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

BUSTOS, D M.; DEL VELIZ, S.; UHART, M.

Mendoza

Congreso; LVII Reunión Anual de SAIB; 2021

SAIB

Adipocytedifferentiation requires the interplay of cell signaling pathways andtranscription factors to be articulated. The Hippo pathway isinvolved in the control of tissue size and shape, through theregulation of proliferation, apoptosis and differentiation of stemcells and cell precursors. TAZ, the transcriptional co-activatorwith PDZ binding motif, is one of the main effectors of the Hippopathway. It has been shown that when the Hippo pathway is inactive,TAZ is dephosphorylated and nuclear. There, TAZ inactivates PPAR𝛾dependent gene transcription. However, when the Hippo pathway isactive, the LATS-1/2 kinases phosphorylate TAZ inducing its retentionin the cytoplasm by 14-3-3 proteins. In our laboratory, we achieveadipocyte differentiation in vitro by adding an adipogenicdifferentiation medium (ADM) that includes Dulbecco's modifiedEagle's medium, 10% fetal bovine serum, synthetic drugs(dexamethasone, IBMX, rosiglitazone), and peptide hormones (insulin).We have previously shown that replacing IBMX in ADM by the GlucagonLike Peptide-1 Analog (GLP-1A) enhanced adipogenic differentiation inmost cells, evidenced as a larger number and size of lipid droplets.In this condition, we found higher levels of Hippo pathway proteins,and both 14-3-3 gamma and beta isoforms on day 7 of differentiation.Here, using the IBMX x GLP1 differentiation cocktail, we studied i)the subcellular localization of TAZ throughout the cell adipogenesisprocess and ii) the adipogenic potential in 3T3-L1 wild-type, and14-3-3𝛾and 14-3-3β silenced cells. We transduced 3T3-L1 cells withlentiviruses containing plasmids with isoform-specific short hairpinribonucleic acids for 14-3-3 (shRNA𝛾or shRNAβ). As these lentiviruses simultaneously express ZsGreen,the levels of infection were monitored. After 3 or 7 days ofdifferentiation, we evaluated the subcellular localization of TAZthrough indirect immunofluorescence and confocal microscopy. Weobserved that in the WT cells, TAZ is more cytoplasmic on day 3 andbecomes diffuse (both nuclear and cytoplasmic) on day 7. In shRNAβcells, TAZ remains diffuse throughout days 3 and 7. However, inshRNA𝛾cells, the subcellular distribution of TAZ is diffuse on day 3 andbecomes cytoplasmic on day 7 of differentiation. Also, adipogenicdifferentiation was affected in different ways by silencing these two14-3-3 isoforms. While 14-3-3β silenced cells showed a decrease inadipogenic differentiation compared to the WT control, the 14-3-3𝛾 silenced cells showed an opposite phenotype, accumulating a largerquantity and size of lipid droplets than the WT control. Our resultssuggest that both 14-3-3𝛾and β isoforms regulate adipogenic differentiation through Hippopathway modulation. More research is needed to understand the exactmechanisms by which each isoform modulates the Hippo pathway.p { margin-bottom: 0.25cm; border: none; padding: 0cm; direction: ltr; line-height: 115%; text-align: left; page-break-inside: auto; orphans: 2; widows: 2; background: transparent; page-break-before: auto; page-break-after: auto }p.western { font-size: 12pt; so-language: en-US }p.cjk { font-size: 12pt; so-language: en-US }p.ctl { font-size: 12pt; so-language: ar-SA }a:link { text-decoration: underline }