Secretagogue-induced pancreatitis in mice devoid of chymotrypsin.

The serine protease chymotrypsin protects the pancreas against pancreatitis by degrading trypsinogen, the precursor to the digestive protease trypsin. Taking advantage of previously generated mouse models with either the Ctrb1 gene (encoding chymotrypsin B1) or the Ctrl gene (encoding chymotrypsin-like protease) disrupted, here we generated the novel Ctrb1-del ×Ctrl-KO strain in the C57BL/6N genetic background, which harbors a naturally inactivated Ctrc gene (encoding chymotrypsin C). The newly created mice are devoid of chymotrypsin yet the animals develop normally, breed well, and show no spontaneous phenotype, indicating that chymotrypsin is dispensable under laboratory conditions. When given cerulein, the Ctrb1-del ×Ctrl-KO strain exhibited markedly increased intrapancreatic trypsin activation and more severe acute pancreatitis, relative to wild-type C57BL/6N mice. After the acute episode, Ctrb1-del ×Ctrl-KO mice spontaneously progressed to chronic pancreatitis while C57BL/6N mice recovered rapidly. The cerulein-induced pancreas pathology in Ctrb1-del ×Ctrl-KO mice was highly similar to that previously observed in Ctrb1-del mice, however, trypsin activation was more robust and pancreatitis severity was increased. Taken together, the results confirm and extend prior observations demonstrating that chymotrypsin safeguards the pancreas against pancreatitis by limiting pathologic trypsin activity. In mice, the CTRB1 isoform, which constitutes about 90% of the total chymotrypsin content, is responsible primarily for the anti-trypsin defenses and protection against pancreatitis, however, the minor isoform CTRL also contributes to an appreciable extent.

Novel chymotrypsin C (CTRC) variants from real-world genetic testing of pediatric chronic pancreatitis cases.

BACKGROUND: Chymotrypsin C (CTRC) protects the pancreas against unwanted intrapancreatic trypsin activity through degradation of trypsinogen. Loss-of-function CTRC variants increase the risk for chronic pancreatitis (CP). The aim of the present study was to characterize novel CTRC variants found during genetic testing of CP cases at a pediatric pancreatitis center. METHODS: We used next-generation sequencing to screen patients. We analyzed the functional effects of CTRC variants in HEK 293T cells and using purified enzymes. RESULTS: In 5 separate cases, we detected 5 novel heterozygous CTRC variants: c.407C>T (p.Thr136Ile), c.550G>A (p.Ala184Thr), c.627Cdup (p.Ser210Leufs∗?, where the naming indicates a frame shift with no stop codon), c.628T>C (p.Ser210Pro), and c.779A>G (p.Asp260Gly). Functional studies revealed that with the exception of p.Ser210Leufs∗?, the CTRC variants were secreted normally from transfected cells. Enzyme activity of purified variants p.Thr136Ile, p.Ala184Thr, and p.Asp260Gly was similar to that of wild-type CTRC, whereas variant p.Ser210Pro was inactive. The frame-shift variant p.Ser210Leufs∗? was not secreted but accumulated intracellularly, and induced endoplasmic reticulum stress, as judged by elevated mRNA levels of HSPA5 and DDIT3, and increased mRNA splicing of XBP1. CONCLUSIONS: CTRC variants p.Ser210Pro and p.Ser210Leufs∗? abolish CTRC function and should be classified as pathogenic. Mechanistically, variant p.Ser210Pro directly affects the amino acid at the bottom of the substrate-binding pocket while the frame-shift variant promotes misfolding and thereby blocks enzyme secretion. Importantly, 3 of the 5 novel CTRC variants proved to be benign, indicating that functional analysis is indispensable for reliable determination of pathogenicity and the correct interpretation of genetic test results.

Mouse model of PRSS1 p.R122H-related hereditary pancreatitis highlights context-dependent effect of autolysis-site mutation.

