1Department of Surgery, Tufts-New England Medical Center and Tufts University School of Medicine, Boston; 2Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts; and 3Drug Discovery, Johnson and Johnson Pharmaceutical Research and Development, Spring House, Pennsylvania
Submitted 30 July 2004 ; accepted in final form 23 September 2004
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ABSTRACT |
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caerulein; mitogen-activated protein kinase; extracellular signal-regulated kinase 1/2
Protease-activated receptor-2 (PAR-2) is one of the four members of the PAR family (8, 11, 18). It is proteolytically activated by trypsin and other trypsin-like serine proteases that cleave the NH2-terminal extracellular domain of the murine receptor at SKGR-SLIGRL, releasing a tethered ligand that contains the activating peptide SLIGRL. This proteolytic cleavage permits the activating peptide to bind, intramolecularly, to activate the receptor. In addition to proteolytic activation, PAR-2 responses can also be nonproteolytically elicited by exposing receptor-bearing cells to soluble preparations of the activating peptide SLIGRL. In contrast to the activating peptide, scrambled-sequence peptides and the reverse activating peptide LRGILS do not cause PAR-2 activation under these conditions (9). In addition to triggering a series of signal-transduction events and a cellular response that varies depending on the cell type, PAR-2 activation is followed by -arrestin-mediated receptor phosphorylation, receptor internalization, and receptor degradation that together result in desensitization to further PAR-2 stimulation and downregulation of PAR-2 levels (9).
PAR-2 is widely expressed in the gastrointestinal tract, and PAR-2-mediated responses in the exocrine pancreas have been previously reported. In the exocrine pancreas, PAR-2 activation has been found to accelerate acinar cell secretion of digestive enzymes and to alter duct cell ion channel function, suggesting that both acinar and duct cells express PAR-2 (13, 17). On the basis of studies involving other tissues, it is also likely that PAR-2 is expressed by pancreatic endothelial cells, resident macrophages, and nociceptive nerves, but studies evaluating PAR-2 expression by these other elements of the exocrine pancreas have not been reported.
It is generally believed that PAR-2-mediated events are proinflammatory (24) and that interference with PAR-2-mediated events has an anti-inflammatory effect (4). Viewed from that perspective, we presumed that genetic deletion of PAR-2 would protect mice from pancreatitis and that pharmacological activation of PAR-2 would worsen pancreatitis.
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MATERIALS AND METHODS |
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Mice. All experiments were performed using wild-type male C57BL/6 mice (2530 g) purchased from Charles River Laboratories (Wilmington, MA) or PAR-2/ mice bred into the C57BL/6 background (6). The animals were housed in temperature-controlled (23 ± 2°C) rooms with a 12:12-h light/dark cycle, fed standard laboratory chow, fasted overnight before each experiment, and given water ad libitum. All experiments conformed to protocols approved by the Institutional Animal Care and Use Committee of the Tufts-New England Medical Center.
Induction of pancreatitis. Secretagogue-induced pancreatitis was elicited by giving mice 12 hourly intraperitoneal injections of caerulein (50 µg/kg per injection). Unless otherwise stated, animals were killed by CO2 asphyxiation 1 h after the final caerulein injection. For measurement of intrapancreatic trypsin activity, animals were killed 30 min after the initial caerulein injection. Mice receiving SLIGRL-NH2 were intravenously injected with 4 µmol/kg SLIGRL-NH2 along with 4 µmol/kg amastatin 1 h before the first caerulein injection and were subsequently given hourly (x12) intraperitoneal injections of SLIGRL-NH2/amastatin along with caerulein. Mice given caerulein and amastatin according to the same protocol, but not SLIGRL-NH2, served as the control group for these experiments.
Severity of pancreatitis. The severity of pancreatitis was evaluated as reported previously (12). Pancreas samples stained with hematoxylin/eosin were examined by an experienced pancreatic morphologist who was not aware of the sample identity, and the extent of acinar cell necrosis, as a percentage of acinar cell mass, was quantitated. Pancreatic edema was quantitated by measuring pancreatic water content and was expressed as a percentage of tissue wet weight. Serum amylase activity and pancreatic trypsin activity were quantitated spectrophotometrically as previously described (15, 23). Bromide-enhanced chemiluminescence was used to quantitate pancreas myeloperoxidase content as described previously (12).
Immunoblot analysis.
Protein extracts were prepared by homogenizing pancreatic tissue with a Tissue Tearor (Biospec Products, Bartlesville, OK) in 10 mM Tris acetate pH 7.5 buffer containing protease inhibitors (Complete; Roche Molecular Biochemicals, Indianapolis, IN) and 1 mM PMSF. The extracted proteins were diluted in Laemmli sample buffer with 5% -mercaptoethanol. After being boiled, 50 µg of protein were resolved in 10% polyacrylamide gels in Tris-glycine-SDS buffer. The proteins were transferred to PVDF membranes, blocked in 5% nonfat dry milk in Tris-buffered saline, pH 7.5, containing 0.05% (wt/vol) Tween 20 (TBST). Blots were incubated at 4°C overnight with the SAM11 anti-PAR-2 antibody or with anti-phospho-ERK1/2 or anti-phospho-JNK antibodies diluted in TBST (0.5 µg/ml). The membranes were washed in TBST and incubated with horseradish peroxidase-conjugated anti-rabbit IgG at 1:5,000 (vol/vol) dilution in TBST-milk for 1 h. After being washed, immunoreactive bands in the membranes were visualized by enhanced chemiluminescence (PerkinElmer Life Sciences, Boston, MA) and quantitated by densitometry. To determine the total ERK1/2 or JNK, membranes were stripped and reblotted with antibodies against ERK1/2 or JNK.
Analysis of data. Results are reported as means ± SE obtained from three or more independent experiments. In all figures, vertical bars denote SE values and the absence of vertical bars indicates that the SE values are too small to depict. Statistical evaluation of data was accomplished by analysis of variance, and P < 0.05 was considered to indicate significant differences.
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RESULTS |
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Parenteral administration of SLIGRL-NH2 causes loss of PAR-2 immunoreactivity. Wild-type mice were given SLIGRL-NH2 (4 µmol/kg) along with amastatin (4 µmol/kg) by tail-vein injection and were killed at varying intervals. Control animals received only amastatin or amastatin with the reverse PAR-2 peptide LRGILS-NH2. The pancreas was removed, and extracted proteins were subjected to immunoblot analysis with anti-PAR-2 antibodies. As shown in Fig. 4, administration of the PAR-2-activating peptide leads to time-dependent loss of PAR-2 immunoreactivity, indicating that PAR-2 has been pharmacologically activated, internalized, and degraded. No change in PAR-2 immunoreactivity is observed when the reverse PAR-2 peptide is administered. The activation and degradation of PAR-2 after administration of a single dose of SLIGRL-NH2 is transient and, within 2 h of SLIGRL-NH2 administration, PAR-2 levels are restored as newly synthesized receptors appear (not shown). With repeated hourly doses of SLIGRL-NH2/amastatin, however, PAR-2 immunoreactivity remains markedly depressed for up to 4 h.
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As shown in Figs. 5 and 6, the severity of pancreatitis is markedly reduced by administration of the PAR-2-activating peptide to wild-type but not to PAR-2/ mice. Microscopic examination of samples show that acinar cell vacuolization, intralobular edema, acinar cell necrosis, and pancreatic edema are each diminished by administration of SLIGRL-NH2/amastatin to wild-type but not to PAR-2/ mice (Fig. 5). Quantitation of the changes related to pancreatitis severity shows that pancreatic edema (i.e., pancreatic water content), inflammation (i.e., myeloperoxidase activity), and acinar cell necrosis are each decreased in caerulein-treated wild-type mice given SLIGRL-NH2/amastatin compared with those given only amastatin but that no decrease in pancreatitis severity is observed when PAR-2/ mice are given the PAR-2-activating peptide (Fig. 6).
