Dataset Information

P47phox-Dependent Reactive Oxygen Species Stimulate Nuclear Translocation of the FoxO1 Transcription Factor During Metabolic Inhibition in Cardiomyoblasts.

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 p47^phox-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 p47^phox-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 p47^phox-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 p47^phox-shRNA successfully knocked-down p47^phox 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 p47^phox-mediated ROS production.

SUBMITTER: Ter Horst EN

PROVIDER: S-EPMC6097050 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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p47phox-Dependent Reactive Oxygen Species Stimulate Nuclear Translocation of the FoxO1 Transcription Factor During Metabolic Inhibition in Cardiomyoblasts.

Ter Horst Ellis N EN Hahn Nynke E NE Geerts Dirk D Musters René J P RJP Paulus Walter J WJ van Rossum Albert C AC Meischl Christof C Piek Jan J JJ Niessen Hans W M HWM Krijnen Paul A J PAJ

Cell biochemistry and biophysics 20180629 3

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 p47<sup>phox</sup>-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. ...[more]

PMID: 29956081

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Project description:Na(V)1.5 is a cardiac voltage-gated Na(+) channel ?subunit and is encoded by the SCN5a gene. The activity of this channel determines cardiac depolarization and electrical conduction. Channel defects, including mutations and decrease of channel protein levels, have been linked to the development of cardiac arrhythmias. The molecular mechanisms underlying the regulation of Na(V)1.5 expression are largely unknown. Forkhead box O (Foxo) proteins are transcriptional factors that bind the consensus DNA sequences in their target gene promoters and regulate the expression of these genes. Comparative analysis revealed conserved DNA sequences, 5'-CAAAACA-3' (insulin responsive element, IRE), in rat, mouse and human SCN5a promoters with the latter two containing two overlapping Foxo protein binding IREs, 5'-CAAAACAAAACA-3'. This finding led us to hypothesize that Foxo1 regulates Na(V)1.5 expression by directly binding the SCN5a promoter and affecting its transcriptional activity. In the present study, we determined whether Foxo1 regulates Na(V)1.5 expression at the transcriptional level and also defined the role of Foxo1 in hydrogen peroxide (H(2)O(2))-mediated Na(V)1.5 suppression in HL-1 cardiomyocytes using chromatin immunoprecipitation (ChIP), constitutively nuclear Foxo1 expression, and RNAi Foxo1 knockdown as well as whole cell voltage-clamp recordings. ChIP with anti-Foxo1 antibody and follow-up semi-quantitative PCR with primers flanking Foxo1 binding sites in the proximal SCN5a promoter region clearly demonstrated enrichment of DNA, confirming Foxo1 recruitment to this consensus sequence. Foxo1 mutant (T24A/S319A-GFP, Foxo1-AA-GFP) was retained in nuclei, leading to a decrease of Na(V)1.5 expression and Na(+) current, while silencing of Foxo1 expression by RNAi resulted in the augmentation of Na(V)1.5 expression. H(2)O(2) significantly reduced Na(V)1.5 expression by promoting Foxo1 nuclear localization and this reduction was prevented by RNAi silencing Foxo1 expression. These studies indicate that Foxo1 negatively regulates Na(V)1.5 expression in cardiomyocytes and reactive oxygen species suppress Na(V)1.5 expression through Foxo1.

Dataset Information

P47phox-Dependent Reactive Oxygen Species Stimulate Nuclear Translocation of the FoxO1 Transcription Factor During Metabolic Inhibition in Cardiomyoblasts.

Publications

p47phox-Dependent Reactive Oxygen Species Stimulate Nuclear Translocation of the FoxO1 Transcription Factor During Metabolic Inhibition in Cardiomyoblasts.

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