Advertisement

Wnt/β-Catenin Signaling Protects Mouse Liver against Oxidative Stress-induced Apoptosis through the Inhibition of Forkhead Transcription Factor FoxO3*

  • Author Footnotes
    1 Both authors contributed equally to this work.
    Guo-Zhong Tao
    Footnotes
    1 Both authors contributed equally to this work.
    Affiliations
    From the Department of Surgery, Stanford University School of Medicine, Stanford, California 94305-5148,
    Search for articles by this author
  • Author Footnotes
    1 Both authors contributed equally to this work.
    Nadja Lehwald
    Footnotes
    1 Both authors contributed equally to this work.
    Affiliations
    From the Department of Surgery, Stanford University School of Medicine, Stanford, California 94305-5148,

    the Department of Surgery, School of Medicine, Heinrich Heine University, 40545 Duesseldorf, Germany,
    Search for articles by this author
  • Kyu Yun Jang
    Affiliations
    From the Department of Surgery, Stanford University School of Medicine, Stanford, California 94305-5148,

    the Department of Pathology and Research Institute of Clinical Medicine, Chonbuk National University Medical School, Jeonbuk 561-180, South Korea,
    Search for articles by this author
  • Joy Baek
    Affiliations
    From the Department of Surgery, Stanford University School of Medicine, Stanford, California 94305-5148,
    Search for articles by this author
  • Baohui Xu
    Affiliations
    From the Department of Surgery, Stanford University School of Medicine, Stanford, California 94305-5148,
    Search for articles by this author
  • M. Bishr Omary
    Affiliations
    the Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109, and
    Search for articles by this author
  • Karl G. Sylvester
    Correspondence
    To whom correspondence should be addressed: Dept. of Surgery, Division of Pediatric Surgery, Stanford University School of Medicine, Hagey Laboratory for Regenerative Medicine, 257 Campus Dr., Stanford, CA 94305-5148. Tel.: 650-804-0597; Fax: 650-725-557;
    Affiliations
    From the Department of Surgery, Stanford University School of Medicine, Stanford, California 94305-5148,

    the Lucile Packard Children's Hospital, Stanford, California 94304
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health, NIDDK, Pilot Grant DK56339 (Digestive Disease Center at Stanford) (to K. G. S.) and National Institutes of Health Grant DK47918 (to M. B. O.). This work was also supported by grants from the Children's Surgical Research Program Fund and the Transplantation and Tissue Engineering Center, Packard Foundation for Children's Health (to K. G. S.) and the YAEL Foundation and the German Research Foundation (Deutsche Forschungsgemeinschaft Grant NL 2509/2-1) (to N. L.).
    1 Both authors contributed equally to this work.
      Numerous liver diseases are associated with extensive oxidative tissue damage. It is well established that Wnt/β-catenin signaling directs multiple hepatocellular processes, including development, proliferation, regeneration, nutrient homeostasis, and carcinogenesis. It remains unexplored whether Wnt/β-catenin signaling provides hepatocyte protection against hepatotoxin-induced apoptosis. Conditional, liver-specific β-catenin knockdown (KD) mice and their wild-type littermates were challenged by feeding with a hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet to induce chronic oxidative liver injury. Following the DDC diet, mice with β-catenin-deficient hepatocytes demonstrate increased liver injury, indicating an important role of β-catenin signaling for liver protection against oxidative stress. This finding was further confirmed in AML12 hepatocytes with β-catenin signaling manipulation in vitro using paraquat, a known oxidative stress inducer. Immunofluorescence staining revealed an intense nuclear FoxO3 staining in β-catenin-deficient livers, suggesting active FoxO3 signaling in response to DDC-induced liver injury when compared with wild-type controls. Consistently, FoxO3 target genes p27 and Bim were significantly induced in β-catenin KD livers. Conversely, SGK1, a β-catenin target gene, was significantly impaired in β-catenin KD hepatocytes that failed to inactivate FoxO3. Furthermore, shRNA-mediated deletion of FoxO3 increased hepatocyte resistance to oxidative stress-induced apoptosis, confirming a proapoptotic role of FoxO3 in the stressed liver. Our findings suggest that Wnt/β-catenin signaling is required for hepatocyte protection against oxidative stress-induced apoptosis. The inhibition of FoxO through its phosphorylation by β-catenin-induced SGK1 expression reduces the apoptotic function of FoxO3, resulting in increased hepatocyte survival. These findings have relevance for future therapies directed at hepatocyte protection, regeneration, and anti-cancer treatment.
      Background: Wnt/β-catenin signaling regulates various hepatocellular processes; however, it remains unexplored whether β-catenin provides hepatocyte protection against oxidative stress-induced apoptosis.
      Results: Mice with β-catenin-deficient hepatocytes demonstrate significantly increased hepatotoxin-induced liver injury.
      Conclusion: Hepatic β-catenin signaling confers hepatocyte protection against oxidative stress-induced apoptosis.
      Significance: Our findings have relevance for potential future therapies directed at hepatocyte protection, regeneration, and anti-cancer treatment.

      Introduction

      Oxidative stress is a common mechanism of progression for many liver diseases (
      • Loguercio C.
      • Federico A.
      Oxidative stress in viral and alcoholic hepatitis.
      • Diesen D.L.
      • Kuo P.C.
      Nitric oxide and redox regulation in the liver. Part I. General considerations and redox biology in hepatitis.
      ,
      • Dey A.
      • Cederbaum A.I.
      Alcohol and oxidative liver injury.
      ,
      • Fausto N.
      Liver regeneration and repair. Hepatocytes, progenitor cells, and stem cells.
      • Kaplowitz N.
      Mechanisms of liver cell injury.
      ). Persistent redox imbalance can cause irreversible modifications of cellular proteins, leading to the permanent loss of function by either degradation of damaged proteins or their progressive aggregation into cytoplasmic inclusions known as Mallory-Denk bodies. This has been observed in many human liver diseases, including alcoholic and nonalcoholic steatohepatitis (
      • Zatloukal K.
      • French S.W.
      • Stumptner C.
      • Strnad P.
      • Harada M.
      • Toivola D.M.
      • Cadrin M.
      • Omary M.B.
      From Mallory to Mallory-Denk bodies. What, how and why?.
      ,
      • Strnad P.
      • Zatloukal K.
      • Stumptner C.
      • Kulaksiz H.
      • Denk H.
      Mallory-Denk-bodies. Lessons from keratin-containing hepatic inclusion bodies.
      ). Although hepatocytes have developed numerous antioxidant defense systems to prevent oxidative stress in the liver, when excessive damage or ineffective repair occurs, cell death (apoptosis) is triggered.
      Wnt/β-catenin signaling is an established critical pathway for hepatic development, regeneration, and carcinogenesis (
      • Thompson M.D.
      • Monga S.P.
      WNT/β-catenin signaling in liver health and disease.
      ,
      • Monga S.P.
      Role of Wnt/β-catenin signaling in liver metabolism and cancer.
      • Behari J.
      • Yeh T.H.
      • Krauland L.
      • Otruba W.
      • Cieply B.
      • Hauth B.
      • Apte U.
      • Wu T.
      • Evans R.
      • Monga S.P.
      Liver-specific β-catenin knockout mice exhibit defective bile acid and cholesterol homeostasis and increased susceptibility to diet-induced steatohepatitis.
      ). We have recently found an additional role for β-catenin regulating hepatic energy metabolism and mitochondrial function (
      • Lehwald N.
      • Tao G.Z.
      • Jang K.Y.
      • Papandreou I.
      • Liu B.
      • Liu B.
      • Pysz M.A.
      • Willmann J.K.
      • Knoefel W.T.
      • Denko N.C.
      • Sylvester K.G.
      β-Catenin regulates hepatic mitochondrial function and energy balance in mice.
      ). Furthermore, we have previously shown a protective role of β-catenin in the ischemic liver through binding to HIF-1α (hypoxia-inducible factor 1α) (
      • Lehwald N.
      • Tao G.Z.
      • Jang K.Y.
      • Sorkin M.
      • Knoefel W.T.
      • Sylvester K.G.
      Wnt-β-catenin signaling protects against hepatic ischemia and reperfusion injury in mice.
      ) to promote cell survival. Another functional cross-talk mechanism has been identified in vitro in mammalian cells and Caenorhabditis elegans between β-catenin and forkhead box transcription factors (FoxOs) in response to oxidative stress (
      • Essers M.A.
      • de Vries-Smits L.M.
      • Barker N.
      • Polderman P.E.
      • Burgering B.M.
      • Korswagen H.C.
      Functional interaction between β-catenin and FOXO in oxidative stress signaling.
      ,
      • Hoogeboom D.
      • Essers M.A.
      • Polderman P.E.
      • Voets E.
      • Smits L.M.
      • Burgering B.M.
      Interaction of FOXO with β-catenin inhibits β-catenin/T cell factor activity.
      ), but to date, this relationship has not been explored in vivo or in the liver.
      In mammalian cells, FoxOs comprise four isoforms (FoxO1, -3, -4, and -6) that critically control fundamental cellular processes, such as metabolism, proliferation, cell cycle arrest, apoptosis, and survival or resistance to cellular stress (
      • van der Horst A.
      • Burgering B.M.
      Stressing the role of FoxO proteins in lifespan and disease.
      ,
      • van der Vos K.E.
      • Coffer P.J.
      FOXO-binding partners. It takes two to tango.
      ). Their activity is tightly regulated by posttranslational modifications, including phosphorylation or acetylation (
      • Calnan D.R.
      • Brunet A.
      The FoxO code.
      ,
      • Salih D.A.
      • Brunet A.
      FoxO transcription factors in the maintenance of cellular homeostasis during aging.
      ). When phosphorylated by Akt or SGK1 (serum/glucocorticoid-regulated kinase 1), a recently identified β-catenin target gene, FoxOs bind to 14-3-3 proteins and are exported out of the nucleus to inhibit their transcriptional activities (
      • Burgering B.M.
      • Kops G.J.
      Cell cycle and death control. Long live Forkheads.
      ). Conversely, FoxOs can translocate to the nucleus in the absence of phosphorylation and increase target gene expression. FoxOs are known to enhance the expression of proapoptotic transcription factors, such as Fas ligand, the Bcl-2-interacting mediator of cell death (
      • Wilk A.
      • Urbanska K.
      • Grabacka M.
      • Mullinax J.
      • Marcinkiewicz C.
      • Impastato D.
      • Estrada J.J.
      • Reiss K.
      Fenofibrate-induced nuclear translocation of FoxO3A triggers Bim-mediated apoptosis in glioblastoma cells in vitro.
      ), and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), which all trigger cell apoptosis (
      • Stahl M.
      • Dijkers P.F.
      • Kops G.J.
      • Lens S.M.
      • Coffer P.J.
      • Burgering B.M.
      • Medema R.H.
      The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2.
      ,
      • Huang H.
      • Tindall D.J.
      Dynamic FoxO transcription factors.
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.
      ).
      Because the ability of hepatocytes to resist excessive oxidative injury is a critical adaptive mechanism in the liver, we questioned whether Wnt/β-catenin signaling is required for hepatocyte protection against oxidative stress-induced apoptosis. In this study, we demonstrate that both hepatocytes with impaired Wnt/β-catenin signaling in vitro and hepatocytes in β-catenin-deficient livers in vivo, show increased liver damage in response to oxidative stress. Furthermore, the nuclear retention of FoxO3 was revealed in β-catenin-deficient hepatocytes, confirming a proapoptotic function of FoxO3 in the liver. Taken together, our findings indicate that Wnt/β-catenin signaling provides hepatocyte protection against oxidative stress-induced apoptosis through the inhibition of FoxO3.

