Initiation of acute pancreatitis in mice is independent of fusion between lysosomes and zymogen granules

Abstract

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.

Keywords: Acute pancreatitis; Cathepsin B; Co-localization; Lysosome; Secretory vesicle; Trypsinogen.

Fig. 1

Rab7 and cathepsin B and L expression in pancreatic tissue and enzyme kinetics in isolated acinar cells stimulated with supramaximal concentrations of CCK. a Immunohistochemical staining in paraffin-embedded slides of pancreatic tissues with intraacinar Rab7 expression in control mice (Rab7 + / +) but no expression in the knockout mice (Rab7 + /cre). b Immunoblot analysis of pancreas homogenates shows an increased expression of cathepsin B and L in the Rab7 knockout mice. c Enzyme kinetics in living isolated acinar cells in response to supramaximal CCK in Rab7-KO (Rab7 cre/ +) and control mice (Rab7 fl/fl). CTSB and CTSL activities are significantly increased in Rab7 KO acinar cells, while trypsin and cell death remain unchanged. d Cathepsin B and L expression in subcellular fractions of the pancreas depends on Rab7. Both pro- and mature CTSB and CTSL are elevated in the zymogen-enriched “heavy fraction” of the Rab7 knockout pancreas. At least three animals were used for these experiments and the measurements were performed in triplicates. Values are mean ± SEM. * denotes p < 0.05

Fig. 2

Initiation and early phase of acute pancreatitis in Rab7 knockout mice and controls. a HE stainings and histoscore indicate no difference in local damage in Rab7-KO mice. b, c Likewise, trypsin, serum amylase and lipase activities are not different at 0 h and 1 h. d In contrast, CTSB and CTSL activities are significantly increased at every time point, showing the same trend compared to control mice. e Subcellular fractionations of pancreatic tissue 1 h after caerulein injection display a significant increase of CTSB activity in the zymogen-enriched, lysosomal, and cytosolic fraction of Rab7 + /cre mice, whereas the lysosomal CTSB activity decreases in controls. f CTSL activity increases as well in all fractions after caerulein induction. g Trypsin activity at 0 h is at basal levels but increases in the zymogen-enriched fraction in both mouse strains upon caerulein. Cathepsin and trypsin activities are presented as relative fluorescence units (RFU) and are normalized for protein amounts. At least seven animals were used for these experiments and the measurements were performed in duplicates. Values are mean ± SEM. * denotes p < 0.05

Fig. 3

Severity of acute pancreatitis is increased at 8 h in Rab7 knockout mice. a HE-stained pancreas and histoscore analysis show an increased severity at 8 h. b Trypsin activity in pancreas homogenates is elevated at 8 h of AP. c Likewise, serum lipase and amylase activities are increased. d Total activities of CTSB and CTSL are elevated in pancreatic homogenates of Rab7 + /cre mice throughout unstimulated and stimulated conditions. At least seven animals were used for each experiment and all experiments were performed in duplicates. Values are means ± SEM. * denotes p < 0.05

Fig. 4

GPN disrupts lysosomal membranes but does not influence trypsin and CTSB activation after induction of secretagogue-induced pancreatitis. a Immunoblots of pancreatic subcellular fractions of WT mice (C57BL/6) show a redistribution of lysosomal proteins LAMP1, LAMP2, CTSB, CTSC from the lysosomal enriched fraction into the cytosolic fraction after GPN injection. b Lysosomal disruption does not prevent activation of trypsin and CTSB localized in the zymogen fraction (ZG) after caerulein injection. In contrast to untreated controls, there is no redistribution of CTSB activity from the lysosomal to the zymogen granule compartment under GPN. c No redistribution of lysosomal proteins is seen in CTSC^−/− mice. d Absence of CTSC prevents the lysosomotropic function of GPN and retains subcellular CTSB distribution and intracellular trypsin activation in CTSC^−/− mice. At least three animals were used for these experiments and the measurements were performed in triplicates. Values are mean ± SEM. * denotes p < 0.05

Fig. 5

Trypsin and cathepsin B activation at onset of caerulein induced acute pancreatitis. a Trypsin activity in pancreatic homogenates increase in both GPN pre-treated and control mice upon caerulein but remain inactivated in CTSB^−/− mice, indicating that trypsinogen activation is dependent on presence of CTSB but not of GPN. b GPN does not permeabilize the membranes of zymogen-enriched fractions from WT pancreas, as even high GPN concentrations do not cause a release of trypsin into the supernatant. A Kruskal–Wallis test and Dunn’s multiple comparisons test was performed for statistical analysis. c In contrast, incubation of lysosome-enriched fractions with 50 nM GPN for 1 h induces a CTSB release from the lysosomes into the extracellular space. Enzyme activities are presented as relative fluorescence units (RFU). d In isolated and GPN pre-incubated wildtype (WT) acinar cells intracellular CTSB activity significantly decreases after 20 min of CCK stimulation but trypsin activity and cell death are unaltered. e CTSB activity does not significantly change after supramaximal CCK stimulation in isolated CTSC^−/− acinar cells following GPN. Trypsin activity and necrosis are also not unaffected. At least three animals were used for each experiment and all experiments were performed in duplicates. Values are means ± SEM. *denotes p < 0.05

Fig. 6

Initiation of acute pancreatitis is still maintained after disruption of lysosomes. a HE-stained pancreas and histoscore analysis demonstrate comparable severity at 1 h. b Serum lipase and amylase activities show no differences after 1 h. c, d Trypsin and CTSB activities in pancreas homogenates of GPN-treated mice are equal to controls. At least seven animals were used for each experiment and all experiments were performed in duplicates. Values are means ± SEM. * denotes p < 0.05