Cell Death in PancreatitisCASPASES PROTECT FROM NECROTIZING PANCREATITIS*

Received for publication, October 17, 2005, and in revised form, December 7, 2005 Published, JBC Papers in Press, December 8, 2005, DOI 10.1074/jbc.M511276200

Olga A. Mareninova‡, Kai-Feng Sung‡§, Peggy Hong‡, Aurelia Lugea‡, Stephen J. Pandol‡, Ilya Gukovsky‡,and Anna S. Gukovskaya‡1

From the ‡Veterans Affairs Greater Los Angeles Healthcare System and Department of Medicine, David Geffen School of Medicine,University of California at Los Angeles, Los Angeles, California 90073 and the §Department of Hepato-Gastroenterology, ChangGung Memorial Hospital, Taipei 333, Taiwan

Mechanisms of cell death in pancreatitis remain unknown.Parenchymal necrosis is a major complication of pancreatitis; also,the severity of experimental pancreatitis correlates directly withnecrosis and inversely with apoptosis. Thus, shifting deathresponses from necrosis to apoptosis may have a therapeutic value.To determine cell death pathways in pancreatitis and the possibilityof necrosis/apoptosis switch, we utilized the differences betweenthe ratmodel of cerulein pancreatitis, with relatively high apoptosisand low necrosis, and the mouse model, with little apoptosis andhigh necrosis.We found that caspases were greatly activated duringcerulein pancreatitis in the rat but not mouse. Endogenous caspaseinhibitor X-linked inhibitor of apoptosis protein (XIAP) underwentcomplete degradation in the rat but remained intact in the mousemodel. Furthermore, XIAP inhibition with embelin triggeredcaspase activation in themousemodel, implicatingXIAP in caspaseblockade in pancreatitis. Caspase inhibitors decreased apoptosisand markedly stimulated necrosis in the rat model, worsening pan-creatitis parameters. Conversely, caspase induction with embelinstimulated apoptosis and decreased necrosis inmousemodel. Thus,caspases not only mediate apoptosis but also protect from necrosisin pancreatitis. One protective mechanism is through degradationof receptor-interacting protein (RIP), a key mediator of “pro-grammed”necrosis.We found thatRIPwas cleaved (i.e. inactivated)in the rat but not themousemodel. Caspase inhibition restored RIPlevels; conversely, caspase induction with embelin triggered RIPcleavage. Our results indicate key roles for caspases, XIAP, and RIPin the regulation of cell death in pancreatitis. Manipulating thesesignals to change the pattern of death responses presents a thera-peutic strategy for treatment of pancreatitis.

Acute pancreatitis is an inflammatory disorder of exocrine pancreas,which carries considerablemorbidity andmortality, and the pathophys-iology of which remains obscure (1). During the past decade, significantprogress has been achieved in our understanding of the inflammatoryresponse in pancreatitis (2–5). By contrast, very little is known about themechanisms mediating another major pathologic response in pancrea-titis, the parenchymal cell death.In experimental models of acute pancreatitis, acinar cells have been

shown to die through both necrosis and apoptosis (6, 7). The apopto-

sis/necrosis ratio varies in different experimentalmodels of pancreatitis.Of note, the severity of experimental pancreatitis directly correlateswith the extent of necrosis and inversely with that of apoptosis (6–12).Mechanisms underlying these differences are not known.Apoptosis and necrosis are two main types of cell death (13–18).

Morphologically, apoptosis is manifested by cell shrinkage and chroma-tin condensation, whereas necrosis is characterized by swelling of thecell and its organelles and rupture of the plasma membrane. Biochem-ical hallmarks of apoptosis, such as activation of specific cysteine pro-teases, the caspases, and internucleosomal DNA fragmentation, areusually absent in necrotic cells. Apoptosis preserves the structural integ-rity of the plasma membrane, whereas the necrotic cell releases its con-stituents, which damage neighboring cells and promote inflammatoryinfiltration in the organ. Therefore, necrotic death is “deadlier” to theorganism than apoptotic death (13–15).There are two distinct pathways of apoptosis (19–21). The extrinsic

pathway is initiated by receptor-induced activation of the initiatorcaspase-8 (or caspase-10) followed by activation of effector caspasessuch as caspase-3. This pathway is typically triggered by “death recep-tors”, e.g. tumor necrosis factor receptor or Fas. In the intrinsic pathway,a critical event is permeabilization of the mitochondrial outer mem-brane, resulting in the release of pro-apoptotic factors such as cyto-chrome c. Once released, cytochrome c forms a complex with Apaf-1and procaspase-9, resulting in caspase-9 activation. Caspase-9 furthercleaves and activates the effectors caspases, (e.g. caspase-3) leading tosubsequent degradation of cellular constituents.Inhibitor of apoptosis proteins (IAPs)2 are an important class of

endogenous proteins that negatively regulate caspase activation (22–24). The X-linked IAP (XIAP) is themost potent among the eightmam-malian IAPs and inhibits themitochondria-driven caspases-9, -3, and -7(23, 25).In contrast with other disease states, for example, myocardial infarc-

tion (26, 27) or ischemic renal failure (28), there is very little knownabout the signaling mechanisms of apoptosis in pancreatitis. We haverecently shown (29) that the apoptotic “machinery” is present in isolatedrat pancreatic acinar cells and can be activated by supramaximal dosesof cholecystokinin-8 (CCK-8), which cause pancreatitis-like changes inacinar cells. In particular, CCK-8 induced mitochondrial cytochrome crelease and activation of caspases-9, -3. and -8.In contrast with apoptosis, the signaling mechanisms mediating

* This work was supported by National Institutes of Health Grant DK59936 (to A. S. G.).The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: Veterans Affairs Greater Los AngelesHealthcare System, West Los Angeles Veterans Affairs Healthcare Center, 11301Wilshire Blvd., Blg. 258, Rm. 340, Los Angeles, CA 90073. Tel.: 310-478-3711 (ext.41525); Fax: 310-268-4578; E-mail: agukovsk@ucla.edu.

2 The abbreviations used are: IAP, inhibitor of apoptosis protein; FLIP, FLICE-inhibitoryprotein; RIP, receptor-interacting protein; Q-VD-OPH, Q-Val-Asp(non-O-methylated)-Oph; XIAP, X-linked inhibitor of apoptosis protein; Z-D-DCB, Z-Asp-2,6-dichloroben-zoyloxymethylketone; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; CCK-8, cho-lecystokinin-8; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonicacid; AMC, 7-amino-4-methylcoumarin; ERK, extracellular signal-regulated kinase; E3,ubiquitin-protein isopeptide ligase.

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necrotic cell death are in general poorly understood. Recent findingsindicate that there are two types of necrosis (15–17). Accidental necro-sis is an unregulated process triggered by severe cellular stress that isoften characterized by depletion of cellular ATP. It is seen as a passiveexplosion of a cell overwhelmed by ion fluxes. By contrast, so-calledprogrammednecrosis (or necrosis-like programmed cell death) ismedi-ated by a coordinated series of signaling events that can be triggered viadeath receptors. For example, depending on conditions the same tumornecrosis factor receptor could induce either apoptosis or necrosis(30, 31). The only so far proven mediator of programmed necrosis isthe receptor-interacting protein kinase (RIP). RIP deficiency rescuescells from tumor necrosis factor-induced programmed necrosis (32–34). The mechanisms of RIP action, as well as its targets, are poorlyunderstood (15–17, 30).Acinar cell necrosis, and in particular, recurrent necrosis, is one of the

most serious complications of acute pancreatitis (1, 35, 36). Based on theabove-described observations that milder forms of experimental pan-creatitis are associated with more apoptosis and the relatively severeforms, withmore necrosis, it has been hypothesized (6–12) that switch-ing from the necrotic pattern of cell death to apoptosis could be bene-ficial in treatment of acute pancreatitis.In the present studywe investigated themechanisms of apoptosis and

necrosis in pancreatitis and explored the possibility of necrosis/apopto-sis switch through manipulating these signaling mechanisms. For thispurpose, we utilized the differences between two related rodent modelsof acute pancreatitis. Pancreatitis induced in rats by supramaximallystimulating doses of the CCK-8 analog, cerulein, is a mild form of thedisease characterized by relatively high extent of apoptosis and lownecrosis (7, 9). By contrast, in mice the same cerulein treatment resultsin a more severe disease with significant necrosis and very little apopto-sis (7). Both are the most commonly used and well characterized in vivomodels of acute pancreatitis (37, 38).We found drastic differences in death-signaling mechanisms,

namely, caspase activation, and XIAP and RIP degradation, between therat and mouse models of cerulein pancreatitis. By manipulating thesemechanisms using pharmacologic inhibitors, we were able to shift thenecrosis/apoptosis ratio, making the two models more like each other.The results identify several critical mediators of acinar cell death thatmay represent targets for therapeutic interventions to attenuate cell-death responses of acute pancreatitis.