Mutation p.R122H in human cationic trypsinogen (PRSS1) is the most frequently identified cause of hereditary pancreatitis. The mutation blocks protective degradation of trypsinogen by chymotrypsin C (CTRC), which involves an obligatory trypsin-mediated cleavage at Arg122. Previously, we found that C57BL/6N mice are naturally deficient in CTRC, and trypsinogen degradation is catalyzed by chymotrypsin B1 (CTRB1). Here, we used biochemical experiments to demonstrate that the cognate p.R123H mutation in mouse cationic trypsinogen (isoform T7) only partially prevented CTRB1-mediated degradation. We generated a novel C57BL/6N mouse strain harboring the p.R123H mutation in the native T7 trypsinogen locus. T7R123H mice developed no spontaneous pancreatitis, and severity parameters of cerulein-induced pancreatitis trended only slightly higher than those of C57BL/6N mice. However, when treated with cerulein for 2 days, more edema and higher trypsin activity was seen in the pancreas of T7R123H mice compared to C57BL/6N controls. Furthermore, about 40% of T7R123H mice progressed to atrophic pancreatitis in 3 days, whereas C57BL/6N animals showed full histological recovery. Taken together, the observations indicate that mutation p.R123H inefficiently blocks chymotrypsin-mediated degradation of mouse cationic trypsinogen, and modestly increases cerulein-induced intrapancreatic trypsin activity and pancreatitis severity. The findings support the notion that the pathogenic effect of the PRSS1 p.R122H mutation in hereditary pancreatitis is dependent on its ability to defuse chymotrypsin-dependent defenses.

Substrate specificity of human chymotrypsin-like protease (CTRL) characterized by phage display-selected small-protein inhibitors.

Chymotrypsin-like protease (CTRL) is one of the four chymotrypsin isoforms expressed in the human exocrine pancreas. Human genetic and experimental evidence indicate that chymotrypsins B1, B2, and C (CTRB1, CTRB2 and CTRC) are important not only for protein digestion but also for protecting the pancreas against pancreatitis by degrading potentially harmful trypsinogen. CTRL has not been reported to play a similar role, possibly due to its low abundance and/or different substrate specificity. To address this problem, we investigated the specificity of the substrate-binding groove of CTRL by evolving the substrate-like canonical loop of the Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), a small-protein reversible chymotrypsin inhibitor to bind CTRL. We found that phage-associated SGPI-2 variants with strong affinity to CTRL were similar to those evolved previously against CTRB1, CTRB2 or bovine chymotrypsin A (bCTRA), indicating comparable substrate specificity. When tested as recombinant proteins, SGPI-2 variants inhibited CTRL with similar or slightly weaker affinity than bCTRA, confirming that CTRL is a typical chymotrypsin. Interestingly, an SGPI-2 variant selected with a Thr29His mutation in its reactive loop was found to inhibit CTRL strongly, but it was digested rapidly by bCTRA. Finally, CTRL was shown to degrade human anionic trypsinogen, however, at a much slower rate than CTRB2, suggesting that CTRL may not have a significant role in the pancreatic defense mechanisms against inappropriate trypsinogen activation and pancreatitis.

Risk of chronic pancreatitis in carriers of the c.180C>T (p.Gly60=) CTRC variant: case-control studies and meta-analysis.

