By A20-Mediated Down-Regulation of Bax Mitochondrial Apoptosis in The

TNF Pretreatment Interferes with Mitochondrial Apoptosis in the Mouse Liver by A20-Mediated Down-Regulation of Bax

This information is current as Gabriele Sass, Noula Dattu Shembade, Florian Haimerl, of September 28, 2021. Nicolas Lamoureux, Said Hashemolhosseini, Andrea Tannapfel and Gisa Tiegs J Immunol 2007; 179:7042-7049; ; doi: 10.4049/jimmunol.179.10.7042

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

TNF Pretreatment Interferes with Mitochondrial Apoptosis in the Mouse Liver by A20-Mediated Down-Regulation of Bax1

Gabriele Sass,2* Noula Dattu Shembade,2† Florian Haimerl,* Nicolas Lamoureux,‡ Said Hashemolhosseini,‡ Andrea Tannapfel,§ and Gisa Tiegs3*

Pretreatment with low doses of the proinflammatory cytokine TNF has been shown to prevent hepatocellular apoptosis and liver damage in inflammatory as well as in ischemia/reperfusion-induced liver injury. The underlying mechanisms of protection have not been elucidated so far. In this study, these mechanisms were investigated in murine hepatocyte cultures as well as in a mouse model of TNF-dependent apoptotic liver damage (galactosamine/TNF model). Our results show that pretreatment with TNF, or application of small-interfering RNA directed against the proapoptotic Bcl2 family member Bax, interfered with the onset of mitochondrial apoptosis in vivo. Knockdown of TNF-␣-induced-protein 3 (A20) restored mitochondrial apoptosis, Bax expression, and liver damage. The underlying mechanism of protection seems to involve a cascade of events, where TNF induces the expression Downloaded from of A20 in hepatocytes, A20 down-modulates Bax expression by interference with transcriptional activation, and the reduced availability of Bax interferes with the onset of mitochondrial apoptosis and the ensuing apoptotic liver damage. In conclusion, we identified Bax and A20 as key players in TNF-induced protection from apoptotic liver damage. Because treatment with TNF itself might be a risk factor for patients, we propose that overexpression of A20 might represent an alternative approach for protection from inflammation related apoptotic liver damage, as well as for TNF preconditioning during transplantation. The Journal of Immunology, 2007, 179: 7042–7049. http://www.jimmunol.org/

n liver diseases, such as viral hepatitis, alcoholic liver dis- TNF induces apoptotic liver damage, which is characterized by the ease, nonalcoholic fatty liver disease, ischemia/reperfusion formation of mitochondrial permeability transition pores, release I injury, or autoimmune hepatitis, activation of the inflamma- of cytochrome c, as well as by the activation of caspases (11–13). tory response is crucial for damage induction. In most of these Contrarily, pretreatment with TNF alone, without transcriptional cases, acute injury seems to be driven by a proinflammatory Th1 inhibition, for 2 to at least 24 h protects mice from apoptotic liver cytokine response. Among these cytokines, TNF, in combination damage subsequently induced by GalN/TNF application (14, 15). with additional pathophysiologic factors, plays a central role in Concerning mechanisms of protection, it has been shown, that, by guest on September 28, 2021 inducing hepatocyte apoptosis (1, 2). However, TNF is also critical similar to TNF preconditioning in hepatic ischemia-reperfusion for hepatocyte proliferation (3) and liver regeneration (4) exerts injury, protection in the GalN/TNF model is dependent on TNF/ beneficial effects during preconditioning against hepatic ischemia/ TNFR1 interaction (16). It has been shown that TNF pretreatment reperfusion injury (5), and recent reports show that anti-TNF strat- does not down-modulate TNFR1 expression (16), ruling out the egies might induce autoimmune diseases (6). possibility that simply a lack of the damage-mediating receptor 4 In the galactosamine (GalN) /TNF mouse model of experimen- might be the mechanism of protection, which has not been eluci- tal hepatitis (1, 7, 8), TNF-induced damage can be investigated. In dated so far. this model, GalN inhibits hepatocyte specific transcription by de- Recently, we performed cDNA arrays to investigate TNF/ pletion of the hepatocellular pool of uracil nucleotides (9). Under TNFR1-dependent gene regulation in the mouse liver (17). Among these conditions and dependent on the presence of TNFR1 (10), the genes repressed by TNF, we detected Bax, a proapoptotic member of the Bcl2 family of proteins, which is involved in the

