SUBCELLULAR LOCALIZATION OF FOXO1 CHANGES IN 3T3L1 PREADIPOCYTE CELLS SILENCED FOR 14-3-3γ PROTEIN

MÜLLER, S.; UHART, M.; DEL VELIZ, S.; BUSTOS, D. M.; RIVERA, L.

Congreso; LVII Reunión Anual de SAIB; 2021

SAIB

Themurine preadipocyte 3T3-L1 cell line is a widely used model for thestudy of adipogenic differentiation. The events that trigger thislineage commitment compress a complex network at both transcriptionallevel and intracellular signaling pathways. To date, an increasingnumber of studies have been carried out revealing the involvement of14-3-3 family proteins on adipogenesis and have been addressed as apotential point of convergence of this intricate process. 14-3-3proteins bind to phospho-serine or phospho-threonine residues intarget proteins, affecting their activity, stability, subcellularlocalization or molecular interactions. In fact, data from our labrevealed higher mRNA and protein levels of the 14-3-3γ isoformduring adipogenesis. We also reported that 14-3-3γ silencingproduced a significant increase in lipid droplets accumulation in3T3-L1 cells, compared to the wild-type.The aim of this work was to study the implication of 14-3-3γ on thesubcellular localization of forkhead box protein O1 (FoxO1), acritical transcription factor for the modulation ofadipogenesis-related genes. Increasing evidence suggests theimportance of its nucleocytoplasmic shuttling which depends onpost-translational modifications (mainly phosphorylation andacetylation) and interactions with 14-3-3. In the early stage ofadipogenesis, FoxO1 upregulates the cell cycle inhibitors p21 andp27, while during terminal differentiation, activated FoxO1 localizesin the nucleus and inhibits transcriptional activity of Peroxisomeproliferator-activated receptor gamma (PPARγ; master regulator ofadipogenesis). To address the possibility of FoxO1 regulation by14-3-3γ, cells were infected with lentiviruses containing shRNA forthe 14-3-3γ paralog. Initially, cells were cultured with DMEM,high-glucose supplemented with 10% serum. Then, adipogenesis wasinduced by adding an adipogenic differentiation medium that containsinsulin, dexamethasone, 3-isobutyl-1-methylxanthine androsiglitazone. Through indirect immunofluorescence and confocalmicroscopy we analyzed the subcellular localization of FoxO1 at the0, 2, 4 and 6 days post-induction. At each time point, 14-3-3γsilencing was confirmed by Western blot. Consistent with previousinvestigations, WT cells showed that FoxO1 shuttles between thecytoplasm and nucleus in an oscillatory pattern during adipogenicdifferentiation. However, FoxO1 is always located in the cytoplasm of14-3-3γ silenced cells. This work suggests that 14-3-3γ could havean inhibitory role on the adipogenic differentiation process throughmodulation of FoxO1 subcellular localization.p { margin-bottom: 0.25cm; line-height: 115%; background: transparent }