Role of Notch Signaling in BMP2-Induced Osteogenic Differentiation of MEFs and Its Mechanism

Abstract: This study was aimed to investigate the role of Notch in bone morphogenetic protein 2 (BMP2)- induced osteogenic differentiation in mouse embryonic fibroblasts (MEFs). The over-express DLL1 (one of the Notch ligands) adenoviruses (adenovirus-delta-like， Ad-DLL1)， dominant-negative mutant of Notch1 (one of the Notch receptors) adenoviruses (adenovirus-dominant-negative mutant of Notch1， Ad-dnNotch1) and specific γ-secretase inhibitor {N-[N-(3，5-difluorophena-cetyl-L-alanyl)]-S-phenylglycine t-butyl ester， DAPT} were used to infect or treat MEFs， respectively. The early osteogenic index alkaline phosphatase (ALP) and late osteogenic index calcium deposits were detected by cytochemical staining and/or activity determination. The expression levels of BMP2 receptors and osteogenesis-related genes， the phosphorylation level of Smad1/5/8， and the transcriptional activity level of Smad-binding element (SBE) were determined by qRT-PCR， Western blot and luciferase reporter assay， respectively. The results showed that， compared with control group， DLL1 could obviously promote BMP2- mediated osteogenic differentiation (P<0.05). DLL1 could also increase the mRNA levels of ALK2 and other BMP2 signaling receptors， the phosphorylation level of Smad1/5/8 and the transcriptional activity level of SBE in MEFs (P<0.05). Correspondingly， dnNotch1 and DAPT exert negative effects on the indexs mentioned above (P<0.05). On the other hand， Hey1， the classical target gene of Notch signal， could promote BMP2-induced osteogenic differentiation. We furthermore found that Hey1 could reverse the inhibitory effect of DAPT on BMP2-induced osteogenic differentiation (P<0.01). These results indicated that Notch signaling could remarkably enhances BMP2- induced osteogenesis in MEFs and might exerts its effect though affecting the activation of BMP2/Smads signaling， Hey1 plays an important role in this process.

CSTR: 32200.14.cjcb.2018.04.0003