*Institute of Experimental and Clinical Pharmacology and Toxicology, University of onset of mitochondrial apoptosis. Activation of Bax is induced Erlangen-Nuremberg, Erlangen, Germany; †Department of Microbiology and Immu- either by tBid, a cleavage product of Bid, a Bcl-2 homology 3 ‡ nology, School of Medicine, University of Miami, Miami, FL 33136; Institute of (BH3) domain-only member of the Bcl2 family, or by mechanisms Biochemistry, University of Erlangen-Nuremberg, Erlangen, Germany; and §Institute of Pathology, University of Bochum, Bochum, Germany independent of Bid cleavage (18). Upon activation, cytosolic mo- Received for publication October 25, 2006. Accepted for publication August 30, 2007. nomeric Bax integrates into the mitochondrial membrane, oli- The costs of publication of this article were defrayed in part by the payment of page gomerizes, forms pores, and facilitates the release of the proapop- charges. This article must therefore be hereby marked advertisement in accordance totic activator cytochrome c from the mitochondrion (19). with 18 U.S.C. Section 1734 solely to indicate this fact. Concomitantly, cytochrome c forms complexes with Apaf-1, 1 This work was supported by the Deutsche Forschungsgemeinschaft, Grant TI 169/7-3. which in turn activate caspase 9, resulting in the subsequent acti- 2 G.S. and N.D.S. contributed equally to this work. vation of the effector caspase 3 (20). 3 Address correspondence and reprint requests to Dr. Gisa Tiegs, Institute of Ex- In this study, we investigated the hypothesis that TNF-induced perimental and Clinical Pharmacology and Toxicology, University of Erlangen- Nuremberg, Fahrstrasse 17, D-91054 Erlangen, Germany. E-mail address: gisa. down-regulation of Bax would be sufﬁcient to interfere with GalN/ [email protected] TNF-induced mitochondrial apoptosis and investigated the underlying 4 Abbreviations used in this paper: GalN, D-galactosamine; siRNA, small-interfering mechanism. Performing knockdown experiments by small-interfer- RNA; HSA, human serum albumin; RU, relative unit; ISEL, in situ end labeling; ing RNA (siRNA) technology, we identiﬁed Bax and the NF-␬B- Act.D, actinomycin D; LDH, lactate dehydrogenase. inhibiting TNF-␣-induced-protein 3 (TNFAIP3; A20) as key play- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 ers in TNF-induced protection from mitochondrial apoptosis. www.jimmunol.org The Journal of Immunology 7043

Materials and Methods 9, and anti-caspase 3 (Cell Signaling Technology; New England Biolabs), Animals anti-cytochrome c (7H8.2C12; BD Biosciences), and anti-Bid, anti-caspase 8, both purchased from Mo BI Tec. Secondary Abs for Western blot anal- C57BL/6 mice (age: 8–10 wk; weight range: 20–25 g) were obtained from ysis were purchased from Sigma-Aldrich and Jackson ImmunoResearch the animal facilities of the Institute of Experimental and Clinical Pharma- Laboratories. cology and Toxicology (University of Erlangen-Nuremberg, Erlangen, Germany). All mice received human care according to the guidelines of the Histochemistry National Institute of Health as well as to the legal requirements in Ger- many. They were maintained under controlled conditions (22°C, 55% hu- Liver tissue was fixed in 4% formalin in PBS and subsequently embedded midity and 12-h day/night rhythm) and fed a standard laboratory chow. in paraffin. Sections were stained for H&E using a standard protocol and analyzed by light microscopy. In situ end labeling (ISEL) for detection of Dosage and application routes apoptotic cells was performed for paraffin-embedded tissue (25).

