Cathepsin L inactivates human trypsinogen, whereas cathepsin L-deletion reduces the severity of pancreatitis in mice

Abstract

Background & aims: Acute pancreatitis is characterized by an activation cascade of digestive enzymes in the pancreas. The first of these, trypsinogen, can be converted to active trypsin by the peptidase cathepsin B (CTSB). We investigated whether cathepsin L (CTSL) can also process trypsinogen to active trypsin and has a role in pancreatitis.

Methods: In CTSL-deficient (Ctsl(-/-)) mice, pancreatitis was induced by injection of cerulein or infusion of taurocholate into the pancreatic duct. Human tissue, pancreatic juice, mouse pancreatitis specimens, and recombinant enzymes were studied by enzyme assay, immunoblot, N-terminal sequencing, immunocytochemistry, and electron microscopy analyses. Isolated acini from Ctsl(-/-) and Ctsb(-/-) mice were studied.

Results: CTSL was expressed in human and mouse pancreas, colocalized with trypsinogen in secretory vesicles and lysosomes, and secreted into pancreatic juice. Severity of pancreatitis was reduced in Ctsl(-/-) mice, whereas apoptosis and intrapancreatic trypsin activity were increased. CTSL-induced cleavage of trypsinogen occurred 3 amino acids toward the C-terminus from the CTSB activation site and resulted in a truncated, inactive form of trypsin and an elongated propeptide (trypsinogen activation peptide [TAP]). This elongated TAP was not detected by enzyme-linked immunosorbent assay (ELISA) but was effectively converted to an immunoreactive form by CTSB. Levels of TAP thus generated by CTSB were not associated with disease severity, although this is what the TAP-ELISA is used to determine in the clinic.

Conclusions: CTSL inactivates trypsinogen and counteracts the ability of CTSB to form active trypsin. In mouse models of pancreatitis, absence of CTSL induces apoptosis and reduces disease severity.

Figure 1. CTSL in the human and mouse pancreas and the Ctsl^−/− pancreas

In panel A, by Western blots with CTSL antibody pro-CTSL, single and heavy chain CTSL in human pancreas and pancreatic juice were detected. Panel B indicates expression and subcellular localization of CTSL in normal human pancreas (red fluorescence) and panel C colocalization of trypsinogen (green) with CTSL (red) in normal mouse pancreas. The pancreas of Ctsl^−/− mice appears completely normal on H&E-stained sections (D, with an islet and duct at the top) and EM (E, with normal arrangement of zymogen granules and mitochondria). Bars indicate 50 μm and asterisks the acinar lumen.

Figure 2. Cell injury and apoptisis in cerulein-induced pancreatitis

Panel A: 3h of pancreatitis in a wild-type animal labeled with antibody against trypsinogen (green) and CTSL (red fluorescence). B: electronmicroscopy of 3h pancreatitis in the Ctsl^−/− mouse. Note whirl-like arrangement of the ER in lower right corner, large autophagic vacuoles, of which one contains a nucleus, and a necrotic cell in the top centre. C: morphometry of acinar tissue necrosis in wild type and Ctsl^−/− animals over 24h of pancreatitis. D: 3-OH-nick-end labeling (TUNEL) of apoptotic nuclei in Ctsl^−/− animals over 24h of pancreatitis. This difference is already present in untreated animals. E: PARP cleavage and caspase-3 activity in pancreatic tissue after 8h of pancreatitis. F: Caspase-3 activity in living pancreatic acini after 60min of supramaximal cerulein stimulation. Bars indicate 1 μm. Values denote means ± SEM for 5 or more measurements.

Figure 3. Disease severity and trypsinogen activation

Deletion of CTSL greatly decreased pancreatitis-associated hyperamylasemia (A), hyperlipasemia (B), as well as serum levels of IL6, MCP-1, and TNFalpha (C) over 24h. E: trypsin activity and F: TAP generation in pancreatic tissue homogenates over 24h of pancreatitis. In strong contrast to serum pancreatic enzyme levels and the extent of tissue necrosis (fig.2), intrapancreatic trypsin activity is greatly increased in Ctsl^−/− animals whereas TAP levels are similar in both groups. Values denote means ± SEM for 5 or more measurements.

Figure 4. Taurocholate-induced pancreatitis

Ctsl^−/− and Ctsl^+/+ animals were sacrificed after 6h of taurocholate-induced pancreatitis. On morphology (A, B), by serum pancreatic enzyme activities (C, D) and according to parameters of systemic inflammation (IL-10:E, Lung myeloperoxidase:F) the disease severity is reduced in the absence of CTSL. Values denote means ± SEM for 5 or more measurements.

Figure 5. Proteolytic processing of trypsinogen

Cleavage of trypsinogen by CTSB and CTSL was studied in vitro. A: CTSL cleaves trypsinogen to a lighter protein that corresponds to active trypsin generated by enterokinase (EK) but is processed much more rapidly than by CTSB. Unlike CTSB-generated trypsin, however, this protein has no trypsin activity (B), nor does CTSL-cleavage generate immunoreactive TAP (C). CTSB and CTSL were used in equimolar amounts. To reach comparable cleavage efficiency a 100-fold CTSB concentration had to be used (inset below CTSB).

Figure 6. Trypsin cleavage by CTSL and effect of pH and Ca⁺⁺

Human cationic trypsinogen or active trypsin were incubated for 3h with CTSL or enterokinase (EK) and submitted to SDS-PAGE followed by N-terminal sequencing. Note that the inactive CTSL-generated protein is three amino acids shorter (IVG) than active trypsin generated by enterokinase (A). Inactivation of human trypsin was measured as residual trypsin activity after incubation with CTSL at the pH indicated (B). Proteolytic cleavage of trypsin by CTSL exhibited a completely different dependence on pH and Ca⁺⁺ compared to CTSL-cleavage of the peptide substrate (C).

Figure 7. Inhibition or deletion of CTSB and CTSL in pancreatic acini

In panel A, pancreatic acini were pre-incubated for 30 minutes with 10 nM CTSL inhibitor (1-Naphthalenylsulfonyl-Ile-Trp-aldehyde) or 1 μM CTSB inhibitor CA-074Me, then stimulated with cerulein. Trypsin activity was compared over 120min. Inhibition of CTSL increased and the inhibition of CTSB decreased intra-acinar cell trypsin activity. In B and C, acini were prepared from wild-type, CTSL-deleted and CTSB-deleted animals. Trypsin activity and TAP were determined after cerulein stimulation for 1h. The absence of CTSL increased whereas absence of CTSB decreased intra-acinar cell trypsin activity. The generation of TAP was reduced in both knock-out strains. Data represent means of 5 or more experiments ± SEM. Significant differences are indicated with asterisks when p was <0.05.