Skeletal satellite cells are adult stem cells located among muscle fibers. Proliferation, migration and subsequent differentiation of these cells are critical steps in the repair of muscle injury. We document in this study the roles and mechanisms through which the NAPDH oxidase complex regulates skeletal satellite cell proliferation. The NADPH oxidase subunits Nox2, Nox4, p22phox, p47phox and p67 phox were detected in primary human and murine skeletal muscle satellite cells. In human satellite cells, NADPH oxidase-fusion proteins were localized in the cytosolic and membrane compartments of the cell, except for p47 phox, which was detected in the nucleus. In proliferating subconfluent satellite cells, both Nox2 and Nox4 contributed to O2- production. However, Nox4 expression was significantly attenuated in confluent cells and in differentiated myotubes. Proliferation of satellite cells was significantly reduced by antioxidants (N-acetylcysteine and apocynin), inhibition of p22phox expression using siRNA oligonucleotides, and reduction of Nox4 and p47phox activities with dominant-negative vectors resulted in attenuation of activities of the Erk1/2, PI-3 kinase/AKT and NFkappaB pathways and significant reduction in cyclin D1 levels. We conclude that NADPH oxidase is expressed in skeletal satellite cells and that its activity plays an important role in promoting proliferation of these cells.

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