Recombinant murine TNF (Innogenetics), dissolved in saline/0.1% human Real-time RT-PCR serum albumin (HSA), was administered to mice i.v. at 10 ␮g/kg alone or at 5 ␮g/kg 30 min before D-galactosamine (GalN; Roth) application. GalN Isolation of total RNA from cells was conducted using the Nucleo Spin was administered i.p. at 700 mg/kg in pyrogen-free saline. For induction of RNA Purification kit (BD Clontech). To analyze altered gene expression, cellular damage, actinomycin D (Act.D; 80 nM; Sigma-Aldrich) was added mRNA was transcribed into cDNA using SuperScript II RNase HϪ reverse to primary hepatocyte cultures 30 min before TNF (40 ng/ml). The NF-␬B transcriptase (Invitrogen Life Technologies). Oligonucleotides for subse- inhibitor gliotoxin (Ref. 21; Alexis Biochemicals) was dissolved in DMSO quent PCR were also obtained from Eurogentec Deutschland. Primer se- and diluted to final concentrations in cell culture medium. siRNA was quences were: ␤-actin: 5Ј-TGG AAT CCT GTG GCA TCC ATG AAA-3Ј purchased from Eurogentec Deutschland and Qiagen. Target sequences for and 5Ј-TAA AAC GCA GCT CAG TAA CAG TCC G-3Ј; Bax: 5Ј-ACA Ј Ј siRNA design were: AGC GAG TGT CTC CGG CGA ATT for Bax; AAC TGT TTG CTG ATG GCA AC-3 and 5 -CTT CTT CCA GAT GGT GAG Downloaded from CAT GCA CCG ATA CAC GCT for A20; AAT ACT CCA ATT GGC C-3Ј; A20: 5Ј-CCA GGT TCC AGA ACA ATG TC-3Ј and 5Ј-CTC CAT GAT GGC for GFP as an irrelevant control gene. siRNA was applied to ACA GAG TTC CTC AC-3Ј; Bid: 5Ј-CGA AGA CGA GCT GCA mice i.p. in PBS at 5 ␮g/mouse (22) 24 h before challenge (siBax) or at 30 GAC-3Ј and 5Ј-CTC GTT TCT AAC CAA GTT CC-3Ј. Real-time RT- ␮g/mouse 3 h before TNF administration (siA20). Control siRNA was PCR was performed using a LightCycler rapid thermal cycler system applied according to the protocol used for siBax or siA20. (Roche Diagnostics) and the LightCycler-FastStart DNA Master SYBR Green I mix (Roche Diagnostics). Reactions were performed in a 10-␮l Analysis of liver enzymes and cellular damage volume. To confirm amplification specificity, PCR products were subjected http://www.jimmunol.org/ Hepatocyte damage was assessed 6 h after GalN/TNF administration by to a melting curve analysis and visualized by gel electrophoresis. measuring plasma enzyme activity of alanine aminotransferase (ALT) (23), using an automated procedure. Cellular damage in primary hepatocytes Cell culture and transfection was measured by lactate dehydrogenase (LDH) activity in culture super- Hepa1–6 cells were plated in 500 ␮l of RPMI 1640 containing 10% FCS natants (S) and the remaining cell monolayer (C) after lysis with 0.1% (Invitrogen Life Technologies) in 24-well plates at a number of 2 ϫ 105 Triton X-100. The percentage of LDH release (toxicity) was calculated ϩ cells/well. Cells were allowed to adhere to culture plates overnight. Pri- from the ratio of S/(S C). mary mouse hepatocytes were isolated by a modification of the two-step Determination of caspase 3 activity collagenase perfusion method of Seglen (43) from adult male BALB/c mice. Digestion step was performed with Liberase Blendzyme 3 recombinant To determine the activation of caspase 3 in liver tissue of mice, liver ho- collagenase (Roche Diagnostics) according to the manufacturer’s instruc- by guest on September 28, 2021 mogenates (50% w/w) were prepared in lysis buffer containing 10 mM tion. Cells were plated in 100 ␮l of RPMI 1640 (Invitrogen Life Technol- HEPES (pH 7.4), 1 mM CHAPS, and 1 mM DTT and analyzed using the ogies) containing 10% basal medium supplement in 96-well tissue-culture colorimetric caspase 3 Assay kit (Sigma-Aldrich) according to the manu- plates (Greiner Bio-One) at a number of 4 ϫ 104 cells/well and maintained facturer’s instructions. at 37°C, 5% (v/v) CO2 and 40% ambient oxygen concentration (v/v). After cells were allowed to adhere to culture plates for 18 h, cell debris and dead Western blot analysis cells were removed by scavenging with Optimem I Medium (Invitrogen Life Technologies). Transfections were performed using Lipofectamine Livers were homogenized in lysis buffer containing 0.5% Nonidet P-40, 2000 (Invitrogen Life Technologies) according to the manufacturer’s in- 137 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl (pH 8.0), 10% glycerol. structions. After 24 h, corresponding wells were prestimulated with recom- Following centrifugation, supernatants were stored at Ϫ80°C. For Western binant murine TNF (40 ng/ml; Innogenetics) for 8 h. Apoptosis was in- blot analysis, 20 ␮g of protein were fractionated by 10% SDS-PAGE and duced by administration of actinomycin D (75 nM) 30 min before blotted onto a nitrocellulose membrane. Western blots were developed us- administration of TNF (40 ng/ml). ing an ECL system (Amersham) according to the manufacturer’s instructions. Semiquantitative evaluation was done using the Gel Doc 2000 Sys- tem (Bio-Rad). Cloning and sequencing Cytochrome c detection assay A full-length expression clone of A20 (pA20) was obtained using the pcDNA3.1/V5-His-TOPO TA Expression kit (Invitrogen Life Technolo- To detect cytochrome c release, mitochondria-free cytosolic fractions were gies) in combination with the AccuPrime Taq Polymerase System (Invitro- prepared as described previously (24). Briefly, tissues were homogenized gen Life Technologies). Sequence analysis was done using the ABI PRISM in ice-cold Mito buffer (20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM 377 DNA sequencer (Applied Biosystems).

MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 250 mM sucrose, 0.1 mM PMSF, 2 mg/ml pepstatin, leupeptin, and aprotinin) in a small glass ho- Luciferase assay mogenizer using a Teflon pestle (50 strokes on ice). The homogenates were ␮ centrifuged at 800 ϫ g for 10 min at 4°C to remove nuclei and cell debris. Hepa1–6 cells were transfected in 24-well plates with 0.4 g of luciferase ␬ ␮ Supernatants were centrifuged at 16,000 ϫ g for 20 min at 4°C two times reporter pB2LUC, containing 2 NF- B-binding sites, or with 0.8 gof to remove mitochondria. Supernatants were used to perform anti- pBaxLUC2.8, containing 2.8 kb of the upstream region of the bax start cytochrome c Western blot analysis. A total of 50 ␮g of protein from the codon cloned in front of firefly luciferase. Twenty-four hours after, trans- cytosolic fraction were loaded onto a 15% SDS-polyacrylamide gel. Fol- fection cells were incubated as described in the figure legends and har- lowing electrophoresis, gels were blotted onto a 0.2-␮m nitrocellulose vested for extract preparation. Luciferase reporter activity was measured by membrane (Protran; Schleicher & Schuell Bioscience). Western blots were a commercial assay (Luciferase Assay System; Promega). developed as described above. Statistical analysis Antibodies The results were analyzed using the Student t test if two groups were The following primary Abs were used for immunohistochemical staining compared and the Dunnett’s test if more groups were tested against a and Western blot analysis: anti-A20 (R20), anti-Bax (D21), anti-Bak control group. If variances were inhomogeneous in the Student’s t test, the (G23), anti-Bad (K17), anti-␤-actin (C11), and anti-GAPDH (V18), all results were analyzed using the Welsh test. All data in this study are ex- purchased from Santa Cruz Biotechnology, anti-Bik (Abgent), anti-caspase pressed as a mean Ϯ SEM. A value of p Յ 0.05 was considered significant. 7044 TNF INTERFERES WITH MITOCHONDRIAL APOPTOSIS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. TNF pretreatment interferes with mitochondrial apopto- FIGURE 2. TNF pretreatment down-regulates Bax expression in sis in vivo. C57BL/6 mice were pretreated with HSA (lanes 1–3)or vivo. A, Real-time RT-PCR for Bax and Bid expression was performed TNF (10 ␮g/kg) (lanes 4–6) for 12 h. Subsequently, liver damage was on livers of mice treated with HSA or TNF (10 ␮g/kg) for 1 h. Data are induced by application of GalN/TNF. Measurements were performed p Յ 0.05 for TNF vs HSA ,ء ;expressed as the mean Ϯ SEM (n ϭ 4 6 h after GalN/TNF challenge. A, Liver damage was measured by de- treated mice). B, C57BL/6 mice were treated with HSA (lanes 1–3)or termination of ALT in plasma. Data are expressed as the mean Ϯ SEM TNF (10 ␮g/kg) (lanes 4–6) for 12 h. Expressions of Bax (21 kDa) and p Յ 0.05 for TNF vs HSA pretreated mice). B and C, Protein ,ء ;n ϭ 5) Bid (22 kDa) were detected by Western blot analysis. C, C57BL/6 mice expressions for inactive caspase 8, uncleaved Bid (22 kDa), monomeric were treated with siRNA directed against GFP (siControl; lanes 1–3)or Bax (21 kDa), release of cytochrome c as well as activation of caspase against Bax (siBax; lanes 4–6) for 24 h. Expression of Bax, Bak, Bad, 9 and activated caspase 3 were detected by Western blot analysis 6 h Bik, and Bid was measured by Western blot analysis in liver after GalN/TNF challenge. homogenates.

Results inactive p22 Bid to tBid (696.51 Ϯ 14.09 RU vs 577.68 Ϯ 21.98 TNF treatment interferes with mitochondrial apoptosis and RU; p Յ 0.05). During activation, e.g., by tBid, Bax monomers down-regulates Bax expression in vivo assemble to oligomers in the mitochondrial membrane (26), which Administration of low doses of TNF, without transcriptional or results in reduced amounts of the Bax monomer. Concomitantly, in translational inhibition, protects mice from liver damage sub- the GalN/TNF model of liver damage, we detected significant ac- sequently induced by GalN/TNF administration (Fig. 1A). To tivation of Bax, as measured by disappearance of the inactive elucidate the mechanism of TNF-induced protection, we mea- monomer (650.12 Ϯ 101.25 RU vs 219.59 Ϯ 11.21 RU; p Յ 0.05), sured downstream signals leading to liver damage in the GalN/ representing a reduction of the Bax monomer by 66%. TNF model and compared them to those in TNF-pretreated, In protected mice (Fig. 1C, lanes 4–6), we found significantly -protected mice. reduced activation of caspase 8 and Bid, as shown by significantly Following induction of liver damage by GalN/TNF administra- reduced cleavage of inactive caspase 8 as well as of p22 Bid to tion, we found activation of Bax-mediated mitochondrial apoptosis tBid. Subsequent events of mitochondrial apoptosis, i.e., release of in comparison to livers of solvent (HSA) treated mice (Fig. 1B). cytochrome c and activation of caspase 9, as well as activation of Densitometric evaluation revealed significant activation of caspase the effector caspase 3 were also found to be attenuated in livers 8, as measured by cleavage of the inactive form (median Ϯ SEM: of TNF-pretreated animals (Fig. 1C). Interestingly, we detected lines 1–3: 355.47 Ϯ 16.97 relative units (RU) vs lines 4–6: almost complete disappearance (Ͼ90%) of the Bax monomer p21 94.57 Ϯ 61.43 RU; p Յ 0.05) as well as significant cleavage of in livers of TNF-pretreated mice (Fig. 1C), while we had expected The Journal of Immunology 7045 Downloaded from http://www.jimmunol.org/

