Induction of the osteocytic phenotype: The influence of oxygen on bone cell maturation and survival
Molecular oxygen is a critical metabolic regulator that is required to maintain the function and viability of most eukaryotic organisms. Oxygen supply in hard tissues is largely uncharacterized but may play important roles during bone cell maturation and survival. The goal of this investigation was to elucidate the influence of oxygen availability on the formation, function, and survival of osteocytes in the development and maintenance of skeletal tissue. The Krogh cylinder model of oxygen diffusion was adapted to predict the oxygen distribution within cortical and cancellous structures of unloaded bones. Substantial oxygen gradients were predicted across mature osteons and trabeculae, which suggested that a drop in oxygen tension accompanies the differentiation of surface lining osteoblasts into subsumed osteocytes. A steep oxygen gradient was predicted during the initial stage of osteon formation, when osteoblasts first line the cutting cone and begin to deposit matrix. In contrast, the largest oxygen gradient was observed in the final stages of trabeculum development. These results suggest oxygen may regulate the dimensions of viable osteons and trabeculae. The effect of oxygen tension on alkaline phosphatase activity and mineralization capacity of MLO-A5 preosteocytes and MLO-Y4 osteocytes was then examined. Culture in low oxygen significantly inhibited alkaline phosphatase activity and mineralization capacity. Hypoxia also significantly reduced MLO-A5 mineral production in three-dimensional alginate bead culture as determined by μCT analysis. Both MLO cell lines responded to hypoxia by upregulating HIF-1α and the HIF-1 target genes GLUT1 and BNIP3. A stable MLO-A5 HIF1α siRNA knockdown clone was developed to determine if HIF-1 mediated the hypoxic inhibition of alkaline phosphatase and mineralization. The loss of HIF1α decreased alkaline phosphatase and mineral formation independent of the oxygen tension. Next, a stable MLO-A5 clone overexpressing ATF4 was developed to determine if the observed reduction in the ATF4 in hypoxia was responsible for the inhibition of the mineralizing phenotype. Cells overexpressing ATF4 evidenced reduced alkaline phosphatase activity and mineral formation independent of oxygen tension in both two- and three-dimensional culture. Many osteocytes of human and rat cortical bone displayed a punctate LC3 staining, which suggests bone cells undergo autophagy in vivo. In addition, a basal autophagic flux that was upregulated by nutrient deprivation, thapsigargin treatment, and culture in low oxygen was noted in MLO cells. Interestingly, thapsigargin treatment resulted in HIF1α stabilization at normoxia. Using HIF1α siRNA MLO-A5 cells, it was determined that HIF1α regulated thapsigargin-induced autophagy. Finally, hypoxia was found to protect MLO-Y4 osteocytes from SNAP-induced apoptosis. This hypoxic protection was independent of HIF-1α, as similar viability was noted between control and HIF1α siRNA MLO-Y4 cells following SNAP treatment. Taken together, these results indicate that oxygen tension and HIF1α regulate the maturation and survival of differentiated bone cells. ^
Biology, Molecular|Biology, Cell
Zahm, Adam M, "Induction of the osteocytic phenotype: The influence of oxygen on bone cell maturation and survival" (2009). ETD Collection for Thomas Jefferson University. AAI3415759.