Reciprocal Regulation of Notch and Nuclear Factor of Activated T-cells (NFAT) c1 Transactivation in Osteoblasts

Notch are transmembrane receptors involved in the determination of cell fate. Nuclear factor of activated T-cells (NFAT) c are transcription factors that control cell differentiation and function. We tested whether Notch and NFAT signaling pathways interacted in osteoblastic cells. Notch signaling was induced in ST-2 cells using vectors expressing Notch1 intracellular domain (NICD), and in Rosa(Notch) osteoblastic cells by Cre recombinase-mediated excision of a loxP-flanked STOP cassette cloned between the Rosa26 promoter and NICD. NFATc1 was induced in Rosa(Notch) osteoblastic cells by transducing an adenoviral vector expressing constitutively active NFATc1. Notch inhibited NFAT transactivation and NFATc1 transcription. In ST-2 cells, suppression of NFAT transactivation by Notch was reversed by constitutively active cGMP-dependent protein kinase type II. NFATc1 inhibited the transactivation of Notch target genes, and competed for binding to DNA with the Notch interacting protein Epstein-Barr virus latency C promoter binding factor-1, suppressor of hairless, Lag-1 (CSL). Co-immunoprecipitation and confocal microscopy demonstrated that NFATc1 and CSL interacted. Studies on the effects of NICD and NFATc1 on the differentiation and function of osteoblastic cells demonstrated that NICD and NFATc1 inhibited expression of osteoblast gene markers in Rosa(Notch) osteoblasts, but only NICD suppressed the commitment of bone marrow stromal cells to the osteoblastic lineage. In conclusion, NICD and NFATc1 reciprocally inhibit their signaling pathways, and form a regulatory network to control their activity in osteoblasts.