Interferon γ Induces Upregulation and Activation of Caspases 1, 3, and 8 to Produce Apoptosis in Human Erythroid Progenitor Cells

Abstract

Interferon γ (IFNγ) induces apoptosis in purified human erythroid colony-forming cells (ECFC) and inhibits cell growth. Fas (APO-1; CD95) and Fas ligand (FasL) mediate apoptosis induced by IFNγ, because Fas is significantly upregulated by IFNγ, whereas Fas ligand is constitutively present in the ECFC and neutralization of FasL greatly reduces the apoptosis. Because conversion of caspases from their dormant proenzyme forms to active enzymes has a critical role in transducing a cascade leading to apoptosis, we performed further studies of the expression and activation of caspases in normal human and IFNγ-treated day-6 ECFC to better understand the mechanism of IFNγ action in producing this cell death. RNase protection assays showed that the caspase-1, -2, -6, -8, and -9 mRNAs were upregulated by IFNγ, whereas the caspase-5 and -7 mRNAs were not increased. Western blots showed that FLICE/caspase-8 was upregulated and activated by 24 hours of incubation with IFNγ. FADD was not similarly altered by incubation with IFNγ. Western blots of ICE/caspase-1, which might be required for amplification of the initial FLICE activation signal, showed that pro-ICE expression significantly increased after treatment with IFNγ for 24 hours and cleavage of pro-ICE also increased. CPP32/apopain/caspase-3, responsible for the proteolytic cleavage of poly (ADP) ribose polymerase (PARP), was also studied and treatment of ECFC with IFNγ resulted in an increased concentration of caspase-3 by 24 hours and a clear induction of enzyme activation by 48 hours, which was identified by the appearance of its p17-kD peptide fragment. The cleavage of PARP was demonstrated by an obvious increase of the 89-kD PARP cleavage product, which was observed at almost the same time as caspase-3 activation in the IFNγ-treated cells, whereas untreated ECFC showed little change. Peptide inhibitors of the caspase proteins, DEVD-fmk, DEVD-cho, YVAD-cho, and IETD-fmk, were incubated with the ECFC to obtain further evidence for the involvement of caspases in IFNγ-induced apoptosis. The activation of FLICE/caspase-8 and CPP32/caspase-3 and cleavage of PARP clearly were inhibited, but the reduction of cell growth due to apoptosis, induced by IFNγ, was only partially blocked by the presence of the inhibitors. These results indicate that IFNγ acts on ECFC not only to upregulate Fas, but also to selectively upregulate caspases-1, -3, and -8, which are activated and produce apoptosis, whereas the concentrations of FasL and FADD are not demonstrably changed.