Enhanced apoptosis is critically involved in many

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1 Tumor Necrosis Factor a Sensitizes Primary Murine Hepatocytes to Fas/CD95-Induced Apoptosis in a Bim- and Bid-Dependent Manner Kathrin Schmich, 1 Rebekka Schlatter, 2 * Nadia Corazza, 3 * Karine Sá Ferreira, 4,5 Michael Ederer, 2 Thomas Brunner, 3,6 Christoph Borner, 4,7,8 and Irmgard Merfort 1 Fas/CD95isacriticalmediatorofcelldeathinmanychronicandacuteliverdiseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the proinflammatory cytokine tumor necrosis factor a (TNFa) fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFa sensitizes primary murine hepatocytes cultured on collagen to Fas ligand (FasL) induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFa. Fas itself is essential for the sensitization, 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 TNFa is clearly dependent on Bid. Moreover, both c- Jun N-terminal kinase (JNK) activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3) only protein, are crucial mediators of TNFa-induced apoptosis sensitization. Bim and Bid activate the mitochondrial amplification loop and induce cytochrome c release, a hallmark of type II apoptosis. The mechanism of TNFa-induced sensitization is supported by a mathematical model that correctly reproduces the biological findings. Finally, our results are physiologically relevant because TNFa also sensitizes to agonistic anti-fas induced liver damage. Conclusion: Our data suggest that TNFa can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3- only proteins Bim and Bid. (HEPATOLOGY 2011;53: ) Enhanced apoptosis is critically involved in many acute and chronic liver diseases, and hepatocytes are the main cell type undergoing massive cell death during liver injury. This process is regulated by a complex network of soluble and cell-associated apoptotic and inflammatory signals. 1 It is therefore increasingly important to obtain insight into the mechanistic interplay of these signals to define new therapeutic strategies. In Abbreviations: ActD, actinomycin D; AST, aspartate aminotransferase; Bak, B cell lymphoma 2 homologous antagonist/killer; Bax, B cell lymphoma 2 associated X protein; Bcl2, B cell lymphoma 2; BH3, B cell lymphoma 2 homology domain 3; c-flip, cellular Fas-associating protein with death domain-like interleukin-1 beta-converting enzyme (FLICE) inhibitory protein; ciap, cellular inhibitor of apoptosis; Diablo, diablo homolog; DISC, death-inducing signaling complex; ELISA, enzyme-linked immunosorbent assay; FADD, Fas-associated death domain; FasL, Fas ligand; FBS, fetal bovine serum; JNK, c-jun N-terminal kinase; KO, knockout; mab, monoclonal antibody; MOMP, mitochondrial membrane permeabilization; mrna, messenger RNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; N2A, neuroblastoma 2A; NF-jB, nuclear factor kappa B; P-JNK, phosphorylated c-jun N-terminal kinase; pbim, phosphorylated Bim; qrt-pcr, quantitative real-time polymerase chain reaction; sibim, small interfering RNA targeting Bim; sirna, small interfering RNA; Smac, second mitochondria-derived activator of caspases; SP600125, anthra[1-9-cd]pyrazol-6(2h)-one; tbid, truncated Bid; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; WT, wild type; XIAP, X-linked inhibitor of apoptosis protein. From the 1 Department of Pharmaceutical Biology and Biotechnology, Albert- Ludwigs- University of Freiburg, Freiburg, Germany, 2 Institute for System Dynamics, University of Stuttgart, Stuttgart, Germany; 3 Biochemical Pharmacology, Department of Biology, University of Konstanz, Konstanz, Germany; 4 Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University of Freiburg, Freiburg, Germany, 5 Faculty of Biology, Albert-Ludwigs-University of Freiburg, Freiburg, Germany, 6 Division of Immunopathology, Institute of Pathology, University of Bern, Bern, Switzerland; 7 Spemann Graduate School of Biology and Medicine, and and 8 Center for Biological Signaling Studies, Albert-Ludwigs-University of Freiburg, Freiburg, Germany. Received January 26, 2010; accepted September 8, This work was supported by the Federal Ministry of Education and Research through a research grant ( D) from HepatoSys (to Kathrin Schmich, Rebekka Schlatter, Karine Sá Ferreira, Michael Ederer, Christoph Borner, and Irmgard Merfort). This work was also supported by the Spemann Graduate School of Biology and Medicine (to Christoph Borner) and by the Bioss Excellence Cluster (to Christoph Borner); both are programs of the Excellence Initiative funded by the German Research Foundation. Karine Sá Ferreira is supported by the German Research Foundation (GRK1104), and Thomas Brunner and Nadia Corazza are supported by the Swiss National Science Foundation ( and ). *These authors contributed equally to this work. 282

