Tumor Necrosis Factor ~ Sensitizes Primary Murine Hepatocytes to FaslCD95-lnduced in a Bim- and Bid-Dependent Manner

Katiuin Schmich, I Rebekka Schlatter/* Nadia Corazza,'* Karine Sa Ferreira,4 ,5 Michael Ederer,2 Thomas Brunner,3,6 Christoph Borner,4.7 ,Hand Irmgard Merfort l

Fas/CD95 is a critical mediator of cell death in many chronic and acute liver diseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the pro inflammatory tumor necrosis factor IX (TNFIX) fails to provoke cell death in isolated hepato­ cytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFIX sensitizes primary murine hepato­ cytes cultured on collagen to (FasL)-induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFIX. Fas itself is essential for the sensi­ tization, but neither Fas up-regulation nor endogenous FasL is responsible for this effect. Although FasL is shown to induce Bid-independent apoptosis in hepatocytes cultured on collagen, the sensitizing effect of TNFIX is clearly dependent on Bid. Moreover, both c­ Jun N-terminal kinase ONK) activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3)-only , are crucial mediators of TN FIX-induced apoptosis sensiti­ zation. Bim and Bid activate the mitochondrial amplification loop and induce cyto­ chrome c release, a hallmark of type II apoptosis. The mechanism of TNFIX-induced sensitization is supported by a mathematical model that correctly reproduces the biolog­ ical findings. Finally, our results are physiologically relevant because TNFIX also sensi­ tizes to agonistic anti-Fas-induced liver damage. Conclusion: Our data suggest that TNFIX can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3- only Bim and Bid. (HEPATOLOGY 2011;53:282-292)

nhanced apoptosis is critically involved in many complex network of soluble and cell-associated apoptotic acute and chronic liver diseases, and hepatocytes and inflammatory signals. I It is therefore increasingly im­ Eare the main cell type undergoing massive cell portant to obtain insight into the mechanistic interplay death during liver injUiy. This process is regulated by a of these signals to define new therapeutic strategies. In

Abbreviations: AuD, actinomycin D; AS?: aspartate aminotrtlnsfertlse; Bak, B cell lymphoma 2 homologous fmtagonist/killer; Bax, B cell lymphoma 2- tlSsociated X protein; Bcl2, B cell lymphoma 2; BI-/3, B cell lymphoma 2 homology domain 3; c-FLIR cellula.r Fas-associffting protein with death domain-like interleukin-1 beta-converting enzyme (FLlCE) i"hibitOlY protein; clAR cellular ; Diablo, diablo homolog; DISC, death-inducing signaling complex; ELISA, enzyme-linked iml1lllnosorbent assay; FADD, his-associated death domain; FasL, Fas ligand; lOBS, fltal bovine serum; JNK, c-Jun N -terminrd kinase; KO, knockout; mAb, monoclollal ftntibocly; MOM!? mitochondrial membrtlne permeabiliZfltion; mRNA, messenger RNA; MTT; 3-(4,5-dimethylthiflzol-2-yl} -2,5- diphenyltetrtlzolilll1l bromide; N2A, neuroblastoma 2A; NF-KB, nuclear jactor kappa B; P-jNK, phosphOlylated c-jun N -terminal kinase; pBim, phoJphOlyiflted Bim; q1?7:PCR, qUftntitfltive retd-time polymerase chilin reaction; siBim, small intelfiring RNA tfllgeting Bim; siRNA, small illtt/firing RNA; Smac, second mitochondria-derived activator of ; 51'600125, anthlYl{1 -9-cd/pyrazol-6{2 H)-one; tBid, truncated Bid; TNF, tumor necrosis foctol; TNFR, tllmor necrosis foctor receptor; WT, wild type; )(JAR X-linked inhibitor ofapoptosis protein. From the I Department of Pharmaceutical Biology and Biotechnolog)\ Albert- Ludwigs- University of fi'eiblllg, Freiburg, Germ({ny, l lnstitute for System Dyn({mics, University ofStuttg({rt, Stuttgart, Germany; ' Biochemical Pharm({cology, Department of Biology, University ofKonstanz, Konstanz, Germany; "Institute of MolecilIaI' Medicine and Cell Rese({rch, Albert-Ludwigs-University of Freibulg, Freibulg, Germany, 5 Faculty of Biology, Albert-Ludwigs- University of Freib'''g, Freiblllg, Germany, (' Division ofImnllmopathology, Imtitllte of Pathology, UnivelJity of Bern, Bern, Switzerland; ;Spemrtrlll Gmdllate School ofBiology and Medicine, and and "Cellter for Biological Signaling Studies, Albert-Ludwigs- University of Freiblllg, Freibltlg, Germ({ny.

This work was SIIpported by the Fedent! Ministly of Educ({tion fmd ResCflrch through a resetlrch gmnt (0313074D) Fom Hep({toSys (to Kathrin Schmich, Rebekkrr Schlatter, IV,rine Sfi Ferreim, Mich({el Edem; Christoph BornCl; fmd Irmgard MCljnrt). This work IVIIS also supported by the Spemfll1n Gradllate School of Biology and Medicine (to Christoph BorrlCl) ({ nd by the Bioss Excellence Cluster (to Christoph BornCl); both {fre programs of the Excellence Initiative fmded by the German Resea rch Foundation. Kllrine Sa' Ferreira is supported hy the German Reseanh Foundation (GRKII04), fmd Thomas Brunner and Nadi({ CoraZZfl are supported by the Swiss National Science Foundation (3 10000- 120427 and 3/0000-121854). 'These flutholJ contributed equally to this work.

