Volume 136, Issue 4, Pages 1423-1434 (April 2009) Antiapoptotic Effect of c-Jun N-terminal Kinase-1 through Mcl-1 Stabilization in TNF- Induced Hepatocyte Apoptosis  Yuzo Kodama, Kojiro Taura, Kouichi Miura, Bernd Schnabl, Yosuke Osawa, David A. Brenner  Gastroenterology  Volume 136, Issue 4, Pages 1423-1434 (April 2009) DOI: 10.1053/j.gastro.2008.12.064 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 jnk2−/− Hepatocytes are resistant to TNF-induced apoptosis. Mouse primary hepatocytes were infected with AdGFP or AdIκB (moi, 10) followed by treatment of TNF (30 ng/mL). (A–C) Wild-type (wt) hepatocytes were analyzed at 8 hours after TNF treatment. (A) Western blot analysis with antibodies against caspase-3, cleaved caspase-3, PARP, and α-tubulin as loading control. Arrow indicates the PARP-cleaved product. (B) Caspase-3 activity was measured. Results are expressed as relative fluorescent units (RFUs). Values are mean ± SD. *P = .00006 (TNF vs control). (C) Cell death was determined and quantified by double staining with Hoechst and propidium iodide as described in “Materials and Methods.” Scale bars = 100 μm. Values are mean ± SD. *P = .0005 (TNF vs control). (D) Expression of phosphorylated and total JNK were assessed in jnk1−/− and jnk2−/− hepatocytes at 0 and 15 minutes after TNF treatment by Western blot analysis with antibodies against phospho-JNK, JNK, and α-tubulin. The p54 and p46 bands are indicated. (E) Caspase-3 activity and (F) apoptosis were quantified in jnk1−/− and jnk2−/− hepatocytes at 8 hours after TNF treatment. Values are mean ± SD. *P = .00006 (E) and .00004 (F) (jnk2−/− vs wild-type). PI, propidium iodide; p-JNK, phosphorylated JNK. Gastroenterology 2009 136, 1423-1434DOI: (10.1053/j.gastro.2008.12.064) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Mcl-1 is increased in jnk2−/− hepatocytes. (A) Expression of antiapoptotic Bcl-2 family members in wild-type (wt), jnk1−/−, and jnk2−/− hepatocytes were assessed by Western blot analysis with antibodies against mouse Mcl-1, Bcl-XL, Bcl-w, A1, and α-tubulin. (B) mcl-1 mRNA expression in wild-type, jnk1−/−, and jnk2−/− hepatocytes was quantified by quantitative real-time PCR. (C) Wild-type, jnk1−/−, and jnk2−/− hepatocytes were infected with AdIκB (moi, 10) and treated with TNF (30 ng/mL) for the indicated hours, and respective lysates were subjected to Western blot analysis with antibodies against mouse Mcl-1, cleaved caspase-3, and α-tubulin. Gastroenterology 2009 136, 1423-1434DOI: (10.1053/j.gastro.2008.12.064) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Mcl-1 blocks TNF-induced apoptosis in primary hepatocytes. (A and B) Wild-type hepatocytes were coinfected with either AdGFP and AdIκB, or AdMcl1-WT and AdIκB at moi of 10 for each adenovirus followed by TNF treatment (30 ng/mL; 8 hours). (A) Western blot analysis was performed with antibodies against Mcl-1, cleaved caspase-3, and α-tubulin. (B) Caspase-3 activity and apoptosis were quantified. Values are mean ± SD. *P = .0008 in caspase-3 activity and .0005 in apoptosis (AdMcl1-WT vs AdGFP). (C) Wild-type hepatocytes were coinfected with AdIκB (moi, 10) and AdMcl1-WT (moi: 0, 0.01, 0.1, 1, and 10) followed by TNF treatment (30 ng/mL; 8 hours). Respective lysates were subjected to Western blot analysis with antibodies against Mcl-1, cleaved caspase-3, and α-tubulin. (D and E) AdIκB-infected (moi, 10) hepatocytes were pretreated with dimethylsulfoxide (DMSO) or MG-132 (10 μmol/L; 1 hour) followed by TNF treatment (30 ng/mL) for the indicated hours. (D) Respective lysates were subjected to Western blot analysis with antibodies against Mcl-1, cleaved caspase-3, and α-tubulin. (E) Caspase-3 activity and apoptosis were quantified at 8 hours after TNF treatment. Values are mean ± SD. *P = .002 in caspase-3 activity and .0001 in apoptosis (MG-132 vs DMSO). Gastroenterology 2009 136, 1423-1434DOI: (10.1053/j.gastro.2008.12.064) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Mcl-1 blocks TNF-induced liver injury. Wild-type mice were infected with AdGFP or AdMcl1-WT (1 × 109 pfu/mouse) by tail vein injection for 2 days. (A) Mcl-1 expressions in whole liver extracts were analyzed by Western blot analysis with antibodies against Mcl-1 and α-tubulin. (B–D) AdGFP-infected or AdMcl1-WT–infected mice were treated with GalN/TNF-α as described in “Materials and Methods,” and liver injury was studied at 6 hours after TNF injection. (B) Serum ALT levels and numbers of TUNEL-positive cells per high power field in the liver sections were assessed. Values are mean ± SD. *P = .001 in ALT and .0003 in TUNEL-positive cells (AdMcl1-WT vs AdGFP). (C) Western blot analysis with antibodies against cleaved caspase-3 and α-tubulin was done. (D) H&E and TUNEL staining of liver