      EXPERIMENTAL PROCEDURES

       Cell Culture, Plasmid, and Transfection

      Mouse non-transformed hepatocyte AML12 cell line was purchased (American Type Culture Collection, Manassas, VA) and cultured in DMEM/F-12 medium supplemented with 10% FCS, at 37 °C with 5% CO2 humidified incubators. AML12 hepatocytes were used to derive stable β-catenin signaling loss (dnTCF), or gain (S33Y) of function mutants or their controls via retroviral transfection and neomycin selection as described previously (
      • Lehwald N.
      • Tao G.Z.
      • Jang K.Y.
      • Sorkin M.
      • Knoefel W.T.
      • Sylvester K.G.
      Wnt-β-catenin signaling protects against hepatic ischemia and reperfusion injury in mice.
      ). To activate canonical Wnt/β-catenin signaling, we used the plasmid pcDNA3S33Y, resulting in robust TCF
      The abbreviations used are: TCF, T cell factor; LV, lentiviral; KD, knockdown; DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine; ROS, reactive oxygen species; DHE, dihydroethidium; PI, propidium iodide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; qRT-PCR, quantitative RT-PCR.
      -dependent transcriptional activation compared with wild-type β-catenin (
      • Kolligs F.T.
      • Hu G.
      • Dang C.V.
      • Fearon E.R.
      Neoplastic transformation of RK3E by mutant β-catenin requires deregulation of Tcf/Lef transcription but not activation of c-myc expression.
      ). To inhibit canonical Wnt/β-catenin signaling, a retroviral expression vector containing a mutant TCF4 expression cassette lacking the β-catenin binding domain retains DNA binding activity and thus functions in a dominant negative fashion as described previously (
      • Lehwald N.
      • Tao G.Z.
      • Jang K.Y.
      • Papandreou I.
      • Liu B.
      • Liu B.
      • Pysz M.A.
      • Willmann J.K.
      • Knoefel W.T.
      • Denko N.C.
      • Sylvester K.G.
      β-Catenin regulates hepatic mitochondrial function and energy balance in mice.
      ,
      • Schwartz D.R.
      • Wu R.
      • Kardia S.L.
      • Levin A.M.
      • Huang C.C.
      • Shedden K.A.
      • Kuick R.
      • Misek D.E.
      • Hanash S.M.
      • Taylor J.M.
      • Reed H.
      • Hendrix N.
      • Zhai Y.
      • Fearon E.R.
      • Cho K.R.
      Novel candidate targets of β-catenin/T-cell factor signaling identified by gene expression profiling of ovarian endometrioid adenocarcinomas.
      ).
      To generate FOXO3-silencing stable hepatocyte cell lines, AML12 cells were transfected with lentiviral (LV) vector FOXO3 shRNA constructs (generously provided by Dr. Anne Brunet) and selected with 2 μg/ml puromycin for 96 h. Empty lentiviral vectors were used as control.

       Animals

      Conditional and liver-specific β-catenin knockdown mice were generated as described previously (
      • Lehwald N.
      • Tao G.Z.
      • Jang K.Y.
      • Sorkin M.
      • Knoefel W.T.
      • Sylvester K.G.
      Wnt-β-catenin signaling protects against hepatic ischemia and reperfusion injury in mice.
      ). The resultant triple transgenic mouse LAP-tTA/tetO-Cre/β-cateninflox/flox (KD) showed an effective β-catenin deletion in KD livers in response to doxycycline removal from the drinking water. Their littermates with genotypes (LAP-tTA/tetO-Cre/β-cateninflox/flox, LAP-tTA/tetO-Cre/β-cateninwt/flox, or LAP-tTA+/tetO-Cre/β-cateninwt/wt) were used as wild-type (WT) controls. Doxycycline-water was given in all breeding cages to prevent any unwanted early deletion of β-catenin during liver development and removed 6–8 weeks after birth. After doxycycline withdrawal for 4 weeks, mice were used for experiments (n = 5). All experiments were conducted under a protocol that was approved by Stanford University School of Medicine Institutional Animal Care and in strict accordance with National Institutes of Health guidelines.

       Oxidative Stress Treatment

      For in vitro oxidative stress treatment, paraquat (Sigma-Aldrich), a known ROS inducer, was added at 1 mm concentration for 24 h to the cells.
      For in vivo oxidative liver damage, mice were fed with a hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 3 weeks to induce chronic oxidative stress in the liver (
      • Hanada S.
      • Snider N.T.
      • Brunt E.M.
      • Hollenberg P.F.
      • Omary M.B.
      Gender dimorphic formation of mouse Mallory-Denk bodies and the role of xenobiotic metabolism and oxidative stress.
      ).

       Liver Injury

      To assess the degree of liver injury, blood was obtained by cardiac puncture, and serum was collected. Serum alanine and aspartate aminotransferase levels were measured using a standard clinical automatic analyzer. Liver tissues were fixed in 10% PBS-buffered formalin, embedded in paraffin, and sectioned, followed by a routine H&E staining.

       Determination of Intracellular ROS Levels

      Dichlorodihydrofluorescein diacetate (Invitrogen) was used to monitor intracellular ROS generation. After incubatation with 10 μmol/liter dichlorodihydrofluorescein diacetate for 30 min, cells were analyzed by flow cytometry. Data were analyzed using FlowJo software and presented as mean fluorescence intensity. In situ ROS detection was performed by dihydroethidium (DHE; Invitrogen) labeling of frozen liver sections with 3 μm DHE at 37 °C for 30 min. Cells were stained with 5 μg/ml of DHE for 15 min to determine intracellular ROS levels. Fluorescent intensity was evaluated on a Spectramax Gemini EM microplate reader with excitation at 310 nm and emission of 610 nm.

