Role of TRAIL and the pro-apoptotic Bcl-2 homolog Bim in acetaminophen-induced liver damage

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1 Role of TRAIL nd the pro-poptotic Bcl-2 homolog Bim in cetminophen-induced liver dmge A Bdmnn 1, A Keough 2, T Kufmnn 3, P Bouillet 4, T Brunner",1,5,6 nd N Corzz",1,6 Acetminophen (N-cetyl-pr-minophenol (), prcetmol) is commonly used nlgesic nd ntipyretic gent. Although considered sfe t therpeutic doses, ccidentl or intentionl overdose cuses cute liver filure chrcterized y centriloulr heptic necrosis with high moridity nd mortlity. Although mny moleculr spects of -induced cell deth hve een descried, no conclusive mechnism hs een proposed. We recently identified TNF-relted poptosis-inducing lignd (TRAIL) nd c-jun kinse (JNK)-dependent ctivtion of the pro-poptotic Bcl-2 homolog Bim s n importnt poptosis mplifiction pthwy in heptocytes. In this study, we, thus, investigted the role of TRAIL, c.jnk nd Bim in -induced liver dmge. Our results demonstrte tht TRAIL strongly synergizes with in inducing cell deth in heptocyte-like cells lines nd primry heptocyte. Furthermore, we found tht strongly induces the expression of Bim in c-jnk-dependent mnner. Consequently, TRAIL- or Bim-deficient mice were sustntilly protected from -induced liver dmge. This study identifies the TRAIL-JNK-Bim xis s novel trget in the tretment of -induced liver dmge nd sustntites its generl role in heptocyte deth. Acetminophen (N-cetyl-pr-minophenol (), prcetmol) is commonly used nlgesic nd ntipyretic gent. 1 Although considered sfe t therpeutic doses, ccidentl or intentionl overdose cuses cute liver filure chrcterized y centriloulr heptic necrosis with high moridity nd mortlity.2 Despite sustntil effort in the pst to investigte the processes involved in -induced liver toxicity, the exct moleculr nd iochemicl mechnisms remin incompletely understood. There is generl consensus tht the formtion of toxic electrophilic metolite y the P45 system, N-cetyl-p-enzoquinone imine (NAPl), is prerequisite for heptocyte injury.3 NAP I is usully detoxified y glutthione in the liver. However, overdose depletes heptic glutthione so much tht NAPl covlently inds to cellulr proteins leding to mitochondril dysfunction. Thus, inhiits mitochondril respirtion nd cuses decrese in ATP levels in liver cells. 4 More recently, in vivo study provided evidence for the formtion of peroxynitrite in the mitochondri nd the induction of mitochondril dmge. 5 It is still under dete whether covlent inding to criticl protein trgets or oxidtive stress s consequence of mitochondril glutthione depletion is the promoting fctor of heptocyte deth. Most proly, however, liver cell deth fter overdose is the consequence of interply etween multiple events. Furthermore, glutthione depletion nd covlent inding to cellulr proteins seems not to e sufficient to induce mitochondril dysfunction resulting in liver filure. In experimentl niml models, the progression nd severity of -induced liver toxicity is not only ssocited with the triggering of the memrne permeility trnsition nd the collpse of mitochondril memrne potentil, ut depends on the interply of other deth-inducing signls, oth within nd outside the heptocytes. The deth mechnism(s) of -induced liver dmge is still mtter of dete. Clerly, typicl signs of oth necrotic nd poptotic cell deth cn e oserved. There is lso significnt overlp etween events normlly ssocited with poptosis nd -induced cell deth. For instnce, mitochondril events generlly linked to poptosis hve een oserved in -induced heptic cell deth, including the proteolytic processing of the pro-poptotic Bcl-2 fmily ldivision of Immunopthology, Institute of Pthology, University of Bem, Bem, Switzerlnd; 2Viscerl nd Trnsplnttion Surgery, University Hospitl, University of Bem, Bern, Switzerlnd; 31nstitute of Phrmcology, University of Bern, Bern, Switzerlnd; 4The Wlter nd Eliz Hll Institute of Medicl Reserch, The Royl Meloume Hospitl, Victori, Melourne, Austrli nd 5Division of Biochemicl Phrmcology, Deprtment of Biology, University of Konstnz, Konstnz, Germny ' Corresponding uthors: T Brunner, Deprtment of Biology, University of Konstnz, Box 66, Konstnz 78457, Germny. Tel: ; Fx: ; E-mil: thoms.runner@uni-konstnz.de or N Corzz, Division of Immunopthology, Institute of Pthology, Murtenstrsse 31, Bern 31, Switzerlnd. Tel: ; Fx: ; E-mil: ndi.corzz@pthology.unie.ch 6These uthors shre senior uthorship of this mnuscript. Keywords: heptocytes; Jun kinse; deth receptor signling; Bcl-2 fmily; prcetmol Arevitions:, N-cetyl pr-minophenol, cetminophen, prcetmol; ATP, denosine triphosphte; Bx, Bcl-2-ssocited X protein; Bcl-2, B cell lymphom gene 2; BH3, Bcl 2 homology domin 3; Bid, BH3-intercting domin deth gonist; Bim, Bcl-2-intercting meditor of cell deth; DISC, deth-inducing signling complex; FADD, Fs-ssocited deth domin; FsL, Fs lignd; JNK, c-jun N-terminl kinse; Fox3, forkhed ox 3; MPT, memrne permeility trnsition; MTT, 3-(4,5-dimethylthizol 2 yl)-2,5-diphenyltetrzolium romide; NAPQI, N cetyl p enzoquinone imine; NK cell, nturl killer cell; NKT cell, nturl killer T cell; ROS, rective oxygen species; TNF, tumor necrosis fctor-; TRAIL, TNF relted poptosis-inducing lignd