EXPERIMENTAL PROCEDURES

Experimental Pancreatitis—Cerulein pancreatitis was induced inmale (200–250 g) Sprague-Dawley rats and male (25–30 g) SwissWeb-ster CD-1 mice by up to seven hourly intraperitoneal injections of 50�g/kg cerulein. Control animals received similar injections of physio-logic saline. Caspase inhibitors Q-VD-OPH (25 mg/kg) and Z-D-DCB(10 mg/kg), or vehicle (Me2SO), were applied in rats as a single intrave-nous injection 30 min before the start of cerulein treatment. XIAPinhibitor embelin (20 mg/kg), or vehicle (Me2SO), was applied in miceas one daily subcutaneous injection for 5 consecutive days; treatmentwith cerulein started 30 min after the last embelin injection. In theceruleinmodels, animals were sacrificed at 30min, 2, 4, and 7 h after thefirst injection. Arginine pancreatitis was induced in rats by two hourlyintraperitoneal injections of 2.5 g/kg L-arginine; controls received sim-ilar injections of saline. In this model, rats were sacrificed 24 h after thefirst injection. Animals were euthanized by CO2-induced asphyxiation,and the blood and pancreas were harvested for measurements.

Serum Amylase and Lipase Measurements—Serum amylase andlipase levels were measured in a Hitachi 707 analyzer (Antech Diagnos-tics, Irvine, CA).

Quantification of Apoptosis—Apoptosis was quantified on pancreatictissue sections stained withHoechst 33258 to visualize nuclear chroma-tin morphology or with TUNEL assay to measure DNA breaks, as wedescribed previously (3, 6, 9, 39). Tissue was fixed in 4% buffered form-aldehyde and embedded in paraffin, and 6 �m-thick sections wereadhered to glass slides. Sections were deparaffinized by washing inHemo-De and hydrated by transferring through graded ethanol. Thesections were stained with 8 �g/ml Hoechst 33258 and examined byfluorescence microscopy. Nuclei with condensed or fragmented chro-matin were considered apoptotic (12, 39). In TUNEL assay (6), tissuesections were stained for breaks in DNA using terminal deoxynucleoti-dyl transferase and fluorescein isothiocyanate-labeled dUTP accordingto the manufacturer’s protocol (Promega, Madison, WI). For these andother quantifications of histologicmeasurements, a total of at least 1000acinar cells was counted on pancreatic tissue sections from each animal.

Quantification of Necrosis—Quantification of necrosis was per-formed on pancreatic tissue sections stainedwithH&E. Cells with swol-len cytoplasm, loss of plasma membrane integrity, and leakage oforganelles into interstitium were considered necrotic.

Quantification of Inflammatory Infiltration—Quantification ofinflammatory infiltration was performed on pancreatic tissue sectionsstained with H&E.

Isolation of Pancreatic Acini—Isolation of pancreatic acini from ratsor mice was performed using a collagenase digestion procedure as wedescribed previously (3, 29, 39). Dispersed pancreatic acini were thenincubated at 37 °C in 199 medium (Invitrogen) in the presence orabsence of 100 nM CCK-8.

Caspase Activities—Caspase activities were measured using a fluoro-genic assay as we described previously (29, 40). Pancreatic tissue oracinar cell samples were homogenized in a lysis buffer containing 150mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.5% Igepal CA-630, and 0.5 mM

EDTA, centrifuged for 15 min at 16,000 � g, and the supernatants werecollected. Proteolytic reactions were carried out at 37 °C in a buffercontaining 25 mM HEPES (pH 7.5), 10% sucrose, 0.1% CHAPS, and 10mM dithiothreitol, using substrates specific for caspase-3 (Ac-DEVD-AMC), caspase-8 (Ac-IETD-AMC), or caspase-9 (Ac-LEHD-AMC).Cleavage of these substrates relieves 7-amino-4-methylcoumarin

(AMC), which emits fluorescent signal with excitation at 380 nm andemission at 440 nm. Fluorescence was calibrated using a standard curvefor AMC. The data are expressed as moles of AMC/mg of protein/min.

Preparation of Tissue and Cell Lysates for Western Blot Analysis—Portions of frozen tissue were homogenized on ice in radioimmuneprecipitation assay buffer supplemented with 1 mM phenylmethylsulfo-nyl fluoride and the protease inhibitor mixture containing pepstatin,leupeptin, chymostatin, antipain, and aprotinin (5 �g/ml of each),rotated for 20min at 4 °C, and centrifuged at 4 °C for 15min at 16,000�g. The supernatants were collected and stored at �80 °C. Dispersedpancreatic acini were washed twice with ice-cold phosphate-bufferedsaline, resuspended in radioimmune precipitation assay buffer, and pro-cessed as described above for tissue samples. Protein concentrationswere determined by the Bio-Rad protein assay (Bio-Rad Laboratories).

Preparation ofMembrane and Cytosolic Fractions—Pancreatic tissueor acinar cell samples were homogenized in a buffer containing 250mM

sucrose, 20 mM HEPES-KOH (pH 7.0), 10 mM KCl, 1 mM EGTA, 2 mM

MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, and the protease inhibitor mixture in a glass Dounce homog-enizer (80 strokes). Nuclei were removed by centrifugation at 1,000 � g

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for 10min at 4 °C. The supernatant was centrifuged for 1 h at 100,000�g, and both the pellet (mitochondria-enriched membrane fraction) andsupernatant (cytosolic fraction) were collected separately and used forWestern blotting.

Western Blot Analysis—was performed on total tissue and cell lysates,or on the membrane and cytosolic fractions, as we described previously(3, 29, 39, 40). Proteins were separated by SDS-PAGE and electro-phoretically transferred to nitrocellulose membranes. Nonspecificbinding was blocked by 1-h incubation of the membranes in 5% (w/v)nonfat dry milk in Tris-buffered saline (pH 7.5). The blots were thenincubated for 2 h or overnight with primary antibodies in the antibodybuffer containing 1% (w/v) nonfat dry milk in TTBS (0.05% (v/v) Tween20 in Tris-buffered saline), washed three times with TTBS, and finallyincubated for 1 h with a peroxidase-labeled secondary antibody in theantibody buffer. The blots were developed for visualization usingenhanced chemiluminescence (ECL) detection kit (Pierce). Band inten-sities in the immunoblots were quantified by densitometry.

Reduction/Alkylation—This was done according to a previous study(41), with minor modifications. Briefly, proteins in tissue lysate werereduced in 0.1 M Tris-HCl (pH 9.0) containing 8 M urea and 0.1 M

dithiothreitol, and incubated at 37 °C for 1 h. Alkylation of reducedproteins was achieved by incubation for 30 min with 0.3 M iodoacet-amide at room temperature in the dark. The samples were then sepa-rated by SDS-PAGE, transferred to nitrocellulose membranes, and sub-jected to Western blot analysis with antibody against cytochrome c.

Antibodies and Reagents—Antibodies against XIAP and p44/42mitogen-activate protein kinase (Erk1/2) were fromCell Signaling (Bev-erly, MA); caspase-3, caspase-8, and FLIPS/L, from Santa Cruz Biotech-nology (Santa Cruz, CA); cytochrome c, RIP, and Pyk2, from BD Bio-sciences (San Diego, CA); COX IV, from Molecular Probes (Eugene,OR); and caspase-9, from Stressgen (San Diego, CA). CCK-8 was fromAmerican Peptide (Sunnyvale, CA); cerulein was from Peninsula Labo-ratories (Belmont, CA). Caspase fluorogenic substrates Ac-IETD-AMC, Ac-DEVD-AMC, and Ac-LEHD-AMC, and the XIAP inhibitorembelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) were fromBiomol (Plymouth Meeting, PA). Caspase inhibitors Z-Asp-2,6-dichlo-robenzoyloxymethylketone (Z-D-DCB) and Q-Val -Asp(non-O-meth-ylated)-OPh (Q-VD-OPH) were from ALEXIS Biochemicals (SanDiego, CA) and Enzyme Systems Products (Livermore, CA), respec-tively. Other reagents were from Sigma.

