Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis

Abstract

Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific.

Keywords: apoptosis; autophagy; cathepsin B (CTSB); cell death; chymotrypsin; pancreas; pancreatitis; trypsin activation.

FIGURE 1.

Subcellular fractions of mouse pancreatic tissue during caerulein-induced pancreatitis (0, 1, and 8 h) were prepared as described under “Materials and Methods.” A, trypsin activity distributed to the zymogen granule fraction after 1 h and to the lysosomal fraction after 8 h. B, activation was absent in CTSB^−/− animals. C and D, chymotrypsin activity followed a similar pattern (C), and activation was abolished in CTSB^−/− mice (D). E and F, CTSB activity predominated in lysosomes under resting conditions, moved to the zymogen granule fraction at 1 h (E), and was absent in CTSB^−/− mice (F). G and H, CTSL activity followed the same redistribution pattern as CTSB (G) but remained unaffected by CTSB deletion (H). Protease activity is shown as relative fluorescent units (RFU). I, on Western blots of fractions, cytosolic marker GAPDH can only be found in cytosolic fraction (Cyt), the lysosomal markers LAMP-2 and lysosomal integral membrane protein distributed to the lysosomal (Lys) and the zymogen (ZG) marker syncollin to the zymogen granule fraction. J, the autophagy marker LC 3-II is redistributed in the lysosomal fraction after 8 h of pancreatitis. K, the same redistribution pattern to the zymogen granule fraction as seen for CTSB activity (substrate AMC-Arg₂) was also found for the processed protein form CTSB (33 kDA) by anti-CTSB antibody (AB) as well as the NS-196-labeled active from of CTSB. All experiments were repeated at least in triplicate, and error bars indicate means ± S.E. *, differences to respective controls statistically significant at the 5% level.

FIGURE 2.

Protease activities were measured in living cells with fluorogenic substrates, and the ratio of change compared with activity changes in homogenates is presented. A and B, trypsin activation followed a similar pattern in living cells (A) and homogenates (B) after incubation with CCK. C–G, for CTSB, the activity increase appeared greater in living cells (C) than in homogenates (D), where it corresponded to the shift from the 44-kDa precursor to the activated 33-kDa form (E) and of the activity marker NS-196 (F). Deletion of the CTSB gene reduced activation (E and F) and neutralization of pH reduced activity of CTSB (G). Data points represent mean ± S.E. from three or more experiments. *, differences to respective control (con) group significant at the 5% level.

FIGURE 3.

Protease activation in response to supramaximal CCK was investigated in living isolated acini using fluorogenic substrates as described under “Materials and Methods” and necrosis via the propidium iodine exclusion technique. A–C, CCK induced a rapid and transient increase in the activities of CTSB (A), trypsin and chymotrypsin (B), and acinar cell necrosis (C). The protease activities were greatly reduced by deletion of CTSB (CTSB^−/−), but necrosis remained unaffected. D–F, a similar reduction in activation of CTSB (D) and trypsin (E) was achieved by neutralizing intracellular pH with 100 μm chloroquine, but no such affect was found for necrosis (F). G–I, inhibiting vacuolar type ATPases with 100 nm bafilomycin A1 correspondingly reduced CTSB activity (G) and trypsin activation (H) but equally failed to affect necrosis (I). Graphs represents mean ± S.E. from three or more experiments. *, denote differences significant at the 5% level.

FIGURE 4.

When neither CTSB deletion nor pH neutralization affected necrosis, we investigated caspase 3 activation and apoptosis at the time point of maximal protease activation in acini following CCK stimulation (30 min). A, pH neutralization with chloroquine (CQ, 100 μm) abolished the activity increase of CTSB (A) as well as labeling with the activity marker NS-196 in Western blots (right panel). con, control. B, similarly, it also blocked activation of caspase 3 in acini. C and D, the vacuolar type ATPase inhibitor bafilomycin A1 (Baf, 100 nm), which prevents acidification of vesicular compartments, had the identical effect of preventing the CTSB activity increase (C) as well as caspase 3 activation and degradation of AIF and the inhibitor of apoptosis protein family member XIAP (D). E and F, serine protease activation of trypsin (E) and chymotrypsin (F) was completely abolished by bafilomycin A1. The graphs represent mean ± S.E. from three or more experiments. *, differences significant at the 5% level.

FIGURE 5.

A, when comparing the effect of CTSB deletion with CTSL deletion, trypsin activation in response to 30-min CCK stimulation was abolished in CTSB^−/− acini, whereas its magnitude was increased in CTSL^−/− acini. B and C, the same pattern was found for chymotrypsin activation (B), but CTSL deletion left the CTSB activity increase unaffected (C). D, CCK-induced caspase 3 activation was abolished in CTSB^−/− acini but increased in acini from CTSL^−/− animals. E, in parallel the degradation of AIF in response to CCK was abolished in CTSB^−/− acini but enhanced in acini from CTSL^−/− animals. *, denote differences significant at the 5% level.

FIGURE 6.

