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Cathepsin-L-Gastroenterology-2010 GASTROENTEROLOGY 2010;138:726–737 Cathepsin L Inactivates Human Trypsinogen, Whereas Cathepsin L-Deletion Reduces the Severity of Pancreatitis in Mice THOMAS WARTMANN,* JULIA MAYERLE,‡ THILO KÄHNE,§ MIKLÓS SAHIN–TÓTH,ʈ MANUEL RUTHENBÜRGER,‡ RAINER MATTHIAS,* ANNE KRUSE,‡ THOMAS REINHECKEL,¶ CHRISTOPH PETERS,¶ F. ULRICH WEISS,‡ MATTHIAS SENDLER,‡ HANS LIPPERT,* HANS–ULRICH SCHULZ,* ALI AGHDASSI,‡ ANNEGRET DUMMER,‡ STEFFEN TELLER,‡ WALTER HALANGK,* and MARKUS M. LERCH‡ *Division of Experimental Surgery, Department of Surgery, Otto-von-Guericke-Universität Magdeburg, Magdeburg; ‡Department of Medicine A, Ernst-Moritz-Arndt- Universität Greifswald, Greifswald; §Institute of Experimental Internal Medicine, Department of Internal Medicine, Otto-von-Guericke-Universität Magdeburg, Magdeburg, Germany; ʈDepartment of Molecular and Cell Biology, Boston University, Goldman School of Dental Medicine, Boston, Massachusetts; and ¶Institut für Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany BACKGROUND & AIMS: Acute pancreatitis is charac- notable example is trypsinogen, which reaches the small terized by an activation cascade of digestive enzymes in the intestine as precursor zymogen, is activated by brush- pancreas. The first of these, trypsinogen, can be converted to border enterokinase, and then acts as the master enzyme active trypsin by the peptidase cathepsin B (CTSB). We in activating other digestive proteases. A pivotal role of investigated whether cathepsin L (CTSL) can also process trypsin in the initiating events of pancreatitis can be trypsinogen to active trypsin and has a role in pancreatitis. assumed because trypsin mutations are the most com- METHODS: In CTSL-deficient (CtslϪ/Ϫ) mice, pancreatitis mon autosomal-dominant changes associated with pan- was induced by injection of cerulein or infusion of tauro- creatitis.1–3 In the absence of enterokinase in the pan- cholate into the pancreatic duct. Human tissue, pancreatic creas, other mechanisms must be operative through juice, mouse pancreatitis specimens, and recombinant en- which a premature and intrapancreatic activation of zymes were studied by enzyme assay, immunoblot, N-ter- trypsinogen can be initiated. One of the best documented minal sequencing, immunocytochemistry, and electron mi- of these mechanisms is trypsinogen activation by cathep- Ϫ Ϫ Ϫ Ϫ croscopy analyses. Isolated acini from Ctsl / and Ctsb / sin B (CTSB), a lysosomal protease. Biochemically, CTSB mice were studied. RESULTS: CTSL was expressed in hu- has long been shown to be a trypsinogen activator,4 and, man and mouse pancreas, colocalized with trypsinogen in in experimental studies, it was demonstrated that inhi- secretory vesicles and lysosomes, and secreted into pancre- bition of CTSB or genetic deletion of the ctsb gene Ϫ Ϫ atic juice. Severity of pancreatitis was reduced in Ctsl / protects not only against premature trypsinogen activa- PANCREAS, AND BILIARY TRACT mice, whereas apoptosis and intrapancreatic trypsin 5 BASIC–LIVER, tion but also against pancreatitis. Although the role of activity were increased. CTSL-induced cleavage of CTSB in trypsinogen activation and pancreatitis is now trypsinogen occurred 3 amino acids toward the C-termi- firmly established,6–8 little is known about a potential nus from the CTSB activation site and resulted in a role of other lysosomal enzymes. truncated, inactive form of trypsin and an elongated Cathepsin L (CTSL) is another member of the papain propeptide (trypsinogen activation peptide [TAP]). This family of cysteine proteinases with similar enzymatic elongated TAP was not detected by enzyme-linked im- properties. CTSL exhibits a much stronger endoproteo- munosorbent assay (ELISA) but was effectively converted lytic activity than CTSB9 and could therefore, if it were to an immunoreactive form by CTSB. Levels of TAP thus expressed in the pancreas, be an even more important generated by CTSB were not associated with disease se- regulator of protease activation in pancreatitis. Recently verity, although this is what the TAP-ELISA is used to established functions of CTSL include distinct steps in determine in the clinic. CONCLUSIONS: CTSL inacti- major histocompatibility complex processing, the matu- vates trypsinogen and counteracts the ability of CTSB ration of enkephalin in neuroendocrine cells, and the to form active trypsin. In mouse models of pancreati- degradation and recycling of growth factor receptors in tis, absence of CTSL induces apoptosis and reduces keratinocytes.10–12 When released into the cytosol, CTSL disease severity. has been suggested to regulate apoptosis and to process nuclear transcription factors.13 cute pancreatitis is thought to be caused by autodi- gestion of the pancreas by its own digestive