282 283 the liver, apoptosis is mainly initiated by the death recep­ expressed on the surface of hepatocytes and is impor­ tor ligands Fas ligand (FasL; CD95L) and tumor necrosis tant to hepatic health and disease. Mice treated with a factor a (TNFa).2 lethal dose of agonisti c anti-Fas antibody die because of After li gand binding, death receptors recruit the mass ive hepatocyte apoptosis and liver failure. l o This adaptor Fas-associated death domain (FADD) and pro­ cell death is dependent on Bid because Bid-defi cient -8 to their intracellul ar fa ce, and this forms the mice are res istant to Fas- induced hepatocellular apopto­ death-inducing signaling complex (DISC).3 By this as­ sis, fulminant hepatitis, and subsequent liver failure. I I sembly, procaspase-8 is autoprocessed and activated, These findings indicate that in vivo hepatocytes die in and it can then trigger two different apoptotic signal­ response to FasL via the rype n signaling pathway? ing pathways. In so-call ed rype I cell s, such as lympho­ However, we have shown recently that isolated primary cytes, active caspase-8 directly cleaves and activates hepatocytes cultured on collagen change their apoptosis procaspase-3 to induce efficient cell death execution." signaling from rype II to the Bid-independent type I In rype II cells, such as hepatocytes, apoptosis induc­ pathway,1 2 and this suggests that the rype JIll decision tion first requires caspase-8-mediated cleavage of depends not only on the expression of endogenous pro­ BH3-inreracting domain death agonist (Bid) into its teins, such as XIAp, but also on external factors. truncated form [truncated Bid (tBid)] . tBid belongs to TNFa is a pleiotropic cytokine that induces a vari­ the subclass of B cel l lymphoma 2 homology domain ery of cellular responses, such as inflammation and cel l 3 (BH3)- only B ce ll lymphoma 2 (Bcl2) family mem­ proliferation, mainly through activation of the nuclear bers (e.g., Bcl2-like protein 11 [Bim], p53-up-regu­ factor kappa B (N F- ICB) signaling cascade. Unlike lated modulator of apoptosis (Puma), and Noxa), FasL, the association of TNFa with its main receptor which se nse apoptotic stimuli and convey the death tumor necrosis factor receptor I (TNFRl) does not signals to B cel l lymphoma 2-associated X protein primarily lead to cel l death in most cel l rypes, includ­ 13 (Bax) and B cell lymphoma 2 homologo us antagonist/ ing hepatocytes . After activation of TNFRl , mem­ killer (Bak) activation on mitochondria. Although it is brane-bound complex I is first formed and rapidly sti ll unclear how this activation occurs,s it has become activates survival transcription factor NF_KB .14 To sig­ well accepted that Bax and Bale are essential for mito­ nal for cell death, a second complex, receptor-free chondrial membrane permeabilization (MOMP) and complex II, has to assemble in the and the release of apoptogenic factors such as cytochrome c recruits FADD and caspase-8 to activate caspase-3/cas­ and second mitochondria-derived activator of caspases pase-7.11 Under normal conditions, complex II forma­ (Smac)/diablo homolog (Diablo).6 Although cyto­ tion is blocked by cellular Fas-associating protein with chrome c activates the apoptotic peptidase activating death domain-like interleukjn-l beta-converting enzyme factor I (APAF- l )/caspase-9 apoptosome, which res ults (FLICE) inhibitory protein (c-FLIP) and NF-ICB sur- in effector caspase-3/caspase-7 activation, SmaclDiablo VIV. al s'i gna Img.' 1)' 1(, H owever, t h'IS reg ul'anon can b'e cir - neutrali zes the caspase-9 and cas pase-3 inhibitor X­ cumvented by yet another TNFa-activated apoptotic linked inhibitor of apoptosis protein (XIAP). Recently, signaling pathway that involves activation of c-J un N ­ XIAP has been shown to determine the rype IIIl FasL terminal kinase ONK). It has been shown that JNK signaling switch in hepatocytes and [3-pancreatic celll mediates TNFa-induced apoptotic signaling by the because a large abundance of XIAP requires neutrali za­ phosphorylation and activation of the BH3-only protein tion of its caspase-3-inhibiting activiry by rype II sig­ Bim. U.17 In agreement with this notion, TNFo:-induced naling to allow effective cell death. S. R hepatocyte apoptosis has recently been reported to FasLlCD95L and its corresponding receptor Fas/ require both Bim and Bid in vivo. I R In this study, active CD95 play pivotal roles in the immune system; they caspase-8 generated tBid, whereas active JNK phospho­ induce the death of infected cell s and obsolete lympho­ rylated and stabilized Bim, and the interplay of both cytes and thereby protect against autoimmuniry and tu­ processes was necessary to induce ful l Bax/Bak activa­ 4 mor develo pment. .? Furthermore, Fas is constitutively tion and hepatocyte apoptosis in response to TNFa.

Address reprint requests to: Irmgftrti Me/jim. Ph.D .. Department o/Phflrmacellticftl l3ioiogy flntll3ioteclmoiogy, Aibert-Llldwigs- Univem'ty 0/ Freib'.tlg, Stefi/ll­ Mcier- Stmle 19. 79104 Freiblllg. Germany. E-mflii: irmgflld. mcljim@p/Jflrlllazic. uni-ji-cib/llg.de; fox: +497612038383.

PotCllt;fti conflict of interest: Dr. Brunner owns stock ill Novtlrtis flnd Roche. 284