2 HEPATOLOGY, Vol. 53, No. 1, 2011 SCHMICH ET AL. 283 the liver, apoptosis is mainly initiated by the death receptor ligands Fas ligand (FasL; CD95L) and tumor necrosis factor a (TNFa). 2 After ligand binding, death receptors recruit the adaptor Fas-associated death domain (FADD) and procaspase-8 to their intracellular face, and this forms the death-inducing signaling complex (DISC). 3 By this assembly, procaspase-8 is autoprocessed and activated, and it can then trigger two different apoptotic signaling pathways. In so-called type I cells, such as lymphocytes, active caspase-8 directly cleaves and activates procaspase-3 to induce efficient cell death execution. 4 In type II cells, such as hepatocytes, apoptosis induction first requires caspase-8 mediated cleavage of BH3-interacting domain death agonist (Bid) into its truncated form [truncated Bid (tbid)]. tbid belongs to the subclass of B cell lymphoma 2 homology domain 3 (BH3) only B cell lymphoma 2 (Bcl2) family members (e.g., Bcl2-like protein 11 [Bim], p53 up-regulated modulator of apoptosis (Puma), and Noxa), which sense apoptotic stimuli and convey the death signals to B cell lymphoma 2 associated X protein (Bax) and B cell lymphoma 2 homologous antagonist/ killer (Bak) activation on mitochondria. Although it is still unclear how this activation occurs, 5 it has become well accepted that Bax and Bak are essential for mitochondrial membrane permeabilization (MOMP) and the release of apoptogenic factors such as cytochrome c and second mitochondria-derived activator of caspases (Smac)/diablo homolog (Diablo). 6 Although cytochrome c activates the apoptotic peptidase activating factor 1 (APAF-1)/caspase-9 apoptosome, which results in effector caspase-3/caspase-7 activation, Smac/Diablo neutralizes the caspase-9 and caspase-3 inhibitor X- linked inhibitor of apoptosis protein (XIAP). Recently, XIAP has been shown to determine the type I/II FasL signaling switch in hepatocytes and b-pancreatic cells 7 because a large abundance of XIAP requires neutralization of its caspase-3 inhibiting activity by type II signaling to allow effective cell death. 5,8 FasL/CD95L and its corresponding receptor Fas/ CD95 play pivotal roles in the immune system; they induce the death of infected cells and obsolete lymphocytes and thereby protect against autoimmunity and tumor development. 4,9 Furthermore, Fas is constitutively expressed on the surface of hepatocytes and is important to hepatic health and disease. Mice treated with a lethal dose of agonistic anti-fas antibody die because of massive hepatocyte apoptosis and liver failure. 10 This cell death is dependent on Bid because Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis, fulminant hepatitis, and subsequent liver failure. 11 These findings indicate that in vivo hepatocytes die in response to FasL via the type II signaling pathway. 7 However, we have shown recently that isolated primary hepatocytes cultured on collagen change their apoptosis signaling from type II to the Bid-independent type I pathway, 12 and this suggests that the type II/I decision depends not only on the expression of endogenous proteins, such as XIAP, but also on external factors. TNFa is a pleiotropic cytokine that induces a variety of cellular responses, such as inflammation and cell proliferation, mainly through activation of the nuclear factor kappa B (NF-jB) signaling cascade. Unlike FasL, the association of TNFa with its main receptor tumor necrosis factor receptor 1 (TNFR1) does not primarily lead to cell death in most cell types, including hepatocytes. 13 After activation of TNFR1, membrane-bound complex I is first formed and rapidly activates survival transcription factor NF-jB. 14 To signal for cell death, a second complex, receptor-free complex II, has to assemble in the cytoplasm and recruits FADD and caspase-8 to activate caspase-3/caspase Under normal conditions, complex II formation is blocked by cellular Fas-associating protein with death domain-like interleukin-1 beta-converting enzyme (FLICE) inhibitory protein (c-flip) and NF-jB survival signaling. 15,16 However, this regulation can be circumvented by yet another TNFa-activated apoptotic signaling pathway that involves activation of c-jun N- terminal kinase (JNK). It has been shown that JNK mediates TNFa-induced apoptotic signaling by the phosphorylation and activation of the BH3-only protein Bim. 13,17 In agreement with this notion, TNFa-induced hepatocyte apoptosis has recently been reported to require both Bim and Bid in vivo. 18 In this study, active caspase-8 generated tbid, whereas active JNK phosphorylated and stabilized Bim, and the interplay of both processes was necessary to induce full Bax/Bak activation and hepatocyte apoptosis in response to TNFa. Address reprint requests to: Irmgard Merfort, Ph.D., Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-University of Freiburg, Stefan- Meier- Straße 19, Freiburg, Germany. irmgard.merfort@pharmazie.uni-freiburg.de; fax: þ Copyright VC 2010 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI /hep Potential conflict of interest: Dr. Brunner owns stock in Novartis and Roche. Additional Supporting Information may be found in the online version of this article.

