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Differential effects of GD3 on Fas- and TNF-α-mediated apoptosis in mouse liver
248A
AASLD ABSTRACTS
HEPATOLOGY O c t o b e r 2 0 0 1
297
298
DIFFERENTIAL EFFECTS OF GD3 ON FAS- AND TNFa-MEDIATED APOPTOSIS IN MOUSE LIVER. Robert F Schwabe, E t s u r o Hatano, Carrie Pur-
NF-tcB INHIBITION SENSITIZES HEPATOCYIES TO TNF-c~-INDUCED APOPTOSIS THROUGH A SUSTAINED ACTIVATION OF JNK AND CJUN. H a i l i n g Liu, C h a u R Lo, M a r k J Czaja, Albert E i n s t e i n Coll of Medicine,
beck, T i n g Q u i a n , J o h n J L e m a s t e r s , U n i v e r s i t y of N o r t h Carolina, C h a p e l Hill, N C ; R y u i c h i W a d a , A k i r a H a h i r a m o t o , R i c h a r d Proia, N I D D K , Bethesda, MD; D a v i d A Brenner, U n i v e r s i t y of N o r t h Carolina, C h a p e l Hill, N C M a n y factors d e t e r m i n e w h e t h e r Fas o r T N F a will i n d u c e a p o p t o s i s in h e p a tocytes. I n particular, the role o f c e r a m i d e s a n d g a n g f l o s i d e G D 3 in a p o p t o s i s i n h e p a t o c y t e s is h i g h l y controversial. A i m s : To assess the role of G D 3 in Fasa n d T N F a - i n d u c e d a p o p t o s i s in h e p a t o c y t e s in v i v o a n d in culture b y c o m p a r i n g G D 3 s y n t h a s e k n o c k o u t m i c e ( G D 3 S k o ) w i t h w i l d type (wt) littermates. M e t h o d s : G D 3 S k o a n d w t m i c e (6-8 w e e k s of age) w e r e treated i n v i v o w i t h the F a s - a g o n i s t a n t i b o d y J o 2 ( 0 . 1 2 5 / , g / k g ) o r v e h i c l e a n d a s s e s s e d for survival. H e p a t o c y t e s f r o m these m i c e w e r e treated w i t h J o 2 p l u s a c t i n o m y c i n D, T N F a p l u s a c t i n o m y c i n D, or a p p r o p r i a t e vehicles to assess the a p o p t o s i s s i g n a l i n g p a t h w a y s . Reactive o x y g e n species (ROS) w e r e m e a s u r e d b y C M D C F - D A fluo r e s c e n c e in a 96-plate fluorescent reader. T h e m i t o c h o n d r i a l p e r m e a b i l i t y t r a n s i t i o n ( M P T ) w a s m e a s u r e d b y s c a n n i n g laser confocal m i c r o s c o p y u s i n g the f l u o r o p h o r e s calcein a n d T M R M . W e s t e r n b l o t t i n g of the S-100 s u p e r n a rant a s s e s s e d c y t o c h r o m e c release into the cytoplasm. Caspase-3 activity w a s m e a s u r e d b y A F P release assays. NF-KB t r a n s c r i p t i o n a l activity w a s m e a s u r e d b y luciferase a s s a y u s i n g a 3x-KB d r i v e n luciferase reporter. IKB k i n a s e activation a n d p65 t r a n s l o c a t i o n w e r e m e a s u r e d b y k i n a s e a s s a y a n d i m m n n o f i u o r e s c e n t staining. Results: J o 2 i n j e c t i o n p r o d u c e d significantly m o r e r a p i d m o r tality i n the w t m i c e t h a n the G D 3 S k o m i c e (p < 0 . 0 1 ). I n c u l t u r e d h e p a t o c y t e s , Fas p l u s a c t i n o m y c i n D p r o d u c e d g r e a t e r a p o p t o t i c cell d e a t h in w t t h a n G D 3 S k o hepatocytes. T N F a p l u s a c t i n o m y c i n D w a s equally effective in i n d u c i n g a p o p t o t i c cell d e a t h i n w t a n d k o hepatocytes. J o 2 i n d u c e d reactive o x y g e n species i n w t b u t n o t G D 3 S k o mice. J o 2 p l u s a c t i n o m y c i n D also i n d u c e d the M P T a n d c y t o c h r o m e c release i n w t b u t n o t G D 3 S k o hepatocytes. F i n a l l y , J o 2 p l u s a c t i n o m y c i n D p r o d u c e d significantly h i g h e r caspase-3 activity in w t t h a n in G D 3 S k o hepatocytes. T h e r e w a s no difference b e t w e e n TNFc¢- a n d J o 2 s t i m u l a t e d NF-KB t r a n s c r i p t i o n a l activity a n d T N