Synergistic Inhibition of Wnt Pathway by HIF-1α and Osteoblast-Specific Transcription Factor Osterix (Osx) in Osteoblasts

Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation. Inhibition of Wnt pathway by Osx highlights the potential for feedback control mechanisms involved in bone formation. Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a master regulator of hypoxia. HIF-1 alpha has been reported to couple angiogenesis to osteogenesis. Our recent study has demonstrated that Osx and HIF-1 alpha cooperatively regulate VEGF expression in osteoblasts. Effects of hypoxia/HIF-1 alpha on osteoblast proliferation and related mechanisms are not well understood. In this study, osteoblast growth under hypoxia was examined. We observed that osteoblast growth was inhibited under hypoxia. To explore possible mechanisms for hypoxia/HIF-1 alpha to inhibit osteoblast proliferation, we tested the effect of hypoxia/HIF-1 alpha on Wnt pathway. Quantitative RT-PCR results revealed that Wnt target genes such as cyclin D1 and c-Myc were downregulated under hypoxia while HIF-1 alpha was upregulated. Treatment of desferrioxamine, a HIF-1 alpha activator, led to further downregulation of expressions of cyclin D1 and c-Myc in osteoblasts. On the contrary, the inhibition of HIF-1 alpha by siRNA in osteoblasts led to the expression increase of cyclin D1 and c-Myc. These data suggest that HIF-1 alpha inhibits Wnt pathway in osteoblasts. To examine the effect of HIF-1 alpha on Wnt pathway, HIF-1 alpha was cotransfected with beta-catenin along with Topflash reporter in transient transfection assay. Our results showed that HIF-1 alpha inhibited beta-catenin-induced Topflash reporter activity. Interestingly, a synergistic interplay was observed between Osx and HIF-1 alpha in the inhibition of beta-catenin-induced Topflash expression. Our findings indicate that Osx and HIF-1 alpha cooperatively inhibit Wnt pathway. This study revealed additional new information of the cooperation between HIF-1 alpha and Osx in osteoblasts.