Title: Knockout of myeloid cell leukemia-1 induces liver damage and increases apoptosis susceptibility of murine hepatocytes
Abstract: HepatologyVolume 49, Issue 2 p. 627-636 Liver Biology/PathobiologyFree Access Knockout of myeloid cell leukemia-1 induces liver damage and increases apoptosis susceptibility of murine hepatocytes† Binje Vick, Binje Vick 1st Department of Medicine, Johannes Gutenberg-University Mainz, GermanySearch for more papers by this authorAchim Weber, Achim Weber Department of Pathology, Institute of Surgical Pathology, University Hospital, Zurich, SwitzerlandSearch for more papers by this authorToni Urbanik, Toni Urbanik 1st Department of Medicine, Johannes Gutenberg-University Mainz, GermanySearch for more papers by this authorThorsten Maass, Thorsten Maass 1st Department of Medicine, Johannes Gutenberg-University Mainz, GermanySearch for more papers by this authorAndreas Teufel, Andreas Teufel 1st Department of Medicine, Johannes Gutenberg-University Mainz, GermanySearch for more papers by this authorPeter H. Krammer, Peter H. Krammer German Cancer Research Center, Tumor Immunology Program, Heidelberg, GermanySearch for more papers by this authorJoseph T. Opferman, Joseph T. Opferman Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, TNSearch for more papers by this authorMarcus Schuchmann, Marcus Schuchmann 1st Department of Medicine, Johannes Gutenberg-University Mainz, GermanySearch for more papers by this authorPeter R. Galle, Peter R. Galle 1st Department of Medicine, Johannes Gutenberg-University Mainz, GermanySearch for more papers by this authorHenning Schulze-Bergkamen, Corresponding Author Henning Schulze-Bergkamen [email protected] 1st Department of Medicine, Johannes Gutenberg-University Mainz, Germany fax: (49) 6131-175687.1st Department of Medicine, Johannes Gutenberg-University Mainz, LangenbeckstraSe 1, 55101 Mainz, Germany===Search for more papers by this author Binje Vick, Binje Vick 1st Department of Medicine, Johannes Gutenberg-University Mainz, GermanySearch for more papers by this authorAchim Weber, Achim Weber Department of Pathology, Institute of Surgical Pathology, University Hospital, Zurich, SwitzerlandSearch for more papers by this authorToni Urbanik, Toni Urbanik 1st Department of Medicine, Johannes Gutenberg-University Mainz, GermanySearch for more papers by this authorThorsten Maass, Thorsten Maass 1st Department of Medicine, Johannes Gutenberg-University Mainz, GermanySearch for more papers by this authorAndreas Teufel, Andreas Teufel 1st Department of Medicine, Johannes Gutenberg-University Mainz, GermanySearch for more papers by this authorPeter H. Krammer, Peter H. Krammer German Cancer Research Center, Tumor Immunology Program, Heidelberg, GermanySearch for more papers by this authorJoseph T. Opferman, Joseph T. Opferman Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, TNSearch for more papers by this authorMarcus Schuchmann, Marcus Schuchmann 1st Department of Medicine, Johannes Gutenberg-University Mainz, GermanySearch for more papers by this authorPeter R. Galle, Peter R. Galle 1st Department of Medicine, Johannes Gutenberg-University Mainz, GermanySearch for more papers by this authorHenning Schulze-Bergkamen, Corresponding Author Henning Schulze-Bergkamen [email protected] 1st Department of Medicine, Johannes Gutenberg-University Mainz, Germany fax: (49) 6131-175687.1st Department of Medicine, Johannes Gutenberg-University Mainz, LangenbeckstraSe 1, 55101 Mainz, Germany===Search for more papers by this author First published: 28 January 2009 https://doi.org/10.1002/hep.22664Citations: 116 † Potential conflict of interest: Nothing to report. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic member of the Bcl-2 protein family. It interacts with proapoptotic Bcl-2 family members, thereby inhibiting mitochondrial activation and induction of apoptosis. Mcl-1 is essential for embryonal development and the maintenance of B cells, T cells, and hematopoietic stem cells. We have recently shown that induction of Mcl-1 by growth factors rescues primary human hepatocytes from CD95-mediated apoptosis. This prompted us to further analyze the relevance of Mcl-1 for hepatocellular homeostasis. Therefore, we generated a hepatocyte-specific Mcl-1 knockout mouse (Mcl-1flox/flox-AlbCre). Deletion of Mcl-1 in hepatocytes results in liver cell damage caused by spontaneous induction of apoptosis. Livers of Mcl-1flox/flox-AlbCre mice are smaller compared to control littermates, due to higher apoptosis rates. As a compensatory mechanism, proliferation of hepatocytes is enhanced in the absence of Mcl-1. Importantly, hepatic pericellular fibrosis occurs in Mcl-1 negative livers in response to chronic liver damage. Furthermore, Mcl-1flox/flox-AlbCre mice are more susceptible to hepatocellular damage induced by agonistic anti-CD95 antibodies or concanavalin A. Conclusion: The present study provides in vivo evidence that Mcl-1 is a crucial antiapoptotic factor for the liver, contributing to hepatocellular homeostasis and protecting hepatocytes from apoptosis induction. (HEPATOLOGY 2009.) Apoptosis, or programmed cell death, regulates tissue development and homeostasis in multicellular organisms. Extrinsic or intrinsic death signals activate proapoptotic pathways, resulting in the activation of caspases and finally in cell death. An important event during the apoptosis process is the permeabilization of the outer mitochondrial membrane. Integrity of the outer mitochondrial membrane is regulated by the Bcl-2 protein family, which is divided into three groups: antiapoptotic members Bcl-2, Bcl-xL, and myeloid cell leukemia-1 (Mcl-1); proapoptotic multidomain members Bax and Bak; and proapoptotic BH3-only proteins. Mitochondrial activation is regulated by selective interactions of Bcl-2 proteins via their Bcl-2 homology (BH) domains.1-4 Expression of Mcl-1 has been found to be induced in cells at various stages of differentiation, in response to specific growth, differentiation, and survival factors. The importance of Mcl-1 during differentiation has been pointed out in Mcl-1 knockout mice, which die at an early stage of embryogenesis.5 In conditional Mcl-1 knockout models, hematopoietic stem cells as well as early-stage B or T cells die due to apoptosis induction.6, 7 Due to its antiapoptotic properties, Mcl-1 is a potential proto-oncogene. Mcl-1 transgenic mice show an increased incidence of B cell lymphomas.8 In addition, enhanced expression of Mcl-1 is observed in a wide range of tumors, including hepatocellular carcinoma.9, 10 To date, little is known about the role of Mcl-1 in nontransformed cells. Mcl-1 expression is indispensable for the survival of hematopoietic stem cells, early stage B and T cells,6, 7 and macrophages.11 We have recently shown that induction of Mcl-1 by hepatocyte growth factor protects primary human hepatocytes from CD95 (APO-1/Fas)-induced apoptosis.12 This observation is in line with studies which have shown that Mcl-1 transgenic mice are rescued from acute liver failure induced by CD95 triggering.13 Therefore, we assume that Mcl-1 is an important antiapoptotic factor for the liver. In this study, we generated hepatocyte-specific Mcl-1 knockout mice. In the absence of Mcl-1, liver homeostasis is critically affected. Spontaneous induction of apoptosis is increased in Mcl-1 negative hepatocytes, resulting in profound liver damage and hepatic fibrosis. Furthermore, hepatocytes lacking Mcl-1 expression are more sensitive to proapoptotic stimuli. Therefore, we conclude that Mcl-1 is a central antiapoptotic factor for hepatocytes. Abbreviations ALT, alanine aminotransferase; AST, aspartate aminotransferase; BrdU, 5-bromo-2-deoxyuridine; ConA, concanavalin A; Mcl-1, myeloid cell leukemia-1. Materials and Methods Generation of Conditional Mcl-1 Knockout Mice. Mcl-1flox/flox mice7 were bred to heterozygous Albumin-Cre mice14 (both C57BL/6 background). Male Mcl-1flox/wt-AlbCre offspring was bred to female Mcl-1flox/flox mice. Mcl-1flox/flox-AlbCre offspring (referred to as Mcl-1−/− mice) were compared to their control littermates with the genotypes Mcl-1flox/wt-AlbCre+/−, Mcl-1flox/flox-AlbCre−/−, or Mcl-1flox/wt-AlbCre−/− (referred to as control or Mcl-1+/+ mice). A scheme of the different genotypes can be found in Opferman et al.7 