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

Abstract

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.

Keywords: pancreatic disease; pancreatic enzymes; pancreatic fibrosis; pancreatic pathology; pancreatitis.

Figure 1.

Effect of the human CPA1 mutation p.N256K on mouse CPA1. HEK 293T cells were transiently transfected with the indicated plasmids and conditioned media and cell lysates were harvested after 48 h. A, Secreted levels of CPA1 in the medium were determined by SDS-PAGE and Coomassie Blue staining (left panel) and CPA1 enzyme activity measurements (right panel, mean ± S.D., n=5). B, Western blot analysis of CPA1 in the culture medium (left panel) and in cell lysates (right panel) with α-tubulin measured as loading control. C, Total RNA prepared from cell lysates was transcribed to cDNA and used to measure markers of ER stress. Splicing of XBP1 was assessed by co-amplification of the spliced (sXBP1) and unspliced (uXBP1) forms followed by agarose gel electrophoresis and ethidium bromide staining. The left panel shows a representative gel with GAPDH measured as loading control. The right panel indicates densitometric evaluation of 4 independent experiments (mean ± S.D.). HSPA5 and DDIT3 mRNA expression was measured by qPCR and expressed as fold change relative to vector-transfected cells (mean ± S.D., n=4).

Figure 2.

Generation of CPA1 N256K mice and expression of the mutant allele. A, The homologous recombination based targeting strategy first yielded an intermediary strain carrying the p.N256K mutation in exon 7 (asterisk) and a neomycin resistance gene (Neo) flanked by loxP sites (gray arrowheads) in intron 6. This strain showed diminished CPA1 expression and was designated as CPA1 null. To produce the CPA1 N256K mice, the neomycin cassette was removed by breeding the CPA1 null mice with a Cre-deleter strain. B, Cpa1 mRNA expression in the pancreas of 1-month-old CPA1 N256K, CPA1 null and C57BL/6N control mice. Quantitative PCR was performed on pancreatic cDNA samples and results were expressed as fold change relative to the average of the C57BL/6N results. Mean values with S.D. (n=4) are shown. C, Western blot analysis of CPA1 protein expression in the pancreas of 1-month-old CPA1 N256K, CPA1 null and C57BL/6N control mice. ERK1/2 was measured as loading control.

Figure 3.

Spontaneous and cerulein-induced secretion of CPA1 protein from isolated acinar cells of CPA1 N256K and C57BL/6N control mice. A, CPA1 secretion to the culture medium after 60 min incubation without or with 30 pM cerulein. Note that sample loading was normalized to the amylase content in the medium, which masks the stimulatory effect of cerulein. B, CPA1 content in cell lysates after 60 min incubation without or with 30 pM cerulein. Sample loading was normalized to total protein (2 μg). Representative western blots and the results of densitometric analyses from three independent experiments are shown.

Figure 4.

Body weight and pancreas weight as a function of age in CPA1 N256K, CPA1 null and C57BL/6N control mice. Individual values with the mean (horizontal bar) are shown. A, Body weight. B, Pancreas weight. Note that some of the body weight differences in the male group reached statistical significance at 3 months and 6 months. Since these small changes have no obvious biological meaning, we did not indicate p values.

Figure 5.

Pancreas histology as a function of age in CPA1 N256K, CPA1 null and C57BL/6N control mice. A, Hematoxylin-eosin stained pancreas sections. Note the progressive pathology in CPA1 N256K mice. B, Higher magnification of a pancreas section from a 12-month old CPA1 N256K mouse showing the typical morphological changes consisting of loss of acini, inflammatory cell infiltration, pseudotubular complexes and some acinar cell vacuolization. C, Quantitation of acinar cell dropout in CPA1 N256K mice as a function of age. Individual values with mean (horizontal bar) are shown.

Figure 6.

Fibrosis and acinar-ductal metaplasia in CPA1 N256K mice. A, Masson’s trichrome staining demonstrates widespread fine fibrotic changes (blue color) surrounding acini in CPA1 N256K mice but not in Cpa1 null mice. B, Hydroxyproline content is increased in pancreata from CPA1 N256K mice versus CPA1 null mice and C57BL/6N controls. Individual values with mean (horizontal bar) are shown. C, Immunohistochemistry for cytokeratin 19 (CK19) and Sox9 demonstrates extensive acinar-ductal metaplasia associated with pseudotubular complexes in CPA1 N256K mice but not in Cpa1 null mice.

Figure 7.

Ultrastructural changes and macrophage infiltration in CPA1 N256K mice. A, Electron micrographs of pancreas sections from 4-month-old CPA1 N256K mice and C57BL/6N controls. CPA1 N256K mice show intact zymogen granules, acinar cell vacuolization, infiltrating macrophages, a pseudotubular complex (middle graph lower right corner), and endoplasmic reticulum whorls (right graph) with no appreciable dilations. Direct magnifications are indicated. B, Immunohistochemistry for the F4/80 marker confirms macrophage infiltration in the pancreas of CPA1 N256K mice.

Figure 8.

Plasma amylase (A) and intra-pancreatic trypsin activity (B) in CPA1 N256K, CPA1 null and C57BL/6N control mice at 1, 3 and 6 months of age. Individual values with mean (horizontal bar) are shown.

Figure 9.

Expression of endoplasmic reticulum stress markers Hspa5 and Ddit3 in CPA1 N256K, CPA1 null and C57BL/6N control mice at 1 month (A), 3 months (B) and 12 months (C) of age. Quantitative PCR was performed on pancreatic cDNA samples and results were expressed as fold change relative to the average of the C57BL/6N results. Individual values with mean (horizontal bar) are shown.