Growth factors are known to favor both proliferation and survival of hepatocytes. In the present study, we investigated if c-FLIPL (cellular FLICE-inhibitory protein, long isoform) could be involved in epidermal growth factor (EGF)-stimulated proliferation of rat hepatocytes since c-FLIPL regulates both cell proliferation and procaspase-8 maturation. Treatment with MEK inhibitors prevented induction of c-FLIPL 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-FLIPL 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-FLIPL 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-FLIPL 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-FLIPL/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.
Keywords: c-FLIPL, cellular FLICE-inhibitory protein, long isoform; CaMKII, calmodulin kinase II; DED, death effector domain; DEVD-AMC, Asp-Glu-Val-Asp-7-amino-4-methylcoumarin; DISC, death-inducing signalling complex; EGF, epidermal growth factor; EGFR, EGF receptor; FADD, Fas-associated death domain protein; FCS, fetal calf serum; GFP, green fluorescent protein; IAP, inhibitor of apoptosis protein; IKK, inhibitor of NF-kB kinase complex; MKP1, MAPK phosphatase 1; NIK, NF-kB inducing kinase; RIP, receptor interacting protein; siRNA, small interference RNA; TCR, T cell receptor; TNF, tumor necrosis factor
Journal Article. 6382 words. Illustrated.
Subjects: Clinical Cytogenetics and Molecular Genetics
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