Caspase-3 feeds back on caspase-8, Bid and XIAP in type I Fas signaling in primary mouse hepatocytes
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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.
KeywordsType I apoptosis Caspase-3 Caspase-8 Caspase inhibitor Feedback loop Bid XIAP
Bifunctional apoptosis regulator
Direct IAP-binding protein
Death-inducing signaling complex
- N2A FasL
Multimerised FasL obtained from stably transfected Neuro2A cells
Poly ADP ribose polymerase
Second mitochondrial-derived activator of caspase
X-chromosome-linked IAP (inhibitor of apoptosis protein)
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.
- 15.Gross A, Yin XM, Wang K, Wei MC, Jockel J, Milliman C, Erdjument-Bromage H, Tempst P, Korsmeyer SJ (1999) Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while Bcl-xL prevents this release but not tumor necrosis factor-R1/Fas death. J Biol Chem 274(2):1156–1163PubMedCrossRefGoogle Scholar
- 29.Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ (1999) Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol 144(2):281–292PubMedCrossRefGoogle Scholar
- 36.Slee EA, Keogh SA, Martin SJ (2000) Cleavage of BID during cytotoxic drug and UV radiation-induced apoptosis occurs downstream of the point of Bcl-2 action and is catalyzed by caspase-3: a potential feedback loop for amplification of apoptosis-associated mitochondrial cytochrome c release. Cell Death Differ 7:556–565PubMedCrossRefGoogle Scholar
- 37.Walter D, Schmich K, Vogel S, Pick R, Kaufmann T, Hochmuth FC, Haber A, Neubert K, McNelly S, von Weizsäcker F, Merfort I, Maurer U, Strasser A, Borner C (2008) Switch from type II to I Fas/CD95 death signaling on in vitro culturing of primary hepatocytes. Hepatology 48:1942–1953PubMedCrossRefGoogle Scholar
- 44.Eyrisch S. Medina-Franco JL, Helms V (2011) Transient pockets on XIAP-BIR2: toward the characterization of putative binding sites of small-molecules XIAP inhibitors. J Mol Model. doi: 10.1007/s00894-011-1217-y