Endogenous Osx in primary osteoblasts is associated with the native <i>VDR</i> promoter <i>in vivo</i>.

<p>Chromatin Immunoprecipitation (ChIP) assays were conducted using primary calvarial osteoblasts isolated from new born wild-type mice. Anti-Osx antibody (a-Osx) was used for ChIP analysis, and IgG was used as a negative control. The precipitated chromatin was analyzed by quantitative real-time PCR. As described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026504#s2" target="_blank">Methods</a>, primer Set 1 corresponds to a segment covering two GC-rich elements within 120 bp the <i>VDR</i> promoter. As a negative control, Primer Set 2 covers a distal 3 kb region of the <i>VDR</i> promoter, which does not contain GC-rich sequences. A paired <i>t</i>-test was performed comparing IgG and a-Osx group.</p

Both Osx and VDR are upregulated during osteoblast differentiation.

<p>MC3T3 osteoblast differentiation experiments were performed in which osteogenic factors were added into the medium, including BMP2, ascorbic acid and β–glycerophosphate. MC3T3 osteoblastic cells were harvested after 0 hr, 8 hr, 24 hr, 72 hr and 104 hr after incubating with differentiation medium. RNA was isolated from cell lysates. RNA levels for VDR and Osx were analyzed by real-time RT-PCR. The level of RNA from 0 hr was normalized to a value of 1. Values were presented as the mean ±S.D.</p

SiRNA-directed knockdown of Osx impairs VDR gene expression in MC3T3 osteoblasts.

<p>(A) RNA expression levels as determined by quantitative real-time RT-PCR. MC3T3 osteoblasts were transfected with siRNA targeting mouse Osx. RNA was isolated 24 h post-transfection and quantitated by real-time RT-PCR. The RNA level from the control siRNA group was normalized to a value of 1. Values were presented as the mean ±S.D. A paired <i>t</i>-test was performed comparing si-control group and si-Osx group. (B) Western analysis of the Osx knockdown. Protein was isolated by acetone precipitation of whole cell lysates and then analyzed by western blot using rabbit anti-VDR or anti-Osx polyclonal antibodies. Beta-actin was used as a loading control.</p

Overexpression of Osx activates VDR gene expression in C2C12 mesenchymal cells.

<p>(A) Western immunoblot analysis of the Dox-regulated Osx-expressing C2C12 cells. Osx expression is turned on in the absence of Dox. Beta-actin was used as a loading control. (B) VDR mRNA levels in a stable, Tet-off C2C12 mesenchymal cell line. RNA was obtained from cultures treated with or without Doxycycline. Osx expression is induced in the absence of Doxycycline in this line. VDR mRNA levels were quantitated by real-time RT-PCR. The VDR RNA level obtained from the cells cultured with Dox was normalized to a value of 1. Values are presented as the mean ±S.D. (C) RNA expression level for the osteoblastic marker gene alkaline phosphatase (ALP). (D) RNA expression level for osteoblastic gene osteocalcin (OC). Conditions are identical to those described in panel B. A paired <i>t</i>-test was performed comparing Dox (−) and Dox (+) groups. *: A star indicates statistical significance compared to Dox (+) group.</p

Osx activates the <i>VDR</i> promoter in a dose-dependent manner.

<p>HEK293 cells were transfected with a 1 kb <i>VDR</i> promoter-luciferase reporter gene without or with increasing amounts of an Osx-expression plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values are presented as the mean ±S.D.</p

Identification of the Osx binding site in the promoter of <i>VDR</i> gene.

<p>(A) Deletion analysis of the <i>VDR</i> promoter-reporter construct. VDR-1 kb, VDR-500 bp, VDR-250 bp and VDR-120 bp promoter-reporter plasmids (300 ng each) were cotransfected with 400 ng of the Osx expression plasmid in HEK293 cells. Twenty-four hours post-transfection, cell extracts were prepared and analyzed for luciferase activity and normalized to β-galactosidase activity. (B) Two GC-rich elements in VDR-120 are responsible for <i>VDR</i> promoter reporter activation by Osx. The promoter mutants VDR-M1, VDR-M2 and VDR-M12 were transfected into HEK293 cells and analyzed as described in panel A. Luciferase activity was normalized by β-galactosidase activity. (C) A diagram of the proximal 120 bp region of the mouse VDR promoter. A 5′ primer and 3′ primer were used to subclone the VDR-120 bp promoter reporter plasmid. M1 refers to point mutations of VDR-M1, and M2 refers to point mutations of VDR-M2. VDR-M12 in (B) contains both M1 and M2.</p

VDR ablation has no effect on Osx gene expression <i>in vivo</i>.

<p>Calvarial RNAs were isolated from E18.5 wild-type or <i>VDR</i>-null embryos. RNA levels for VDR and Osx were analyzed by real-time RT-PCR. The level of each RNA from wild type calvaria was normalized to a value of 1 shown in the mean±S.D. A paired <i>t</i>-test was performed comparing wild-type and VDR-null group.</p

Osx ablation reduces VDR gene expression <i>in vivo</i>.

<p>Calvaria RNAs were isolated from E18.5 <i>Osx</i> wild-type and <i>Osx</i>-null embryos. RNA expression levels for Osx, osteocalcin (OC), Runx2 and VDR were analyzed by real-time RT-PCR. The level of each RNA from <i>Osx</i>-null calvaria was normalized to a value of 1. *: A star indicates statistical significance compared to Osx wild type group.</p

Osx-regulated expression of VDR is important in Osx-induced osteoblast differentiation.

<p>(A) SiRNA directed against VDR knocks down VDR expression. C2C12 mesenchymal cells stably expressing Osx were transfected with VDR siRNA at different concentrations. RNA was isolated and specific RNA levels were measured by real-time RT-PCR. (B,C) Cells were transfected with 300 nM of siRNA targeting VDR, and Osx expression was induced by removing Dox. Osteoblast marker gene expression was examined including (B) ALP and (C)osteocalcin (OC). The RNA level from control siRNA group (Si-C) was normalized to a value of 1. Values were presented as the mean ±S.D. *: A star indicates statistical significance compared to Dox (+) group (p<0.05). **: Two stars indicate statistical significance compared to si-control (Si-C) (p<0.05). (D) Western analysis of the VDR knockdown. C2C12 stable cells without Dox were transfected with 300 nM of siRNA targeting VDR. Protein was isolated from whole cell lysates of control siRNA group (si-C) and VDR siRNA group (si-VDR) and then analyzed by western blot using rabbit anti-VDR, anti-ALP or anti-OC polyclonal antibodies. Beta-actin was used as a loading control.</p