H ere we show that a similar Bim/Bid interplay is total, cytosoli c, und mitochondrial lysates, western used by TNFa to se nsiti ze primary mouse hepatocytes blotting, quantification of neuroblastoma 2A (N2A) to FasL-induced apoptosis in vitro. We also demon­ FasL, quantification of Vl q TNFa-neutralizing anti­ strate this sensitizing effect toward anti-Fas-induced body, o EVDase, also known as caspase-3 like assay, liver damage in vivo. Although TNFa itself is nona­ assay, 3-(4,5-dimethylthiazol -2-yl) -2,5-di phenyl tetrazo­ poptotic, it markedly enhances FasL-induced hepato­ lium bromide (MIT) viabili ty assay, Cell Death Detec­ cyte apoptosis via both the JNK/Bim and Bid signal­ tion enzyme-linked immunosorbent assay (ELISA) , ing pathways. These data co nfirm that TNFa is RNA isolation, complementary DNA synthesis and capable not only of engaging the JNK/Bim apoptotic qRT-PCR, and cytochrome c ELISA. pathway but also of restoring type II signaling on col­ lagen-cultured primary hepatocytes. This crosstalk is supported by a systems biology approach because we Results present a qualitative mathematical model that correctly TNFrJ. Preincubation of Primary Mouse reproduces the biological findings. Hepatocytes Increases FasL-Induced Caspase-3 Activation and Cell Death in a Materials and Methods Time-Dependent Manner Isolation and Cultivation of Primary Mouse Hep­ We previously reported that FasL induces the apo­ ptosis of coll agen-cultured primary murine hepatocytes atocytes. Primary hepatocytes were isolated from 8- to I 1 I via the type I signaling pathway, but only to a moder­ l2-week-old wild-type (WT), Bid- - , XlAP- - , Fas - - , or FasLgld /gld C57BLl6 mice with the collagenase perfusion ate extent. 12 In this study, we focused on the crosstalk of FasL with the proinflammatory cytokine TNFex. We technique (see the supporting information for details) . preincubated collagen-cultured primary murine hepa­ Induction of Hepatitis and Histology. Young, tocytes with 25 ng/mL TNFa for 12 hours, and this adult WT C57BLl6 mice were injected intravenously was followed by a treatment with 50 ng/mL FasL for with TNFa (40 ftg/kg of body weight; Peprotech), and 6 hours. As expected, untreated and TNFa-treated he­ this was followed by an intravenous injection with an patocytes showed a typical binuclear morphology and anti-Fas antibody (clone J02; BD Bioscience-Pharmin­ no signs of cel l death over an incubation period of 18 gen) at a dose of 80 ltg/kg of body weight 2 hours hours (Fig. 1A) . In contrast, as previously reported, later. Liver damage was assessed 5 hours later by the cells treated with FasL for 6 hours showed hallmarks measurement of the serum aspartate aminotransferase of apoptosis such as cell shrinkage and plas ma mem­ (AST) levels with a commercially available kit (505-0r, 12 brane blebbing. When the ce ll s were preincubated Teco Diagnostics). Five-micrometer liver tissue sections with TNFa for 12 hours before the FasL treatment, were stained with hematoxylin and eosin for histological they underwent a significantly hi gher degree of apo­ assessment. All animals were handled and housed under ptosis (Fig. lA). T hese findings could be confirmed by specific pathogen-free conditions, and animal experi­ the measurement of the effector caspase-3/caspase-7 ac­ ments were reviewed and approved by the animal exper­ tivity in response to the different treatments. As shown imentation review board of the Stare of Bern. in Fig. lB, the longer the hepatocytes were cultured RNA Interference. Smart pools of mouse Bim small (J 2, 24, or 48 hours), the more caspase-3/caspase-7 ac­ interfering RNA (siRNA) duplexes and nontargeting tivity they displayed with a 6-hour FasL treatment. If control duplexes were purchased from Dharmacon during this culturing the cell s were exposed to TNFa, (ON-TARGETplus SMARTpool); the Lipofectamine the caspase-3/caspase-7 activities further increased and RNAiMAX transfection reagent was obtained from were consistently higher than those with FasL alone. In vitrogen. For siRNA transfection, cells were reverse­ Importantly, a minimum preincubation time of-approxi­ transfected with 10 nM siRNA with Lipofectamine in mately 2.5 to 3 hours was needed for TNFa to exe rt its the Opti-MEM medium according to the manufac­ sensitization on FasL-induced caspase-3/caspase-7 activa­ turer's instructions. Effective knockdown was verifi ed tion, and this indicated that the TN Fa effect was not by quantitative real-time polymerase chai n reaction immediate (Fig. 1 C). We also tested the dose depend­ (qRT-PCR) and immunoblotting after di fferent times ence of the sensitization and found that varying the (Supporting Fig. 1) . TNFa concentrations from 10 to 50 ng/mL did not Other Experimental Procedures. Other experimen­ modulate the preincubation time required for sensitiza­ tal procedures are described in detail in the supporting tion (Supporting Fig. 2). Moreover, the sensitization inform ation. These include th e mice, preparation of was clearly caspase-dependent because cell death (as 285

A

phase contrast

80 B control C 40 _ control ~ FasL o TNF D TNFa 60 -~ FasL 30 • TNFa+FasL _ TNF+FasL

~ 40 ~ 20

20 10

0 0 12+6 24+6 48+6 6 0.2+6 0.5+6 1 +6 2+6 3+6 4+6 12+6 time [h] time [h]

Fig. 1. TNFo. sensitizes primary hepatocytes to FasL-induced caspase-3/caspase-7 activation and apoptosis in a tim e-dependent manner. (A) Phase contrast microsco py of primary murine hepatocytes that were untreated (control) or treated with TNFo. (25 ngjmL), N2A FasL (50 ngjmL), or a combination of the two for the indicated times (origina l magnification, 200 x ). (B) Caspase-3/caspase-7 activity determined by a fluoro ­ genic DEVDase assay of ce lls treated with TNF~ FasL, or TNFo. and FasL after the indicated times. Fa sL was added during the last 6 hours of the total treatment time (1 8, 30, and 54 hours, respectively). (C) Kinetics of caspase-3/caspase-7 activation of cells preincubated for different times with TNFo. (FasL treatment for 6 hours). Values represent means of at lea st three independent experiments and standard deviations. *P < 0.001 versus FasL-treated ce lls and #P < 0.05 versus FasL-treated cells (Student t test). measured by the MTT assay) was effectively blocked in optosis sensitization effect of TNFIX is specific for this the presence of a pancaspase inhibitor (quinoli ne-Va l­ cytokine, needs a certain time threshold (as shown in Asp O-phenoxy, non- O-methlyated (q VD-OPh); Sup­ Fig. 1C), and involves a molecular mechanism that porting Fig. 3). cannot be engaged by FasL. To completely exclude the Sensitization of FasL-Induced Apoptosis by TNF(X implication of growth factors, we tested the role of fe­ tal bovine serum (FBS) in the sensitization effect. As Is Specific for TNFrr. and the Opposite Sensitization shown in Supporting Fig. 4, FBS neither enhanced nor Cannot Be Observed. Because other factors in the inhibited the sensitization of FasL-induced apoptosis growth medium may modulate FasL-induced apoptosis by TNF~ but primary hepatocytes turned out to be signaling, we first co nfirmed that the sensitizing effect more se nsitive toward FasL-induced apoptosis in the is specifically mediated by TNFcx. We therefore added 12 presence of FBS (see also Walter et aI. ). TNFIX- neutralizing antibodies produced by the VI q hybridoma cell line (J 00 J1L of the culture superna­ TNF(X Sensitization Is Not Mediated via Tran­ tant) to the primary hepatocytes 30 minutes before scriptional Up-Regulation of Fas 01' FasL but Inter­ TNFIX and FasL stimulation. TNFIX-neutralizing anti­ feres With the Fas Signaling Pathway. To uncover bodies effectively prevented the se nsitization because the molecular mechanism of the TNFIX sensitization, caspase-3/caspase-7 activity did not increase beyond we tested various poss ibilities for TN FIX crosstalk with that measured with FasL alone (Fig. 2A). We then the Fas/FasL system. First, we compared apoptosis tes ted the inverse scenario (i .e., whether FasL was also between WT and Fas 1- hepatocytes to inves tigate I able to sensitize hepatocytes to TNFIX- induced apopto­ the role of Fas. As shown in Fig. 3A, Fas - - hepato­ sis). For that purpose, cells were first treated with cytes did not show any caspase-3/caspase-7 activation FasL, and 2 hours later, TNFIX was added for a total in response to FasL or se nsitization by TNFcx. In con­ of 4 hours before the measurement of active caspase-31 trast, caspase-3/caspase-7 activity levels were unchanged I caspase-7. As demonstrated in Fig. 2B, FasL-induced between WT and Fas - - cel ls when they were treated caspase-3/caspase-7 activity could not be further with TNFlX/actinomycin 0 (ActO), and this indicated increased by TNFcx. This finding co nfirms that the ap- that TNFIX-mediated sensitization to FasL-induced 286