       Immunofluorescence Staining

      For immunofluorescence staining, frozen livers were sectioned for subsequent triple immunofluorescence staining after acetone fixation (
      • Tao G.Z.
      • Looi K.S.
      • Toivola D.M.
      • Strnad P.
      • Zhou Q.
      • Liao J.
      • Wei Y.
      • Habtezion A.
      • Omary M.B.
      Keratins modulate the shape and function of hepatocyte mitochondria. A mechanism for protection from apoptosis.
      ). FoxO3 antibody and SignalSilence® siRNA (Cell Signaling Technology, Inc) were used. Images were obtained with a fluorescence microscope at ×200 or ×400 magnification.

       Apoptosis and Cell Death Assay

      For apoptosis detection, a polyclonal antibody K18D237 (AnaSpec, Fremont, CA), which recognizes a keratin 18 (K18) fragment generated by specific caspase digestion during apoptosis, was used for immunoblotting of cell lysates or immunofluorescence staining on liver tissue. K18D237 reactivity is indicative of cells undergoing apoptosis (
      • Weerasinghe S.V.
      • Moons D.S.
      • Altshuler P.J.
      • Shah Y.M.
      • Omary M.B.
      Fibrinogen-γ proteolysis and solubility dynamics during apoptotic mouse liver injury. Heparin prevents and treats liver damage.
      ,
      • Tao G.Z.
      • Li D.H.
      • Zhou Q.
      • Toivola D.M.
      • Strnad P.
      • Sandesara N.
      • Cheung R.C.
      • Hong A.
      • Omary M.B.
      Monitoring of epithelial cell caspase activation via detection of durable keratin fragment formation.
      • Habtezion A.
      • Toivola D.M.
      • Asghar M.N.
      • Kronmal G.S.
      • Brooks J.D.
      • Butcher E.C.
      • Omary M.B.
      Absence of keratin 8 confers a paradoxical microflora-dependent resistance to apoptosis in the colon.
      ). For cell death analysis after paraquat treatment, cells were trypsinized, washed, and measured using a FACScan after staining with propidium iodide (PI). Data were analyzed using FlowJo software and presented as percentage of total cells.

       Cell Proliferation and Viability Assay

      The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed according to the manufacturer's instructions (Roche Applied Science).

       Luciferase Reporter Assay

      The Dual-Light reporter gene assay (Applied Biosystems, Foster City, CA) was performed using pMegaTOPFLASH, pMegaFOPFLASH, or LacZ plasmid as described previously (
      • Lehwald N.
      • Tao G.Z.
      • Jang K.Y.
      • Sorkin M.
      • Knoefel W.T.
      • Sylvester K.G.
      Wnt-β-catenin signaling protects against hepatic ischemia and reperfusion injury in mice.
      ).

       Western Blot Analysis

      Total proteins (60 μg) from liver lysates were separated on SDS-polyacrylamide gels followed by immunoblotting with the indicated primary antibodies at 4 °C overnight. Antibodies used were as follows: FoxO1, FoxO3, and FoxO4 (Cell Signaling Technology, Beverly, MA); K18D237 (AnaSpec, Fremont, CA); β-catenin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); K19 (Developmental Studies Hybridoma Bank); and SGK1 and SGK2 (Abcam, Cambridge, MA). Anti-β-actin (Abcam, Cambridge, MA) served as a loading control.

       Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

      Total RNAs were isolated using the RNeasy Mini Kit (Qiagen Sciences, Valencia, CA) and reverse-transcribed, followed by a gene expression assay using the Applied Biosystems Prism 7900HT sequence detection system by SYBR Green technology (Applied Biosystems, Foster City, CA). Data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences used are as follows: mouse Bim, 5′-CGACAGTCTCAGGAGGAACC (forward) and 5′-CCTTCTCCATACCAGACGGA (reverse); p27, 5′-GGACCAAATGCCTGACTCGT (forward) and 5′-CGCTTCCTCATCCCTGGAC (reverse); β-catenin, 5′-GTCAGCTCGTGTCCTGTGAA (forward) and 5′-GATCTGCATGCCCTCATCTA (reverse); cyclin D1, 5′-TGGAGCCCCTGAAGAAGAG (forward) and 5′-AAGTGCGTTGTGCGGTAGC (reverse); c-Myc, 5′-CTGTTTGAAGGCTGGATTT (forward) and 5′-TCGAGGTCATAGTTCCTGTT (reverse); Axin2, 5′-ACACATGCAGAAATGGGTCA (forward) and 5′-ACGTACGGTGTAGCCTTTGG (reverse); and GAPDH, 5′-GACGGCCGCATCTTCTTGT (forward) and 5′-CACACCGACCTTCACCATTTT (reverse).

       Statistics

      All results are expressed as mean ± S.D. and represent data from three independent experiments with duplicates in each treatment group. Statistical significance was determined by Student's t test, and significance was defined as follows: *, p < 0.05; **, p < 0.01. n.s. (see Figs. 1, 2, and 5) indicates not significant.
      Figure thumbnail gr1
      FIGURE 1.Wnt/β-catenin signaling protects hepatocytes against oxidative stress-induced cell death. AML12 hepatocytes with Wnt/β-catenin signaling loss (dnTCF) or gain (S33Y) of function were created via retroviral infection. A, β-catenin/TCF reporter activity is significantly increased in S33Y mutants with a mild increase in total β-catenin protein. dnTCF hepatocytes demonstrate impaired TCF signal transduction. B, S33Y mutants show increased proliferation as measured by the MTT assay. C, dnTCF cells demonstrated increased intracellular ROS levels as measured by dichlorodihydrofluorescein diacetate (DCF-DA) staining (10 μm) followed by FACS analysis. D, hepatocytes were challenged with paraquat (1 mm) for 24 h for oxidative stress treatment. After trypsinization, cells were incubated with PI, followed by FACS analysis. E, S33Y hepatocytes are highly resistant to paraquat-induced apoptosis, as determined by Western blot for caspase-cleaved K18D237. K18 and β-actin served as controls. Note that dnTCF hepatocytes are most susceptible and S33Y mutants are least susceptible to paraquat-induced cell death and apoptosis. F, dnTCF cells demonstrated increased intracellular ROS levels as measured by DHE staining (5 μg/ml). RLU, relative light units; hrs, hours; MFI, mean fluorescence intensity; RFI, relative fluorescence intensity. *, p < 0.05; **, p < 0.01; n.s., not significant. Error bars, S.D.
      Figure thumbnail gr2
      FIGURE 2.Conditional β-catenin KD mice are more susceptible to oxidative stress-induced liver injury. WT and liver-specific β-catenin KD mice were fed with a normal control diet or DDC diet for 3 weeks. A, qRT-PCR shows a significant reduction in β-catenin mRNA in KD livers after 4 weeks of doxycycline withdrawal. The immunoblot shows significant knockdown in β-catenin protein in KD hepatocytes. B, hepatocellular injury, as evidenced by elevated serum transaminase levels (serum alanine (ALT) and aspartate aminotransferase (AST)), was significantly increased in KD mice after DDC treatment. C, severe chronic liver damage and increased ROS were detected in KD mice after the DDC diet. Representative liver histology of untreated or DDC-treated livers. H&E staining reveals increased liver damage in β-catenin-deficient hepatocytes with disruption of the normal sinusoidal architecture with inflammatory infiltration, necroinflammation (arrows), bile duct proliferation (white line), and cholestasis. Intracellular ROS levels were detected by DHE staining. Double-immunostaining for hepatocyte/cholangiocyte marker K18 and K18D237 demonstrated increased apoptosis in KD livers. Immunostaining for cholangiocyte marker K19 reveals increased atypical ductular proliferation. D, immunoblot of total liver lysates shows increased caspase-cleaved K18D237 and K19 in DDC-treated KD mice. *, p < 0.05; n.s., not significant. Error bars, S.D.
      Figure thumbnail gr5
      FIGURE 5.Knockdown of FoxO3 results in hepatocyte resistance to paraquat-induced apoptosis. A, AML12 hepatocytes were transfected with lentiviral control (LV-cont) or FoxO3 shRNA (shFoxO3) and selectd by puromycin. Lysates of resultant stable cell lines were used for immunoblot analysis, using the antibodies to the indicated antigens. B, stable transfected hepatocytes were treated with paraquat (1 mm) for 24 h. After trypsinization and PI labeling, cell death was assessed by FACS analysis. PI FACS analysis (top) and immunoblot for K18D237 apoptosis detection (bottom) demonstrate decreased cell death and less apoptosis in shFoxO3-KD hepatocytes after paraquat treatment. C, cell viability was determined using the MTT assay. D, mRNAs extracted from WT and KD hepatocytes were used for qRT-PCR to determine proapopotic Bim expression. Note that shFoxO3 hepatocytes demonstrate decreased cell death, less apoptosis, and increased survival after oxidative stress. E, FoxO3 knockdown was achieved via siRNA transfection in hepatocytes with both active (normal) and inhibited (dnTCF) β-catenin signaling. Less apoptosis and ROS were detected in dnTCF hepatocytes with FoxO3 knockdown by double-immunostaining for K18D237 and K18 and DHE staining. F, for quantitative analysis of apoptosis, the percentage of positive K18D237 cells in immunostaining was counted and graphed in bars. Note that knockdown of FoxO3 blocks ∼60% of apoptosis in dnTCF hepatocytes. *, p < 0.05; **, p < 0.01; n.s., not significant. Error bars, S.D.