F a - i n d u c e d I-KB k i n a s e activation a n d p65 translocation in w t a n d G D 3 S k o hepatocytes. C o n c l u s i o n s : G D 3 is r e q u i r e d for effective F a s - i n d u c e d a p o p t o s i s in h e p a t o c y t e s in v i v o a n d in culture. G D 3 is r e q u i r e d for the s i g n a l i n g p a t h w a y p r o x i m a l to the g e n e r a tion of ROS a n d the M P T , b u t does n o t m o d u l a t e NF-KB activity. T h i s is the first e v i d e n c e that G D 3 plays a role i n F a s - i n d u c e d b u t n o t T N F c M n d u c e d a p o p t o s i s in h e p a t o c y t e s
Bronx, NY Inhibition of NF-KB activation converts the hepatocyte response to tumor necrosis factor-o~ (TNF-a) from cellular proliferation to one of apoptotic cell death. This finding together with the fact that hepatocyte resistance to TNF-a killing is dependent on active gene expression, have suggested that protection from TNF-a cytotoxicity results from NF-KBmediated transcriptional up-regulation of an anti-apoptotic gene(s). However, the mechanism by which NF-~B inactivation sensitizes hepatocytes to TNF-a-induced apoptosis remains unknown. We hypothesized that the protective effects of NF-~cB activation may result from an inhibitory effect of this transcription factor on the pro-apoptotic c-Jnn N-terminal kinase (JNK) signaling cascade. To test this hypothesis, we examined both the effects of NF-KB inhibition on TNF-a-inducedJNK activation, and the involvement of JNK in TNF-a toxicity. Studies were performed in the non-transformed, rat hepatocyte cell line RALA255-10G infected with the/3-galactosidase-expressing adenovirus Ad5LacZ as a control, or the adenovirus Ad5IKB. Ad5IKB expresses a mutant Ir~B that irreversibly binds NF-KB, preventing its activation. Previous studies have demonstrated that Ad5LacZ-infected cells are resistant to TNF-a toxicity, while cells infected with Ad5I~cB undergo caspase-dependent apoptosis. JNK activation in Ad5LacZ- and Ad5IKB-infected hepatocytes was measured by immune complex kinase assay. TNF-a treatment of Ad5IKB-infected cells led to a significantly greater and more prolonged JNK activation than in Ad5LacZ-infected cefis. The major substrate of JNK is c-Jun, which is the principle subunit of the transcriptional regulator AP-1. To determine whether the increased JNK activity associated with NF-KB inhibition altered cellular gene expression, AP-1 activity was measured by transient transfections with an AP-l-driven luciferase reporter gene. By luciferase assay, AP-1 activity was 2.5-fold greater in Ad5I~B-infected cells than Ad5LacZ-infected cells 4 h after TNF-a treatment. To determine whether increased c-Jun activity mediated hepatocyte death from TNF-a, the effect of blocking c-Jun function on cell death was examined. Cells were coinfected with Ad5IKB together with either Ad5LacZ or Ad5TAM, which expresses a truncated c-Jun that acts as a dominant negative and blocks c-Jun function. Coinfection with Ad5TAM decreased cell death after 6 h of TNF-a treatment by 53% as determined by MTT assay. Quantification of the numbers of apoptotic cells under fluorescent microscopy following costaining with acridine orange and ethidium bromide revealed that the percentage of apoptotic Ad5IKB/Ad5LacZ-coinfected cells increased from 3.7 to 24% after 6 h of TNF-a treatment. In contrast, the percentage of apoptotic TNF-atreated Ad5IKB/Ad5TAM-coinfected cells was only 9.1%. By FACS analysis of propidium iodide stained cells, Ad5TAM also decreased the percentage of hypoploid cells following TNF-a treatment from 16.9 to 5.9%, suggesting that c-Jnn was critical for caspase activation. Western blotting confirmed that TNF-a-treated Ad5IKB/Ad5LacZ cells had detectable