A–C, to test whether these effects on apoptosis were cathepsin-dependent or digestive protease-dependent, we used the serine protease inhibitor nafamostat (Naf, 100 μm), which completely inhibited trypsin activation in acini (A) as well as chymotrypsin activation (B) but did not affect CTSB activation (C). D–F, the fact that caspase 3 activation as well as degradation of AIF and XIAP were abolished by nafamostat (D and E) indicates that CTSB-induced trypsinogen activation and not CTSB activation per se mediates caspase 3 activation, whereas nafamostat has no in vitro effect on caspase 3 activity (F). con, control. G and H, a specific trypsin (try) inhibitor, N166, shows similar effects to nafamostat. Trypsin activity is completely abolished (G), whereas cathepsin B activity was not affected (H). I, inhibition of trypsin results in reduced caspase 3 activity. The inhibitory capacity of the trypsin inhibitor was tested in living acinar cells. J–L, trypsin activity is completely blocked upon CCK stimulation (J), whereas cathepsin B activity (K) and necrosis (L) were not influenced. M, to investigate the organ specificity of the mechanism, we repeated the experiments in hepatocytes. Apoptosis was induced in freshly isolated murine hepatocytes via two different pathways: stimulation of cells with 200 ng/ml Fas Ligand (FasL) for 6 h and heating cells for 1 h at 43 °C. Both methods led to an increased caspase 3 activity, but only heat-induced apoptosis led to degradation of AIF. Additional treatment of cells with 100 μm nafamostat did not affect caspase 3 activation or degradation of AIF or XIAP. This indicates that serine protease-induced apoptosis is an acinar specific effect. The graphs represents mean ± S.E. from three or more experiments. *, differences significant at the 5% level.

FIGURE 7.

A, the optimal catalytic activity of cathepsin B is limited to acidic conditions (∼pH 5), but also under physiological pH 7.5, as in cytoplasm, CTSB shows a third of maximal catalytic activity, and CTSB activity is presented in relative fluorescence units (RFU). B, in subcellular fractions of pancreatic tissue, increased CTSB activity 8 h after caerulein stimulation can be found in cytoplasmic fractions. This increased activity is only stable under acidic conditions and is completely abolished at pH 7 or higher. C, cytoplasmic fractions from CTSB-deleted animals functioned as negative controls. D and E, in contrast to cathepsin B, trypsin activity in cytoplasmic fractions after 8 h of caerulein stimulation is stable under neutral pH conditions (D), whereas CTSB-deleted animals display markedly decreased trypsin activity (E). CTSB activity was measured in cytoplasmic and zymogen fractions. F, in the zymogen fraction we detected a stably increased CTSB activity, even at neutral pH, at 8 h of caerulein treatment (F), resembling enzyme activity measurement of recombinant cathepsin B (A). Adding the cytosolic fraction to the zymogen fraction resulted in inhibition of CTSB activity at pH values between 6.5 and 8 but not under acidic conditions. These findings suggest that the cytoplasmic fraction contains endogenous inhibitors for CTSB that are pH-dependent. G, Western blotting of cystatin B and C showed a strong signal in the cytoplasmic fractions (Cyt) but only weak in zymogen (ZG) and lysosomal fraction (Lys), that was stable over the time course of caerulein-induced pancreatitis (1h and 8h). The graphs represents mean ± S.E. from three or more experiments. *, differences significant at the 5% level.

FIGURE 8.

A–C, to study whether trypsin-induced caspase 3 activation translates into apoptosis in vivo, we used two models of experimental pancreatitis and measured apoptosis by TUNEL assay in tissue sections. After 8 h of caerulein-induced pancreatitis, caspase 3 activity was greatly reduced in pancreatic homogenates of CTSB^−/− pancreatitis animals, and when TUNEL-positive acinar cells were quantitated (examples are presented in B; scale bars represent 50 μm), the reduction in apoptosis was even greater (C). D–F, in the duct ligation-induced pancreatitis model, the deletion of CTSB did not affect pancreatic injury markers such as serum amylase activity (D) and lipase activity (E), but the number of apoptotic cells was greatly reduced (F). The graphs represents mean ± S.E. from three or more experiments. *, differences significant at the 5% level.

FIGURE 9.

Model for cathepsin-induced pancreatic apoptosis. Cathepsin B is activated in response to a pathophysiological stimulus such as supramaximal CCK within a secretory vesicle-containing subcellular compartment of acinar cells. Cathepsin L acts as its antagonist. Both have opposing effects on the intracellular activation and activity of trypsin, the critical component in caspase 3-mediated apoptosis. Cathepsin B and cathepsin L activities are in an equilibrium of activation and degradation under physiological conditions (A). This balance between CTSB and CTSL is disturbed during pancreatitis (B), and the activation of CTSB, together with a change in intracellular pH, leads to a dominance of CTSB activity over CTSL activity. This results in a net increase in trypsinogen activation over trypsin degradation. This trypsinogen activation induces the serine protease cascade, which is directly or indirectly responsible for acinar cell apoptosis.