14-3-3 GAMMA OR BETA KNOCKDOWN AFFECTS 3T3-L1 ADIPOGENIC DIFFERENTIATION THROUGH HIPPO PATHWAY MODULATION

DEL VELIZ, SAMANTA; UHART, MARINA; BUSTOS, DIEGO M.

Congreso; LVII Congreso de la SAIB; 2020

SAIB

Adipocyte differentiation requires the interplay of cell signaling pathways and transcription factors to be articulated. The Hippopathway is involved in the control of tissue size and shape, through the regulation of proliferation, apoptosis and differentiationof stem cells and cell precursors. TAZ, the transcriptional co-activator with PDZ binding motif, is one of the main effectorsof the Hippo pathway. It has been shown that when the Hippo pathway is inactive, TAZ is dephosphorylated and nuclear.There, TAZ inactivates PPARγ dependent gene transcription. However, when the Hippo pathway is active, the LATS-1/2kinases phosphorylate TAZ inducing its retention in the cytoplasm by 14-3-3 proteins. In our laboratory, we achieve adipocytedifferentiation in vitro by adding an adipogenic differentiation medium (ADM) that includes Dulbecco's modified Eagle'smedium, 10% fetal bovine serum, synthetic drugs (dexamethasone, IBMX, rosiglitazone), and peptide hormones (insulin). Wehave previously shown that replacing IBMX in ADM by the Glucagon-Like Peptide-1 Analog (GLP-1A) enhanced adipogenicdifferentiation in most cells, evidenced as a larger number and size of lipid droplets. In this condition, we found higher levelsof Hippo pathway proteins, and both 14-3-3 gamma and beta isoforms on day 7 of differentiation. Here, using the IBMX xGLP1 differentiation cocktail, we studied i) the subcellular localization of TAZ throughout the cell adipogenesis process andii) the adipogenic potential in 3T3-L1 wild-type, and 14-3-3γ and 14-3-3β silenced cells. We transduced 3T3-L1 cells withlentiviruses containing plasmids with isoform-specific short hairpin ribonucleic acids for 14-3-3 (shRNAγ or shRNAβ). Asthese lentiviruses simultaneously express ZsGreen, the levels of infection were monitored. After 3 or 7 days of differentiation,we evaluated the subcellular localization of TAZ through indirect immunofluorescence and confocal microscopy. We observedthat in the WT cells, TAZ is more cytoplasmic on day 3 and becomes diffuse (both nuclear and cytoplasmic) on day 7. InshRNAβ cells, TAZ remains diffuse throughout days 3 and 7. However, in shRNAγ cells, the subcellular distribution of TAZis diffuse on day 3 and becomes cytoplasmic on day 7 of differentiation. Also, adipogenic differentiation was affected indifferent ways by silencing these two 14-3-3 isoforms. While 14-3-3β silenced cells showed a decrease in adipogenicdifferentiation compared to the WT control, the 14-3-3 silenced cells showed an opposite phenotype, accumulating a largerquantity and size of lipid droplets than the WT control. Our results suggest that both 14-3-3γ and β isoforms regulateadipogenic differentiation through Hippo pathway modulation. More research is needed to understand the exact mechanismsby which each isoform modulates the Hippo pathway.