Chymotrypsin C (CTRC) is a digestive serine protease produced by the pancreas that regulates intrapancreatic trypsin activity and provides a defensive mechanism against chronic pancreatitis (CP). CTRC exerts its protective effect by promoting degradation of trypsinogen, the precursor to trypsin. Loss-of-function missense and microdeletion variants of CTRC are found in around 4% of CP cases and increase disease risk by approximately 3-7-fold. In addition, a commonly occurring synonymous CTRC variant c.180C>T (p.Gly60=) was reported to increase CP risk in various cohorts but a global analysis of its impact has been lacking. Here, we analyzed the frequency and effect size of variant c.180C>T in Hungarian and pan-European cohorts, and performed meta-analysis of the new and published genetic association data. When allele frequency was considered, meta-analysis revealed an overall frequency of 14.2% in patients and 8.7% in controls (allelic odds ratio (OR) 2.18, 95% confidence interval (CI) 1.72-2.75). When genotypes were examined, c.180TT homozygosity was observed in 3.9% of CP patients and in 1.2% of controls, and c.180CT heterozygosity was present in 22.9% of CP patients and in 15.5% of controls. Relative to the c.180CC genotype, the genotypic OR values were 5.29 (95% CI 2.63-10.64), and 1.94 (95% CI 1.57-2.38), respectively, indicating stronger CP risk in homozygous carriers. Finally, we obtained preliminary evidence that the variant is associated with reduced CTRC mRNA levels in the pancreas. Taken together, the results indicate that CTRC variant c.180C>T is a clinically relevant risk factor, and should be considered when genetic etiology of CP is investigated.

Defective binding of SPINK1 variants is an uncommon mechanism for impaired trypsin inhibition in chronic pancreatitis.

The serine protease inhibitor Kazal type 1 (SPINK1) protects the pancreas from intrapancreatic trypsin activation that can lead to pancreatitis. Loss-of-function genetic variants of SPINK1 increase the risk for chronic pancreatitis, often by diminishing inhibitor expression or secretion. Variants that are secreted normally have been presumed to be pathogenic because of defective trypsin inhibition, but evidence has been lacking. Here, we report quantitative studies on the inhibition of human trypsins by wildtype SPINK1 and seven secreted missense variants. We found that tyrosine sulfation of human trypsins weakens binding of SPINK1 because of altered interactions with Tyr43 in the SPINK1 reactive loop. Using authentic sulfated human trypsins, we provide conclusive evidence that SPINK1 variants N34S, N37S, R65Q, and Q68R have unimpaired inhibitory activity, whereas variant P55S exhibits a small and clinically insignificant binding defect. In contrast, rare variants K41N and I42M that affect the reactive-site peptide bond of SPINK1 decrease inhibitor binding by 20,000- to 30,000-fold and three- to sevenfold, respectively. Taken together, the observations indicate that defective trypsin inhibition by SPINK1 variants is an uncommon mechanism in chronic pancreatitis. The results also strengthen the notion that a decline in inhibitor levels explains pancreatitis risk associated with the large majority of SPINK1 variants.

Chronic progression of cerulein-induced acute pancreatitis in trypsinogen mutant mice.

T7K24R mice carry mutation p.K24R in mouse cationic trypsinogen (isoform T7), which is analogous to the human hereditary pancreatitis-associated mutation p.K23R. The mutation renders trypsinogen more prone to autoactivation. We recently reported that T7K24R mice exhibit increased severity of acute pancreatitis induced by repeated cerulein injections. The objective of the present study was to test whether trypsinogen mutant mice are prone to develop chronic pancreatitis, as observed in patients. We characterized the natural course of cerulein-induced pancreatitis in T7K24R mice and the C57BL/6N parent strain from the acute episode to 3 months post-attack. As expected, an acute episode of pancreatitis in C57BL/6N mice was followed by rapid recovery and histological restitution. In stark contrast, T7K24R mice developed progressive chronic pancreatitis with acinar cell atrophy, persistent macrophage infiltration, and diffuse fibrosis. The nadir of pancreas damage occurred on days 5-6 after the acute episode and was accompanied by digestive dysfunction. Remarkably, histological recovery was markedly delayed and permanent, chronic changes were still detectable 1-3 months after the acute pancreatitis episode. We conclude that during cerulein-induced acute pancreatitis in T7K24R mice, trypsin triggers an autonomous inflammatory program resulting in chronic disease progression, even after the cessation of cerulein-mediated injury. We propose that this uniquely trypsin-dependent mechanism explains the development of hereditary chronic pancreatitis in humans. Trypsin inhibition during acute attacks should prevent or delay progression to chronic disease.

Loss-of-function variant in chymotrypsin like elastase 3B (CELA3B) is associated with non-alcoholic chronic pancreatitis.