3 284 SCHMICH ET AL. HEPATOLOGY, January 2011 Here we show that a similar Bim/Bid interplay is used by TNFa to sensitize primary mouse hepatocytes to FasL-induced apoptosis in vitro. We also demonstrate this sensitizing effect toward anti-fas induced liver damage in vivo. Although TNFa itself is nonapoptotic, it markedly enhances FasL-induced hepatocyte apoptosis via both the JNK/Bim and Bid signaling pathways. These data confirm that TNFa is capable not only of engaging the JNK/Bim apoptotic pathway but also of restoring type II signaling on collagen-cultured primary hepatocytes. This crosstalk is supported by a systems biology approach because we present a qualitative mathematical model that correctly reproduces the biological findings. Materials and Methods Isolation and Cultivation of Primary Mouse Hepatocytes. Primary hepatocytes were isolated from 8- to 12-week-old wild-type (WT), Bid /,XIAP /,Fas /, or FasL gld/gld C57BL/6 mice with the collagenase perfusion technique (see the supporting information for details). Induction of Hepatitis and Histology. Young, adult WT C57BL/6 mice were injected intravenously with TNFa (40 lg/kg of body weight; Peprotech), and this was followed by an intravenous injection with an anti-fas antibody (clone Jo2; BD Bioscience-Pharmingen) at a dose of 80 lg/kg of body weight 2 hours later. Liver damage was assessed 5 hours later by the measurement of the serum aspartate aminotransferase (AST) levels with a commercially available kit (505-OP, Teco Diagnostics). Five-micrometer liver tissue sections were stained with hematoxylin and eosin for histological assessment. All animals were handled and housed under specific pathogen-free conditions, and animal experiments were reviewed and approved by the animal experimentation review board of the State of Bern. RNA Interference. Smart pools of mouse Bim small interfering RNA (sirna) duplexes and nontargeting control duplexes were purchased from Dharmacon (ON-TARGETplus SMARTpool); the Lipofectamine RNAiMAX transfection reagent was obtained from Invitrogen. For sirna transfection, cells were reversetransfected with 10 nm sirna with Lipofectamine in the Opti-MEM medium according to the manufacturer s instructions. Effective knockdown was verified by quantitative real-time polymerase chain reaction (qrt-pcr) and immunoblotting after different times (Supporting Fig. 1). Other Experimental Procedures. Other experimental procedures are described in detail in the supporting information. These include the mice, preparation of total, cytosolic, und mitochondrial lysates, western blotting, quantification of neuroblastoma 2A (N2A) FasL, quantification of V1q TNFa-neutralizing antibody, DEVDase, also known as caspase-3 like assay, assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay, Cell Death Detection enzyme-linked immunosorbent assay (ELISA), RNA isolation, complementary DNA synthesis and qrt-pcr, and cytochrome c ELISA. Results TNFa Preincubation of Primary Mouse Hepatocytes Increases FasL-Induced Caspase-3 Activation and Cell Death in a Time-Dependent Manner We previously reported that FasL induces the apoptosis of collagen-cultured primary murine hepatocytes via the type I signaling pathway, but only to a moderate extent. 12 In this study, we focused on the crosstalk of FasL with the proinflammatory cytokine TNFa. We preincubated collagen-cultured primary murine hepatocytes with 25 ng/ml TNFa for 12 hours, and this was followed by a treatment with 50 ng/ml FasL for 6 hours. As expected, untreated and TNFa-treated hepatocytes showed a typical binuclear morphology and no signs of cell death over an incubation period of 18 hours (Fig. 1A). In contrast, as previously reported, cells treated with FasL for 6 hours showed hallmarks of apoptosis such as cell shrinkage and plasma membrane blebbing. 12 When the cells were preincubated with TNFa for 12 hours before the FasL treatment, they underwent a significantly higher degree of apoptosis (Fig. 1A). These findings could be confirmed by the measurement of the effector caspase-3/caspase-7 activity in response to the different treatments. As shown in Fig. 1B, the longer the hepatocytes were cultured (12, 24, or 48 hours), the more caspase-3/caspase-7 activity they displayed with a 6-hour FasL treatment. If during this culturing the cells were exposed to TNFa, the caspase-3/caspase-7 activities further increased and were consistently higher than those with FasL alone. Importantly, a minimum preincubation time of approximately 2.5 to 3 hours was needed for TNFa to exert its sensitization on FasL-induced caspase-3/caspase-7 activation, and this indicated that the TNFa effect was not immediate (Fig. 1C). We also tested the dose dependence of the sensitization and found that varying the TNFa concentrations from 10 to 50 ng/ml did not modulate the preincubation time required for sensitization (Supporting Fig. 2). Moreover, the sensitization was clearly caspase-dependent because cell death (as

