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Abstract
Reactive oxygen species (ROS) control forkhead box O (FOXO) transcription factor activity by influencing their nuclear translocation. However, knowledge of the ROS cellular source(s) 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 protein nuclear translocation, including that of transcription factors. In the current study, involvement of p47phox-dependent production of ROS in the nuclear translocation of FOXO1 was analyzed in H9c2 cells following 4 h of metabolic inhibition (MI), which mimics the effects of ischemia. Nuclear translocation of FOXO1 was determined by quantitative digital-imaging fluorescence and western blot analysis. Subsequently, the effect of inhibiting p47phox-dependent ROS production by short hairpin RNA (shRNA) transfection on FOXO1 translocation was analyzed by digital-imaging microscopy. MI induced a significant translocation of FOXO1 into the nucleus. Transfection with p47phox-shRNA successfully knocked-down p47phox expression, reduced nuclear nitrotyrosine production, an indirect marker for ROS production, and inhibited the nuclear translocation of FOXO1 following MI. With these results, we show for the first time that nuclear import of FOXO1 induced by MI in H9c2 depends critically on p47phox-mediated ROS production.
Highlights
Mammalian cells produce reactive oxygen species (ROS) in response to a multitude ofphysiological conditions and thereby regulate cell fate decisions [1]
When subjected to metabolic inhibition (MI), cytosolic FOXO1 expression decreased to 25 ± 4% in comparison to non-MI condition (P < 0.001, Fig. 2a) whereas nuclear FOXO1 expression increased to 135 ± 25% (P < 0.01, Fig. 2a)
These results were validated using western blot analysis of isolated nuclear and cytosol fractions of H9c2 cells. This showed that following MI, cytosolic FOXO1 protein expression level was reduced to 63 ± 24% (Fig. 2c; P < 0.01)
Summary
Mammalian cells produce reactive oxygen species (ROS) in response to a multitude of (patho)physiological conditions and thereby regulate cell fate decisions [1]. In the last two decades it has become recognized that these ROS, rather than being by-products of signaling events, act as second messengers in cellular signaling through the reversible oxidation of cysteine residues in signal transduction proteins, which alters their activity [2, 3]. These signal transduction proteins include protein kinases, phosphatases, and transcription factors. A number of redox-sensitive transcription factors have been described that are either activated or inactivated through redox modifications, including NF-κB, c-Jun and Nrf2 [4,5,6,7,8].
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