Cathepsin B mediated protease activation increases acinar cell apoptosis Medizinische Klinik der königlichen Universität Greifswald 1856 1456 M. Sendler1, D. John1, F.U. Weiss1, M. Persike1,A. Aghdassi1,T. Wartmann2, W. Halangk2, N. Schaschke3, J. Mayerle1, M.M. Lerch1 1Department of Medicine A, Ernst-Moritz-Arndt-University Greifswald, Germany, 2Division of Experimental Surgery, Otto-von-Guericke University Magdeburg, Germany, 3Max-Planck-Institute for Biochemistry Martinsried, Germany Introduction Cathepsin-B (CTSB) is the major intracellular trypsionogen activator in the pancreas and during experimental pancreatitis. Already under physiological conditions trypsinogen and CTSB are co-localised in the acinar cell but premature digestive protease activation only develops in response to a pathological stimulus. Here we have investigated the underlying mechanisms. Results – Cathepsin B is activated by CCK stimulation and activity depends on intracellular pH A Increase of active CTSB by CCK B Activity of CTSB in homogenates C Activity of CTSB in living acinar cells A Acidification of acinar cells occurs along with CTSB activity B Effect of Chloroquin C Effect of Baffilomycin A1 control CCK 10-7M control + CQ CCK 10-7M + CQ Cathepsin B time in min Cathepsin B activity relativ to control 20 40 60 5 10 15 25 * Trypsin activity relative to control Trypsin 4 8 12 16 control + Baf CCK 10-7M + Baf 0min 5min 10min 15min 20min Lyso Sensor AMC-Arg-Arg CTSB merge 25kDa 35kDa CTSB+/+ CTSB-/- NS196 CCK + - Cathepsin B activity in acinar cell homogenate 50 100 150 200 250 * 1,5 fold time in min Cathepsin B activity relative to control 2 4 6 8 10 12 14 16 20 40 60 control 10-7M CCK * 10 - 15 fold CTSB GAPDH 30 min 10-7 M CCK control Fig. 1: Cathepsin B is activated upon supramaximal CCK stimulation (A). The NS196 inhibitor, which recognices only the active form of CTSB shows a stronger band in western blot analysis of acinar cell homogenates. CTSB activity increases in acinar cell homogenates upon supramaximal stimulation of CCK by a factor 1,5 (B). In contrast to homogenates in living acinar cells the activity increase is 10 fold higher (C). On one hand CTSB gets activated, and on the other hand in vivo conditions modulate the activity. Fig. 2: Acidification, shown by LysoSensor, is clearly located at the apical pole of acinar cells. The activity signal of Cathepsin B is located in this area (A). Neutralization of the lysosomal compartment by Chloroquin inhibits Cathepsin B activity and Trypsinogen activation (B). Bafilomycin A1, a V-ATPase inhibitor, has an equal effect as chloroquin (C). This indicates the necessity of acidification for CTSB activity and pancreatic protease activation. Results – Cathepsin B during Caerulein pancreatitis A Subcellular fractionation of the pancreas during Caerulein pancreatitis in CTSB+/+ and CTSB-/- mice A A Apoptotic cell death is reduced in CTSB-/- mice during Caerulein pancreatitis Apoptotic cell death is reduced in CTSB-/- mice during Caerulein pancreatitis Trypsin activity in CTSB+/+ Trypsin activity in CTSB-/- CTSB+/+ CTSB-/- H&E Tunnel assay Tunnel assay % of tunnel positv cells 0,0 0,5 1,0 1,5 2,0 2,5 3,0 Caspase 3 Caspase 3 activity in RFU/protein 5 10 15 20 25 30 * Trypsin activity in RFU/mg protein 200 400 600 800 1000 1200 1400 0h 1h 8h 200 400 600 800 1000 1200 1400 Trypsin activity in RFU/mg protein 0h 1h 8h ZG Lys Cyt GAPDH – cytosolic marker LAMP-2 – lysosomal marker Syncollin – zymogene marker Zymogene granules Lysosomes Cytosol Cathepsin B activity in CTSB+/+ Cathepsin B activity in CTSB-/- active Cathepsin B in CTSB+/+ Cathepsin B activity in RFU/mg protein 200 400 600 800 1000 0h 1h 8h 200 400 600 800 1000 0h 1h 8h Pro Cathepsin B Cathepsin B 40kDa 35kDa 25kDa NS196 Densitometry of NS196 western blot signal 100 200 300 400 500 600 700 0h 1h 8h * B * Apoptotic cell death is also reduced in CTSB-/- in a second model of pancreatitis CTSB+/+ CTSB-/- 1,2 Cathepsin B activity in RFU/mg protein 0,8 Tunnel positive cells in % CTSB+/+ CTSB-/- 0,4 * 0,0 Anti -CTSB AK Fig. 3: Subcellular fractionation was preformed in CTSB+/+ and CTSB-/- mice by density centrifugation, western blot was used to proof purity. After 1h of Caerulein pancreatitis the activity of CTSB is increasing in the heavy zymogene fraction where trypsinogen gets activated. Western blots of CTSB shows a loss of pro-Cathepsin B in this fraction. Later time points of pancreatitis shows a reorganization of lysosomal compartment and a different compartment of trypsinogen activation. total ligation of pancreatic duct CTSB-/- CTSB+/+ Fig. 4: Apoptotic cell death pathways depend on the presence of Cathepsin B. Tunnel positive cells after 8h of Caerulein pancreatitis are reduced in CTSB-/- mice as well as Caspase 3 activity in pancreas homogenate (A). Also 24h after duct ligation, which represents a CCK independent second model of pancreatitis, apoptosis is reduced in CTSB-/- mice (B). Results – Cathepsin B and Cathepsin L regulate Apoptosis A Activity of pancreatic proteases in acinar cells of wild type, CTSB-/- and CTSL-/- mice B Apoptosis in acinar cells of wild type, CTSB-/- and CTSL-/- mice WT CTSB-/- CTSL-/- Cathepsin B Cathepsin B activity/protein 100 200 300 400 500 * Trypsin Trypsin activity/protein 600 800 Chymotrypsin Chymotrypsin activity/protein 1000 2000 3000 4000 5000 6000 7000 Caspase 3 activity/ protein Caspase 3 1500 AIF Elastase Elastase activity/protein 1200 1600 amylase 0,0 0,4 0,8 1,2 1,6 Apoptosis inducing factor (AIF) Densitometry of AIF/amylase CTSB CTSL Trypsinogen activation Trypsin degradation Protease cascade Enzyme activity Apoptosis 30 min 10-7 M CCK control Fig. 5: Isolated acinar cells of WT, CTSB-/- and CTSL-/- mice were stimulated with CCK. Pancreatic protease activation depends entirely on the presence of active cathepsin B, which is highly increased in the absence of cathepsin L (A). Activity of cathepsin B is independent of Cathepsin L, in contrast to Caspase 3 which is highly increased in CTSL-/- mice (B). These findings suggest a protease activity dependent induction of apoptosis in acinar cells. summary and conclusion The increase in CTSB activity following supramaximal CCK-stimulation is a strictly intravesicular process, depends on an acidic intravesicular pH, but neither on reactive-oxygen-species generation nor on cytosolic CTSB-inhibitors. Acinar cell necrosis can develop independently of either CTSB activity or overall digestive protease activation, whereas induction of apoptosis involves CTSB activity. .