4 HEPATOLOGY, Vol. 53, No. 1, 2011 SCHMICH ET AL. 285 Fig. 1. TNFa sensitizes primary hepatocytes to FasL-induced caspase-3/caspase-7 activation and apoptosis in a time-dependent manner. (A) Phase contrast microscopy of primary murine hepatocytes that were untreated (control) or treated with TNFa (25 ng/ml), N2A FasL (50 ng/ml), or a combination of the two for the indicated times (original magnification, 200). (B) Caspase-3/caspase-7 activity determined by a fluorogenic DEVDase assay of cells treated with TNFa, FasL, or TNFa and FasL after the indicated times. FasL was added during the last 6 hours of the total treatment time (18, 30, and 54 hours, respectively). (C) Kinetics of caspase-3/caspase-7 activation of cells preincubated for different times with TNFa (FasL treatment for 6 hours). Values represent means of at least three independent experiments and standard deviations. *P < versus FasL-treated cells and #P < 0.05 versus FasL-treated cells (Student t test). measured by the MTT assay) was effectively blocked in the presence of a pancaspase inhibitor (quinoline-val- Asp O-phenoxy, non O-methlyated (qvd-oph); Supporting Fig. 3). Sensitization of FasL-Induced Apoptosis by TNFa Is Specific for TNFa, and the Opposite Sensitization Cannot Be Observed. Because other factors in the growth medium may modulate FasL-induced apoptosis signaling, we first confirmed that the sensitizing effect is specifically mediated by TNFa. We therefore added TNFa-neutralizing antibodies produced by the V1q hybridoma cell line (100 ll of the culture supernatant) to the primary hepatocytes 30 minutes before TNFa and FasL stimulation. TNFa-neutralizing antibodies effectively prevented the sensitization because caspase-3/caspase-7 activity did not increase beyond that measured with FasL alone (Fig. 2A). We then tested the inverse scenario (i.e., whether FasL was also able to sensitize hepatocytes to TNFa-induced apoptosis). For that purpose, cells were first treated with FasL, and 2 hours later, TNFa was added for a total of 4 hours before the measurement of active caspase-3/ caspase-7. As demonstrated in Fig. 2B, FasL-induced caspase-3/caspase-7 activity could not be further increased by TNFa. This finding confirms that the apoptosis sensitization effect of TNFa is specific for this cytokine, needs a certain time threshold (as shown in Fig. 1C), and involves a molecular mechanism that cannot be engaged by FasL. To completely exclude the implication of growth factors, we tested the role of fetal bovine serum (FBS) in the sensitization effect. As shown in Supporting Fig. 4, FBS neither enhanced nor inhibited the sensitization of FasL-induced apoptosis by TNFa, but primary hepatocytes turned out to be more sensitive toward FasL-induced apoptosis in the presence of FBS (see also Walter et al. 12 ). TNFa Sensitization Is Not Mediated via Transcriptional Up-Regulation of Fas or FasL but Interferes With the Fas Signaling Pathway. To uncover the molecular mechanism of the TNFa sensitization, we tested various possibilities for TNFa crosstalk with the Fas/FasL system. First, we compared apoptosis between WT and Fas / hepatocytes to investigate the role of Fas. As shown in Fig. 3A, Fas / hepatocytes did not show any caspase-3/caspase-7 activation in response to FasL or sensitization by TNFa. In contrast, caspase-3/caspase-7 activity levels were unchanged between WT and Fas / cells when they were treated with TNFa/actinomycin D (ActD), and this indicated that TNFa-mediated sensitization to FasL-induced

5 286 SCHMICH ET AL. HEPATOLOGY, January 2011 apoptosis required Fas. Therefore, we next tested whether sensitization could be due to up-regulation of endogenous Fas by TNFa. However, the qrt-pcr analysis did not reveal any induction of Fas messenger RNA (mrna) in response to TNFa (data not shown). Besides Fas, TNFa could up-regulate endogenous FasL and thereby amplify the FasL-induced apoptotic response. To test this hypothesis, we analyzed TNFa sensitization in FasL gld/gld hepatocytes, which express a mutant form of FasL that cannot bind Fas. As shown in Fig. 3B, the loss of endogenous FasL production did not significantly reduce the enhanced caspase-3/caspase-7 activation because of TNFa preincubation of the FasL-treated cells. These findings indicate that TNFa impinges on the intracellular FasL signaling pathway rather than the regulation of Fas or FasL in order to sensitize primary hepatocytes to FasL-induced apoptosis. Bid but Not XIAP Is Critical for TNFa Sensitization. Because our findings so far suggested direct crosstalk between TNFa and Fas signaling, we performed a detailed analysis of the components of the two signaling pathways. We recently reported that FasL-induced apoptosis of collagen-cultured primary mouse hepatocytes occurred independently of Bid. This was in contrast to apoptosis induced by TNFa/ ActD, which still required Bid (type II signaling). 12 We therefore tested whether this was also the case for the sensitization effect of TNFa on FasL-induced apoptosis. 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 TNFa and FasL was entirely abolished (Fig. 4A). Both cell death (based on the MTT assay; Supporting Fig. 5A) and apoptosis-associated DNA fragmentation (Supporting Fig. 5B) were reduced in Bid / hepatocytes versus WT cells when they were treated with TNFa and FasL, and this supported the caspase data. Additionally, Bid was processed into its active form (tbid) in cells treated with TNFa and FasL, whereas TNFa alone did not lead to any tbid formation (Fig. 4B). However, TNFa induced Fig. 2. The sensitizing effect is specifically mediated by TNFa. (A) Caspase-3/caspase-7 activity of cells treated with TNFa, FasL, or a combination of the two with or without the TNFa-neutralizing antibody. #P < 0.05 versus FasL-treated cells (Student t test). (B) Cells treated in the opposite order (first with FasL for 2 hours and then with TNFa for another 4 hours versus FasL or TNFa alone for 6 hours). Values represent means of at least three independent experiments and standard deviations. Abbreviation: mab, monoclonal antibody. Fig. 3. Fas (but not newly synthesized FasL) is essential for TNFamediated sensitization. Caspase-3/caspase-7 activity assays of (A) WT and Fas / hepatocytes and (B) WT and FasL gld/gld hepatocytes treated with TNFa (25 ng/ml), FasL (50 ng/ml), or a combination of the two for 12 and 6 hours. Values represent means of at least three independent experiments and standard deviations. *P < versus FasLtreated cells and #P < 0.05 versus FasL-treated cells (Student t test).

