatory response. In most cases, cellular defense mechanisms within the pancreas limit the extent of the inflammatory response such that the injury is mild and restricted to the pancreas with complete recovery expected (29). However, in some cases the inflammatory response propagates and becomes systemic, leading most often to acute respiratory distress syndrome (ARDS) or, in the most severe cases, multiple organ failure and death (5). The ultimate outcome of acute pancreatitis depends upon the balance of competing cellular mechanisms that either expand or restrict the local and systemic responses. The cellular mechanisms responsible for the initiation, propagation, and limitation of the inflammatory response in acute pancreatitis are not well understood.

Rapid events that occur in acinar cells early after stress or injury include the activation of a variety of signaling mechanisms and intracellular activation of digestive enzymes within pancreatic acinar cells. We hypothesized that these rapid early events are translated in to long-term responses by their abilities to stimulate the acinar cell expression of specific genes that determine the ultimate severity of pancreatitis. Support for this hypothesis comes from previous studies indicating that the transcription factor NF-{kappa}B is activated early in acute pancreatitis (25, 51), that its activity leads to acinar cell expression of cytokines and chemokines (21, 24, 25), and that adenoviral-mediated gene transfer of an active subunit of NF-{kappa}B into the pancreas mimics many of the inflammatory aspects of this disease (10). However, attempts to reduce NF-{kappa}B activation pharmacologically have not led to consistent conclusions (51). Furthermore, NF-{kappa}B is unlikely to be solely responsible for the regulation of gene expression during acute pancreatitis. Moreover, no study has been undertaken to comprehensively analyze the alterations in gene expression that occur early during the initiation of acute pancreatitis.

In the present study, we investigated alterations in gene expression in the rat pancreas associated with experimental acute pancreatitis using Affymetrix GeneChips to analyze ~8,000 genes. To discover genes mediating events in the initiation phase of acute pancreatitis, we selected those genes that were commonly expressed at early times in two rapid-onset experimental models of acute pancreatitis, the caerulein and taurocholate models. To reduce the contributions of infiltrating leukocytes, we focused on genes that were also induced in isolated pancreatic acini in vitro. This comparison yielded 51 genes, many of which have not previously been associated with acute pancreatitis, including ATF3, BRF1, C/EBPß, CGRP, EGR-1, ephrinA1, villin2, ferredoxin, latexin, lipocalin, MKP-1, NGFI-B, RhoA, tissue factor (TF), and syndecan. To validate our approach, we further investigated the role of the transcription factor EGR-1 in caerulein-induced acute pancreatitis. EGR-1 expression correlated with the development of secretagogue-induced acute pancreatitis. Furthermore, the levels of the inflammation-related genes IL-6, MCP-1, PAI, TF, ICAM-1, and systemic parameters of inflammation were reduced during the initiation of acute pancreatitis in EGR-1-deficient mice. These data demonstrate the importance of EGR-1 in acute pancreatitis and provide a list of novel molecules that are also likely to influence the severity of the disease. These data support the hypothesis that early acinar cell gene expression is important for the subsequent severity of acute pancreatitis and provide novel insight into the mechanism involved in the initiation of acute pancreatitis.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals and treatments.
All experiments were conducted with the consent of the University Committee for the Use of Experimental Animals at the University of Michigan. For secretagogue-induced pancreatitis in rats, male Wistar rats weighing 190–250 g were fasted overnight (16 h), then they were injected intraperitoneally with 1 ml of a supramaximal dose of caerulein (20 µg/kg), JMV-180 (1 mg/kg), or bombesin (50 µg/kg). In the taurocholate model, the rats were fasted overnight and then subjected to general anesthesia with ketamine (87 mg/kg) and xylazine (13 mg/kg). After laparotomy, the rats underwent temporary proximal occlusion of the bile duct at the level of the bifurcation of the right and left bile duct with a vascular occlusion clip, and a 30-gauge needle connected with PE-10 tubing was inserted into the common bile duct at the level of duodenum and secured by a second vascular occlusion clip. Taurocholic acid (T-4009, Sigma, St. Louis, MO; 50 µl of 1% solution dissolved in 0.9% sodium chloride) was injected over 10 s. For the analysis of the role of EGR-1 in acute pancreatitis, experiments were conducted with either wild-type (C57BL/6) or EGR-1-deficient mice, that have been previously described (34). Mice received hourly injections with 0.1 ml of either saline or caerulein (20 µg/kg) for 6 h. The animals were euthanized by an overdose of pentobarbital at indicated times posttreatment, and tissue samples were obtained for histology, assessment of edema, and preparation of RNA.

Isolation of acini and in vitro treatments.
The preparation of rat pancreatic acini was performed as previously described (26). The dispersed acini were aliquoted and treated with caerulein (100 nM) in HEPES-buffered Ringer solution (pH 7.5) supplemented with 0.2% glucose, Eagle minimal essential amino acids, 2 mM glutamine, SBTI (0.1 mg/ml), and 0.5% BSA 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.