_ WT formed a detailed analys is of the components of the o TNFmAb twO signaling pathways. We recently reported that FasL-induced apoptosis of co ll agen-cultured primary . mouse hepatocytes occurred independently of Bid. T his was in contrast to apoptosis induced by TNFC(/ ActO, which still required Bid (type II signaling). 12 We therefore tes ted whether this was also the case for the sensitization effect of TNFC( on FasL-induced apo­ I ptosis. Indeed, although Bid- - hepatocytes showed the same caspase-3/caspase-7 activation in response to FasL that WT cells showed, the increased caspase-3/caspase-7 activation due to treatment with T NFC( and FasL was entirely abolished (Fig. 4A). Both cell death (based on the MIT assay; Supporting Fig. 5A) and apoptosis-asso­ ciated DNA fragmentation (Supporting Fig. 5B) were I reduced in Bid- - hepatocytes versus WT cells when they were treated with TNFC( and FasL, and this sup­ ported the caspase data. Additionall y, Bid was processed into its active form (tBid) in cells treated with TNFct and FasL, whereas TNFC( alone did not lead to any tB id formation (Fig. 4B) . However, T NFct induced

A Fig. 2. The se nsitizing effect is specifically med iated by TNFa. (A) Ca spase-3/caspase- 7 activity of cells treated with TNFCI, FasL, or a com bination of the two with or without the TN Fa.-neutralizing antibody. ffP < 0.05 versus FasL-treated cells (Student t test). (8) Cells treated in the opposite order (first with FasL for 2 hou rs and then with TNFa. for another 4 hours versus FasL or TNFo: alone for 6 hours). Valu es rep rese nt mea ns of at least th ree ind epende nt experim ents and stand ­ ard deviations. Abbreviation : mAb, monoclonal antibody. apoptosis required Fas. Therefore, we next tested whether sensitization could be due to up-regul ation of endogenous control TNF FasL TN F+FasL TNF+ActD Fas by TNFct However, the qRT-PCR analys is did not _ WT reveal any induction of Fas messe nger RNA (mRNA) in response to TNFC( (data no t shown). Besides Fas, D FasLgldlgld # TNFC( could up-regul ate endogenous FasL and thereby ampli fY the FasL-induced apoptotic response. To test this hypothesis, we analyzed TNFC( sensitization in gld/" ld C F'as L " hepatocytes, w h'I CI 1 express a mutant rorm 0 f FasL that cannot bind Fas. As shown in Fig. 3B, the loss of endogenous FasL production did not signifi­ can t1 y reduce the enhanced caspase-3/ caspase-7 activa­ tion because of TNFC( preincubatio n of the FasL-treated cells. T hese findings indicate that TNFC( impinges on co ntrol TN F FasL TNF+FasL the intracel lular FasL signali ng pathway rather than the regul ation of Fas or FasL in order to sensitize primary Fi g. 3. Fas (but not newly synth esize d Fas L) is essential for TNFo:­ mediated se nsitizatio n. Caspase-3/caspase-7 activity assays of (A) 'NT hepatocytes to FasL-induced apoptosis. and Fas / hepatocytes and (8) 'NT and FasLgld/gld hepatocytes Bid but Not XIAP Is Critical for TNFrx Sensitiza­ treated with TNFo: (25 ng!mL), FasL (50 ng!mL), or a combination of th e two fo r 12 and 6 hou rs. Values represent means of at least three ind e­ tion. Because our findings so fa r suggested direct pendent experim ents and standard deviations. ' P < 0.001 versus FasL­ crosstalk between TNFC( and Fas signaling, we per- treated ce lls and ffP < 0.05 versus FasL-treated ce lls (Stud ent t test). 287

_ WT tured hepatocytes, it is absolutely crucial for the TNFa sensitization of this process. XlAP is an endogenous inhibitor of caspase-9 and effector caspase-3/caspase-7 and restrains apoptosis along the type I pathway unless it is neutralized by apoptogenic factors emanating from mitochondria. Accordingly, as we previously showed, XlAP- /- hepa­ tocytes ex hibited 10-fold higher caspase-3/caspase-7 activity in response to FasL than WT cells (Supporting Fig. 6). T his activity was not further increased by TNFa preincubation. However, a slight sensitization was seen at low FasL doses (10-20 ng/mL). This indi­ B 12+6 12+6 12+6 24+6 24+6 24+6 TNFa 25ng/mL[hj cates that deletion of XlAP does not abrogate the FasL 50 ng/mLl6hj

