Over two fold upregulation of AbMole (-)-Tetramisole 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. The repressive regulation of NELL-1 by Osterix may seem paradoxical given that both are known to be pro-osteoblastic, with many reports having shown that Osterix and NELL-1 can positively regulate osteoblast differentiation. However, in reality, this is not uncommon. For instance, Osterix, a proosteogenic regulator, negatively regulates the Wnt signaling pathway which is known to play a crucial role in the control of bone mass. Osterix inhibits the Wnt signaling pathway through several mechanisms, including binding to and activating the Wnt antagonist DKK1 promoter, or interrupting TCF binding to its DNA elements and then suppressing downstream b-catenin activity. 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. What is known is that NELL-1 is a critical component in regulating osteoblastic differentiation, and that both Runx2 and Osterix are involved in its transcriptional regulation and osteogenic function. Runx2, a positive regulator of NELL-1, is highly expressed during transition from AbMole Nodakenin mesenchymal cells to preosteoblasts and immature osteoblasts. NELL-1 may be an effector of a large portion of Runx29s role, as it is a key downstream functional mediator in this process. Osterix negative regulation of NELL-1, which is also tightly regulated by Runx2, may result from a delicate balancing of various driving forces in this regulatory network, modulating NELL-1 expression levels as needed at different developmental time points. Moreover, the overexpression of NELL-1 also affects Runx2 expression levels or bioactivity reciprocally, adding to the complexity of the regulatory network. We expect that the regulatory relationship between NELL-1 and Osterix presented here from our in vitro studies is likely also true in vivo.