6 HEPATOLOGY, Vol. 53, No. 1, 2011 SCHMICH ET AL. 287 Fig. 4. Loss of Bid abrogates the sensitizing effect, and Bid is processed to its active form tbid upon treatment with TNFa and FasL. (A) Caspase-3/caspase-7 activity of WT and Bid / hepatocytes treated with TNFa (25 ng/ml), FasL (50 ng/ml), or a combination of the two for 12 and 6 hours. Means of at least three independent experiments and standard deviations are shown. *P < versus FasL-treated cells (Student t test). (B) Anti-tBid/Bid western blot analysis of total extracts of cells treated with TNFa and/or FasL for the indicated times or additionally preincubated with the JNK inhibitor SP (20 lm). Actin is shown as the loading control. (C) Time course analysis of Bid mrna expression in response to TNFa (25 ng/ml) as measured by qrt-pcr (TaqMan). Values represent means of at least three independent experiments and standard deviations. increased expression of the Bid protein (Fig. 4B) and mrna (Fig. 4C) and this further strengthened a crucial role of this protein in the sensitizing mechanism. Bid processing was also observed with FasL alone, but this did not contribute to apoptosis induction (Fig. 4A). Thus, our results show that although Bid is not required for FasL-induced apoptosis on collagen-cultured hepatocytes, it is absolutely crucial for the TNFa sensitization of this process. XIAP 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, XIAP / hepatocytes exhibited 10-fold higher caspase-3/caspase-7 activity in response to FasL than WT cells (Supporting Fig. 6). This activity was not further increased by TNFa preincubation. However, a slight sensitization was seen at low FasL doses (10-20 ng/ml). This indicates that deletion of XIAP does not abrogate the TNFa sensitization to FasL-induced apoptosis. Importantly, XIAP protein (Supporting Fig. 7) and mrna (Supporting Fig. 16C) remained nearly unchanged during TNFa preincubation. Thus, XIAP turned out to be dispensable for the sensitizing effect of TNFa. TNFa Activates JNK, and JNK Inhibition Blocks Apoptosis Sensitization by TNFa. Activation of JNK has been implicated in TNFa-induced apoptosis in several cell types, including hepatocytes. 19,20 We therefore monitored the active phosphorylated form of JNK by anti phospho-jnk western blot analysis in primary mouse hepatocytes treated with TNFa. TNFa/ActD, which is known to induce apoptosis by prolonged JNK activation, 21 was included as a positive control. We found that TNFa induced early phosphorylation of JNK in the first 30 minutes, although this was not as 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 primary hepatocytes with the JNK inhibitor anthra[1-9- cd]pyrazol-6(2h)-one (SP600125; 25lM), which was followed by FasL or a consecutive treatment with TNFa and FasL. Strikingly, JNK inhibition could effectively block the sensitizing effect of TNFa on caspase-3/caspase-7 activation because DEVDase activity levels in the presence of SP600125, TNFa, and FasL were essentially the same as those with FasL alone (Fig. 5B). This decrease in caspase-3/caspase-7 activity resulted in a significant reduction in actual cell death and apoptosis (Supporting Fig. 8), and this supported the role of JNK in the sensitization. In contrast, the p38 mitogen-activated protein kinase inhibitor RN3503 (10 lm) had no effect (Supporting Fig. 9), and this indicated that JNK (but not p38 mitogen-activated protein kinase) was crucially involved in apoptosis sensitization by TNFa. Bim Is Essential for the Sensitizing Effect of TNFa. It has recently been reported that Bid and Bim are both essential for TNFa-mediated hepatocyte apoptosis in vivo. 18 Furthermore, it is known that the

7 288 SCHMICH ET AL. HEPATOLOGY, January 2011 proapoptotic activity of Bim can be regulated by JNKmediated phosphorylation. 17,22 Consequently, we studied the role of Bim in the TNFa sensitization mechanism by down-regulating Bim expression by sirna. The Bim mrna and protein were effectively downregulated by small interfering RNA targeting Bim (sibim); this was verified by qrt-pcr (Supporting Fig. 1A) and western blot analysis (Supporting Fig. 1B), respectively. Strikingly, although control sirna did not affect caspase-3/caspase-7 activity levels in cells 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 hepatocytes. Because JNK is also crucial for this effect and the inhibition of JNK could not abrogate tbid formation (Fig. 4B), we suggest that the implication of Bim involves its JNK-mediated phosphorylation, as previously shown. 