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J. Biol. Chem., Vol. 280, Issue 28, 26467-26476, July 15, 2005

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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^*

Claudia Piccoli, Roberto Ria, Rosella Scrima, Olga Cela, Annamaria D'Aprile, Domenico Boffoli, Franca Falzetti, Antonio Tabilio¶, and Nazzareno Capitanio¶||

From the Department of Biomedical Science, University of Foggia, Foggia, Italy 71100 and the Department of Clinical and Experimental Medicine, Hematology and Clinical Immunology Section, University of Perugia, Perugia, Italy 06100

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 . 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.

Received for publication, January 3, 2005 , and in revised form, April 29, 2005.

^* This work was supported by the University of Foggia Funds for Research "Quota progetti 2002-2003." The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Both authors contributed equally to this study.

^|| To whom correspondence should be addressed: Dept. of Biomedical Science, University of Foggia, viale L. Pinto OO.RR. 71100 Foggia, Italy. Tel.: 39-0881-711148; Fax: 30-0881-714745; E-mail: n.cap{at}unifg.it.

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