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. 2011;6(9):e24638.
doi: 10.1371/journal.pone.0024638. Epub 2011 Sep 13.

NELL-1, an Osteoinductive Factor, Is a Direct Transcriptional Target of Osterix

Free PMC article

NELL-1, an Osteoinductive Factor, Is a Direct Transcriptional Target of Osterix

Feng Chen et al. PLoS One. .
Free PMC article


NELL-1 is a novel secreted protein associated with premature fusion of cranial sutures in craniosynostosis that has been found to promote osteoblast cell differentiation and mineralization. Our previous study showed that Runx2, the key transcription factor in osteoblast differentiation, transactivates the NELL-1 promoter. In this study, we evaluated the regulatory involvement and mechanisms of Osterix, an essential transcription factor of osteoblasts, in NELL-1 gene expression and function. Promoter analysis showed a cluster of potential Sp1 sites (Sp1/Osterix binding sites) within approximately 70 bp (from -71 to -142) of the 5' flanking region of the human NELL-1 transcriptional start site. Luciferase activity in our NELL-1 promoter reporter systems was significantly decreased in Saos-2 cells when Osterix was overexpressed. Mutagenesis study demonstrated that this suppression is mediated by the Sp1 sites. The binding specificity of Osterix to these Sp1 sites was confirmed in Saos-2 cells and primary human osteoblasts by EMSA in vitro and ChIP assay in vivo. ChIP assay also showed that Osterix downregulated NELL-1 by affecting binding of RNA polymerase II to the NELL-1 promoter, but not by competing with Runx2 binding to the OSE2 sites. Moreover, NELL-1 mRNA levels were significantly decreased when Osterix was overexpressed in Saos-2, U2OS, Hela and Glioma cells. Correspondingly, knockdown of Osterix increased NELL-1 transcription and osteoblastic differentiation in both Saos-2 cells and primary human osteoblasts. These results suggest that Osterix is a direct transcriptional regulator with repressive effect on NELL-1 gene expression, contributing to a delicate balance of regulatory effects on NELL-1 transcription with Runx2, and may play a crucial role in osteoblast differentiation and mineralization. These findings also extend our understanding of the molecular mechanism of Runx2, Osterix, and NELL-1 and demonstrate their crosstalk during osteogenesis.

Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflicts: Bone Biologics, Inc. licensed Nell-1 related patents from UCLA. C.S., K.T. and X. Zhang are founders of Bone Biologics, Inc. and inventors of the related patents. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials. Patent numbers and Dates: 7.052.856 on 30-May-2006, 7.544.486 on 09-Jun-2009, 7.687.462 on 30-Mar-2010.