Histology.
For routine histology, tissues were fixed for 2 h at room temperature with 4% formaldehyde, prepared from paraformaldehyde, in phosphate-buffered saline (PBS), pH 7.4, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Sections (5 µm-thick) were viewed with a Nikon Eclipse TE200 microscope. To identify the sites of EGR-1 expression, immunofluorescence of EGR-1 was examined using anti-EGR-1 antibody (sc-110; Santa Cruz Biotechnology, Santa Cruz, CA) on samples fixed as above then cryoprotected with 20% sucrose/PBS, and frozen in isopentane cooled in liquid nitrogen. Cryostat sections (5 µm-thick) were incubated in PBS containing 5% normal goat serum and 0.2% Triton X-100 for 30 min, and then for 1.5 h in rabbit anti-EGR-1 diluted 1:500 with 2% goat serum plus 0.2% Triton X-100. Sections were then rinsed in PBS, incubated for 30 min in goat anti-rabbit IgG conjugated to Cy3 (1:200 dilution in the same buffer used for primary antibody dilution), rinsed in PBS, and mounted under coverslips with a 3:1 mixture of glycerol and PBS containing 4 mg/ml p-phenylenediamine. Sections were viewed with a Leitz Aristoplan microscope. All images were recorded digitally and processed using Photoshop software (Adobe, Mountain View, CA).

Preparation of cRNA and GeneChip hybridization.
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA), cleaned on a RNeasy spin column (Qiagen, Valencia, CA), and then used to generate cRNA probes. Preparation of cRNA, hybridization, and scanning of the rat U34A microarrays were performed according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA). Briefly, 5 µg of total RNA was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (Superscript Choice System, Invitrogen) with an oligo(dT)24 primer containing a T7 RNA polymerase promoter site added 3' of the poly-T (Genset, La Jolla, CA). Following second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled cRNA was purified by using RNeasy spin columns (Qiagen). Then, 15 µg of each cRNA was fragmented at 94°C for 35 min in fragmentation buffer [40 mM Tris acetate (pH 8.1), 100 mM potassium acetate, 30 mM magnesium acetate] and then used to prepare 300 µl of hybridization cocktail (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween 20) containing 0.1 mg/ml of herring sperm DNA (Promega, Madison, WI), 500 µg/ml acetylated BSA (GIBCO-BRL), and a mixture of control cRNAs for comparison of hybridization efficiency between arrays and for relative quantitation of measured transcript levels. Prior to hybridization, the cocktails were heated to 94°C for 5 min, equilibrated at 45°C for 5 min and then clarified by centrifugation (16,000 g) at room temperature for 5 min. Aliquots of each sample (10 µg of fragmented cRNA in 200 µl of hybridization cocktail) were hybridized to RG-U34A arrays at 45°C for 16 h in a rotisserie oven set at 60 rpm. The arrays were then washed with nonstringent wash buffer (6 x SSPE) at 25°C, followed by stringent wash buffer [100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween 20] at 50°C, stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR), washed again with 6x SSPE, and stained with biotinylated anti-streptavidin IgG, followed by a second staining with streptavidin-phycoerythrin and a third washing with 6x SSPE. The arrays were scanned using the GeneArray scanner (Affymetrix).

Data analysis.
Probe intensity values were extracted from the array images using GeneChip 4.0 software (Affymetrix). Probe intensity values were then normalized using a control GeneChip with low background values as a standard. Probe pairs for which either the perfect match (PM) or the mismatched (MM) features were saturated in the image of the standard or for which PM-MM was smaller than -1,000 on the standard were excluded from the analysis. For saturated PM features on other GeneChips, the ratios of nonsaturating PM values for the GeneChip divided by the standard are averaged for a probe set by taking the anti-logarithm of the mean of the log ratios. This factor was multiplied by the PM values of the standard to impute values for the GeneChip under consideration. The MM values were imputed similarly. Probe set expression values were then computed by dropping the top and bottom 25% of the PM-MM data for each probe set on each GeneChip and averaging the remaining differences. Variation in the overall distribution of probe set expression values between GeneChips was removed by applying quantile normalization, in which a linear spline with 99 evenly spaced knots is fit to the quantile-quantile plots for the entire set of 8,800 probe sets and the resulting transformation applied. Values were normalized to the quantiles of the standard GeneChip, which had been scaled to have average probe set values of 1,500 units. We calculated fold changes between groups of samples by first replacing mean expression values below 10 units to a value of 10 to avoid negative values or spuriously large fold changes. For statistical tests, we first log transformed each normalized probe set expression value, x, to log[max(x + 100,0) + 100], which we found stabilized the within-group variances between high- and low-expression probe sets. To compare normal samples to in vivo caerulein and taurocholate samples, we performed a one-way analysis of variance, modeling the log-transformed values for each probe set as having separate means for each group. Comparison between pairs of groups were performed using the resulting simple contrast tests that are equivalent to ordinary two-sample t-tests except that the variance is estimated using the data from all groups. To exhibit the data for individual samples, the probe set values, or expression values below 100 units were changed to a value of 100 to avoid spurious results, were divided by the mean of all samples and converted to log base 2 values, then displayed using TreeView software (Stanford University). (Software to obtain probe set intensity measures, quantile normalize, and computed probe set annotation are available as part of the supplementary material at http://dot.ped.med.umich.edu:2000/pub/Panc_pancreatitis/index.html).