BACKGROUND: Genetic alterations in digestive enzymes have been associated with chronic pancreatitis (CP). Recently, chymotrypsin like elastase 3B (CELA3B) emerged as a novel risk gene. Thus, we evaluated CELA3B in two European cohorts with CP. METHODS: We analyzed all 8 CELA3B exons in 550 German non-alcoholic CP (NACP) patients and in 241 German controls by targeted DNA sequencing. In addition, we analyzed exons 6 and 7 by Sanger sequencing and the c.129+1G>A variant by melting curve analysis in 1078 further German controls. As replication cohort, we investigated up to 243 non-German European NACP patients and up to 1665 controls originating from Poland, Hungary, and Sweden. We assessed the cellular secretion and the elastase activity of recombinant CELA3B variants. RESULTS: In the German discovery cohort, we detected a splice-site variant in intron 2, c.129+1G>A, in 9/550 (1.64%) CP patients and in 5/1319 (0.38%) controls (P=0.007, OR=4.4, 95% CI=1.5-13.0). In the European replication cohort, this variant was also enriched in patients (9/178 [5.06%]) versus controls (13/1247 [1.04%]) (P=0.001, OR=5.1, 95% CI=2.1-12.0). We did not find the two previously reported codon 90 variants, p.R90C and p.R90L. CONCLUSIONS: Our data indicate that CELA3B is a susceptibility gene for CP. In contrast to previous reports suggesting that increased CELA3B activity is associated with CP risk, the splice-site variant identified here is predicted to cause diminished CELA3B expression. How reduced CELA3B function predisposes to pancreatitis remains to be elucidated.

Variants in the pancreatic CUB and zona pellucida-like domains 1 (CUZD1) gene in early-onset chronic pancreatitis - A possible new susceptibility gene.

OBJECTIVE: Non-alcoholic chronic pancreatitis (NACP) frequently develops in the setting of genetic susceptibility associated with alterations in genes that are highly expressed in the pancreas. However, the genetic basis of NACP remains unresolved in a significant number of patients warranting a search for further risk genes. DESIGN: We analyzed CUZD1, which encodes the CUB and zona pellucida-like domains 1 protein that is found in high levels in pancreatic acinar cells. We sequenced the coding region in 1163 European patients and 2018 European controls. In addition, we analyzed 297 patients and 1070 controls from Japan. We analyzed secretion of wild-type and mutant CUZD1 from transfected cells using Western blotting. RESULTS: In the European cohort, we detected 30 non-synonymous variants. Using different prediction tools (SIFT, CADD, PROVEAN, PredictSNP) or the combination of these tools, we found accumulation of predicted deleterious variants in patients (p-value range 0.002-0.013; OR range 3.1-5.2). No association was found in the Japanese cohort, in which 13 non-synonymous variants were detected. Functional studies revealed >50% reduced secretion of 7 variants, however, these variants were not significantly enriched in European CP patients. CONCLUSION: Our data indicate that CUZD1 might be a novel susceptibility gene for NACP. How these variants predispose to pancreatitis remains to be elucidated.

Novel p.G250A Mutation Associated with Chronic Pancreatitis Highlights Misfolding-Prone Region in Carboxypeptidase A1 (CPA1).