17,22,23 TNFa Sensitization Involves Restoration of Type II Signaling in Collagen-Cultured Hepatocytes. Both Bid and Bim relay apoptotic signals to the activation of Bax/Bak, which in turn triggers MOMP and the release of cytochrome c and other apoptogenic factors (type II signaling). We therefore tested whether TNFa-mediated sensitization to FasL-induced apoptosis involved cytochrome c release. For that purpose, we prepared cytosolic and mitochondrial fractions from TNFatreated, FasL-treated, or TNFa/FasL-treated hepatocytes, verified their purity by western blot analysis (Supporting Fig. 11), and determined the concentration of cytosolic Fig. 5. TNFa activates JNK, and both JNK and its downstream target Bim are crucial for the sensitization. (A) Western blot analysis of P- JNK after treatment with TNFa alone or ActD for the indicated times. (B) Caspase-3/caspase-7 activity in response to TNFa and FasL with or without pretreatment with the JNK inhibitor SP (25 lm) for 12 and 6 hours. Means of at least three independent experiments and standard deviations are shown. *P < versus FasL-treated cells (Student t test). (C) Caspase-3/caspase-7 activity of primary mouse hepatocytes not transfected or transfected with control sirna or Bim sirna (10 nm) after treatment with TNFa (25 ng/ml), FasL (50 ng/ml), or both for 12 and 6 hours. Values represent means of at least three independent experiments and standard deviations. *P < versus FasL-treated cells, **P < 0.01 versus FasL-treated cells, and #P < 0.05 versus Bim sirna transfected cells treated with TNFa and FasL (Student t test). Abbreviation: P-JNK, phosphorylated c-jun N-terminal kinase. Fig. 6. TNFa sensitization induces JNK-dependent and Bid-dependent cytochrome c release. The cytochrome c concentration was determined by ELISA in the cytosol of WT and Bid / hepatocytes after treatment with TNFa (25 ng/ml), FasL (50 ng/ml), or both with or without pretreatment with the JNK inhibitor SP (25 lm) for 12 and 6 hours. Means of at least three independent experiments and standard deviations are shown. *P < versus FasL-treated cells and **P < 0.01 versus cells treated with SP600125, TNFa, and FasL.

8 HEPATOLOGY, Vol. 53, No. 1, 2011 SCHMICH ET AL. 289 Fig. 7. TNFa preincubation sensitizes mice to anti-fas induced liver damage. (A) Liver aminotransferase (AST) serum levels in mice treated with Jo2 (agonistic anti-fas antibody; 80 lg/kg of body weight) alone for 5 hours or pretreated for 2 hours with TNFa (40 lg/kg of body weight) before Jo2 administration. Values represent means and standard deviations (four mice per group). One of two representative experiments is shown. (B) Histological analysis of liver sections from mice injected with Jo2 alone or injected with TNFa 2 hours before Jo2 administration. Low-power and high-power magnifications of representative samples are shown. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] cytochrome c by ELISA. As shown in Fig. 6, cytochrome c could indeed be detected in the cytosol of hepatocytes treated with TNFa and FasL, whereas neither TNFa nor FasL alone promoted any cytochrome c release, as previously described.12 Importantly, cytochrome c release did not occur in TNFa/FasL-treated Bid / hepatocytes or when JNK was inhibited (Fig. 6), and this supported the notion that Bid and JNK were involved in the sensitization mechanism. These results indicate that TNFa enhances FasL-induced apoptosis of collagen-cultured primary hepatocytes by activating a Bid-dependent and Bim-dependent type II apoptosis pathway. We also investigated whether antiapoptotic Bcl2 family members were modulated during TNFa sensitization, but neither B cell lymphoma extra large (Bcl-XL) nor myeloid cell leukemia sequence 1 (Mcl-1) levels were up-regulated or downregulated (Supporting Fig. 12). TNFa Sensitizes Mice In Vitro to Anti-Fas Induced Liver Damage. To test whether the sensitizing effect in cultured hepatocytes could also be observed in vivo, mice were injected with recombinant murine TNFa followed by anti-fas antibody (Jo2), and liver damage was assessed by the measurement of AST levels. Strikingly, these first experiments revealed an increase in AST levels (Fig. 7A) and tissue damage, which was shown by an enhancement of apoptotic cells (Fig. 7B) when mice were challenged with TNFa and Jo2 versus Jo2 administration alone. Before final conclusions can be drawn, further experiments have to be performed. Nevertheless, these results indicate that the sensitizing effect reported here could be physiologically and clinically relevant. Mathematical Modeling Confirms the Mechanism of Sensitization by TNFa. A qualitative mathematical model of the crosstalk between TNFa and FasL signaling was built to further analyze the sensitizing mechanism. The model is based on ordinary differential equations using mass action kinetics, and its structure is illustrated in Fig. 8A. TNFa and FasL are considered possible model inputs that activate their respective