RESULTS

In rat andmouse cerulein pancreatitis, wemeasured time-dependentchanges in the extent of apoptosis and necrosis in pancreas (Fig. 1). Inthe ratmodel, the extent of apoptosis was unchanged at 30min after thestart of cerulein treatment and increased �33-fold at 4 h and �16-fold

at 7 h. The decrease in apoptosis at 7 h could be due to an increasedclearance of apoptotic cells by inflammatory cells, the number of whichin pancreas increases with time. Compared with the rat model, therewas much less apoptosis in mouse cerulein pancreatitis, and theincrease in apoptosis was minimal. Necrosis time dependentlyincreased in both models of cerulein pancreatitis. In the rat model, theincrease in necrosis was only detected at 4 and 7 h, whereas in themousemodel necrosis was already evident at 30 min. At all time points, necro-sis in the rat was several times less than in the mouse model. Fig. 1illustrates the reciprocal pattern of cell-death responses in the rat versusmouse cerulein pancreatitis. For example, at 7 h the extent of apoptosisin the rat model was �8-fold higher and that of necrosis, �5-fold lowerthan in the mouse.

Apoptotic Signaling Pathways in Cerulein Pancreatitis

Caspases Are Greatly Activated in the Rat but Not Mouse CeruleinPancreatitis—Wemeasured the effects of pancreatitis on both the effec-tor caspase-3 and initiator caspases-9 and -8 (Fig. 2). In rat pancreas,caspase-3 antibody recognized only one procaspase-3 band (�34 kDa),whereas in themouse pancreas this antibody detected two bands of�34and �37 kDa (Fig. 2A, left panel), which may reflect different phospho-rylation states of procaspase-3 (18, 42). In the course of cerulein pan-creatitis in the rat, caspase-3 underwent time-dependent processingmanifest by a decrease in the 34-kDa proform and the appearance of thecleaved �17-kDa product, which represents the active form ofcaspase-3 (29, 43). Caspase-3 processing was first evident at 30 min. Incontrast, we detected no processing of caspase-3 in mouse ceruleinpancreatitis (Fig. 2A, right panel).In parallel with the observed processing of procaspase-3, pancreatic

caspase-3 activity (measured with a fluorogenic assay) greatly increasedin rat cerulein pancreatitis compared with untreated or control, saline-injected animals (Fig. 2B). This increase leveled off at�15-fold between2 and 4 h of cerulein treatment. No increase in caspase-3 activity wasdetected in the mouse model (Fig. 2B).Caspase-9 processing (29, 43, 44) was detected in rat cerulein pancre-

atitis by time-dependent accumulation of the�40-kDa cleaved product(already evident at 30 min) and concomitant decrease in the �50-kDaproform (Fig. 2A, left panel). In contrast, no processing of pancreaticcaspase-9 was detected in the mouse model. Measurements ofcaspase-9 activity paralleled those of its processing: caspase-9 activityincreased up to �10-fold in rat cerulein pancreatitis whereas no activa-tion of caspase-9 was detected in the mouse model (Fig. 2B).In both rat and mouse pancreas, caspase-8 antibody recognized two

bands likely corresponding to the 2main procaspase-8 isoforms (45). Inrat cerulein pancreatitis caspase-8 processingwas alreadymanifest at 30

FIGURE 1. The extent of apoptosis (left) andnecrosis (right) in the rat and mouse models ofcerulein pancreatitis. Pancreatitis was induced inrats and mice by up to seven hourly intraperito-neal injections of cerulein (CR, 50 �g/kg); controlanimals received similar injections of saline. Ani-mals were sacrificed at indicated times after thefirst cerulein injection. Induction of pancreatitiswas confirmed by elevated levels of serum amy-lase and lipase, and by changes in pancreatic his-tology on tissue sections stained with H&E (as illus-trated in subsequent figures). Apoptosis wasmeasured on pancreatic tissue sections by TUNELassay; necrosis was measured on tissue sectionsstained with H&E. Values are means � S.E. from atleast three animals per group.

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min, resulting in accumulation of the �50-kDa cleaved product (theintermediate form). We did not detect the completely processedcaspase-8 (the p16 product (45)). It has been shown (46) that caspase-8can be activated not only through its cleavage to the p16 form but alsothrough oligomerization of the intermediate products. Despite incom-plete processing, the activity of caspase-8 greatly (up to 20-fold)increased in rat cerulein pancreatitis. As with the other caspases, wedetected neither processing nor activation of caspase-8 in mouse cer-ulein pancreatitis (Fig. 2).The results in Fig. 2 show that both initiator (caspases-8 and -9) and

effector (caspase-3) caspases are greatly activated in rat cerulein pan-creatitis. In contrast, neither caspase processing nor their increasedactivity was detected in the mouse model.Activation of both initiator and effector caspases occurred early in rat

cerulein pancreatitis, with processing and increased activity being evi-

dent at 30 min of cerulein treatment. The kinetics of activation wasdifferent for the initiator and effector caspases: the activities ofcaspase-9 and -8 increased almost linearly during the whole period ofobservation, whereas the increase in caspase-3 activity leveled off at 4 h.To determine whether the differences in caspase activation found in

the rat and mouse models of cerulein pancreatitis occur at the level ofacinar cells, we compared the effects of CCK-8 on caspase activities inacinar cells isolated from rat and mouse pancreas. As we showed previ-ously (29), in rat pancreatic acinar cells supramaximal CCK-8 caused arapid and pronounced activation of all 3 caspases (Fig. 3). By contrast, inmouse pancreatic acinar cells supramaximalCCK-8 had no effect on theactivities of caspases-3, -9, and -8 (Fig. 3). These results indicate that the“caspase block” in mouse cerulein pancreatitis occurs in acinar cells.Of note, the procedure of acinar cell isolation from the pancreas

induces various stresses in isolated acini (47). In particular, basal caspase

FIGURE 2. Caspases-3, -9, and -8 are activated in the rat but not mouse model of cerulein pancreatitis. Pancreatitis was induced in rats and mice as described in Fig. 1 legend.Animals were sacrificed at indicated times after the first cerulein (CR) injection. A, processing of caspases-3, -9, and -8 in pancreatic tissue was measured by Western blot analysis(cleaved products are indicated by arrows). Blots were re-probed for ERK1/2 to confirm equal protein loading. The experiments were repeated with similar results on at least threeanimals in each group. Numbers to the left in this and other figures are size markers in kilodaltons. B, caspase activities were measured in pancreatic tissue homogenates by afluorogenic assay using substrates specific for caspase-3 (DEVD-AMC), caspase-9 (LEHD-AMC), and caspase-8 (IETD-AMC). For each caspase, the activity was normalized on that incontrol, saline-treated animals. Caspase activities in control animals did not change with time. Values are means � S.E. from at least three animals per group.

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activities are greater in isolated rat acinar cells than in pancreatic tissuefrom untreated or saline-treated rats (29).

Cytochrome c Release Occurs in Both Models of Cerulein Pancreatitis—One possible reason for the observed differences in caspase activationbetween the rat and mouse models could be a blockade of mitochondrialcytochrome c release upstreamof caspase-9 inmouse cerulein pancreatitis.We found, however, that pancreatic cytochrome c release occurred in bothmodels (Fig. 4A), as was manifested by time-dependent decrease in themembrane 14-kDa cytochrome c on Western blot performed on mem-brane fractions from pancreatic tissue. Concomitantly, cytochrome cimmunoreactivity increased in the cytosolic fractions from pancreatic tis-sue in both models (Fig. 4A). Surprisingly, in the rat model we observed atime-dependent decrease in the cytosolic 14-kDa cytochrome c band,which was accompanied by accumulation of a �30-kDa band. This bandwas detected by both monoclonal (BD Bioscience, Fig. 4A) and polyclonal(Santa Cruz Biotechnology, not shown) cytochrome c antibodies; further,preincubation of the polyclonal antibody with blocking peptide preventedrecognition of both 14- and 30-kDa bands, indicating that both bands cor-respond to cytochrome c (data not shown).

