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

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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.

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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)

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Acknowledgments

We are particularly grateful to Rebekka Schlatter, Institute for System Dynamics, University of Stuttgart, Germany, Ulrich Maurer and Dorothée Walter, University of Freiburg, Germany for their useful comments and constructive advice on the manuscript. We also thank Adriano Fontana, University Clinic Zurich, Switzerland for the N2A FasL cells, John Silke, La Trobe University, Melbourne, Australia for the XIAP−/− mice and David Huang, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia, for the monoclonal anti-Bid antibody. We gratefully acknowledge support from The Virtual Liver Network which is sponsored by the German Federal Ministry of Education and Research to KF, CK, JT and CB, and from the National Institutes of Health (NIH)—grant R01 EB005011 to MB. CB is also supported by the Excellence Initiative of the German Federal and State Governments (GSC-4, Spemann Graduate School of Biology and Medicine, SGBM).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Institute of Molecular Medicine and Cell Research, University of Freiburg, Stefan-Meier-Str. 17, 79104, Freiburg, Germany

    Karine Sá Ferreira, Karin Neubert, Angelika Haber & Christoph Borner

  2. GRK 1104, From Cells to Organs: Molecular Mechanisms of Organogenesis, Faculty of Biology, University of Freiburg, Schaenzlestr. 1, 79104, Freiburg, Germany

    Karine Sá Ferreira & Christoph Borner

  3. Institute for Physics, University of Freiburg, Hermann-Herder-Str. 3, 79104, Freiburg, Germany

    Clemens Kreutz & Jens Timmer

  4. Freiburg Center for Systems Biology (ZBSA), University of Freiburg, Habsburgerstr. 49, 79104, Freiburg, Germany

    Clemens Kreutz & Jens Timmer

  5. Internal Medicine, University Hospital of Freiburg, Hugstetterstr. 55, 79106, Freiburg, Germany

    Sabine MacNelly

  6. Department of Pathology, School of Medicine, Stanford University, 300 Pasteur Drive, Stanford, CA, 94305, USA

    Matthew Bogyo

  7. Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Albertstr. 19, 79104, Freiburg, Germany

    Jens Timmer

  8. BIOSS Centre for Biological Signalling Studies, University of Freiburg, Hebelstr. 25, 79104, Freiburg, Germany

    Jens Timmer & Christoph Borner

  9. Department of Clinical and Experimental Medicine, Linköping University, Universitetssjukhuset, 581 85, Linköping, Sweden

    Jens Timmer

  10. Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Albertstrasse 19A, 79104, Freiburg, Germany

    Christoph Borner

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  1. Karine Sá Ferreira
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10495_2011_691_MOESM1_ESM.tif

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)

Supplementary material 2 (TIFF 4466 kb)

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Ferreira, K.S., Kreutz, C., MacNelly, S. et al. Caspase-3 feeds back on caspase-8, Bid and XIAP in type I Fas signaling in primary mouse hepatocytes. Apoptosis 17, 503–515 (2012). https://doi.org/10.1007/s10495-011-0691-0

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