Inhibition of peroxisome proliferator-activated receptor-γ in steroidinduced adipogenic differentiation of the bone marrow mesenchymal stem cells of rabbit using small interference RNA

BACKGROUND: Steroids inhibit osteogenic differentiation and decrease bone formation while concomitantly inducing adipose deposition in osteocytes. This leads to the fatty degeneration and necrosis of bone cells commonly seen in osteonecrosis of the femoral head. The peroxisome proliferator-activated receptor-γ (PPARγ) is an adipogenic transcription factor linked to the development of this disease and responsible for inducing adipogenesis over osteogenesis in bone marrow mesenchymal stem cells (BMSCs). The aim of this study was to assess whether adipogenic differentiation could be suppressed, and thus osteogenic potential retained, by inhibiting PPARγ expression in BMSCs. METHODS: Cells from the bone marrow of New Zealand rabbits were treated with 10(-7) mol/L dexamethasone and infected with one of three small interference RNA (siRNA) adenovirus vectors (S1, S2, and S3) or non-targeting control siRNA (Con) and compared with dexamethasone-treated (model) and untreated (normal) cells. Cells were grown for 21 days and stained with Sudan III for adipocyte formation. At various time points, cells were also assessed for changes in PPARγ, osteocalcin (OC), Runx2, alkaline phosphatase (ALP) activity, and triglyceride (TG) content. RESULTS: Dexamethasone-treated model and control groups showed a significant increase in fatty acid-positive staining, which was inhibited in cells treated with PPARγ siRNA-treated, similar to normal untreated cells. All three siRNA groups significantly inhibited PPARγ mRNA and protein, adipocyte number, and TG content compared with the dexamethasone-treated model and control groups, matching that seen in normal cells. OC and Runx2 mRNA and protein, as well as ALP activity, were significantly higher in cells treated with siRNA against PPARγ, similar to that seen in the normal cells. These osteogenic markers were significantly lower in the dexamethasone-treated cell cultures. CONCLUSIONS: The siRNA adenovirus vector targeting PPARγ can efficiently inhibit steroid-induced adipogenic differentiation in rabbit BMSCs and retain their osteogenic differentiation potential.