Gamma or beta knockdown affects 3T3-L1 adipogenic differentiation through hippo pathway modulation

• Autores: del Veliz, Samanta; Uhart, Marina; Bustos, Diego Martin
• Año de publicación: 2021
• Idioma: inglés
• Tipo de recurso: documento de conferencia
• Materia: 14-3-3; ADIPOGENESIS; 3T3-L1; TAZ
• Institución: Consejo Nacional de Investigaciones Científicas y Técnicas

Adipocyte differentiation requires the interplay of cell signaling pathways and transcription factors to be articulated. The Hippo pathway is involved in the control of tissue size and shape, through the regulation of proliferation, apoptosis and differentiation of stem cells and cell precursors. TAZ, the transcriptional co-activator with PDZ binding motif, is one of the main effectors of the Hippo pathway. It has been shown that when the Hippo pathway is inactive, TAZ is dephosphorylated and nuclear. There, TAZ inactivates PPAR dependent gene transcription. However, when the Hippo pathway is active, the LATS-1/2 kinases phosphorylate TAZ inducing its retention in the cytoplasm by 14-3-3 proteins. In our laboratory, we achieve adipocyte differentiation in vitro by adding an adipogenic differentiation medium (ADM) that includes Dulbecco's modified Eagle'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 Glucagon Like Peptide-1 Analog (GLP-1A) enhanced adipogenic differentiation in most 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 adipogenesis process and ii) the adipogenic potential in 3T3-L1 wild-type, and 14-3-3 and 14-3-3β silenced cells. We transduced 3T3-L1 cells with lentiviruses containing plasmids with isoform-specific short hairpin ribonucleic acids for 14-3-3 (shRNA or shRNAβ). As these 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 observed that in the WT cells, TAZ is more cytoplasmic on day 3 and becomes diffuse (both nuclear and cytoplasmic) on day 7. In shRNAβ cells, TAZ remains diffuse throughout days 3 and 7. However, in shRNA cells, the subcellular distribution of TAZ is diffuse on day 3 and becomes cytoplasmic on day 7 of differentiation. Also, adipogenic differentiation was affected in different ways by silencing these two 14-3-3 isoforms. While 14-3-3β silenced cells showed a decrease in adipogenic differentiation compared to the WT control, the 14-3-3 silenced cells showed an opposite phenotype, accumulating a larger quantity and size of lipid droplets than the WT control. Our results suggest that both 14-3-3 and β isoforms regulate adipogenic differentiation through Hippo pathway modulation. More research is needed to understand the exact mechanisms by which each isoform modulates the Hippo pathway.
Fil: del Veliz, Samanta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina
Fil: Uhart, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina
Fil: Bustos, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina
LVII Reunión Anual de Sociedad Argentina de Investigación Bioquímica
Mendoza
Argentina
Sociedad Argentina de Investigación en Bioquimica y Biología Molecular