9 290 SCHMICH ET AL. HEPATOLOGY, January 2011 Fig. 8. Mathematical modeling is able to reproduce the sensitizing effect. (A) Illustration of the model structure. (B-D) Simulation results for WT hepatocytes after stimulation with (B) TNFa, (C) FasL, or (D) both for 12 and 6 hours. Invisible curves are zero. (E) Simulation results for WT, Bid /, and XIAP / hepatocytes after stimulation with FasL or with TNFa and FasL for 12 and 6 hours versus the measurement results of this study. Abbreviations: KO, knockout; cytcfree, cytochrome c released to the cytosol. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] pathways to converge on Bax/Bak activation. We assume that phosphorylated Bim (pbim) and tbid act similarly on Bax/Bak activation but with different parameters (v 6 and v 12 ). Both can also be neutralized by Bcl2 family members (Bcl2). In the model, the release of cytochrome c is realized via a step function triggering 100% release at a threshold of 90% Bax/Bak activation. The model equations, parameter values, and sensitivity analysis are provided in the supporting information. Simulation results for WT hepatocytes after treatment with TNFa, FasL, or TNFa and FasL are shown in Fig. 8B-D. Analogous simulations are provided in the supporting information for Bid / and XIAP / cells (Supporting Figs. 13 and 14). In Fig. 8E, the simulation results for caspase-3 activation are compared to the respective measurements for WT and XIAP / and Bid / hepatocytes. Overall, the model is able to accurately reproduce the observed sensitizing effect in all studied genotypes. Discussion TNFa is a proinflammatory cytokine that plays a crucial role in both liver regeneration 24 and liver cell apoptosis during disease states. 1 In this article, we report that TNFa sensitizes primary mouse hepatocytes to FasL-induced apoptosis in a Bid-dependent and Bim-dependent manner. We further show that this crosstalk involves JNK activation and most likely Bim phosphorylation, cleavage of Bid, and, consequently, activation of the type II mitochondrial pathway and results in cytochrome c release and effector caspase-3/ caspase-7 activation. Controversial results have so far been reported concerning the crosstalk of TNFa and FasL in apoptosis induction. On the one hand, TNFa has been shown to confer resistance to Fas-induced cell death in eosinophilic acute myeloid leukemia cells because of its NF-jB mediated antiapoptotic functions. 25 In this respect, we analyzed some typical antiapoptotic NF-jB target genes such as cellular inhibitor of apoptosis 2 (ciap2), c-flip, and XIAP, but we found that they were only moderately up-regulated (if ever) in response to TNFa (see Supporting Fig. 16). ciap1 protein was not at all detected in hepatocytes (see also Walter et al. 12 ; data not shown). On the other hand, several studies have indicated that TNFa positively regulates Fas-mediated apoptosis. In one case, TNFa could even overcome the Fas resistance of human lung fibroblasts 26 by allowing more FADD adaptor to bind to Fas and therefore increase DISC formation and FasLmediated apoptotic signaling. In contrast to human lung fibroblasts, primary mouse hepatocytes do not seem to have impaired DISC formation because they are quite sensitive to FasL-induced apoptosis. To obtain evidence for the physiological relevance of TNFa/FasL crosstalk, Costelli et al. 27 used gene targeting to show that a loss of TNFR1 and TNFR2 protects mice from anti-fas antibody induced liver injury. Our results confirm these findings and demonstrate that TNFa is necessary for efficient FasL-mediated hepatocyte apoptosis. However, the exact mechanism of the interplay of the two pathways was not unraveled in the

10 HEPATOLOGY, Vol. 53, No. 1, 2011 SCHMICH ET AL. 291 previous study. It was shown that liver tissue levels of Fas and FasL as well as Fas expression on the hepatocyte surface were unchanged, but Bcl2 was up-regulated upon TNFR1 and TNFR2 depletion; this indicates that TNFa may regulate Bcl2 family members. 27 This again is consistent with our finding that neither Fas up-regulation nor endogenous FasL is critical for the TNFa sensitizing effect, and changes in members of the Bcl2 protein family could be the underlying mechanisms for the involvement of the type II mitochondrial pathway in the sensitization process. On the other hand, it is widely accepted that TNFa fails to induce apoptosis in hepatocytes under normal conditions because of activation of the NF-jB survival pathway. Inhibition of this pathway restores apoptosis, and one mechanism involves the inducement of sustained activation of JNK. 21,28 This prolonged JNK activation has been shown to be crucial for TNFamediated hepatocyte apoptosis but not for Fas. 20 Our findings confirm that TNFa alone does not induce hepatocyte apoptosis but, under transcriptional arrest with ActD, leads to sustained JNK activation critical for apoptosis. Interestingly, TNFa also induces early transient JNK activation, which by itself does not directly induce apoptosis but is critical for TNFamediated sensitization to FasL-induced apoptosis. Several reports have indicated that JNK modulates the proapoptotic activity of the BH3-only protein Bim by phosphorylation. 