active, cleaved forms of caspase-3 and -7, while caspase cleavage was virtually absent in TNF-a-treated Ad5IKB/Ad5TAM-infected cells. Ad5TAM coinfection also prevented the TNF-a-induced release of cytochrmne c from mitochondria as determined by immunoblotting. Thus, the mechanism by which NF-KB inactivation sensitizes hepatocytes to TNF-a killing is through sustained activation of JNK and c-Jun, c-Jnn exerts a pro-apoptotic function critical for activation of the mitochondrial death pathway, causing release of cytochrome c, and resultant caspase activation. The protective effect of NF-KB activation is therefore likely mediated by increased expression of a gene(s) that down-regulates JNK.
299
300
THE ADAPTER MOLECULE MORT1/FADD IS INDISPENSABLE FOR CD95 (FAS) AND CD120A (TNF-R1) MEDIATED APOPTOSIS OF HEPATOCYTES IN V I V O . M a r c u s S c h u c h m a n n , U n i v e r s i t y of Mainz, M a i n z G e r -
CATHEPSIN B MEDIATES ITS OWN RELEASE FROM LYSOSOMES DURING TUMOR NECROSIS FACTOR-c~ (TNF-c0-MEDIATED APOPTOSIS.
m a n y ; E u g e n e E V a r f o l o m e e v , W e i z m a n n Institute, R e h o v o t Israel; C h r i s t i n e W a l d m a n n , D e n n i s Strand, Peter R Galle, A n s g a r W Lohse, U n i v e r s i t y of Mainz, M a i n z G e r m a n y Background: Disruption of the apoptotic program is considered an important cause for liver disease. It has become clear that receptor mediated apoptosis is of particular importance in this context with CD95 (Fas) and CD120a (TNF-R1), the most prominent cell death receptors and members of the TNF receptor superfamily, involved. Although mainly expressed on lymphocytes, in the liver they seem to have an additional important parenchymal arena and are involved in a series of liver pathologies. The death signal is induced upon ligand binding by recruitment of caspasesvia the adapter molecule Mortl/FADD to the receptor and their subsequent activation. Interestingly the adapter molecule Mortl/FADD seems also to be crucial for signaling of a number of other death receptors such as the Trail receptors DR4 and DR5 Methods: To investigate the role of MortI/FADD in the process we generated transgenic mice expressing an N-terminal truncated form of the adapter molecule under control of the albumin promoter which should exert a dominant negativefunction. Purified and linearized cDNAwas injected in pro-nuclei of fertilized oocytesof CB6FI mice (BalbC x C57 BL6). By southern blotting analysis 3 out of 87 animals tested were identified as founders from which 2 independent strains (#21 and #77) were established. Resuhs: Mice looked grossly normal and breeding was not different compared to wildtype linermates. Expression of the transgene completely protected 3-4 months old animals from liver failure induced by the anti-CD95 antibody Jo2, while control animals died as expected 3-6 hours after i.p. injection of 15ug antibody from acute hemorrhagic liver failure (Fig.). In a model of TNF mediated hepatotoxicity animals were challenged i.p. with 20mg D-galactosamineand 20ug LPS in 500 ul PBS. In contrast to controls, after 6 hours, the transgenic animals had only mildly elevated liver enzymes (mean ALT 724 vs. i00 U/L). Histology proved only moderate inflammatory changes in the transgenic animals while a severe hemorrhagic hepatitis was observed in control animals. Discussion: Our experiments with a transgenic animal model expressing a dominant negative form of the adapter molecule Mortl/FADD in the liver provides evidence that Morti/ FADDis indispensable for CD95 and CD120a mediated hepatic injury. This is not only of great importance for targeting future therapies of liver diseases, but might also serve as an intriguing model to study other causes of liver injury.
