Mathematical modeling reveals modulation of both nuclear influx and efflux of Foxo1 by the IGF-I/PI3K/Akt pathway in skeletal muscle fibers

Abstract

Wimmer RJ, Liu Y, Schachter TN, Stonko DP, Peercy BE, Schneider MF. Mathematical modeling reveals modulation of both nuclear influx and efflux of Foxo1 by the IGF-I/PI3K/Akt pathway in skeletal muscle fibers. Am J Physiol Cell Physiol 306: C570-C584, 2014. First published January 22, 2014; doi:10.1152/ajpcell.00338.2013.-Foxo family transcription factors contribute to muscle atrophy by promoting transcription of the ubiquitin ligases muscle-specific RING finger protein and muscle atrophy F-box/atrogin-1. Foxo transcriptional effectiveness is largely determined by its nuclear-cytoplasmic distribution, with unphosphorylated Foxo1 transported into nuclei and phosphorylated Foxo1 transported out of nuclei. We expressed the fluorescent fusion protein Foxo1-green fluorescent protein (GFP) in cultured adult mouse flexor digitorum brevis muscle fibers and tracked the time course of the nuclear-to-cytoplasmic Foxo1-GFP mean pixel fluorescence ratio (N/C) in living fibers by confocal imaging. We previously showed that IGF-I, which activates the Foxo kinase Akt/PKB, caused a rapid marked decline in N/C, whereas inhibition of Akt caused a modest increase in N/C. Here we develop a two-state mathematical model for Foxo1 nuclear-cytoplasmic redistribution, where Foxo phosphorylation/dephosphorylation is assumed to be fast compared with nuclear influx and efflux. Cytoplasmic Foxo1-GFP mean pixel fluorescence is constant due to the much larger cytoplasmic than nuclear volume. Analysis of N/C time courses reveals that IGF-I strongly increased unidirectional nuclear efflux, indicating similarly increased fractional phosphorylation of Foxo1 within nuclei, and decreased unidirectional nuclear influx, indicating increased cytoplasmic fractional phosphorylation of Foxo1. Inhibition of Akt increased Foxo1 unidirectional nuclear influx, consistent with block of Foxo1 cytoplasmic phosphorylation, but did not decrease Foxo1 unidirectional nuclear efflux, indicating that Akt may not be involved in Foxo1 nuclear efflux under control conditions. New media change experiments show that cultured fibers release IGF-I-like factors, which maintain low nuclear Foxo1 in the medium. This study demonstrates the power of quantitative modeling of observed nuclear fluxes.Akt1; Foxo1; IGF-I; skeletal muscle TRANSCRIPTION OF A GIVEN GENE is controlled by numerous regulatory proteins, including transcription factors, which are primary activators of transcription, as well as numerous other activators, coactivators, repressors, and corepressors (12, 15a). These factors combine to form macromolecular transcriptional regulatory complexes assembled on the promoter regions of the gene in question (16). To participate in such regulatory complexes, the regulatory molecules must be resident in the nucleus. Thus an important aspect of transcriptional regulation is control of the nuclear-cytoplasmic distribution of these regulatory molecules, and the mechanisms controlling nuclearcytoplasmic movements of transcriptional regulatory molecules can thus be important dete...

Green tea component EGCG, insulin and IGF-1 promote nuclear efflux of atrophy-associated transcription factor Foxo1 in skeletal muscle fibers

“…In addition, it allows for adjustments of laser intensity when the nuclear fluorescence signal becomes saturated. Previous studies have shown that under the conditions of the present work the level of Foxo1-GFP cytoplasmic fluorescence remains constant over the course of our experiments [22,26]. To compensate for variations in starting N/C ratios, the data were normalized to the average of the first three time points (recorded under “control” conditions) or the first two time points (recorded under “control” conditions).…”

“…All experimental procedures use individual muscle fibers dissociated from flexor digitorum brevis (FDB) skeletal muscle isolated from adult (4 – 8 wk old) female CD1 mice, in protocols approved by the University of Maryland Institutional Animal Care and Use Committee, and as previously described [15,22,26]. Figure 1 summarizes the experimental procedures used for culture and Foxo1-GFP expression (left side) and the general imaging protocol at day 3 (right side) used to acquire data shown below.…”

“…The culture dish containing 4 compartments of Foxo1-GFP expressing fibers was placed on an Olympus IX70 inverted microscope with Olympus FLUOVIEW 500 laser scanning confocal imaging system previously described [22,26]. The excitation wavelength was 488 nm, which with a 505 nm long pass emission filter monitored the GFP fluorescence.…”

“…Within the nucleus, phosphorylation of Foxo1 by activated Akt results in Foxo1 unbinding from DNA, exposing the nuclear export signal (NES) for translocation out of the nucleus [5,24]. A predominate pathway implicated for the deactivation of Foxo1 transcriptional effects is the IGF receptor / phosphatidylinositol 3-kinase (PI3K) / Akt (PKB) pathway, which causes phosphorylation of Foxo1 in both cytoplasm and nuclei [26], and a reduction in nuclear localization [9,28]. When Foxo1 is activated and localized inside the nucleus it induces gene transcription by binding to DNA sites with its conserved 100 residue DNA binding domain known as the forkhead domain [20,22].…”

Smoking, aging, and expression of proteins related to the FOXO3 signaling pathway in lung tissues

“…FOXO proteins are transcription factors that regulate the expression of numerous target genes related to cell cycle progression, apoptosis, longevity and aging, stress response, and metabolism. FOXO proteins are thought to be related to various aging-related signal transduction pathways, including the Akt (Wimmer et al, 2014), c-Jun N-terminal kinase (van den Berg et al, 2013), and IkB kinase pathways (Salminen and Kaarniranta, 2010), which play key roles in the regulation of aging and longevity. However, the contribution of the FOXO subfamily proteins to age-related changes in lung tissue and to senile emphysema remains unclear.…”