Quantitative RT-PCR.
Total RNA was isolated and purified as described above. The total RNA was reversed transcribed using Moloney murine leukemia virus (MulV) reverse transcriptase kit (Promega). PCR was performed using specific primers (Table 1). To correct for quantitative differences between samples and possible PCR artifacts, primers specific for acidic ribosomal phosphoprotein (ARP0) were used as internal controls in each sample. Briefly, 1 µg of total RNA was denatured for 5 min at 100°C and cooled for 5 min at 4°C; then 5 U of MulV reverse transcriptase was added to a total volume of 20 µl, and reverse transcription was conducted for 45 min at 42°C. For standard RT-PCR, 1 µl of the RT products were amplified by PCR using PCR master mixture (Promega). Amplification was performed on a thermal cycler (Bio-Rad) for 27–32 cycles (denaturation, 1 min at 95°C; annealing, 1 min at 56°C; and extension, 1 min at 72°C). The PCR products were separated on 1.8% agarose gels, and photomicrographs were taken of the ethidium bromide-stained gels. For real-time quantitative RT-PCR, SYBR green I was used to monitor the PCR products on the I-Cycler thermal cycler and IQ real-time PCR detection system (Bio-Rad) as previously described (30).


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  		Table 1. Primers used for RT PCR and quantitative RT-PCR

 
Analysis of the severity of acute pancreatitis.
Severity of acute pancreatitis was evaluated histologically and by measurements of pancreatic edema, pancreatic myeloperoxidase (MPO) activity, lung MPO activity, and serum amylase. Pancreatic edema was evaluated by measuring the wet-to-dry weight ratio as described previously (21). The results were calculated and expressed as a water index (wet weight/dry weight). MPO activity in pancreas and lung was determined according to Schierwagen et al. (46). Serum concentration of amylase was measured in 10 µl serum from each animal using the Phadebas amylase test as described previously (21).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Caerulein and taurocholate treatments lead to rapid onset of acute pancreatitis and share common effects on gene expression.
Treatment of rats either with high-dose caerulein or retrograde delivery of taurocholate led to histological evidence of pancreatic acinar cell disruption which was detectable within 1 h (data not shown) and more pronounced after 3–4 h (Fig. 1, A–C). Increased levels of tissue edema were also observed (Fig. 1E). These data establish that acute pancreatitis was induced in both of these models. The level of severity in the taurocholate model depends upon the concentration and volume of infusate, such that this approach has been used as a severe hemorrhagic model of acute pancreatitis (1). However, in the current study, to more closely mimic clinically relevant acute pancreatitis, a small volume of comparatively low concentration of taurocholate was utilized, which resulted in a relatively mild edematous pancreatitis with no evidence of hemorrhage (Fig. 1C). In contrast to high concentrations of caerulein, treatment with a high-dose JMV-180 (Fig. 1, D and E) did not disturb normal pancreatic histology or cause significant changes in tissue edema, as has been previously established (44). Similarly, the secretagogue bombesin did not cause acute pancreatitis (data not shown), as has been previously reported (22).



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  		Fig. 1. Acute pancreatitis is induced in rats by caerulein and taurocholate but not JMV-180 treatments. Rats were either untreated (A) or treated with caerulein (20 µg/kg ip) for 4 h (B) or with taurocholate (1%, intraductal injection) for 3 h (C) or with JMV-180 (1 mg/kg ip) for 4 h (D), and samples were prepared for histology. Both caerulein and taurocholate-treated animals showed histological disruption of the pancreas including enlarged tissue spaces and acinar cell vacuolization, whereas those treated with JMV-180 did not. E: measurement of tissue water also indicated significant increases in animals after treatments with caerulein (C) or taurocholate (T) but not JMV-180 (P < 0.05, n = 3–4).

 
To identify genes whose expression was correlated with the onset of acute pancreatitis we utilized Affymetrix RG-U34A GeneChips possessing 8,800 probe sets which surveyed ~7,000 known genes and ~1,000 expressed sequence tags (ESTs). We utilized two different rapid-onset models of acute pancreatitis, caerulein and taurocholate. To focus on early events, analysis was confined to the first 3–4 h after treatments. To focus on gene expression within pancreatic cells, we also utilized a selection criterion involving an in vitro preparation in which all gene expression originated from pancreatic cells. For this in vitro preparation, pancreatic acini from normal pancreas were stressed by the combination of physical isolation using collagenase digestion and mechanical shearing and in vitro treatment with caerulein for 2 h. Gene expression in the in vitro stressed pancreatic cells was compared with normal pancreas. Thus comparisons were made between gene expression levels in the pancreata of rats (3 for each group) that were either untreated or treated with one of three different treatments of caerulein (1, 2 or 4 h), one of three different treatments of JMV-180 (1, 2, or 4 h), one of two taurocholate treatments (1 h, 3 h), or those stressed in vitro. We made statistical comparisons between groups using the criteria that P < 0.5 for statistical tests, and, to further decrease the proportion of false positives, we further required that fold changes be >3.

To identify genes induced in pancreatic cells early in the caerulein pancreatitis model, we selected probe sets based upon three comparisons. We first compared gene expression levels between untreated (normal) and caerulein-treated rats (Fig. 2A). As expected, caerulein treatment increased the expression of many genes (336 probe sets increased in at least one caerulein treatment time point). To eliminate genes that were induced as part of the physiological response to secretagogue stimulation, we further required that expression for at least one caerulein time point be greater than the corresponding JMV-180 treatment time point (365 probe sets). Genes that were induced by caerulein compared with both normal controls and JMV-180-treated animals (216 probe sets) were considered specific to caerulein pancreatitis. Finally, we excluded genes that were not also induced in the in vitro stress model, which reduced the number of genes by 41% to 127 probe sets (Fig. 2A).



