Objective       To determine the role of Notch1 in the osteogenic differentiation of immortalized mouse embryonic fibroblasts (iMEF) induced by bone morphogenetic proteins 9 (BMP9).       Methods       iMEF cells were treated with dominant negative mutant adenovirus AdRdnNotch1/dnNotch1 and BMP9. And the cells treated by the adenovirus vector containing red fluorescence protein (RFP) served as control. The early osteogenic index alkaline phosphatase (ALP) was observed by ALP staining and its activity was determined. While the late osteogenic indexes osteopontin (OPN) and osteocalcin (OCN) were measured with qRT-PCR. The protein expression of p-Smad1/5/8 and Smad binding element (SBE) activity were studied by Western blotting and luciferase detection. The effects of dnNotch1 on the cell proliferation

cycle and apoptosis were determined with crystal violet staining and flow cytometry.       Results       Compared with the control cells

the ALP activity was significantly decreased in 3

5 and 7 d after the treatment of dnNotch1 combined with BMP9 in a dose-dependent manner (P0.05). On the 11th day

the expression of OPN and OCN was obviously decreased (P0.05)

but the level of Smad1/5/8 had no remarkable change though the protein level of p-Smad1/5/8 and the activity of SBE were inhibited significantly (P0.05). Crystal violet staining and flow cytometric analysis indicated that dnNotch1 suppressed cell proliferation induced by BMP9 at the early stage

but had no effect on cell apoptosis.       Conclusion      dnNotch1 can competitively inhibit the Notch1 signal

and thus significantly decrease the osteogenesis induced by BMP9

which might be due to suppressing the activity of BMP signal pathway and the early proliferation of iMEF cells

suggesting that Notch1 may play a promoting role in BMP9-induced osteogenesis.