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MAPKs are activated during caerulein-induced pancreatitis, and the nuclear translocation of activated ERK1/2 is decreased by administration of the PAR-2-activating peptide.
Wild-type mice were injected with caerulein along with amastatin either with or without SLIGRL-NH2. After 30 min, the pancreata were removed and the nuclear fraction was separated from the cytosolic fraction. Proteins were subjected to immunoblot analysis with anti-phospho-ERK1/2 antibody. As shown in Fig. 7, total ERK1/2 in whole cells is not altered at this time but the level of phospho-ERK1/2 is markedly increased, indicating that it is activated during the initial 30 min of supramaximal secretagogue stimulation. Nuclear translocation of phospho-ERK1/2 as well as total ERK1/2 is observed, but the nuclear translocation of the total and activated forms is markedly reduced by administration of SLIGRL-NH2. In other studies using anti-JNK and anti-phospho-JNK antibodies, activation (i.e., phosphorylation) and nuclear translocation of JNK following supramaximal stimulation with caerulein was also observed, but PAR-2 activation did not alter the nuclear translocation (not shown). Activation and nuclear translocation of the proinflammatory transcription factors activator protein-1 (AP-1) and nuclear factor (NF)-B were also evaluated. Although both factors are activated and translocated to the nucleus within 30 min of supramaximal secretagogue stimulation, our studies showed that PAR-2 activation does not alter this response (not shown).
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DISCUSSION |
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We have employed an experimental model of pancreatitis that is induced by the repeated administration of supramaximally stimulating doses of the cholecystokinin analog caerulein to test our hypothesis. This secretagogue-induced model of pancreatitis has been extensively employed by us as well as many other groups to define the early events and severity determinants in pancreatitis. It is the most well-characterized of the various pancreatitis models and, although its mechanism of induction clearly differs from that which is likely to trigger clinical pancreatitis, its widespread application to studies of pancreatitis is based on the generally held beliefs that secretagogue-induced pancreatitis involves many of the same biochemical and cell biological events that are responsible for the clinical disease and that factors that regulate the severity of caerulein-induced pancreatitis are also likely to regulate the severity of clinical pancreatitis. Furthermore, since it is not possible to access the pancreas of patients during the early stages of clinical pancreatitis, studies dealing with the cell biology of acute pancreatitis and the factors that regulate pancreatitis severity must, for pragmatic reasons, be performed using experimental models of the disease. Of the various models that might be used, the secretagogue-induced model in mice is ideal for such studies because it is an easily elicited and highly reproducible model of severe, necrotizing pancreatitis.
To test our hypothesis, we have used mice with genetic deletion of PAR-2, but contrary to our expectations that genetic deletion of PAR-2 would reduce the severity of secretagogue-induced pancreatitis, we have found that genetic deletion of PAR-2 actually leads to a marked worsening of the disease. As shown in Figs. 1 and 2, the magnitude of pancreatic edema, acinar cell vacuolization, acinar cell injury/necrosis, and intrapancreatic inflammation are all increased in PAR-2-deficient mice when those animals are compared with the PAR-2-sufficient control group. The hyperamylasemia of pancreatitis is not altered by PAR-2 deletion, but as noted by many groups including our own, the magnitude of hyperamylasemia does not correlate well with the severity of pancreatitis.
Our finding that PAR-2 deletion worsens the severity of experimental pancreatitis strongly suggests that the presence of PAR-2 protects mice against pancreatitis and that PAR-2 exerts this protective effect by mediating events that reduce the extent of acinar cell injury/necrosis and pancreatic inflammation. If valid, this conclusion would predict that 1) in wild-type animals, PAR-2 is activated during pancreatitis, and 2) pharmacological activation of PAR-2 in wild-type animals will reduce the severity of pancreatitis. Our studies have confirmed both of those predictions.
