Sclerostin is a direct target of osteoblast-specific transcription factor osterix

Abstract

Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation and bone formation. Osx knock-out mice lack bone completely. Recent findings that Osx inhibits Wnt signaling provide a feedback control mechanism involved in bone formation. Mechanisms of Osx inhibition on Wnt signaling are not fully understood. Our results in this study revealed that the expression of a Wnt antagonist Sclerostin (Sost) was downregulated in Osx-null calvaria. Overexpression of Osx in stable C2C12 mesenchymal cell line resulted in Sost upregulation. Transient transfection assay showed that Osx activated 1kb Sost promoter reporter activity in a dose-dependent manner. To define Sost promoter activated by Osx, we made a series of deletion mutants of Sost constructs, and narrowed down the minimal region to the proximal 260bp. Gel shift assay indicated that Osx bound to GC-rich site within this minimal region, and that point mutations of this binding site disrupted Osx binding. Moreover, the same point mutations in 260bp Sost promoter reporter disrupted the promoter activation by Osx, suggesting that the GC-rich binding site was responsible for Sost promoter activation by Osx. To further examine physical association of Osx with Sost promoter in vivo, Chromatin immunoprecipitation (ChIP) assays were performed using primary osteoblasts from mouse calvaria. Osx was found to associate with endogenous Sost promoter. Taken together, these findings support our hypothesis that Sost is a direct target of Osx. This provides a new additional mechanism through which Osx inhibits Wnt signaling during bone formation.

Figure 1

Effect of Osx on Sost expression. (A) Fold change in RNA levels from E18.5 wild-type and Osx-null mice embryos. Calvaria RNAs were isolated from E18.5 Osx wild-type and Osx-null embryos. RNA levels for Osx, Osteocalcin, Runx2, and Sost were analyzed by real-time RT-PCR. The level of each RNA from Osx-null calvaria was normalized to a value of 1. (B) Fold change of RNA levels for Sost gene expression in C2C12 stable cells using Tet-off system. RNA was isolated from C2C12 stable cells. In C2C12 stable cell line, Osx expression was induced in the absence of tetracycline. RNA levels were measured by real-time RT-PCR. The RNA level from Tet-present cells was normalized to a value of 1. Values were presented as the mean ± SD.

Figure 2

Osx activates Sost promoter activity in a dose-dependent manner. HEK293 cells were transfected with 1 kb Sost promoter luciferase reporter without or with increasing amounts of Osx as indicated. Luciferase activity was normalized by β-galactosidase activity. Values were presented as the mean ± SD.

Figure 3

Osx binding site is located within 260 bp of Sost promoter. (A) Deletion analysis of Sost promoter reporter activated by Osx. Sost-1 kb, Sost-540 bp, Sost-260 bp, and Sost-106 bp Sost promoter reporter plasmids were constructed. Each plasmid was cotransfected with Osx expression plasmid in HEK293 cells. Luciferase activity was normalized by β-galactosidase activity. (B) The GC-rich element in Sost-260-M1 is responsible for Sost promoter reporter activation by Osx. Sost-260-M1 and Sost-260-M2 point mutants were constructed. Each plasmid was cotransfected with Osx expression plasmid in HEK293 cells. Luciferase activity was normalized by β-galactosidase activity.

Figure 4

Osx associates with Sost promoter. (A) Osx directly binds to Sost GC-rich sequence in EMSA. In the gel shift assays, P³²-labeled oligo corresponding to Sost GC-rich sequence identified within 260 bp of Sost promoter region was incubated with Osx. Baculovirus-expressed Osx was used as the protein resource. Protein–DNA complexes were resolved on a non-denaturing polyacrylamide gel and visualized by autoradiography. (B) Osx associates with native Sost promoter in ChIP assay. Calvarial cells were isolated and cultured from wild-type new born mice. Anti-Osx antibody was used for ChIP analysis, and IgG was used as a negative control. The precipitated chromatin was analyzed by quantitative real-time PCR. Primer set 1 corresponds to a segment of the Sost promoter containing GC-rich element between 106 bp and 260 bp. As a negative control, Primer set 2 covers a distal region of the Sost promoter, which does not contain GC-rich sequence.