dn Mort/FADD transgenic Mice {lin~ 721+77l anti C D 9 5 i n d u c e d l i v e r failure
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so
T g mlce
4o
Wt mice
hours
N a t h a n W W e r n e b u r g , M E Guicciardi, H a j i m e H i g n c h i , Steven F Bronk, G r e g o r y J Gores, M a y o Clinic, Rochester, M N Tumor necrosis factor-alpha (TNF-e0 has been implicated as a hepatotoxin in a wide variety of human inflammatory liver diseases. We have recently demonstrated that cathepsin B, a cysteine protease localized in intracellular vesicles, contributes to TNF-a-mediated hepatocyte apoptosis and liver injury (J Clin Invest 2000; 106: 1127). These data also suggested that cathepsin B must be released from a vesicular compartment to cause cell death. However, the nature of the vesicles releasing cathepsin B, the specificity of the vesicle breakdown, and the mechanism responsible for cathepsin B release were not elucidated in these prior studies. Insight into the cellular compartment(s) and processes resulting in cathepsin B release into the cytosol may provide innovative treatment strategies for TNF-a-mediated liver injury. Thus, the AIMS of the present study were: 1) to identify the vesicles responsible for cathepsin B release; and 2) to examine the mechanisms mediating vesicle permeabilization. METHODS: The rat hepatoma McNtcp.24 cell line and isolated hepatocytes from wild-type ( + / + ) and cathepsin B knockout ( - / - ) mice were used for this study. McNtcp.24 cells were transfected with a cathepsin B-GFP expression vector to evaluate the intracellular localization of this protease. Cathepsin B-GFP expressing cells were incubated with LysoTracker red DND-99 (15 nM, 1 hour) to identify acidic vesicles, cotransfected with a LAMP-CFP vector to mark lysosomes and incubated with texas red-dextran 3000 (100/*M, 2 hours) to label endosomes. Confocal microscopy was utilized to determine probe compartmentation and colocalization. Apoptosis was induced with TNF-a (28 ng/ml) plus actinomycin D (AcD, 0.2/*g/ml). RESULTS: Cathepsin B-GFP colocalized with LysoTracker red verifying that cathepsin B is stored in acidic vesicles. LAMP-CFP, but not texas red-dextran, colocalized with cathepsin B-GFP demonstrating that the protease was in primary lysosomes but not endosomes or secondary endosomes. Following treatment with TNF-ogAcD, punctate cathepsin B-GFP fluorescence became diffuse consistent with its redistribution from lysosomes into the cytosol. However, release of soluble cytosolic constituents by digitonin-permeabilization revealed a residual population of intact cathepsin B-GFP containing vesicles; thus, cathepsin B release occurs in only a subpopulation of lysosomes. Treatment with TNF-cy./AcD also induced partial release of LysoTracker red; but in separate experiments, texas red-dextran fluorescence was retained within endosomes. These data indicate that permeabilization of acidic vesicles is selective involving lysosomes but not endosomes. Remarkably, LysoTracker red release from lysosomes into the cytosoI was not observed in cathepsin B - / - murine hepatocytes following TNF-a treatment, whereas Iysosomes in cathepsin B + / + -treated cells readily released this fluorescent dye. CONCLUSIONS: A unique subpopulation of lysosomes release cathepsin B during TNF-a-mediated apoptosis. Cathepsin B appears to be involved in its own release from lysosomes as cathepsm B-/- mouse hepatocyte lysosomes remain intact during TNF-o~ treatment. These data support the existence of a novel paradigm for protease signaling in cell biology, namely', protease-mediated membrane permeabilization. Given a role for cathepsin B in lysosomal permeabflization, cathepsin B inhibitors may prove useful in attenuating TNF-a-mediated liver injury.
