Chondrocytes are known to express Sp7/Osterix (Osx) to varying degrees, but the role of Osx in chondrocytes is still unknown. In the current study, we investigated the role of the Osx gene using the clonal mouse embryonic cell line ATDC5, which retains the properties of the chondroprogenitor. ATDC5 cells express Osx; therefore, the effects of Osx gene silencing with shRNA lentiviral particles on chondrocyte marker gene expression and alkaline phosphatase (ALP) activity were investigated. At confluency, gene silencing down-regulated expression of the Sox trio (Sox5, 6, 9), DIx5 and Alp mRNA, as well as ALP enzyme activity. Bone morphogenetic protein 2 (BMP2) is known to induce Osx gene expression in chondrocytes, and stimulation with BMP2 rescued Osx expression accompanied by up-regulation of Alp expression and ALP enzyme activity in a dose-dependent manner. To clarify the role of Osx in chondrocyte differentiation, cells induced to differentiate by 10 mu g/ml insulin for 21 days were examined. Gene silencing inhibited the expression of the hypertrophic chondrocyte marker gene, type X collagen (Col X), and attenuated up-regulation of DIx5 and Alp mRNA, as well as ALP enzyme activity. Taken together, these results suggest that Osx is involved in chondrogenic gene activation and is a positive regulator of the chondrocyte differentiation. (C) 2010 Elsevier Inc. All rights reserved.