2 2 memer Bid, the trnsloction of Bid nd Bx to the mitochondri nd the relese of cytochrome c. 6 In greement with the role of BcI-2 fmily memers in -induced heptocyte deth is tht mitochondril Bx trnsloction ccelertes DNA frgmenttion nd cell deth in induced liver dmge. 7 Moreover, criticl role of the stress kinse c-jun N-terminl kinse (Jun kinse, JNK) hs een demonstrted in oth -induced liver dmges nd other forms of heptocyte poptosis.9-11 Thus, tretment leds to JNK ctivtion, nd dministrtion of JNK inhiitors hve protective effect on -induced heptocyte deth nd ssocited liver dmge. Similrly, role of JNK hs een postulted in TN FIX 12 nd Fs lignd-medited heptocyte poptosis nd heptitis. 9 JNK is likely involved in deth lignd-induced heptocyte poptosis y the phosphoryltion nd ctivtion of downstrem poptosis effector molecules. In greement with this notion, we hve recently shown tht JNK phosphoryltes the pro-poptotic Bcl-2 homolog Bim, nd tht JNK-ctivted Bim criticlly regultes Fs-induced heptocyte poptosis. 9 Interestingly, we hve seen tht Fs-induced heptocyte poptosis is synergisticlly enhnced y the TNF-relted poptosis-inducing lignd (TRAIL) receptor signling pthwy vi TRAIL-induced JNK ctivtion nd Bim phosphoryltion. Similrly, Kufmnn et l. 13 demonstrted tht D-glctosmine/lipopolyscchride-induced heptitis in mice, proceeding vi TNF-IX-medited heptocyte poptosis, lso depends on JNK nd Bim. Finlly, eneficil effect of JNK inhiition hs lso een oserved in heptic ischemi-reperfusion injury.14 Given the criticl role of JNK nd Bim in vrious forms of heptocyte poptosis, nd the similrity etween induced liver dmge nd heptitis medited y the poptosis pthwy, we explored in this study the role of deth receptor-induced JNK nd ssocited Bim ctivtion in -induced liver dmge. We here demonstrte tht -induced heptocyte deth is enhnced y the TRAIL signling pthwy. Similrly, -induced liver dmge is reduced in TRAIL-deficient mice. Tretment of mice, isolted heptocytes or heptom cell lines with results in n increse in Bim expression, which is dependent on induced JNK ctivtion. Finlly, we demonstrte tht -induced liver dmge is significntly ttenuted in Bim-deficient mice. These findings demonstrte novel role of the BH3-only protein Bim in -induced liver dmge, nd further ccentute the role of JNK nd Bim in heptocyte cell deth. Results -induced deth is synergisticlly enhnced y TRAIL. We hve previously shown tht TRAIL synergisticlly enhnces Fs-induced poptosis in heptocytes in JNK-Bim-dependent mnner.9 We thus tested whether -induced heptocyte cell deth is lso enhnced y tretment the cells with TRAIL. Isolted primry humn heptocytes (hhep) were stimulted with incresing doses of, in the presence or sence of low doses of TRAIL. Figure 1 illustrtes tht lone filed to induce C ;; 3 '" 4> 2 Qj u u 1 1: u VI -1 ;; 8 '" 4> 6 Qj u u4 1:. 2 c- VI hhep 5 1 (mm) IHH -o-conlrol... 3ng/ml TRAIL... 3ng/ml TRAIL 1 2 (mm) Annexin V (log) HepG2 -o-conlrol ng/ml TRAIL... 3ng/ml TRAIL d "3 >< 2 >.... :;::;.., 4> If) o 1 2 (mm) IHH - TRAIL - LL:iJ Figure 1 TRAIL synergizes with in inducing cell deth. Humn heptocytes (hhep) (), the heptom cell line HepG2 () or immortlized humn heptocytes (IHH) (c) were preincuted with uffer control or incresing concentrtions of TRAIL, prior stimultion with different doses of. Cell deth ws ssessed y MIT ssy (-c) in primry heptocytes, HepG2 nd IHH cells, y DEVD clevge ssy (d) or Annexin V stining (e) in controllhh cells or IHH cells treted with 1 mm, 3 ng TRAIL or the comintion thereof, respectively. Men vlues ± S.D. of triplictes re shown for MIT nd DEVDse ssys, which were repeted three times, yielding similr results. A typicl experiment out of two is shown for Annexin V stining significnt cell deth in primry hheps. Similrly, TRAIL lone, t 3 nd 3 ng/ml, did not promote ny deth-inducing ctivities. However, comined tretment of cells with TRAIL nd incresing doses of synergisticlly induced cell deth in primry heptocytes. Similr results were lso confirmed in the heptom cell line HepG2 nd immortlized humn heptocytes (IHHs) (Figures 1 nd c). The synergistic induction of cell deth in IHH ws prlleled y incresed cspse 3 ctivity (DEVDse ctivity) nd phosphtidyl serine externliztion. These dt confirm tht TRAIL synergisticlly enhnces -induced cell deth in heptocytes nd heptocyte-like cells in vitro.

