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

Abstract

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.

Keywords: Genetics; Inflammation; Intrapancreatic; Mouse Model.

Figure 1.

Effect of mutation p.K24R on the activation of mouse cationic trypsinogen (isoform T7). (A) The activation peptide sequences of human (PRSS1) and mouse (T7) cationic trypsinogen. Position of analogous mutations p.K23R and p.K24R are highlighted in red. (B) Autoactivation of purified wild-type (empty circles) and p.K24R mutant (solid circles) T7 trypsinogen. Data points represent mean  ±  standard deviation (n = 3). (C) Activation of purified wild-type and p.K24R mutant T7 trypsinogen by cathepsin B. See Methods for experimental details. Data points represent mean  ±  standard deviation (n = 3).

Figure 2.

Cerulein-induced acute pancreatitis in T7K24R mice. (A) Representative pictures of the pancreas of C57BL/6N and T7K24R mice given 12 hourly injections of saline or cerulein. (B) Pancreas mass of C57BL/6N and T7K24R mice given saline or cerulein, expressed as percent of body mass. (C) Pancreatic water content of T7K24R and C57BL/6N mice treated with saline or cerulein, expressed as percent wet pancreas weight. Individual data points with mean (horizontal bar) and standard deviation are shown. See Methods for experimental details.

Figure 3.

Cerulein-induced acute pancreatitis in T7K24R mice. (A) Plasma amylase activity in C57BL/6N and T7K24R and mice given 12 hourly injections of saline or cerulein. (B) Pancreatic myeloperoxidase (MPO) content of C57BL/6N and T7K24R mice treated with saline or cerulein. (C) Lung MPO content of C57BL/6N and T7K24R mice treated with saline or cerulein. Individual data points with mean (horizontal bar) and standard deviation are shown. See Methods for experimental details.

Figure 4.

Histology of cerulein-induced acute pancreatitis in T7K24R mice. (A) Representative hematoxylin-eosin stained histological sections of the pancreas from saline and cerulein-treated mice. Scale bars correspond to 100 μm. (B) Histology scoring for edema, inflammatory cell infiltration, and acinar cell necrosis in cerulein-treated mice. Mean values with standard deviation are shown. See Methods for details.

Figure 5.

Histology of pancreatitis induced by sustained cerulein stimulation in T7K24R mice. Mice were treated with saline or cerulein for 2 days (8 hourly injections per day) and sacrificed 3 days or 10 days after the last injection. Representative hematoxylin-eosin stained histological sections of the pancreas from cerulein-treated mice are shown. The pancreas of mice given saline was normal (not shown). Scale bars correspond to 50 μm.

Figure 6.

Pancreas mass and pancreas fibrosis after sustained cerulein stimulation in T7K24R mice. Mice were treated with saline or cerulein for 2 days (8 hourly injections per day) and sacrificed 3 days or 10 days after the last injection. (A) Pancreas mass (as percent of body mass) of C57BL/6N and T7K24R mice given saline or cerulein. Individual data points with mean (horizontal bar) and standard deviation are shown. (B) Masson’s trichrome staining of pancreas sections from cerulein-treated T7K24R mice 3 days and 10 days after the last injection. Blue color indicates fibrosis. Scale bars correspond to 50 μm.

Figure 7.

Intrapancreatic protease activation in T7K24R mice. Trypsin (A) and chymotrypsin (B) activities were measured 30 minutes after a single saline or cerulein injection. Individual data points with mean (horizontal bar) and standard deviation are shown. See Methods for experimental details.