Role of the RAM domain and ankyrin repeats on notch signaling and activity in cells of osteoblastic lineage

Abstract

Notch proteins belong to a family of single pass transmembrane receptors that are activated after interactions with the membrane-bound ligands Delta and Jagged/Serrate. We determined the pathways responsible for the inhibitory effects of Notch on osteoblastogenesis and the contributions of the RAM domain and ankyrin repeats to this process in cells of the osteoblastic lineage.

Introduction: Notch receptors play a role in osteoblast differentiation. Activation of Notch results in its cleavage and the release of its intracellular domain (NICD), which interacts with the CBF1/RBP-Jkappa, Suppressor of Hairless, Lag-1 (CSL) family of transcription factors. The interaction is presumably mediated by the RBP-Jkappa-associated module (RAM) of NICD, although the role of the ankyrin repeats is uncertain.

Materials and methods: To determine the contributions of the RAM domain and ankyrin repeats to the inhibitory effects of Notch on osteoblastogenesis, ST-2 and MC3T3-E1 cells were transfected or transduced with vectors expressing NICD, RAM (NICD DeltaRAM), and ankyrin (NICD DeltaANK) deletion mutants.

Results: Notch increased the transactivation of transiently transfected 12xCSL-Luc constructs, containing 12 repeats of an RBP-Jkappa/CSL binding site, and of the hairy and E (spl) (HES)-1 promoter. Deletion of the ankyrin repeats resulted in the loss of 12xCSL-Luc and HES-1 promoter transactivation, whereas deletion of the RAM domain caused a partial loss of 12xCSL-Luc and sustained HES-1 promoter transactivation. NICD overexpression inhibited osteocalcin mRNA levels and alkaline phosphatase activity in ST-2 cells, and deletion of the ankyrin repeats, and to a lesser extent of the RAM domain, resulted in loss of the NICD inhibitory effect. NICD inhibited Wnt signaling and deletion of ankyrin repeats or the RAM domain restored Wnt signaling activity.

Conclusions: The RAM domain and ankyrin repeats are required for Notch signaling and activity, and the CSL pathway is central to the inhibitory effect of Notch on osteoblastogenesis.