Quantitative proteomics reveals possible mechanisms of regulation of adipogenic differentiation in 3T3-L1 cells

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

Congreso; 58vo Congreso de la Sociedad Argentina de Bioq y Biol Molecular; 2022

The murine preadipocyte 3T3-L1 cell line is a widely used model for the study of adipogenic differentiation. During its differentiation, a complex network of signaling processes takes place, including many unknown interactions. Currently, a growing number of studies reveal the participation of the 14-3-3 family proteins in adipogenesis, and these proteins have been recognized as a possible point of convergence of this intricate process. 14-3-3 proteins bind to phospho-serine or phospho-threonine residues in target proteins, affecting their activity, stability, subcellular localization or molecular interactions. Previous results in our laboratory revealed higher levels of mRNA and protein of the 14-3-3γ isoform during adipogenesis. We also reported that 14-3-3γ silencing produced a significant increase in lipid droplet accumulation in 3T3-L1 cells, compared to the wild-type, both in untreated or adipogenic induced cells.Therefore, the aim of this work was to examine the proteome and study how 14-3-3γ participates and regulates other proteins during the adipogenic differentiation process in 3T3-L1 cells. To do this, we infected cells with lentiviruses containing shRNA for the 14-3-3γ paralog; the silencing of this protein was confirmed by Western blot. Initially, cells were cultured with high-glucose DMEM and supplemented with 10% fetal bovine serum. Then, adipogenesis was induced by adding an adipogenic differentiation medium (ADM) containing insulin, dexamethasone, 3-isobutyl-1-methylxanthine, and rosiglitazone. Four biological independent protein samples of wild-type and 14-3-3γ-silenced cells were analyzed by mass spectrometry at day four of differentiation. Over 2750 individual proteins were identified in each cell lysate.14-3-3γ silenced samples expressed a significant increase in adipogenesis-relevant proteins, such as C/EBPβ and GLUT1, and proteins related to the oxidative stress. Interestingly, the expression of the enzyme PARP3 was decreased compared to the wild-type (p < 0.01). Subsequent bioinformatic analysis indicated that PARP3 has two binding sites for 14-3-3γ. PARP3 is an enzyme primarily involved in many cellular processes, such as DNA repair and programmed cell death. Recently it was discovered to be involved in the inhibition of C/EBPβ activity and consequently, cellular adipogenesis. Our results showed that 14-3-3γ could be an inhibitor of the adipogenesis process. This work suggests a possible regulatory mechanism, completely unknown until now, which involves the binding of 14-3-3γ to PARP3 and the indirect modulation of C/EBPβ activity