SP6001 25 25 ~M TNFa sensitization to FasL-induced apoptosis. Impor­ tantly, XlAP protein (Supporting Fig. 7) and mRNA (Supporting Fig. 16C) remained nearly unchanged during TNFa preincubation. Thus, XlAP turned out to be dispensable for the sensitizing effect of TNFa. TNF(J. Activates JNK, and JNK Inhibition Blocks Apoptosis Sensitization by TNFa. Activation of JNK has been implicated in TNFa-induced apoptosis in C 10 several cell types, including hepatocytes. 19,20 We there­ fore monitored the active phosphorylated form of JNK -+- TNFa I 8 by anti-phospho-JNK western blot analysis in primary Q) t; mouse hepatocytes treated with TNFa. TNFa/ActD, .S: 6 g'0 which is known to induce apoptosis by prolonged JNK activation,21 was included as a positive control. We « 4 ex:Z found that TNFa induced early phosphorylation of E '0 JNK in the first 30 minutes, although this was not as 00 2 high as that with TNFa/ ActD after 6 or 8 hours (Fig. 5A) . To investigate the significance of this early JNK activation for apoptosis sensitization, we preincubated 'l­ time [hJ primary hepatocytes with the JNK inhibitor anthra[1-9- cdJpyrazol-6(2H)-one (SP600125; 25{IM), which was Fig. 4. Loss of Bid abrogates the sensitizin g effect, and Bid is pro­ cessed to its active form tBid upon treatment with TNFCI. and FasL. (A) followed by FasL or a consecutive treatment with TNFa easpase-3/caspase-7 activity of WT and Bid - / - hepatocytes treated and FasL. Strikingly, JNK inhibition could effectively with TNFCI. (25 ngjmL). FasL (50 ngjmL), or a combi nation of the two block the sensitizing effect of TNFa on caspase-3/cas­ for 12 and 6 hours. Means of at least three independent experim ents and standard deviations are shown. 'P < 0.001 versus FasL-treated pase-7 activation because DEVDase activity levels in the ce lls (Student t test). (B) Anti-tBid/Bid western blot ana lysis of total presence of SP600 125, TNFa, and FasL were essentially extracts of ce lls treated with TNFCI. and/or FasL for the indicated times the same as those with FasL alone (Fig, 5B), This or additiona lly preincubated with the JNK inhibitor SP600125 (20 decrease in caspase-3/caspase-7 activity resulted in a sig­ pM). Actin is shown as the loading contro l. (e) Time course ana lysis of Bid mRNA expression in response to TNFCI. (25 ngjmL) as meas­ nificant reduction in actual cell death and apoptosis ured by qRT-peR (TaqMan). Values rep resent means of at least three (Supporting Fig, 8), and this supported the role of JNK ind ependent experiments and standard deviations. in the sensitization, In contrast, the p38 mitogen-acti­ vated protein kinase inhibitor RN3503 (10 {1M) had no increased expression of the Bid protein (Fig. 4B) and effect (Supporting Fig, 9), and this indicated that JNK mRNA (Fig. 4C) and this further strengthened a crucial (but not p38 mitogen-activated protein kinase) was cru­ role of this protein in the sensitizing mechanism. Bid cially involved in apoptosis sensitization by TNFa. processing was also observed with FasL alone, but this Bim Is Essential for the Sensitizing Effect of did not contribute to apoptosis induction (Fig. 4A). TNFa. It has recently been reported that Bid and Bim Thus, our results show that although Bid is not are both essential for TNFa-mediated hepatocyte apopto­ 8 required for FasL-induced apoptosis on collagen-cul- sis in vivo. I Furthermore, it is known that the 288

regulated by small interfering RNA targeting Bim 10 20 30 3SO 3SO 480 480 TNFa 25 ng/mL[minl

ActO 0.4 ~glmL {hi (siBim); this was verified by qRT-PCR (Supporting ~----~~~--~~--~~~~----. +-p54 P·JNK Fig, lA) and western blot analysis (Supporting Fig. 1B), respectively, Strikingly, although control siRNA ~;=~i=:::::;======~ +- p46 did not affect caspase-3/caspase-7 activity levels in ce lls JNK treated with TNFa and FasL, siBim significantly reduced them to the levels measured with FasL alone (Fig. 5C). In addition, the loss of Bim resulted in decreased apoptosis"associated DNA fragmentation and cytotoxicity upon treatment with TNFa and FasL (Supporting Fig, 10). Thus, both Bid and Bim seem to be required for the sensitization effect of TNFa on the FasL-induced apoptosis of primary mouse hepato­ cytes. Because JNK is also crucial for this effect and the inhibition of JNK could not abrogate tBid forma­ tion (Fig. 4B), we suggest that the implication of Bim involves its JNK-mediated phosphorylation, as previ- ously shown. 17"2,- ,-':3

TNFrx Sensitization Involves Restoration of TYpe II Signaling in Collagen-Cultured Hepatocytes. Both Bid and Bim relay apoptotic signals to the activation #,' _ control of Bax/Bak, which in turn triggers MOMP and CJ TNF the release of cytochrome c and other apoptogenic fac­ ~ FasL tors (type II signaling). We therefore tested whether _ TNF+FasL TNFa-mediated sensitization to FasL-induced apoptosis involved cytochrome c release. For that purpose, we pre­ pared cytosolic and mitochondrial fractions from TNFa­ treated, FasL-treated, or TNFalFasL-treated hepatocytes, verified their purity by western blot analysis (Supporting Fig. 11), and determined the concentration of cytosolic

150 _ WT Fig, 5, TNFIX activates JNK, and both JNK and its downstream target :::::J E o Bid·l- Bim are crucia l for the sensitization, (A) Western blot ana lysis of P­ 0, JNK after treatment with TNFIX alon e or ActD for the indi cated times, oS 100 (B) easpase-3/caspase-7 activity in response to TNFIX and FasL with l.l Ql or without pretreatment with the JNK inhibitor SP600125 (25 11M) for E 12 and 6 hours, Means of at least three independent experiments o 50 and standard deviations are shown, 'P < 0,001 versus FasL-treated .cu ce lls (Student t test) , (e) easpase-3/caspase-7 activity of primary o mouse hepatocytes not transfected or transfected with control siRNA % or Bim siRNA (10 nM) after treatment with TNFIX (25 ngfmL), FasL (50 ngfmL), or both for 12 and 6 hours, Values represent means of at least three independent experim ents and standard deviations, 'P < 0,001 versus FasL-treated ce lls, " P < 0,01 versus FasL-treated ce ll s, and ffP < 0,05 versus Bim siRNA-transfected ce lls treated with TNFIX and FasL (Student t test), Abbreviation: P-JNK, phosphorylated c-Jun N-terminal kinase, Fig, 6, TNFIX sensitization induces JNK-dependent and Bid -depend­ ent cytochrome c release, The cytochrome c concentration was deter­ proapoptotic activity of Bim can be regulated by JNK­ mined by ELISA in the of WT and Bid - I - hepatocytes after mediated phosphorylation, J 7,22 Consequently, we stud­ treatment with TNFIX (25 ngfmL), FasL (50 ngfmL), or both with or ied the ro le of Bim in the TNFa sensitization mecha­ without pretreatment with the JNK inhibitor SP600125 (25 11M) for 12 and 6 hours, Means of at least three in dependent experiments and nism by down-regulating Bim expression by siRNA. standard deviations are shown, 'P < 0,001 versus Fa sL-treated cells The Bim mRNA and protein were effectively down- and "P < 0,01 versus cells treated with SP600125, TNFCI, and FasL. 289

