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Intracellular autoactivation of human cationic trypsinogen mutants causes reduced trypsinogen secretion and acinar cell death.

ABSTRACT: Mutations in the activation peptide of human cationic trypsinogen have been found in patients with chronic pancreatitis. Previous biochemical studies demonstrated that mutations p.D19A, p.D22G, and p.K23R strongly stimulate trypsinogen autoactivation. In the present study, we characterized the cell biological effects of these mutants using human embryonic kidney 293T and AR42J rat acinar cells. We found that relative to wild-type trypsinogen, secretion of the mutants from transfected cells was markedly decreased. This apparent secretion defect was completely rescued by inhibition of autoactivation via (1) inclusion of the small molecule trypsin inhibitor benzamidine in the growth medium; or (2) cotransfection with the physiological trypsin inhibitor SPINK1; or (3) by mutation of the catalytic Ser(200) residue in trypsinogen. In contrast, extracellularly added SPINK1 or other nonpermeable proteinaceous trypsin inhibitors did not restore normal secretion of the mutants, indicating that intracellular autoactivation is responsible for the observed secretion loss. Acinar cells expressing the p.D22G mutant detached from the culture plate over time, became terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive, and exhibited elevated levels of the proapoptotic transcription factor CHOP. The observations indicate that activation peptide mutants of human cationic trypsinogen undergo autoactivation intracellularly, which leads to decreased trypsinogen secretion and eventual acinar cell death. The results thus define a novel pathological pathway for parenchymal injury in hereditary chronic pancreatitis.

SUBMITTER: Kereszturi E

PROVIDER: S-EPMC2785183 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Intracellular autoactivation of human cationic trypsinogen mutants causes reduced trypsinogen secretion and acinar cell death.

Kereszturi Eva E Sahin-Tóth Miklós M

The Journal of biological chemistry 20090929 48

Mutations in the activation peptide of human cationic trypsinogen have been found in patients with chronic pancreatitis. Previous biochemical studies demonstrated that mutations p.D19A, p.D22G, and p.K23R strongly stimulate trypsinogen autoactivation. In the present study, we characterized the cell biological effects of these mutants using human embryonic kidney 293T and AR42J rat acinar cells. We found that relative to wild-type trypsinogen, secretion of the mutants from transfected cells was m ...[more]

PMID: 19801634

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Project description:Hereditary pancreatitis (HP) is an autosomal dominant disease that displays the features of both acute and chronic pancreatitis. Mutations in human cationic trypsinogen (PRSS1) are associated with HP and have provided some insight into the pathogenesis of pancreatitis, but mechanisms responsible for the initiation of pancreatitis have not been elucidated and the role of apoptosis and necrosis has been much debated. However, it has been generally accepted that trypsinogen, prematurely activated within the pancreatic acinar cell, has a major role in the initiation process. Functional studies of HP have been limited by the absence of an experimental system that authentically mimics disease development. We therefore developed a novel transgenic murine model system using wild-type (WT) human PRSS1 or two HP-associated mutants (R122H and N29I) to determine whether expression of human cationic trypsinogen in murine acinar cells promotes pancreatitis. The rat elastase promoter was used to target transgene expression to pancreatic acinar cells in three transgenic strains that were generated: Tg(Ela-PRSS1)NV, Tg(Ela-PRSS1*R122H)NV and Tg(Ela-PRSS1*N29I)NV. Mice were analysed histologically, immunohistochemically and biochemically. We found that transgene expression is restricted to pancreatic acinar cells and transgenic PRSS1 proteins are targeted to the pancreatic secretory pathway. Animals from all transgenic strains developed pancreatitis characterised by acinar cell vacuolisation, inflammatory infiltrates and fibrosis. Transgenic animals also developed more severe pancreatitis upon treatment with low-dose cerulein than controls, displaying significantly higher scores for oedema, inflammation and overall histopathology. Expression of PRSS1, WT or mutant, in acinar cells increased apoptosis in pancreatic tissues and isolated acinar cells. Moreover, studies of isolated acinar cells demonstrated that transgene expression promotes apoptosis rather than necrosis. We therefore conclude that expression of WT or mutant human PRSS1 in murine acinar cells induces apoptosis and is sufficient to promote spontaneous pancreatitis, which is enhanced in response to cellular insult.

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Intracellular autoactivation of human cationic trypsinogen mutants causes reduced trypsinogen secretion and acinar cell death.

Publications

Intracellular autoactivation of human cationic trypsinogen mutants causes reduced trypsinogen secretion and acinar cell death.

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