      RESULTS

       β-Catenin Signaling Provides Hepatocyte Protection against Oxidative Stress-induced Cell Death

      Given the established critical roles of Wnt signaling in hepatocyte proliferation (
      • Tan X.
      • Behari J.
      • Cieply B.
      • Michalopoulos G.K.
      • Monga S.P.
      Conditional deletion of β-catenin reveals its role in liver growth and regeneration.
      ,
      • Tan X.
      • Apte U.
      • Micsenyi A.
      • Kotsagrelos E.
      • Luo J.H.
      • Ranganathan S.
      • Monga D.K.
      • Bell A.
      • Michalopoulos G.K.
      • Monga S.P.
      Epidermal growth factor receptor. A novel target of the Wnt/β-catenin pathway in liver.
      ) and redox balance (
      • Lehwald N.
      • Tao G.Z.
      • Jang K.Y.
      • Sorkin M.
      • Knoefel W.T.
      • Sylvester K.G.
      Wnt-β-catenin signaling protects against hepatic ischemia and reperfusion injury in mice.
      ,
      • Hoogeboom D.
      • Essers M.A.
      • Polderman P.E.
      • Voets E.
      • Smits L.M.
      • Burgering B.M.
      Interaction of FOXO with β-catenin inhibits β-catenin/T cell factor activity.
      ,
      • Funato Y.
      • Michiue T.
      • Asashima M.
      • Miki H.
      The thioredoxin-related redox-regulating protein nucleoredoxin inhibits Wnt-β-catenin signalling through dishevelled.
      ,
      • Hoogeboom D.
      • Burgering B.M.
      Should I stay or should I go. β-Catenin decides under stress.
      • Kaidi A.
      • Williams A.C.
      • Paraskeva C.
      Interaction between β-catenin and HIF-1 promotes cellular adaptation to hypoxia.
      ), we reasoned that Wnt/β-catenin signaling might be important for hepatocyte survival and protection against oxidative liver injury. To investigate the effects of Wnt/β-catenin signaling on cell survival, we generated β-catenin mutants from mouse AML12 hepatocytes with β-catenin loss (dnTCF) or gain (S33Y) of function (
      • Lehwald N.
      • Tao G.Z.
      • Jang K.Y.
      • Papandreou I.
      • Liu B.
      • Liu B.
      • Pysz M.A.
      • Willmann J.K.
      • Knoefel W.T.
      • Denko N.C.
      • Sylvester K.G.
      β-Catenin regulates hepatic mitochondrial function and energy balance in mice.
      ,
      • Lehwald N.
      • Tao G.Z.
      • Jang K.Y.
      • Sorkin M.
      • Knoefel W.T.
      • Sylvester K.G.
      Wnt-β-catenin signaling protects against hepatic ischemia and reperfusion injury in mice.
      ,
      • Kolligs F.T.
      • Hu G.
      • Dang C.V.
      • Fearon E.R.
      Neoplastic transformation of RK3E by mutant β-catenin requires deregulation of Tcf/Lef transcription but not activation of c-myc expression.
      ,

      Deleted in proof,

      ). S33Y mutants demonstrate heightened TCF signaling, mild increase in β-catenin protein, and increased proliferation (Fig. 1, A and B). Conversely, dnTCF hepatocytes show impaired TCF signal transduction and decreased proliferation. Loss of function hepatocytes also demonstrated increased intracellular ROS levels as measured by dichlorodihydrofluorescein diacetate FACS analysis (Fig. 1C). In order to determine whether Wnt/β-catenin signaling has a protective effect against oxidative liver injury, AML12 hepatocytes were stressed in vitro with paraquat, a known ROS inducer. In response to paraquat, S33Y mutants demonstrate resistance against oxidative stress-induced cell death as measured by PI FACS analysis (Fig. 1D). Conversely, dnTCF mutants were more susceptible to cell death in response to oxidative stress when compared with controls (Fig. 1D). Consistently, a similar result was observed by Western blot using K18D237 antibody for apoptosis detection (
      • Tao G.Z.
      • Li D.H.
      • Zhou Q.
      • Toivola D.M.
      • Strnad P.
      • Sandesara N.
      • Cheung R.C.
      • Hong A.
      • Omary M.B.
      Monitoring of epithelial cell caspase activation via detection of durable keratin fragment formation.
      ). Paraquat treatment itself caused remarkable apoptosis in control hepatocytes compared with untreated cells (Fig. 1E). However, significantly increased apoptosis was detected in hepatocytes with reduced TCF signaling. β-Catenin stabilized mutants (S33Y) appeared resistant to paraquat treatment, showing only a mild increase in apoptosis (Fig. 1E). Measuring intracellular ROS production by DHE staining, no difference in ROS levels could be detected in untreated cells (Fig. 1F). However, following Paraquat treatment, dnTCF hepatocytes demonstrated increased ROS levels compared with control cells, whereas S33Y mutants revealed significantly lower ROS levels, suggesting an important role for β-catenin in regulating hepatic redox balance.

       β-Catenin KD Mice Are More Susceptible to Oxidative Liver Damage

      Next, we questioned whether this protective effect of Wnt/β-catenin signaling may also affect chronic oxidative liver injury in vivo. To investigate the effects of Wnt/β-catenin signaling on oxidative liver damage, we utilized the previously described, conditional hepatocyte-specific β-catenin knockdown mouse (
      • Lehwald N.
      • Tao G.Z.
      • Jang K.Y.
      • Sorkin M.
      • Knoefel W.T.
      • Sylvester K.G.
      Wnt-β-catenin signaling protects against hepatic ischemia and reperfusion injury in mice.
      ). To determine if β-catenin knockdown exacerbates oxidative injury in vivo, we subjected KD mice and littermates to a DDC-containing diet for 3 weeks. DDC is a porphyrinogenic agent that causes chronic oxidative liver damage and Mallory-Denk body formations (
      • Snider N.T.
      • Weerasinghe S.V.
      • Singla A.
      • Leonard J.M.
      • Hanada S.
      • Andrews P.C.
      • Lok A.S.
      • Omary M.B.
      Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation.
      ). Effective β-catenin deletion in response to doxycycline withdrawal for 4 weeks was confirmed by mRNA and protein analysis (Fig. 2A). Liver injury was assessed by serum transaminase levels and histology. Untreated KO and WT mice did not show any significant difference in transaminase levels (Fig. 2B), liver histology (Fig. 2C), or apoptosis (Fig. 2D). In response to DDC treatment, a dramatic elevation of transaminases was observed for both genotypes when compared with untreated groups. However, significant transaminase elevation was detected in KD mice compared with wild-type controls, suggesting an exaggerated oxidative hepatocellular injury in β-catenin-deficient livers. This finding corresponded to the histology results demonstrating significant more liver damage with disruption of the normal sinusoidal architecture with inflammatory infiltration, necroinflammation, bile duct proliferation, and cholestasis in KD hepatocytes (Fig. 2C). Immunoblot and immunostaining for caspase-cleaved K18 also indicated increased hepatocellular apoptosis following the DDC diet in β-catenin-deficient mice (Fig. 2, C and D). DHE staining for the in situ detection of ROS (Fig. 2C) revealed a pronounced increase in KD livers when compared with controls after the DDC diet. Furthermore, KD livers demonstrate increased K19 expression, as determined by immunostaining (Fig. 2C) and by immunoblot analysis (Fig. 2D), consistent with increased atypical ductular proliferation. Together, these results show that β-catenin has a protective role during chronic oxidative liver injury that reduces the degree of hepatocellular injury.

