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.

Preclinical testing of dabigatran in trypsin-dependent pancreatitis.

Pancreatitis, the inflammatory disorder of the pancreas, has no specific therapy. Genetic, biochemical, and animal model studies revealed that trypsin plays a central role in the onset and progression of pancreatitis. Here, we performed biochemical and preclinical mouse experiments to offer proof of concept that orally administered dabigatran etexilate can inhibit pancreatic trypsins and shows therapeutic efficacy in trypsin-dependent pancreatitis. We found that dabigatran competitively inhibited all human and mouse trypsin isoforms (Ki range 10-79 nM) and dabigatran plasma concentrations in mice given oral dabigatran etexilate well exceeded the Ki of trypsin inhibition. In the T7K24R trypsinogen mutant mouse model, a single oral gavage of dabigatran etexilate was effective against cerulein-induced progressive pancreatitis, with a high degree of histological normalization. In contrast, spontaneous pancreatitis in T7D23A mice, which carry a more aggressive trypsinogen mutation, was not ameliorated by dabigatran etexilate, given either as daily gavages or by mixing it with solid chow. Taken together, our observations showed that benzamidine derivatives such as dabigatran are potent trypsin inhibitors and show therapeutic activity against trypsin-dependent pancreatitis in T7K24R mice. Lack of efficacy in T7D23A mice is probably related to the more severe pathology and insufficient drug concentrations in the pancreas.

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.

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.

Loss of chymotrypsin-like protease (CTRL) alters intrapancreatic protease activation but not pancreatitis severity in mice.

The digestive enzyme chymotrypsin protects the pancreas against pancreatitis by reducing harmful trypsin activity. Genetic deficiency in chymotrypsin increases pancreatitis risk in humans and pancreatitis severity in mice. Pancreatic chymotrypsin is produced in multiple isoforms including chymotrypsin B1, B2, C and chymotrypsin-like protease (CTRL). Here we investigated the role of CTRL in cerulein-induced pancreatitis in mice. Biochemical experiments with recombinant mouse enzymes demonstrated that CTRL cleaved trypsinogens and suppressed trypsin activation. We generated a novel CTRL-deficient strain (Ctrl-KO) using CRISPR-Cas9 genome engineering. Homozygous Ctrl-KO mice expressed no detectable CTRL protein in the pancreas. Remarkably, the total chymotrypsinogen content in Ctrl-KO mice was barely reduced indicating that CTRL is a low-abundance isoform. When given cerulein, Ctrl-KO mice exhibited lower intrapancreatic chymotrypsin activation and a trend for higher trypsin activation, compared with C57BL/6N mice. Despite the altered protease activation, severity of cerulein-induced acute pancreatitis was similar in Ctrl-KO and C57BL/6N mice. We conclude that CTRL is a minor chymotrypsin isoform that plays no significant role in cerulein-induced pancreatitis in mice.

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.

Natural single-nucleotide deletion in chymotrypsinogen C gene increases severity of secretagogue-induced pancreatitis in C57BL/6 mice.

Genetic susceptibility to chronic pancreatitis in humans is frequently associated with mutations that increase activation of the digestive protease trypsin. Intrapancreatic trypsin activation is an early event in experimental acute pancreatitis in rodents, suggesting that trypsin is a key driver of pathology. In contrast to trypsin, the pancreatic protease chymotrypsin serves a protective function by mitigating trypsin activation through degradation. In humans, loss-of-function mutations in chymotrypsin C (CTRC) are common risk factors for chronic pancreatitis; however, the pathogenic effect of CTRC deficiency has not been corroborated in animal models yet. Here we report that C57BL/6 mice that are widely used for genetic manipulations do not express functional CTRC due to a single-nucleotide deletion in exon 2 of the Ctrc gene. We restored a functional Ctrc locus in C57BL/6N mice and demonstrated that in the novel Ctrc+ strain the severity of cerulein-induced experimental acute and chronic pancreatitis was significantly ameliorated. Improved disease parameters were associated with reduced intrapancreatic trypsin activation suggesting a causal link between CTRC-mediated trypsinogen degradation and protection against pancreatitis. Taken together with prior human genetic and biochemical studies, the observations provide conclusive evidence for the protective role of CTRC against pancreatitis.

