HIF-1α Inhibits Wnt Signaling Pathway by Activating Sost Expression in Osteoblasts

The nature of the cellular and molecular mechanisms for the transition of avascular cartilage replacement with bone during endochondral ossification remains poorly understood. One of the driving forces is hypoxia. As a master regulator of hypoxia, hypoxia-inducible factor-1 alpha (HIF-1 alpha) has been reported to couple angiogenesis to osteogenesis. Our recent study has demonstrated that osteoblast growth is inhibited under hypoxia and that HIF-1 alpha cooperates with Osterix (Osx) to inhibit Wnt pathway. However, molecular mechanisms for inhibitory effects of HIF-1 alpha on Wnt pathway are not well understood. In this study, our quantitative RT-PCR results revealed that the expression of a Wnt antagonist Sclerostin (Sost) was upregulated in osteoblasts during hypoxia while HIF-1 alpha was upregulated. Treatment of desferrioxamine (DFO), a HIF-1 alpha activator, led to further increase of Sost expression, suggesting that HIF-1 alpha may activate Sost expression. The regulation of Sost gene expression by HIF-1 alpha was then investigated. We performed loss-of-function experiments to examine Sost expression by using siRNA approach against HIF-1 alpha, and found that the inhibition of HIF-1 alpha by siRNA in osteoblasts led to the decrease of Sost expression. To address transcriptional regulation of Sost gene by HIF-1 alpha, transient transfection assay was performed and showed that HIF-1 alpha activated Sost-1 kb promoter reporter activity in a dose-dependent manner. To narrow down the minimal region of Sost promoter activated by HIF-1 alpha, we generated a series of deletion mutants of Sost constructs. It was demonstrated that Sost-260 was the minimal region of Sost promoter for HIF-1 alpha activation and that Sost-106 construct, which lack hypoxia response element, abolished HIF-1 alpha-mediated Sost reporter activation. Gel shift assay showed that HIF-1 bound to the promoter sequence of Sost directly. These findings support our hypothesis that HIF-1 alpha activates Sost expression. This study provides a novel molecular mechanism through which HIF-1 alpha inhibits Wnt signaling in osteoblasts.