Role of NF-kappa B in regulation of apoptosis of erythroid progenitor cells.
Erythropoietin (EPO) and interferon-gamma (IFN-gamma) added to human erythroid progenitor cells purified from peripheral blood (erythroid colony-forming cells; ECFC) significantly reduces apoptosis as assessed by flow cytometry (FCM) using annexin V. To clarify the role of NF-kappaB in the regulation of the apoptosis of erythroid progenitor cells, cyclosporin A (CsA), which blocks dissociation of the NF-kappaB complex, was added to serum-free cultures of ECFC. CsA induced the apoptosis of ECFCs in the presence of EPO or IFN-gamma, but at different magnitudes. In the presence of a relatively low concentration of CsA (10 microm), apoptosis was induced only in cultures with EPO. The direct involvement of NF-kappaB was then assessed by Western blotting and confocal microscopy. In the presence of EPO, NF-kappaB was abundant both in the cytoplasm and in the nucleus, and nuclear expression was diminished after adding CsA. In contrast, NF-kappaB was undetectable in the nucleus in the presence of IFN-gamma. The effect of CsA on mitochondrial function was investigated by determining the DeltaPsim and reactive oxygen species production. CsA disturbed the transmembrane potential in the presence of either EPO or IFN-gamma, although the viability of the cells was maintained in the presence of IFN-gamma plus CsA. These results indicate that IFN-gamma reduced the apoptosis of erythroid progenitor cells through a unique signaling pathway that is independent of NF-kappaB translocation, and which is not mediated by modulating mitochondrial function, whereas EPO reduced apoptosis through NF-kappaB translocation to the nucleus.
Sae-Ung
N
N
Department of Medicine and Bioregulatory Science, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
Matsushima
T
T
Choi
I
I
Abe
Y
Y
Winichagoon
P
P
Fucharoen
S
S
Nawata
H
H
Muta
K
K
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001693:Biological Transport, Active / Q000187:drug effects; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D003114:Colony-Forming Units Assay; D016572:Cyclosporine / Q000494:pharmacology; D015672:Erythroid Precursor Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D004921:Erythropoietin / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins; D051020:bcl-X Protein
C494811:BCL2L1 protein, human; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D051020:bcl-X Protein; D004921:Erythropoietin; D016572:Cyclosporine