However, the exact role Osterix plays, along with RNA polymerase II, in the negative regulation of NELL-1 with and without Runx2 induction remains unclear and warrants further study. Notably, there has been no evidence to date that Osterix and Runx2 TWS119 GSK-3 inhibitor interact with each other directly to alter their DNA binding and promoter transactivating activities . To determine how Osterix repressive transcriptional regulation of NELL-1 affects its osteogenic activity, we performed in vitro osteoblastic differentiation studies with either overexpression or specific siRNA knockdown of Osterix in Saos2 as well as in normal primary human osteoblast cells. Expectedly, the mRNA expression of NELL-1 was severely inhibited by overexpression of Osterix. Notably, NELL-1 repression was associated with the early transient decrease of Ocn and Opn mRNA indicating some level of impairment of NELL-1 osteoinductive capacity. In line with these findings, over two fold upregulation of NELL-1 mRNA along with increase of Opn at the early phase, and increase of Ocn and mineralization at the late stage of osteoblastic differentiation were observed after Osterix knock down by specific siRNA. Interestingly, the different pattern of Opn expression between Saos-2 osteosarcoma cells and normal primary human osteoblast cells suggests a more complicated role for Osterix in osteoblastic differentiation at different maturation stages of human osteoblasts. Taken together, these data definitively demonstrate the functional impact and significance of Osterix repression of NELL-1. Furthermore, the forced expression of NELL-1 remarkably reduced Osterix mRNA levels in Saos-2 cells , demonstrating reciprocal repression of Osterix by NELL-1. This further confirmed our previous study on MC3T3 cells that showed transduction of AdNELL-1 inhibited Osterix mRNA expression without affecting Runx2 mRNA levels . Studies on the inter-relationship among various factors involved in the transcriptional regulatory network of osteogenesis are few in number and provide only limited answers likely owing to the high complexity of this area of study.