Novel Pathogenic PRSS1 Variant p.Glu190Lys in a Case of Chronic Pancreatitis.

Mutations in the PRSS1 (serine protease 1) gene encoding human cationic trypsinogen cause hereditary pancreatitis or may be associated with sporadic chronic pancreatitis. The mutations exert their pathogenic effect either by increasing intra-pancreatic trypsinogen activation (trypsin pathway) or by causing proenzyme misfolding and endoplasmic reticulum stress (misfolding pathway). Here we report a novel heterozygous c.568G>A (p.Glu190Lys) variant identified in a case with chronic pancreatitis. The parents of the index patient had no history of pancreatitis but were unavailable for genetic testing. Functional characterization revealed 2.5-fold increased autoactivation of the mutant trypsinogen relative to wild type. Unlike many other clinically relevant PRSS1 mutations, p.Glu190Lys did not alter the chymotrypsin C (CTRC)-dependent degradation of trypsinogen nor did it increase CTRC-mediated processing of the trypsinogen activation peptide. Cellular secretion of the mutant protein was unchanged indicating normal folding behavior. Based on the genetic and functional evidence, we classify the p.Glu190Lys PRSS1 variant as likely pathogenic, which stimulates autoactivation of cationic trypsinogen independently of CTRC.

Chymotrypsin Reduces the Severity of Secretagogue-Induced Pancreatitis in Mice.

Intrapancreatic activation of the digestive proteases trypsin and chymotrypsin is an early event in the development of pancreatitis. Human genetic studies indicate that chymotrypsin controls trypsin activity via degradation, but there is no evidence of this from animal models. We used CRISPR-Cas9 to disrupt the chymotrypsinogen B1 gene (Ctrb1) in C57BL/6N mice and induced pancreatitis in CTRB1-deficient and C57BL/6N (control) mice by administration of cerulein. CTRB1-deficient mice given cerulein had significant increases in intrapancreatic trypsin activity and developed more severe pancreatitis compared with control mice. CTRB1 therefore protects against secretagogue-induced pancreatitis by reducing trypsin activity. Protease inhibitors developed for treatment of pancreatitis should be designed to target trypsin but not chymotrypsin.

Tighter Control by Chymotrypsin C (CTRC) Explains Lack of Association between Human Anionic Trypsinogen and Hereditary Pancreatitis.

The human pancreas expresses two major trypsinogen isoforms, cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2). Mutations in PRSS1 cause hereditary pancreatitis by altering cleavage of regulatory nick sites by chymotrypsin C (CTRC) resulting in reduced trypsinogen degradation and increased autoactivation. Despite 90% identity with PRSS1 and a strong propensity for autoactivation, mutations in PRSS2 are not found in hereditary pancreatitis suggesting that activation of this isoform is more tightly regulated. Here, we demonstrated that CTRC promoted degradation and thereby markedly suppressed autoactivation of human anionic trypsinogen more effectively than previously observed with cationic trypsinogen. Increased sensitivity of anionic trypsinogen to CTRC-mediated degradation was due to an additional cleavage site at Leu-148 in the autolysis loop and the lack of the conserved Cys-139-Cys-206 disulfide bond. Significant stabilization of anionic trypsinogen against degradation was achieved by simultaneous mutations of CTRC cleavage sites Leu-81 and Leu-148, autolytic cleavage site Arg-122, and restoration of the missing disulfide bridge. This stands in stark contrast to cationic trypsinogen where single mutations of either Leu-81 or Arg-122 resulted in almost complete resistance to CTRC-mediated degradation. Finally, processing of the trypsinogen activation peptide at Phe-18 by CTRC inhibited autoactivation of anionic trypsinogen, although cationic trypsinogen was strongly stimulated. Taken together, the observations indicate that human anionic trypsinogen is controlled by CTRC in a manner that individual natural mutations are unlikely to increase stability enough to promote intra-pancreatic activation. This unique biochemical property of anionic trypsinogen explains the lack of association of PRSS2 mutations with hereditary pancreatitis.

Gut region-specific accumulation of reactive oxygen species leads to regionally distinct activation of antioxidant and apoptotic marker molecules in rats with STZ-induced diabetes.