3 We next investigted the relevnce of TRAIL-medited enhncement of -induced cell deth lso in vivo. Wildtype (WT) C57BI/6 mice or TRAIL-deficient mice were treted with overdose of (4 mg/kg ody weight) nd liver dmge ws ssessed 5 h lter y tissue histology nd serum trnsminses. Figure 2 illustrtes tht induced severe tissue destruction with lrge res of dmged nd necrotic heptocytes in WT nimls. In contrst, histologicl ltertions were reduced in -treted TRAIL-deficient mice. Reduced liver dmge of TRAIL-deficient mice compred with WT nimls fter tretment ws lso confirmed y trnsminse ctivity in the ser. Although tretment of WT nimls with resulted in strong increse in serum AL T ctivity, reduced levels were seen in -treted TRAIL-deficient mice. These dt indicte tht endogenous TRAIL contriutes to -induced heptocyte deth nd liver dmge lso in vivo. induces expression of 8im. Our previous studies hve demonstrted n importnt role of Bim in heptocyte poptosis. 9 We thus investigted whether or TRAIL would ffect Bim expression in primry heptocytes nd heptocyte-like cell lines. Anlysis of Bim expression y western lot reveled tht tretment of primry hhep (Figure 3), HepG2 (Figure 3) nd IHH cells (Figure 3c) with resulted in strong induction of Bim expression. In contrst, no increse in Bim levels were seen in primry hheps nd HepG2 cells fter tretment with TRAIL, nd only slight induction ws oserved in IHH cells. Comined tretment of cells (HepG2 nd IHH), with TRAIL nd did not further increse expression of Bim. In greement with these in vitro dt, incresed Bim protein levels were lso oserved in liver smples from -treted mice (Figure 3d). To define in more detil whether induces n incresed Bim protein levels either y stiliztion of the protein or y promoting Bim gene expression, we nlyzed Bim mrna expression fter tretment with nd/or TRAIL y quntittive RT-PCR. TRAIL ws found to e n Bim EL Tuulin I (mm) TRAIL Bim EL Tuulin TRAIL 1 2 I C Bim EL :=:=====: JNKI TRAIL Figure 3 induces Bim protein expression. Humn heptocytes (), HepG2 () or IHH (c) cells were treted with, TRAIL or oth for 6 h, nd Bim protein expression ws nlyzed y western lot. (d) Liver smples of PBS- or -treted wild-type mice were nlyzed for the expression of Bim y western lot. JNK or tuulin ws used to normlize protein lodings 2 ;2 j:' 1...J......,'".\ Ii. A ). A )..,,v,,v PBS Figure 2 TRAIL is required for efficient -induced liver dmge in vivo. Wild type (WT) or TRAIL-deficient mice (TRAIL- /- ) were treted with PBS s control or (4 mg/kg ody weight), nd liver dmge ws nlyzed y histology () nd trnsminse levels (AST) in serum () fter 5 h. Pooled experimentl dt from 8 to 12 mice per group re shown

4 4 "' z 3 :: E 2 I:: :J ".!: " :E. TRAIL C "' z 8 :: E 6 I:: 4 :J ".!: 2 " :E TRAIL hhep IHH o TRAIL d HepG , o -L-F---"T"""--I PBS Figure 4 induces Bim trnscription. Humn heptocytes (), HepG2 () or IHH (e) cells were treted with with 1 mm, 3 ng TRAIL or the comintion thereof for 6 h, nd Bim expression ws nlyzed y quntittive RT-PCR. (d) Wild-type mice were treted with PBS or, nd Bim expression ws nlyzed in liver smples y quntittive RT-PCR fter 5 h. GAPDH ws used to normlize Bim expression levels. Men vlues ± S.D. of triplictes re shown for fold induction of mrna levels. Pooled dt from 6 to 7 mice per group re shown. p =.11 (unpired Student's I-test) inefficient inducer of Bim mrna expression in primry heptocytes nd IHH cells, nd modertely induced Bim expression in HepG2 cells. In contrst, strongly promoted Bim mrna expression in ll cells tested (Figures 4-c). No synergy etween nd TRAIL in promoting Bim mrna expression ws detected. Bim mrna levels were lso significntly incresed in the liver of -treted WT mice compred with PBS-treted control mice (Figure 4d). induces Bim promoter ctivity in JNKdependent mnner. JNK hs een previously implicted in the trnscriptionl control of Bim expression vi n AP-1- dependent pthwy. Furthermore, JNK ctivity hs een shown to prticipte in -induced liver disese In ccordnce with these reports, we demonstrted tht lone induced roust JNK ctivtion s mesured y the detection of phospho-jnk y western lot (Figure S). -induced JNK ctivtion ws comprle to tht induced y phorol 12-myristte 13-cette used s positive control. To investigte the role of JNK in induced Bim gene expression we used luciferse reporter construct contining.8 k of the Bim proximl promoter. 