All animals were bred at the animal facility of the University of Mainz, had free access to water and food under standard conditions with a 12-hour dark/light cycle, and received humane care. All experiments were done in accordance with German federal law and were approved by the Local Committee for Experimental Animal Research. Animal Genotyping. For genotyping, ear biopsies were digested overnight at 56°C with proteinase K (Calbiochem, Schwalbach, Germany) in buffer containing 100 mM Tris-HCl pH 7.6, 50 mM ethylene diamine tetraacetic acid (EDTA) pH 8.0, 0.5% sodium dodecyl sulfate. Afterward, proteinase K was heat-inactivated at 96°C for 3 minutes, and the solution was diluted with 10 volume of water. One μL of the solution was used for polymerase chain reaction (PCR)-based genotyping. PCR was performed using the following primers: Mcl-1 flox: 5′-CTGAGAGTTGTACCGGACAA-3′ and 5′-GCAGTACAGGTTCAAGCCGATG-3′; Mcl-1 deleted (Δ): 5′-CTGAGAGTTGTACCGGACAA-3′ and 5′-ACGCTCTTTAAGTGTTTGGCC-3′; Cre: 5′-GCACTGATTTCGACCAGGTT-3′and 5′-CCCGGCAAAACAGGTAGTTA-3′; Actin (internal control for Cre PCR): 5′-TGTTACCAACTGGGACGACA-3′and 5′-GACATGCAAGGAGTGCAAGA-3′. PCR was performed in a standard thermocycler and analyzed on 2% agarose gels. Aminotransferase Levels. About 100 μL of blood was collected from the tail vein. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in the Institute of Clinical and Laboratory Medicine at the University Hospital Mainz by standard procedures. Acute Liver Damage. Mice were injected intraperitonally with 0.5 mg/kg body weight of an anti-mouse CD95 monoclonal antibody (Jo2; BD Pharmingen, Heidelberg, Germany)15 or with 25 mg/kg body weight concanavalin A (ConA, Sigma) into the tail vein. After the indicated time points, mice were sacrificed, blood was collected, and livers were shock-frozen in liquid nitrogen or transferred into 4% phosphate buffered saline (PBS)-buffered formalin (55 mM Na2HPO4, 12 mM NaH2PO4, 4% formalin, pH 7.4) until further analysis. Isolation of Hepatocytes. Hepatocytes were isolated by a two-step perfusion technique.16 Briefly, mice were anesthetized with 2.5% Avertin (2,2,2-tribromoethanol; Sigma) and livers were perfused in situ with about 100 mL of buffer I containing 140 mM NaCl, 7 mM KCl, and 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) pH 7.4. Livers were then perfused with 50 mL of buffer II containing 70 mM NaCl, 7 mM KCl, 5 mM CaCl2, 100 mM HEPES pH 7.6, and 0.5 mg/mL collagenase (Serva, Heidelberg, Germany). Thereafter, livers were mechanically disrupted in buffer I, and cells were suspended and washed twice with Dulbecco's modified Eagle's medium (DMEM; Gibco, Karlsruhe, Germany). Tissue Lysis and Western Blotting. About 20 mg of shock-frozen liver tissue were minced, transferred into ice-cold lysis buffer containing 20 mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.5% Triton-X 100, and 1× protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), and incubated on ice for 15 minutes. Cell debris was removed by centrifugation (10,000g at 4°C). Protein concentration was determined by Dc Protein Assay (Bio-Rad, Munich, Germany), and equal amounts of protein were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Immunodetection was performed using the following primary antibodies: Mcl-1 (Rockland, Gilbertsville, PA), Bcl-xL (H-62; Santa Cruz Biotechnology, Heidelberg, Germany), Bak (BD Pharmingen), Bax (Cell Signaling Technology, Frankfurt, Germany), Bid (Cell Signaling Technology), and α-tubulin (Sigma). Peroxidase-conjugated species-specific secondary antibodies (Santa Cruz Biotechnology) were used at a dilution of 1:10,000. Bound antibody was visualized using chemiluminescent substrate (PerkinElmer, Zaventem, Belgium) and exposure to Fuji Medical X-Ray film. All western blots were performed for at least two male and two female mice at each age indicated. Caspase Assay. Caspase-3 activity was measured as previously described.17 DNA and RNA Isolation. For DNA isolation, about 20 mg of shock-frozen liver tissue were lysed overnight with proteinase K (see above). For isolation of total RNA, about 20 mg of shock-frozen liver tissue were homogenized