Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Abstract

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state.

Notch activation promotes osteoblast mineralization by inhibition of apoptosis

“…During intramembranous ossification, mature osteoblasts synthesize and secrete large amounts of type I collagen, non-collagenous proteins, enzymes, and growth factors, which comprise major Coordinated early proliferation of pre-committed osteoblasts and differentiation of mature osteoblasts is required for generation of adequate numbers of functional cells to promote later mineralization during normal growth and development of the skeleton (Karsenty, 2008). It has been suggested that JAG1 exerts diverse effects on mesenchymal stromal cells (MSCs) osteoblastic differentiation depending on the timing of treatment and the stage of stromal cells (Kaur, Ahn, Hankenson, & Ashley, 2017;Wang, Liu, Buhl, & Rowe, 2005). Here we show that Notch signaling in the osteoblasts is essential for early proliferation, later differentiation, and subsequent mineralization.…”

Suppression of Notch Signaling in Osteoclasts Improves Bone Regeneration and Healing

“…Osteoclast precursors were cultured and differentiated as previously described . Briefly, bone marrow from femurs and tibias were flushed using α‐minimum essential medium (α‐MEM) and centrifuged.…”

“…Notch signaling was stimulated using immobilized Jagged1 as previously described . Goat immunoglobulin G (Jackson Immunology, West Grove, PA) and Jagged‐1, a Notch ligand (R&D Systems, Minneapolis, MN) were made in PBS at a concentration of 10 µg/ml and added to 48‐well plates.…”

“…[27][28][29] Osteoclast Culture and Maintenance Osteoclast precursors were cultured and differentiated as previously described. 21,30 Briefly, bone marrow from femurs and tibias were flushed using α-minimum essential medium (α-MEM) and centrifuged. Cells were later incubated overnight to allow adherent cells to attach to the plate.…”

“…Notch signaling was stimulated using immobilized Jagged1 as previously described. 21,30 Goat immunoglobulin G (Jackson Immunology, West Grove, PA) and Jagged-1, a Notch ligand (R&D Systems, Minneapolis, MN) were made in PBS at a concentration of 10 µg/ml and added to 48-well plates. Wells were then washed using PBS and later 26,000 osteoclast precursors were seeded (24 h pre-exposure to either MCSF alone or both MCSF and RANKL).…”