There was no obvious difference seen in Runx2 binding to NELL-1 promoter with and without Osterix forced expression . Interestingly, the general transcription factor RNA polymerase II binding to NELL-1 promoter was significantly decreased in the Osterix overexpression group , indicating one possible mechanism for Osterix negative regulation of NELL-1 promoter activity. The data showed that Osterix forced expression 1056634-68-4 decreases NELL-1 mRNA levels in Saos-2 cells . To further demonstrate the effect of suppression, we also analyzed other osteoblastic marker mRNA levels after Osterix overexpression in Saos-2 cells and primary human osteoblasts. Interestingly, some markers such as Ocn and Opn expression levels also decreased following the decrease of NELL-1 expression at 2 days posttransfection . However, by 7 days post-transfection, Ocn and Opn expression levels showed no significant difference between the pCtr and pOsx groups in Saos-2 cells. Moreover, Ocn expression level also decreased in a similar fashion as Nell-1 at 2 days post-transfection in primary human osteoblasts , but Opn expression patterns were different between Saos-2 osteosarcoma cells and normal primary human osteoblast cells, which may indicate that overexpression of Osterix plays a transient and more complicated role with variable effects on bone marker gene levels at different stages of maturation of human osteoblasts. To further confirm Osterix suppression of NELL-1 expression, we inhibited Osterix mRNA level using siRNA in Saos-2 cells and primary human osteoblasts. Data showed that NELL-1 mRNA levels increased almost 3 fold 2 days after Osterix siRNA transfection at which time Osterix mRNA expression levels were decreased by 80% in Saos-2 cells . Ocn and Opn expression also increased slightly 2 days after transfection. At posttransfection day 7, when Osterix mRNA levels were still less than 30%, NELL-1 mRNA levels continued to be elevated. NELL-1 and Ocn mRNA levels also increased in a similar pattern at 7 days posttransfection . To further confirm Osterix regulation of NELL-1 in mature osteoblast cells, these experiments were performed in human primary osteoblasts. Although the inhibition efficiency of Osx-siRNAs in this cell line is less than that in Saos-2 cells at Day 2, NELL-1 mRNA levels showed significant increase along with significant changes in other bone markers after 7 days post Osterix siRNA transfection .