A pancreatitis-associated chymotrypsinogen C mutant elicits endoplasmic reticulum stress and apoptosis

Background and Aims: We recently identified chymotrypsinogen C (CTRC) as a novel susceptibility gene for chronic pancreatitis in humans (Nat Genet 2008; 40:78–82). Pancreatitis-associated CTRC mutants exhibit diminished secretion due to intracellular retention and degradation. We hypothesized that mutation-induced CTRC retention may result in endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR). Since prolonged activation of the UPR can lead to apoptotic cell death, a direct link may be established between CTRC mutants and acinar cell death in chronic pancreatitis. The aim of the present study was to test this hypothesis using the disease-associated p.A73T CTRC mutant.

Methods: Wild type and p.A73T mutant human CTRC were expressed in the AR42J rat acinar cell line and primary mouse acinar cells using adenovirus mediated transfection. Levels of UPR markers (BiP, spliced XBP1, and CHOP) were determined in cell lysates by western blot and RT-PCR analysis. As indicators of apoptotic cell death, caspase-3/7 activity and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) were also assessed.

Results: Cells expressing the p.A73T mutant exhibited elevated BiP mRNA and protein levels and increased splicing of XBP1, compared to cells expressing wild type CTRC. Cells transfected with the p.A73T mutant became detached over time and showed significantly increased caspase-3/7 activity and TUNEL staining, consistent with apoptotic cell death. Finally, marked induction of the transcription factor CHOP, a known mediator of ER stress induced apoptosis, was also observed in cells expressing the p.A73T mutant.

Conclusions: The results demonstrate that the p.A73T pancreatitis-associated CTRC mutant causes ER stress, activates the UPR and elicits apoptosis via induction of the pro-apoptotic transcription factor CHOP in pancreatic acinar cells. The findings suggest a novel disease mechanism in chronic pancreatitis which is unrelated to trypsin activity.