       β-Catenin-deficient Livers Exhibit Nuclear Retention of FoxO3 in Response to Oxidative Stress

      Given these observations in β-catenin-deficient mice and the previously reported cross-talk between β-catenin and FoxO in C. elegans and cultured cells (
      • Essers M.A.
      • de Vries-Smits L.M.
      • Barker N.
      • Polderman P.E.
      • Burgering B.M.
      • Korswagen H.C.
      Functional interaction between β-catenin and FOXO in oxidative stress signaling.
      ,
      • Hoogeboom D.
      • Essers M.A.
      • Polderman P.E.
      • Voets E.
      • Smits L.M.
      • Burgering B.M.
      Interaction of FOXO with β-catenin inhibits β-catenin/T cell factor activity.
      ), we sought further evidence for the effect of β-catenin deletion on FoxO3 signal transduction in response to oxidative stress in the liver. Immunofluorescence staining of untreated liver tissues revealed a primarily nuclear expression pattern of FoxO3 (Fig. 3, A and D), suggesting that FoxO3 functions physiologically in normal liver homeostasis, probably in its ascribed role of lipid metabolism (
      • Zhang K.
      • Li L.
      • Qi Y.
      • Zhu X.
      • Gan B.
      • DePinho R.A.
      • Averitt T.
      • Guo S.
      Hepatic suppression of Foxo1 and Foxo3 causes hypoglycemia and hyperlipidemia in mice.
      ,
      • Tao R.
      • Wei D.
      • Gao H.
      • Liu Y.
      • DePinho R.A.
      • Dong X.C.
      Hepatic FoxOs regulate lipid metabolism via modulation of expression of the nicotinamide phosphoribosyltransferase gene.
      ). Under oxidative stress, however, FoxO3 mainly translocates to the cytoplasm and membranes (Fig. 3, B and E). Interestingly, nuclear expression of FoxO3, which suggests persistent activity, has been detected in β-catenin-deficient livers despite DDC treatment (Fig. 3, C and F). The specificity of FoxO3 liver staining was confirmed by negatively stained livers of FoxO3 knock-out mice (data not shown).
      Figure thumbnail gr3
      FIGURE 3.Nuclear retention of FoxO3 in DDC-treated β-catenin KD livers. Wild-type and β-catenin KD mice were fed with a normal control diet or DDC diet for 3 weeks. Liver cryosections were fixed by acetone and triple-stained with FoxO3 (red), K8 (green), and DAPI (blue). K8 served as an epithelial marker that specifically stained the cytoplasms of hepatocytes or cholangiocytes. Immunofluorescence images were obtained with a fluorescence microscope. Single-color staining images of FoxO3 (A–C) and merged three-color images (D–F) are presented at ×400 magnification. Note that FoxO3 expression was retained in the nucleus in β-catenin KD hepatocytes despite DDC treatment.
      Together, these data indicate that under oxidative stress, β-catenin deletion results in nuclear retention of FoxO3, which is consistent with recent findings in colon cancer cells in which accumulation of nuclear FoxO3 resulted in apoptotic cell death (
      • Tenbaum S.P.
      • Ordonez-Moran P.
      • Puig I.
      • Chicote I.
      • Arques O.
      • Landolfi S.
      • Fernandez Y.
      • Herance J.R.
      • Gispert J.D.
      • Mendizabal L.
      • Aguilar S.
      • Cajal S.R.
      • Schwartz Jr., S.
      • Vivancos A.
      • Espin E.
      • Rojas S.
      • Baselga J.
      • Tabernero J.
      • Munoz A.
      • Palmer H.G.
      β-Catenin confers resistance to PI3K and AKT inhibitors and subverts FOXO3a to promote metastasis in colon cancer.
      ,
      • Yan Y.
      • Lackner M.R.
      FOXO3a and β-catenin co-localization. Double trouble in colon cancer?.
      ).

       Impaired SGK1 Expression in β-Catenin-deficient Livers Is Accompanied by Up-regulation of Proapoptotic FoxO3 Target Genes

      To investigate whether the observed nuclear FoxO3 expression was a result of impaired Wnt/β-catenin signaling, we next sought to determine SGK1 expression. SGK1 was recently identified as a novel β-catenin target gene, which can phosphorylate and therefore inhibit FoxO3-induced apoptosis in different organ systems (
      • Shin I.
      • Bakin A.V.
      • Rodeck U.
      • Brunet A.
      • Arteaga C.L.
      Transforming growth factor β enhances epithelial cell survival via Akt-dependent regulation of FKHRL1.
      ,
      • Dehner M.
      • Hadjihannas M.
      • Weiske J.
      • Huber O.
      • Behrens J.
      Wnt signaling inhibits Forkhead box O3a-induced transcription and apoptosis through up-regulation of serum- and glucocorticoid-inducible kinase 1.
      ). β-Catenin KD livers demonstrate decreased target gene expression for Cyclin D1, c-Myc, and Axin2 at the mRNA level compared with wild-type controls (Fig. 4A). SGK1 protein did not show any difference in untreated WT and KD livers as detected by Western blot. However, SGK1 expression was significantly reduced in β-catenin-deficient livers in response to oxidative stress (Fig. 4B). SGK1 protein was mildly induced in WT livers in response to the DDC diet. SGK2 protein did not show a difference between KD and WT untreated or DDC-treated livers (Fig. 4B). However, β-catenin protein expression was found to be reduced in WT and KD livers in response to DDC (Fig. 4B). Together, these findings suggest that sustained nuclear expression of FoxO3 is probably a result of impaired SGK1 expression in β-catenin KD livers in response to DDC treatment, suggesting an important role of SGK1 in regulating β-catenin and FoxO3 signaling pathways. To further investigate whether the persistent nuclear FoxO3 expression results in increased apoptosis, we measured apoptotic FoxO3 target gene expression. Our results show a significant increase in expression of Bim and p27, known proapoptotic genes that are regulated by FoxO3, in β-catenin-deficient livers (Fig. 4C). Taken together, our results suggest that Wnt/β-catenin signaling is important for SGK1 activation under oxidative stress conditions to promote FoxO3 inactivation by nuclear exclusion to inhibit hepatocellular apoptosis.
      Figure thumbnail gr4
      FIGURE 4.Impaired SGK1 expression results in up-regulation of the proapoptotic FoxO3 target genes in β-catenin-deficient livers. A, impaired β-catenin target gene expression in KD livers as measured by qRT-PCR. B, immunoblot for SGK1, SGK2, and β-catenin was performed on liver lysates (60 μg of total protein) from both WT and KD mice, treated with or without DDC diet. β-Actin was used as a loading control. C, mRNA extracted from DDC-treated livers was used for qRT-PCR to detect mRNA expression levels of p27 and Bim, proapoptotic FoxO3 target genes. β-Catenin KD livers demonstrate increased p27 and Bim expression. *, p < 0.05. Error bars, S.D.

       FoxO3 Mediates Oxidative Stress-induced Apoptosis in Hepatocytes

      Because we have observed a heightened accumulation of nuclear FoxO3 together with increased Bim and p27 expression in the absence of β-catenin, we reasoned that FoxO3 is an important molecular regulator for hepatocellular apoptosis under oxidative stress. To further investigate the molecular mechanism of hepatocyte death under oxidative stress, we silenced FoxO3 in hepatocytes using shRNA to establish the overall effect on oxidative stress-induced apoptosis. FoxO3 protein expression was specifically reduced (Fig. 5A), whereas its isoforms FoxO1 and FoxO4 were unaffected in shFoxO3-KD cells. After treatment with paraquat for oxidative stress induction, cell death and cell viability were assessed in shFoxO3-KD and LV control hepatocytes by FACS analysis and MTT assay. shFoxO3-KD hepatocytes showed less cell death than LV control cells (Fig. 5B). Consistently, as shown by immunoblot of K18D237 (bottom), apoptosis was much less detected in shFoxO3-KD when compared with LV controls. Moreover, cell viability as measured by the MTT assay was lower in shFoxO3-KD hepatocytes than LV controls during basal conditions, but after paraquat treatment, shFoxO3-KD cells demonstrated increased cell survival relative to controls (Fig. 5C). These results indicate that FoxO3 is required in hepatocytes for oxidative stress-induced apoptosis. This result was further supported by increased expression of Bim, a known FoxO3 target for apoptosis (Fig. 5D). Although Bim expression was very similar at a low level in untreated LV control and shFoxO3-KD cells, Bim mRNA showed a dramatic increase in LV control cells in response to paraquat treatment, whereas in shFoxO3-KD hepatocytes, Bim expression was maintained at a low level (Fig. 5D). These findings suggest that FoxO3 has an important role for hepatocyte apoptosis under oxidative stress through up-regulation of the proapoptotic gene Bim. To provide further evidence that increased apoptosis in β-catenin KD liver (Fig. 2, B—D) is mediated by the FoxO3 activation (Fig. 3), we performed a FoxO3 siRNA knockdown in control and dnTCF hepatocytes. The knockdown was confirmed by FoxO3 immunofluorescence staining and biochemically (data not shown). Our results clearly show that in response to oxidative stress, dnTCF hepatocytes demonstrate increased apoptosis, which can be significantly ameliorated by FoxO3 knockdown (Fig. 5, E and F). Together, these findings suggest that β-catenin protects hepatocytes from oxidative stress-induced/FoxO3-mediated apoptosis by inactivation of the proapoptotic FoxO3.

