Physical and Functional Interactions Between Runx2 and HIF-1α Induce Vascular Endothelial Growth Factor Gene Expression

Angiogenesis and bone formation are intimately related processes. Hypoxia during early bone development stabilizes hypoxia-inducible factor-1 alpha (HIF-1 alpha) and increases angiogenic signals including vascular endothelial growth factor (VEGF). Furthermore, stabilization of HIF-1 alpha by genetic or chemical means stimulates bone formation. On the other hand, deficiency of Runx2, a key osteogenic transcription factor, prevents vascular invasion of bone and VEGF expression. This study explores the possibility that HIF-1 alpha and Runx2 interact to activate angiogenic signals. Runx2 over-expression in mesenchymal cells increased VEGF mRNA and protein under both normoxic and hypoxic conditions. In normoxia, Runx2 also dramatically increased HIF-1 alpha protein. In all cases, the Runx2 response was inhibited by siRNA-mediated suppression of HIF-1 alpha and completely blocked by the HIF-1 alpha inhibitor, echinomycin. Similarly, treatment of preosteoblast cells with Runx2 siRNA reduced VEGF mRNA in normoxia or hypoxia. However, Runx2 is not essential for the HIF-1 alpha response since VEGF is induced by hypoxia even in Runx2-null cells. Endogenous Runx2 and HIF-1 alpha were colocalized to the nuclei of MC3T3-E1 preosteoblast cells. Moreover, HIF-1 alpha and Runx2 physically interact using sites within the Runx2 RUNT domain. Chromatin immunoprecipitation also provided evidence for colocalization of Runx2 and HIF-1 alpha on the VEGF promoter. In addition, Runx2 stimulated HIF-1 alpha-dependent activation of an HRE-luciferase reporter gene without requiring a separate Runx2-binding enhancer. These studies indicate that Runx2 functions together with HIF-1 alpha to stimulate angiogenic gene expression in bone cells and may in part explain the known requirement for Runx2 in bone vascularization. J. Cell. Biochem. 112: 3582-3593, 2011. (C) 2011 Wiley Periodicals, Inc.