The Influence of Tetracycline Inducible Targeting Rat PPARγ Gene Silencing on the Osteogenic and Adipogenic Differentiation of Bone Marrow Stromal Cells

Peroxisome proliferator-activated receptor gamma (PPAR gamma) has been considered as the master regulator for adipogenesis of bone marrow stromal cells (BMSCs). However, there are few reports regarding the effect of PPAR gamma gene silencing on osteogenic and adipogenic differentiation in rat BMSCs, and no reports about tissue targeting and conditional knockdown of PPAR gamma gene. In this study, we construct rat PPAR gamma gene shRNA Tet-on lentiviral vector, the lentiviral vector facilitated tetracycline (which has the characteristics of bone targeting)-inducible knockdown specific to PPAR gamma gene, and transfect it into BMSCs, the silencing effects induced by tetracycline is significant. The expression of the adipogenic factors adipocyte determination and differentiation-dependent factor 1 (ADD1) and recombinant CCAAT/enhancer binding protein alpha (C/EBP alpha) were decreased as measured by RT-PCR and Western blot assay following PPAR gamma silencing. In contrast, expression of the osteogenic genes encoding collagen I and Cbfa1/Runx2 were increased. In adipogenic medium, PPAR gamma-shRNA transfection reduced the lipid droplet count as measured by Oil red O staining when compared to the control groups. In osteogenic medium, PPAR gamma-shRNA increased the activity of alkaline phosphatase and the amount of calcium deposition as measured by Alizarin red S staining. These results suggest that the rat PPAR gamma gene shRNA Tet-on lentiviral vector decreases adipogenic differentiation and promotes osteogenic differentiation in BMSCs induced by tetracycline.