Inborn mutations in the digestive protease carboxypeptidase A1 (CPA1) gene may be associated with hereditary and idiopathic chronic pancreatitis (CP). Pathogenic mutations, such as p.N256K, cause intracellular retention and reduced secretion of CPA1, accompanied by endoplasmic reticulum (ER) stress, suggesting that mutation-induced misfolding underlies the phenotype. Here, we report the novel p.G250A CPA1 mutation found in a young patient with CP. Functional properties of the p.G250A mutation were identical to those of the p.N256K mutation, confirming its pathogenic nature. We noted that both mutations are in a catalytically important loop of CPA1 that is stabilized by the Cys248-Cys271 disulfide bond. Mutation of either or both Cys residues to Ala resulted in misfolding, as judged by the loss of CPA1 secretion and intracellular retention. We re-analyzed seven previously reported CPA1 mutations that affect this loop and found that all exhibited reduced secretion and caused ER stress of varying degrees. The magnitude of ER stress was proportional to the secretion defect. Replacing the naturally occurring mutations with Ala (e.g., p.V251A for p.V251M) restored secretion, with the notable exception of p.N256A. We conclude that the disulfide-stabilized loop of CPA1 is prone to mutation-induced misfolding, in most cases due to the disruptive nature of the newly introduced side chain. We propose that disease-causing CPA1 mutations exhibit abolished or markedly reduced secretion with pronounced ER stress, whereas CPA1 mutations with milder misfolding phenotypes may be associated with lower disease risk or may not be pathogenic at all.

Inactivation of mesotrypsin by chymotrypsin C prevents trypsin inhibitor degradation.

Mesotrypsin is an unusual human trypsin isoform with inhibitor resistance and the ability to degrade trypsin inhibitors. Degradation of the protective serine protease inhibitor Kazal type 1 (SPINK1) by mesotrypsin in the pancreas may contribute to the pathogenesis of pancreatitis. Here we tested the hypothesis that the regulatory digestive protease chymotrypsin C (CTRC) mitigates the harmful effects of mesotrypsin by cleaving the autolysis loop. As human trypsins are post-translationally sulfated in the autolysis loop, we also assessed the effect of this modification. We found that mesotrypsin cleaved in the autolysis loop by CTRC exhibited catalytic impairment on short peptides due to a 10-fold increase in Km , it digested ß-casein poorly and bound soybean trypsin inhibitor with 10-fold decreased affinity. Importantly, CTRC-cleaved mesotrypsin degraded SPINK1 with markedly reduced efficiency. Sulfation increased mesotrypsin activity but accelerated CTRC-mediated cleavage of the autolysis loop and did not protect against the detrimental effect of CTRC cleavage. The observations indicate that CTRC-mediated cleavage of the autolysis loop in mesotrypsin decreases protease activity and thereby protects the pancreas against unwanted SPINK1 degradation. The findings expand the role of CTRC as a key defense mechanism against pancreatitis through regulation of intrapancreatic trypsin activity.

Evolutionary expansion of polyaspartate motif in the activation peptide of mouse cationic trypsinogen limits autoactivation and protects against pancreatitis.

The activation peptide of mammalian trypsinogens typically contains a tetra-aspartate motif (positions P2-P5 in Schechter-Berger numbering) that inhibits autoactivation and facilitates activation by enteropeptidase. This evolutionary mechanism protects the pancreas from premature trypsinogen activation while allowing physiological activation in the gut lumen. Inborn mutations that disrupt the tetra-aspartate motif cause hereditary pancreatitis in humans. A subset of trypsinogen paralogs, including the mouse cationic trypsinogen (isoform T7), harbor an extended penta-aspartate motif (P2-P6) in their activation peptide. Here, we demonstrate that deletion of the extra P6 aspartate residue (D23del) increased the autoactivation of T7 trypsinogen threefold. Mutagenesis of the P6 position in wild-type T7 trypsinogen revealed that bulky hydrophobic side chains are preferred for maximal autoactivation, and deletion-induced shift of the P7 Leu to P6 explains the autoactivation increase in the D23del mutant. Accordingly, removal of the P6 Leu by NH2-terminal truncation with chymotrypsin C reduced the autoactivation of the D23del mutant. Homozygous T7D23del mice carrying the D23del mutation did not develop spontaneous pancreatitis and severity of cerulein-induced acute pancreatitis was comparable with that of C57BL/6N controls. However, sustained stimulation with cerulein resulted in markedly increased histological damage in T7D23del mice relative to C57BL/6N mice. Furthermore, when the T7D23del allele was crossed to a chymotrypsin-deficient background, the double-mutant mice developed spontaneous pancreatitis at an early age. Taken together, the observations argue that evolutionary expansion of the polyaspartate motif in mouse cationic trypsinogen contributes to the natural defenses against pancreatitis and validate the role of the P6 position in autoactivation control of mammalian trypsinogens.NEW & NOTEWORTHY Unwanted autoactivation of the digestive protease trypsinogen can result in pancreatitis. The trypsinogen activation peptide contains a polyaspartate motif that suppresses autoactivation. This study demonstrates that evolutionary expansion of these aspartate residues in mouse cationic trypsinogen further inhibits autoactivation and enhances protection against pancreatitis.