FIGURE 4. TNF-induced down-modulation of Bax in vitro is dependent on A20. A, Hepa1–6 cells were incubated in the presence of TNF (100

ng/ml) for 30 min up to 8 h. Expression of Bax and A20 was measured by by guest on September 28, 2021 real-time RT-PCR. Data are expressed as the mean Ϯ SEM (n ϭ 3–5; p Յ 0.05 for TNF vs untreated cells). B, Hepa1–6 cells were transfected ,ء with an expression clone for murine A20 (pA20) or an empty control vector for 12 or 24 h. Subsequently, expression of A20 (upper part) and FIGURE 3. Down-modulation of Bax protects mice from apoptotic Bax (lower part) was measured by real-time RT-PCR. Data are expressed p Յ 0.05 for cells transfected with pA20 ,ء ;liver damage. C57BL/6 mice were pretreated with siRNA directed as the mean Ϯ SEM (n ϭ 3 against GFP (siControl; lanes 1–3) or against Bax (siBax; lanes 4–6) vs vector control-transfected cells). for 24 h, and subsequently challenged with GalN/TNF. A, Liver damage was measured 6 h after challenge by determination of ALT in plasma. p Յ 0.05 for siControl ,ء ;Data are expressed as the mean Ϯ SEM (n ϭ 4 vs siBax pretreated mice). B, Release of cytochrome c and activation of caspase 9 were measured by Western blot analysis 6 h after challenge. C, chondrial apoptosis by reducing Bax expression and in contrast by Livers of mice were stained using the H&E method (upper part) as well as interfering with activation of residual Bax by preventing Bid the ISEL method for detection of apoptosis (lower part; apoptotic cells are activation. stained in brown). Down-modulation of Bax by siRNA protects mice from apoptotic liver damage this process to be interrupted or even reverted in case of protection, To determine whether down-modulation of Bax expression, as as seen for caspase 8 and Bid cleavage (compare Fig. 1, B and C). observed following TNF treatment (Fig. 2, A and B), actually In contrast, Bax expression was signiﬁcantly reduced (median Ϯ interferes with mitochondrial apoptosis, we designed siRNA di- SEM: lines 1–3: 526.9 Ϯ 14.8 RU vs lines 4–6: 53.97 Ϯ 22.59 rected speciﬁcally against murine Bax (siBax). In comparison RU; p Յ 0.05) in livers of TNF-protected mice, which was obvi- to control siRNA (siControl), siBax down-modulated Bax ex- ously not due to activation, because subsequent events in mito- pression in vivo by ϳ70% (Fig. 2C), while expression of other chondrial apoptosis were missing (Fig. 1C). This prompted us to proapoptotic members of the Bcl2 family of proteins (Bak, Bad, investigate the effect of TNF pretreatment alone on Bax expres- Bik, or Bid) was not affected (Fig. 2C). Down-modulation of sion. In cDNA arrays (17) as well as in real-time RT-PCR (Fig. Bax by siRNA protected mice from GalN/TNF-induced liver 2A), we observed reduced expression of Bax while expression of damage (Fig. 3A) and reduced subsequent events in mitochon- Bid was not affected. We therefore determined the impact of pro- drial apoptosis, such as release of cytochrome c and activation tective TNF treatment on expression of Bax and Bid on protein of caspase 9 (Fig. 3B). The protective effect of siBax against level. Our results show that Bax, but not Bid, protein expression apoptosis induced by GalN/TNF could also be demonstrated in was markedly reduced following TNF pretreatment (Fig. 2B). liver sections stained with H&E (Fig. 3C, upper part)orby These results indicate that TNF might affect Bax-induced mito- using ISEL to determine DNA damage (Fig. 3C, lower part). 7046 TNF INTERFERES WITH MITOCHONDRIAL APOPTOSIS

TNF-induced A20 expression contributes to Bax down-modulation in vitro Because liver damage induction by TNF exclusively occurs in the presence of hepatocyte transcriptional inhibition, there must be a TNF-inducible protein which in turn down-regulates Bax expression. This additional player in TNF tolerance could be A20, a protein which has been shown to be inducible in hepatocytes (27) and to protect from mitochondrial apoptosis (28). To investigate a possible connection between TNF, A20 induction and Bax down- modulation, we incubated Hepa1–6 cells in the presence of TNF for different periods of time and measured A20 as well as Bax expression by real-time RT-PCR. TNF induced the expression of A20 within 30 min for at least 3 h, while it decreased the expres- FIGURE 5. Overexpression of A20 or down-regulation of Bax inter- sion of Bax from 3 h onward for at least 8 h (Fig. 4A). To inves- feres with TNF-induced protection in primary hepatocytes. Primary mouse tigate whether these processes were related, A20 was overex- hepatocytes were transfected with siRNA directed against GFP (siControl) pressed in Hepa1–6 cells by transient transfection of an A20 or against Bax (siBax) alone or in combination with an expression clone for expression clone for 12 or 24 h (Fig. 4B), resulting in signiﬁcantly murine Bax (pBax) or an empty control vector. After 24 h, corresponding increased A20 expression (Fig. 4B, upper part) and decreased ex- wells were stimulated with TNF (40 ng/ml) for 8 h. Apoptosis was induced Downloaded from by administration of Act.D (75 nM) 30 min before TNF (40 ng/ml). LDH pression of Bax mRNA (Fig. 4B, lower part). These results indi- release was measured after 18 h of incubation to calculate cytotoxicity. cate a connection between A20 expression and expressional down- .p Յ 0.05 for cells regulation of Bax ,ء ;Data are expressed as the mean Ϯ SEM (n ϭ 20 incubated with Act.D/TNF vs cells pretreated with TNF; n ϭ 10–20; #, p Յ 0.05 for cells pretreated with TNF vs cells pretreated with TNF after Down-regulation of A20 or overexpression of Bax interferes transfection with pBax and siA20, either alone or in combination). with TNF-induced protection in primary hepatocytes

