HIF-1 alpha plays an essential role in BMP9-mediated osteoblast differentiation through the induction of a glycolytic enzyme, PDK1

Bone homeostasis is regulated by bone morphogenic proteins (BMPs), among which BMP9 is one of the most osteogenic. Here, we have found that BMP9 rapidly increases the protein expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha) in osteoblasts under normoxic conditions more efficiently than BMP2 or BMP4. A combination of BMP9 and hypoxia further increased HIF-1 alpha protein expression. HIF-1 alpha protein induction by BMP9 is not accompanied by messenger RNA (mRNA) increase and is inhibited by the activation of prolyl hydroxylase domain (PHD)-containing protein, indicating that BMP9 induces HIF-1 alpha protein expression by inhibiting PHD-mediated protein degradation. BMP9-induced HIF-1 alpha protein increase was abrogated by inhibitors of phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) kinase, indicating that it is mediated by PI3K-AKT signaling pathway. BMP9 increased mRNA expression of pyruvate dehydrogenase kinase 1 (PDK1), a glycolytic enzyme, and vascular endothelial growth factor-A (VEGF-A), an angiogenic factor, in osteoblasts. Notably, BMP9-induced mRNA expression of PDK1, but not that of VEGF-A, was significantly inhibited by small interference RNA-mediated knockdown of Hif-1 alpha. BMP9-induced matrix mineralization and osteogenic marker gene expressions were significantly inhibited by chemical inhibition and gene knockdown of either Hif-1 alpha or Pdk-1, respectively. Since increased glycolysis is an essential feature of differentiated osteoblasts, our findings indicate that HIF-1 alpha expression is important in BMP9-mediated osteoblast differentiation through the induction of PDK1.