Unlike the rat model, in mouse cerulein pancreatitis the intensity ofthe cytosolic 14-kDa cytochrome c band only increased with time (Fig.4A, right panel). The �30-kDa band was also present in the cytosoliccytochrome c immunoblots frommouse pancreatitis tissue; its intensityincreased much more slowly than in the rat model and was only prom-inent at 7 h (Fig. 4A).The nature of the 30-kDa form of cytosolic cytochrome c remains to

be determined.One possible explanation is that this form represents thecytochrome c dimer. It has been reported that cytochrome c could formoligomers and that its oligomerization is potentiated by the neuronalprotein �-synuclein (48). �-Synuclein derived from nerves could bepresent in pancreatic tissue homogenates and promote cytochrome coligomerization. The more cytochrome c released from the mitochon-dria during the course of pancreatitis, the greater the extent of its oli-gomerization in the cytosol and thus the intensity of the 30-kDa band.To demonstrate that the 30-kDa cytochrome c bandwas associatedwitholigomerization, protein samples were subjected to a reduction/alkyla-tion procedure (41) prior to SDS-PAGE. The reduction/alkylation pro-cedure abrogated the 30-kDa band (Fig. 4D), indicating that this bandresults from complex formation involving disulfide linkages.The presence of the 30-kDa band complicated the comparison of

cytosolic cytochrome c levels between the rat and mouse models. Weestimated the changes in cytosolic cytochrome c levels bymeasuring thesum of the intensities of both 14- and 30-kDa bands. The densitometricquantification (Fig. 4B) showed that the total cytochrome c accumula-

tion in the cytosol was more rapid in the rat model, reaching the maxi-mal level by 2–4 h of cerulein treatment. However, by 7 h the increasesin total cytosolic cytochrome cwere of similar magnitude in both mod-els of cerulein pancreatitis.We further measured cytochrome c release in isolated pancreatic

acinar cells. In rat acinar cells, as we showed previously (29), supramaxi-mal CCK-8 induced a decrease in membrane cytochrome c with a con-comitant increase in the cytosolic 14-kDa cytochrome c (Fig. 4C). Asimilar decrease in the membrane cytochrome c with concomitantincrease in the cytosolic cytochrome c was observed in isolated mousepancreatic acinar cells treated with CCK-8 (Fig. 4C). Of note, we onlydetected the 14-kDa cytosolic cytochrome c, but not the 30-kDa band,in both rat and mouse acinar cells. Whether the absence of the 30-kDacytosolic cytochrome c band in isolated pancreatic acinar cells is due tothe absence of the nerve-derived �-synuclein (or other factors facilitat-ing cytochrome c oligomerization) remains to be determined.The results in Figs. 2–4 show that cytochrome c release from mito-

chondria occurs in bothmodels of cerulein pancreatitis; however, in themousemodel it does not translate into caspase activation. This indicatesthat the caspase block we found in the mouse model is not due to ablockade of cytochrome c release. Therefore, we next investigated theeffect of pancreatitis on endogenous protein inhibitors of caspases.

XIAP Mediates the Caspase Block in Mouse Cerulein Pancreatitis—We first evaluated the effect of pancreatitis on c-FLIP, a cytosolic pro-tein with homology to caspase-8 that acts as a dominant-negative inhib-itor of caspase-8 activation (49). There are two splice isoforms of c-FLIP,one of which, the c-FLIPS (short isoform), completely blocks caspase-8processing, whereas the other, c-FLIPL (long isoform), allows partialprocessing of caspase-8 (49–51). We found that both c-FLIPS andc-FLIPL were markedly up-regulated in both rat and mouse ceruleinpancreatitis (Fig. 5A). The up-regulation of c-FLIP isoforms occurredfaster in the rat model, but the magnitude of the response was compa-rable in both models. Thus, changes in c-FLIP cannot account for thedifferences in caspase activation between rat and mouse models of cer-ulein pancreatitis (if anything, c-FLIP up-regulation was greater in therat).We next asked whether the blockade of the mitochondria-driven

caspase-9 (and subsequently, caspase-3) activation in the mouse modeloccurs downstream of cytochrome c release, at the level of IAPs. West-ern blot analysis showed the presence of XIAP, a keymember of the IAPfamily (22–24), in both rat and mouse pancreas (Fig. 5). However, theeffect of cerulein pancreatitis on XIAPwas drastically different betweenthe rat and mouse models. XIAP was rapidly and fully degraded in therat model but its level remained unchanged in mouse cerulein pancre-

FIGURE 3. CCK-8 activates caspases in the rat but not mouse pancreatic acinar cells. Pancreatic acinar cells isolated from rat or mouse were incubated for indicated times with 100nM CCK-8. Caspase activities were measured by a fluorogenic assay using substrates specific for caspase-3 (DEVD-AMC), caspase-9 (LEHD-AMC), and caspase-8 (IETD-AMC). Values aremeans � S.E. (n � 3).

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atitis (Fig. 5A). These results suggest that XIAP degradation is onemechanism allowing for caspase activation in rat cerulein pancreatitis,whereas sustained high levels of XIAP in themousemodel block caspaseactivation downstream of cytochrome c.To test that the observed differences in XIAP degradation are not

species-specific, we measured XIAP changes in another rat model ofacute pancreatitis that induced by L-arginine. Arginine pancreatitis is asevere disease characterized by much greater (�20%) necrosis than therat ceruleinmodel (52, 53).Western blot analysis showed no increase inXIAP degradation in rat arginine pancreatitis (Fig. 5B), demonstratingthat the differences in XIAP behavior we observed between rat andmouse cerulein pancreatitis are not species-specific.To evaluate the role of XIAP in the regulation of caspase activities in

pancreatitis we applied the pharmacologic XIAP inhibitor embelin (54).Embelin is a molecule derived from the Japanese Ardisia herb, whichwas shown to have anti-tumor activities (55, 56) and was also used as acontraceptive (57). Recently, it has been shown (54) that embelin bindsto the BIR3 domain in XIAP (the binding site for caspase-9), preventingXIAP interaction with caspase-9 and thus allowing for caspase-9 acti-vation. Embelin activated caspase-9 and induced apoptosis in prostatecancer cells that display high levels of XIAP but had minimal effect onnormal prostate cells with low levels of XIAP (54).In mouse cerulein pancreatitis, treatment with embelin markedly

increased the activities of caspase-9 and -3 and (to a lesser extent)caspase-8 (Fig. 5C). The stimulation of caspase-9 with embelin wasmore reproducible than stimulation of caspases-3 and -8. Caspase-9activity was increased in all 9 embelin-treated mice with cerulein pan-creatitis, whereas caspases-3 and -8 were significantly activated in six

out of these ninemice. This can be explained by the fact that embelin, bypreventing XIAP interactionwith BIR3 domain, directly stimulates onlycaspase-9 (22–24). The increased activity of caspases-3 and -8 (whichcan be activated by caspase-3 (58)) is likely the result of caspase-9 stim-ulation. Embelin did not affect caspase activities in control, saline-treated mice (Fig. 5C). The results in Fig. 5 indicate that XIAP plays animportant role in the regulation of caspases in cerulein pancreatitis.

Caspases Mediate Apoptosis and Protect from Necrosis inCerulein Pancreatitis

To determine the role of caspases in cell-death responses of pancre-atitis, we first measured the effects of caspase inhibition on apoptosisand necrosis in the rat model of cerulein pancreatitis in which caspaseswere greatly activated (Fig. 2). For this purposewe used broad-spectrumpeptide caspase inhibitors Z-D-DCB (59) and Q-VD-OPH (28, 60). Wedid not apply another broad spectrum caspase inhibitor, the commonlyused Z-VAD-fmk (61), because in experiments on isolated acinar cellswe found that Z-VAD-fmk, in addition to caspases, also inhibitedcathepsin B activity (data not shown). By contrast, neither Z-D-DCBnor Q-VD-OPH affected cathepsin B in acinar cells (not shown).Q-VD-OPH completely, and Z-D-DCB partially, inhibited activation of

caspase-3 (Fig. 6A) and caspases-9 and -8 (data not shown) in rat ceruleinpancreatitis. Increasing Z-D-DCB concentration did not further increasethe extent of caspase inhibition (not shown). Both inhibitors decreasedpancreatic apoptosis in rat cerulein pancreatitis as measured by bothHoechst 33258 and TUNEL staining (Fig. 6B). Importantly, althoughQ-VD-OPH completely prevented caspase activation in rat cerulein pan-creatitis, it only inhibited apoptosis by�50% (Fig. 6,A andB). These results

FIGURE 4. Cytochrome c release occurs in bothrat and mouse cerulein pancreatitis. A, B, and D,pancreatitis was induced in rats and mice asdescribed in the Fig. 1 legend. Animals were sacri-ficed at indicated times after the first cerulein (CR)injection. C, isolated rat and mouse pancreatic aci-nar cells were incubated with or without 100 nM

CCK-8 for 3 h. Cytochrome c (cyt c) was measuredby Western blot analysis in membrane and cytoso-lic fractions from pancreatic tissue or pancreaticacinar cells. Blots were re-probed with an antibodyagainst complex IV cytochrome c oxidase (COX IV),an integral mitochondrial inner membrane pro-tein, to assess the quality of separating mitochon-dria-enriched membrane fractions from cytosolicfractions. Blots from cytosolic fractions were fur-ther re-probed with an antibody against Pyk-2 ortubulin to confirm equal protein loading. Theexperiments were repeated at least three timeswith similar results. B, combined intensity of thecytochrome c bands (14 kDa plus 30 kDa) on theimmunoblots of cytosolic fractions from pancre-atic tissue of cerulein- or saline-treated animalswas quantified by densitometry and normalized tothat of Pyk2 band (i.e. loading control) in the samesample. The mean ratio of cytochrome c/Pyk2intensities in cerulein-treated animals at a giventime point was further normalized to that in saline-treated animals at the same time point. Values aremeans � S.E. (n � 4). D, pancreatic tissue from a ratwith cerulein pancreatitis was obtained at 7 h afterthe first cerulein injection. Proteins in the cytosolicfraction from this tissue were subjected or not toreduction/alkylation as described under “Experi-mental Procedures,” separated by SDS-PAGE, andanalyzed by Western blot with the antibodyagainst cytochrome c. The experiment wasrepeated with similar results.