To further investigate the hypothesis of a connection between http://www.jimmunol.org/ These results indicate that indeed down-modulation of Bax ex- A20-, Bax-, and TNF-induced protection, we isolated primary pression, as observed following TNF pretreatment, is able to mouse hepatocytes and induced cellular damage by incubation protect from liver damage. with the transcriptional inhibitor Act.D in combination with TNF. by guest on September 28, 2021

FIGURE 6. A20 induces TNF tolerance by down-modulating Bax expression. C57BL/6 mice were pretreated with siRNA directed against GFP (siControl; lanes 1–6) or siRNA directed against A20 (siA20; lanes 7–9) as well as with HSA (lanes 1–3)or TNF (10 ␮g/kg) (lanes 4–9) for 12 h. Sub- sequently, liver damage was induced by application of GalN/TNF. Measurements were performed 6 h after GalN/TNF challenge. Liver damage was measured by (A) determination of ALT in plasma and (B) caspase-3 activity in liver homogenates by ELISA. Data are expressed as the mean Ϯ SEM (n ϭ p Յ 0.05 for HSA vs TNF pretreated ,ء ;5 mice; #, p Յ 0.05 for siControl vs siA20 pretreated mice). C, A20 was detected by Western blot analysis 6 h after GalN/TNF challenge. D, Expression of caspase 8, Bid (22 kDa), and Bax (21 kDa) as well as release of cytochrome c and activation of caspase 9 were measured by Western blot analysis also 6 h after challenge. E, Signs of apoptosis were detected by H&E staining (upper part) and the ISEL staining method (lower part; apoptotic cells are stained in brown). The Journal of Immunology 7047

by activation of caspase 3 (Fig. 6B). A20 knockdown partially restored activation of caspase 8 and Bid, and almost completely restored Bax expression, release of cytochrome c and activation of caspase 9 (Fig. 6D). Restoration of apoptosis by siA20 in GalN/ TNF-treated mice could also be observed in liver slices stained with H&E (Fig. 6E, upper part) as well as in liver slices where DNA damage was determined by ISEL staining (Fig. 6E, lower part). These experiments indicate that TNF-induced expression of A20 is responsible for down-regulation of Bax expression and also for reduced activation of residual Bax.

TNF-induced expression of A20 results in decreased NF-␬B activation and subsequently in reduced Bax expression In an attempt to explain how A20 might be able to interfere with Bax expression, we performed additional in vitro experiments. Be- cause A20 is known to interfere with NF-␬B activation (29, 30), we ﬁrst investigated the impact of NF-␬B inhibition on Bax expression. Real-time RT-PCR analysis revealed that NF-␬B inhibi-

tion by gliotoxin (21) interfered with Bax expression starting at Downloaded from concentrations of 100 nM (Fig. 7A), while concentrations of gliotoxin lower than 1 ␮M were not toxic to the cells during 24 h of incubation time, as measured by release of lactate dehydrogenase (data not shown). The effect of gliotoxin incubation on NF-␬B FIGURE 7. Inhibition of NF-␬B activation interferes with Bax expres- activity was conﬁrmed in luciferase assays using a construct con-