AASLD ABSTRACTS
HEPATOLOGY O c t o b e r 2 0 0 1
297
298
DIFFERENTIAL EFFECTS OF GD3 ON FAS- AND TNFa-MEDIATED APOPTOSIS IN MOUSE LIVER. Robert F Schwabe, E t s u r o Hatano, Carrie Pur-
NF-tcB INHIBITION SENSITIZES HEPATOCYIES TO TNF-c~-INDUCED APOPTOSIS THROUGH A SUSTAINED ACTIVATION OF JNK AND CJUN. H a i l i n g Liu, C h a u R Lo, M a r k J Czaja, Albert E i n s t e i n Coll of Medicine,
beck, T i n g Q u i a n , J o h n J L e m a s t e r s , U n i v e r s i t y of N o r t h Carolina, C h a p e l Hill, N C ; R y u i c h i W a d a , A k i r a H a h i r a m o t o , R i c h a r d Proia, N I D D K , Bethesda, MD; D a v i d A Brenner, U n i v e r s i t y of N o r t h Carolina, C h a p e l Hill, N C M a n y factors d e t e r m i n e w h e t h e r Fas o r T N F a will i n d u c e a p o p t o s i s in h e p a tocytes. I n particular, the role o f c e r a m i d e s a n d g a n g f l o s i d e G D 3 in a p o p t o s i s i n h e p a t o c y t e s is h i g h l y controversial. A i m s : To assess the role of G D 3 in Fasa n d T N F a - i n d u c e d a p o p t o s i s in h e p a t o c y t e s in v i v o a n d in culture b y c o m p a r i n g G D 3 s y n t h a s e k n o c k o u t m i c e ( G D 3 S k o ) w i t h w i l d type (wt) littermates. M e t h o d s : G D 3 S k o a n d w t m i c e (6-8 w e e k s of age) w e r e treated i n v i v o w i t h the F a s - a g o n i s t a n t i b o d y J o 2 ( 0 . 1 2 5 / , g / k g ) o r v e h i c l e a n d a s s e s s e d for survival. H e p a t o c y t e s f r o m these m i c e w e r e treated w i t h J o 2 p l u s a c t i n o m y c i n D, T N F a p l u s a c t i n o m y c i n D, or a p p r o p r i a t e vehicles to assess the a p o p t o s i s s i g n a l i n g p a t h w a y s . Reactive o x y g e n species (ROS) w e r e m e a s u r e d b y C M D C F - D A fluo r e s c e n c e in a 96-plate fluorescent reader. T h e m i t o c h o n d r i a l p e r m e a b i l i t y t r a n s i t i o n ( M P T ) w a s m e a s u r e d b y s c a n n i n g laser confocal m i c r o s c o p y u s i n g the f l u o r o p h o r e s calcein a n d T M R M . W e s t e r n b l o t t i n g of the S-100 s u p e r n a rant a s s e s s e d c y t o c h r o m e c release into the cytoplasm. Caspase-3 activity w a s m e a s u r e d b y A F P release assays. NF-KB t r a n s c r i p t i o n a l activity w a s m e a s u r e d b y luciferase a s s a y u s i n g a 3x-KB d r i v e n luciferase reporter. IKB k i n a s e activation a n d p65 t r a n s l o c a t i o n w e r e m e a s u r e d b y k i n a s e a s s a y a n d i m m n n o f i u o r e s c e n t staining. Results: J o 2 i n j e c t i o n p r o d u c e d significantly m o r e r a p i d m o r tality i n the w t m i c e t h a n the G D 3 S k o m i c e (p < 0 . 0 1 ). I n c u l t u r e d h e p a t o c y t e s , Fas p l u s a c t i n o m y c i n D p r o d u c e d g r e a t e r a p o p t o t i c cell d e a t h in w t t h a n G D 3 S k o hepatocytes. T N F a p l u s a c t i n o m y c i n D w a s equally effective in i n d u c i n g a p o p t o t i c cell d e a t h i n w t a n d k o hepatocytes. J o 2 i n d u c e d reactive o x y g e n species i n w t b u t n o t G D 3 S k o mice. J o 2 p l u s a c t i n o m y c i n D also i n d u c e d the M P T a n d c y t o c h r o m e c release i n w t b u t n o t G D 3 S k o hepatocytes. F i n a l l y , J o 2 p l u s a c t i n o m y c i n D p r o d u c e d significantly h i g h e r caspase-3 activity in w t t h a n in G D 3 S k o hepatocytes. T h e r e w a s no difference b e t w e e n TNFc¢- a n d J o 2 s t i m u l a t e d NF-KB t r a n s c r i p t i o n a l activity a n d T N F a - i n d u c e d I-KB k i n a s e activation a n d p65 translocation in w t a n d G D 3 S k o hepatocytes. C o n c l u s i o n s : G D 3 is r e q u i r e d for effective F a s - i n d u c e d a p o p t o s i s in h e p a t o c y t e s in v i v o a n d in culture. G D 3 is r e q u i r e d for the s i g n a l i n g p a t h w a y p r o x i m a l to the g e n e r a tion of ROS a n d the M P T , b u t does n o t m o d u l a t e NF-KB activity. T h i s is the first e v i d e n c e that G D 3 plays a role i n F a s - i n d u c e d b u t n o t T N F c M n d u c e d a p o p t o s i s in h e p a t o c y t e s
