The ARF Tumor Suppressor Regulates Bone Remodeling and Osteosarcoma Development in Mice
Figure 1
Loss of the Arf tumor suppressor resulted in increased bone formation and enhanced osteoblast differentiation.
a) µCT analysis of tibias from 8-week-old female Arf+/+ versus Arf-/- littermates (n = 6/genotype) mice. Bone mineral density (p = 0.0289), trabecular bone volume (BV/TV) (p = 0.0074), trabecular number (p = 0.0117) and trabecular thickness (p = 0.0252) were significantly increased in Arf-/- tibias. b) Bone formation was visualized by double calcein labeling 7d and 2d prior to sacrifice and visualized in the calveria. The distance between labels is directly proportional to bone formation. Arf-/- mice showed a significant increase in bone formation rate per bone surface (p = 0.0211). c) Serum osteocalcin levels were significantly increased in Arf-/- mice (p = 0.0376). d) Long bones of 8-week-old Arf-/- mice expressed increased transcripts of OB differentiation markers by quantitative RT-PCR (n = 3/genotype). Data is represented as fold change following normalization to cyclophilin levels. RUNX2 = cbfa1, OSX = osterix, ALP = alkaline phosphatase, OCN = osteocalcin. Significance (*) indicates p<0.05 by Student's T-test. e) In vitro differentiation of OB from bone marrow stromal cells under osteogenic conditions (β-glycerophosphate and ascorbic acid). Cells were co-stained for alkaline phosphatase (ALP) expression (purple) and mineralization (Alizarin Red) at indicated days. Note the nearly complete mineralization (red stain) of Arf-/- wells present at each day as compared to punctate areas of mineralization present in Arf+/+ wells. Representative of >3 independent experiments.