in 1 mL TRI-Reagent (Sigma), and further isolated according to the manufacturer's instructions. RNA concentration was measured in a NanoDrop photometer (Peqlab, Erlangen, Germany), and 1 μg of total RNA was subjected to reverse transcription using Oligo-dT primers. Isolated DNA and complementary DNA (cDNA) were used for PCR (see above) and real-time (RT) PCR approaches, respectively. RT Quantitative PCR. Relative target messenger RNA (mRNA) expression was analyzed by RT-quantitative PCR using the QuantiTect SYBR Green PCR Kit and QuantiTect primers (Qiagen, Hilden, Germany) for murine Mcl-1, interleukin-6 (IL-6), tumor necrosis factor (TNF), collagen-1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The relative increase in reporter fluorescent dye emission was monitored. The level of target mRNA, relative to GAPDH, was calculated using the formula: Relative target mRNA/GAPDH mRNA expression = 1/2 ∧ [ct target – ct GAPDH] * 100, where ct is defined as the number of the cycle in which emission exceeds threshold levels. Histology and Immunohistochemistry. Liver specimens were fixed in 4% PBS-buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin as well as Sirius red using standard histological techniques. In addition, slides were immunostained for activated caspase-3 (rabbit polyclonal antibody, 1:300 dilution; Cell Signaling Technology) and Ki67 (monoclonal rabbit clone SP6, 1:100 dilution; NeoMarkers) using the Ventana Discovery automated staining system with an iView DAB kit (Ventana, Tucson, AZ), replacing the secondary antibody with a donkey anti-rabbit biotinylated antibody (Jackson 711-065-152, 1:80 dilution; Jackson ImmunoResearch). All sections were counterstained with hematoxylin. The whole section was evaluated for the number of positive hepatocytes, and pictures were taken from representative high-power fields (at 200× magnification). Detection of Cell Proliferation. Mice were exposed to 0.8 mg/mL bromodeoxyuridine (BrdU; Roche) in drinking water for 60 hours. Mice were sacrificed and livers were shock-frozen in liquid nitrogen. Tissue sections (5 μm) were prepared and BrdU-positive cells were stained with the In Situ Cell Proliferation Kit (Roche) according to the manufacturer's instructions. Afterward, nuclei were counterstained with 2 μg/mL 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Karlsruhe, Germany) for 15 minutes at room temperature. Sections were analyzed for fluorescein isothiocyanate (FITC) and DAPI staining with a fluorescence microscope using corresponding filters. From each mouse, two independent sections were stained and seven microscopic fields from each section were analyzed by counting FITC-positive and DAPI-positive nuclei. Detection of Apoptosis. Mice were sacrificed and livers were shock-frozen in liquid nitrogen. Tissue sections (5 μm) were prepared and transferred into 4% PBS-buffered formalin. Apoptosis was detected by staining cell nuclei with DNA strand breaks (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling [TUNEL] technology) using the In Situ Cell Death Detection Kit, Fluorescein (Roche), according to the manufacturer's instructions. Counterstaining and further procedure was performed as described above. Data Analysis. All graphs show both single and median values from at least three independent experiments. Histological images show representative results. Data were analyzed by Mann-Whitney U test using SPSS software, with P < 0.05 considered significant. Results Deletion of Mcl-1 in Hepatocytes of Mcl-1flox/flox-AlbCre Mice. After breeding Mcl-1flox/flox mice to Mcl-1flox/wt-AlbCre mice, offspring were screened for deletion of Mcl-1 and expression of Cre (Fig. 1A). All animals positive for Cre and for a floxed Mcl-1 allele were also positive for the deleted Mcl-1 fragment in liver lysates. Figure 1Open in figure viewerPowerPoint Deletion of Mcl-1 in hepatocytes of Mcl-1flox/flox-AlbCre mice. (A) DNA from ear biopsies (left) or liver specimens (right) was isolated. PCR was performed using primers for wild-type (wt), floxed, or deleted (Δ) Mcl-1, Cre, and actin as internal control. (B) Liver lysates of Mcl-1flox/+, Mcl-1flox/flox, Mcl-1flox/+-AlbCre