16 Although this Bim promoter construct ws constitutively ctive in control-treted cells, reltively low concentrtions of (2.5 mm) induced the Bim reporter construct _ Time(min) g 1... l '2 :J 5 '13.= ;... PMA c:.. 1 d ",8 z E 8 6 l I:: 6 '2 :J tl4 '13 4 :J ".=.S 2 Ctrl. 2 " :Eo - Inh (11M) Inh (IlM) e Time(min) BimEL I - I Tuulin r.1-;--:::-"!!"""'=="'=-::=-==:;1 AP: I I I: : : _ 3 '---...'----:p""g"l"2--- g -- BimWT )( ; 2 '2 :J '13 1 :::I o (mm) PMA 3 1 Figure 5 -induced Bim trnscription is JNK- nd Fox3-dependent. () IHH cells were stimulted with (1 mm) or, s positive control, phorol12- myristte 13-cette (3 ng/ml) nd phospo-jnk nd tuulin protein levels were nlyzed t different time points y western lot. () IHH cells were trnsfected with the wild-type (WT) Bim reporter construct, pre-treted with JNK V inhiitor (1 11M) nd stimulted with (1mM) or phorol12-myristte 13-cette (3ng/ml). (e) Similrly, IHH cells were trnsfected with the WT Bim reporter construct, pretreted with different concentrtions of the JNK inhiitor V nd stimulted with (1 mm). Luciferse ctivity ws mesured nd normlized to fl-glctosidse. (d) IHH were pretreted with JNK inhiitor V nd stimulted with (1 mm). Bim mrna expression ws mesured y quntittive RT-PCR (6 h). (e) IHH cells were treted s descried ove nd Bim were nlyzed t different time points y western lot. (I) IHH cells were trnsfected with empty vector (pgl2), the wild-type (Bim WT) or the Fox3 mutnt Bim reporter constructs (Bim Fox3), nd stimulted with (1 mm) for 6 h. Luciferse ctivity ws mesured nd normlized to fl-glctosidse ctivity. Men vlues ± S.D. of triplictes re shown for reltive luciferse units nd fold induction of mrna levels. The experiments hve een repeted three times, yielding similr results (Figure 5). Higher concentrtions further induced Bim promoter ctivity y out fourfold ove levels of unstimulted cells (Figures 5 nd c). Inhiition of JNK, y the phrmcologicl inhiitor, resulted in strong inhiition of

5 2x - - 4x 2 ;j &., :; 1 & (.-'./:' >:./:'. o/ -PBS o/ Figure 6 Bim is required for efficient -induced liver dmge in vivo. Wild-type (WT) or Bim-deficient mice (Bim - I- ) were treted with PBS s control or 4 mg/kg ody weight, nd liver dmge ws nlyzed y histology () nd trnsminse levels (AST) in serum (). Pooled dt from 1 to 12 mice per group re shown... p =.47 (unpired Student's (-test) -induced Bim promoter ctivity (Figures 5 nd c), Bim mrna expression (Figure 5d) nd Bim protein expression (Figure 5e). These findings indicte tht JNK hs role in induced Bim expression in liver cells. Fox3 hs lso een reported to e involved in the regultion of Bim expression nd ssocited poptosis in vrious forms of liver disese. 17 In greement with this proposed role of Fox3, we lso found tht -induced Bim promoter ctivity in IHH cells ws reduced when Fox3 inding sites were mutted in the Bim promoter, indicting tht -induced Bim expression is lso regulted in prt y the trnscription fctor Fox3 (Figure 5f). -induced liver dmge is 8im-dependent. Given the potent Bim-inducing ctivity of in vivo nd in vitro, we nlyzed the role of Bim in -induced liver dmge in vivo. Wild-type nd Bim-deficient C57BI/6 mice were treted with n overdose of (4 mg/kg ody weight) nd liver dmge ws nlyzed y histologicl ltertions nd serum trnsminse levels 5 h lter. While in WT nimls tretment resulted in the extensive heptic cell deth nd lrge necrotic lesions descried erlier (Figure 6), tissue dmge ws sustntilly reduced in Bim-deficient mice, indicting tht Bim is n importnt determinnt of -induced liver dmge. These findings were confirmed when nlyzing serum trnsminse levels. While tretment of WT mice resulted in strong increse of AL T levels, significntly reduced levels were seen in drug-treted Bim-deficient mice. These findings support criticl role of Bim in -induced heptocyte deth nd liver dmge. Discussion Owing to its unique position, the liver is frequent trget of dmge induced y drugs, xenoiotics nd oxidtive