title: Kinetics of nuclear-cytoplasmic translocation of Foxo1 and Foxo3A in adult skeletal muscle fibers.
abstract: In skeletal muscle, the transcription factors Foxo1 and Foxo3A control expression of proteins that mediate muscle atrophy, making the nuclear concentration and nuclear-cytoplasmic movements of Foxo1 and Foxo3A of therapeutic interest in conditions of muscle wasting. Here, we use Foxo-GFP fusion proteins adenovirally expressed in cultured adult mouse skeletal muscle fibers to characterize the time course of nuclear efflux of Foxo1-GFP in response to activation of the insulin-like growth factor-1 (IGF-1)/phosphatidylinositol-3-kinase (PI3K)/Akt pathway to determine the time course of nuclear influx of Foxo1-GFP during inhibition of this pathway and to show that Akt mediates the efflux of nuclear Foxo1-GFP induced by IGF-1. Localization of endogenous Foxo1 in muscle fibers, as determined via immunocytochemistry, is consistent with that of Foxo1-GFP. Inhibition of the nuclear export carrier chromosome region maintenance 1 by leptomycin B (LMB) traps Foxo1 in the nucleus and results in a relatively rapid rate of Foxo1 nuclear accumulation, consistent with a high rate of nuclear-cytoplasmic shuttling of Foxo1 under control conditions before LMB application, with near balance of unidirectional influx and efflux. Expressed Foxo3A-GFP shuttles ∼20-fold more slowly than Foxo1-GFP. Our approach allows quantitative kinetic characterization of Foxo1 and Foxo3A nuclear-cytoplasmic movements in living muscle fibers under various experimental conditions.
authors.affiliation: Dept. of Biochemistry and Molecular Biology, Univ. of Maryland School of Medicine, 108 North Greene St., Baltimore MD 21201-1503, USA. mschneid@umaryland.edu
authors.forename: Tova Neustadt
authors.initials: TN
authors.lastname: Schachter
authors.forename: Tiansheng
authors.initials: T
authors.lastname: Shen
authors.forename: Yewei
authors.initials: Y
authors.lastname: Liu
authors.forename: Martin F
authors.initials: MF
authors.lastname: Schneider
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005260:Female; D000071161:Forkhead Box Protein O1; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D007150:Immunohistochemistry; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000145:classification / Q000166:cytology / Q000378:metabolism*; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
chemical_list: D005231:Fatty Acids, Unsaturated; D000071161:Forkhead Box Protein O1; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; C474033:FoxO3 protein, mouse; C467200:Foxo1 protein, mouse; C519530:insulin-like growth factor-1, mouse; D007334:Insulin-Like Growth Factor I; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; C038753:leptomycin B