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indicate an equally important contribution of caspase-dependent and -in-dependent pathways to apoptosis in this model of pancreatitis.Caspase inhibition increasednecrosis in rat ceruleinpancreatitis�3-fold

(Figs. 6C and 7A). It also worsened other parameters of pancreatitis,namely, the increases in serum amylase and lipase (Fig. 7, B and C) andinflammatory cell infiltration in the pancreas (Fig. 7D).To further elucidate the role of caspases in the regulation of cell-death

responses, we measured the effect of the XIAP inhibitor embelin on

apoptosis and necrosis in the mouse model of cerulein pancreatitis, inwhich caspases are silent. As shown in Fig. 5, in this model embelininduced activation of caspases-9, -3, and -8. Embelin caused a �3-foldincrease in apoptosis (Fig. 8A); furthermore, caspase activation byembelin resulted in a significant decrease in necrosis and normalizationof pancreatic histology in mouse cerulein pancreatitis (Fig. 8, B and C).The results in Figs. 6–8 show that caspases not onlymediate apopto-

sis but also protect from necrosis in cerulein pancreatitis. Caspase inhi-

FIGURE 5. XIAP is degraded in rat but not mousecerulein pancreatitis, and XIAP inhibitor trig-gers caspase activation in the mouse model. Aand C, pancreatitis was induced in rats and mice asdescribed in Fig. 1 legend. Animals were sacrificedat indicated times after the first cerulein (CR) injec-tion. B, pancreatitis was induced in rats by twohourly intraperitoneal injections of L-arginine(L-Arg, 2.5 g/kg); controls received similar injec-tions of saline. Rats were sacrificed 24 h after thefirst injection. In A and B, the levels of XIAP andc-FLIP isoforms, c-FLIPS and c-FLIPL, were meas-ured in pancreatic tissue by Western blot analysis.Blots were re-probed for ERK1/2 to confirm equalprotein loading. The experiments were repeatedwith similar results on at least three animals ineach group. In C, mice received 5 daily subcutane-ous injections of the XIAP inhibitor embelin (20mg/kg) or vehicle. 30 min after the last embelininjection, pancreatitis was induced by sevenhourly intraperitoneal injections of cerulein (CR);control mice received saline instead of cerulein.Mice were sacrificed at 7 h after the first ceruleininjection. Caspase activities were measured inpancreatic tissue homogenates by a fluorogenicassay using substrates specific for caspase-9(LEHD-AMC), caspase-3 (DEVD-AMC), andcaspase-8 (IETD-AMC). For each caspase, the activ-ity was normalized on that in control mice (i.e.mice that received vehicle instead of embelin andsaline instead of cerulein). Values are means � S.E.(n � 9). *, p � 0.05 compared with cerulein alone.

FIGURE 6. Caspase inhibitors decrease apoptosis and stimulate necrosis in rat cerulein pancreatitis. Rats received intravenous injection of caspase inhibitors Z-D-DCB (10mg/kg) or Q-VD-OPH (25 mg/kg), or vehicle, followed in 30 min by four hourly intraperitoneal injections of cerulein (CR, 50 �g/kg) or saline. Rats were sacrificed 4 h after the firstcerulein injection. A, caspase-3 activity was measured in pancreatic tissue homogenates by a fluorogenic assay using specific DEVD-AMC substrate. B, apoptosis was measured onpancreatic tissue sections stained with Hoechst 33258 or TUNEL. C, necrosis was measured on pancreatic tissue sections stained with H&E. Values are means � S.E. (n � 4). *, p � 0.05compared with cerulein alone.

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bition stimulated necrosis, whereas their activation inhibited necrosis.Fig. 9 summarizes the data on the effects of modulating caspase activityon necrosis/apoptosis ratio in the two models of cerulein pancreatitis.At the indicated time points, the necrosis/apoptosis ratio is �120-foldgreater in themouse than in the ratmodel. Caspase inhibition increasedthis ratio in rat cerulein pancreatitis (Fig. 6); conversely, caspase induc-tion with embelin decreased the necrosis/apoptosis ratio in mouse cer-ulein pancreatitis (Fig. 8) by stimulating apoptosis and inhibiting necro-

sis. Thus, manipulating caspase activities caused necrosis/apoptosisswitch andmade the patterns of cell-death responses in the twomodelsmore similar (Fig. 9).

RIP Degradation by Caspases Correlates with Low Necrosis inExperimental Pancreatitis

Compared with apoptosis, the signaling pathwaysmediating necrosisaremuch less understood (15–17). RIP kinase has recently emerged as a

FIGURE 7. Caspase inhibition worsens rat cerulein pancreatitis. Rats received intravenous injections of caspase inhibitors Z-D-DCB (10 mg/kg) or Q-VD-OPH (25 mg/kg), or vehicle,followed in 30 min by four hourly intraperitoneal injections of cerulein (CR, 50 �g/kg) or saline. Rats were sacrificed 4 h after the first cerulein injection. A, pancreatic tissue from ratswith cerulein pancreatitis, which did or did not receive the caspase inhibitor Q-VD-OPH, was stained with H&E and analyzed by light microscopy. Areas of necrosis are indicated byarrows. B and C, serum amylase and lipase were measured by enzymatic assay. D, inflammatory cell infiltration was measured on pancreatic tissue sections stained with H&E. Valuesare means � S.E. (n � 4). *, p � 0.05 compared with cerulein alone.

FIGURE 8. XIAP inhibitor embelin stimulates apoptosis and inhibits necrosis in mouse cerulein pancreatitis. Mice received 5 daily subcutaneous injections of the XIAP inhibitorembelin (20 mg/kg) or vehicle. 30 min after the last embelin injection pancreatitis was induced by seven intraperitoneal injections of cerulein (CR); control mice received saline insteadof cerulein. Animals were sacrificed at 7 h after the first cerulein injection. A and B, apoptosis and necrosis were measured on pancreatic tissue sections stained with Hoechst 33258and H&E, respectively. Values are means � S.E. (n � 4). *, p � 0.05 compared with cerulein alone. C, pancreatic tissue sections were stained with H&E and analyzed by light microscopy.

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key mediator of necrosis-like programmed cell death (15–17, 32–34).We found that the effect of cerulein pancreatitis on RIP was drasticallydifferent between the rat and mouse models (Fig. 10A). In rat ceruleinpancreatitis, RIP underwent rapid and full cleavage to a �47-kDa prod-uct. In contrast, there was very little cleavage of RIP in themousemodel(Fig. 10A). To test that RIP cleavage is not species-specific, wemeasuredthat RIP was not cleaved in rat arginine pancreatitis (Fig. 10B); thus, RIPbehaves differently in the two rat models of acute pancreatitis.RIP can be cleaved (and thus inactivated) by caspases-8 and -3 (62–

64). To determine whether RIP cleavage observed in cerulein pancrea-titis is mediated by caspases, we measured the effects of caspase inhib-itors and embelin on RIP. Caspase inhibition with Q-VD-OPHmarkedly decreased RIP cleavage in rat cerulein pancreatitis (Fig. 10C).Conversely, stimulation of caspase activity by embelin induced RIPcleavage in mouse cerulein pancreatitis (Fig. 10D). These results indi-cate that caspases mediate RIP degradation in cerulein pancreatitis.Thus, RIP cleavage directly correlates with caspase activation and

apoptosis, and inversely, with the extent of necrosis in the experimentalmodels of acute pancreatitis we applied. Such correlations suggest thatRIP cleavage (i.e. inactivation) could be one mechanism through whichcaspases inhibit necrosis.

