shRNA-MEDIATED KNOCKDOWN OF 14-3-3γ REVEALS ITS ESSENTIAL ROLE IN REGULATING ADIPOGENIC DIFFERENTIATION OF UCMSCs

RIVERA, LAUTARO; SANTONI, DIEGO; BUSTOS, D.M.; UHART, MARINA

Congreso; Congreso SAIB 2019; 2019

Human umbilical cord-derived mesenchymal stem cells (UCMSCs) are self-renewing multipotent progenitors that can differentiate into cells ofthe mesoderm-lineage, which makes them attractive targets for regenerative medicine. However, the lack of understanding of the molecularmechanisms that regulate cell differentiation is currently an obstacle to such applications. In this sense, 14-3-3 proteins have received tremendousattention since these phospho-serine/threonine binding proteins have a pivotal role in the regulation of metabolism and signal transductionnetworks. A recently published work in our lab has shown that both at the mRNA and protein level, 14-3-3 β and γ were the isoforms that mostchanged after adipogenic differentiation of 3T3-L1, a well-established murine pre-adipocyte cell line. Since we have developed a method forUCMSCs isolation and also obtained the efficient adenoviral transduction of these cells, the aim of this study was to analyze the effect of decreasedexpression of 14-3-3γ by using shRNA on the adipogenic potential of UCMSCs. The recombinant adenoviruses (Adv) were E1/E3-deleted type 5Advs expressing shRNA for 14-3-3γ, under the control of the small nuclear RNA (snRNA) U6 promoter. The Pac I-digested vector was used totransfect 293A cells to produce Adv-shRNA 14-3-3γ stock. Then, the Advs were amplified by infecting the 293A cells with the crude viral lysate.UCMSCs were isolated and expanded from Wharton Jelly of the human umbilical cord, using a culture explant method. For transductionexperiments, cells at 80?90% confluence were incubated with Adv-shRNA 14-3-3γ for 2 h at 37oC, and then the transduction media was replacedwith standard growth media (high glucose DMEM; 10% FBS). To investigate the effects of 14-3-3γ in adipogenesis, we induced UCMSCs(transduced or not- with Adv-shRNA 14-3-3γ) with adipogenic differentiation medium (ADM; an optimized drug cocktail that includes highglucose DMEM, 10% FBS, Dexamethasone, Insulin, Rosiglitazone, and IBMX) for 10 days. We also used untreated and Adv-GFP transducedcells as controls. Lipid droplets accumulation was examined using Oil Red O staining. UCMSCs transduced with Adv-shRNA 14-3-3γ exhibitedan increased lipid droplets accumulation, compared to control cells. These data suggest an essential role for 14-3-3γ in the process of adipogenesisin UCMSCs. Our finding proposes the existence of a previously unknown regulatory mechanism of UCMSCs adipogenic differentiation, and that14-3-3γ could be an interesting candidate to be evaluated as a therapeutic target molecule to treat chronic diseases, such as obesity and type 2 diabetes.