Activation of caspases occurs downstream from radical oxygen species production, Bcl-xL down-regulation, and early cytochrome C release in apoptosis induced by transforming growth factor β in rat fetal hepatocytes

Authors

  • Blanca Herrera,

    1. Departamento de Bioquímica y Biología Molecular, Instituto de Bioquímica, Centro Mixto CSIC/UCM, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain
    Search for more papers by this author
  • Margarita Fernández,

    1. Departamento de Bioquímica y Biología Molecular, Instituto de Bioquímica, Centro Mixto CSIC/UCM, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain
    Search for more papers by this author
  • Alberto M. Álvarez,

    1. Centro de Citometría de Flujo y Microscopía Confocal, Universidad Complutense de Madrid, Madrid, Spain
    Search for more papers by this author
  • César Roncero,

    1. Departamento de Bioquímica y Biología Molecular, Instituto de Bioquímica, Centro Mixto CSIC/UCM, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain
    Search for more papers by this author
  • Manuel Benito,

    1. Departamento de Bioquímica y Biología Molecular, Instituto de Bioquímica, Centro Mixto CSIC/UCM, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain
    Search for more papers by this author
  • Joan Gil,

    1. Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Hospitalet de Llobregat, Barcelona, Spain.
    Search for more papers by this author
  • Isabel Fabregat Ph.D.

    Corresponding author
    1. Departamento de Bioquímica y Biología Molecular, Instituto de Bioquímica, Centro Mixto CSIC/UCM, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain
    • Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad Complutense de Madrid, Ciudad Universitaria, 28040 Madrid, Spain. fax: (34) 913941779
    Search for more papers by this author

Abstract

Most of the morphologic changes that are observed in apoptotic cells are caused by a set of cysteine proteases (caspases) that are activated during this process. In previous works from our group we found that treatment of rat fetal hepatocytes with transforming growth factor β1 (TGF-β1) is followed by apoptotic cell death. TGF-β1 mediates radical oxygen species (ROS) production that precedes bcl-xL down-regulation, loss of mitochondrial transmembrane potential, release of cytochrome c, and activation of caspase-3 (Herrera et al., FASEB J 2001;15:741-751). In this work, we have analyzed how TGF-β1 activates the caspase cascade and whether or not caspase activation precedes the oxidative stress induced by this factor. Our results show that TGF-β1 activates at least caspase-3, -8, and -9 in rat fetal hepatocytes, which are not required for ROS production, glutathione depletion, bcl-xL down-regulation, and initial cytochrome c release. However, caspase activation mediates cleavage of Bid and Bcl-xL that could originate an amplification loop on the mitochondrial events. An interesting result is that transmembrane potential disruption occurs later than the initial cytochrome c release and is mostly blocked by the pan-caspase inhibitor Z-VAD.fmk, indicating that the decrease in mitochondrial transmembrane potential (ΔΨm) may be a consequence of caspase activity rather than the mechanism by which TGF-β induces cytochrome c efflux. Finally, although Z-VAD.fmk completely blocks nucleosomal DNA fragmentation, it only delays cell death, which suggests that activation of the apoptotic program by TGF-β in fetal hepatocytes inevitably leads to death, with or without caspases. (HEPATOLOGY 2001;34:548-556.)

Ancillary