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  		Fig. 2. The relationships between genes induced in the caerulein and taurocholate models of acute pancreatitis are indicated by Venn diagrams. Affymetrix probe sets with fold changes greater than 3 (P < 0.05) at any of the experimental time points were selected. A: to identify genes induced in the caerulein model, expression levels were compared between caerulein-treated animals and untreated animals (C vs. N) and also between caerulein- and JMV-180-treated animals (C vs. JMV). To limit the selection to genes expressed within pancreatic cells, a further selection criterion involved a comparison to genes induced by stressing acini in vitro (in vitro vs. N). B: to identify genes induced in the taurocholate model, expression levels were compared between taurocholate-treated animals and untreated animals (T vs. N), and this list was reduced by requiring that the genes also be induced in acini stressed in vitro (in vitro vs. N). C: finally, to determine genes that were common to both models of acute pancreatitis, we compared genes specific to the caerulein model and those specific to the taurocholate model.

 
To identify genes associated with the taurocholate model of acute pancreatitis, we utilized two comparisons. We first compared gene expression between normal rats and rats treated with taurocholate and selected genes whose expression was induced at either of the time points analyzed (Fig. 2B). Next, to limit our analysis to genes expressed within pancreatic cells, we again utilized the comparison with pancreatic acini stressed in vitro, which reduced the final list by 47% to 100 probe sets (Fig. 2B).

To identify genes that were commonly induced in pancreatic cells during the initiation of acute pancreatitis, we compared the genes specifically associated with pancreatic cells in the caerulein and taurocholate models (Fig. 2C). Many genes induced by caerulein (52 probe sets) or taurocholate (25 probe sets) were specific to those individual models and were not selected in the other model. However, there were 75 probe sets that were selected for both models and which represented 51 known genes and 5 ESTs because of some redundancy on the GeneChip. Identical analysis of 1,000 data sets with randomly permuted labels gave only 0.36 qualifying probe sets on average (maximum 12) where we have obtained 75, indicating that less than 1 of these 75 probe sets is expected to be a false-positive finding. (Data on the full set of genes induced in both models of acute pancreatitis is available as part of the supplementary material.)

To indicate the expression levels of the genes induced in the individual animals in both models of acute pancreatitis, the genes with meaningful names were organized using average linkage clustering and displayed as a heat map (Fig. 3). This analysis showed that groups of genes were differentially expressed in the individual samples and that highly reproducible changes in mRNA levels occurred within each of the treated groups. These data also indicated that the expression levels of these genes fell into obvious temporal patterns including genes that were induced either early or late.



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  		Fig. 3. Multiple genes are induced early in acute pancreatitis and show a temporal pattern. The level of genes expressed early in two models of acute pancreatitis, as defined by the comparisons in the Venn diagrams shown in Fig. 2, A and B, were calculated as fold change from the mean of the levels of all in vivo samples and were displayed using colors to represent relative levels of gene expression, with the brightest red indicating the highest level of expression and green depicting low levels. Individual animals are represented by the vertical columns with normal (N), caerulein (Caer), JMV-180 (JMV), and taurocholate (Taur) after 1, 2, 3, or 4 h (represented by the number following the group designation) followed by the sample number. Genes are represented as horizontal rows, and the Affymetrix probe set name, the gene symbol or UniGene ID numbers, and name(s) of the genes are indicated. The genes were also placed into functional categories, and whether the individual genes were induced in the caerulein model (C) the taurocholate (T) or both (B) is indicated. Also indicated in columns are the mean fold changes in gene expression at each experimental time point compared with normal pancreas for caerulein at 1, 2, or 4 h (C1, C2, C4), JMV-180 at 1, 2, or 4 h (J1, J2, J4) or taurocholate at 1 or 3 h (T1, T3). Gene names in red represent genes previously associated with acute pancreatitis.

 
Genes expressed during the initiation of acute pancreatitis fall into several functional categories.
Both in vivo models of acute pancreatitis shared the common effect of inducing the expression of 21 genes coding for regulators of gene expression, including subunits of several transcription factors, coactivators of transcription, and regulators of RNA processing and stability (Fig. 3). This functional category included the greatest number of genes increased in this study (41%). Most of these genes have not previously been associated with acute pancreatitis. The majority of genes coding for transcription factor subunits were rapidly and transiently induced, showing high expression within the first hour and returning to control levels within 3–4 h (Fig. 3). However, some members of this functional category were more delayed in expression, including C/EBPß, nuclear protein 1, BTG3, and HNF3{gamma}. In general, there was agreement between the relative levels of induction of the various gene regulators in both models of acute pancreatitis, although larger effects were generally observed in the caerulein model (Fig. 3).

Several genes coding for molecules that are either secreted into pancreatic juice, or into extracellular spaces, were induced early after induction of experimental acute pancreatitis (Fig. 3). These genes code for a variety of molecules that are either protective or contribute to the expansion of the inflammatory response, and several have not previously been associated with acute pancreatitis. Expression of the genes coding for secreted molecules tended to occur at later times than did the expression of genes coding for transcription factors, and most were not increased before 2 or 3 h after treatments (Fig. 3). However, MCP-1, Gro-{alpha}, PAI-1, and Reg III were rapidly induced in both pancreatitis models.