DISCUSSION

Inflammation and parenchymal cell death are hallmarks of pancrea-titis (1, 2). In the past decade, significant progress has been achieved inunderstanding the mechanisms of the inflammatory response of pan-creatitis (2–4, 9, 39, 65, 66). In contrast, the mechanisms of cell-deathresponses of pancreatitis remain largely unexplored. To determine themechanisms mediating necrosis and apoptosis in pancreatitis, in thepresent study we analyzed death-signaling pathways in rat and mousecerulein pancreatitis, the most commonly used and well characterizedmodels of acute pancreatitis (37, 38). Cerulein, an analog of CCK-8,interacts with CCK receptors on the pancreatic acinar cell (38, 67). Boththe rat and mouse models display key responses of acute pancreatitissuch as the dysregulation of digestive enzymes’ secretion (e.g. increasedlevels of serum amylase and lipase); premature, intrapancreatic trypsin-ogen activation; activation of transcription factor NF-�B resulting inup-regulation of pro-inflammatory cytokines and chemokines; inflam-matory cell infiltration; and parenchymal cell death (2, 3, 5, 9, 38, 66).However, the patterns of death responses differ in these two models ofacute pancreatitis induced by the same treatment. Rat cerulein pancre-

atitis is a mild disease characterized by low necrosis and relatively highapoptosis, whereas the mouse cerulein model is a more severe diseasewith high necrosis and very little apoptosis. Indeed, our data show thatthe necrosis/apoptosis ratio in the mouse model is �120-fold higherthan in the rat model. (It is worth noting that actual rates of apoptosiscould be higher, because the remnants of cells dying through apoptosis(i.e. apoptotic bodies) are rapidly phagocytosed (68).) Indeed, weshowed (9) that depletion of the inflammatory cells increased apoptosisin the rat cerulein pancreatitis up to 17%. By contrast, phagocytosis ofnecrotic cells’ remnants is much less efficient (69).We utilized the differences in cell-death responses between the two

rodent models of cerulein pancreatitis to elucidate the mechanismsmediating necrosis and apoptosis in pancreatitis and to explore thepossibility of manipulating the death responses for therapeutic pur-poses. We found that both the effector caspase-3 and initiatorcaspases-9 and -8 are rapidly and greatly activated in rat cerulein pan-creatitis. By contrast, there was no caspase activation in the mousemodel. Similarly, in vitro supramaximal CCK-8 induced caspase activa-tion in acinar cells isolated from rat but not mouse pancreas. Thus, onedifference in cell-death responses between the rat andmouse models ofcerulein pancreatitis is the caspase block in mouse pancreatic acinarcells.Cytochrome c release occurred in both models of cerulein pancrea-

titis. This indicates thatmitochondrial damage is induced in experimen-tal acute pancreatitis, suggesting a role for themitochondrial pathway in

FIGURE 9. Modulation of necrosis/apoptosis ratio in cerulein pancreatitis bycaspases. Pancreatic necrosis/apoptosis ratios were calculated for cerulein pancreatitisin rats (rat CR), mice (mouse CR), rats treated with the caspase inhibitor Q-VD-OPH, andmice treated with the XIAP inhibitor embelin, using the data from Figs. 6 and 8. In rats, thedata are for the 4-h time point; in mice, data represent the 7-h time point. The necrosis/apoptosis ratio in rat cerulein pancreatitis without inhibitors was considered as 1.0.

FIGURE 10. RIP is degraded in rat but not mouse cerulein pancreatitis. RIP cleavage ismediated by caspases. A, C, and D, pancreatitis was induced in rats and mice as describedin Fig. 1 legend. Animals were sacrificed at indicated times after the first cerulein (CR)injection. B, pancreatitis was induced in rats by two hourly intraperitoneal injections ofL-arginine (L-Arg, 2.5 g/kg); controls received similar injections of saline. Rats were sacri-ficed 24 h after the first injection. In C, rats received intravenous injections of the caspaseinhibitor Q-VD-OPH (25 mg/kg) or vehicle, followed in 30 min by four hourly intraperito-neal injections of cerulein (50 �g/kg) or saline. Rats were sacrificed 4 h after the firstcerulein injection. In D, mice received five daily subcutaneous injections of the XIAPinhibitor embelin (20 mg/kg) or vehicle. 30 min after the last embelin injection pancre-atitis was induced by seven hourly intraperitoneal injections of cerulein (CR); controlmice received saline instead of cerulein. Mice were sacrificed at 7 h after the first ceruleininjection. The levels of RIP were measured in pancreatic tissue by Western blot analysis.Blots were re-probed for ERK1/2 to confirm equal protein loading. The experiments wererepeated with similar results on at least three animals in each group.

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parenchymal cell death. Because cytochrome c release was comparablebetween the rat andmousemodels (and similarly, supramaximalCCK-8induced cytochrome c release in both rat and mouse pancreatic acinarcells), the caspase block in mouse cerulein pancreatitis is not due to ablockade of cytochrome c release. We, therefore, evaluated the roles oftwo key endogenous protein caspase inhibitors, c-FLIP and XIAP (22–24, 49–51, 70), in the regulation of caspase activation in ceruleinpancreatitis.Both isoforms of c-FLIP, the endogenous caspase-8 inhibitor (49–

51), were similarly up-regulated in rat and mouse cerulein pancreatitis,suggesting a role for c-FLIP in the regulation of death responses in acutepancreatitis. However, because c-FLIP up-regulationwas comparable inboth models, it cannot account for the differences in caspase-8 activa-tion between rat and mouse cerulein pancreatitis.In contrast with c-FLIP, the effect of cerulein pancreatitis on XIAP

differed drastically between the rat and mouse models. XIAP wasdegraded rapidly and fully in rat cerulein pancreatitis but remainedintact in the mouse model. Furthermore, XIAP inhibitor embelin (54)removed the caspase block observed in mouse cerulein pancreatitis.Embelin is a compound from the Japanese Ardisia herb (Herba ardisiaejaponicae) used as a key ingredient in several traditional Chinese anti-cancer recipes as well as a contraceptive (54–57). Embelin has recentlybeen shown (54) to prevent the inhibition of caspase-9 (but notcaspase-3) by XIAP and is currently in phase I of clinical trials for cancertreatment (71).Of interest, although embelin only prevents XIAP binding to and

inhibition of caspase-9 (54), it induced activation of all three caspasesmeasured, i.e. caspases-3, -9, and -8. Caspase-3 is a downstream targetof caspase-9 (21). In turn, activation of caspase-8 with embelin could bethrough the caspase-33 caspase-8 amplification pathway (58), provid-ing evidence for this pathway in pancreatitis.Our results indicate that XIAP is a key caspase regulator in pancrea-

titis. In particular, high XIAP levels maintained in mouse cerulein pan-creatitis block caspase activation in this model, whereas XIAP degrada-tion renders caspase activation in rat cerulein pancreatitis. Mechanismof XIAP degradation in pancreatitis is yet to be determined. XIAP is anubiquitin ligase (E3 ligase) that can promote its own degradation (72);XIAP degradation can also be mediated by Smac/DIABLO, anotherubiquitin ligase (25). XIAP knock-out mice have been generated previ-ously (73); however, XIAP deficiency had no effect on caspase activationdue to a compensatory up-regulation of other IAPs such as c-IAP1 and-2 (73).Of note, caspase activation induced by embelin (e.g. 3-fold for

caspase-9) wasmuch less than that observed in rat cerulein pancreatitis.This suggests the involvement of other mechanisms, in addition toXIAP, in mediating the caspase block in mouse cerulein pancreatitis.To determine the role of caspases in the regulation of cell-death

responses in cerulein pancreatitis we used two approaches, namely,caspase inhibition in the rat model and caspase induction (with embe-lin) in the mouse model. Specific caspase inhibitors decreased apopto-sis; conversely, caspase inductionwith embelinmarkedly increased apo-ptosis in the mouse model. These results indicate that caspases mediateapoptosis in cerulein pancreatitis. This conclusion is not obvious,because recent findings established that in many situations apoptosisdevelops without caspase activation (14, 16, 18, 74, 75). Indeed, our dataindicate that caspase-dependent and -independent pathways playequally important roles in apoptosis in cerulein pancreatitis. Caspaseactivation in the rat model was completely prevented by Q-VD-OPH,but apoptosis was only inhibited by 50%. Caspase-independent apopto-sis in pancreatitis could be mediated by mitochondria-derived pro-ap-optotic factors such as EndoG, Omi, or AIF (14, 16, 18).We found that caspases not only mediate apoptosis but also protect

from necrosis in cerulein pancreatitis. Caspase inhibition markedlystimulated necrosis in the ratmodel; conversely, caspase induction withembelin decreased necrosis in the mouse model. Similarly, our in vitrodata (29) showed that caspase inhibition stimulated necrosis in isolatedrat pancreatic acinar cells. These results provide an explanation for theobservations of an inverse correlation between necrosis and apoptosisin experimental models of acute pancreatitis (Fig. 1 and Refs. 6–12).Further, caspase inhibition in the ratmodel not only increased necro-

sis but also worsened other parameters of cerulein pancreatitis, i.e.serum levels of amylase and lipase, inflammatory infiltration in the pan-creas, and histological changes. Conversely, embelin treatmentimproved pancreatic histology.Mechanismsmediating necrosis are, in general,much less established

