Figure 7. A constitutive activation of Notch signaling rescues BMP9-induced ALP activity that is diminished by Rmst silencing. (A) Subconfluent iMADs were co-infected with Ad-BMP9, Ad-GFP, Ad-simRmst, and/or Ad-NICD1. Quantitative measurement of relative ALP activity was determined at 3, 5 and 7 days after infection. Assays were done in triplicate. “*”, p<0.05, “**”, p<0.01, when Ad-BMP9+Ad-simRmst group vs. Ad-BMP9+Ad-NICD1+Ad-simRmst group. (B) A working model for the role of Rmst-miRNA-Notch regulatory loop in mediating BMP9-induced osteogenesis through Notch signaling. While BMP9 can induce osteogenic differentiation directly through Notch or other mediators, lncRNA Rmst provides an important delicate modulation of this process. The expression of Notch receptors and ligands is normally suppressed by a panel of miRNAs. BMP9 induces lncRNA Rmast, which subsequently sponges out those Notch-targeting miRNAs, leading to the de-suppression of Notch signaling and facilitating bone formation. The constitutive Notch activator NICD1 can bypass the Rmst-miRNA loop and directly activate Notch downstream events.