Volume 134, Issue 5, Pages 1507-1520 (May 2008) Mechanism of Mitochondrial Glutathione-Dependent Hepatocellular Susceptibility to TNF Despite NF-κB Activation  Montserrat Marí, Anna Colell, Albert Morales, Francisco Caballero, Anna Moles, Anna Fernández, Oihana Terrones, Gorka Basañez, Bruno Antonsson, Carmen García–Ruiz, José C. Fernández–Checa  Gastroenterology  Volume 134, Issue 5, Pages 1507-1520 (May 2008) DOI: 10.1053/j.gastro.2008.01.073 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 mGSH depletion sensitizes to TNF. (A) Compartmentalized GSH from primary cultured mouse hepatocytes were analyzed after HP treatment with or without GSH ethyl ester (GSH-EE) (5 mmol/L). The transient effect of HP on mGSH is shown on the right. (B) Time course of ROS generation after TNF challenge (100 ng/mL) in control and mGSH-depleted hepatocytes with or without GSH-EE pretreatment. (C) Cell death was determined 16 hours after TNF challenge by staining with Höechst-33258 and propidium iodide. (D) Activity of glutathione peroxidase in mouse liver mitochondria and representative Western blots of peroxiredoxin-III (Prx-III) and thioredoxin-2 (Trx-2) in the presence of 4-acetoamido-4′-maleimidylstilbene-2-2′-disulfonic acid (AMS), a thiol-reactive alkylating agent that modifies reduced protein thiols, showing that Prx-III and Trx-2 are fully reduced compared with tert-butylhydroperoxide (tBOOH). Data are mean ± SD, (A) n = 5 and *P = 1.74 × 10−6 and #P ≤ .0009; (B) n = 5 and *P ≤ 1.16 × 10−5 vs TNF; (C) n=5, *P ≤ .001 vs TNF-treated hepatocytes and #P ≤ .001 vs HP + TNF-treated hepatocytes. Gastroenterology 2008 134, 1507-1520DOI: (10.1053/j.gastro.2008.01.073) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Mitochondrial downstream events by TNF occur only when mGSH levels are depleted. (A) Representative fluorescence microscopy images of mouse hepatocytes labeled with calcein and tetramethylrhodamine methyl ester (TMRM) after treatment with TNF (100 ng/mL) for 2 hours with or without HP pretreatment. Time-dependent TMRM fluorescence of hepatocytes incubated with TNF with or without HP pretreatment. Data are mean ± SD of n = 5, *P < .00034 vs TNF-treated hepatocytes. (B) Cytochrome c and Smac/Diablo release were determined by immunoblot 3 hours after TNF with or without HP pretreatment. (C) Representative fluorescence microscopy of hepatocytes labeled with TMRM and PhiPhiLux after TNF exposure. (D) Time-dependent caspase-3 activation in extracts of hepatocytes after TNF incubation with or without HP pretreatment using a specific caspase-3 fluorescent substrate. Data are represented as mean ± SD of n = 5, *P < .0054 vs control hepatocytes. Gastroenterology 2008 134, 1507-1520DOI: (10.1053/j.gastro.2008.01.073) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 NF-κB and JNK activation by TNF. (A) Representative electrophoretic mobility shift assay showing the DNA binding activity of NF-κB in nuclear extracts of mouse hepatocytes treated with TNF with or without HP pretreatment or Bay 11-7085, which blocks NF-κB activation. (B) Translocation of the p65 subunit to the nuclei examined by Western blot and by immunofluorescence microscopy showing a punctate pattern induced by TNF regardless of HP pretreatment. (C) Time-dependent analyses by real-time RT-PCR of κB-dependent genes cIAP1 and MnSOD. (D) JNK phosphorylation was determined by immunoblot at different time points after TNF (100 ng/mL) exposure with or without HP or actinomycin D (ActD) pretreatment. Gastroenterology 2008 134, 1507-1520DOI: (10.1053/j.gastro.2008.01.073) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Premitochondrial TNF signaling in primary mouse hepatocytes. (A) Representative immunoblot of procaspase-8 processing by TNF with or without HP pretreatment. (B) tBid Western blot analyzed 2 hours after TNF challenge in control and mGSH-depleted hepatocytes. (C) Bax translocation to mitochondria, MOM, and mitoplasts, as well as its resistance to alkali treatment with Na2CO3, was assessed by immunoblot 2 hours after TNF challenge. (D) Bax oligomerization in MOM was determined after EGS cross-linking as detailed in Supplementary Methods (see supplementary material online at www.gastrojournal.org). (E) Expression of Bax and Bak 48 hours after transfection of mouse hepatocytes with siRNA targeting Bax or Bax using scrambled siRNA as control. Cell death was assessed in siRNA-transfected hepatocytes 16 hours after exposure to TNF in mGSH-depleted hepatocytes. Data are mean ± SD of n = 3, *P = .0002. (F) Representative Bax and Bak levels in mitochondria from rat and mouse hepatocytes. Gastroenterology 2008 