      DISCUSSION

      The molecular mechanism of oxidative stress-induced liver damage involves the activation of multiple signaling pathways. The present study provides the first in vivo evidence that Wnt/β-catenin signaling plays an essential role for liver protection against oxidative stress-induced cell death through cross-talk with FoxO3 to inhibit hepatocyte apoptosis. Interestingly, we found that β-catenin KD livers showed impaired cytoplasmic translocation and therefore retained nuclear expression of FoxO3 combined with up-regulated proapoptotic target gene expression of Bim and p27. Our data further suggest that SGK1, a known β-catenin target gene, is a molecular link between these two signaling pathways to inhibit FoxO3-mediated cell death.
      Oxidative stress is closely associated with almost all human liver diseases and has emerged as a key player in the development and progression of pathological conditions (
      • Loguercio C.
      • Federico A.
      Oxidative stress in viral and alcoholic hepatitis.
      ,
      • Diesen D.L.
      • Kuo P.C.
      Nitric oxide and redox regulation in the liver. Part I. General considerations and redox biology in hepatitis.
      ,
      • Thompson M.D.
      • Monga S.P.
      WNT/β-catenin signaling in liver health and disease.
      ). Although hepatocytes have developed efficient molecular mechanisms to strictly regulate intracellular ROS levels and maintain the balance between oxidant and antioxidant molecules, extensive oxidative stress resulting from an imbalance between the generation of ROS and the antioxidant defense capacity of hepatocytes can cause cellular damage or cell death. Oxidative stress was thought to trigger cell death through the modification of critical cellular components, including lipids, proteins, and DNA. However, recently, it has been shown that oxidative stress-induced death mechanisms are far more complex, with the involvement of different signal transduction pathways, such as the mitogen-activated protein kinase ERK1/2, JNK, NF-κB, or FoxO pathways (
      • Huang H.
      • Tindall D.J.
      Dynamic FoxO transcription factors.
      ,
      • Czaja M.J.
      Cell signaling in oxidative stress-induced liver injury.
      ,
      • Nejak-Bowen K.
      • Kikuchi A.
      • Monga S.P.
      β-Catenin-NF-κB interactions in murine hepatocytes. A complex to die for.
      ). The molecular mechanisms that link oxidative stress are still not fully understood. Moreover, cell signaling varies depending on the type, intensity, and duration of oxidative stress. However, continued investigation of the molecular mechanisms of the cellular stress response can provide novel approaches to prevent liver damage in clinical conditions of increased oxidative stress.
      Wnt/β-catenin signaling appears to be required for hepatocyte survival by either activating cellular antioxidant defense systems, activating survival pathways, or suppressing apoptotic cell death signaling pathways (
      • Kim P.J.
      • Plescia J.
      • Clevers H.
      • Fearon E.R.
      • Altieri D.C.
      Survivin and molecular pathogenesis of colorectal cancer.
      ,
      • Tapia J.C.
      • Torres V.A.
      • Rodriguez D.A.
      • Leyton L.
      • Quest A.F.
      Casein kinase 2 (CK2) increases survivin expression via enhanced β-catenin-T cell factor/lymphoid enhancer binding factor-dependent transcription.
      ). Our data show that Wnt/β-catenin signaling is important for the reduction of oxidative stress-induced cell death through the suppression of apoptotic cell signaling by FoxO3. Mice with β-catenin deletion demonstrated increased hepatocellular injury, elevated ROS, and more apoptosis. Others have recently reported that β-catenin KO mice demonstrated decreased apoptosis in response to LPS injection, which was explained through increased NF-κB activation in the KO mice to induce hepatocyte survival (
      • Nejak-Bowen K.
      • Kikuchi A.
      • Monga S.P.
      β-Catenin-NF-κB interactions in murine hepatocytes. A complex to die for.
      ). Therefore, previous findings (
      • Nejak-Bowen K.
      • Kikuchi A.
      • Monga S.P.
      β-Catenin-NF-κB interactions in murine hepatocytes. A complex to die for.
      ) together with our data indicate that various injury stimuli can result in different signal pathway activation leading to different cellular functions. In our study, ROS mediates Wnt/β-catenin signaling. We have previously shown that Wnt/β-catenin signaling is strongly impacted by cellular redox balance, resulting in decreased transcriptional activity (
      • Lehwald N.
      • Tao G.Z.
      • Jang K.Y.
      • Sorkin M.
      • Knoefel W.T.
      • Sylvester K.G.
      Wnt-β-catenin signaling protects against hepatic ischemia and reperfusion injury in mice.
      ).
      Another intriguing finding in this study was the observation that FoxO3 relocation from the nucleus into the cytoplasm in response to oxidative stress was impaired in β-catenin-deficient livers. FoxO3 was mainly retained in the nucleus, which was distinct from the observation in wild-type mice. Our results further showed that under homeostatic conditions without significant cell mitosis as exists in the liver, FoxO3 is located in the nucleus. The nuclear expression of FoxO3 probably relates to its role in metabolic control. Conversely, under stress, FoxO3 is shuttled into the cytoplasm to promote cellular adaptation and survival. Furthermore, our data suggest that this is regulated by β-catenin as a molecular modulator because its kinase and known target gene SGK1 is strongly diminished in the absence of β-catenin. This mechanism of Wnt signaling activation as an activator of SGK1 leading to nuclear exclusion of FoxO3 has been reported previously in colon cancer cells (
      • Dehner M.
      • Hadjihannas M.
      • Weiske J.
      • Huber O.
      • Behrens J.
      Wnt signaling inhibits Forkhead box O3a-induced transcription and apoptosis through up-regulation of serum- and glucocorticoid-inducible kinase 1.
      ), but herein we provide the first demonstration of this mechanism in the liver. Because, in the absence of β-catenin, FoxO3 shuttling is impaired in response to oxidative stress, we speculate that SGK1 is the molecular link between β-catenin and phosphorylation of FoxO3 to inactivate the apoptotic function of FoxO. SGK1 is known to play a protective role by promoting cell survival under stress conditions and inhibiting apoptosis (
      • Leong M.L.
      • Maiyar A.C.
      • Kim B.
      • O'Keeffe B.A.
      • Firestone G.L.
      Expression of the serum- and glucocorticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells.
      ). The effect of the β-catenin/SGK1 signaling axis resembles the insulin/PI3K/Akt signaling pathway because they are both known to phosphorylate FoxO, resulting in nuclear exclusion of FoxO proteins, thereby inhibiting FoxO-dependent transcription (
      • Huang H.
      • Tindall D.J.
      Dynamic FoxO transcription factors.
      ,
      • Xie X.W.
      • Liu J.X.
      • Hu B.
      • Xiao W.
      Zebrafish foxo3b negatively regulates canonical Wnt signaling to affect early embryogenesis.
      ). Furthermore, it was recently reported that in addition to FoxO3, FoxO1 can also be regulated by activated β-catenin, leading to decreased inflammatory ischemia/reperfusion injury in mice via the Akt/β-catenin/FoxO1 signaling axis (
      • Kamo N.
      • Ke B.
      • Busuttil R.W.
      • Kupiec-Weglinski J.W.
      PTEN-mediated Akt/β-catenin/Foxo1 signaling regulates innate immune responses in mouse liver ischemia/reperfusion injury.
      ).
      In the present study, we provide novel in vivo evidence that FoxO3 serves a proapoptotic role in the injured liver because its knockdown resulted in decreased hepatocyte death in response to oxidative stress. The balance between proapoptotic and prosurvival actions of FoxOs depends on mechanisms not yet completely understood. In the presence of stressful stimuli, FoxOs reside in the nucleus and actively transcribe target gene expression. Their function differs, depending on the intensity of the oxidative stress; FoxOs can either promote apoptotic signaling cascades like Bim, Fas ligand, or TRAIL under high stress or antagonize oxidative stress by activating stress resistance genes (i.e. SOD, CAT, or GADD45) (
      • Essers M.A.
      • de Vries-Smits L.M.
      • Barker N.
      • Polderman P.E.
      • Burgering B.M.
      • Korswagen H.C.
      Functional interaction between β-catenin and FOXO in oxidative stress signaling.
      ,
      • Calnan D.R.
      • Brunet A.
      The FoxO code.
      ), reduce DNA damage, or induce cell cycle arrest for cells under low stress as described before (
      • Essers M.A.
      • de Vries-Smits L.M.
      • Barker N.
      • Polderman P.E.
      • Burgering B.M.
      • Korswagen H.C.
      Functional interaction between β-catenin and FOXO in oxidative stress signaling.
      ). Overexpression of FoxO3 within human colon carcinoma cells or activation of FoxO3 in cultured hepatocytes by the saturated free fatty acid palmitic acid, a known oxidative stress inducer (
      • Listenberger L.L.
      • Ory D.S.
      • Schaffer J.E.
      Palmitate-induced apoptosis can occur through a ceramide-independent pathway.
      ,
      • Brookheart R.T.
      • Michel C.I.
      • Schaffer J.E.
      As a matter of fat.
      ), stimulated the expression of Bim, a proapoptotic target gene product, and caused apoptotic cell death (
      • Dehner M.
      • Hadjihannas M.
      • Weiske J.
      • Huber O.
      • Behrens J.
      Wnt signaling inhibits Forkhead box O3a-induced transcription and apoptosis through up-regulation of serum- and glucocorticoid-inducible kinase 1.
      ). In line with these reports, our studies provide evidence that FoxO3 imparts a proapoptotic function by activating downstream Bim, thereby leading to increased hepatocyte injury.
      Taken together, we provide in vitro and in vivo evidence in support of a model in which β-catenin signaling provides hepatocyte protection through the inhibition of FoxO3-mediated apoptosis (Fig. 6). Under homeostatic conditions, activation of FoxO3 is required for the control of cellular metabolism, including glycolysis (
      • Khatri S.
      • Yepiskoposyan H.
      • Gallo C.A.
      • Tandon P.
      • Plas D.R.
      FOXO3a regulates glycolysis via transcriptional control of tumor suppressor TSC1.
      ) or fatty acid oxidation (
      • Zhang K.
      • Li L.
      • Qi Y.
      • Zhu X.
      • Gan B.
      • DePinho R.A.
      • Averitt T.
      • Guo S.
      Hepatic suppression of Foxo1 and Foxo3 causes hypoglycemia and hyperlipidemia in mice.
      ,
      • Tao R.
      • Wei D.
      • Gao H.
      • Liu Y.
      • DePinho R.A.
      • Dong X.C.
      Hepatic FoxOs regulate lipid metabolism via modulation of expression of the nicotinamide phosphoribosyltransferase gene.
      ). Under oxidative stress, β-catenin signaling activates downstream SGK1 in wild-type hepatocytes, which inactivates FoxO3 by phosphorylation, leading to cytoplasmic translocation of FoxO3. This inhibits oxidative stress-induced hepatocyte apoptosis. In the absence of β-catenin, however, FoxO3 is retained in the nucleus in its active form as result of impaired SGK1 expression to trigger apoptosis. Our study provides new evidence for potential future therapeutic strategies targeting liver injury protection, repair, and regeneration.
      Figure thumbnail gr6
      FIGURE 6.Summary model describing the role of Wnt/β-catenin signaling for hepatocyte protection against oxidative liver injury. Under normal conditions, nuclear FoxO3 may function in the control of cellular metabolic homeostasis. Under oxidative stress, Wnt/β-catenin signaling provides hepatocyte protection by activating downstream SGK1, which phosphorylates FoxO3 and thereby inactivates its function to inhibit oxidative stress-induced, FoxO3/Bim-mediated apoptosis to promote hepatocyte survival. In the absence of β-catenin, FoxO3 cannot be effectively phosphoryated by SGK1 and remains in the active form in the nucleus to trigger Bim-mediated apoptosis.