Figure 1
Figure 1. Schematic of the human and mouse Nell-1 promoters (not drawn to scale).
In silico analysis identified a cluster of potential Sp1/Osterix binding sites within approximately 70 bp of the 5′ flanking region of the human NELL-1 gene. Within the cluster, Site A contains three overlapping potential Sp1 sites from −71 to −96 bp and Site B contains a single Sp1 site from −133 to −142 bp. Four OSE2 sites to which Runx2 binds in human NELL-1 promoter are also shown (labeled A, B, C and H1). A similar pattern is seen in mouse Nell-1 promoter. Multiple putative Sp1 sites (labeled Site a∼e) are located before three OSE2 sites (labeled 1, m1 and 2). The relative location of primers for CHIP assay are indicated on the human 2.2 kb promoter.
Figure 2
Figure 2. Dose-dependent NELL-1 expression response to Osterix.
(A) Human NELL-1 promoter is responsive to Osterix in a dose-dependent manner shown by reporter assay. Graph detailing the luciferase activity in Saos-2 cells co-transfected with either the p2213WT-Luc or the p325WT-Luc promoter-luciferase constructs as well as 0, 0.25, 0.5, or 1 ug of Osterix expression vector (pOsx). Data are expressed as a percentage of the luciferase activity of the p325WT-Luc construct in the absence of control vector (pCtr). (B) Osterix overexpression decreased NELL-1 mRNA level in Saos-2 cells at 2 days and 7 days post-transfection. Western blot showed Osterix protein levels at 2 days and 7 days post-transfection. (*p<0.05) (C) Osterix transcriptional repression of NELL-1 promoter in osteoblastic and non-osteoblastic cells. A graph detailing the luciferase activity of p325WT-Luc 2 days post-transfection in Saos-2, U2OS, Hela and Glioma cell lines co-transfected with pCtr or pOsx. (*p<0.05)
Figure 3
Figure 3. Both Site A and Site B are functional for Osterix-mediated NELL-1 promoter activity suppression.
(A) Schematic of p325WT-Luc, p325mut all-Luc, p325mutSiteA, and p325mutSiteB constructs. Blue represents mutated Sp1 sites. (B) Graph depicting promoter activity on Saos-2 cells cotransfected with pOsx as well as p325WT-Luc or mutated NELL-1 promoter-luciferase constructs (p325mut all-Luc, p325mutSiteA-Luc, and p325mutSiteB-Luc, respectively). Data are reported as percent activity of control cells transfected with pCtr. (*p<0.05)
Figure 4
Figure 4. Osterix specifically binds to Sp1 sites in the NELL-1 promoter in vitro and in vivo.
(A) EMSA of Saos-2 nuclear proteins transfected with pCtr or pOsx binding to SiteA (containing three proximal Sp1 sites) and SiteB probes. Supershifts with specific Osterix antibody indicate the specific Osterix-DNA complexes. (B) EMSA depicting primary human osteoblast cell (hOB) nuclear proteins transfected with pOsx binding to the SiteA probes, with competition by 20x and 200x unlabeled SiteA and 200x unlabeled mutated SiteA (MutA) oligonucleotides. Note that the MutA probes failed to bind nuclear proteins. The same pattern is also seen by competition of SiteB element and MutB. (C–D) Osterix binds to endogenous Sp1 sites of the human NELL-1 promoter in Saos-2 (C) and hOB (D) cells. The NELL-1 -1 kb primer set covers 1 kb proximal promoter region containing all Sp1/Osterix binding sites and three OSE2 sites. The NELL-1 -2 kb primer set covers NELL-1 promoter region from −1 kb to −2 kb where no Sp1/Osterix binding site but one OSE2 site exists. The qPCR products depict DNA amplified from Chromatin Immunoprecipitation with cells utilizing Control IgG and Osterix antibody. Input DNA represents positive genomic DNA control.
Figure 5
Figure 5. Osterix down regulates Runx2-induced NELL-1 promoter activity.
(A) Graph depicting promoter activity in Saos-2 cells after co-transfection with control empty pcDNA3.1 vector (pCtr), pcDNA-Runx2 (pRunx2) or pRunx2 plus pOsx expression vectors as well as p2213-Luc, p325-Luc or p325mut-Luc NELL-1 promoter-luciferase constructs. Data are reported as fold changes in comparison to control cells transfected with pCtr and p325WT-Luc constructs. (p<0.05) (B) Osterix affects binding of RNA polymerase II to NELL-1's promoter, but does not compete with Runx2 binding of OSE2 sites. The NELL-1 -1 kb primer set used covers 1 kb proximal promoter region where all Sp1/Osterix binding sites and three OSE2 sites are located. The qPCR products depict DNA amplified from Chromatin Immunoprecipitation with Saos-2 cells transfected with pCtr or pOsx utilizing Control IgG, Osterix antibody, Runx2 antibody or RNA polymerase II antibody. Input DNA represents positive genomic DNA control. (*p<0.05)
Figure 6
Figure 6. Osterix affects NELL-1 and some bone marker genes expression in Saos-2 cells (A) and primary human osteoblast (hOB) (B).
Real time PCR analysis of Nell-1, OCN and OPN transcripts in Saos-2 cells transiently transfected with Control (pCtr) or Osterix expression vector (pOsx) at 2 days and 7 days (top panel). The effects with transcient transfection of Negative control siRNA (NC-siRNA) or Osterix siRNA mixture (Osx-siRNA) were revealed by Real time PCR analysis at 2 days and 7 days (bottom panel). (C) Diagram of the regulatory relationship between Runx2, Osterix and NELL-1. Runx2 positively regulates Osterix and NELL-1. Osterix negatively controlled NELL-1 expression in this study. NELL-1 was also shown to positively affect Runx2 through phosphorylation and negatively feedback on Osterix during osteogenesis.

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