Characterization of Dkk1 gene regulation by the osteoblast-specific transcription factor Osx

Bone formation is a developmental process involving the differentiation of mesenchymal stem cells to osteoblasts. Osterix (Osx) is an osteoblast-specific transcription factor required for bone formation and osteoblast differentiation. Previous observation that Osx inhibits Wnt signaling pathway provides a novel concept of feedback control mechanisms involved in bone formation. Wnt antagonist Dickkopf-1 (Dkk1) plays an important role on skeletal development and bone remodeling. Osx has been shown to activate the Dkk1 promoter; however, the detailed mechanism of Osx regulation on Dkk1 expression is not fully understood. In this study, quantitative real-time RT-PCR results demonstrated that Dkk1 expression was downregulated in Osx-null calvaria at two different points of E15.5 and E18.5 in mice embryos. Overexpression of Osx resulted in upregulation of Dkk1 expression in Tet-off stable C2C12 cell line. Inhibition of Osx expression by siRNA led to downregulation of Dkk1 in osteoblasts. These data suggest that Osx may target Dkk1 directly. To define minimal region of Dkk1 promoter activated by Osx, we made a series of deletion mutants of Dkk1 promoter constructs, and narrowed down the minimal region to the proximal 250 bp by transient transfection assay. It was shown that two GC-rich binding sites within this minimal region of Dkk1 promoter were required for the Dkk1 promoter activation by Osx. Importantly, quantitative chromatin immunoprecipitation (ChIP) assays were performed to show that endogenous Osx associated with native Dkk1 promoter in primary osteoblasts. Taken together, these findings support our hypothesis that Dkk1 is a direct target of Osx.

► Dkk1 expression is down-regulated in the absence of Osx.
► Overexpression of Osx activates Dkk1 expression.
► Inhibition of Osx by siRNA results in repression of Dkk1 expression.
► GC-rich sequences of Dkk1 promoter are responsible for Osx activation.
► Osx associates with native Dkk1 promoter in vivo.