Bronx, NY Inhibition of NF-KB activation converts the hepatocyte response to tumor necrosis factor-o~ (TNF-a) from cellular proliferation to one of apoptotic cell death. This finding together with the fact that hepatocyte resistance to TNF-a killing is dependent on active gene expression, have suggested that protection from TNF-a cytotoxicity results from NF-KBmediated transcriptional up-regulation of an anti-apoptotic gene(s). However, the mechanism by which NF-~B inactivation sensitizes hepatocytes to TNF-a-induced apoptosis remains unknown. We hypothesized that the protective effects of NF-~cB activation may result from an inhibitory effect of this transcription factor on the pro-apoptotic c-Jnn N-terminal kinase (JNK) signaling cascade. To test this hypothesis, we examined both the effects of NF-KB inhibition on TNF-a-inducedJNK activation, and the involvement of JNK in TNF-a toxicity. Studies were performed in the non-transformed, rat hepatocyte cell line RALA255-10G infected with the/3-galactosidase-expressing adenovirus Ad5LacZ as a control, or the adenovirus Ad5IKB. Ad5IKB expresses a mutant Ir~B that irreversibly binds NF-KB, preventing its activation. Previous studies have demonstrated that Ad5LacZ-infected cells are resistant to TNF-a toxicity, while cells infected with Ad5I~cB undergo caspase-dependent apoptosis. JNK activation in Ad5LacZ- and Ad5IKB-infected hepatocytes was measured by immune complex kinase assay. TNF-a treatment of Ad5IKB-infected cells led to a significantly greater and more prolonged JNK activation than in Ad5LacZ-infected cefis. The major substrate of JNK is c-Jun, which is the principle subunit of the transcriptional regulator AP-1. To determine whether the increased JNK activity associated with NF-KB inhibition altered cellular gene expression, AP-1 activity was measured by transient transfections with an AP-l-driven luciferase reporter gene. By luciferase assay, AP-1 activity was 2.5-fold greater in Ad5I~B-infected cells than Ad5LacZ-infected cells 4 h after TNF-a treatment. To determine whether increased c-Jun activity mediated hepatocyte death from TNF-a, the effect of blocking c-Jun function on cell death was examined. Cells were coinfected with Ad5IKB together with either Ad5LacZ or Ad5TAM, which expresses a truncated c-Jun that acts as a dominant negative and blocks c-Jun function. Coinfection with Ad5TAM decreased cell death after 6 h of TNF-a treatment by 53% as determined by MTT assay. Quantification of the numbers of apoptotic cells under fluorescent microscopy following costaining with acridine orange and ethidium bromide revealed that the percentage of apoptotic Ad5IKB/Ad5LacZ-coinfected cells increased from 3.7 to 24% after 6 h of TNF-a treatment. In contrast, the percentage of apoptotic TNF-atreated Ad5IKB/Ad5TAM-coinfected cells was only 9.1%. By FACS analysis of propidium iodide stained cells, Ad5TAM also decreased the percentage of hypoploid cells following TNF-a treatment from 16.9 to 5.9%, suggesting that c-Jnn was critical for caspase activation. Western blotting confirmed that TNF-a-treated Ad5IKB/Ad5LacZ cells had detectable active, cleaved forms of caspase-3 and -7, while