Adipocyte Differentiation of SOD2−/− Mouse Bone Marrow Stromal Cells Is Associated with Decreased Antioxidant Reserves and Is Reversed by the Antioxidant WR2721 (Amifostine).

Cell lines from homozygous deletion recombinant negative manganese superoxide dismutase (SOD2 −/−) mice have intrinsic ionizing radiation sensitivity that is reversed by expression of the transgene for human SOD2 (Radiation Research154:365, 2000). This study tests whether redox status influences adipocyte differentiation potential of bone marrow stromal cells by comparing the differentiation potential of SOD2−/− and SOD2 +/+ bone marrow stromal cell lines. Cells were cultured in basal medium (Dulbecco’s Modified Eagle’s Medium, 1% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/mL streptomycin +/− adipocytogenic supplements (10 μg/ml insulin, 1 μM dexamethasone, 100 mM indomethacin)). Adipocytogenesis was assessed by spectrophotometric content of oil red-O dye and by RT-PCR for peroxisome proliferator-activated receptor-gamma (PPARγ) and lipoprotein lipase (LPL). Glutathione peroxidase (GPX) activity or glutathione (GSH) levels were measured by a Glutathione Peroxidase, Cellular Assay Kit or Glutathione Assay Kit, respectively, by Calbiochem, Inc. SOD2+/+ cells developed adipocytes only when treated with adipocytogenic supplements and expressed PPARγ and LPL at day 5. SOD2−/− cells cultured in basal conditions demonstrated constitutive adipocytogenesis. The level of GPX activity in SOD2−/− cells (43.2 ± 3.5 mU/ml) was 52.7% (p<0.001) of that in SOD2+/+ cells (82.0 ± 2.3). The level of GSH in SOD2−/− cells (173 ± 2 μM) was 78.6% (p=0.0089) of that in SOD2+/+ cells (220 ± 4 μM). Three day treatment of SOD2−/−with 4 mM WR2721 resulted in 22% reduction in oil red-O content. In addition, in SOD2−/− cells cultured in adipocytogenic medium, 3-day treatment in 4 mM WR2721 resulted in 46% reduction in oil red-O content and undetectable expression of PPARγ and LPL. In conclusion, WR2721 limits constitutive adipocytogenesis and inhibits induced adipocytogenesis in SOD2−/− cells. These data provide evidence for the involvement of the cellular redox pathway in adipocyte differentiation of bone marrow stromal cells.