A

Jo2 TNFa+Jo2

B 20x 100x

Fig. 7. TNFa preincubation se nsiti zes Jo2 mice to anti-Fas-induced liver damage. (A) Liver aminotransferase (AST) serum levels in mice treated with J02 (agonistic anti-Fas antibody; 80 fig/kg of body weight) alone for 5 hours or pretreated for 2 hours with TNFa (40 fig/kg of body weight) before J02 adm inistration. Values represent means and sta ndard deviations (four mice per group). One of two repre­ sentative experiments is shown. (8) His­ to logical ana lysis of liver sections from mice injected with J02 alone or injected TNFa+Jo2 with TNFa 2 hours before J02 administra ­ tion. Low-power and high -power magnifi­ cations of represe ntative sa mples are shown. [Color figure can be viewed in the online issue, which is ava ilab le at wileyonlinelibrary.com.] cytochrome c by ELISA. As shown in Fig. 6, cytochrome observed in vivo, mice were injected with recombinant c could indeed be detected in the cytosol of hepatocytes murine TNFIX followed by anti-Fas antibody 002), treated with TNFIX and FasL, whereas neither TNFIX nor and liver damage was assessed by the measurement of FasL alone promoted any cytochrome c release, as previ­ AST levels. Strikingly, these first experiments revealed ously described. 12 Importantly, cytochrome c release did an increase in AST levels (Fig. 7 A) and tissue damage, I not occur in TNFIX/FasL-treated Bid- - hepatocytes or which was shown by an enhancement of apoptotic when JNK was inhibited (Fig. 6), and this supported the cells (Fig. 7B) when mice were chall enged with TNFIX notion that Bid and JNK were involved in the sensitiza­ and J02 versus J02 administration alone. Before final tion mechanism. These results indicate that TNFIX conclusions can be drawn, further experiments have to enhances FasL-induced apoptosis of collagen-CLutured pri­ be performed. Nevertheless, these results indicate that mary hepatocytes by activating a Bid-dependent and the sensitizing effect reported here could be physiologi­ Bim-dependent type II apoptosis pathway. We also inves­ cally and clinically relevant. tigated whether antiapoptotic Bel2 family members were Mathematical Modeling Confirms the Mechanism modulated during TNFIX sensitization, but neither B cell of Sensitization by TNF(1. A qualitative mathematical lymphoma extra large (Bel-XL) nor myeloid cell leukemia model of the crosstalk between TNFIX and FasL signal­ sequence 1 (Mel-I) levels were up-regulated or down­ ing was built to further analyze the sensitizing mecha­ regulated (Supporting Fig. 12). nism. The model is based on ordinary differential TNF(I. Sensitizes Mice In Vitro to Anti-Fas­ equations using mass action kinetics, and its structure Induced Liver Damage. To test whether the sensitiz­ is illustrated in Fig. 8A. TNFIX and FasL are consid­ ing effect in cultured hepatocytes could also be ered possible model inputs that activate their respective 290

A TNF FAS B 100 80 ~------..... ,. ...., l: 60

ITI '" " ~ ~~=~~;;;;;;~ V Va 0 2 ~ 6 8 14 16 18 20 v" time [h] time[h] fpBlm: Bel2 fiBId: 0 100 E 100 :.~' .. : :.. v 'P. : 801+... ----- ...... , 80 l: ~ l:oo 40 ~ fu fi ~ '" 20 '" 20

O ~O ~2~4~6~8~1 ~0 ~12~14~16~18~2~0 14 16 18 20 time [h] time [h] B·O: pJNK[t} pElim[t] Bc/2[t] Ca'(t] tElid[t] E: wild type XIAP KO Bid KO I FasL: BaxBak'[t] CytC.,,[t] XIAP[t] C3'[t] dashed , circle I TNF+FasL: Joined , star

Fig. 8. Mathematical mod elin g is able to reproduce th e se nsitizing effect. (A) Illustration of the mod el structure. (B·D) Simulation results for wr hepatocytes after stimulation with (B) TN FCI, (C) FasL, or (D) both for 12 and 6 hours. Invisible curves are zero. (E) Simul ation res ults for wr, Bid I ,and XIAP I hepatocytes after stimulation with Fa sL or with TNFa and Fa sL for 12 and 6 hou rs versus the measure ment results of this study. Abbreviations: KO, kn oc kout; cytcfree, cytochrome c released to the cytosol. [Color figure can be viewed in th e onlin e iss ue, which is ava il· ab le at wileyonlinelibrary.com.] pathways ~o co nverge on Bax/Bak activation. We results in cytochrome c release and effector caspase-3/ assume that phosphorylated Bim (pBim) and tBid act caspase-7 activation. Controversial results have so far similarly on Bax/Bak activation but with different pa­ been reported concerning the crosstalk of TNFa and rameters (vG and vd. Both can also be neutralized by FasL in apoptosis induction. On the one hand, TN Fa Bcl2 family members (Bcl 2). In the model, the release has been shown to confer resistance to Fas-induced cell of cytochrome c is realized via a step nmction triggering death in eosinophilic acute myeloid leukemia cell s 100% release at a threshold of 90% Bax/Bak activation. because of its NF-KB-mediated antiapoptotic func­ The model equations, parameter values, and sensitiviry tions.25 In this respect, we analyzed some ryp ical antia­ analysis are provided in the supporting information. poptotic NF-KB target such as cellular inhibitor Si mulation results for WT hepatocytes after treat­ of apoptosis 2 (cIAP2), c-FLIp, and XlAl~ but we ment with TNFa, FasL, or TNFa and FasL are shown found that they were only moderately up-regulated (if in Fig. 8B-D. Analogous simulations are provided in ever) in response to TNFa (see Supporting Fig. 16). I the supporting information for Bid- - and XlAP- I­ clAPl protein was not at all detected in hepatocytes (see 12 cells (Supporting Figs. 13 and 14) . In Fig. 8E, the also Walter et aI. ; data not shown). On the other simulation results for caspase-3 activation are com­ hand, several studies have indicated that TNFa positively pared to the respective measurements for WT and regulates Fas-mediated apoptosis. In one case, TNFa I XlAP- I- and Bid- - hepatocytes. Overall, the model could even overcome the Fas resistance of human lung is able to accurately reproduce the observed sensitizing fi broblasts 2G by allowing more FADD adaptor to bind to effect in all studied genorypes. Fas and therefore increase DISC formation and FasL­ mediated apoptotic signaling. In contrast to human lung Discussion fibroblasts, primary mouse hepatocytes do not seem to have impaired DISC formation because they are quite TNFa is a proinflammatory cytokine that plays a se nsitive to FasL-induced apoptosis. crucial ro le in both liver regeneration24 and liver cell To obtain evidence for the physiological rel evance of 27 apoptosis during disease states. I In this article, we TNFa/FasL crosstalk, Costelli et al. used ge ne target­ report that TNFa sensitizes primary mouse hepato­ ing to show that a loss of TNFRI and TNFR2 pro­ cytes to FasL-induced apoptosis in a Bid-dependent tects mice from anti-Fas antibody-induced liver injury. and Bim-dependent manner. We further show that this O ur results confirm these findings and demonstrate that crosstalk involves JNK activation and most likely Bim TNFa is necessary for efficient FasL-mediated hepato­ phosphorylation, cleavage of Bid, and, consequently, cyte apoptosis. However, the exact mechanism of the activation of the rype I1 mitochondrial pathway and interplay of the two pathways was not unraveled in the 29 1