than those for apoptosis. Recent findings indicate that there are twodistinct types of necrosis (15–17). Accidental necrosis is triggered bysevere cellular stress that is often characterized by depletion of ATP. Bycontrast, the so-called programmed necrosis, or necrosis-like pro-grammed cell death, is mediated by specific signals. Our data show thatnecrosis inmouse cerulein pancreatitis develops rapidly: by 30min afterstart of cerulein treatment, the extent of necrosis in thismodel increasedfrom zero to � 5% (i.e. to the level observed in fully developed ratcerulein pancreatitis). At this time point, there was no ATP decrease(data not shown, and Refs. 76 and 77) and no inflammatory infiltrationin the pancreas (5, 38, 78). These data provide evidence for the involve-ment of programmed necrosis in cerulein pancreatitis.Our results further suggest the involvement of RIP, a mediator of

programmed necrosis (15–17, 32–34), in the regulation of cell-deathresponses of pancreatitis. We found that the behavior of RIP drasticallydiffered between the two models of cerulein pancreatitis. RIP under-went rapid and complete cleavage in the rat model but remained intactin the mouse model. RIP degradation in rat cerulein pancreatitis wasinhibited by caspase inhibitors, whereas caspase inductionwith embelin

FIGURE 11. Death-signaling pathways in cer-ulein pancreatitis. Initial injury triggers cyto-chrome c release and caspase activation in cer-ulein pancreatitis. In the mouse model, caspaseactivation downstream of cytochrome c is inhib-ited by maintaining high levels of XIAP; in contrast,XIAP degradation allows for caspase activation inthe rat model. Strong caspase activation results inrelatively high extent of apoptosis in the ratmodel, whereas caspase blockade results in lowapoptosis and high necrosis in the mouse model.One mechanism through which caspases inhibitnecrosis in pancreatitis could be through cleavage(i.e. inactivation) of RIP; this mechanism does notoperate in the mouse model because of thecaspase block.

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triggered RIP cleavage in themousemodel. These data indicate that RIPis regulated by caspases in cerulein pancreatitis.The level of intact RIP directly correlated with the extent of necrosis

in cerulein pancreatitis. Maximal RIP cleavage was observed in rat cer-ulein pancreatitis that has little necrosis, whereas there was no RIPdegradation in the mouse cerulein and rat arginine pancreatitis, themodels characterized by high necrosis.The results of the present study show that acinar cell death in pan-

creatitis is regulated by a number of signaling mechanisms the balanceofwhich determines the pattern of parenchymal cell death, i.e. apoptosisversus necrosis. These mechanisms, and their different involvement inthe rat versusmousemodels of cerulein pancreatitis, are depicted in Fig.11. Caspase activation is a major factor in this balance, switching thecell-death response toward apoptosis and away from necrosis. Ourresults indicate XIAP as a key negative regulator of caspases in pancre-atitis.Maintaining intactXIAP levels (and, possibly, levels of other IAPs)is one mechanism that prevents caspase activation, thus promotingnecrosis and aggravating pancreatitis. Further, caspase activationresulting from XIAP inhibition not only stimulated apoptosis in mousecerulein pancreatitis but also triggered the cleavage (i.e. inactivation) ofRIP, a key mediator of necrosis-like programmed cell death (15–17).This could be one mechanism through which caspases inhibit necroticdeath in pancreatitis.The findings in the present study raise a number of further questions

about the mechanisms of death responses in pancreatitis. In particular,do the signaling mechanisms that we found to regulate cell-deathresponses of cerulein pancreatitis (i.e. caspases, XIAP, and RIP) alsooperate in other experimental models of acute pancreatitis, such as theCDE (choline-deficient, ethionine-supplemented) diet or duct ligationmodels (37), as well as in human disease? What pathways mediatecaspase-independent apoptosis in pancreatitis?What is themechanismof XIAP degradation? Is there a role for other IAPs in the regulation ofcell death in pancreatitis? What are the downstream targets of RIP inpancreatitis?In sum, our results demonstrate key roles for caspases, XIAP, and RIP

in the regulation of cell-death responses of pancreatitis. They show howmanipulating death-signaling mechanisms changes the necrosis/apo-ptosis pattern in experimental pancreatitis. These signals representpotential therapeutic targets in the treatment of pancreatitis, especiallyto prevent or attenuate necrosing pancreatitis.

Acknowledgments—We thankDr. Vay LiangW.Go for helpful discussion, andMohammad Shahsahebi for help in preparing the figures.

REFERENCES1. Raraty, M. G., Connor, S., Criddle, D. N., Sutton, R., and Neoptolemos, J. P. (2004)

Curr. Gastroenterol. Rep. 6, 99–1032. Bhatia, M., Brady, M., Shokuhi, S., Christmas, S., Neoptolemos, J. P., and Slavin, J.

(2000) J. Pathol. 190, 117–1253. Gukovsky, I., Gukovskaya, A. S., Blinman, T. A., Zaninovic, V., and Pandol, S. J. (1998)

Am. J. Physiol. 275, G1402–G14144. Norman, J. (1998) Am. J. Surg. 175, 76–835. Steer, M. L. (1999) Baillieres Best. Pract. Res. Clin. Gastroenterol. 13, 213–2256. Gukovskaya, A. S., Perkins, P., Zaninovic, V., Sandoval, D., Rutherford, R., Fitzsim-

mons, T., Pandol, S. J., and Poucell-Hatton, S. (1996) Gastroenterology 110, 875–8847. Kaiser, A. M., Saluja, A. K., Sengupta, A., Saluja, M., and Steer, M. L. (1995) Am. J.

Physiol. 269, C1295–C13048. Saluja, A., Hofbauer, B., Yamaguchi, Y., Yamanaka, K., and Steer, M. (1996) Biochem.

Biophys. Res. Commun. 220, 875–8789. Sandoval, D., Gukovskaya, A., Reavey, P., Gukovsky, S., Sisk, A., Braquet, P., Pandol,

S. J., and Poucell-Hatton, S. (1996) Gastroenterology 111, 1081–109110. Bhatia, M., Wallig, M. A., Hofbauer, B., Lee, H. S., Frossard, J. L., Steer, M. L., and

Saluja, A. K. (1998) Biochem. Biophys. Res. Commun. 246, 476–483

11. Bhatia, M. (2004) Am. J. Physiol. 286, G189–G19612. Gukovskaya, A. S., and Pandol, S. J. (2004) Pancreatology 4, 567–58613. Fiers, W., Beyaert, R., Declercq, W., and Vandenabeele, P. (1999) Oncogene 18,

7719–773014. Assuncao, G. C., and Linden, R. (2004) Eur. J. Biochem. 271, 1638–165015. Edinger, A. L., and Thompson, C. B. (2004) Curr. Opin. Cell Biol. 16, 663–66916. Leist, M., and Jaattela, M. (2001) Nat. Rev. Mol. Cell. Biol. 2, 589–59817. Proskuryakov, S. Y., Konoplyannikov, A.G., andGabai, V. L. (2003)Exp. Cell Res. 283,

1–1618. Reed, J. C., Doctor, K. S., and Godzik, A. (2004) Sci. STKE. 2004, re919. Lee, H. C., and Wei, Y. H. (2000) J. Biomed. Sci. 7, 2–1520. Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K. J., Debatin, K.M.,

Krammer, P. H., and Peter, M. E. (1998) EMBO J. 17, 1675–168721. Wolf, B. B., and Green, D. R. (1999) J. Biol. Chem. 274, 20049–2005222. Bratton, S. B., Walker, G., Srinivasula, S. M., Sun, X. M., Butterworth, M., Alnemri,

E. S., and Cohen, G. M. (2001) EMBO J. 20, 998–100923. Salvesen, G. S., and Duckett, C. S. (2002) Nat. Rev. Mol. Cell Biol. 3, 401–41024. Bratton, S. B., Lewis, J., Butterworth, M., Duckett, C. S., and Cohen, G.M. (2002)Cell

Death Differ. 9, 881–89225. Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000) Cell 102, 33–4226. Hayakawa, K., Takemura, G., Kanoh, M., Li, Y., Koda, M., Kawase, Y., Maruyama, R.,

Okada, H., Minatoguchi, S., Fujiwara, T., and Fujiwara, H. (2003) Circulation 108,104–109

27. von Harsdorf, R. (2004) Circ. Res. 95, 554–55628. Melnikov, V. Y., Faubel, S., Siegmund, B., Lucia, M. S., Ljubanovic, D., and Edelstein,

C. L. (2002) J. Clin. Invest. 110, 1083–109129. Gukovskaya, A. S., Gukovsky, I., Jung, Y., Mouria, M., and Pandol, S. J. (2002) J. Biol.