stress. Although the liver hs sophisticted detoxifiction system, overdoses of heptotoxic gents my eventully led to liver cell deth, cute liver dmge nd liver filure. is well-chrcterized liver-dmging sustnce, whose heptotoxic ctivities hve een widely studied. Nevertheless, so fr investigtions hve filed to identify single criticl pthwy involved in -induced liver dmge, nd rther suggest multitude of fctors nd signling events leding to the oserved effects in vitro nd in vivo. Thus, -induced liver dmge is the interply of severl distinct deth mechnisms. Our study, identifying TRAIL, JNK nd Bim s criticl elements in -induced heptocyte cell deth in vitro nd in vivo, hs dded new elements into this scheme of underlying mechnisms of -induced liver dmge. It lso provides interesting new links etween known pthwys of -induced heptocyte deth nd other forms of heptitis. In prticulr, JNK hs een previously ssocited with -induced nd other forms of heptocyte deth. B,1 Our findings tht induces JNK ctivtion, nd tht JNK ctivity is criticl for -induced Bim expression further supports n importnt role of this stress kinse pthwy in the regultion of liver cell deth. Recent findings suggest tht -induced JNK ctivtion my e medited vi n ASK1 - dependent mnner.1b Interestingly, we hve lso previously demonstrted regultory role of JNK ctivtion in deth lignd-induced heptocyte poptosis. JNK ws found to e criticl for the phosphoryltion nd ctivtion of Bim in response to Fs nd TNF receptor ctivtion. 9,13 Similrly, we descrie here criticl role of JNK in -induced Bim induction. In contrst to our previous results on deth receptor-induced heptitis, our present dt suggest tht -induced JNK ctivtion my e rther involved in ctivtion of the Bim promoter nd induction of Bim expression. tretment induced strong increse in Bim promoter ctivity, mrna nd protein expression, which ws significntly inhiited y JNK inhiitor. Although phosphoryltion of Bim hs een oserved in some circumstnces, the induction of Bim expression ppers to e the min pthwy y which JNK is involved in -induced heptocyte deth. Our study lso sustntites relevnt role for Bim in -induced liver dmge, nd further supports mjor function of this Bcl-2 homolog protein in heptocyte cell deth in generl. Although Fs- nd TNF receptor-induced

6 6 heptocyte cell deth, prototypicl type II cell, hs een suggested to depend on cspse 8-medited clevge of the BH3-only protein Bid to tbid nd susequent mplifiction of the poptosis signl vi the mitochondril pthwy, our own results nd those y others demonstrte tht Bim is lso n essentil component of this pthwy. Asence of Bim lmost completely rogted nti-fs ntiody-induced heptitis, nd synergized with lck of Bid in protecting from TNF-exmedited liver dmge. 9,13 Similrly, we find here tht Bim is n importnt component of the -initited heptotoxic signling pthwy. Although we demonstrte here lso n importnt mplifying role of TRAIL in -induced heptocyte deth in vitro nd liver dmge in vivo, our results re not fully conclusive s how TRAIL is involved in these processes. Clerly, we filed to find strong evidence tht TRAIL signling my e involved in the induction of Bim expression. In generl, TRAIL did either not induce Bim expression or ws only wek trigger. Thus, TRAIL my directly or indirectly contriute to -induced Bim expression depending on vrying circumstnces. Amplifiction of the deth signl in vivo nd ssocited heptocyte deth y TRAIL my enhnce inflmmtory responses, which could further contriute to induction of Bim expression. Our previous results on Fs-induced heptocyte deth rther suggest, however, tht TRAIL-induced signling pthwys my e more criticl for the ctivtion of Bim y phosphoryltion. For instnce, sence of TRAIL led to reduced JNK ctivtion nd Bim phosphoryltion. Similrly, TRAIL my e involved in ctivting -induced Bim y JNK-medited phosphoryltion. 