Apoptosis

, Volume 17, Issue 5, pp 503–515

Caspase-3 feeds back on caspase-8, Bid and XIAP in type I Fas signaling in primary mouse hepatocytes

  • Karine Sá Ferreira
  • Clemens Kreutz
  • Sabine MacNelly
  • Karin Neubert
  • Angelika Haber
  • Matthew Bogyo
  • Jens Timmer
  • Christoph Borner
Original Paper

Abstract

The TNF-R1 like receptor Fas is highly expressed on the plasma membrane of hepatocytes and plays an essential role in liver homeostasis. We recently showed that in collagen-cultured primary mouse hepatocytes, Fas stimulation triggers apoptosis via the so-called type I extrinsic signaling pathway. Central to this pathway is the direct caspase-8-mediated cleavage and activation of caspase-3 as compared to the type II pathway which first requires caspase-8-mediated Bid cleavage to trigger mitochondrial cytochrome c release for caspase-3 activation. Mathematical modeling can be used to understand complex signaling systems such as crosstalks and feedback or feedforward loops. A previously published model predicted a positive feedback loop between active caspases-3 and -8 in both type I and type II FasL signaling in lymphocytes and Hela cells, respectively. Here we experimentally tested this hypothesis in our hepatocytic type I Fas signaling pathway by using wild-type and XIAP-deficient primary hepatocytes and two recently characterized, selective caspase-3/-7 inhibitors (AB06 and AB13). Caspase-3/-7 activity assays and quantitative western blotting confirmed that fully processed, active p17 caspase-3 feeds back on caspase-8 by cleaving its partially processed p43 form into the fully processed p18 species. Our data do not discriminate if p18 positively or negatively influences FasL-induced apoptosis or is responsible for non-apoptotic aspects of FasL signaling. However, we found that caspase-3 also feeds back on Bid and degrades its own inhibitor XIAP, both events that may enhance caspase-3 activity and apoptosis. Thus, potent, selective caspase-3 inhibitors are useful tools to understand complex signaling circuitries in apoptosis.

Keywords

Type I apoptosis Caspase-3 Caspase-8 Caspase inhibitor Feedback loop Bid XIAP 

Abbreviations

BAR

Bifunctional apoptosis regulator

DIABLO

Direct IAP-binding protein

DISC

Death-inducing signaling complex

FasL

Fas ligand

N2A FasL

Multimerised FasL obtained from stably transfected Neuro2A cells

PARP

Poly ADP ribose polymerase

Q-VD-OPh

Quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methylketon

Smac

Second mitochondrial-derived activator of caspase

wt

Wild-type

XIAP

X-chromosome-linked IAP (inhibitor of apoptosis protein)

Supplementary material

10495_2011_691_MOESM1_ESM.tif (1 mb)
Supplementary Fig. 1 The general caspase inhibitor Q-VD-OPh (QVD) blocks not only FasL-induced caspase-3 but also caspase-8 processing and activation. a DEVDase assay and b anti-caspase-3, -caspase-8, -XIAP and –Bid western blotting of wt primary mouse hepatocytes challenged with 100 ng/mL N2A FasL in the presence or absence of 25 μM Q-VD-OPh. Actin served as a loading control. Note that the p43 caspase-8 fragment is not formed in the presence of Q-VD-OPh indicating that this inhibitor also blocks caspase-8 autoprocessing at the DISC (in contrast to AB06 which only blocks caspase-3, but p43 caspase-8 formation is maintained, see Fig. 3) (TIFF 1046 kb)
10495_2011_691_MOESM2_ESM.tif (4.4 mb)
Supplementary material 2 (TIFF 4466 kb)

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Karine Sá Ferreira
    • 1
    • 2
  • Clemens Kreutz
    • 3
    • 4
  • Sabine MacNelly
    • 5
  • Karin Neubert
    • 1
  • Angelika Haber
    • 1
  • Matthew Bogyo
    • 6
  • Jens Timmer
    • 3
    • 4
    • 7
    • 8
    • 9
  • Christoph Borner
    • 1
    • 2
    • 8
    • 10
  1. 1.Institute of Molecular Medicine and Cell ResearchUniversity of FreiburgFreiburgGermany
  2. 2.GRK 1104, From Cells to Organs: Molecular Mechanisms of Organogenesis, Faculty of BiologyUniversity of FreiburgFreiburgGermany
  3. 3.Institute for PhysicsUniversity of FreiburgFreiburgGermany
  4. 4.Freiburg Center for Systems Biology (ZBSA)University of FreiburgFreiburgGermany
  5. 5.Internal MedicineUniversity Hospital of FreiburgFreiburgGermany
  6. 6.Department of Pathology, School of MedicineStanford UniversityStanfordUSA
  7. 7.Freiburg Institute for Advanced Studies (FRIAS)University of FreiburgFreiburgGermany
  8. 8.BIOSS Centre for Biological Signalling StudiesUniversity of FreiburgFreiburgGermany
  9. 9.Department of Clinical and Experimental MedicineLinköping UniversityLinköpingSweden
  10. 10.Spemann Graduate School of Biology and Medicine (SGBM)University of FreiburgFreiburgGermany

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