We studied the transcriptional regulation of NELL-1, a craniosynostosis-related 
gene. We identitifed three OSE2 elements in the NELL-1 promoter that are 
directly bound and transactivated by Runx2. Forced expression of Runx2 induces 
NELL-1 expression in rat calvarial cells.
INTRODUCTION: We previously reported the upregulation of NELL-1 in human 
craniosynostosis and the overexpression of Nell-1 in transgenic animals that 
induced premature suture closure associated with increased osteoblast 
differentiation. To study the transcriptional regulation of NELL-1, we analyzed 
the 5' flanking region of the human NELL-1 gene. We identified three osteoblast 
specific binding elements 2 (OSE2) sites (A, B, and C) within 2.2 kb upstream of 
the transcription start site and further studied the functionality of these 
sites.
MATERIALS AND METHODS: An area of 2.2 kb and a truncated 325 bp, which lacked 
the three OSE sites, were cloned into a luciferase reporter gene, and 
co-transfected with Runx2 expression plasmid. The three OSE2 sites were 
individually mutated and co-transfected with Runx2 expression plasmid into Saos2 
cells. Gel shifts and supershifts with Runx2 antibodies were used to determine 
specific binding to OSE2 sites. CHIP assays were used to study in vivo binding 
of Runx2 to the Nell-1 promoter. Runx2 expression plasmid was transfected into 
wildtype and Runx2(-/-) calvarial cells. Nell-1, osteocalcin, and Runx2 
expression levels were measured using RT-PCR.
RESULTS: Addition of Runx2 dose-dependently increased the luciferase activity in 
the human NELL-1 promoter-luciferase p2213. The p325 truncated NELL-1 construct 
showed significantly lower basal level of activity. Nuclear extract from Saos2 
cells formed complexes with site A, B, and C probes and were supershifted with 
Runx2 antibody. Mutation of sites A, B, and C significantly decreased basal 
promoter activity. Furthermore, mutation of sites B and C had a blunted response 
to Runx2, whereas mutation of site A had a lesser effect. Runx2 bound to NELL-1 
promoter in vivo. Transfection of Runx2 in rat osteoblasts upregulated Nell-1 
and Ocn expression, and in Runx2 null calvarial cells, both Nell-1 and Ocn 
expression were rescued.
CONCLUSIONS: Runx2 directly binds to the OSE2 elements and transactivates the 
human NELL-1 promoter. These results suggest that Nell-1 is likely a downstream 
target of Runx2. These findings may also extend our understanding of the 
molecular mechanisms governing the pathogenesis of craniosynostosis.