, Volume 17, Issue 5, pp 503–515 | Cite as

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


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.


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



Bifunctional apoptosis regulator


Direct IAP-binding protein


Death-inducing signaling complex


Fas ligand

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.

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)


  1. 1.
    Meier P, Finch A, Evan G (2000) Apoptosis in development. Nature 407:796–801PubMedCrossRefGoogle Scholar
  2. 2.
    Kanzler S, Galle PR (2000) Apoptosis and the liver. Semin Canc Biol 10:173–184CrossRefGoogle Scholar
  3. 3.
    Osagawara J, Watanabe-Fukunaga R, Adachi M, Matsuzawa A, Kasugai T, Kitamura Y, Itoh N, Suda T, Nagata S (1993) Lethal effect of the anti-Fas antibody in mice. Nature 364:806–809CrossRefGoogle Scholar
  4. 4.
    Galle PR, Krammer PH (1998) CD95-induced apoptosis in human liver disease. Semin Liver Dis 18:141–151PubMedCrossRefGoogle Scholar
  5. 5.
    Canbay A, Friedman S, Gores GJ (2004) Apoptosis: the nexus of liver injury and fibrosis. Hepatology 39:273–278PubMedCrossRefGoogle Scholar
  6. 6.
    Ni R, Tomita Y, Matsuda K, Ichihara A, Ishimura K, Ogasawara J, Nagata S (1994) Fas-mediated apoptosis in primary cultured mouse hepatocytes. Exp Cell Res 215:332–337PubMedCrossRefGoogle Scholar
  7. 7.
    Schüngel S, Buitrago-Molina LE, devi Nalapareddy P, Lebofsky M, Manns MP, Jaeschke H, Gross A, Vogel A (2009) The strength of the Fas ligand signal determines whether hepatocytes act as type 1 or type 2 cells in murine livers. Hepatology 50:1558–1566PubMedCrossRefGoogle Scholar
  8. 8.
    Yin XM, Wang K, Gross A, Zhao Y, Zinkel S, Klocke B, Roth KA, Korsmeyer SJ (1999) Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400:886–891PubMedCrossRefGoogle Scholar
  9. 9.
    Kaufmann T, Tai L, Ekert PG, Huang DCS, Norris F, Lindemann RK, Johnstone RW, Dixit VM, Strasser A (2007) The BH3-only protein Bid is dispensable for DNA damage- and replicative stress-induced apoptosis or cell-cycle arrest. Cell (129):423–433CrossRefGoogle Scholar
  10. 10.
    Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME (1998) Two CD95 (APO-1/Fas) signaling pathways. EMBO J 17:1675–1687PubMedCrossRefGoogle Scholar
  11. 11.
    Peter ME, Krammer PH (2003) The CD95(APO-1/Fas) DISC and beyond. Cell Death Differ 10:26–35PubMedCrossRefGoogle Scholar
  12. 12.
    Chang DW, Xing Z, Capacio VL, Peter ME, Yang X (2003) Interdimer processing mechanism of procaspase-8 activation. EMBO J 22:4132–4142PubMedCrossRefGoogle Scholar
  13. 13.
    Medema JP, Scaffidi C, Kischkel FC, Schevchenko A, Mann M, Krammer PH, Peter ME (1997) FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J 16:2794–2804PubMedCrossRefGoogle Scholar
  14. 14.
    Muzio M, Stockwell BR, Stennicke HR, Salvesen GS, Dixit VM (1998) An induced proximity model for caspase-8 activation. J Biol Chem 273:2926–2930PubMedCrossRefGoogle Scholar
  15. 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
  16. 16.
    Peter ME, Budd RC, Desbarats J, Hedrick SM, Hueber AO, Newell MK, Owen LB, Pope RM, Tschopp J, Wajant H, Wallach D, Wiltrout RH, Zörnig M, Lynch DH (2007) The CD95 receptor: apoptosis revisited. Cell 129:447–450PubMedCrossRefGoogle Scholar
  17. 17.
    Green DR, Kroemer G (2004) The pathophysiology of mitochondrial cell death. Science 305:626–629PubMedCrossRefGoogle Scholar
  18. 18.
    Desbarats J, Newell MK (2000) Fas engagement accelerates liver regeneration after partial hepatectomy. Nat Med 6:920–923PubMedCrossRefGoogle Scholar
  19. 19.
    Kang TB, Ben-Moshe T, Varfolomeev EE, Pewzner-Jung Y, Yogev N, Jurewicz A, Waisman A, Brenner O, Haffner R, Gustafsson E, Ramakrishnan P, Lapidot T, Wallach D (2004) Caspase-8 serves both apoptotic and nonapoptotic roles. J Immunol 173:2976–2984PubMedGoogle Scholar
  20. 20.
    Kang TB, Oh GS, Scandella E, Bolinger B, Ludewig B, Kovalenko A, Wallach D (2008) Mutation of a self-processing site in caspase-8 compromises its apoptotic but not its nonapoptotic functions in bacterial artificial chromosome-transgenic mice. J Immunol 181:2522–2532PubMedGoogle Scholar
  21. 21.
    Barbero S, Mielgo A, Torres V, Teitz T, Shields DJ, Mikolon D, Bogyo M, Barilà D, Lahti JM, Schlaepfer D, Stupack DG (2009) Caspase-8 association with the focal adhesion complex promotes tumor cell migration and metastasis. Cancer Res 69(9):3755–3763PubMedCrossRefGoogle Scholar
  22. 22.
    Kovalenko A, Kim JC, Kang TB, Raiput A, Bogdanoy K, Dittrich-Breiholz O, Kracht M, Brenner O, Wallach D (2009) Caspase-8 deficiency in epidermal keratinocytes triggers an inflammatory skin disease. J Exp Med 206:2161–2177PubMedCrossRefGoogle Scholar
  23. 23.