and Mcl-1flox/flox-AlbCre mice at the age of 4 and 8 weeks, were analyzed by western blot for Mcl-1 expression and for α-tubulin as loading control. (C) mRNA from total livers or from isolated hepatocytes of Mcl-1flox/flox-AlbCre and control mice at the age of 4 or 8 weeks was isolated and transcribed into cDNA. Mcl-1 and GAPDH expression were analyzed by RT-PCR, and Mcl-1/GAPDH ratio was calculated. Both single (squares) and median values (bars) are presented. **, P < 0.01; ***, P < 0.001; n.s., not significant. We analyzed Mcl-1 expression in liver lysates of 4-week-old and 8-week-old mice. Mcl-1 protein expression was significantly reduced in liver lysates of Mcl-1flox/flox-AlbCre mice compared to Mcl-1flox/wt mice, but was only slightly reduced in Mcl-1flox/wt-AlbCre mice (Fig. 1B). RT-PCR also showed a significant reduction in Mcl-1 mRNA expression in total liver lysates of Mcl-1flox/flox-AlbCre mice (4 weeks: 27%; 8 weeks: 15%; n = 8, P < 0.001), and a slight reduction in Mcl-1 mRNA expression in Mcl-1flox/wt-AlbCre mice (4 weeks: 81%, n.s.; 8 weeks: 71%; n = 5, P < 0.05) compared to Mcl-1flox/flox mice (n = 6; Fig. 1C). To analyze if the remaining Mcl-1 expression observed was originating from hepatocytes or from nonparenchymal cells, we isolated hepatocytes from Mcl-1flox/flox-AlbCre mice and found that Mcl-1 mRNA expression was reduced to 4% of that of control hepatocytes (n = 4, P < 0.05; Fig. 1C). Deletion of Mcl-1 expression was equally efficient both in male and in female mice (data not shown). Next, we asked whether other Bcl-2 family members might be regulated in hepatocytes to compensate for the loss of Mcl-1 expression. Expression of Bcl-xL, Bid, Bax, and Bak, however, were not altered in liver lysates of Mcl-1flox/flox-AlbCre mice (Supporting Fig. 1). Basal Liver Damage and Hepatic Fibrosis in Mcl-1flox/flox-AlbCre Mice. We analyzed the effect of a loss of Mcl-1 expression for liver homeostasis. Interestingly, livers of Mcl-1flox/flox-AlbCre mice appeared much smaller, and liver weight was profoundly reduced (64%-78% compared to control littermates, P < 0.001; Fig. 2A,B). Body weight, however, was only slightly decreased (Fig. 2A). Furthermore, aspartate and alanine aminotransferase (AST and ALT) values, which are indicators of hepatocyte injury, were highly increased in these mice. Eight-week-old Mcl-1flox/flox-AlbCre mice show a 7.6-fold increase in serum ALT levels (median: 280 U/L versus 40 U/L in control; P < 0.001) and a 5.7-fold increase in serum AST levels (median: 340 U/L versus 80 U/L in control; P < 0.001; Fig. 2C). We also analyzed 4-week-old animals and again found a significant increase in serum aminotransferase levels (Fig. 2C). Interestingly, AST and ALT levels were only slightly, yet still significantly, enhanced in 4-month-old animals (Fig. 2C). In addition, we determined serum bilirubin values. There was no increase in bilirubin values in Mcl-1flox/flox-AlbCre mice in the age of 8 weeks to 4 months, demonstrating that liver function was not impaired (data not shown). Figure 2Open in figure viewerPowerPoint Liver damage in Mcl-1flox/flox-AlbCre mice. (A) Four-week-old to 16-week-old Mcl-1flox/flox-AlbCre (Mcl-1−/−) or control (Mcl-1+/+) mice were analyzed for body weight and liver weight. (B) Macroscopic appearance of representative livers from 8-week-old female Mcl-1+/+ and Mcl-1−/− mice. (C) Serum of Mcl-1+/+ and Mcl-1−/− mice between 4 and 16 weeks of age was collected. Serum AST and ALT levels were measured. Both single (squares) and median values (bars) are presented. Values beyond scaling are marked separately. *, P < 0.05; ***, P < 0.001; n.s., not significant. Because Mcl-1flox/flox-AlbCre mice reveal a basal liver damage, we asked whether this was due to spontaneous induction of apoptosis in hepatocytes. Therefore, we analyzed liver histology and stained liver sections of 4-week-old mice for active caspase-3 as a marker for the execution phase of apoptosis. Among Mcl-1−/− hepatocytes, 1%-3% of cells were stained positive for active caspase-3. In contrast, control liver sections revealed hardly any or no caspase-3 positive hepatocytes (Fig. 