      Acknowledgments

      We are very grateful to Drs. Valerie Renault, Victoria Rafalski, Ashley Webb, and Anne Brunet (Department of Genetics, Stanford University) for sharing reagents and insightful discussion.

      REFERENCES

        • Loguercio C.
        • Federico A.
        Oxidative stress in viral and alcoholic hepatitis.
        Free Radic. Biol. Med. 2003; 34: 1-10
        • Diesen D.L.
        • Kuo P.C.
        Nitric oxide and redox regulation in the liver. Part I. General considerations and redox biology in hepatitis.
        J. Surg. Res. 2010; 162: 95-109
        • Dey A.
        • Cederbaum A.I.
        Alcohol and oxidative liver injury.
        Hepatology. 2006; 43: S63-S74
        • Fausto N.
        Liver regeneration and repair. Hepatocytes, progenitor cells, and stem cells.
        Hepatology. 2004; 39: 1477-1487
        • Kaplowitz N.
        Mechanisms of liver cell injury.
        J. Hepatol. 2000; 32: 39-47
        • Zatloukal K.
        • French S.W.
        • Stumptner C.
        • Strnad P.
        • Harada M.
        • Toivola D.M.
        • Cadrin M.
        • Omary M.B.
        From Mallory to Mallory-Denk bodies. What, how and why?.
        Exp. Cell Res. 2007; 313: 2033-2049
        • Strnad P.
        • Zatloukal K.
        • Stumptner C.
        • Kulaksiz H.
        • Denk H.
        Mallory-Denk-bodies. Lessons from keratin-containing hepatic inclusion bodies.
        Biochim. Biophys. Acta. 2008; 1782: 764-774
        • Thompson M.D.
        • Monga S.P.
        WNT/β-catenin signaling in liver health and disease.
        Hepatology. 2007; 45: 1298-1305
        • Monga S.P.
        Role of Wnt/β-catenin signaling in liver metabolism and cancer.
        Int. J. Biochem. Cell Biol. 2011; 43: 1021-1029
        • Behari J.
        • Yeh T.H.
        • Krauland L.
        • Otruba W.
        • Cieply B.
        • Hauth B.
        • Apte U.
        • Wu T.
        • Evans R.
        • Monga S.P.
        Liver-specific β-catenin knockout mice exhibit defective bile acid and cholesterol homeostasis and increased susceptibility to diet-induced steatohepatitis.
        Am. J. Pathol. 2010; 176: 744-753
        • Lehwald N.
        • Tao G.Z.
        • Jang K.Y.
        • Papandreou I.
        • Liu B.
        • Liu B.
        • Pysz M.A.
        • Willmann J.K.
        • Knoefel W.T.
        • Denko N.C.
        • Sylvester K.G.
        β-Catenin regulates hepatic mitochondrial function and energy balance in mice.
        Gastroenterology. 2012; 143: 754-764
        • Lehwald N.
        • Tao G.Z.
        • Jang K.Y.
        • Sorkin M.
        • Knoefel W.T.
        • Sylvester K.G.
        Wnt-β-catenin signaling protects against hepatic ischemia and reperfusion injury in mice.
        Gastroenterology. 2011; 141: 707-718
        • Essers M.A.
        • de Vries-Smits L.M.
        • Barker N.
        • Polderman P.E.
        • Burgering B.M.
        • Korswagen H.C.
        Functional interaction between β-catenin and FOXO in oxidative stress signaling.
        Science. 2005; 308: 1181-1184
        • Hoogeboom D.
        • Essers M.A.
        • Polderman P.E.
        • Voets E.
        • Smits L.M.
        • Burgering B.M.
        Interaction of FOXO with β-catenin inhibits β-catenin/T cell factor activity.
        J. Biol. Chem. 2008; 283: 9224-9230
        • van der Horst A.
        • Burgering B.M.
        Stressing the role of FoxO proteins in lifespan and disease.
        Nat. Rev. Mol. Cell Biol. 2007; 8: 440-450
        • van der Vos K.E.
        • Coffer P.J.
        FOXO-binding partners. It takes two to tango.
        Oncogene. 2008; 27: 2289-2299
        • Calnan D.R.
        • Brunet A.
        The FoxO code.
        Oncogene. 2008; 27: 2276-2288
        • Salih D.A.
        • Brunet A.
        FoxO transcription factors in the maintenance of cellular homeostasis during aging.
        Curr. Opin. Cell Biol. 2008; 20: 126-136
        • Burgering B.M.
        • Kops G.J.
        Cell cycle and death control. Long live Forkheads.
        Trends Biochem. Sci. 2002; 27: 352-360
        • Wilk A.
        • Urbanska K.
        • Grabacka M.
        • Mullinax J.
        • Marcinkiewicz C.
        • Impastato D.
        • Estrada J.J.
        • Reiss K.
        Fenofibrate-induced nuclear translocation of FoxO3A triggers Bim-mediated apoptosis in glioblastoma cells in vitro.
        Cell Cycle. 2012; 11: 2660-2671
        • Stahl M.
        • Dijkers P.F.
        • Kops G.J.
        • Lens S.M.
        • Coffer P.J.
        • Burgering B.M.
        • Medema R.H.
        The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2.
        J. Immunol. 2002; 168: 5024-5031
        • Huang H.
        • Tindall D.J.
        Dynamic FoxO transcription factors.
        J. Cell Sci. 2007; 120: 2479-2487
        • Brunet A.
        • Bonni A.
        • Zigmond M.J.
        • Lin M.Z.
        • Juo P.
        • Hu L.S.
        • Anderson M.J.
        • Arden K.C.
        • Blenis J.
        • Greenberg M.E.
        Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.
        Cell. 1999; 96: 857-868
        • Khatri S.
        • Yepiskoposyan H.
        • Gallo C.A.
        • Tandon P.
        • Plas D.R.
        FOXO3a regulates glycolysis via transcriptional control of tumor suppressor TSC1.
        J. Biol. Chem. 2010; 285: 15960-15965
        • Kolligs F.T.
        • Hu G.
        • Dang C.V.
        • Fearon E.R.
        Neoplastic transformation of RK3E by mutant β-catenin requires deregulation of Tcf/Lef transcription but not activation of c-myc expression.
        Mol. Cell. Biol. 1999; 19: 5696-5706
        • Schwartz D.R.
        • Wu R.
        • Kardia S.L.
        • Levin A.M.
        • Huang C.C.
        • Shedden K.A.
        • Kuick R.
        • Misek D.E.
        • Hanash S.M.
        • Taylor J.M.
        • Reed H.
        • Hendrix N.
        • Zhai Y.
        • Fearon E.R.
        • Cho K.R.
        Novel candidate targets of β-catenin/T-cell factor signaling identified by gene expression profiling of ovarian endometrioid adenocarcinomas.
        Cancer Res. 2003; 63: 2913-2922
        • Listenberger L.L.
        • Ory D.S.
        • Schaffer J.E.
        Palmitate-induced apoptosis can occur through a ceramide-independent pathway.
        J. Biol. Chem. 2001; 276: 14890-14895
      1. Deleted in proof,

