Notch ligands disseminate the osteogenic signals of bone morphogenetic protein

Abstract

Bone morphogenetic proteins (BMP) play a well-established role in osteoblastogenesis while the role of canonical Notch signaling in osteoblastogenesis is less-well understood. To determine common regulatory mechanisms between Notch-mediated and BMP-mediated human osteoblastic differentiation, three independent human mesenchymal stem/progenitor cells (hMSC) were stimulated in parallel with either the Notch ligand Jagged-1 or BMP2, and bulk RNA sequencing was performed. At FDR<10%, 2906 and 342 genes were differentially expressed between BMP2 and Jagged-1 treated cells, respectively. Genes associated with osteoblast differentiation, such as SPP1, SOST, DKK1, Osterix (SP7) and DMP1 were induced in the BMP2-treated cells, but not in the Jagged-1-treated cells. 45 genes were commonly upregulated, and 69 genes were commonly downregulated (FC>1.5). Ingenuity pathway analysis of commonly upregulated genes showed upregulation of Notch signaling; Regulation of the Epithelial-Mesenchymal Transition Pathway; Osteoarthritis Pathway; RhoA signaling; and Th2 pathway as top biological processes. It was notable that two of the top five commonly upregulated genes belonged to the Notch signaling pathway (HEY2 and HES4), and additionally both induction protocols induced JAG1 and NOTCH3. A direct regulatory role of Notch pathway activation was established during BMP2 mediated osteoblastogenesis by using DAPT, a gamma-secretase inhibitor, which showed that terminal differentiation could be abolished by blocking Notch activation for up to 24 hours after BMP stimulation. This result was also supported by impaired osteoblastogenesis of RBPJ silenced cells. On the contrary, co-stimulation with BMP2 and Jagged-1 synergistically enhanced alkaline phosphatase (ALPL) expression and extracellular matrix mineralization. A requirement for JAG1 was demonstrated during BMP2-dependent osteoblastogenesis, since JAG1 siRNA transfected cells showed diminished ALPL expression and extracellular calcium deposition. Further, exogenous Jagged-1 administration could rescue the differentiation defect of JAG1-silenced hMSCs, since JAG1-silenced cells that received exogenous Jagged-1 (and additionally stimulated with BMP2) showed restored expression of ALPL and calcium deposition. Rescue of JAG1 deficiency in hMSCs could also be restored with another Jagged ligand, Jagged-2, suggesting Notch ligand promiscuity in affecting BMP2-induced osteogenesis. In vivo evidence for BMP2 and Notch intersection was also demonstrated since systemic treatment with the gamma-secretase inhibitor, diabenzazepine, which blocks Notch signaling, resulted in impaired BMP-induced bone formation in a calvarial defect at day 42. Taken together, these data advance our current understanding of basic mechanisms regulating bone formation and highlight the role of aberrant Notch signaling associated with human skeletal disorders, such as those seen in Alagille syndrome. Various in vivo experiments are currently underway to evaluate the effect of Cre-mediated recombination of Rbpj, Jag1 and Jag2 within the cells of the skeletal lineage on murine intramembranous and endochondral bone regeneration.