caspase cleavage was virtually absent in TNF-a-treated Ad5IKB/Ad5TAM-infected cells. Ad5TAM coinfection also prevented the TNF-a-induced release of cytochrmne c from mitochondria as determined by immunoblotting. Thus, the mechanism by which NF-KB inactivation sensitizes hepatocytes to TNF-a killing is through sustained activation of JNK and c-Jun, c-Jnn exerts a pro-apoptotic function critical for activation of the mitochondrial death pathway, causing release of cytochrome c, and resultant caspase activation. The protective effect of NF-KB activation is therefore likely mediated by increased expression of a gene(s) that down-regulates JNK.
299
300
THE ADAPTER MOLECULE MORT1/FADD IS INDISPENSABLE FOR CD95 (FAS) AND CD120A (TNF-R1) MEDIATED APOPTOSIS OF HEPATOCYTES IN V I V O . M a r c u s S c h u c h m a n n , U n i v e r s i t y of Mainz, M a i n z G e r -
CATHEPSIN B MEDIATES ITS OWN RELEASE FROM LYSOSOMES DURING TUMOR NECROSIS FACTOR-c~ (TNF-c0-MEDIATED APOPTOSIS.
m a n y ; E u g e n e E V a r f o l o m e e v , W e i z m a n n Institute, R e h o v o t Israel; C h r i s t i n e W a l d m a n n , D e n n i s Strand, Peter R Galle, A n s g a r W Lohse, U n i v e r s i t y of Mainz, M a i n z G e r m a n y Background: Disruption of the apoptotic program is considered an important cause for liver disease. It has become clear that receptor mediated apoptosis is of particular importance in this context with CD95 (Fas) and CD120a (TNF-R1), the most prominent cell death receptors and members of the TNF receptor superfamily, involved. Although mainly expressed on lymphocytes, in the liver they seem to have an additional important parenchymal arena and are involved in a series of liver pathologies. The death signal is induced upon ligand binding by recruitment of caspasesvia the adapter molecule Mortl/FADD to the receptor and their subsequent activation. Interestingly the adapter molecule Mortl/FADD seems also to be crucial for signaling of a number of other death receptors such as the Trail receptors DR4 and DR5 Methods: To investigate the role of MortI/FADD in the process we generated transgenic mice expressing an N-terminal truncated form of the adapter molecule under control of the albumin promoter which should exert a dominant negativefunction. Purified and linearized cDNAwas injected in pro-nuclei of fertilized oocytesof CB6FI mice (BalbC x C57 BL6). By southern blotting analysis 3 out of 87 animals tested were identified as founders from which 2 independent strains (#21 and #77) were established. Resuhs: Mice looked grossly normal and breeding was not different compared to wildtype linermates. Expression of the transgene completely protected 3-4 months old animals from liver failure induced by the anti-CD95 antibody Jo2, while control animals died as expected 3-6 hours after i.p. injection of 15ug antibody from acute hemorrhagic liver failure (Fig.). In a model of TNF mediated hepatotoxicity animals were challenged i.p. with 20mg D-galactosamineand 20ug LPS in 500 ul PBS. In contrast to controls, after 6 hours, the transgenic animals had only mildly elevated liver enzymes (mean ALT 724 vs. i00 U/L). Histology proved only moderate inflammatory changes in the transgenic animals while a severe hemorrhagic hepatitis was observed in control animals. Discussion: Our experiments with a transgenic animal model expressing a dominant negative form of the adapter molecule Mortl/FADD in the liver provides evidence that Morti/ FADDis indispensable for CD95 and CD120a mediated hepatic injury. This is not only of great importance for targeting future therapies of liver diseases, but might also serve as an intriguing model to study other causes of liver injury.
