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

被引:157
作者
Piccoli, C
Ria, R
Scrima, R
Cela, O
D'Aprile, A
Boffoli, D
Falzetti, F
Tabilio, A
Capitanio, N
机构
[1] Univ Foggia, Dept Biomed Sci, I-71100 Foggia, Italy
[2] Univ Perugia, Dept Clin & Expt Med, Hematol & Clin Immunol Sect, I-06100 Perugia, Italy
关键词
D O I
10.1074/jbc.M500047200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 O-2(radical anion) 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 O-2 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.
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收藏
页码:26467 / 26476
页数:10
相关论文
共 73 条
[11]   Integrative molecular and developmental biology of adult stem cells [J].
Bunting, KD ;
Hawley, RG .
BIOLOGY OF THE CELL, 2003, 95 (09) :563-578
[12]   Mitochondrial free radical generation, oxidative stress, and aging [J].
Cadenas, E ;
Davies, KJA .
FREE RADICAL BIOLOGY AND MEDICINE, 2000, 29 (3-4) :222-230
[13]   A replicating module as the unit of mitochondrial structure and functioning [J].
Capaldi, RA ;
Aggeler, R ;
Gilkerson, R ;
Hanson, G ;
Knowles, M ;
Marcus, A ;
Margineantu, D ;
Marusich, M ;
Murray, J ;
Oglesbee, D ;
Remington, SJ ;
Rossignol, R .
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2002, 1555 (1-3) :192-195
[14]   Vitamin C inhibits granulocyte macrophage-colony-stimulating factor-induced signaling pathways [J].
Cárcamo, JM ;
Bórquez-Ojeda, O ;
Golde, DW .
BLOOD, 2002, 99 (09) :3205-3212
[15]   Homologs of gp91phox:: cloning and tissue expression of Nox3, Nox4, and Nox5 [J].
Cheng, GJ ;
Cao, ZH ;
Xu, XX ;
Van Meir, EG ;
Lambeth, JD .
GENE, 2001, 269 (1-2) :131-140
[16]   Modeling pO2 distributions in the bone marrow hematopoietic compartment.: I.: Krogh's model [J].
Chow, DC ;
Wenning, LA ;
Miller, WM ;
Papoutsakis, ET .
BIOPHYSICAL JOURNAL, 2001, 81 (02) :675-684
[17]  
Cottler-Fox Michele H, 2003, Hematology Am Soc Hematol Educ Program, P419
[18]  
COX JA, 1987, J IMMUNOL, V138, P1884
[19]   The NADPH oxidase of professional phagocytes - prototype of the NOX electron transport chain systems [J].
Cross, AR ;
Segal, AW .
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2004, 1657 (01) :1-22
[20]   Expansion of human SCID-repopulating cells under hypoxic conditions [J].
Danet, GH ;
Pan, Y ;
Luongo, JL ;
Bonnet, DA ;
Simon, MC .
JOURNAL OF CLINICAL INVESTIGATION, 2003, 112 (01) :126-135