Notch signaling negatively regulates BMP9-induced osteogenic differentiation of mesenchymal progenitor cells by inhibiting JunB expression

Although interaction between BMP and Notch signaling has been demonstrated to be crucial for osteogenic differentiation of mesenchymal stem cells (MSCs), the precise molecular mechanism remains unknown. Here, we show that Notch intracellular domain (NICD) overexpression inhibits BMP9-induced C3H10T1/2 cell osteogenesis in vivo and in vitro. Our results show that activated Notch signaling results in down-regulation of Runx2 and early osteogenesis differentiation factors, without affecting p-Smad1/5/8 expression, and that blocking Notch signaling with DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) significantly increases p-Smad1/5/8 expression. Interestingly, Notch signaling also regulates the cell cycle by increasing PCNA (proliferation cell nuclear antigen) and CyclinD1 expression. Furthermore, similar results were obtained by ectopic bone formation and histological analyses, indicating that Notch signaling activation significantly inhibits BMP9-induced MSC osteogenic, cartilage and adipogenic differentiation. Moreover, we are the first to show that Notch regulates by suppressing JunB synthesis and that the negative effect of Notch is partially reversed by treatment with the JunB activator TPA (12-O-tetradeca-noylphorbol-13-acetate). Our findings demonstrate that Notch signaling significantly enhances cell proliferation but inhibits MSC osteogenic differentiation induced by BMP9 via JunB protein suppression rather than by BMP/Smad signaling regulation.