1. 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 *
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. .