14-3-3γ silencing impairs osteogensis differentiation of human adipose derived-mesenchymal stem cells

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

Congreso; LVII Congreso de la SAIB; 2021

SAIB

14-3-3 proteins constitute a family of regulatory molecules that participate in a plethora of cellular processes mainly throughprotein-protein interactions. Even though 14-3-3 protein family members show some functional redundancy, there is growingevidence that indicates evolutionary and biochemical diversity. Consistent with the literature, previous research from ourlaboratory showed that expression levels of 14-3-3 paralogs are independently regulated during the adipogenesis andosteogenesis of human adipose derived-mesenchymal stem cells (hASCs). In the current work, we used a validated approachto isolate hASCs and studied the implication of 14-3-3γ on the osteogenic commitment of these cells. To address this purpose,we delivered a 14-3-3γ shRNA construct into hASCs by pAd-BLOCKiT, an adenoviral vector containing a human U6promoter, and examined the effect on the differentiation potential into osteoblasts. The latter was evaluated by: i) measuringalkaline phosphatase (ALP) activity, an early-stage osteoblast differentiation biomarker, and ii) detecting Runt-relatedtranscription factor 2 (Runx2, master regulator of bone formation) protein levels. Cells were either maintained for 14 dayswith standard growth media (control, low glucose DMEM; 5% FBS) or induced with an osteogenic differentiation medium(ODM; an optimized drug cocktail that includes dexamethasone, β-glycerophosphate, and 2-phospho-L-ascorbic acid). Ourresults clearly showed a decrease in both Runx2 protein levels and ALP activity in 14-3-3γ depleted hASCs. This also accordswith our earlier observations, which showed that reduced expression of 14-3-3γ had a negative impact on the osteoblastictransdifferentiation of NIH3T3-L1 cells. Taken together, these findings suggest a regulatory role for 14-3-3γ in hASCdifferentiation to the osteogenic lineage.