Abstract 10134: S-nitrosylation of PPAR? Regulates the Balance of Adipogenesis and Osteogenesis in Mesenchymal Stem Cells
Introduction: Circulating bone marrow-derived mesenchymal stem cells (BMMSCs) can undergo adipogenic or osteogenic differentiation to form ectopic aggregates of fat and bone seen in atherosclerotic plaques. We assessed the role of S-nitrosoglutathione reductase (GSNOR) in BMMSC differentiation. We hypothesized that S-nitrosylation mediated-nitric oxide (NO) signaling shifts the balance between adipogenic and osteogenic differentiation of BMMSCs by altering peroxisome proliferator-activated receptor γ (PPARγ) activity.
Methods: BMMSCs were isolated from WT and GSNOR–/– mice and functional and gene expression assays performed following differentiation. S-nitrosylation of PPARγ, a master regulator of adipogenesis and osteogenesis, was assessed by SNO-RAC assay and transcriptional activity was mearsured by luciferase reporter assay and chromatin immunoprecipitation (ChIP).
Results: GSNOR–/– BMMSCs had higher baseline expression of the osteogenic markers Osteopontin, Osteocalcin and Runx2, suggesting a predisposition toward an osteogenic lineage. GSNOR–/– cells exhibited enhanced osteogenic differentiation after exposure to osteogenic media, as measured by greater calcium deposition and higher expression of the Osteopontin (10.9-fold, P<0.05). Conversely, GSNOR–/– BMMSCs grown in adipogenic media exhibited reduced fat droplet formation and expression of the adipogenic markers PPARγ (8.4-fold, P<0.05) and fatty acid binding protein 4 (FABP4; 2.7-fold, P<0.05). Treatment of GSNOR–/– BMMSCs with L-NAME, a nitric oxide synthase inhibitor, decreased osteogenesis without affecting adipogenesis. The abundance of nitrosylated PPARγ was greater at baseline in GSNOR–/– cells (1.4-fold, P<0.05) which in turn led to a lower binding affinity for its downstream target FABP4. PPARγ binding to FABP4 was also reduced in WT BMMSCs treated with GSNO (8.2-fold, P<0.05). Similarly, 293T cells transfected with a PPARγ reporter exhibited decreased transcriptional activity of PPARγ when treated with GSNO (1.3-fold, P<0.05).
Conclusion: The absence of GSNOR-mediated denitrosylation shifts the osteogenic/adipogenic balance toward osteogenesis by altering the transcriptional activity and post-translational modification of PPARγ.
- © 2013 by American Heart Association, Inc.