A Study On NELL-1’s Functional Mechanism And Interacting Proteins Identification In Osteoblasts

Nel-like protein1(NELL-1) is a novel secreted protein associated with premature fusion of cranial sutures in craniosynostosis. As a potent growth factor, NELL-1is highly specific to the osteochondral lineage, and has demonstrated robust induction of bone in multiple in vivo models from rodents to pre-clinical large animals. This study aimed at contributing to the understanding of NELL-1’s functional mechanism through various aspects including identification of its regulatory factor and interaction protein. Promoter analysis showed a cluster of potential Spl sites (Spl/Osterix binding sites) within approximately70bp (from-71to-142) of the5’flanking region of the human NELL-1transcriptional start site. Luciferase activity in our NELL-1promoter reporter systems was significantly decreased in Saos2cells when Osterix was overexpressed. Mutagenesis study demonstrated that this suppression is mediated by the Sp1sites. The binding specificity of Osterix to these Spl sites was confirmed in Saos2cells and primary human osteoblasts by EMSA in vitro and ChIP assay in vivo. ChIP assay also showed that Osterix downregulated NELL-1by affecting binding of RNA polymerase Ⅱ to the NELL-1promoter, but not by competing with Runx2binding to the OSE2sites. Moreover, NELL-1mRNA levels were significantly decreased when Osterix was overexpressed in Saos2, U2OS, Hela and Glioma cells. Correspondingly, knockdown of Osterix increased NELL-1transcription and osteoblastic differentiation in both Saos2cells and primary human osteoblasts. These results suggest that Osterix is a direct transcriptional regulator with repressive effect on NELL-1gene expression, contributing to a delicate balance of regulatory effects on NELL-1transcription 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-1and demonstrate their crosstalk during osteogenesis. We constructed a T7phage display system expressing proteins or peptides from Saos2cDNA library and identified apoptosis related protein3(APR3), a membrane associated protein known as a proliferation suppressor, as a binding protein of NELL-1by biopanning. Immunoprecipitation and confocal investigation showed that NELL-1and APR3co-overexpression indeed have a physical interaction and colocalized on the nuclear envelope of human osteoblasts. NELL-1significantly inhibited proliferation of osteoblasts co-transfected with APRS through further down-regulation of Cyclin D1. The co-expression of NELL-1and APR3enhanced Ocn and Bsp expression and mineralization. RNAi of APR3significantly reduced the differentiation effect of NELL-1. These findings suggest that the effects of NELL-1on osteoblastic differentiation and proliferation are partly through binding to APR3.