3A). To further validate this result, we also determined caspase-3 activity fluorometrically in liver lysates. Again, we found that caspase-3 activity was significantly enhanced in Mcl-1−/− hepatocytes (Fig. 3B). Furthermore, we analyzed DNA strand breaks to determine apoptosis rates. TUNEL assays revealed that five-fold more hepatocytes were positive for DNA strand breaks in Mcl-1−/− livers compared to controls (P < 0.001; Fig. 3C). Figure 3Open in figure viewerPowerPoint Livers of Mcl-1flox/flox-AlbCre mice show higher apoptosis rates. (A) Liver sections of 4-week-old Mcl-1+/+ and Mcl-1−/− mice were stained for active caspase-3 and with eosin/hematoxylin. (B) Total liver lysates were analyzed for active caspase-3 activity using fluorometric substrates. (C) Liver sections were stained for DNA strand breaks using TUNEL technology, and cell nuclei were counterstained using DAPI. The ratio of TUNEL-positive nuclei was calculated. Both single (squares) and median values (bars) are presented. The bar corresponds to 100 μm. ** P < 0.01, *** P < 0.001. RLU indicates relative light units. Next, we tested whether reduced liver mass in Mcl-1flox/flox-AlbCre mice is due to increased apoptosis rates or could also be caused by reduced proliferation of hepatocytes. To determine proliferation of Mcl-1−/− hepatocytes, 4-week-old mice received BrdU in the drinking water for 60 hours. The amount of BrdU-positive nuclei was about 2.5-fold increased in Mcl-1−/− hepatocytes compared to controls (17.5 versus 7%, P < 0.01; Fig. 4B). Furthermore, we analyzed Ki67 expression, another marker for cell proliferation. Again, expression was profoundly increased in Mcl-1−/− hepatocytes compared to controls (Fig. 4A). Therefore, we conclude that the proliferation rate of Mcl-1−/− hepatocytes is enhanced, presumably to partially compensate for the loss of hepatocytes due to apoptosis. Figure 4Open in figure viewerPowerPoint Livers of Mcl-1flox/flox-AlbCre mice show higher proliferation rates. (A) Liver sections of 4-week-old Mcl-1+/+ and Mcl-1−/− mice were stained for Ki67 and with eosin/hematoxylin. (B) Mcl-1+/+ and Mcl-1−/− mice received BrdU (0.8 mg/mL for 60 hours) in their drinking water. Liver sections were stained for BrdU incorporation, and cell nuclei were counterstained using DAPI. The ratio of BrdU-positive nuclei was calculated. Both single (squares) and median values (bars) are presented. The bar corresponds to 100 μm. **, P < 0.01. Then, we analyzed IL-6 and TNF expression in liver lysates of 16-week-old mice, to determine if the increase in apoptosis induction in Mcl-1−/− hepatocytes leads to an inflammatory response. We could, however, observe no difference in IL-6 or TNF mRNA expression (data not shown). Next, we asked whether hepatic fibrosis occurs as a response to chronically increased apoptosis rates in Mcl-1−/− livers. Therefore, we analyzed mRNA expression of collagen-1. Interestingly, collagen-1 expression was significantly enhanced in Mcl-1−/− livers, indicating fibrosis induction (P < 0.01; Fig. 5A). In addition, we stained Mcl-1−/− liver sections with Sirius Red. Mcl-1−/− mice revealed a mild degree of pericellular fibrosis at the age of 8 weeks, and even more at the age of 16 weeks, which was not observable in control littermates (Fig. 5B). Figure 5Open in figure viewerPowerPoint Fibrosis induction in Mcl-1flox/flox-AlbCre mice. (A) mRNA from total liver lysates of 16-week-old Mcl-1+/+ and Mcl-1−/− mice was isolated and transcribed into cDNA. Collagen-1 and GAPDH expression were analyzed by RT-PCR, and collagen/GAPDH ratio was calculated. Both single (squares) and median values (bars) are presented. **, P < 0.01. (B) Liver sections of 8-week-old or 16-week-old Mcl-1+/+ and Mcl-1−/− mice were stained with Sirius red for collagen deposition. The bar corresponds to 100 μm. Effect of Mcl-1 Deletion on CD95-Mediated or T Cell–Mediated Liver Damage. CD95-mediated apoptosis of hepatoctytes contributes to the pathophysiology of various liver diseases.18 Hepatocytes constitutively express CD95, and the liver is highly susceptible to CD95-induced apoptosis.18, 19 To analyze the effect of Mcl-1 deletion on CD95 