previous study. It was shown that live r tissue levels of findings have been observed for TNF-related apoptosis­ Fas and FasL as well as Fas expression on the hepatocyte inducing ligand (TRAIL), which enhances Fas-induced surface were unchanged, but Bel2 was up-regulated hepatocyte apoptosis and liver damage via activation of upon TNFRI and TNFR2 depletion; this indicates the JNK-Bim axis 23 ; this suggests some ove rlapping that TNFa may regulate Bel2 family members.27 T his effects of different TNF family members. Our results again is consistent with our finding that nei ther Fas with cultured primary mutine hepatocytes support the up-regulation nor endogenous FasL is critical for the aforementioned mechanism. TNFa preincubation led to TNFa sensitizing effect, and changes in members of JNK activation, and the inhibition of JNK and the loss the Bel2 protein family could be the underlying mech­ of Bim abolished the sensitizing effect; however, FasL­ anisms for the involvement of the type II mitochon­ induced apoptosis remained unchanged. In addition, sen­ drial pathway in the sensitization process. sitization was mitigated by the loss of Bid. In our study, On the other hand, it is widely accepted that TNFa TNFa needs to crosstalk with Fas to exert its apoptosis­ fails to induce apoptosis in hepatocytes under normal sensitizing effect. We recently reported the unexpected conditions because of activation of the NF-/(B survival finding that in co llagen-cultured primary mouse hepato­ pathway. Inhibition of this pathway restores apoptosis, cytes, Fas signaling switches from a rype II, Bid-de­ and one mechanism involves the inducement of sus­ pendent apoptotic signaling pathway to a rype I, Bid­ 2L 28 tained activation of JNK. • This prolonged JNK independent apoptotic signaling pathway. As shown activation has been shown to be crucial for TNFa­ here, TN Fa is obviously able to res tore the rype II sig­ mediated hepatocyte apoptosis but not for Fas. 20 Our naling pathway by a so far unknown mechanism. It findings confirm that TNFa alone does not induce he­ wi ll be crucial to identifY these crosstalk points patocyte apoptosis but, under transcriptional arrest between TNFa and FasL signaling. Our data suggest with ActD, leads to sustained JNK activation critical that Bim and Bid may be part of these points. Both for apoptosis. Interestingly, TNFa also induces early act by triggering Bax/Bak-mediated MOMP and cyto­ transient JNK activation, which by itself does not chrome c rel ease, but perhaps this occurs efficiently directly induce apoptosis but is critical for TNFa­ only when both are indeed present and activated. mediated sensitization to FasL-induced apoptosis. Sev­ TNFa would activate Bim via JNK and regulate Bid eral reports have indicated that JNK modulates the in a so far unknown way such that it becomes required proapoptotic activity of the BH3-only protein Bim by for FasL-induced apoptosis. This would explain why 17 22 phosphorylation. . ,23 T his specific phosphorylation TNFa-induced sensitization is impeded in both Bim causes either the rel ease of Bim from its sequestration knockdown and Bim - /- hepatocytes. We therefore sug­ to the microtubular dynein motor complex or the sta­ gest that Bim and Bid can only cooperatively activate bilization of the Bim protein; both can induce Bax/ the mitochondrial amplification loop in hepatocytes and Bak-dependent apoptosis. However, regulatory phos­ that this is crucial for the observed increased sensitiviry phorylation of Bim by other kinases such as extracellu­ to FasL-induced apoptosis. lar signal-regul ated kinase can induce the opposite T he presented mathematical model accurately repro­ effect and lead to proteasomal degradation and protec­ duces the sensitizing effect and will promote further tion from apoptosis. 29 H ence, the regulation and out­ directions for future research. Sensitivity analysis reveals come of Bim phosphorylation have to be further elarified the sensitizing mechanisms to be very robust, although in hepatocytes through, for example, the identification the model contains only the most important players. of the exact phosphorylation sites and the expression of Most critical interactions for the crosstalk model after phosphorylation-defective Bim mutants. T he role of TNFa and FasL stimulation are the ones associated JNK- mediated Bim phosphorylation in hepatocyte apo­ with Bid and also all reactions associated with Bim (see ptosis has recently been substantiated in vivo. 18 The the supporting information for the model equations). authors showed that lipopolysaccharide/galactosamine­ XIAP has a prominent role as a caspase-3 buffer, and treated mice died because of TNFa-mediated fatal hepa­ the function of Bel2 family members has turned out to titis and demonstrated that this apoptosis was dependent be essential for the model because the sensitizing effect on Bid and Bim. Bim was shown to be phosphorylated is completely disrupted otherwise (Supporting Fig. 15) . by JNK and, co nsequently, redistributed from microtu­ Consequentl y, it would be of special interest to further buies to the cytosol; there, it induced apoptosis in coop­ analyze the specific function and interplay of pBim and eration with caspase-8-cleaved tBid. Remarkably, only other members of the Bel2 fami ly. the loss of both Bid and Bim protected mice from li po­ Because many chronic liver diseases in which FasL polysaccharide/ galactosamine-induced hepati tis. Simil ar levels are elevated are associated with chronic 292 inflammation, the herein reported TNF/FasL crosstalk 10. O gasawara J, W.ltanabe-F" kunaga R, Ad achi M , Matsuzawa A, Kas uga i might be of clinical rel evance. Our first in vivo studies T, Kitamura Y, et al. Lethal effect of the anti-Fas antibody in mi ce. Nature 1993;364:806- 809. showing TNFex sensitizati on toward anti-Fas-induced 11. Yin XM , Wang K, G ross A, Zhao Y, Z in kel 5, Klocke B, et al. Bid­ li ver damage strengthen this ass umption. Elevated defi cient mice are resistant to Fas-induced hepatocell ular apoptosis. T N F levels due to inflammatory processes might affect Nature 1999;400:886- 89 1. 