p47phox-dependent reactive oxygen species stimulate nuclear translocation of the transcription factor FOXO1 in ischemia-challenged H9c2 cells

Reactive oxygen species (ros) control forkhead box o (foxo) transcription factors by influencing their nuclear translocation. However, knowledge of the cellular source(s) of ros involved herein remains scarce. Recently, we have shown p47phox-dependent activation of ros-producing nadph oxidase (nox) at the nuclear pore in h9c2 rat cardiomyoblasts in response to ischemia. This localizes nox perfectly to affect proteins translocating in or out of the nucleus, including transcription factors. Therefore, in this study involvement of p47phox-dependent production of ros in the nuclear translocation of foxo1 was analyzed in h9c2 cells under ischemia. The effect of ischemia on h9c2 cardiomyoblasts was mimicked by metabolic inhibition. Nuclear translocation of foxo1 was determined via quantitative digital imaging fluorescence and Western blot analysis. Subsequently, the effect of inhibiting of p47phox-dependent ros production via short hairpin rna (shrna) transfection on foxo1 translocation was analyzed by digital-imaging microscopy. Ischemia induced a significant translocation of foxo1 into the nucleus. Transfection with p47phox-shrna successfully inhibited p47phox expression, reduced the generation of ros at the nucleus and inhibited the nuclear translocation of foxo1. Our data for the first time show that ischemia-induced nuclear import of foxo1 in h9c2 depends critically on p47phox-mediated ros production.