A role for caspase-8 and c-FLIP_L in proliferation and cell-cycle progression of primary hepatocytes

Growth factors are known to favor both proliferation and survival of hepatocytes. In the present study, we investigated if c-FLIP_L (cellular FLICE-inhibitory protein, long isoform) could be involved in epidermal growth factor (EGF)-stimulated proliferation of rat hepatocytes since c-FLIP_L regulates both cell proliferation and procaspase-8 maturation. Treatment with MEK inhibitors prevented induction of c-FLIP_L by EGF along with total inhibition of DNA replication. However, EGF failed to inhibit processing of procaspase-8 in the presence of EGF suggesting that c-FLIP_L does not play its canonical anti-apoptotic role in this model. Downregulation of c-FLIP expression using siRNA oligonucleotides strongly reduced DNA replication but did not result in enhanced apoptosis. Moreover, intermediate cleavage products of c-FLIP_L and caspase-8 were found in EGF-treated hepatocytes in the absence of caspase-3 maturation and cell death. To determine whether the Fas/FADD/ caspase-8/c-FLIP_L complex was required for this activity, Fas, procaspase-8 and Fas-associated death domain protein (FADD) expression or function was inhibited using siRNA or constructs encoding dominant negative mutant proteins. Inhibition of any of these components of the Fas/FADD/caspase-8 pathway decreased DNA replication suggesting a function of these proteins in cell-cycle arrest. Similar results were obtained when the IETD-like caspase activity detectable in EGF-treated hepatocytes was inhibited by the pan-caspase inhibitor, z-ASP. Finally, we demonstrated co-immunoprecipitation between EGFR and Fas within 15 min following EGF stimulation. In conclusion, our results indicate that the Fas/FADD/c-FLIP_L/caspase-8 pathway positively controls the G₁/S transition in EGF-stimulated hepatocytes. Our data provide new insights into the mechanisms by which apoptotic proteins participate to mitogenic signals during the G₁ phase.