Abstract
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.
Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starch-based three-dimensional scaffolds