134, 1507-1520DOI: (10.1053/j.gastro.2008.01.073) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 TNF-induced mROS after mGSH depletion predominantly target cardiolipin. (A) Cardiolipin levels (CLs) were determined 2 hours after TNF challenge with or without HP pretreatment. (B) Levels of oxidized cardiolipin in mouse hepatocytes after TNF exposure with or without HP pretreatment. Representative HP-TLC displaying the presence of cardiolipin hydroperoxides (CL-OOH and CL-2OOH) in control and mGSH-depleted hepatocytes treated with TNF for 3 hours. (C) Competition assay based on the competition for cytochrome c between intact CL or CLOOH-containing liposomes and pure solid-phase CL. Data are mean ± SD; (A) n = 5, *P = .0009; (B) n = 5, *P = 1.5 × 10−5; (C) n = 4, ‡P ≤ .0008; (D) n = 3, *P ≤ .0028. Gastroenterology 2008 134, 1507-1520DOI: (10.1053/j.gastro.2008.01.073) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Cardiolipin peroxidation promotes BAX-driven membrane permeabilization. (A) Representative TLC analysis of cardiolipin peroxidation. (B) Dose-dependent effect of peroxidized cardiolipin on the vesicular FD-70 release induced by Bax and tBid (50 nmol/L each) in LUVs in which PC was substituted by increasing amounts of cardiolipin (Control) or oxidized cardiolipin (Perox-CL). Data represent mean values and standard errors of 3–6 measurements. (C) Time-dependent effect of cardiolipin oxidation on the release of fluorescein-labeled dextran of 70 kDa (FD-70) from LUVs induced by Bax and tBid (arrow) and effect of cardiolipin oxidation on the Bcl-2 inhibition (dose-response assay) of Bax + tBid-mediated vesicular FD-70 release. (D) Effect of cardiolipin oxidation on the release of vesicular ANTS/DPX induced by various pore-forming agents and dose-dependence of vesicular FD-70 release induced by octylglucoside-oligomerized Bax. Data represent mean ± standard error, n = 3. (E) Effect of oxidized cardiolipin on the insertion of oligomeric BAX into phospholipid monolayers at different surface pressures. Critical surface pressure values were 37.7 mN/m (Control) and 37.4 mN/m (Perox-CL); both values exceed that estimated for a membrane bilayer (30–35 mN/m). (F) Effect of cardiolipin oxidation on lipid polymorphism. Kinetics of bilayer-to-hexagonal lipid-phase transition in LUVs were assessed by changes in NBD-DOPE fluorescence intensity as described in detail in the Supplementary Methods (see supplementary material online at www.gastrojournal.org). Gastroenterology 2008 134, 1507-1520DOI: (10.1053/j.gastro.2008.01.073) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 ASMase is required for TNF-induced cardiolipin oxidation. (A) Time course of ROS generation after TNF challenge (100 ng/mL) in control and mGSH-depleted mouse hepatocytes from wild-type and ASMase−/− hepatocytes and selective mGSH depletion by HP in ASMase−/− hepatocytes. (B) Cardiolipin (CL) and oxidized cardiolipin (CLOOH) levels in mitochondria from ASMase−/− hepatocytes treated 3 hours after TNF challenge with or without HP pretreatment. (C) Representative fluorescence microscopy images from wild-type and ASMase−/− hepatocytes labeled with calcein and TMRM, with or without HP treatment, after treatment with TNF (100 ng/mL) for 2 hours. (D) Caspase-3 activation examined by confocal microscopy of PhiPhiLux fluorescence 4 hours after TNF challenge in wild-type and ASMase−/− hepatocytes with or without HP treatment. (E) Bax translocation to mitochondria and Bid truncation in ASMase−/− hepatocytes in response to TNF with or without HP pretreatment. (F) Hepatocellular survival after TNF challenge in wild-type and ASMase−/− hepatocytes with mGSH depletion. Data are mean ± SD; (A) n = 5, *P ≤ .0014 and #P = 1.38 × 10−5; (F) n = 4, *P = .0011. Gastroenterology 2008 134, 1507-1520DOI: (10.1053/j.gastro.2008.01.073) Copyright © 2008 AGA Institute Terms and Conditions

Figure 8 ASMase−/− mice with mGSH depletion by cholesterol loading are resistant to TNF-mediated steatohepatitis. (A) Hypercholesterolemic diet (HC) feeding for 48 hours induces depletion of mitochondrial GSH stores in both ASMase+/+ and ASMase−/− mice. (B) Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined 24 hours after intravenous TNF injection. (C) Representative H&E slides (×10) and myeloperoxidase (MPO) (×40) staining from ASMase+/+ and ASMase−/− mice 24 hours after TNF challenge with or without HC feeding. Data are mean ± SEM; (A) n = 5, *P = .0003 and #P = .00027; (B) n = 4–5 animals/group, *P < .0001. Gastroenterology 2008 134, 1507-1520DOI: (10.1053/j.gastro.2008.01.073) Copyright © 2008 AGA Institute Terms and Conditions