12. Wa lter 0 , Schmich K, Vogel 5, Pick R, Kaufmann T, Hoch muth FC, many acute and chro ni c live r diseases by enhancing et al. Switch fro m rype II to I Fas/C D95 death signaling on in vitro FasL-induced apoptosis signaling and, therefore, might culturing of primary hepatocy tes. H EI'ATO I.OGY 2008;48: 1942- 1953. constitute a poss ibl e therapeutic target. 13. V;" folo meev EE, Ashkenazi A. Tumor necrosis factor: an apoptosis Ju N Kie? Cel l 2004; 11 6:4 9 1-497. Acknowledgment: T he authors thank Fritz von 14. M icheau 0 , Tscho pp J. In d uctio n of T N I' receptor [-mediated apopto­ Weizsacker and Sabine M acNeily (Department of In­ sis via tWO seq uenri al signaling complexes. Cell 2003; 11 4: 18 1- 190. 15. Irmler M, T ho me M, Hahne M, Schneider I ~ Hofmann K, Steiner V, ternal Medicine II, Unive rsity Hospital, Freiburg, Ger­ et al. Inhibition of death recepto r signals by cellular FLI P. Nature many) for the isolation of primary murine hepatocytes 1997;388: 190- 195. and Ka rin N eubert (Institute of Molecular Medicine 16. Ka rin M , Lin A. NF-kappaB at the cross roads of life and death. Nat and Cell Research, Freiburg, Germany) for providing l mmunoI 2002;3:22 1- 227. and quanti fYing N 2A FasL. T hey are grateful to Mar­ 17. Putcha GV, Le 5, Frank 5, Besirli CG, Clark K, C hu B, et al. ) N K­ mediated B1M phospho ry lation po tentia tes BAX-dependent apoptosis. kus Simon (Max-Planck Insti tute, Freiburg, Germany) Neuron 2003;38:899- 91 4. for the Fas -/- and FasUld/gld mice, to Andreas Strasser 18. Kaufm ann T, Jost pJ , Pellegri ni M , Puthalakarh H , G ugasyan R, Ger­ (Walter and Eliza H all Jn stitute of Medical Research, o ndakis 5, et al. Fata l hepatitis med iated by tu mor necrosis fa ctor Melbourne, Australia) for the Bid- /- mice, to John Silke T N Fa lpha req ui res caspase-8 and involves the B1-I 3-o nl y proreins Bid (La Trobe University, Melbourne, Australia) for the and Bi m. Immunity 2009;30:56- 66. 19. Deng Y, Ren X, Ya ng L, Lin Y, Wu X. A J NK-dependent parh way is XlAP- /- mice and the mouse clAPI antibody, to Peter required fo r TNFalpha-induced apoptosis. Cell 2003; 11 5:61- 70. H . Krammer (Germ an Cancer Research Center, H eidel­ 20. Schwabe R1~ Uchinami H, Q ian "C Bennett 13 L, Lemasters )J, Brenner berg, Germany) for the hybridoma cell line producing DA. Diffe rential reguiremelll fo r c-Ju n N H2-rerm inal kinase in TNFalpha­ T NF monoclonal antibody VI q, and to David Huang and Fas-mediated apoptosis in hepatocytes. fo AS EB ) 2004; 18:720-722. (Walter and Eliza Hall Institute of Medical Research, 2 1. Lin A. Activation of the J N K signaling pathway: breaking the brake o n Parkville, Australia) for the monoclonal Bid antibody apoptosis. Bioessays 2003;25 : 17- 24. 22. Lei K, Davis RJ. ) NK phospho rylation of Bim-relared members of the Bcl2 family induces Bax-dependent apoptosis. Proc Nat! Acad Sci U S A 2003; 10 0:2432- 2437. 23. Corazza N, Jakob 5, Schaer C , Frese S, Keogh A, Stroka 0 , et al. References TRAIL receptor-med iated J NK activatio n and Bi m phospho ryla tion I. Malh i H , Gores GJ. Cellular and molecular mechanisms of li ver injury. criti cally regul ate Fas-med iated li ver damage and Ierhali ty. J C lin In vest GastroenterolOb'Y 2008; 134: 164 1- 1654. 2006; 11 6:2493- 2499. 2. Canbay A, Fried man 5, Gores GJ. Ap optosis: the nexus of li ver inj ury 24. Michalopoul os G K. Liver regeneratioll . ) Cell physiol 2007;2 13:286-300. and fibrosis. H Ei'ATOLOC Y 2004;39: 273- 278. 25. Q in Y, Auh 5, Blo kh L, Lo ng C , Gagno n I, Hamann KJ. TNF-alpha 3. Sa ,ffidi C. Ful da 5, Srini vasa n A, Friesen C. l.i F, Tomaselli KJ, et al. T\vo ind uces transient res islal1 ce to Fas ~ in d ll ce d apoprosis in eosin oph ili c C D95 (A PO- IIFns) signa ling pathways . EMBO J 1998; 17:1675- 1687. acute myeloid leukemia cel ls. Cel l Mol Immunol 2007;4:43- 52. 4. Krammer PH. C D95's deadl y miss ion in the immune system. Nature 26. Frankel SK, Cosgrove G p, C ha 51, Cool C D, Wynes M W, Edelman 2000;407:789- 795. BL, et al. TNF-alpha sensitizes no rmal and fibrotic human lung fibro­ 5. Youle Rj, Strasser A. T he BC L-2 protein fam ily: opposing activities blasrs to Fas-induced apoptosis. Am J Res pir Cell Mol Bioi 2006;34: that med iate cell death. Nat Rev Mol Cel l Bioi 2008;9:47- 59. 293- 304. 6. Fletcher )1 , H uang DC. Conrroll ing the cel l dea th med iators Bax and 27. Costelli P, Aoki P, Zingaro B, Carbo N, Reffo P, Lopez-Soriano F) , Bak: puzzles and co nund ru ms. Cell Cycle 2008;7:39-44. et al. Mice lacking TNFalpha receptors 1 and 2 are resistalll to death 7. JOS t pJ , G rabow 5, G ray 0 , McKenzie MD, Nachbur U, H uang D C, and ful minant li ve r inj ury ind uced by ago nistic anri-Fas antibody. Cell et al. XIAp d iscriminates between ty pe I and ty pe II FAS-induced apo­ Death D iffer 2003; 10:997- 1004. ptosis. Nature 2009;460: 10 35- 10 39. 28. Liu H , Lo C R, Czaja MJ. N F-kappaB inhib itio n sensitizes hepatOcytes 8. Gree n DR, Kroemer G. T he pathophysiology of mi tochond ri al cel l to T N F-ind ucecl apoptosis thro ugh a sustained activatio n of J N K and death. Science 2004;305:626- 629. c-Jun. H EP"TOI.OG Y 2002;35:772- 778. 9. Strasser A, Jost pJ , Nagata S. T he many roles of FAS recepto r signaling 29. Ew ings KE, W iggins C M, Cook SJ. Bim and the pro-survival BcI-2 in the immune sys tem. Im muni ty 2009;30: 180- 192. pro teins: opposites attract, ERK repels. Cel l Cycl e 2007;6:2236- 2240.