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

MULLER, SERGIO; DEL VELIZ, SAMANTA; RIVERA, LAUTARO; UHART, MARINA; BUSTOS, DIEGO M

Congreso; LVII Congreso de la SAIB; 2021

The murine preadipocyte 3T3-L1 cell line is a widely used model for the study of adipogenic differentiation. The events thattrigger this lineage commitment compress a complex network at both transcriptional level and intracellular signaling pathways.To date, an increasing number of studies have been carried out revealing the involvement of 14-3-3 family proteins onadipogenesis and have been addressed as a potential point of convergence of this intricate process. 14-3-3 proteins bind tophospho-serine or phospho-threonine residues in target proteins, affecting their activity, stability, subcellular localization ormolecular interactions. In fact, data from our lab revealed higher mRNA and protein levels of the 14-3-3γ isoform duringadipogenesis. We also reported that 14-3-3γ silencing produced a significant increase in lipid droplets accumulation in 3T3-L1 cells, compared to the wild-type. The aim of this work was to study the implication of 14-3-3γ on the subcellular localizationof forkhead box protein O1 (FoxO1), a critical transcription factor for the modulation of adipogenesis-related genes. Increasingevidence suggests the importance of its nucleocytoplasmic shuttling which depends on post-translational modifications(mainly phosphorylation and acetylation) and interactions with 14-3-3. In the early stage of adipogenesis, FoxO1 upregulatesthe cell cycle inhibitors p21 and p27, while during terminal differentiation, activated FoxO1 localizes in the nucleus andinhibits transcriptional activity of Peroxisome proliferator-activated receptor gamma (PPARγ; master regulator ofadipogenesis). To address the possibility of FoxO1 regulation by 14-3-3γ, cells were infected with lentiviruses containingshRNA for the 14-3-3γ paralog. Initially, cells were cultured with DMEM, high-glucose supplemented with 10% serum. Then,adipogenesis was induced by adding an adipogenic differentiation medium that contains insulin, dexamethasone, 3-isobutyl-1-methylxanthine and rosiglitazone. Through indirect immunofluorescence and confocal microscopy we analyzed thesubcellular localization of FoxO1 at the 0, 2, 4 and 6 days post-induction. At each time point, 14-3-3γ silencing was confirmedby Western blot. Consistent with previous investigations, WT cells showed that FoxO1 shuttles between the cytoplasm andnucleus in an oscillatory pattern during adipogenic differentiation. However, FoxO1 is always located in the cytoplasm of 14-3-3γ silenced cells. This work suggests that 14-3-3γ could have an inhibitory role on the adipogenic differentiation processthrough modulation of FoxO1 subcellular localization.