Mutation That Promotes Activation of Trypsinogen Increases Severity of Secretagogue-Induced Pancreatitis in Mice.

BACKGROUND & AIMS: Mutations in the human serine protease 1 gene (PRSS1), which encodes cationic trypsinogen, can accelerate its autoactivation and cause hereditary or sporadic chronic pancreatitis. Disruption of the locus that encodes cationic trypsinogen in mice (T7) causes loss of expression of the protein, but only partially decreases the severity of secretagogue-induced acute pancreatitis and has no effect on chronic pancreatitis. We investigated whether trypsinogen becomes pathogenic only when its activation is promoted by mutation. METHODS: We generated mice with knock-in of the p.K24R mutation (called T7K24R mice), which is analogous to human PRSS1 mutation p.K23R. We gave T7K24R and C57BL/6N (control) mice repeated injections of cerulein to induce pancreatitis. Plasma amylase activity, pancreatic edema, and myeloperoxidase content in pancreas and lungs were quantified. We expressed mutant and full-length forms of PRSS1 in Escherichia coli and compared their autoactivation. RESULTS: The p.K24R mutation increased autoactivation of T7 5-fold. T7K24R mice developed no spontaneous pancreatitis. T7K24R mice given cerulein injections had increased pancreatic activation of trypsinogen and more edema, infiltration of lung and pancreas by inflammatory cells, and plasma amylase activity compared with control mice given cerulein injections. Injection of cerulein for 2 days induced progressive pancreatitis in T7K24R mice, but not in control mice, with typical features of chronic pancreatitis. CONCLUSIONS: Introduction of a mutation into mice that is analogous to the p.K23R mutation in PRSS1 increases pancreatic activation of trypsinogen during secretagogue-induced pancreatitis. Higher pancreatic activity of trypsin increases the severity of pancreatitis, even though loss of trypsin activity does not prevent pancreatitis in mice.

Mouse model suggests limited role for human mesotrypsin in pancreatitis.

Mesotrypsin is a low-abundance human trypsin isoform with a unique evolutionary mutation that conferred resistance to trypsin inhibitors and restricted substrate specificity. Mesotrypsin degrades the serine protease inhibitor Kazal type 1 (SPINK1) and thereby might increase risk for pancreatitis. Here, we report a mouse model designed to test the role of mesotrypsin in pancreatitis. We introduced the human mesotrypsin evolutionary signature mutation into mouse cationic trypsinogen (isoform T7), resulting in a Gly to Arg change at the corresponding position 199. In biochemical experiments using purified proteins, the p.G199R T7 mutant recapitulated all salient features of human mesotrypsin. T7G199R mice developed normally with no spontaneous pancreatitis or other obvious phenotypic changes. Cerulein-induced acute pancreatitis in C57BL/6N and T7G199R mice showed similar severity with respect to inflammatory parameters and acinar cell necrosis while plasma amylase activity was higher in T7G199R mice. Neither SPINK1 degradation nor elevated intrapancreatic trypsin activation was apparent in T7G199R mice. The results indicate that in T7G199R mice the newly created mesotrypsin-like activity has no significant impact on cerulein-induced pancreatitis. The observations suggest that human mesotrypsin is unimportant for pancreatitis; a notion that is consistent with published human genetic studies.

Common calcium-sensing receptor (CASR) gene variants do not modify risk for chronic pancreatitis in a Hungarian cohort.