9 Disturing in this hypothesis is the fct tht strong JNK ctivtion, s mesured y detection of phospho JNK, ws much stronger in response to thn TRAIL tretment. Clerly, though, TRAIL signling cn ctivte the JNK pthwy, nd differentil ctivtion of JNK1 or JNK2 y nd TRAIL my result in -medited nd JNK-dependent Bim expression, or TRAIL-induced nd JNK-medited Bim phosphoryltion. Although our results re not fully conclusive in this respect, they support synergistic role of TRAIL, JNK nd Bim in -induced liver dmge. An open question is the cellulr source of TRAIL in vivo. TRAIL expression hs een previously demonstrted in vrious liver-homing leukocytes, including Kupffer cells nd NK cells nd NKT cells. 19 2o Interestingly, immune cells nd inflmmtion hve een implicted in -induced liver dmge, prticulrly s n mplifying element in this form of liver disese. -induced mcrophge ctivtion nd susequent mplifiction of tissue dmge hs een shown to involve toll-like receptor 9 nd NALP3 inflmmsome ctivtion. 2 1 Similr processes my lso result in TRAIL expression in Kupffer cells nd mplifiction of -induced liver dmge. Although our dt support role of the TRAIL-JNK-Bim xis in rther poptotic form of cell deth in response to, -induced heptocyte cell deth clerly shows lso more necrosis-like fetures. Currently it is unknown whether necrosis nd poptosis represent two independent events, whether poptosis occurs in some cells nd necrosis in others, or whether the two pthwy re intimtely linked. Although the sence of TRAIL nd Bim likely hs strong inhiitory effect on the poptosis pthwy, it should not ffect -induced necrosis if occurring independently. Histology of -treted liver tissue from oth TRAIL nd Bim deficient mice reveled strongly reduced liver dmge nd necrotic res, suggesting tht -induced poptosis nd necrosis depend on ech other. Detiled exmintion, however, lso revels tht surviving heptocytes still show fetures of stressed cells, for exmple, strong vcuoliztion. Thus, locking poptosis my strongly reduce tissue dmge, however, my not e sufficient to lock -induced cellulr dmge. Nevertheless, our present dt illustrte tht inhiition of either TRAIL or Bim results in sustntil meliortion of disese pthology. Thus, selective inhiition of this signl trnsduction pthwy my represent novel nd successful therpy in the tretment of overdose-ssocited liver dmge. Current tretment of poisoning involved dministrtion of N-cetylcysteine, which helps to uild up cellulr glutthione levels nd thus to detoxify NAPQI. This tretment trgets, however, not the cell deth-inducing pthwy per se ut seeks to remove the trigger of tissue destruction, which my e often too lte. Trgeting criticl signling elements in -induced liver cell deth my thus represent more efficient strtegy. Mterils nd Methods Cell nd cell lines. HepG2 ( heptom cell line) nd IHH were kindly provided y JF Dufour (Institute for Clinicl Phrmcology, Inselspitl, University of Bern). Cells were cultured in IMDM medium, contining 5% FCS, 1% L-glutmine nd.1% gentmicin. Primry hheps were isolted s descried previously from the liver tissue of consented ptients undergoing liver surgery. Humn heptocytes were enzymticlly dissocited from humn liver smples using two-step enzymtic microperfusion technique with collgense nd kept on ice in suspension. 22 Humn heptocytes were cultured in Willims E medium contining 1% FCS, 1% L'glutmine,.1% gentmicin,.1 % insulin/protinin nd.5% trnsferrin. Cell deth ssy (MTT). HepG2 nd IHH were grown in logrithmic phse, hrvested nd plted in 96-well flt ottom pltes. Primry hheps were directly seeded into collgen-coted 96-well flt ottom pltes. After overnight dherence, medium ws chnged nd cells were treted with incresing concentrtions of recominnt humn TRAIL (untgged form, R&D Systems, Schonenuch/Bsel, Switzerlnd) nd (prcetmol, Sigm Aldrich, St. Louis, MO, USA) for 16 h. Cell viility ws then ssessed y 3 (4,5 dimethylthizol-2-yl) 2,5 diphenyltetrzolium romide Mn ssy (Sigm-Aldrich) nd normlized to untreted controls. 