Hypoxic downregulation of cellular proliferation and loss of phenotype stability in human osteoblasts is mediated by HIF-1α

Both, skeletal development and fracture healing depend on an orchestrated sequence of cellular growth and differ- entiation processes. Regional changes in tissue oxygen tension were proposed as key regulators of osteoblast proliferation and phenotype. Hypoxia results in the stabilization of hypoxia-inducible factor-1 (HIF-1), thus influencing expression of a mul- titude of genes required for cellular adaptation. In the present study we dissected the effects of HIF-1 on cellular proliferation and gene expression of primary human osteoblasts. Primary human osteoblasts were studied by transfecting siRNA and plasmids coding for human HIF-1. Gene expression was analyzed by western blot and quantitative PCR. Functional assays were performed to study HIF-1 function, i.e. proliferation and cell cycle analysis. As previously reported exposure to hypoxia led to a stabilization of HIF-1 on protein level and resulted in reduced rates of proliferation and osteocalcin expression. Furthermore, the expression of the proproliferative gene survivin was significantly reduced (p < 0.01). Knock down of HIF-1 attenuated hypoxic downregulation of proliferation (p < 0.05), and osteocalcin (p < 0.05) as well as survivin (p < 0.05) expression significantly. Importantly, the isolated overexpression of HIF-1 impaired proliferative activity and led to significantly reduced rates of expression of osteocalcin (p < 0.05) and survivin (p < 0.01). The present study shows that HIF-1 might reduce proliferation and survivin expression in primary human osteoblasts inde- pendently from cellular hypoxia. Furthermore, HIF-1 promoted the loss of the characteristic osteoblastic marker, osteocalcin in vitro. These findings underline the important role of HIF-1 in bone physiology and pathophysiology. Modulating HIF-1 function in hypoxic environments could be of value for future therapeutic approaches.