FIG. 4.
Loss of p53 alleviates hypoxia-mediated repression of bone formation. (A and B) MEFs (p53+/+ and p53−/−) were treated with hypoxia (Hyp) or adriamycin (Adr) for 8 h as previously described, and qRT-PCR was then carried out for (A) Runx 2 and (B) procollagen, type 1, alpha 1. The error bars indicate standard deviations. N, normoxia. (C) HCT116p53+/+ and HCT116p53−/− were transfected with a Runx 2 reporter construct (p2800-luc), allowed to recover for 16 h, and exposed to hypoxia (0.02% O2) for 16 h. pCMV-renilla was also transfected into all cells as an internal transfection control; the levels of firefly luciferase/Renilla luciferase are shown. (D) Primary neonatal mouse calvarial osteoblast cultures were established from p53+/+ (+/+), p53+/− (+/−), and p53−/− (−/−) neonatal mice and exposed to 24 h of 20% or 0.02% O2 as previously described (39). The cultures underwent in vitro osteogenic differentiation in 20% O2 via medium supplementation with 1 μM dexamethasone, 5 mM beta-glycerophosphate, and 100 μg/ml ascorbic acid every 2 days. At day 28, mineralized matrix deposition was assessed by bone nodule staining via the von Kossa method for calcium phosphates (39).