sections. Gastroenterology 2009 136, 1423-1434DOI: (10.1053/j.gastro.2008.12.064) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Increased Mcl-1 in jnk2−/− hepatocytes is mediated by JNK1 activity. (A) JNK activities were determined in wild-type (wt), jnk1−/−, and jnk2−/− hepatocytes with or without TNF treatment (30 ng/mL; 30 minutes) by in vitro kinase assay with c-Jun as a substrate. JNK activity was measured by Western blot analysis for phospho-cJun. Equivalent loading of c-Jun protein was confirmed by Western blot analysis with anti–c-Jun antibody. (B and C) jnk2−/− Hepatocytes were treated with DMSO or SP600125 (20 μmol/L) for 12 hours. (B) Western blot analysis with antibodies against mouse Mcl-1, Bcl-XL, and α-tubulin antibodies. (C) mcl-1 mRNA expressions were measured by quantitative real-time PCR. P-c-Jun, phospho-c-Jun; SP6, SP600125. Gastroenterology 2009 136, 1423-1434DOI: (10.1053/j.gastro.2008.12.064) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 JNK phosphorylates and stabilizes Mcl-1 in primary hepatocytes. (A) Wild-type hepatocytes were infected with AdGFP, AdMcl1-WT, or AdMcl1-S121A/T163A followed by treatment of DMSO, SP600125 (20 μmol/L), SB220025 (20 μmol/L), U0126 (20 μmol/L), BIO (20 μmol/L), or R-roscovitine (20 μmol/L) for 6 hours. Mcl-1 protein was immunoprecipitated with anti–Mcl-1 antibody, and its phosphorylation was assessed by Western blot analysis with anti–phospho-serine/threonine antibody. Equivalent precipitation of Mcl-1 protein was confirmed by Western blot analysis with anti–Mcl-1 antibody. (B) Hepatocytes were infected with AdMcl1-WT or AdMcl1-S121A/T163A, followed by treatment with DMSO or SP600125 (20 μmol/L; 12 hours), and respective lysates were subjected to Western blot analysis with antibodies against Mcl-1 and α-tubulin. (C) Hepatocytes were infected with AdMcl1-WT or AdMcl1-S121A/T163A, followed by cycloheximide treatment (10 μg/mL) for the indicated hours. Respective lysates were subjected to Western blot analysis with antibodies against Mcl-1 and α-tubulin. (D) Band intensity in panel C was measured by densitometry. Mcl-1:α-tubulin ratios were calculated, and Mcl-1 expressions were presented as a percentage of time 0. Values are mean ± SD. *P = .004 (Mcl1-S121A/T163A vs Mcl1-WT). Gastroenterology 2009 136, 1423-1434DOI: (10.1053/j.gastro.2008.12.064) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Deletion of mcl-1 in jnk2−/− hepatocytes increased TNF-induced apoptosis. mcl-1f/fjnk2−/− hepatocytes were coinfected with either AdGFP and AdIκB or AdCre and AdIκB at moi of 10 for each adenovirus. (A) Expression levels of mcl-1 and bcl-XL mRNA were quantified by quantitative real-time PCR. (B–D) Apoptosis was induced by TNF (30 ng/mL) treatment for the indicated hours. (B) Western blot analysis with antibodies against mouse Mcl-1, cleaved caspase-3, and α-tubulin was performed. (C) GFP expression (driven by AdGFP or AdCre) and Hoechst staining in identical field were analyzed by fluorescent microscopy. Scale bars = 100 μm. (D) Caspase-3 activity and apoptosis were quantified at 8 hours after TNF treatment. Values are mean ± SD. *P = .0001 in caspase-3 activity and .0008 in apoptosis (AdCre vs AdGFP). Gastroenterology 2009 136, 1423-1434DOI: (10.1053/j.gastro.2008.12.064) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 Deletion of mcl-1 in jnk2−/− mouse liver increased GalN/TNF-induced injury. (A–C) Liver injury was induced in wild-type (wt) or jnk2−/− mice by GalN/TNF administration. (A) Serum ALT levels and numbers of TUNEL-positive cells per high power field were assessed at 6 hours after TNF injection. Values are mean ± SD. *P = .004 in ALT and .00004 in TUNEL-positive cells (jnk2−/− vs wild-type). (B) H&E and TUNEL staining were shown at 6 hours after TNF injection. (C) Whole liver extracts from wild-type or jnk2−/− mice at 0, 2, 4, and 6 hours after TNF-α treatment were subjected to Western blot analysis with antibodies against mouse Mcl-1, cleaved caspase-3, and α-tubulin. (D–F) mcl-1f/fjnk2−/− mice were infected with AdGFP or AdCre (1 × 109 pfu/mouse) by tail vein injection. Two days later, liver injury was induced by of GalN/TNF administration. (D) Western blot analysis at the indicated hours after TNF treatment was done with antibodies against Mcl-1, cleaved caspase-3, and α-tubulin. (E) Serum ALT levels and numbers of TUNEL-positive cells per high power field were measured at 6 hours after TNF injection. Values are mean ± SD. *P = .023 in ALT and .0004 in TUNEL-positive cells (jnk2−/− vs wild-type). (F) H&E and TUNEL staining at 6 hours after TNF injection. (B and F) Scale bars = 100 μm. Gastroenterology 2009 136, 1423-1434DOI: (10.1053/j.gastro.2008.12.064) Copyright © 2009 AGA Institute Terms and Conditions