Matrix metalloproteinase 13 (MMP13) is a direct target of osteoblast-specific transcription factor osterix (Osx) in osteoblasts

Abstract

Osterix (Osx) is an osteoblast-specific transcription factor required for bone formation and osteoblast differentiation from mesenchymal stem cells. In Osx-null mice, no bone formation occurs. Matrix metalloproteinase 13 (MMP13) is a member of the matrix metalloproteinase family and plays an important role in endochondral ossification and bone remodeling. Transcriptional regulation of MMP13 expression in osteoblasts is not well understood. Here, we provide several lines of evidence which show that MMP13 is a direct target of Osx in osteoblasts. Calvaria obtained from Osx-null embryos displayed dramatic reductions in MMP13 expression compared to wild-type calvaria. Stable overexpression of Osx stimulated MMP13 expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of MMP13 expression. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the MMP13 promoter in a dose-dependent manner. To define the region of the MMP13 promoter that was responsive to Osx, a series of MMP13 promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 80 bp of the MMP13 promoter. Additional point mutant analysis was used to identify one GC-rich region that was responsible for MMP13 promoter activation by Osx. Gel Shift Assay showed that Osx bound to MMP13 promoter sequence directly. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native MMP13 promoter in primary osteoblasts in vivo. Taken together, these data strongly support a direct regulatory role for Osx in MMP13 gene expression in osteoblasts. They further provide new insight into potential mechanisms and pathways that Osx controls bone formation.

Figure 1. Osx ablation reduces MMP13 gene expression in vivo.

Calvaria RNAs were isolated from E18.5 Osx wild-type and Osx-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 Osx-null calvaria was normalized to a value of 1. Values are presented as the mean ± S.D.

Figure 2. Overexpression of Osx activates MMP13 gene expression in C2C12 mesenchymal cells.

(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.

Figure 3. siRNA-directed knockdown of Osx impairs MMP13 gene expression in MC3T3 osteoblasts.

RNA expression levels were determined by quantitative real-time RT-PCR. MC3T3 osteoblasts were transfected with siRNA targeting mouse Osx. RNA was isolated 24 hr 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. si-C: Lamin A/C Control siRNA as a non-specific control; si-Hif1a: siRNA against Hif1a as another control; si-Osx: si-RNA against Osx.

Figure 4. Effect of Osx on MMP13 promoter activity.

(A) Osx activates the MMP13 promoter in a dose-dependent manner. HEK293 cells were transfected with a 1 kb MMP13 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 MMP13 promoter activity. HEK293 cells were transfected with a 1 kb MMP13 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 MMP13 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.

Figure 5. Identification of the Osx binding site in the promoter of MMP13 gene.

(A) Deletion analysis of the MMP13 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 MMP13 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.

Figure 6. Endogenous Osx in primary osteoblasts is associated with the native MMP13 promoter in vivo.

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 MMP13 promoter. As a negative control, Primer Set 2 covers a distal 3 kb region of the MMP13 promoter, which does not contain GC-rich sequences.

Figure 7. Osx binds to MMP13 promoter oligos in Gel Shift Assay.

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.