9 Annexln V stining. Afterdifferenttretments, HepG2 nd IHH ce ll deth ws monitored y Annexin V-FITC stining (ebioscience, Sn Diego, CA, USA) t different time points depending on the experiment. Briefly, the cells were hrvested, centrifuged nd resuspended in 5 ml Annexin V inding uffer (1 mm Hepes ph 7.4, 15 mm NCI, 5 mm KCI, 1 mm MgCI 2 nd 1.5 mm CCI 2 ) contining Annexin V FITC. The cells were stined for 1 min in the drk, wshed two times with Annexin V inding uffer, resuspended into 2 ml Annexin V inding uffer nd nlyzed on FACScn flow cytometer using Cell Quest softwre (Becton Dickinson, Heidelerg, Germny). The percentge of Annexin V-FITC positive ce lls ws th en clculted using the Flow Jo softwre (FlowJo LLC Softwre pckge, Ashlnd, OR, USA). Cspse ctivity ssy (DEVDse ctivity). Cells were treted with TRA IL nd/or s indicted, nd hrvested fter 6 h. Cells were then wshed nd lysed for 1min on ice with 2ml PBS contining 1% Triton X 1. After centrifugtion for 5 min t 16 x gt 4 C, superntnt ws hrvested nd 5 ml were mixed with 15 ml Hepes uffer (1 mm Hepes, ph 7.5, 2% glycerol,.5 mm EDTA, 5mM Dn) contining 1mM Ac-DEVD AFC (Alexis Biochemicls,

7 Sn Diego, CA, USA). Rections were incuted for 1 h t 37 C, nd enzymtic ctivity ws mesured on spectrofluorometer (4 nm excittion, 55 nm emission). Bckground fluorescence ws mesured y incuting cspse sustrte with lysis uffer. Western lot nlysis. Cells were treted s indicted, efore lysis in cell lysis uffer contining 15 mm NCl, 1% NP-4,.5% deoxycholic cid,.1% SDS nd 5 mm Tris, ph 7.4. Cell Iystes were seprted y electrophoresis nd trnsferred to nitrocellulose. JNK, phosphorylted JNK nd Bim were detected y incution of memrnes with the respective primry ntiodies (nti-jnk nd ntiphospho JNK, Cell Signling Technology, Allschwil, Switzerlnd; nti-bim ntiody dilution, Sigm-Aldrich) nd corresponding horserdish peroxidse-lelled secondry ntiodies. Signls were visulized y enhnced chemiluminescence nd detected in Fujifilm LAS 4 imging system (Fujifilm Corportion, Aksk, Jpn). Quntittive PCR. Humn nd mouse Bim mrna expression ws detected y quntittive RT-PCR s descried erlier. 16 Briefly, cells were lysed in TAl regent (Sigm-Aldrich) nd RNA ws isolted. RNA ws DNse-treted (Promeg, Mdison, WI, USA) nd 2 mg of RNA were reverse trnscried using high cpcity kit (Applied Biosystems, Rotkreuz Zug, Switzerlnd). Rel-time PCR ws performed in n Applied Biosystems Rel-time PCR 75 mchine using SYBR green nd Quntitec primer ssys (QIAGEN, Hilden, Germny). GAPDH ws used to normlize Bim expression levels. 81m promoter ssy. The murine im po.8 reporter ws generted y cloning.8 k upstrem of the trnscription strt site of the murine im gene (Gennk AL8595) into the pgl2-sic plsmid (Promeg). The two FOXO sites present in im po.8 promoter construct (FOXO inding site 1 ggcc - ggg; position to p upstrem of im exonl; FOXO inding site 2 gtcc - gt9!92c; position to p) were modified y site-directed mutgenesis to generte im po.8 (mutl, 2)-pGL2-sic luciferse reporter construct. Wild-type nd mutted im reporter constructs, nd (I-glctosidse expression vector for trnsfection control, were co-trnsfected into IHH cells using Amx Nuleofection kit (Amx, Cologne, Germny). Cells were plted on 1 cm plte for 1 dy, hrvested nd distriuted on 96-well flt ottom plte. Cells were then incuted with incresing concentrtions of uffer control, (1 mm) or TRAIL (3 nglml) for 6 h. In some experiments, cells were preincuted with different concentrtions of the JNK V Inhiitor (Cliochem, Drmstdt, Germny). Cells were wshed once with PBS, nd then ceillystes were prepred y dding lysis uffer directly to the cells for 2 min t RT on rocking tle. Lystes were trnsferred to V-ottom plte nd centrifuged for 1 min with 2 x 9 t RT. A totl volume of 4 ml ws used for the reporter ssy nd 4ml for the (I-glctosidse ssy. Luciferse ctivity ws quntified using Orion Microplte Luminometer (Pforzheim, Germny). In vivo experiments. After 24 h fsting period, dult TRAIL-deficient (TRAIL- /- ), Bim-deficient (Bim - /- ) mice nd WT C57BU6 mice were injected Lp. with 4ml PBS or 1mg (4mg/kg ody weight) dissolved in wrm PBS. Mice were killed t the indicted time points, nd serum nd liver smples were collected. Liver smples were either snp-frozen in liquid nitrogen for susequent isoltion of mrna nd proteins, or fixed in 4% pr formldehyde in PBS for prffin emedding nd histology. Serum AST levels were mesured using commercilly ville kit (TECO Dignostics, Anheim, CA, USA). All niml experiments were reviewed nd pproved y the Animl Experimenttion Review Bord of the Stte of Bern. Sttisticl nlysis. Sttisticl differences were nlyzed using unpired twotiled Student's