    Keller N, Mares J, Zerbe O, Grütter MG (2009) Structural and biochemical studies on procaspase-8: new insights on initiator caspase activation. Structure 17:438–448PubMedCrossRefGoogle Scholar
  24. 24.
    Hughes MA, Harper N, Butterworth M, Cain K, Cohen GM, MacFarlane M (2009) Reconstitution of the death-inducing signaling complex reveals a substrate switch that determines CD95-mediated death or survival. Mol Cell 35:265–279PubMedCrossRefGoogle Scholar
  25. 25.
    Strasser A, Jost PJ, Nagata S (2009) The many roles of FAS receptor signaling in the immune system. Immunity 30:180–192PubMedCrossRefGoogle Scholar
  26. 26.
    Oberst A, Pop C, Tremblay AG, Blais V, Denault JB, Salvesen GS, Green DR (2010) Inducible dimerization and inducible cleavage reveal a requirement for both processes in caspase-8 activation. J Biol Chem 285(22):16632–16642PubMedCrossRefGoogle Scholar
  27. 27.
    Blanc C, Deveraux QL, Krajewski S, Jänicke RU, Porter AG, Reed JC, Jaggi R, Marti A (2000) Caspase-3 is essential for procaspase-9 processing and cisplatin-induced apoptosis of MCF-7 breast cancer cells. Cancer Res 60:4386–4390PubMedGoogle Scholar
  28. 28.
    Fujita E, Egashira J, Urase K, Kuida K, Momoi T (2001) Caspase-9 processing by caspase-3 via a feedback amplification loop in vivo. Cell Death Differ 8:335–344PubMedCrossRefGoogle Scholar
  29. 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
  30. 30.
    Van de Craen M, Declercq W, Van den brande I, Fiers W, Vandenabeele P (1999) The proteolytic procaspase activation network: an in vitro analysis. Cell Death Differ 6:1117–1124PubMedCrossRefGoogle Scholar
  31. 31.
    Eissing T, Conzelmann H, Gilles ED, Allgöwer F, Bullinger E, Scheurich P (2004) Bistability analyses of a caspase activation model for receptor-induced apoptosis. J Biol Chem 279(35):36892–36897PubMedCrossRefGoogle Scholar
  32. 32.
    Angeli D, Ferrell JE, Sontag ED (2004) Detection of multistability, bifurcations, and hysteresis in a large class of biological positive-feedback systems. Proc Natl Acad Sci USA 101(7):1822–1827PubMedCrossRefGoogle Scholar
  33. 33.
    Ferrell JE (2002) Self-perpetuating states in signal transduction: positive feedback, double-negative feedback and bistability. Curr Opin Cell Biol 14(2):140–148PubMedCrossRefGoogle Scholar
  34. 34.
    Legewie S, Blüthgen N, Herzel H (2006) Mathematical modeling identifies inhibitors of apoptosis as mediators of positive feedback and bistability. PLoS Comput Biol 2(9):e120PubMedCrossRefGoogle Scholar
  35. 35.
    Tang D, Lahti JM, Kidd VJ (2000) Caspase-8 activation and Bid cleavage contribute to MCF7 cellular execution in a caspase-3-dependent manner during staurosporine-mediated apoptosis. J Biol Chem 275(13):9303–9307PubMedCrossRefGoogle Scholar
  36. 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. 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
  38. 38.
    Berger AB, Witte MD, Denault JB, Sadaghiani AM, Sexton KMB, Salvesen GS, Bogyo M (2006) Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes. Mol Cell 23:509–521PubMedCrossRefGoogle Scholar
  39. 39.
    Klingmüller U, Bauer A, Bohl S, Nickel PJ, Breitkopf K, Dooley S et al (2006) Primary mouse hepatocytes for systems biology approaches: a standardized in vitro system for modelling of signal transduction pathways. Syst Biol (Stevenage) 153:433–447CrossRefGoogle Scholar
  40. 40.
    Shimizu M, Fontana A, Takeda Y, Yagita H, Yoshimoto T, Matsuzawa A (1999) Induction of antitumor immunity with Fas/APO-1 ligand (CD95L)-transfected neuroblastoma Neuro-2a cells. J Immunol 162:7350–7357PubMedGoogle Scholar
  41. 41.
    Huber W, von Heydebreck A, Sültmann H, Poustka A, Vingron M (2002) Variance stabilization applied to microarray data calibration and to the quantification of differential expression. Bioinformatics 18(Suppl 1):S96–S104PubMedCrossRefGoogle Scholar
  42. 42.
    Pop C, Salvesen GS (2009) Human caspases: activation, specificity, and regulation. J Biol Chem 284(33):21777–21781PubMedCrossRefGoogle Scholar
  43. 43.
    Würstle ML, Laussmann MA, Rehm M (2010) The caspase-8 dimerization/dissociation balance is a highly potent regulator of caspase-8, -3, -6 signaling. J Biol Chem 285(43):33209–33218PubMedCrossRefGoogle Scholar
  44. 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
  45. 45.
    Jost PJ, Grabow S, Gray D, McKenzie MD, Nachbur U, Huang DCS, Bouillet P, Thomas HE, Borner C, Silke J, Strasser A, Kaufmann T (2009) XIAP discriminates between type I and type II FAS-induced apoptosis. Nature 460(20):1035–1039PubMedCrossRefGoogle Scholar
  46. 46.
    Madesh M, Antonsson B, Srinivasula SM, Alnemri ES, Hajnóczky G (2002) Rapid kinetics of tBid-induced cytochrome c and Smac/DIABLO release and mitochondrial depolarization. J Biol Chem 277(7):5651–5659PubMedCrossRefGoogle Scholar

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

Personalised recommendations