Genes belonging to several additional functional groups, including stress-related molecules, intracellular signaling molecules, structural molecules, and metabolism or other functional categories, were also induced in acute pancreatitis (Fig. 3). Stress-related molecules belonging to several important groups, including antioxidants and heat shock proteins, were induced early during acute pancreatitis (Fig. 3). Several of these molecules were previously known to be induced during caerulein-induced pancreatitis, but others have not previously been associated with this disease. Acute pancreatitis is associated with disruption of the acinar cell cytoskeleton (31, 45). Therefore, it is not surprising that genes encoding several structural molecules were induced as a response to acinar cell injury that occurs during acute pancreatitis. Expression of these genes tended to occur later, and the levels continued to increase throughout the duration of the experiments (Fig. 3). Most of these genes have not been described previously as being induced during acute pancreatitis.

EGR-1 gene disruption attenuates pancreatic inflammatory gene expression.
EGR-1 is a transcription factor that has previously been suggested to be a master switch regulating inflammatory parameters (62) but has not previously been associated with acute pancreatitis. We observed that EGR-1 mRNA levels were rapidly elevated to high levels by both caerulein treatment (90-fold within 1 h) and taurocholate (30-fold within 1 h). In comparison, EGR-1 levels were also induced by JMV-180, but to a much lesser extent and much more slowly (7-fold at 4 h). To validate our gene profiling strategy, we further analyzed the expression of EGR-1 during secretagogue-induced acute pancreatitis in rats. EGR-1 mRNA was induced by caerulein but not by bombesin, a secretagogue that does not cause acute pancreatitis (Fig. 4, A and B) or by JMV-180 (1 mg·kg-1·h-1) (data not shown). EGR-1 mRNA levels were elevated within 30 min of caerulein treatment and remained highly elevated for at least 2 h before returning toward basal after 4 h. EGR-1 protein was also detected by Western blotting after caerulein, but not bombesin, treatment. Western blots indicated the presence of EGR-1 within 2 h of caerulein treatment, and levels remained elevated for at least 4 h (Fig. 4C). Immunocytochemistry confirmed the expression of EGR-1 within pancreatic acinar cells after caerulein treatment (Fig. 5). A low level of EGR-1 immunofluorescence was observed in control pancreas. Treatment with high-dose caerulein for 4 h led to strong induction of EGR-1 as indicated by increased fluorescent staining in acinar cells.



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  		Fig. 4. EGR-1 is expressed early in caerulein-induced pancreatitis. Rats were either administered saline (control) or treated with bombesin (500 µg·kg-1·h-1 ip for 2 h) or caerulein (20 µg/kg) for indicated times. RNA levels were estimated by RT-PCR (A), and quantitative levels from three independent experiments were determined using real-time PCR (B). Protein levels of pancreatic EGR-1 after various treatments were determined by Western blotting using an anti-EGR-1 antibody (C).

 


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  		Fig. 5. Immunofluorescence localization of EGR-1 in pancreas from control animals and animals receiving caerulein 4 h prior to death (A and C, respectively). Corresponding Nomarski images are shown in B and D. EGR-1 was expressed at very low levels in acinar cells in control animals (A), whereas a diffuse, moderately intense fluorescence was seen in acinar cells from caerulein-treated animals (C).

 
To determine the role of EGR-1 in gene expression associated with acute pancreatitis, we utilized quantitative RT-PCR to determine the levels of several known inflammation-related genes including MCP-1, IL-6, Gro-{alpha}, TF, and PAI-1, in wild-type and EGR-1-deficient mice 6 h after caerulein treatment (Fig. 6). Caerulein treatment induced expression of each of these molecules in wild-type mice as expected from our microarray data. However, EGR-1-deficient mice showed significantly lower levels of each of these inflammation-related molecules with the exception of Gro-{alpha}.



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  		Fig. 6. EGR-1-deficient animals show reduced expression of a number of inflammation-associated genes after caerulein treatment. Wild-type mice were treated with saline (WT-) or caerulein (6 hourly injections of 20 µg/kg) (WT+), and changes in the levels of various inflammation-associated genes were compared with those observed in EGR-1-deficient animals treated with caerulein (KO+). Levels of mRNA for tissue factor (TF) (A), plasminogen activator inhibitor (PAI) (B), MCP-1 (C), IL-6 (D), ICAM-1 (E), and Gro-{alpha} (F) were analyzed by real-time quantitative PCR from 3 independent experiments (bar graphs), and representative RT-PCR results are also shown (insets). For all data, values are means ± SE for 3 independent experiments. *P < 0.05 vs. control.

 
To determine whether these changes in inflammatory gene expression influenced the severity of acute pancreatitis, we examined wild-type and EGR-1-deficient mice after caerulein treatment for 6 h. Histological examination of the pancreas indicated that the extent of edema, pancreatic acinar cell vacuoles, and infiltration of inflammatory cells were not significantly different in wild-type and EGR-1-deficient mice (Fig. 7, A–C). Pancreatic edema and infiltration of neutrophils as analyzed by assays of water content and MPO activity (Fig. 7D) also appeared equivalent in wild-type and EGR-1-deficient mice. However, neutrophilic infiltration of the lungs was significantly reduced in EGR-1-deficient mice as indicated by reduced septal thickening observed histologically (Fig. 8, A–C) and by a significant decrease in lung MPO levels (Fig. 8D).



