Release of Cathepsin B in Cytosol Causes Cell Death in Acute Pancreatitis

Abstract

Background & aims: Experimental studies in acute pancreatitis (AP) suggest a strong association of acinar cell injury with cathepsin B-dependent intracellular activation of trypsin. However, the molecular events subsequent to trypsin activation and their role, if any, in cell death is not clear. In this study, we have explored intra-acinar events downstream of trypsin activation that lead to acinar cell death.

Methods: Acinar cells prepared from the pancreas of rats or mice (wild-type, trypsinogen 7, or cathepsin B-deleted) were stimulated with supramaximal cerulein, and the cytosolic activity of cathepsin B and trypsin was evaluated. Permeabilized acini were used to understand the differential role of cytosolic trypsin vs cytosolic cathepsin B in activation of apoptosis. Cell death was evaluated by measuring specific markers for apoptosis and necrosis.

Results: Both in vitro and in vivo studies have suggested that during AP cathepsin B leaks into the cytosol from co-localized organelles, through a mechanism dependent on active trypsin. Cytosolic cathepsin B but not trypsin activates the intrinsic pathway of apoptosis through cleavage of bid and activation of bax. Finally, excessive release of cathepsin B into the cytosol can lead to cell death through necrosis.

Conclusions: This report defines the role of trypsin in AP and shows that cytosolic cathepsin B but not trypsin activates cell death pathways. This report also suggests that trypsin is a requisite for AP only because it causes release of cathepsin B into the cytosol.

Keywords: Apoptosis; Bid Cleavage; Cytosolic Cathepsin B; Necrosis.

Figure-1. Secretagogue induced AP results in release of Cathepsin-B and other lysosomal-enzymes into the cytosol

After appropriate in vitro and in vivo treatment rat acinar cells were fractionated into cytosol and membrane fraction with Streptolysin O and presence of Cathepsin-B in the cytosol was evaluated. (A) Representative western blot demonstrating that supramaximal caerulein stimulation leads to increase in cytosolic cathepsin-B. Actin was used as loading control. (B) In vitro Supramaximal caerulein and carbachol but not maximal caerulein, carbachol or maximal or supramaximal CCK-JMV 180 leads to increase in cytosolic cathepsin-B activity. (C) Supramaximal caerulein stimulation In vitro leads to increase in cytosolic activity of the lysosomal enzyme aryl sulfatase. (D) Induction of AP in rats with supramaximal caerulein or arginine treatment leads to increase in cytosolic cathepsin-B activity. (E) Immunofluorescence evidence of leakage of cathepsin-B into the cytosol in caerulein model of AP (1 dose of 10 μg/kg caerulein with sacrifice after 2 hours) in rats. Cathepsin-B demonstrates punctate green staining in pancreatic acini of control animal while as a result of induction of caerulein pancreatitis cathepsin-B was released into cytosol and gave diffuse staining. (F) Cathespin B immunohistochemistry in pancreatic section from normal donor pancreas or patients with chronic pancreatitis is shown. While cathepsin-B in normal pancreas demonstrates punctate staining, we observed increased number of cells with diffuse cytoplasmic staining in patients with chronic pancreatitis (black arrows). Values of all enzyme activity are expressed as percent of total (enzymatic activity obtained in the supernatant after lysing the cells with sonication) after normalizing to protein content. All data in (B), (C) and (D) represents mean ± SEM of triplicates of three individual experiments. * p≤ 0.05 as compared to controls, which were not stimulated with secretagogue or L- arginine.

Figure-2. Supramaximal caerulein stimulation leads to leakage of cathepsin-B from the co-localized organelles into the cytosol

Supramaximal caerulein stimulation of rat pancreatic acini results in leakage of (A) amylase, (B) trypsin and (C) cathepsin-B into the cytoplasm suggesting that the cytosolic cathepsin-B is arising from organelles containing both lysosomal-enzymes, zymogens and active trypsin i.e. co-localized organelles. Inhibition of co-localization by pre-treatment with the PI-3 kinase inhibitors Wortmannin and Ly492004, leads to significant reduction in cytosolic amylase, trypsin and cathepsin-B activity again suggesting that co-localized organelles are the source of the cytosolic Cathepsin-B. (D) HSP70 induction by prior thermal stress or sodium arsenite prevents cytosolic cathepsin-B increase in response to supramaximal caerulein. (E) Attenuation of cytosolic calcium reduces cytosolic cathepsin-B activity following supramaximal caerulein. (F) Cytosolic cathepsin-B activity normalized to LDH is increased in mice pancreatic acini treated with supramaximal caerulein. However, supramaximal caerulein is unable to cause release of Cathepsin-B into the cytosol of trypsinogen-7 (T7KO) mice. Values of enzyme activity in A-E are expressed as percent of total (enzymatic activity obtained in the supernatant after lysing the cells with sonication) after normalizing protein content. Cathepsin-B activity is normalized to LDH content in F. All data represents mean ± SEM of triplicates of three individual experiments. * p< 0.05 as compared to caerulein only stimulation.

