Apoptosis and Oncotic NecrosisApoptosis vs Oncotic Necrosis Initiation Execution Apoptosis Apoptosis...

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Apoptosis and Oncotic Necrosis: The final common pathways of liver injury Hartmut Jaeschke Department of Pharmacology, Toxicology & Therapeutics University of Kansas Medical Center Kansas City, KS
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Transcript of Apoptosis and Oncotic NecrosisApoptosis vs Oncotic Necrosis Initiation Execution Apoptosis Apoptosis...

  • Apoptosis and Oncotic Necrosis:The final common pathways of liver injury

    Hartmut Jaeschke

    Department of Pharmacology, Toxicology & Therapeutics

    University of Kansas Medical CenterKansas City, KS

  • Fibrosis: Response to Cell Injury

    Rockey & Friedman, 2006

    Chronic Cell Injury: Apoptosis and Oncotic Necrosis

  • • cellular condensation (cell shrinkage)

    • membrane blebbing, but no loss of integrity

    • aggregation of chromatin at the nuclear membrane

    • formation of membrane bound vesicles (apoptotic bodies)

    • no disintegration of organelles; organelles remain intact

    • swelling of the cell and lysis

    • loss of membrane integrity

    • flocculation of chromatin

    • no vesicle formation, complete lysis

    • disintegration (swelling) of cell organelles

    Morphology

    Apoptosis Oncotic Necrosis

  • Hepatocellular Apoptosis

    H & E

    Galactosamine(500 mg/kg; 6 hr)

  • Hepatocellular Oncotic Necrosis and Inflammation

    H & E

    Bile Duct Ligation3 days

  • Apoptotic Cell Death: Caspases

    Proenzyme (zymogen)

    prodomain large subunit linker domain small subunit

    Peptidase1 496

    DED DED Pro-Caspase 8Initiator Caspase

    Death effector domainDED

    Peptidase1 277 Pro-Caspase 3

    Effector Caspase

    Cysteine-dependentaspartase domainPeptidase

    PeptidaseCARD1 416 Pro-Caspase 9

    Initiator Caspase

    Caspase recruitment domainCARD

  • Activation of CaspasesProenzyme(zymogen)

    Active Caspase(tetramer)

    prodomain large subunit linker domain small subunit

  • Activation of CaspasesProenzyme(zymogen)

    Active Caspase(tetramer)

    prodomain large subunit linker domain small subunit

    0 20 40 90 120 Time (min)

    p32

    p11Fas-AbJo-2

    Proenzyme

    Active Fragment

  • 0

    2 0 0

    4 0 0

    6 0 0

    Activation of Caspases

    0 20 40 90 120 Time (min)

    p32

    p11

    (ΔF/min/mg protein)

    600

    400

    200

    0

    *

    *

    *

  • DDDD DD

    FAS-L FAS-L

    DDDDDD

    FAD

    DFA

    DD

    FAD

    DFA

    DD

    Cas8

    FAS-L

    DDDDDD

    FAD

    DFA

    DD

    FAD

    DFA

    DD

    Cas10

    Cas

    p10

    Casp 6

    Casp

    8Bid

    BaxBak

    C

    C

    C

    C

    C

    C

    C

    CC

    C

    C

    CC

    C

    Casp 7Casp 3A

    paf1

    Apa

    f1

    Cas

    p9

    Cas

    p9

    C

    CC

    C

    CAD

    Endo

    GEn

    doG

    CAD

    Endo

    G

    AIF

    AIF

    Dia

    blo

    Diablo

    C

    Bak Bak

    Bax

    Bax

    CaspaseSubstrates

    CAD

    CARD

    C

    C

    CARD

    DISC

    FAS-LFAS-L

    dATP

    dATP

    Fas-mediated Apoptosis Signaling in Hepatocytes

    CAD ICADCAD

  • Bid

    Bcl-2

    Bak

    C

    C

    CC

    C

    C

    C

    C

    C

    C

    C

    C Casp 3

    C

    C

    CC

    EndoG EndoG

    EndoG

    EndoG

    AIF

    Diablo

    C

    Caspase 3 SubstratesAIF

    cIAPDiablo

    Intrinsic Pathway of Apoptosis

    Ca

    Ca

    Ca

    Ca

    AIF

    Diablo

    Diablo C

    AIF

    C

    C

    C

    C

    C

    C

    C C

    CC

    C

    C

    BaxBakBax

    Bak

    AIFDiablo

    CaCa

    Ca

    Ca

    CaCa

    CaCa

    CaCa

    PTP

    Bcl-XL

    Bcl-2 Bax

    Bax

    Bax

    28S

    eIF2α

    PTP

    Cathepsin

    C

    Apa

    f1CARD

    Apaf1CAR

    D

    Apaf1 CARD

    Apaf1

    CARDCasp 9

    Casp

    9Casp

    9

    C

    CC

    BaxBakBax

    Bak

    CaCaCa

    Ca

    Casp 9

    C C

    m-Calpain

    Casp 12

    p53Bax

  • Caspase Targets in ApoptosisCasp10

    Casp 6

    Casp 8

    Casp 7

    Casp 3

    Apa

    f1

    Apa

    f1

    Cas

    p9

    Cas

    p9

    CARDC

    CARD

    CARD Casp 2

    MDM2Fodrin

    Prese-nelin2 Gelsolin

    Actin Lamin A

    DNA fragmentation andchromatin condensation

    CytoskeletonCell shape and membrane

    blebbing

    CAD

    Trans-glutaminase

    Topo-isomerase

    PARPDNA-PKEndoGICAD

    Keratin-18

    Gas2

    Cell cycle and other

    p21

    FAK

    hnRNP

    β-catenin

    NuMASREB1

    Calpastatin

    EMAP II

    Rock-1

  • DNA Fragmentation

    1 2 3 4 5 6 7 8 9 10 11 12 13

    MW Controls G/ET

    0

    200

    400

    600

    800

    1000

    (% Vmax)