The calcium-sensing receptor (CASR) is expressed in the pancreas where it might regulate calcium concentrations in pancreatic secretions. Two independent studies reported conflicting results claiming that commonly occurring missense variants of the CASR gene are risk factors for chronic pancreatitis (CP). Here, we attempted to replicate the association between CASR variants and CP. We analyzed 337 patients and 840 controls from the Hungarian National Pancreas Registry either by direct sequencing of exon 7 and the flanking noncoding regions or by TaqMan SNP genotyping assays. We identified two common missense variants, c.2956G>T (p.A986S), and c.2968A>G (p.R990G), three low-frequency variants, c.3031C>G (p.Q1011E), c.2610G>A (p.E870=) and c.∗60T>A, and 8 rare variants including the novel variant c.1895G>A (p.G632D). When allelic or genotype distributions were considered, none of the CASR variants associated with CP. Subgroup analysis of nonalcoholic versus alcoholic patients revealed no disease association either. Our results demonstrate that common CASR variants do not modify the risk for CP and should not be considered as genetic risk factors in the clinical setting.

Ethanol feeding accelerates pancreatitis progression in CPA1 N256K mutant mice.

Alcoholic pancreatitis is a multifactorial, progressive, inflammatory disorder of the pancreas. Alcohol initiates pancreatitis and promotes its progression in the context of genetic susceptibility and/or other environmental risk factors such as smoking. Genetic mutations can cause digestive enzyme misfolding, which induces endoplasmic reticulum (ER) stress and elicits pancreatitis. Here, we tested the hypothesis that alcohol synergizes with misfolding in promoting ER stress and thereby accelerates chronic pancreatitis progression. To this end, we fed an ethanol-containing diet to CPA1 N256K mice, which carry the human p.N256K CPA1 mutation and develop spontaneous chronic pancreatitis. Inexplicably, CPA1 N256K mice suffered generalized seizures after 2-3 wk of ethanol feeding, which resulted in high mortality and the early termination of the study. Analysis of CPA1 N256K mice euthanized after 3-3.5 wk of ethanol feeding revealed more severe chronic pancreatitis associated with significantly increased Hspa5 [ER chaperone immunoglobulin heavy chain-binding protein (BiP)] mRNA levels when compared with CPA1 N256K mice on a control liquid diet. In contrast, ethanol feeding of C57BL/6N mice for 4 wk increased Hspa5 levels to a lesser degree and caused no pancreatitis. We conclude that ethanol feeding synergizes with the misfolding CPA1 mutant in promoting ER stress and thereby accelerates progression of chronic pancreatitis in CPA1 N256K mice.NEW & NOTEWORTHY Alcoholic pancreatitis is a multifactorial, progressive, inflammatory disorder of the pancreas. This study demonstrates that alcohol synergizes with digestive enzyme misfolding in promoting endoplasmic reticulum stress and thereby accelerates progression of chronic pancreatitis.

Genetics, Cell Biology, and Pathophysiology of Pancreatitis.

Since the discovery of the first trypsinogen mutation in families with hereditary pancreatitis, pancreatic genetics has made rapid progress. The identification of mutations in genes involved in the digestive protease-antiprotease pathway has lent additional support to the notion that pancreatitis is a disease of autodigestion. Clinical and experimental observations have provided compelling evidence that premature intrapancreatic activation of digestive proteases is critical in pancreatitis onset. However, disease course and severity are mostly governed by inflammatory cells that drive local and systemic immune responses. In this article, we review the genetics, cell biology, and immunology of pancreatitis with a focus on protease activation pathways and other early events.

Measuring digestive protease activation in the mouse pancreas.