        • Hanada S.
        • Snider N.T.
        • Brunt E.M.
        • Hollenberg P.F.
        • Omary M.B.
        Gender dimorphic formation of mouse Mallory-Denk bodies and the role of xenobiotic metabolism and oxidative stress.
        Gastroenterology. 2010; 138: 1607-1617
        • Tao G.Z.
        • Looi K.S.
        • Toivola D.M.
        • Strnad P.
        • Zhou Q.
        • Liao J.
        • Wei Y.
        • Habtezion A.
        • Omary M.B.
        Keratins modulate the shape and function of hepatocyte mitochondria. A mechanism for protection from apoptosis.
        J. Cell Sci. 2009; 122: 3851-3855
        • Weerasinghe S.V.
        • Moons D.S.
        • Altshuler P.J.
        • Shah Y.M.
        • Omary M.B.
        Fibrinogen-γ proteolysis and solubility dynamics during apoptotic mouse liver injury. Heparin prevents and treats liver damage.
        Hepatology. 2011; 53: 1323-1332
        • Tao G.Z.
        • Li D.H.
        • Zhou Q.
        • Toivola D.M.
        • Strnad P.
        • Sandesara N.
        • Cheung R.C.
        • Hong A.
        • Omary M.B.
        Monitoring of epithelial cell caspase activation via detection of durable keratin fragment formation.
        J. Pathol. 2008; 215: 164-174
        • Habtezion A.
        • Toivola D.M.
        • Asghar M.N.
        • Kronmal G.S.
        • Brooks J.D.
        • Butcher E.C.
        • Omary M.B.
        Absence of keratin 8 confers a paradoxical microflora-dependent resistance to apoptosis in the colon.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 1445-1450
        • Tan X.
        • Behari J.
        • Cieply B.
        • Michalopoulos G.K.
        • Monga S.P.
        Conditional deletion of β-catenin reveals its role in liver growth and regeneration.
        Gastroenterology. 2006; 131: 1561-1572
        • Tan X.
        • Apte U.
        • Micsenyi A.
        • Kotsagrelos E.
        • Luo J.H.
        • Ranganathan S.
        • Monga D.K.
        • Bell A.
        • Michalopoulos G.K.
        • Monga S.P.
        Epidermal growth factor receptor. A novel target of the Wnt/β-catenin pathway in liver.
        Gastroenterology. 2005; 129: 285-302
        • Funato Y.
        • Michiue T.
        • Asashima M.
        • Miki H.
        The thioredoxin-related redox-regulating protein nucleoredoxin inhibits Wnt-β-catenin signalling through dishevelled.
        Nat. Cell Biol. 2006; 8: 501-508
        • Hoogeboom D.
        • Burgering B.M.
        Should I stay or should I go. β-Catenin decides under stress.
        Biochim. Biophys. Acta. 2009; 1796: 63-74
        • Kaidi A.
        • Williams A.C.
        • Paraskeva C.
        Interaction between β-catenin and HIF-1 promotes cellular adaptation to hypoxia.
        Nat. Cell Biol. 2007; 9: 210-217
        • Snider N.T.
        • Weerasinghe S.V.
        • Singla A.
        • Leonard J.M.
        • Hanada S.
        • Andrews P.C.
        • Lok A.S.
        • Omary M.B.
        Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation.
        J. Cell Biol. 2011; 195: 217-229
        • Zhang K.
        • Li L.
        • Qi Y.
        • Zhu X.
        • Gan B.
        • DePinho R.A.
        • Averitt T.
        • Guo S.
        Hepatic suppression of Foxo1 and Foxo3 causes hypoglycemia and hyperlipidemia in mice.
        Endocrinology. 2012; 153: 631-646
        • Tao R.
        • Wei D.
        • Gao H.
        • Liu Y.
        • DePinho R.A.
        • Dong X.C.
        Hepatic FoxOs regulate lipid metabolism via modulation of expression of the nicotinamide phosphoribosyltransferase gene.
        J. Biol. Chem. 2011; 286: 14681-14690
        • Tenbaum S.P.
        • Ordonez-Moran P.
        • Puig I.
        • Chicote I.
        • Arques O.
        • Landolfi S.
        • Fernandez Y.
        • Herance J.R.
        • Gispert J.D.
        • Mendizabal L.
        • Aguilar S.
        • Cajal S.R.
        • Schwartz Jr., S.
        • Vivancos A.
        • Espin E.
        • Rojas S.
        • Baselga J.
        • Tabernero J.
        • Munoz A.
        • Palmer H.G.
        β-Catenin confers resistance to PI3K and AKT inhibitors and subverts FOXO3a to promote metastasis in colon cancer.
        Nat. Med. 2012; 18: 892-901
        • Yan Y.
        • Lackner M.R.
        FOXO3a and β-catenin co-localization. Double trouble in colon cancer?.
        Nat. Med. 2012; 18: 854-856
        • Shin I.
        • Bakin A.V.
        • Rodeck U.
        • Brunet A.
        • Arteaga C.L.
        Transforming growth factor β enhances epithelial cell survival via Akt-dependent regulation of FKHRL1.
        Mol. Biol. Cell. 2001; 12: 3328-3339
        • Dehner M.
        • Hadjihannas M.
        • Weiske J.
        • Huber O.
        • Behrens J.
        Wnt signaling inhibits Forkhead box O3a-induced transcription and apoptosis through up-regulation of serum- and glucocorticoid-inducible kinase 1.
        J. Biol. Chem. 2008; 283: 19201-19210
        • Czaja M.J.
        Cell signaling in oxidative stress-induced liver injury.
        Semin. Liver Dis. 2007; 27: 378-389
        • Nejak-Bowen K.
        • Kikuchi A.
        • Monga S.P.
        β-Catenin-NF-κB interactions in murine hepatocytes. A complex to die for.
        Hepatology. 2013; 57: 763-774
        • Kim P.J.
        • Plescia J.
        • Clevers H.
        • Fearon E.R.
        • Altieri D.C.
        Survivin and molecular pathogenesis of colorectal cancer.
        Lancet. 2003; 362: 205-209
        • Tapia J.C.
        • Torres V.A.
        • Rodriguez D.A.
        • Leyton L.
        • Quest A.F.
        Casein kinase 2 (CK2) increases survivin expression via enhanced β-catenin-T cell factor/lymphoid enhancer binding factor-dependent transcription.
        Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 15079-15084
        • Leong M.L.
        • Maiyar A.C.
        • Kim B.
        • O'Keeffe B.A.
        • Firestone G.L.
        Expression of the serum- and glucocorticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells.
        J. Biol. Chem. 2003; 278: 5871-5882
        • Xie X.W.
        • Liu J.X.
        • Hu B.
        • Xiao W.
        Zebrafish foxo3b negatively regulates canonical Wnt signaling to affect early embryogenesis.
        PloS One. 2011; 6: e24469
        • Kamo N.
        • Ke B.
        • Busuttil R.W.
        • Kupiec-Weglinski J.W.
        PTEN-mediated Akt/β-catenin/Foxo1 signaling regulates innate immune responses in mouse liver ischemia/reperfusion injury.
        Hepatology. 2013; 57: 289-298
        • Brookheart R.T.
        • Michel C.I.
        • Schaffer J.E.
        As a matter of fat.
        Cell Metab. 2009; 10: 9-12