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

Feng Chen,Xuan Zou,Chia Soo,Shan Sun,Xinli Zhang,Robert Chiu,Janette N Zara,Kang Ting,Cymbelin T Culiat,Jean-Marc Vanacker

doi:10.1371/journal.pone.0024638

Abstract

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.

Highlights

Through promoter analyses, we recently established NELL-1, a Nel-like molecule-1 [1,2], as a novel direct transcriptional target of runt homology domain transcription factor-2 (Runx2) [3]
In silico analysis of the human NELL-1 promoter identified a cluster of potential Sp1 sites (Sp1/Osterix binding sites) within approximately 70 bp of the 59 flanking region of the human NELL1 gene
To better illustrate the spatial distribution of Sp1 sites in both human and mouse Nell-1 promoters, the locations of previously identified Runx2 binding sites osteoblast specific binding elements 2 (OSE2) are displayed in Fig. 1, with three functional sites labeled A, B, C and a cryptic site H1 in the human NELL-1 promoter, and two sites labeled site 1 and 2 and a cryptic site m1 in the mouse promoter region

Summary

Introduction

We recently established NELL-1, a Nel-like molecule-1 [1,2], as a novel direct transcriptional target of runt homology domain transcription factor-2 (Runx2) [3]. The overexpression of NELL-1 was originally found in pathologically fusing and fused sutures in nonsyndromic unilateral coronal synostosis (UCS) patients [4], and CMV-Nell-1 overexpression mice exhibited CS-like phenotypes that ranged from simple to compound synostoses [5] These findings highly suggest that NELL-1 is a CS-associated factor with preferential osteogenic effects on cells of the osteochondral lineage. N-ethyl-N-nitrosourea (ENU)-induced Nell-1 deficient mice revealed major abnormalities in the skeletal system such as decreased calvarial bone mineralization and decreased vertebral disc volume, and perinatal death due to respiratory failure secondary to a deformed cartilaginous ribcage [6] This Nell-1 deficient mouse model in addition to the overexpression transgenic mouse model further supports the critical role of Nell-1 in the Runx regulatory network of osteogenesis, the precise mechanism of action of Nell-1 remains unknown [7,8]

Methods

Results

Conclusion