Notch Suppresses Nuclear Factor of Activated T Cells (Nfat) Transactivation and Nfatc1 Expression in Chondrocytes

Notch1 to Notch4 transmembrane receptors determine cell fate, and release of the Notch intracellular domain (NICD) in the cytoplasm induces gene expression. Notch regulates endochondral ossification, but it is not clear whether Notch interacts with signals controlling chondrocyte differentiation. Nuclear factor of activated T cells (Nfatc) transcription factors regulate chondrogenesis, and we asked whether Notch modifies Nfat signaling in chondrocytes. Notch was induced in teratocarcinoma ATDC5 chondrogenic cells infected with a retroviral vector, where the cytomegalovirus (CMV) promoter directs NICD expression. NICD suppressed chondrocyte differentiation and inhibited Nfat transactivation and Nfatc1 expression. Notch was activated in chondrocytes from Rosa(Notch) mice, where the Rosa26 promoter is upstream of a loxP-flanked STOP cassette and NICD. To excise the STOP cassette and express NICD, Rosa(Notch) chondrocytes were infected with an adenoviral vector where the CMV promoter directs Cre expression (Ad-CMV-Cre). Notch1 and Notch2 mediate the effects of Notch in skeletal cells, and to inhibit Notch signaling, chondrocytes from mice homozygous for Notch1 and Notch2 alleles targeted with loxP sites were infected with Ad-CMV-Cre. NICD suppressed chondrogenic nodules formation and expression of selected chondrocyte gene markers, induced Col10a1 and Mmp13, and suppressed Nfat transactivation and Nfatc1 expression, whereas inactivation of Notch1 and Notch2 did not affect chondrocyte differentiation. To investigate Nfatc1 function in chondrocytes, Nfatc1 was induced in Rosa(Notch) chondrocytes overexpressing NICD or controls. Nfatc1 suppressed chondrocyte differentiation and opposed Col10a1 induction by Notch. In conclusion, Notch suppresses Nfat transactivation in chondrocytes and Notch and Nfatc1 regulate chondrocyte differentiation. (Endocrinology 154: 762-772, 2013)