Figure-3. Supramaximal caerulein in vitro and in vivo and L- arginine in vivo leads to apoptosis in rat pancreatic acini

(A) Supramaximal caerulein stimulation of rat acini in vitro leads to caspase-3 activation. Caspase-3 activity was measured using Caspase-Glo® 3/7 Assay from promega. Supramaximal caerulein stimulation of acini also leads to increase in (B) green fluorescence (yellow arrow) and (C) Apo Trace fluorescence after dye extraction suggesting occurrence of apoptosis. (D) In vivo induction of AP by either supramaximal caerulein or L-arginine leads to increase in caspase-3 activation. Caerulein induced acinar cell apoptosis, as measured by (E) Apo Trace fluorescence or (F) caspase-3 activity, is decreased in acini pre-treated with either cathepsin-B inhibitor CA074-me or trypsin inhibitor benzamidine. Values are expressed as percent of control and normalized to per mg protein and all data represents mean ± SEM of triplicates of three individual experiments. * p≤ 0.05 as compared to controls, which were not stimulated with caerulein or L-arginine.

Figure-4. Cytosolic cathepsin-B but not trypsin is responsible for acinar cell apoptosis

Introduction of exogenous Cathepsin-B but not trypsin into SLO permeabilized normal rat pancreatic acinar cells leads to caspase-3 activation. Caspase-3 activity was noticed over a wide range of doses of cathepsin-B while no activity was seen with any given dose of trypsin. Values are expressed as percent of total and normalized to per mg protein. All data represents mean ± SEM of triplicates of three individual experiments.

Figure-5. Cathepsin-B and trypsin are required for activation of caspase 3 and acinar cell death

(A) Supramaximal caerulein stimulation reduced WT pancreatic acinar cell viability, whereas CBKO and T7KO acini were protected against caerulein induced cell death. Pretreatment of pancreatic acini with cathepsin-B inhibitor CA074me (10 μM for 15 minutes) also protected acini against cell death caused by supramaximal caerulein. Acini were stimulated using either maximal or supramaximal concentrations of caerulein for 3hrs. Cell viability was quantitated using MultiTox-Fluor Multiplex Cytotoxicity Assay from Promega. (B) Supramaximal caerulein activated caspase-3 in WT acini, but CA074-me pretreatment protected WT acini against caspase-3 activation. In CBKO acini which lack cathepsin-B or T7KO acini which do not show trypsin activation no caspase-3 activity was detected in acinar cells after supramaximal stimulation with caerulein. (C) Exogenous cathepsin-B activates caspase-3 in permeabilized WT or CTSB^-/- pancreatic acinar cells. Supramaximal caerulein stimulation does not augment caspase-3 activation over and above that induced by addition of exogenous cathepsin-B to permeabilized WT and CTSB^-/- mice pancreatic acini. (D) Non-permeable CA074 treatment blocks exogenous cathepsin-B-induced caspase-3 activation in permeabilized WT mice pancreatic acinar cells. WT mice pancreatic acini were either mock- or pre-incubated for 20 min with 10 μM permeable CA074-me, followed by treatments for 1 h with 10^-7 M caerulein after SLO permeabilization. The cells were subsequently mock-incubated or incubated with CA074 for 20 min followed by exposure to exogenous cathepsin-B for 3hrs. Caspase-3 activity was then quantified and normalized to DNA content.

Figure-6. Cathepsin-B activates the mitochondrial apoptosis pathway. Excessive amount of cytosolic cathepsin-B causes necrotic cell death

(A) Caerulein hyperstimulation leads to Bid cleavage in WT but not in CTSB^-/- mice. WT or CTSB^-/- mice were injected intraperitoneally with caerulein (50 μg/kg/injection, for 3 hourly injections, animals were sacrificed 30 min or 60 min after injection and pancreatic tissues probed with Bid and GAPDH antibodies. (B) Quantification of bid cleavage from three independent experiments is shown. (C) Cytosolic cathepsin-B induces Bax activation in response to supramaximal caerulein stimulation. WT and CTSB^-/- mice pancreatic acini were untreated or treated with 10^-10 M, or 10^-7 M caerulein for 3h. Cell lysates (400 μg) were immunoprecipitated with 0.2μg of the mouse anti-Bax antibody (BD Pharmingen), and the immunoprecipitates were examined by immunoblot analysis with active Bax monoclonal antibody 6A7. (D) Caerulein stimulation of pancreatic acinar cells caused release of cytochrome c into the cytosol of WT mice. This caerulein induced Cathepsin-B release was prevented by pre-treatment with Cathepsin-B inhibitor CA074-me. GAPDH was used as loading control for cytosolic fraction and COX IV for mitochondrial fraction. Cytosolic and membrane fractions of the acini were separated after permeabilization with SLO. Role of cathepsin-B induced cytochrome C was further evaluated by evaluating effect of addition of extrinsic cathepsin-B to the permeabilized acini with and without neutralizing anti-cytochrome c antibody. While addition of extrinsic cathepsin-B to the permeabilized acini in presence of control isotype specific antibody causes caspase-3 activation, neutralization of the cytochrome c prevents cathepsin-B induced caspase 3 activation. (E) LLOMe leads to release of cathepsin-B into the cytosol of acinar cells in a dose dependent fashion. At lower doses there is activation of caspase-3 which decreases at higher dose of LLOMe with corresponding increase in LDH release suggesting shift from apoptosis to necrosis. (F) While higher dose of LLOMe is able to induce necrosis in acini from wild type mice, no necrosis was observed in cathepsin-B knockout mice when treated with LLOMe. Results are expressed as fold change compared with untreated controls. Each value is the mean of triplicate samples. Representative data of three independent experiments are shown.

Figure 7

Schematic representation of the new paradigm of cell death in experimental AP.