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  		Fig. 7. The severity of pancreas damage during caerulein-induced acute pancreatitis is unchanged in EGR-1-deficient mice. Histology of the pancreas was determined in wild-type (A and B) or EGR-1-deficient mice (C) treated with either saline (A) or caerulein (6 hourly injections of 20 µg/kg) (B and C). Caerulein treatment of wild-type and EGR-1-deficient mice leads to histological alterations of the pancreas associated with acute pancreatitis, including increased interstitial spaces, acinar cell vacuolization, and infiltration of leukocytes. Analysis of pancreatic edema and myeloperoxidase (MPO) activity (D) confirmed the lack of significant differences in parameters of pancreatitis in EGR-1-deficient (KO) vs. wild-type (WT) mice. For all data, values are means ± SE for 3–5 independent experiments.

 


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  		Fig. 8. The severity of lung damage during caerulein-induced acute pancreatitis is reduced in EGR-1-deficient mice. Histology of the lung was determined in wild-type (A and B) or EGR-1-deficient mice (C) treated with either saline (A) or caerulein (6 hourly injections of 20 µg/kg) (B and C). Caerulein treatment of wild-type mice lead to histological alterations of the lung associated with acute pancreatitis, including increased septal thickness and infiltration of leukocytes. Analysis of lung MPO activity (D) confirmed the significant decrease in lung inflammation in EGR-1-deficient (KO) vs. wild-type (WT) mice. Values are means ± SE for 3–5 independent experiments. *P < 0.05 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
Acute pancreatitis involves several separate processes, including acinar cell damage, thrombosis, increased vascular permeability, and local and systemic inflammation. These disease processes are initiated by pancreatic acinar cells in response to stress. The mechanisms mediating these events are not well understood. Many acinar cell responses, especially the most rapid, entail the activation of proteins by posttranslational modifications, including stress kinases (20, 58), trypsinogen (49), and the transcription factor NF-{kappa}B (27). However, we hypothesized that alterations in gene expression within pancreatic acinar in response to injury play an important role during the development of acute pancreatitis. To test this hypothesis, we identified genes whose expression was commonly induced in two different in vivo models within the first few hours after the initiation of pancreatic injury. Among the earliest genes induced were several transcription factors, including EGR-1. These transcription factors were further hypothesized to be key elements that regulate the subsequent expression of secreted, signaling, and effecter molecules that ultimately determine the severity of acute pancreatitis. Supporting this hypothesis, we demonstrated the importance of EGR-1 in the expression of inflammation-related genes and a systemic inflammatory response during the initiation of acute pancreatitis using EGR-1-deficient mice. Our data support a crucial role for acinar cell gene expression in determining the severity of acute pancreatitis, suggest a number of important genes whose abilities to either restrict or expand the inflammatory response are likely key to this process, and identify EGR-1 as a central regulator of pro-inflammatory gene expression in acute pancreatitis.

The current study focused on the early initiation phase of acute pancreatitis, as genes expressed during this phase are likely to instigate cascades of further responses. To achieve this objective, we utilized two well-characterized experimental models of acute pancreatitis that are rapid in onset. The importance of focusing on the earliest time points was underscored by the observation that a number of genes induced after 1 h returned to baseline after 2 or 4 h, such that these molecules would have been missed at later time points. The focus on early times utilized in the current study differs from a previous study that investigated pancreatic gene expression 12 h after caerulein treatment (13). Furthermore, the current study identified genes induced commonly in two different in vivo models to reveal genes of general importance in acute pancreatitis. There were many genes that were induced only in one and not in the other model. Such genes are less likely to be generally involved in acute pancreatitis and may reflect specific responses to specific insults. Ultimately, studies conducted at various intervals and with multiple models will be useful for full elucidation of the mechanisms involved in the initiation, propagation, and recovery from this disease.

The genes expressed in early acute pancreatitis, observed in the current study, most likely arose from pancreatic acinar cells, as the selection strategy utilized in this study included the requirement that gene expression be elevated in isolated pancreatic acini stressed in vitro. This in vitro model consists of >95% acinar cells and was prepared from a pancreas taken from an unstressed animal, thus eliminating the possibility of changes in gene expression due to infiltrating inflammatory cells. Several of the genes selected in this study are known to be expressed in acinar cells from previous research including, MCP-1 (4, 39), Gro-{alpha} (25, 61), IL-6 (12, 25, 38), fos (36, 64), jun (36), trypsinogen (42, 59), pancreatitis associated protein I (8), and metallothionein (17). Furthermore, immunohistological examination verified that EGR-1 expression occurred within pancreatic acinar cells. However, it remains possible that other minor pancreatic cell types may account for some of the observed changes in gene expression, and further studies to localize the molecules identified in this study will be necessary to fully determine their cellular origins.

The selection strategy utilized in this study identified genes commonly induced in two different models of acute pancreatitis. However, these genes are not necessarily expressed exclusively during acute pancreatitis. Thus several of the selected genes, including metallothionein and pancreatitis-associated-proteins, were also strongly induced by the JMV-180, which does not cause pancreatitis. Therefore, the presence of a gene on this list does not indicate that the gene product is either necessary or sufficient for causing pancreatitis, but only that it is expressed as part of the complex pattern of gene expression that occurs during the initiation of this disease. Further studies will be required to understand the specific roles of the individual genes.

