The transcriptional level in multiple human osteoblast-like and non-osteoblastic cell lines, and verified that this inhibitory effect on NELL-1 expression with and without Runx2 induction involves Osterix direct binding of Sp1 sites in the NELL-1 promoter in a human osteosarcoma cell line, Saos2. We also verified that Nell-1 has inhibitory effects on Osterix expression during osteoblastic differentiation reciprocally. Taken together, we conclude that a delicate balance of regulatory effects on Nell-1 transcription by Osterix and Runx2 is crucial, and these novel findings provide new insights into the underlying mechanism of Nell-19s action during osteochondral differentiation. In addition to the Saos2 cells, the transcriptional repression of Nell-1 promoter by Osterix was also detected in other human cell lines including an immature osteoblast, U2OS cells and two non-osteoblastic cell lines, Hela and Glioma cells. Similar results in these cell lines compared to results in Saos2 cells further suggest that the transcriptional repression of NELL-1 promoter by Osterix exists in human cells irrespective of tissue origin or degree of osteoblastic maturity. NELL-1 is a novel osteoinductive factor under direct transcriptional regulation of Runx2, the master transcription factor of osteogenesis. Osterix is another essential transcription factor for osteoblast differentiation and bone formation directly downstream of Runx2. In this study we sought to determine the regulatory and functional relationship between these two downstream targets of Runx2, in particular to validate the functional characteristics of potential Osterix binding sites in the human NELL-1 promoter revealed by in silico analysis. Our data showed that Osterix exhibits repressive instead of assumed inductive effect on NELL-1 expression at the transcriptional level by binding directly to Sp1 sites in the NELL-1 promoter region; a surprising finding given the fact that NELL-1 and Osterix are both considered pro-osteogenic factors. This adds NELL-1 as a member of Osterix regulated molecules that include Col 1a, Col 11a2, DKK1 and IL-1a. Like IL1-a, NELL-1 is also negatively regulated by Osterix. In addition, we also found that the Sp1 binding elements in the human NELL-1 promoter, identified as two AbMole Indinavir sulfate clusters, Site A and B, have similar capacity to be fully occupied by Osterix to mediate repression. The release of this repression can occur only when Site A and B are mutated simultaneously. The definitive mechanisms underlying the activating or inhibitory effects of Osterix on target promoters of these molecules remain unclear. Interestingly, basic transcription element B1, a Sp1-like AbMole Pyriproxyfen protein, has been found to activate transcription on promoters containing multiple GC boxes but act as a repressor on promoters containing only a single GC box. This differential effect on multiple versus single GC box in gene promoters also applies to Osterix direct targets including activation of Col 11a2 and DKK1 which both have multiple binding sites, and repression of IL-1a which has a single.