Characterization of mitochondrial and extra-mitochondrial oxygen consuming reactions in human hematopoietic stem cells. Novel evidence of the occurrence of NAD(P)H oxidase activity.
This study was aimed to characterize the mitochondrial and extra-mitochondrial oxygen consuming reactions in human CD34+ hematopoietic stem cells. Cell samples were collected by apheresis following pre-conditioning by granulocyte colony-stimulating factor and isolated by anti-CD34 positive immunoselection. Polarographic analysis of the CN-sensitive endogenous cell respiration revealed a low mitochondrial oxygen consumption rate. Differential absorbance spectrometry on whole cell lysate and two-dimensional blue native-PAGE analysis of mitoplast proteins confirmed a low amount of mitochondrial respiratory chain complexes thus qualifying the hematopoietic stem cell as a poor oxidative phosphorylating cell type. Confocal microscopy imaging showed, however, that the intracellular content of mitochondria was not homogeneously distributed in the CD34+ hematopoietic stem cell sample displaying a clear inverse correlation of their density with the expression of the CD34 commitment marker. About half of the endogenous oxygen consumption was extra-mitochondrial and completely inhibitable by enzymatic scavengers of reactive oxygen species and by diphenylene iodinium. By spectral analysis, flow cytometry, reverse transcriptase-PCR, immunocytochemistry, and immunoprecipitation it was shown that the extra-mitochondrial oxygen consumption was contributed by the NOX2 and NOX4 isoforms of the O2-*. producer plasma membrane NAD(P)H oxidase with low constitutive activity. A model is proposed suggesting for the NAD(P)H oxidase a role of O2 sensor and/or ROS source serving as redox messengers in the activation of intracellular signaling pathways leading (or contributing) to mitochondriogenesis, cell survival, and differentiation in hematopoietic stem cells.
Piccoli
Claudia
C
Department of Biomedical Science, University of Foggia, Foggia, Italy 71100.
Ria
Roberto
R
Scrima
Rosella
R
Cela
Olga
O
D'Aprile
Annamaria
A
Boffoli
Domenico
D
Falzetti
Franca
F
Tabilio
Antonio
A
Capitanio
Nazzareno
N
D018952:Antigens, CD34 / Q000096:biosynthesis; D002460:Cell Line; D002478:Cells, Cultured; D004579:Electron Transport; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D019255:NADPH Oxidases / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D010101:Oxygen Consumption; D010766:Phosphorylation; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017382:Reactive Oxygen Species; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013053:Spectrophotometry
D018952:Antigens, CD34; D017382:Reactive Oxygen Species; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019255:NADPH Oxidases; D010100:Oxygen