As shown in Fig. 3, pancreatic PAR-2 immunoreactivity is rapidly lost following the administration of a single, supramaximally stimulating dose of caerulein. Theoretically, loss of PAR-2 immunoreactivity could reflect the nonspecific digestive changes of pancreatitis, but this is unlikely because PAR-2 immunoreactivity is lost very shortly after the administration of caerulein, before evidence of cell injury can be detected. On the other hand, it is much more likely that the loss of PAR-2 immunoreactivity that we have observed reflects trypsin-induced activation of the receptor, an event that is known to be followed by receptor phosphorylation, arrestin-mediated receptor internalization, and receptor degradation (7). Our observations, therefore, lead us to conclude that, in wild-type mice, PAR-2 is activated during the early stages of pancreatitis, and we are led to speculate that the worsening of pancreatitis, which is noted in PAR-2-deficient animals, results from the loss of protection against pancreatitis that is otherwise afforded to wild-type animals by PAR-2 activation.
We performed a series of experiments designed to define the conditions under which PAR-2 could be pharmacologically activated before and during the onset of secretagogue-induced pancreatitis. In addition to proteolytic activation of PAR-2, the tethered ligand receptor can be nonproteolytically activated by exposure to the activating peptide SLIGRL-NH2. As shown in Fig. 4, pancreatic PAR-2 immunoreactivity is rapidly lost following the parenteral administration of the activating peptide, but PAR-2 immunoreactivity is not lost if the reverse peptide LRGILS-NH2, which does not activate PAR-2, is administered.
To achieve long-lasting PAR-2 activation in our studies, we gave animals repeated doses of SLIGRL-NH2 along with the aminopeptidase inhibitor amastatin and, as shown in Figs. 5 and 6, the severity of secretagogue-induced pancreatitis is reduced by repeated administration of SLIGRL-NH2/amastatin. Pancreatic edema (i.e., pancreatic water content), pancreatic inflammation (i.e., myeloperoxidase activity within the pancreas), and pancreatic acinar cell necrosis are all markedly diminished by administration of the PAR-2-activating peptide to wild-type animals. To be certain that the effects of SLIGRL-NH2 administration reflect the effects of the PAR-2-activating peptide on PAR-2-bearing cells, we carried out a control series of identical experiments using PAR-2-deficient animals, and as shown in Fig. 6, administration of SLIGRL-NH2 to those animals did not alter the severity of pancreatitis. Together, our observations have led us to conclude that pharmacological activation of PAR-2, achieved by parenteral administration of the PAR-2-activating peptide, reduces the severity of secretagogue-induced pancreatitis in wild-type mice and that, in pancreatitis, PAR-2 activation is an anti-inflammatory event.
We have examined the following three mechanisms by which PAR-2 activation might reduce the severity of pancreatitis: 1) that PAR-2 activation might interfere with the intrapancreatic activation of digestive enzyme zymogens; 2) that PAR-2 activation might interfere with the activation and/or nuclear translocation of the proinflammatory transcription factors AP-1 or NF-B; and 3) that PAR-2 activation might interfere with the activation and/or nuclear translocation of MAPKs. Intrapancreatic activation of trypsinogen and other digestive zymogens is a critical event in the evolution of secretagogue-induced and other models of pancreatitis, and in previous studies, we have found that interventions that prevent trypsinogen activation lead to a reduction in pancreatitis severity (20). However, as shown in Fig. 6, pharmacological activation of PAR-2 does not reduce the level of trypsin activity within the pancreas during secretagogue-induced pancreatitis, and thus it is unlikely that PAR-2 protects against pancreatitis by interfering with intrapancreatic zymogen activation. In other studies (not shown), we found that the intrapancreatic activation and nuclear translocation of both AP-1 and NF-
B, which occur during secretagogue-induced pancreatitis (20) are not altered by pharmacological activation of PAR-2, and based on these findings, we have concluded that PAR-2 activation protects against pancreatitis by mechanisms that do not involve either AP-1 or NF-
B.
