Stem cell factor increases the expression of FLIP that inhibits IFNγ-induced apoptosis in human erythroid progenitor cells

Abstract

Interferon γ (IFNγ) acts on human erythroid colony-forming cells (ECFCs) to up-regulate Fas, without a demonstrable change of Fas ligand (FasL) or Fas-associated DD-containing protein (FADD) expression and activates caspase-8 plus caspase-3, which produce apoptosis. Our previous data showed that stem cell factor (SCF) reduced the inhibitory effect of IFNγ on human ECFCs when both factors were present in the cultures. However, the mechanism by which SCF prevents IFNγ-induced apoptosis in ECFCs is unclear. In this study we used highly purified human ECFCs to investigate the mechanism of the effect of SCF on IFNγ-induced apoptosis. Because the binding of FasL to Fas is the first step of the apoptosis cascade and IFNγ strongly up-regulates Fas expression, we added FasL (50 ng/mL) to the cultures with IFNγ to accentuate the IFNγ-induced activation of caspase-8 and caspase-3 plus subsequent apoptosis. SCF (100 ng/mL) clearly inhibited the activation of caspase-8 and caspase-3 induced by IFNγ and/or FasL, and it also reduced apoptosis as measured by the terminal dUTP nick-end labeling (TUNEL) assay. SCF did not decrease the surface expression of Fas on the ECFCs. FADD-like interleukin 1 β (IL-1β)–converting enzyme (FLICE)–inhibitory protein (FLIP) has been reported to interact with FADD and/or caspase-8 at the death-inducing signaling complex (DISC) level following Fas stimulation and acts as a dominant-negative caspase-8. SCF increased FLIP mRNA and protein expression, concomitant with reduced apoptosis, whereas IFNγ and/or FasL did not change FLIP expression. Reduction of FLIP expression with antisense oligonucleotides decreased the capacity of SCF to inhibit IFNγ-induced apoptosis, demonstrating a definite role for FLIP in the SCF-induced protection of ECFCs from IFNγ-initiated apoptosis.