CBF1 (RBP-Jκ) promotes osteogenic potentials of Kusa-A1 cells in vivo

Objective: Evolutionarily conserved transcription factor CBF1, also known as RBP-Jκ, has been shown to be the primary nuclear mediator of Notch signaling pathway. The aim of this study was to investigate the role of CBF1 during the osteogenic differentiation of mouse mesenchymal progenitor cell, Kusa-A1 in vivo. Methods: Stable CBF1-expressing cell line, Kusa-A1/CBF1 was generated using Flp-In mammalian expression system, during which a control cell, Kusa-A1/host was generated as well. C3H/HeN mice were used for the in vivo osteogenesis assays. The cells were subcutaneously injected into the abdomen of mice. Thirty days after inoculation the animals were sacrificed and soft X-ray photos were taken to examine the calcification. The bone-like tissues originating from the injected cells were dissected and processed for histological analysis. Non-decalcified frozen sections were used for Von Kossa's staining and decalcified frozen sections were stained with ALP, TRAP and HE methods. Results: Establishment of Kusa-A1/CBF1 was proved successful by RT-PCR and Western-blot. Thirty days after inoculation, although both Kusa-A1/CBF1 and Kusa-A1/host formed nodular masses in mice, the masses formed by Kusa-A1/CBF1 was larger in size and weighed more than those formed by Kusa-A1/host. In histology, Kusa-A1/CBF1 formed more compact structure of trabecular bone-like tissues than Kusa-A1/host did. The bone-like tissues formed by Kusa-A1/CBF1 also exhibited more calcium deposits and stronger ALP staining than those by Kusa-A1/host. Multinucleated TRAP-positive cells were only observed in the samples formed by Kusa-A1/CBF1 but not in those formed by Kusa-A1/host. Conclusion: CBF1 may exert a positive role in osteogenic differentiation of Kusa-A1 cells, thus promoting osteogenic activities of Kusa-A1 cells. (This study was supported in part by Japan-China Sasakawa Medical Fellowship)