title: HIF-1α inhibits Wnt signaling pathway by activating Sost expression in osteoblasts.
abstract: 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-1α (HIF-1α) has been reported to couple angiogenesis to osteogenesis. Our recent study has demonstrated that osteoblast growth is inhibited under hypoxia and that HIF-1α cooperates with Osterix (Osx) to inhibit Wnt pathway. However, molecular mechanisms for inhibitory effects of HIF-1α 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-1α was upregulated. Treatment of desferrioxamine (DFO), a HIF-1α activator, led to further increase of Sost expression, suggesting that HIF-1α may activate Sost expression. The regulation of Sost gene expression by HIF-1α was then investigated. We performed loss-of-function experiments to examine Sost expression by using siRNA approach against HIF-1α, and found that the inhibition of HIF-1α by siRNA in osteoblasts led to the decrease of Sost expression. To address transcriptional regulation of Sost gene by HIF-1α, transient transfection assay was performed and showed that HIF-1α activated Sost-1 kb promoter reporter activity in a dose-dependent manner. To narrow down the minimal region of Sost promoter activated by HIF-1α, 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-1α activation and that Sost-106 construct, which lack hypoxia response element, abolished HIF-1α-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-1α activates Sost expression. This study provides a novel molecular mechanism through which HIF-1α inhibits Wnt signaling in osteoblasts.
authors.affiliation: Laboratory of Bone Tissue Engineering, Beijing Research Institute of Traumatology and Orthopaedics, Beijing JiShuiTan Hospital, Beijing, China.
authors.forename: Dafu
authors.initials: D
authors.lastname: Chen
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.forename: Zhiyu
authors.initials: Z
authors.lastname: Zhou
authors.forename: Chengai
authors.initials: C
authors.lastname: Wu
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Xing
authors.forename: Xuenong
authors.initials: X
authors.lastname: Zou
authors.forename: Wei
authors.initials: W
authors.lastname: Tian
authors.forename: Chi
authors.initials: C
authors.lastname: Zhang
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015153:Blotting, Western; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism*; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D060449:Wnt Signaling Pathway / Q000235:genetics / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D006023:Glycoproteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D036341:Intercellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; C486030:Sost protein, mouse