Effect of Osx on <i>MMP13</i> promoter activity.

<p>(<b>A</b>) Osx activates the <i>MMP13</i> promoter in a dose-dependent manner. HEK293 cells were transfected with a 1 kb <i>MMP13</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. (B) Jab1 does not activate <i>MMP13</i> promoter activity. HEK293 cells were transfected with a 1 kb <i>MMP13</i> promoter-luciferase reporter gene without or with increasing amounts of a Jab1-expression plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values are presented as the mean ± S.D. (C) MMP13 promoter activity is inhibited in the presence of Osx siRNA. MC3T3 osteoblastic cells were transfected with a 1 kb <i>MMP13</i> promoter-luciferase reporter gene with siRNA Control or Osx siRNA as indicated. Luciferase activity was normalized by β-galactosidase activity. Values are presented as the mean ± S.D.</p

Endogenous Osx in primary osteoblasts is associated with the native <i>MMP13</i> promoter in vivo.

<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 Methods, primer Set 1 corresponds to a segment covering the GC-rich element within 80 bp <i>MMP13</i> promoter. As a negative control, Primer Set 2 covers a distal 3 kb region of the <i>MMP13</i> promoter, which does not contain GC-rich sequences.</p

Overexpression of Osx activates MMP13 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) MMP13 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 stable cell line. MMP13 mRNA levels were quantitated by real-time RT-PCR. The MMP13 RNA level obtained from the cells cultured with Dox was normalized to a value of 1. Values are presented as the mean ± S.D.</p

Osx ablation reduces MMP13 gene expression in vivo.

<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 MMP13 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. Values are presented as the mean ± S.D.</p

Identification of the Osx binding site in the promoter of <i>MMP13</i> gene.

<p>(A) Deletion analysis of the <i>MMP13</i> promoter-reporter constructs. MMP13-1 kb, MMP13-540 bp, MMP13-210 bp and MMP13-80 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) The GC-rich element in MMP13-80 is responsible for <i>MMP13</i> promoter reporter activation by Osx. The promoter mutant MMP13-80-M was transfected into HEK293 cells and analyzed as described in panel A. Luciferase activity was normalized by β-galactosidase activity.</p

Osx binds to MMP13 promoter oligos in Gel Shift Assay.

<p>DNA oligonucleotides of MMP13 were labeled by Biotin. Osx protein and biotin-labeled DNA probe were incubated. Protein-DNA complexes were separated on 4% polyacrylamide gels, and visualized by a Chemiluminescent Nucleic Acid detection Module. Two hundred-fold molar excess of unlabeled MMP13 promoter oligos (lane 3) were used. Baculovirus-expressed Osx was used as the protein resource.</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

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

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

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