dn Mort/FADD transgenic Mice {lin~ 721+77l anti C D 9 5 i n d u c e d l i v e r failure
"~
so
T g mlce
4o
Wt mice
hours
N a t h a n W W e r n e b u r g , M E Guicciardi, H a j i m e H i g n c h i , Steven F Bronk, G r e g o r y J Gores, M a y o Clinic, Rochester, M N Tumor necrosis factor-alpha (TNF-e0 has been implicated as a hepatotoxin in a wide variety of human inflammatory liver diseases. We have recently demonstrated that cathepsin B, a cysteine protease localized in intracellular vesicles, contributes to TNF-a-mediated hepatocyte apoptosis and liver injury (J Clin Invest 2000; 106: 1127). These data also suggested that cathepsin B must be released from a vesicular compartment to cause cell death. However, the nature of the vesicles releasing cathepsin B, the specificity of the vesicle breakdown, and the mechanism responsible for cathepsin B release were not elucidated in these prior studies. Insight into the cellular compartment(s) and processes resulting in cathepsin B release into the cytosol may provide innovative treatment strategies for TNF-a-mediated liver injury. Thus, the AIMS of the present study were: 1) to identify the vesicles responsible for cathepsin B release; and 2) to examine the mechanisms mediating vesicle permeabilization. METHODS: The rat hepatoma McNtcp.24 cell line and isolated hepatocytes from wild-type ( + / + ) and cathepsin B knockout ( - / - ) mice were used for this study. McNtcp.24 cells were transfected with a cathepsin B-GFP expression vector to evaluate the intracellular localization of this protease. Cathepsin B-GFP expressing cells were incubated with LysoTracker red DND-99 (15 nM, 1 hour) to identify acidic vesicles, cotransfected with a LAMP-CFP vector to mark lysosomes and incubated with texas red-dextran 3000 (100/*M, 2 hours) to label endosomes. Confocal microscopy was utilized to determine probe compartmentation and colocalization. Apoptosis was induced with TNF-a (28 ng/ml) plus actinomycin D (AcD, 0.2/*g/ml). RESULTS: Cathepsin B-GFP colocalized with LysoTracker red verifying that cathepsin B is stored in acidic vesicles. LAMP-CFP, but not texas red-dextran, colocalized with cathepsin B-GFP demonstrating that the protease was in primary lysosomes but not endosomes or secondary endosomes. Following treatment with TNF-ogAcD, punctate cathepsin B-GFP fluorescence became diffuse consistent with its redistribution from lysosomes into the cytosol. However, release of soluble cytosolic constituents by digitonin-permeabilization revealed a residual population of intact cathepsin B-GFP containing vesicles; thus, cathepsin B release occurs in only a subpopulation of lysosomes. Treatment with TNF-cy./AcD also induced partial release of LysoTracker red; but in separate experiments, texas red-dextran fluorescence was retained within endosomes. These data indicate that permeabilization of acidic vesicles is selective involving lysosomes but not endosomes. Remarkably, LysoTracker red release from lysosomes into the cytosoI was not observed in cathepsin B - / - murine hepatocytes following TNF-a treatment, whereas Iysosomes in cathepsin B + / + -treated cells readily released this fluorescent dye. CONCLUSIONS: A unique subpopulation of lysosomes release cathepsin B during TNF-a-mediated apoptosis. Cathepsin B appears to be involved in its own release from lysosomes as cathepsm B-/- mouse hepatocyte lysosomes remain intact during TNF-o~ treatment. These data support the existence of a novel paradigm for protease signaling in cell biology, namely', protease-mediated membrane permeabilization. Given a role for cathepsin B in lysosomal permeabflization, cathepsin B inhibitors may prove useful in attenuating TNF-a-mediated liver injury.