sion. A, Hepa1–6 cells were incubated in the presence of 10–1000 nM taining luciferase under the control of a promoter containing 3 http://www.jimmunol.org/ gliotoxin for 2 h. Subsequently, real-time RT-PCR analysis for Bax ex- NF-␬B-binding sites (pB2Luc; Fig. 7B). Incubation with TNF pression was performed. Data are expressed as the mean Ϯ SEM (n ϭ 4; (Fig. 7C) or with 100 nM gliotoxin (Fig. 7D) was able to interfere p Յ 0.05 for gliotoxin vs untreated cells). B, Hepa1–6 cells were trans- with the expression of a construct containing a full-length Bax ,ء fected with the luciferase reporter construct pB2LUC under the control of promoter region fused to the luciferase gene (pBaxLuc2.8). Fi- ␬ an NF- B-dependent promoter for 24 h. Subsequently, cells were incu- nally, expression of pBaxLuc2.8 was significantly lower in a cell bated with the NF-␬B inhibitor gliotoxin (100 nM) for 2 h. Data are ex- Յ line stably overexpressing A20 (Fig. 7E). These results indicate ء Ϯ ϭ pressed as the mean SEM (n 6; , p 0.05 for gliotoxin incubated ␬ vs untreated transfected cells). C, Hepa1–6 cells were transfected with a that NF- B inhibition, as performed by an inhibitor or by overex- luciferase expression vector under the control of the Bax promoter (2.8 kb pression of A20, is able to interfere with Bax promoter activity. by guest on September 28, 2021 upstream of the start codon; pBaxLuc2.8) for 24 h. Subsequently, cells Therefore, the mechanism of TNF-induced protection seems to were incubated with TNF (100 ng/ml) for 24 h. Data are expressed as the involve induction of A20 expression, A20-mediated inhibition of p Յ 0.05 for TNF incubated vs untreated cells). NF-␬B activation and, subsequently, reduced expression of the ,ء ;mean Ϯ SEM (n ϭ 3 D, Hepa1–6 cells were transfected with pBaxLuc2.8 for 24 h. Subse- proapoptotic Bax gene. quently, cells were incubated with gliotoxin for 2 h. Data are expressed as p Յ 0.05 for gliotoxin incubated vs untreated Discussion ,ء ;the mean Ϯ SEM (n ϭ 6 cells). E, Hepa1–6 cells or the A20 overexpressing Hepa1–6-based cell TNF, as a proinflammatory cytokine, plays a pivotal role in several line STA20 were transfected with pBaxLuc2.8 for 24 h. Data are expressed severe human diseases such as rheumatoid arthritis or Crohn’s dis- -p Յ 0.05 for STA20 vs Hepa1–6). Lucif ,ء ;as the mean Ϯ SEM (n ϭ 6 erase activity in B–E was measured immediately following cell extract ease (31). TNF is also implicated in apoptotic liver damage, seen preparation. Luciferase activity in all samples was normalized by protein during viral hepatitis, inflammatory hepatitis, endotoxemia-in- content. Basal activity of the respective promoter/luciferase construct in duced liver failure, and ischemia/reperfusion-induced liver damage Hepa1–6 cells was regarded as 1. LRA, Luciferase reporter assay. (2), and therefore promotes inflammation-related processes resulting in apoptotic and necrotic destruction of organs. Accordingly, therapy of the above-mentioned syndromes involves blockade of We found that pretreatment with TNF-protected hepatocytes from TNF action, either by application of anti-TNF Abs (infliximab) or damage, while this protective effect was counteracted by either soluble TNFR2 (etanercept) (31). However, in December 2004, the overexpressing Bax or interfering with A20 expression. This result Food and Drug Administration revised the labeling of the anti- was most obvious when both incubation regimens were combined TNF therapeutic remicade to include a warning on severe hepatic (Fig. 5). Our results show that TNF-induced protection is also reactions (www.fda.gov/medwatch/SAFETY/2004/Remicade), achievable in vitro and seems to depend on the presence of A20 as and a recent report implicates a connection between anti-TNF ther- well as on the absence of Bax. apy and the induction of autoimmune diseases (6), suggesting that TNF exerts also beneficial effects. Moreover, it has been shown A20 induces TNF tolerance by down-modulating Bax expression that TNF-preconditioning protects from ischemia-reperfusion in- in vivo jury (5), a finding which could improve the outcome of transplan- To investigate the contribution of A20 to TNF-induced tolerance tations in patients. Unfortunately, preconditioning with TNF itself and its connection to Bax expression in vivo, we administered would bear a high potential of risk, because TNF is a proinflam- siA20 or siControl to TNF-pretreated mice and subsequently mea- matory cytokine and involved in apoptotic organ destruction (2). sured GalN/TNF-induced liver damage. Down-modulation of A20 Therefore, we intended to provide knowledge on mechanisms and expression was ϳ90% efficient in vivo (Fig. 6C). A20 knockdown mediators of protection induced by TNF, to identify novel protec- in TNF-pretreated mice restored their sensitivity toward GalN/ tive factors for future gene therapy that might have similar effects TNF-induced apoptosis and liver damage (Fig. 6A) as manifested as TNF preconditioning, but circumvent its risks. 7048 TNF INTERFERES WITH MITOCHONDRIAL APOPTOSIS

against Bax mRNA, we could in fact demonstrate that a lack of Bax itself, and not only a lack of Bax activation, can interfere with the onset of mitochondrial apoptosis. This result prompted us to investigate signaling pathways leading from TNF to reduction of Bax expression in vitro as well as in vivo. TNF induces apoptotic liver damage in mice only in the presence of hepatocyte-specific transcriptional inhibition. Without such inhibition, injection of TNF alone protects mice from liver injury. Hence, there must be at least one TNF-inducible protein in hepatocytes that protects from detrimental TNF effects. We identified the NF-␬B-inducible protein A20 (36) as the missing link. A20, a zinc finger protein originally discovered in endothelial cells (37), has been shown to protect from TNF-induced apoptosis in vitro (38) as well as in vivo (27), and to stabilize the mitochondrial membrane potential (28). As an immediate early gene, A20 is inducible by NF-␬B-activating stimuli, such as TNF or LPS (36, 39), within a very short period of time, due to a constitutive association of the general transcription apparatus (40). In contrast, in a feed-