The complex changes in gene expression observed in the current study are likely accounted for by interactions occurring between several transcription factors. Eukaryotic genes employ complex enhancers that integrate the input of multiple transcription factors, acting in a concerted manner. In acute pancreatitis, it has been observed that activation of the transcription factor NF-{kappa}B plays an important role in the development of an inflammatory response (10, 21, 24, 25, 51). Activation of NF-{kappa}B does not involve increased mRNA expression and therefore was not directly observed in the current study. The transcription factor AP-1 has also been implicated in acute pancreatitis (57), and components of AP-1, including c-jun and c-fos, were elevated in the current study in accord with earlier reports (36, 64). Importantly, the current study greatly expands the number of transcription factors that are likely important in the course of acute pancreatitis. Specifically, this study indicates for the first time the induction of the transcription factors ATF3, C/EBPß, EGR-1, NGFI-B, and Kruppel-like factors 4 and 6.

To validate our profiling strategy, we investigated EGR-1, a transcription factor not previously associated with acute pancreatitis. EGR-1, also known as nerve growth factor induced-A (NGFI-A), Krox-24, ZIF268, ETR103, and TIS8, is a phosphorylated zinc-finger-containing transcription factor often associated with cell growth stimulation (18, 62). However, the gene for EGR-1, located on the q31.1 "cytokine cluster" region of chromosome 5 in humans, is rapidly and transiently induced by a large number of stressful stimuli as well as growth factors and cytokines. In fact, EGR-1 has been proposed as a "master switch" of gene expression underlying coordinated responses to various types of injury (62). We observed that high levels of EGR-1 expression correlated with the development of acute pancreatitis. Thus much larger increases in expression were observed in rats treated with high concentrations of caerulein compared with those treated with either bombesin or JMV-180, secretagogues that do not cause acute pancreatitis. Moreover, we observed that inflammation-related gene expression was attenuated in mice with a homozygous deletion of EGR-1. Although these effects were not unexpected due to previous studies on the role of EGR-1 in other tissues, this represents the first demonstration of a role for EGR-1 in the initiation of acute pancreatitis.

To further investigate the role of EGR-1 in acute pancreatitis, we examined the effects of caerulein treatments in a mouse model deficient in EGR-1 gene expression (34). EGR-1-deficient mice had attenuated responses to treatments with high concentrations of caerulein on the expression of several inflammation-related molecules, including TF, intercellular adhesion molecule-1 (ICAM-1), plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). EGR-1 activity has previously been found to induce the expression of MCP-1 (62), ICAM-1 (62), PAI-1 (53), and TF (63) in other cell types. TF is the key cellular initiator of the coagulation protease cascade, and TF-induced local coagulation reduces blood flow and contributes to ischemia in a variety of inflammatory diseases (37). PAI-1 is the physiological inhibitor of plasmin, a protease associated with tissue repair and remodeling (7). PAI-1 has previously been observed to be induced in portions of necrotizing pancreas removed from human patients with pancreatitis (14). IL-6 is a cytokine with both pro- and anti-inflammatory properties (41) whose levels are increased during acute pancreatitis (35). A recent study conducted in IL-6-deficient mice indicated an increase in the severity of acute pancreatitis in these animals, suggesting that IL-6 may play a predominately protective effect in this setting (11). MCP-1 is a chemokine previously observed to be highly induced in acute pancreatitis (4, 21). ICAM-1 (CD54) is a cell surface glycoprotein that plays an important role in mediating leukocyte adhesion to endothelial and epithelial cells and has been shown to be increased in pancreatic acinar cells during acute pancreatitis and to play a key role in mediating neutrophilic infiltration (15, 65). Therefore, EGR-1 is a key regulator for several genes known, or likely, to play important roles in acute pancreatitis.

Despite the pronounced effects of EGR-1 deficiency on caerulein-induced expression of a variety of pro-inflammatory genes, the effects of EGR-1 deficiency on parameters of acute pancreatitis were small. This observation is most likely explained by the specific characteristics of the EGR-1-deficient mouse model. These mice possess complex adaptive responses to the germ line deletion of this important inflammation-related transcription factor, including alterations in immune cell development and antibody production (3, 47). Previously, these adaptive responses have helped explain the observation that these EGR-1-deficient mice had levels of airway inflammation similar to that of wild-type mice, despite a decrease in TNF-{alpha} production following allergen stimulation (47). Therefore, the systemic inflammatory responses in these genetically modified animals may not fully reflect the role of EGR-1 in normal animals. Despite these caveats, a significant decrease was noted in the level of pancreatitis-associated lung inflammation in EGR-1-deficient animals. Reduced lung inflammation has previously been found that in other animal models with reduced levels of pro-inflammatory molecules, including CCR-1 chemokine receptors (19), GM-CSF (16), or CINC (6). Thus these data support the recent suggestion that the major influence of pro-inflammatory molecules is on the systemic, rather than the pancreatic, parameters of acute pancreatitis (50). These systemic effects are highly clinically relevant because it is the systemic, rather than the local, effects of acute pancreatitis that are primarily responsible for the morbidity and mortality of the disease (5).

