A role for caspase-8 and c-FLIPL 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 G(1)/S transition in EGF-stimulated hepatocytes. Our data provide new insights into the mechanisms by which apoptotic proteins participate to mitogenic signals during the G(1) phase.
Gilot
David
D
INSERM U620, Rennes, France. david.gilot@rennes.inserm.fr
Serandour
Anne-Laure
AL
Ilyin
Guennady P
GP
Lagadic-Gossmann
Dominique
D
Loyer
Pascal
P
Corlu
Anne
A
Coutant
Alexandre
A
Baffet
Georges
G
Peter
Marcus E
ME
Fardel
Olivier
O
Guguen-Guillouzo
Christiane
C
D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000818:Animals; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism*; D049109:Cell Proliferation; D002478:Cells, Cultured; D004261:DNA Replication; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors; D053200:Fas-Associated Death Domain Protein; D016193:G1 Phase; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D008297:Male; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D016196:S Phase; D015398:Signal Transduction; D019014:fas Receptor / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D004791:Enzyme Inhibitors; C505533:FADD protein, human; C505535:Fadd protein, rat; D053200:Fas-Associated Death Domain Protein; D047908:Intracellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D019014:fas Receptor; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D048369:MAP Kinase Kinase 1; C505530:CASP8 protein, human; C505528:Casp8 protein, rat; D053181:Caspase 8; D020169:Caspases