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

Most of the morphologic changes that are observed in apoptotic cells are ca used by a set of cysteine proteases (caspases) that are activated during th is process. In previous works from our group we found that treatment of rat fetal hepatocytes with transforming growth factor beta1 (TGF-beta1) is fol lowed by apoptotic cell death. TGF-beta1 mediates radical oxygen species (R OS) production that precedes bcl-x(L) down-regulation, loss of mitochondria l transmembrane potential, release of cytochrome c, and activation of caspa se-3 (Herrera et al., FASEB J 2001;15:741-751). In this work, we have analy zed how TGF-beta1 activates the caspase cascade and whether or not caspase activation precedes the oxidative stress induced by this factor. Our result s show that TGF-beta1 activates at least caspase-3, -8, and -9 in rat fetal hepatocytes, which are not required for ROS production, glutathione deplet ion, bcl-xL down-regulation, and initial cytochrome c release. However, cas pase activation mediates cleavage of Bid and Bcl-x(L) that could originate an amplification loop on the mitochondrial events. An interesting result is that transmembrane potential disruption occurs later than the initial cyto chrome c release and is mostly blocked by the pan-caspase inhibitor Z-VAD.f mk, indicating that the decrease in mitochondrial transmembrane potential ( ATM) may be a consequence of caspase activity rather than the mechanism by which TGF-beta induces cytochrome c efflux. Finally, although Z-VAD.fmk com pletely blocks nucleosomal DNA fragmentation, it only delays cell death, wh ich suggests that activation of the apoptotic program by TGF-beta in fetal hepatocytes inevitably leads to death, with or without caspases.