VEGF expression in an osteoblast-like cell line is regulated by a hypoxia response mechanism

Angiogenesis is essential for the increased delivery of oxygen and nutrient s required for the reparative processes of bone healing. Vascular endotheli al growth factor (VEGF), a potent angiogenic growth factor, has been implic ated in this process. We have previously shown that hypoxia specifically an d potently regulates the expression of VEGF by osteoblasts. However, the mo lecular mechanisms governing this interaction remain unknown. In this study , we hypothesized that the hypoxic regulation of VEGF expression by osteobl asts occurs via an oxygen-sensing mechanism similar to the regulation of th e erythropoietin gene (EPO). To test this hypothesis, we examined the kinet ics of oxygen concentration on osteoblast VEGF expression. In addition, we analyzed the effects of nickel and cobalt on the expression of VEGF in oste oblastic cells because these metallic ions mimic hypoxia by binding to the heme portion of oxygen-sensing molecules. Our results indicated that hypoxi a potently stimulates VEGF mRNA expression. In addition, we found that nick el and cobalt both stimulate VEGF gene expression in a similar time- and do se-dependent manner, suggesting the presence of a hemelike oxygen-sensing m echanism similar to that of the EPO gene. Moreover, actinomycin D, cyclohex imide, dexamethasone, and mRNA stabilization studies collectively establish ed that this regulation is predominantly transcriptional, does not require de novo protein synthesis, and is not likely mediated by the transcriptiona l activator AP-1. These studies demonstrate that hypoxia, nickel, and cobal t regulate VEGF expression in osteoblasts via a similar mechanism, implicat ing the involvement of a heme-containing oxygen-sensing molecule. This may represent an important mechanism of VEGF regulation leading to increased an giogenesis in the hypoxic microenvironment of healing bone.