Analysis of the NADPH oxidase components during differentiation of HL-60 cells to eosinophilic lineage.
HL-60 cells were induced to differentiate into eosinophil-like cells with sodium butyrate after passage under mild alkaline condition. The differentiating cells gradually possessed the Luxol-fast-blue (LFB) staining-positive granules and the capacity to produce superoxide. The increase in the amounts of cytochrome b-558 paralleled the superoxide anion generating activity. Immunoblot analysis demonstrated that p47-phox cytosolic oxidase protein appeared 1 day after differentiation, and increased up to 7 days. On the other hand, p67-phox cytosolic oxidase protein appeared in 3 days, and increased gradually up to 7 days. The oxidase activity did not appear until p67-phox protein was expressed in the cytosol during eosinophilic differentiation, indicating that p67-phox protein is likely to be a key protein of cytosolic factors also in eosinophilic differentiating cells. The amounts of p47-phox and p67-phox translocated to the plasma membrane in response to phorbol myristate acetate (PMA) increased with increasing amounts of cytochrome b-558 in the membrane. Our data demonstrate that the appearance of NADPH oxidase activity during eosinophilic differentiation is dependent on the levels of p47-phox and p67-phox cytosolic proteins translocated to the plasma membrane and the amount of cytochrome b-558 in the membrane as observed with neutrophils and monocytes.
Miyamoto
D
D
Department of Biochemistry, School of Medicine, Juntendo University, Tokyo, Japan.
Someya
A
A
Nunoi
H
H
Nagaoka
I
I
Yamashita
T
T
D002454:Cell Differentiation; D002460:Cell Line; D003573:Cytochrome b Group / Q000032:analysis; D004789:Enzyme Activation; D004804:Eosinophils / Q000201:enzymology*; D006801:Humans; D015151:Immunoblotting; D009000:Monocytes / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis* / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000032:analysis; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology; D010750:Phosphoproteins / Q000032:analysis; D013347:Subcellular Fractions / Q000201:enzymology
D003573:Cytochrome b Group; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase