Hypoxia induces BMP-2 expression via ILK, Akt, mTOR, and HIF-1 pathways in osteoblasts

It has been shown that hypoxia stimulation regulates bone formation, maintenance, and repair. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. However, the effects of hypoxia exposure on BMP-2 expression in cultured osteoblasts are largely unknown. Here we found that hypoxia stimulation increasedmRNAand protein levels of BMP-2 by qPCR, Western blot and ELISA assay in osteoblastic cells MG-63, hFOB and bone marrow stromal cells M2-10B4. Integrin-linked kinase (ILK) inhibitor (KP-392), Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-[(R)-2-O-methyl-3- O-octadecylcarbonate]) or mammalian target of rapamycin (mTOR) inhibitor (rapamycin) inhibited the potentiating action of hypoxia. Exposure to hypoxia increased the kinase activity of ILK and phosphorylation of Akt and mTOR. Furthermore, hypoxia also increased the stability and activity of HIF-1 protein. The binding of HIF-1α to the HRE elements after exposure to hypoxia was measured by EMSA assay. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that both ILK/Akt and mTOR signaling pathway were potentially required for hypoxia-induced HIF-1a activation and subsequent BMP-2 up-regulation. Taken together, our results provide evidence that hypoxia enhances BMP-2 expression in osteoblasts by an HIF-1a-dependent mechanism involving the activation of ILK/Akt and mTOR pathways.