    00 20 40 90 120

    600

    400

    200

    800

    1000 *

    *

    *

    Time (min)

    DNA LadderAnti-Histone ELISA

    CADCAD

  • DNA Strand Breaks: TUNEL Assay

    Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling

    GalactosamineGalactosamine (500 mg/kg; 6 hr)(500 mg/kg; 6 hr)

  • Characteristic Features of Oncotic Necrosis

    • Morphology: cell swelling, cell contents release, karyolysis

    Mechanism is dependent on the insult

  • Oncotic Necrosis: Acetaminophen- induced Hepatotoxicity

    Cell Swelling, Karyolysis

    Cell Contents Release : Plasma ALT > 3000 U/L)

  • DNA Strandbreaks during OncoticNecrosis

    TUNEL Assay

    CV

    4 h APAP 6 h APAP

  • DNA Strandbreaks: Oncotic Necrosis vs Apoptosis

    TUNEL Assay

    CV

    6 h Gal/ET 6 h APAP

  • DNA Fragmentation:Apoptosis vs Oncotic Necrosis

    1 2 3 4 5 6 7 8 9 10 11 12 13

    MW Controls G/ET

    DNA LadderAnti-Histone ELISA

    APAP

    DN

    A F

    ragm

    enta

    tion

    (% c

    ontro

    l)

    0

    500

    1000

    1500

    2000

    C G/E G/E ZVAD

    AAP AAP ZVAD

    AAP GSH

    *

    * *#

    #

  • 1 2 3 4 5 6 7 8 9

    0 0.5 1 2 3 4 6 G/E

    Acetaminophen (h)

    p32

    p11

    Caspase-3 Processing during Acetaminophen Toxicity

    Lawson et al., Toxicol Appl Pharmacol 156: 179-86, 1999 Gujral et al., Toxicol Sci 67: 322-8, 2002

  • BaxBak

    C

    C

    C

    C

    C

    C

    C

    CC

    C

    C

    CC

    C

    C

    CC

    C

    EndoG

    Endo

    GEn

    doG

    EndoG

    Endo

    G

    EndoG

    AIF

    AIF

    Dia

    blo

    Diablo

    C

    Bak Bak

    Bax

    Bax

    AIF

    C

    DiabloAIF

    EndoG

    DNA Fragmentation: Nuclear Translocation of Mitochondrial Intermembrane Proteins

    Diablo

    C C

    Nucleus

    Mitochondria

    Control

    5 mM AAP, 6 h

    Endonuclease G

    Bajt et al., Toxicol Sci 94: 217-225,2006

  • Cytochrome c

    Bax

    BidtBid

    Contr

    olAP

    AP 2

    h

    APAP

    4 h

    Contr

    olAP

    AP 2

    hAP

    AP 4

    h

    Mitochondria Cytosol

    APAP-induced Bax and tBid Translocation to Mitochondria

  • APAP Hepatotoxicity in Bax Gene Knockout Mice

    Pla

    sma

    ALT

    (U/L

    )

    0

    1000

    2000

    3000

    4000

    5000

    6000

    7000

    *

    WT Bax-/-

    Bajt et al., J Pharmacol Exp Therap, in press, 2007

  • APAP Hepatotoxicity in Bax Gene Knockout Mice

    Pla

    sma

    ALT

    (U/L

    )

    0

    1000

    2000

    3000

    4000

    5000

    6000

    7000

    *

    WT Bax-/-

    Bajt et al., J Pharmacol Exp Therap, in press, 2007

    WT

    Bax-/-

  • NAPQI APAPGSH ↓

    Protein Arylation

    P4501.

    2.

    Bax

    Cyt c / SmacAIF Endonuclease G

    ONOO-

    Nucleus

    DNA-Strandbreaks

    ATP ↓

    CaspaseActivation

    ATP ↓

    PARP Activation

    DNA Repair

    DNA-Fragmentation

    Chromatin Condensation

    NAD+ Depletion

    ATP ↓ ⇐ MPT

    ?Mechanism of Mechanism of APAP ToxicityAPAP Toxicity

    Toxicol Sci 89: 31-41, 2006

    O2 –NO

    Bax MPT

    Protein Nitration

  • Apoptosis vs Oncotic Necrosis

    Initiation Execution

    Apoptosis ApoptosisATP high

    Apoptosis Secondary NecrosisATP low

    Oncosis Oncotic NecrosisATP low

    OncosisATP high Secondary Apoptosis ?

    Same process or replaced by different mode of cell death?

  • Cell Death Connection to Fibrosis

    Apoptotic Bodies

    PhagocytosisHSC

    KC

    TGFβ-1

    TNF-α

    Canbay et al., Lab Invest 83:655,2003

  • Cell Death Connection to Fibrosis

    Phagocytosis

    HSC

    KC

    TGFβ-1

    TNF-α

    HMGB1

    Cell Debris

    HSC

    KC

    TLR4TGFβ-1

    TNF-α

  • Fibrosis: Response to Cell Injury

    Rockey & Friedman, 2006

    Apoptosis

    Oncotic Necrosis