Glucocorticoids inhibit notch target gene expression in osteoblasts

Glucocorticoids in excess suppress osteoblast function and cause osteoporosis. We demonstrated that cortisol induces the expression of selected Notch receptors in osteoblasts, revealing a potential mechanism for the skeletal effects of glucocorticoids. However, it remains to be determined whether increased expression of Notch receptors results into enhanced signaling. Following activation of Notch, its intracellular domain (NICD) binds to the DNA-associated protein recombination signal binding protein for immunoglobulin kappa-J region (RBPJ) and induces the expression of target genes such as Hey1, Hey2, and HeyL. To determine whether glucocorticoids modulate Notch signaling in the skeleton, 1 month old wild-type mice were administered prednisolone or placebo and sacrificed after 72 h, and gene expression was analyzed in femoral bone. Prednisolone induced Tsc22d3, a glucocorticoid target gene, and suppressed Hey1 and HeyL expression, which is indicative of inhibited Notch receptor activity or direct Hey downregulation. To determine the mechanisms of Hey suppression, wild-type osteoblast-enriched cells were seeded on the Notch cognate ligand Delta-like (DLL)1 or transfected with constructs expressing the NOTCH1 NICD fragment and exposed to either cortisol or vehicle. Cortisol opposed the induction of mRNA and heterogeneous nuclear RNA for Hey1, Hey2, and HeyL by DLL1, but had no effect on mRNA stability, indicating that glucocorticoids inhibit Hey expression by transcriptional mechanisms. Transactivation studies and electrophoretic mobility shift assays revealed that cortisol did not oppose RBPJ-mediated transcription or RBPJ/DNA interactions, respectively. In conclusion, glucocorticoids suppress expression of Hey1, Hey2, and HeyL in osteoblasts by RBPJ-independent transcriptional mechanisms.