Chem. 277, 22595–2260430. Kuida, K., Zheng, T. S., Na, S., Kuan, C., Yang, D., Karasuyama, H., Rakic, P., and

Flavell, R. A. (1996) Nature 384, 368–37231. Vercammen, D., Beyaert, R., Denecker, G., Goossens, V., Van Loo, G., Declercq, W.,

Grooten, J., Fiers, W., and Vandenabeele, P. (1998) J. Exp. Med. 187, 1477–148532. Chan, F. K., Shisler, J., Bixby, J. G., Felices, M., Zheng, L., Appel, M., Orenstein, J.,

Moss, B., and Lenardo, M. J. (2003) J. Biol. Chem. 278, 51613–5162133. Holler, N., Zaru, R., Micheau, O., Thome, M., Attinger, A., Valitutti, S., Bodmer, J. L.,

Schneider, P., Seed, B., and Tschopp, J. (2000) Nat. Immunol. 1, 489–49534. Lin, Y., Choksi, S., Shen, H. M., Yang, Q. F., Hur, G. M., Kim, Y. S., Tran, J. H.,

Nedospasov, S. A., and Liu, Z. G. (2004) J. Biol. Chem. 279, 10822–1082835. Bassi, C., Butturini, G., Falconi, M., Salvia, R., Frigerio, I., and Pederzoli, P. (2003)

Pancreatology 3, 128–13236. Connor, S., and Neoptolemos, J. P. (2004)World J. Gastroenterol. 10, 1697–169837. Lerch, M. M., and Adler, G. (1994) Int. J. Pancreatol. 15, 159–17038. Gorelick F. S., AdlerG., andKernH. F. (1993) inThe Pancreas: Biology, Pathology, and

Disease, (Go, V.W., DiMagno, E.P., Gardner, J. D., Lebenthal, E., Reber, H. A., andScheele, G. A., eds) pp. 64–69, Raven, New York

39. Gukovskaya, A. S., Gukovsky, I., Zaninovic, V., Song, M., Sandoval, D., Gukovsky, S.,and Pandol, S. J. (1997) J. Clin. Invest. 100, 1853–1862

40. Vaquero, E. C., Edderkaoui, M., Nam, K. J., Gukovsky, I., Pandol, S. J., and Guk-ovskaya, A. S. (2003) Gastroenterology 125, 1188–1202

41. Soares, R. V., Liu, B., Oppenheim, F. G., Offner, G. D., and Troxler, R. F. (2002) Arch.Oral Biol. 47, 591–597

42. Alvarado-Kristensson, M., and Andersson, T. (2005) J. Biol. Chem. 280, 6238–624443. Sun, X. M., MacFarlane, M., Zhuang, J., Wolf, B. B., Green, D. R., and Cohen, G. M.

(1999) J. Biol. Chem. 274, 5053–506044. Johnson, C. R., and Jarvis, W. D. (2004) Apoptosis 9, 423–42745. Scaffidi, C., Medema, J. P., Krammer, P. H., and Peter, M. E. (1997) J. Biol. Chem. 272,

26953–2695846. Martin, D. A., Siegel, R. M., Zheng, L., and Lenardo, M. J. (1998) J. Biol. Chem. 273,

4345–434947. Blinman, T. A., Gukovsky, I., Mouria, M., Zaninovic, V., Livingston, E., Pandol, S. J.,

and Gukovskaya, A. S. (2000) Am. J. Physiol. 279, C1993–C200348. Kakimura, J., Kitamura, Y., Takata, K., Kohno, Y., Nomura, Y., and Taniguchi, T.

(2001) Eur. J. Pharmacol. 417, 59–6749. Peter, M. E. (2004) Biochem. J. 382, e1–e350. Krueger, A., Schmitz, I., Baumann, S., Krammer, P. H., and Kirchhoff, S. (2001) J. Biol.

Chem. 276, 20633–2064051. Thome, M., and Tschopp, J. (2001) Nat. Rev. Immunol. 1, 50–5852. Hegyi, P., Rakonczay, Z., Jr., Sari, R., Gog, C., Lonovics, J., Takacs, T., and Czako, L.

(2004)World J. Gastroenterol. 10, 2003–200953. Tashiro, M., Schafer, C., Yao, H., Ernst, S. A., and Williams, J. A. (2001) Gut 49,

241–25054. Nikolovska-Coleska, Z., Xu, L., Hu, Z., Tomita, Y., Li, P., Roller, P. P., Wang, R., Fang,

X., Guo, R., Zhang, M., Lippman, M. E., Yang, D., andWang, S. (2004) J. Med. Chem.47, 2430–2440

55. Chitra, M., Sukumar, E., Suja, V., and Devi, C. S. (1994) Chemotherapy 40, 109–113

Necrosis/Apoptosis Switch in Experimental Acute Pancreatitis

3380 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 6 • FEBRUARY 10, 2006

by guest on February 10, 2020http://w

ww

.jbc.org/D

ownloaded from

56. Sumino, M., Sekine, T., Ruangrungsi, N., Igarashi, K., and Ikegami, F. (2002) Chem.Pharm. Bull. (Tokyo) 50, 1484–1487

57. Gupta, S., Sanyal, S. N., and Kanwar, U. (1989) Contraception 39, 307–32058. Tang, D., Lahti, J. M., and Kidd, V. J. (2000) J. Biol. Chem. 275, 9303–930759. Kasahara, Y., Tuder, R. M., Taraseviciene-Stewart, L., Le Cras, T. D., Abman, S.,

Hirth, P. K.,Waltenberger, J., and Voelkel, N. F. (2000) J. Clin. Invest. 106, 1311–131960. Caserta, T. M., Smith, A. N., Gultice, A. D., Reedy, M. A., and Brown, T. L. (2003)

Apoptosis 8, 345–35261. Schotte, P., Declercq, W., Van Huffel, S., Vandenabeele, P., and Beyaert, R. (1999)

FEBS Lett. 442, 117–12162. Kim, J. W., Choi, E. J., and Joe, C. O. (2000) Oncogene 19, 4491–449963. Lin, Y., Devin, A., Rodriguez, Y., and Liu, Z. G. (1999) Genes Dev. 13, 2514–252664. Slee, E. A., Adrain, C., and Martin, S. J. (2001) J. Biol. Chem. 276, 7320–732665. Chen, X., Ji, B., Han, B., Ernst, S. A., Simeone, D., and Logsdon, C. D. (2002) Gastro-

enterology 122, 448–45766. Norman, J. G. (1999) Digestion 60, Suppl. 1, 57–6067. Jensen, R. T., Wank, S. A., Rowley, W. H., Sato, S., and Gardner, J. D. (1989) Trends

Pharmacol. Sci. 10, 418–42368. Krieser, R. J., and White, K. (2002) Curr. Opin. Cell Biol. 14, 734–73869. Henson, P. M., Bratton, D. L., and Fadok, V. A. (2001) Nat. Rev. Mol. Cell. Biol. 2,

627–63370. Ekert, P. G., Silke, J., and Vaux, D. L. (1999) Cell Death Differ. 6, 1081–108671. Reed, J. C., and Pellecchia, M. (2005) Blood 106, 408–41872. MacFarlane, M., Merrison, W., Bratton, S. B., and Cohen, G. M. (2002) J. Biol. Chem.

277, 36611–3661673. Harlin, H., Reffey, S. B., Duckett, C. S., Lindsten, T., and Thompson, C. B. (2001)Mol.

Cell. Biol. 21, 3604–360874. Adams, J. M. (2003) Genes Dev. 17, 2481–249575. Jaattela, M., and Tschopp, J. (2003) Nat. Immunol. 4, 416–42376. Luthen, R., Niederau, C., and Grendell, J. H. (1995) Am. J. Physiol. 268, G592–G60477. Luthen, R. E., Niederau, C., Ferrell, L. D., and Grendell, J. H. (1995) Pancreas 11,

141–14678. Gukovskaya, A. S., Vaquero, E., Zaninovic, V., Gorelick, F. S., Lusis, A. J., Brennan,

M. L., Holland, S., and Pandol, S. J. (2002) Gastroenterology 122, 974–984

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Ilya Gukovsky and Anna S. GukovskayaOlga A. Mareninova, Kai-Feng Sung, Peggy Hong, Aurelia Lugea, Stephen J. Pandol,

PANCREATITISCell Death in Pancreatitis: CASPASES PROTECT FROM NECROTIZING

doi: 10.1074/jbc.M511276200 originally published online December 8, 20052006, 281:3370-3381.J. Biol. Chem. 

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