Intrapancreatic activation of digestive proteases, trypsin and chymotrypsin in particular, is a hallmark of pancreatitis. In experimental rodent models, protease activation is routinely measured from pancreatic homogenates using fluorogenic peptide substrates. Here we investigated the optimal conditions for the determination of intrapancreatic trypsin and chymotrypsin activation elicited by a single intraperitoneal injection of cerulein in C57BL/6N mice. We found that these protease assays were significantly improved by using lower amounts of pancreatic homogenate and exclusion of bovine serum albumin from the assay buffer. Furthermore, pancreatic homogenates had to be freshly prepared and assayed; as freezing and thawing stimulated protease activation. Finally, replacement of the widely used Boc-Gln-Ala-Arg-AMC trypsin substrate with Z-Gly-Pro-Arg-AMC reduced the background activity in saline-treated control mice and thereby increased the extent of cerulein-induced trypsin activation. Using the optimized protocol, we reproducibly measured 20-fold and 200-fold increases in the intrapancreatic trypsin and chymotrypsin activity, respectively, in mice given cerulein.

International Consensus Guidelines for Risk Factors in Chronic Pancreatitis. Recommendations from the working group for the international consensus guidelines for chronic pancreatitis in collaboration with the International Association of Pancreatology, the American Pancreatic Association, the Japan Pancreas Society, and European Pancreatic Club.

BACKGROUND: Chronic pancreatitis (CP) is a complex inflammatory disease with remarkably impaired quality of life and permanent damage of the pancreas. This paper is part of the international consensus guidelines on CP and presents the consensus on factors elevating the risk for CP. METHODS: An international working group with 20 experts on CP from the major pancreas societies (IAP, APA, JPS, and EPC) evaluated 14 statements generated from evidence on four questions deemed to be the most clinically relevant in CP. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach was used to evaluate the level of evidence available per statement. To determine the level of agreement, the working group voted on the 14 statements for strength of agreement, using a nine-point Likert scale in order to calculate Cronbach's alpha reliability coefficient. RESULTS: Strong consensus and agreement were obtained for the following statements: Alcohol, smoking, and certain genetic alterations are risk factors for CP. Past history, family history, onset of symptoms, and life-style factors including alcohol intake and smoking history should be determined. Alcohol consumption dose-dependently elevates the risk of CP up to 4-fold. Ever smokers, even smoking less than a pack of cigarettes per day, have an increased risk for CP, as compared to never smokers. CONCLUSIONS: Both genetic and environmental factors can markedly elevate the risk for CP. Therefore, health-promoting lifestyle education and in certain cases genetic counselling should be employed to reduce the incidence of CP.

Human CPA1 mutation causes digestive enzyme misfolding and chronic pancreatitis in mice.

OBJECTIVE: Chronic pancreatitis is a progressive, relapsing inflammatory disorder of the pancreas, which often develops in the background of genetic susceptibility. Recently, loss-of-function mutations in CPA1, which encodes the digestive enzyme carboxypeptidase A1, were described in sporadic early onset cases and in hereditary pancreatitis. Mutation-induced misfolding of CPA1 and associated endoplasmic reticulum (ER) stress was suggested as potential disease mechanism; however, in vivo evidence has been lacking. The objective of the present study was to create a mouse model that recapitulates features of CPA1-associated chronic pancreatitis. DESIGN: We knocked-in the most frequently occurring p.N256K human CPA1 mutation to the mouse Cpa1 locus. Mutant mice were characterised with respect to pancreas pathology and ER stress and compared with C57BL/6N and CPA1 null control mice. RESULTS: In the CPA1 N256K mutant mice, we observed hallmarks of chronic pancreatitis that included progressive acinar cell atrophy, inflammatory cell infiltration, fibrosis and acinar-ductal metaplasia. In contrast, similarly to the C57BL/6N mice, the CPA1 null control strain exhibited no signs of pancreatic disease. Mutation p.N256K induced misfolding of mouse CPA1 and resulted in elevated expression of ER stress markers Hspa5 (BiP) and Ddit3 (CHOP) both in cell culture and mutant mice. CONCLUSION: The results offer categorical evidence that CPA1 mutations elicit enzyme misfolding and cause chronic pancreatitis via an ER stress-related mechanism.