␬ Downloaded from FIGURE 8. Interactions of TNF, A20 and Bax. TNF induced protection back loop, A20 inhibits the activation of NF- B and other tran- seems to involve a mechanism by which TNF induces the expression of scription factors (29, 30, 41). A20 is a hepatoprotective (27), TNF- A20 in hepatocytes, A20 down-modulates Bax expression by interference inducible protein in hepatocytes and has been shown to protect with e.g., NF-␬B activation, and reduced availability of Bax interferes with cells, which are deficient in NF-␬B activation, from TNF-induced the onset of mitochondrial apoptosis and the ensuing apoptotic liver dam- apoptosis by disrupting the recruitment of the death domain-sig- age. In addition to this process TNF pretreatment interferes with activation naling molecules TRADD and receptor-interacting protein (RIP) of Bid, a prerequisite for activation of residual Bax protein. Cyt c: cyto- to the receptor signaling complex (42). Our results show that TNF http://www.jimmunol.org/ chrome c. induces A20 expression before Bax down-modulation, and that A20 overexpression is able to interfere with Bax expression on transcriptional level. Using siRNA against A20 or Bax overex- It has been shown that TNF pretreatment does not down-regu- pression in vitro, we were able to reverse TNF-induced protection late the amount of TNFR1 on hepatocytes (16), the receptor which from cellular damage in primary hepatocytes. Moreover, we could is necessary for induction of damage, as well as for induction of demonstrate in vivo that interference with A20 expression restored tolerance. Furthermore, it has been shown that neither IL-1, nor Bax expression and subsequent events in mitochondrial apoptosis, heme oxygenase-1, nor inducible NO synthase are mediators of including cytochrome c release and caspase 9 as well as caspase 3 TNF tolerance (16), although inducible NO synthase-derived NO activation. Concerning the mechanism by which A20 is able to by guest on September 28, 2021 contributes to endotoxin tolerance (32), which therefore is clearly interfere with Bax expression, and knowing that A20 inhibits distinguishable from TNF tolerance. Nevertheless, because under NF-␬B activation (29, 30), we investigated the dependence of Bax experimental conditions, e.g., in mouse models or in cell culture, expression on NF-␬B activation. Using an NF-␬B inhibitor and a TNF exerts its proapoptotic effects only in the presence of tran- fusion construct of the Bax promoter and luciferase, we could scriptional inhibition, TNF tolerance seems to depend on the ac- indeed demonstrate that Bax expression is inducible by NF-␬B. tivation of yet unknown TNF-inducible cytoprotective proteins. To A20 also seems to be able to interfere with the activation of identify those proteins, we performed cDNA arrays on livers of AP1 (29). Therefore, it might be possible that A20-induced in- TNF-treated mice (17). We found that in fact numerous potentially terference with the activity of other transcription factors might antiapoptotic genes were induced, but, more intriguing, TNF also also contribute to TNF-induced hepatoprotection. reduced the expression of proapoptotic members of the Bcl2 fam- Taken together, our results show that TNF-induced protection ily at the transcriptional level. Among these genes we identified seems to involve a mechanism by which TNF induces the expres- Bax, which is a key factor in the onset of mitochondrial apoptosis. sion of A20 in hepatocytes, A20 down-modulates Bax expression In the initial process of mitochondrial apoptosis, TNF/TNFR1 in- by interference with, e.g., NF-␬B activation, and that the reduced teraction results in the recruitment of TNFR-associated death do- availability of Bax interferes with the onset of mitochondrial ap- main-containing protein (TRADD) leading to NF-␬B activation, or optosis and the ensuing apoptotic liver damage (Fig. 8). In addition to an interaction with Fas-associated death domain-containing pro- to this process, we observed an effect of TNF pretreatment on tein (FADD) resulting in activation of caspase 8 (33). Activation of activation of Bid, which would be a prerequisite for activation of caspase 8 promotes cleavage of Bid to tBid, which subsequently is residual Bax protein. With respect to human immune-mediated able to activate Bax (18), while NF-␬B activation is able to induce liver damage, e.g., hepatic ischemia/reperfusion injury, or un- cytoprotective proteins (Refs. 34 and 35; Fig. 8). Indeed, we found wanted side effects of anti-TNF therapy, substitution of hepato- that Bid is activated in our mouse model, and that TNF pretreat- protective proteins, such as A20 or other TNF-inducible cytopro- ment seems to interfere with its activation, while it does not in- tective proteins, might be a promising gene therapeutic approach. fluence its expression. Upon activation, cytosolic Bax disappears It is also possible from the therapeutic point of view that mole- from the cytoplasm, integrates into the mitochondrial membrane, cules, other than those responsive to TNF, could be beneficial in oligomerizes, forms pores, and facilitates the release of the pro- protection of the liver. Knockdown technologies such as the use of apoptotic activator cytochrome c from the mitochondrion (19), re- siRNA could possibly be used in the future to repress TNF-induc- sulting in subsequent activation of the effector caspase 3 (Ref. 20; ible proapoptotic gene products such as Bax to prevent immune Fig. 8). We observed that TNF pretreatment, besides interference activation-related apoptotic liver damage. Despite the obvious ex- with events upstream events in Bax activation, was very efficient in perimental advantages of siRNA technology that affords the quick, interfering with Bax expression. 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