Transcription factors and pro-inflammatory genes were not the only genes observed to be induced early in the initiation phase of acute pancreatitis in the current study. Many other genes were induced that could be suggested to play either protective or damage inducing roles. Interestingly, often genes with directly opposing functions were both induced. For example, expression of mRNA for several cytokine/chemokines that are pro-inflammatory mediators were observed in the current study in accord with previous studies, including mcp-1 (4, 21, 39), gro-{alpha} (25, 61), and IL-6 (25, 38, 54). The expression of these genes was concurrent with the expression of several molecules that serve to limit pro-inflammatory signals. For example, an EST (UniGene: Rn.12550) which likely represents I{kappa}B-{alpha} (98% similarity), an inhibitory subunit of NF-{kappa}B, was rapidly induced, in accordance with previous studies (25, 51). The current study also observed increased expression of two members of the tristetraprolin (TTP) family of CCCH tandem zinc finger (TZF) proteins, BRF1 and TIS11, which play anti-inflammatory roles by decreasing the stability of RNAs for several cytokines including TNF-{alpha} (33, 52, 56). The rapid expression of BRF1, also known as CMG1, TIS11B, ERF1, or Berg36, has been observed in other models of inflammation (9) but has not previously been observed in acute pancreatitis.

Our discovery of a common set of genes expressed in two different in vivo models of acute pancreatitis helps explain the observation that many aspects of the disease are similar irrespective of the specific etiological factor. The induction of a common set of genes during multiple models of acute pancreatitis also suggests the possibility of activation of a common set of upstream signaling mechanisms. Although these signaling pathways are not the subject of the current work, speculation about their identities can be made based upon previous studies. Both caerulein at high concentrations (60) and taurocholate (32) have been reported to cause supraphysiological increases in pancreatic acinar cell intracellular Ca2+. Blocking the increase in intracellular Ca2+ with chelators blocks caerulein-induced cellular effects associated with acute pancreatitis including activation of intracellular trypsin (43) and NF-{kappa}B (28, 55). Therefore, one common mechanism involved in the initiation of pancreatitis in these two models may be a supraphysiological increase in intracellular Ca2+. Caerulein (20) and taurocholate (32) also induce the activation of stress kinases, including Jun kinase. Jun kinase has been shown to be important in caerulein-induced acute pancreatitis (20, 58). Interestingly, several of the transcription factors induced early in the onset of the disease are known to be regulated by activation of stress kinase pathways including EGR-1 (23), NGFB-1 (48), ATF3 (66), C/EBP (2), and AP-1 (40). Thus activation of stress kinases provides a potential link between the earliest known signaling events in acute pancreatitis and the more long-term consequences that stem from changes in gene expression.

In summary, the current study utilized gene array technology to identify genes induced in pancreatic cells early in two different models of rapid-onset experimental acute pancreatitis. We observed that a complex program of gene expression was rapidly initiated early in the course of this disease. Taken together with previous work in the field, the data from the current study suggest a model in which early signaling events lead to activation of transcription factors, including EGR-1, which regulate the expression of specific cellular and secreted molecules (Fig. 9). Some of the induced molecules tend to limit, whereas others tend to further exacerbate, the severity of the disease. The ultimate outcome of acute pancreatitis depends upon the balance of these opposing forces, and a comprehensive model for the mechanisms involved in the initiation, propagation, and restitution of acute pancreatitis will need to take into account changes in expression of both protective and injury promoting genes. Interventions that increase the expression of the protective genes or decrease the expression of genes that precipitate a systemic inflammatory response might be expected to ameliorate the disease, and investigation of the genes identified in the current study may lead to the development of improved therapies. Furthermore, several of the genes identified in the current study are secreted factors, and some may prove to be valuable as prognostic indices for this disease. Perhaps most importantly, this study provides a framework upon which to understand the mechanisms involved in the development of the many facets of acute pancreatitis and should serve as a foundation for further investigation.



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  		Fig. 9. Changes in gene expression that occur during the initiation of acute pancreatitis are incorporated into a model. Pancreatic acinar cells under the influence of various stressors show increased activity of a number of signaling mechanisms including increased intracellular Ca2+ and activation of stress kinases. These signaling pathways induce of a large number of transcription factors that act alone and in concert to influence the expression of specific genes. Many of these genes tend to limit the severity of the disease, including those for anti-inflammatory, vasodilator, anti-coagulant, protease inhibitor, and antioxidant molecules. Other genes tend to exacerbate the severity of the disease, including pro-inflammatory cytokine/chemokines, coagulation activators, and proteases. The balance between the actions of protective and damage-inducing genes determines the ultimate severity of acute pancreatitis.

 

   DISCLOSURES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-52067, the University of Michigan Gastrointestinal Peptide Center Grant DK-34933, and the NIDDK Biotechnology Center Grant DK-58771.


   ACKNOWLEDGMENTS
 
We thank Drs. J. A. Williams, D. Simeone, and C. Binkley for critically reading the manuscript and Barbara Lamb for expert assistance with the Affymetrix GeneChips.


   FOOTNOTES
 
Address for reprint requests and other correspondence: C. D. Logsdon, Dept. of Physiology, Univ. of Michigan, Ann Arbor, MI 48109 (E-mail: clogsdon{at}umich.edu).

10.1152/physiolgenomics.00174.2002.

* B. Ji and X. Chen contributed equally to this work. Back


   REFERENCES
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 DISCUSSION
 DISCLOSURES
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