UNLABELLED: The aim of this study was to seek possible links between the regionality along the digestive tract and the accumulation of reactive oxygen species, the effectiveness of the antioxidant defense system and the sensitivity to the types of demise in different gut regions of rats with streptozotocin-induced diabetes. Significant changes were observed in the oxidative status and in the activity of the antioxidant defense system in the diabetic colon; the peroxynitrite production was doubled, the level of hemoxygenase-2 protein was increased 11-fold and the expression of anti-apoptotic bcl-2 was also increased. The segment-specific vulnerability of the gastrointestinal tract induced by hyperglycemia was also confirmed by electron microscopy, demonstrating the presence of severe necrosis in the colon of the diabetic rats. No remarkable histopathological alterations were seen in the duodenum of the diabetic animals and there were likewise no significant changes in the production of peroxynitrite in their duodenum, whereas the level of the free radical scavenger metallothionein-2 was increased ∼300-fold. CONCLUSION: The spatially restricted vulnerability observed along the digestive tract could originate from a high level of oxidative stress via peroxynitrite production.

Impact of acute arsenic and cadmium exposure on the expression of two haeme oxygenase genes and other antioxidant markers in common carp (Cyprinus carpio).

The aim was to study the effects of cadmium (Cd) and arsenic (As) on haeme oxygenases (HOs) and other oxidative stress biomarkers, and their roles in macromolecule damage in liver and kidney of common carp (Cyprinus carpio L.). HOs play a critical role in the defence system against oxidative damage, producing biliverdin and carbon monoxide with important free radical scavenging properties. However, increased HO activity in haeme degradation may also lead to a pro-oxidant effect through the liberation of Fe-modifying Cd and As toxicity. The response of an organism to exposure to toxic metals is in many cases brought about by changes at the level of gene expression. In this study, the genes ho-1 and ho-2 of the common carp were identified, and the changes in gene expressions were analysed from the aspect of Cd and As accumulation. Both ho-1 and ho-2 are transcriptionally induced by Cd and As, but their inductions differ in time course, dose response and tissue specificity. The expression of ho1 was mostly affected by As, primarily in the liver (45-fold), whereas it was enhanced with higher efficacy by Cd in the kidney (25-fold). The cellular redox status and the damage of lipid molecules were monitored via the ratio of reduced to oxidized glutathione, the levels of H2 O2 and lipid peroxidation, and the activities of superoxide dismutase (SOD) and catalase (CAT).

Gut region-specific rearrangement of the cellular and subcellular compartments of nitric oxide synthase isoforms after chronic ethanol consumption in rats.

We recently provided evidence of cell-type-specific differences in the subcellular distributions of the three nitric oxide synthase (NOS) isoforms in the myenteric neurons, enteric smooth muscle cells and the capillary endothelium of the rat duodenum. We hypothesized that the presence of three NOS isoforms in the same type of cells with differences in subcellular compartmentalization might reflect a functional plasticity. Therefore, investigation of the possible rearrangement of cellular and subcellular NOS compartments in different gut segments following chronic ethanol treatment was the aim of this study. Rats were randomly divided into two groups and received water or 20% ethanol solution, preceded by short periods of adaptation with 10% and 15% ethanol. After 8 weeks, segments of duodenum, ileum and colon of the control and the alcohol-treated rats were processed for post-embedding immunohistochemistry and RT-PCR. The quantitative differences in the numbers of gold particles indicative of the different NOSs and their relative mRNA levels between the two experimental groups varied greatly, depending on the gut segment, and also on the cellular and subcellular compartments investigated. The chronic ethanol administration had the opposite effect on the quantitative distribution of the neuronal and endothelial NOS labelling gold particles in the different cellular compartments and resulted in subcellular rearrangement of NOS labels along the gastrointestinal tract. The intestinal region-specific rearrangement of the cellular and subcellular NOS compartments may possibly result in functional plasticity and help to maintain the optimum NO level under pathological conditions.

Major distinctions in the antioxidant responses in liver and kidney of Cd(2+)-treated common carp (Cyprinus carpio).