triggering on hepatocytes, we treated mice with the agonistic CD95-antibody Jo2. After Jo2 administration, 8-week-old Mcl-1−/− mice had significantly elevated serum AST and ALT levels compared to control (AST: four-fold; ALT: nine-fold, P < 0.001; Fig. 6A). Histology, immunohistochemical staining for active caspase-3, and analysis of caspase-3 activity in total liver lysates revealed that Mcl-1−/− livers had both enhanced apoptosis levels and more severe architectural damage after Jo2-treatment (Fig. 6C,D,E). Additionally, we treated older animals, which had only slightly enhanced basal aminotransferase levels (Fig. 2C), with Jo2. Again, Mcl-1flox/flox-AlbCre mice had significantly increased serum aminotransferase levels after Jo2-administration compared to control (P < 0.05; Fig. 6B). Figure 6Open in figure viewerPowerPoint Mcl-1flox/flox-AlbCre mice are more susceptible toward CD95-mediated liver damage. Eight-week-old (A, C-E) or 24-week-old (B) Mcl-1+/+ and Mcl-1−/− mice were treated with Jo2 (0.5 mg/kg) administered intraperitoneally. After 3 hours, mice were sacrificed. (A, B) Blood was collected and serum AST and ALT levels were determined. Both single (squares) and median values (bars) are presented. (C) Liver sections were stained with eosin/hematoxylin and for active caspase-3. The bar corresponds to 100 μm. (D) Total liver lysates were analyzed for caspase-3 activity. (E) Apoptosis rate and architectural damage were analyzed within hematoxylin/eosin-stained sections and were clustered into four groups (0: no, 1: low, 2: moderate, 3: high apoptosis rate/architectural damage). * P < 0.05; ** P < 0.01; *** P < 0.001. RLU: relative light units. ConA-induced hepatitis is frequently used as a model for autoimmune liver disease and is dependent on T cell activity as well as CD95 signaling.20, 21 We tested whether Mcl-1 deletion in hepatocytes influences T cell–mediated liver injury in vivo. Eight-week-old mice were injected with 25 mg/kg body weight ConA into the tail vein. Mcl-1−/− mice showed an increased liver damage after ConA treatment compared to control (AST: three-fold increase; ALT: six-fold increase; P < 0.05; Fig. 7). We conclude that Mcl-1 deletion renders hepatocytes more susceptible to apoptosis stimuli, e.g., death receptor ligands and activated T cells. Figure 7Open in figure viewerPowerPoint Mcl-1flox/flox-AlbCre mice are more susceptible to T cell–mediated liver damage. Eight-week-old Mcl-1+/+ and Mcl-1−/− mice were treated with ConA (25 mg/kg) intravenously. After 4 hours, mice were sacrificed. Blood was collected and serum AST and ALT levels were determined. Both single (squares) and median values (bars) are presented. *, P < 0.05. Discussion The aim of this study was to investigate the role of Mcl-1 for liver cell homeostasis and susceptibility of hepatocytes to apoptosis stimuli. Our results clearly demonstrate that Mcl-1 is an important antiapoptotic factor in the liver and contributes to hepatocyte survival, e.g., after death receptor activation. Mcl-1 was first described in the human myeloid leukemia cell line ML-1, where it is induced early during phorbol ester-induced differentiation.22 Further studies revealed that Mcl-1 is promoting viability rather than proliferation. In a vast number of cell types, transformed or untransformed, and after diverse stress or growth signals, Mcl-1 expression is rapidly induced and rescues cells from apoptosis induction. By contrast, Mcl-1 degradation is a prerequisite for cells to die.23 Therefore, Mcl-1 expression is crucial in developing and differentiating cells, and in cells facing constant stress signals (like cancer cells). Mouse models revealed that Mcl-1 expression is crucial for embryonal development,5 and for the viability of hematopoietic (stem) cells.6, 7, 11 So far, little is known about the importance of Mcl-1 in fully differentiated liver cells. We have recently shown that Mcl-1 is induced by growth factors in primary human hepatocytes and contributes to the survival of primary human hepatocytes and hepatocellular carcinoma cells in vitro.9, 12, 17 Thus, we hypothesized that Mcl-1 might also be a crucial factor for liver cell