enzymes. Abbreviations used in this paper: CTSB, cathepsin B; CTSL, cathepsin A L; TAP, trypsinogen activation peptide. Physiologically, most digestive proteases are secreted as © 2010 by the AGA Institute precursor zymogens and only acquire activity after cleav- 0016-5085/10/$36.00 age and activation by proteolytic processing. The most doi:10.1053/j.gastro.2009.10.048 February 2010 CATHEPSIN L IN PANCREATITIS 727 In the present study using human and mouse material, we found that CTSL is abundantly expressed in the exocrine pancreas, sorted into the lysosomal as well as the secretory pathway of acinar cells, and secreted into pan- creatic juice. In mice, in which the ctsl gene was deleted, experimental pancreatitis was significantly less severe and involved a dramatic shift to cell injury via apoptosis. Surprisingly, and in complete contrast to CTSB, CTSL was found to very effectively inactivate trypsinogen and trypsin in vivo, in isolated acini, and in vitro. Materials and Methods Please refer to Supplementary Materials and Methods for more detailed descriptions. Induction of Experimental Pancreatitis in Cathepsin L-Deficient Mice CTSL-deficient (CtslϪ/Ϫ) mice were generated by gene targeting in embryonic day 14 mouse embryonic stem cells as described by Roth et al.14 The CtslϪ/Ϫ mice lack CTSL activity in all organs but do not show pheno- typic alterations of the pancreas (Figure 1). Cerulein pancreatitis was induced by 7 hourly injections of supra- maximal (50 ␮g/kg/body weight/intraperitoneal) cer- ulein injections and taurocholate-induced pancreatitis by infusion of 50 ␮L 2% taurocholate into the pancreatic 5,15 duct as previously described. Figure 1. CTSL in the human and mouse pancreas and the CtslϪ/Ϫ pancreas. In panel A, by Western blots with CTSL antibody pro-CTSL, Preparation of Serum and Tissue Samples single and heavy chain CTSL in human pancreas and pancreatic juice were detected. Panel B indicates expression and subcellular localiza- Mice were killed at intervals between 1 and 24 tion of CTSL in normal human pancreas (red fluorescence) and panel C hours after the first injection of cerulein and 6 hours colocalization of trypsinogen (green) with CTSL (red) in normal mouse after intraductal taurocholate application. Blood and tis- pancreas. The pancreas of CtslϪ/Ϫ mice appears completely normal on sue were harvested as previously reported.5 Pancreatic H&E-stained sections (D, with an islet and duct at the top) and electron microscopy (E, with normal arrangement of zymogen granules and acini were prepared using purified collagenase (Serva, mitochondria). Bars indicate 50 ␮m and asterisks the acinar lumen. Heidelberg, Germany).5,15 BASIC–LIVER, BILIARY TRACT PANCREAS, AND Biochemical Assays Trypsin and trypsinogen after enterokinase ac- tivation were measured at 37°C using 64 ␮mol/L Human and Animal Material for Morphology BOC-Gln-Ala-Arg-7-amido-4-methylcoumarin as a sub- and Morphometry strate.5,15 Serum amylase and lipase activities were deter- Human material from donor pancreas as well as mined enzymatically by commercially available assays pancreatic juice from controls and pancreatitis patients (Roche-Hitachi, Basel, Switzerland). Trypsinogen activa- was obtained as previously described16 under an ethics tion peptide (TAP) was assayed using an enzyme immu- committee-approved protocol and with informed patient noassay (Biotrin, Dublin, Ireland), and protein concen- consent. For animal experiments, we collected tissue sam- trations were determined according to Bradford. Serum ples at selected time intervals of pancreatitis as previously cytokines were measured by fluorescence-activated cell reported.5,8 For experiments involving the detection of sorter analysis using a bead-based mouse immunoassay apoptotic cells, free 3’OH-DNA termini were labeled (Becton-Dickinson, Franklin Lakes, NJ). Caspase-3 activ- using the terminal deoxynucleotidyl-transferase (TdT) ity was measured fluorometrically using Rodamin110- method with fluorescein-labeled digoxigenin nucleo- DEVD as substrate (Invitrogen, Eugene, OR). Lung my- tides.5,8 Methods for electron microscopy and the quan- eloperoxidase was measured using O-dianisidine and tification of areas of necrosis by morphometry are de- 5 8 H2O2 as previously reported. scribed in detail elsewhere. 728 WARTMANN ET AL GASTROENTEROLOGY Vol. 138, No. 2 Proteolytic Processing of Trypsin and as well as heavy chain CTSL (Figure 1A). Detection of Trypsinogen by CTSB and CTSL CTSL in pancreatic juice is direct evidence that some The human cationic trypsin (PRSSI1) expression CTSL is sorted into the secretory pathway of the pancreas plasmid was constructed as previously reported and and actively secreted into the ducts. Immunofluorescence expressed in Escherichia coli Rosetta
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