Osterix is Required for Sonic Hedgehog-Induced Osteoblastic MC3T3-E1 Cell Differentiation

It has been shown that hedgehog (Hh) signaling plays an important role during bone development. However, the mechanism(s) by which Hh stimulates osteoblast differentiation are not fully elucidated. This study was performed to examine if Sonic hedgehog (Shh) affects osteoblast differentiation in osteoblastic MC3T3-E1 cells and determine the exact role of osterix (Osx) involved in Hh-induced osteoblast differentiation. Our real-time RT-PCR result shows that Shh significantly induced osteoblast differentiation by up-regulation of osteocalcin, alkaline phosphatase, bone sialoprotein, Type I collagen, runt-related transcription factor 2 (Runx2), and Osx RNA expression in MC3T3-E1 cells. ALP protein activity, Osx protein expression, as well as Osx promoter activity was also enhanced by Shh treatment in cell culture. Interestingly, Shh-induced Osx up-regulation was only slightly affected by knocking down Runx2 using siRNA in cells within 3 days of culture, however, knocking down Osx expression in cells totally blocked Shh-induced osteoblast differentiation. These findings demonstrate for the first time that Shh stimulates early osteoblast differentiation mainly through up-regulation of the expression of Osx in osteoblastic MC3T3-E1 cells in both Runx2-dependent and Runx2-independent manner.