17,22,23 This specific phosphorylation causes either the release of Bim from its sequestration to the microtubular dynein motor complex or the stabilization of the Bim protein; both can induce Bax/ Bak-dependent apoptosis. However, regulatory phosphorylation of Bim by other kinases such as extracellular signal-regulated kinase can induce the opposite effect and lead to proteasomal degradation and protection from apoptosis. 29 Hence, the regulation and outcome of Bim phosphorylation have to be further clarified in hepatocytes through, for example, the identification of the exact phosphorylation sites and the expression of phosphorylation-defective Bim mutants. The role of JNK-mediated Bim phosphorylation in hepatocyte apoptosis has recently been substantiated in vivo. 18 The authors showed that lipopolysaccharide/galactosaminetreated mice died because of TNFa-mediated fatal hepatitis and demonstrated that this apoptosis was dependent on Bid and Bim. Bim was shown to be phosphorylated by JNK and, consequently, redistributed from microtubules to the cytosol; there, it induced apoptosis in cooperation with caspase-8 cleaved tbid. Remarkably, only the loss of both Bid and Bim protected mice from lipopolysaccharide/galactosamine-induced hepatitis. Similar findings have been observed for TNF-related apoptosisinducing ligand (TRAIL), which enhances Fas-induced hepatocyte apoptosis and liver damage via activation of the JNK-Bim axis 23 ; this suggests some overlapping effects of different TNF family members. Our results with cultured primary murine hepatocytes support the aforementioned mechanism. TNFa preincubation led to JNK activation, and the inhibition of JNK and the loss of Bim abolished the sensitizing effect; however, FasLinduced apoptosis remained unchanged. In addition, sensitization was mitigated by the loss of Bid. In our study, TNFa needstocrosstalkwithfastoexertitsapoptosissensitizing effect. We recently reported the unexpected finding that in collagen-cultured primary mouse hepatocytes, Fas signaling switches from a type II, Bid-dependent apoptotic signaling pathway to a type I, Bidindependent apoptotic signaling pathway. As shown here, TNFa is obviously able to restore the type II signaling pathway by a so far unknown mechanism. It will be crucial to identify these crosstalk points between TNFa and FasL signaling. Our data suggest that Bim and Bid may be part of these points. Both act by triggering Bax/Bak-mediated MOMP and cytochrome c release, but perhaps this occurs efficiently only when both are indeed present and activated. TNFa would activate Bim via JNK and regulate Bid in a so far unknown way such that it becomes required for FasL-induced apoptosis. This would explain why TNFa-induced sensitization is impeded in both Bim knockdown and Bim / hepatocytes. We therefore suggest that Bim and Bid can only cooperatively activate the mitochondrial amplification loop in hepatocytes and that this is crucial for the observed increased sensitivity to FasL-induced apoptosis. The presented mathematical model accurately reproduces the sensitizing effect and will promote further directions for future research. Sensitivity analysis reveals the sensitizing mechanisms to be very robust, although the model contains only the most important players. Most critical interactions for the crosstalk model after TNFa and FasL stimulation are the ones associated with Bid and also all reactions associated with Bim (see the supporting information for the model equations). XIAP has a prominent role as a caspase-3 buffer, and the function of Bcl2 family members has turned out to be essential for the model because the sensitizing effect is completely disrupted otherwise (Supporting Fig. 15). Consequently, it would be of special interest to further analyze the specific function and interplay of pbim and other members of the Bcl2 family. Because many chronic liver diseases in which FasL levels are elevated are associated with chronic

11 292 SCHMICH ET AL. HEPATOLOGY, January 2011 inflammation, the herein reported TNF/FasL crosstalk might be of clinical relevance. Our first in vivo studies showing TNFa sensitization toward anti-fas induced liver damage strengthen this assumption. Elevated TNF levels due to inflammatory processes might affect many acute and chronic liver diseases by enhancing FasL-induced apoptosis signaling and, therefore, might constitute a possible therapeutic target. Acknowledgment: The authors thank Fritz von Weizsäcker and Sabine MacNelly (Department of Internal Medicine II, University Hospital, Freiburg, Germany) for the isolation of primary murine hepatocytes and Karin Neubert (Institute of Molecular Medicine and Cell Research, Freiburg, Germany) for providing and quantifying N2A FasL. 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