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-1α (HIF-1α) is a master regulator of hypoxia. HIF-1α has been reported to couple angiogenesis to osteogenesis. Our recent study has demonstrated that Osx and HIF-1α cooperatively regulate VEGF expression in osteoblasts. Effects of hypoxia/HIF-1α 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-1α to inhibit osteoblast proliferation, we tested the effect of hypoxia/HIF-1α 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-1α was upregulated. Treatment of desferrioxamine, a HIF-1α activator, led to further downregulation of expressions of cyclin D1 and c-Myc in osteoblasts. On the contrary, the inhibition of HIF-1α by siRNA in osteoblasts led to the expression increase of cyclin D1 and c-Myc. These data suggest that HIF-1α inhibits Wnt pathway in osteoblasts. To examine the effect of HIF-1α on Wnt pathway, HIF-1α was cotransfected with β-catenin along with Topflash reporter in transient transfection assay. Our results showed that HIF-1α inhibited β-catenin-induced Topflash reporter activity. Interestingly, a synergistic interplay was observed between Osx and HIF-1α in the inhibition of β-catenin-induced Topflash expression. Our findings indicate that Osx and HIF-1α cooperatively inhibit Wnt pathway. This study revealed additional new information of the cooperation between HIF-1α and Osx in osteoblasts.