Roles of Notch and Nov signaling on regeneration of hard tissues and a development of their clinical application

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Notch is a transmembrane receptor that plays a crucial role in differentiation of stem cells. Its role in the formation of mesenchymal tissues has also been implicated although detailed mechanism has not yet been analyzed. To elucidate the function of Notch signaling in osteogenesis, we transfected the constitutively active Notch 1 (Notch intracellular domain, NICD) into two different osteoblastic mesenchymal cell lines, Kusa-A1 and KusaO, and established stable transformants (KusaA1NICD and KusaONICD) to examine the Notch signaling. NICD generally suppressed the expression of osteogenic marker genes, calcium deposition and in vitro mineralization in both Kusa-A1 and KusaO. The promoter activities of the Cbfa1 and the Ose2 element were attenuated by NICD. In vivo bone formation of Kusa-A1 was significantly suppressed by NICD. These results suggest that Notch signaling functions as a general coordinator of osteogenesis through cell-cell interactions.Nephroblastoma overexpressed gene (Nov : CCN3) is a cysteine-rich protein that is overexpressed in avian nephroblastomas, and it is thought to be involved in the control of cell proliferation and development of various tissues. As Nov has recently reported to bind Notch, we evaluated the Nov signaling in the osteoblastic mesenchymal cell lines. Transfection of Nov expression vector to Kusa-A1 induced downregulation of osteoblastic markers. On the centrally, Notch related genes were upregulated. DNA microarray revealed that Nov expression was highly induced in KusaA1NICD. Therefore, Nov may induce the Notch signaling, which further induced Nov expression, and this cycle may be essential to keep the cells in immature condition.