t-test. P-vlues <.5 were considered significnt. Conflict of Interest The uthors declre no conflict of interest. from the Swiss Ntionl Science Foundtion to TB nd NC, nd the EUore nd Vleri Rossi Foundtion to NC. 1. Rumck BH. Acetminophen misconceptions. Hepto/ogy 24; 4: Lee WM. Acetminophen nd the U.S. Acute Liver Filure Study Group: lowering the risks 1 heptic filure. Hepto/ogy 24; 4: Msuuchi Y, Sud C, Horie T. Involvement of mitochondril permeility trnsition in cetminophen-induced liver injuly in mice. J Hepto/25 ; 42: lhh Burchm PC, Hnnn AW. Acetminophen toxicity results in site-specific mitochondril dmge in isolted mouse heptocytes. J Bioi Chem 1991 ; 266: Cover C, Mnsouri A, Knight TR, Bjt ML, Lemsters JJ, Pessyre D et l. Peroxynitriteinduced mitochondril nd endonuclese-medited nucler DNA dmge in cetminophen heptotoxicity. J Phrmcol Exp Ther25; 315: EI-Hssn H, Anwr K, Mcns-Pirrd P, Crtree M, Chow SC, Johnson VL et l. Involvement of mitochondri in cetminophen-induced poptosis nd heptic injury: roles of cytochrome c, Bx, Bid, nd cspses. Toxicol Appl Phrmcol 23; 191 : Bjt ML, Frhood A, Lemsters JJ, Jeschke H. Mitochondril x trnsloction ccelertes DNA frgmenttion nd cell necrosis in murine model of cetminophen heptotoxicity. J Phrmcol Exp Ther 28; 324: Gunwn BK, Liu ZX, Hn, Hnw N, Grde WA, Kplowitz N. c-jun N-terminl kinse plys mjor role in murine cetminophen heptotoxicity. Gstroenterology26; 131: Corzz N, Jko S, ScherC, Frese S, Keogh A, Strok et l. TRAIL receptor-medited JNK ctivtion nd Bim phosphoryltion criticlly regulte Fs-medited liver dmge nd lethtity. J Clin Invest 26; 116: 249:> Mlhi H, Bronk SF, Werneurg NW, Gores GJ. Free ftty cids induce JNK-dependent heptocyte lipopoptosis. J Bioi Chem 26; 281 : Tkmur M, Mtsud Y, Ymgiw S, Tmur Y, Hond Y, Suzuki K et l. An inhiitor of c-jun NH2-terminl kinse, SP6125, protects mice from D-glctosminel lipopolyscchride-induced heptic filure y modulting BH3-only proteins. Life Sci 27; 8: Ds M, Sio G, Jing F, Rincon M, Flvell RA, Dvis RJ. Induction of heptitis y JNKmedited expression of TNF-lph. Cell 29; 136: Kufmnn T, Jost PJ, Pellegrini M, Puthlkth H, Gugsyn R, Gerondkis Set l. Ftl heptitis medited y tumor necrosis fctor TNFlph requires cspse-8 nd involves the BH3-only proteins Bid nd Bim. Immunity 29; 3: Llcun L, Mri M, Grci-Ruiz C, Fernndez-Chec JC, Morles A. Criticl role of cidic sphingomyelinse in murine heptic ischemi-reperfusion injury. Heptology 26; 44: Kiessling MK, Linke B, Brechmnn M, Suss, Krmmer PH, Gulow K. Inhiition of NF-kppB induces switch from CD95L-dependent to CD95L-independent nd JNKmedited poptosis in T cells. FEBS Leff 21; 584: Bouillet P, Zhng LC, Hung DC, We GC, Bottem CD, Shore P et l. Gene structure lterntive splicing, nd chromosoml locliztion of pro-poptotic Bcl-2 reltive Bim. Mmm Genome 21; 12: 16:> Brreyro FJ, Koyshi S, Bronk SF, Werneurg NW, Mlhi H, Gores GJ. Trnscriptionl regultion of im y FOX3A medites heptocyte lipopoptosis. J Bioi Chem 27; 11 : Nkgw H, Med S, Hiki Y, Ohme T, Shit W, Yni A et l. Deletion of poptosis signl-regulting kinse 1 ttenutes cetminophen-induced liver injury y inhiiting c-jun N-terminl kinse ctivtion. Gstroenterology 28; 135: Ahlenstiel G, Titerence RH, Koh C, Edlich B, Feld JJ, Rotmn Y et l. Nturl killer cells re polrized towrd cytotoxicity in chronic heptitis C in n interferon-lf-dependent mnner. Gstroenterology 21; 138: e321-e Berz N, Mlto Y, Snder LE, AI-Msoudi M, Freimuth J, Riethmcher et t. Heptocyte-specific NEMO deletion promotes NKiNKT cell- nd TRAIL-dependent liver dmge. J Exp Med29 ; 26: Imed AB, Wtne A, Sohil MA, Mhmood S, Mohmdnejd M, Sutterwl FS et l. Acetminophen-induced heptotoxicity in mice is dependent on TIr9 nd the Nlp3 inflmmsome. J Clin Invest 29; 119: Rencurel F, Foretz M, Kufmnn MR, Strok, Looser R, Leclerc I et l. Stimultion of AMP-ctivted protein kinse is essentil for the induction of drug metolizing enzymes y phenoritl in humn nd mouse liver. Mol Phrmcol 26; 7: Acknowledgements. We thnk ImmunexiGenetech for providing the TRAILdeficient mice, Andres Strsser nd Georg Hcker for th e Bim-deficient mice nd Jen-Frn9is Dufour for IHH cells. This work ws supported y grnts received