Studies previously reported by others have indicated that pancreatic JNK and ERK1/2 are each activated during the early stages of secretagogue-induced pancreatitis (5, 19), and we have confirmed these findings (Fig. 7). In addition, we have found that ERK1/2 is translocated to the nucleus during pancreatitis, where, on the basis of studies in many other systems, it presumably acts to regulate a large number of cellular events (1, 14). In our studies, we have found that pharmacological activation of PAR-2 during pancreatitis does not alter the activation (i.e., phosphorylation) of either ERK1/2 or JNK or the nuclear translocation of activated JNK. On the other hand, we have found that PAR-2 activation does profoundly reduce the nuclear translocation of ERK1/2 and phospho-ERK1/2 (Fig. 7).
Recently, DeFea and co-workers (7) have shown that, in neutrophils, PAR-2 activation causes ERK1/2 to associate with the PAR-2/arrestin complex. As a result, ERK1/2 translocation to the nucleus is prevented by PAR-2 activation, and this causes the activated ERK1/2 to be redirected to the plasmalemma. A similar mechanism may explain the effects of PAR-2 activation on the intracellular trafficking of activated ERK1/2 within the pancreas during pancreatitis. Regardless of the mechanisms involved, however, our findings that PAR-2 activation reduces the severity of pancreatitis and interferes with nuclear translocation of activated MAPKs suggests that the two events are interrelated, i.e., that PAR-2 activation protects against pancreatitis by trapping ERK1/2 within the cytoplasmic compartment and that this alteration in MAPK trafficking downregulates proinflammatory events and/or upregulates anti-inflammatory events that are critical to pancreatitis.
It is perhaps important to point out that the studies reported in this communication were performed under in vivo conditions, that our knockout mice had global deletion of PAR-2, and that parenteral administration of the PAR-2-activating peptide was likely to cause PAR-2 activation in many, if not all, PAR-2-bearing cell types. Thus, although our studies clearly indicate that PAR-2 activation exerts a protective effect on pancreatitis, we are unable to identify the cell type responsible for that protective effect. The functional expression of PAR-2 on pancreatic acinar cells and the fact that trypsinogen is activated within pancreatic acinar cells during the early stages of pancreatitis certainly support the notion that it is activation of acinar cell PAR-2 that exerts the protective effect on pancreatitis, but studies employing cell type-specific silencing of PAR-2 and/or in vitro studies using isolated acinar cells will be needed to confirm this.
Finally, it may be of interest to consider the potential survival advantage that accompanies PAR-2 expression by pancreatic acinar cells. Studies by our group (10) and others have shown that, even in the absence of pancreatitis, small amounts of digestive zymogens, including trypsinogen, become activated within pancreatic acinar cells. It has been assumed that the gland is protected against injury from these inappropriately activated enzymes by the presence of potent trypsin inhibitors within the secretory compartment of acinar cells and by the fact that, before their secretion, the zymogens are sequestered from the cytoplasmic space by being enclosed within membrane-bound organelles. From a purely teleological standpoint, however, it is also tempting to speculate that a trypsin receptor-mediated anti-inflammatory response might convey a considerable survival advantage by dampening the potentially injurious effects of intrapancreatic trypsinogen activation.
In summary, we have shown that genetic deletion of PAR-2 in mice worsens the severity of experimental pancreatitis and that pharmacological activation of mouse PAR-2 has the opposite effect. As this manuscript was being submitted for publication, Namkung et al. (16) reported studies that indicated that pharmacological activation of rat PAR-2 could reduce the severity of secretagogue-induced pancreatitis in those animals, but their studies did not address the effects of PAR-2 deletion or the mechanisms responsible for the protective effects of PAR-2 activation. Our own studies have suggested that PAR-2 activation may reduce the severity of pancreatitis by interfering with the intracellular trafficking and action of the MAPK ERK1/2. Regardless of the mechanisms involved, however, these studies suggest that interventions that trigger PAR-2 activation may beneficially effect the severity of pancreatitis and that they may be of value in the treatment and/or prevention of the clinical disease.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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