This study is related to the accumulation of Cd(2+), its effects on oxidative stress biomarkers and its role in macromolecule damage in liver and kidney of common carp. We present evidence of an increased ratio of reduced to oxidized glutathione (GSH/GSSG) in both organs after 10 mg/L Cd(2+) exposure, with different underlying biological mechanisms and consequences. In the liver, the expressions and/or activities of superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase increased to cope with the Cd(2+)-generated toxic effects during the first 48 h of treatment. In contrast, none of these selected antioxidant markers was significantly altered in the kidney, whereas the expression of glutathione synthetase was upregulated. These results suggest that the major defense mechanism provoked by Cd(2+) exposure involves the regeneration of GSH in the liver, while its de novo synthesis predominates in the kidney. High levels of accumulation of Cd(2+) and peroxynitrite anion (ONOO(-)) were detected in the kidney; the major consequences of ONOO(-) toxicity were enhanced lipid peroxidation and GSH depletion. The accumulation of ONOO(-) in the kidney suggests intensive production of NO and the development of nitrosative stress. In the liver the level of hydrogen peroxide was elevated.

Gut region-specific diabetic damage to the capillary endothelium adjacent to the myenteric plexus.

OBJECTIVE: Damage in the capillaries supplying the MP has been proposed as a critical factor in the development of diabetic enteric neuropathy. We therefore investigated connections between STZ-induced diabetes and the BM morphology, the size of caveolar compartments, the width of TJs, the transport of albumin, and the quantitative features of Cav-1 and eNOS expression in these microvessels. METHODS: Gut segments from diabetic rats were compared with those from insulin-treated diabetics and those from controls. The effects of diabetes on the BM, the caveolar compartments, and the TJs were evaluated morphometrically. The quantitative features of the albumin transport were investigated by postembedding immunohistochemistry. The diabetes-related changes in Cav-1 and eNOS expression were assessed by postembedding immunohistochemistry and molecular method. RESULTS: Thickening of the BM, enlargement of the caveolar compartments, opening of the junctions, enhanced transport of albumin, and overexpression of Cav-1 and eNOS were documented in diabetic animals. Insulin replacement in certain gut segments prevented the development of these alterations. CONCLUSIONS: These data provide morphological, functional, and molecular evidence that the endothelial cells in capillaries adjacent to the MP is a target of diabetic damage in a regional manner.

Effects of Kupffer cell blockade on the hepatic expression of metallothionein and heme oxygenase genes in endotoxemic rats with obstructive jaundice.

AIMS: Heme oxygenase (HO) and metallothionein (MT) genes are rapidly upregulated in the liver by pro-inflammatory cytokines and/or endotoxin as protection against cellular stress and inflammation. Gadolinium chloride (GdCl3)-induced Kupffer cell blockade has beneficial consequences in endotoxemia following bile duct ligation. Herein we further characterized the effects of Kupffer cell inhibition on the activation of the antioxidant defense system (HO and MT gene expressions, and antioxidant enzyme activities) in response to endotoxemia and obstructive jaundice. MAIN METHODS: The isoform-specific expression of MT and HO genes was assessed (RT-PCR) in rat livers following 3-day bile duct ligation, 2-h lipopolysaccharide treatment (1mg/kg) or their combination, with or without GdCl3 pretreatment (10 mg/kg, 24h before endotoxin). Lipid peroxidation, DNA damage and hepatic antioxidant enzyme activities were also assessed. KEY FINDINGS: All these challenges induced similar extents of DNA damage, whereas the lipid peroxidation increased only when endotoxemia was combined with biliary obstruction. The MT and HO mRNA levels displayed isoform-specific changes: those of MT-1 and HO-2 did not change appreciably, whereas those of MT-2 and HO-1 increased significantly in 2-h endotoxemia, with or without obstructive jaundice. Among the enzymes reflecting the endogenous protective mechanisms, the catalase and copper/zinc-superoxide dismutase levels decreased, while that of Mn-SOD slightly increased. Interestingly, GdCl3 alone induced lipid peroxidation, DNA damage and MT-2 expression. In response to GdCl3, HO-1 induction was significantly lower in each model. SIGNIFICANCE: Despite its moderate hepatocellular toxicity, the ameliorated stress-induced hepatic reactions provided by GdCl3 may contribute to its protective effects.