RANKL subcellular trafficking and regulatory mechanisms in osteocytes

Abstract

The receptor activator of the NF-kB ligand (RANKL) is the central player in the regulation of osteoclastogenesis, and the quantity of RANKL presented to osteoclast precursors is an important factor determining the magnitude of osteoclast formation. Because osteoblastic cells are thought to be a major source of RANKL, the regulatory mechanisms of RANKL subcellular trafficking have been studied in osteoblastic cells. However, recent reports showed that osteocytes are a major source of RANKL presentation to osteoclast precursors, prompting a need to reinvestigate RANKL subcellular trafficking in osteocytes. Investigation of molecular mechanisms in detail needs well-designed in vitro experimental systems. Thus, we developed a novel co-culture system of osteoclast precursors and osteocytes embedded in collagen gel. Experiments using this model revealed that osteocytic RANKL is provided as a membrane-bound form to osteoclast precursors through osteocyte dendritic processes and that the contribution of soluble RANKL to the osteoclastogenesis supported by osteocytes is minor. Moreover, the regulation of RANKL subcellular trafficking, such as OPG-mediated transport of newly synthesized RANKL molecules to lysosomal storage compartments, and the release of RANKL to the cell surface upon stimulation with RANK are confirmed to be functional in osteocytes. These results provide a novel understanding of the regulation of osteoclastogenesis.

Magnitude-dependent response of osteoblasts regulated by compressive stress

“…The up-regulated osteoclastic differentiation was further demonstrated by increased TRAP activity in RAW264.7 cells co-cultured with MC3T3-E1 cells exposed to 2 g/cm 2 . As RANKL binds to RANK as a membrane-bound form34, it was not detected in the supernatants. Expression of Opg increased significantly at gene and protein levels when the stress increased to 5 g/cm 2 and TRAP activity was inhibited at the same magnitude.…”

Parathyroid hormone: anabolic and catabolic actions on the skeleton

“…Accordingly, in mice with conditional deletion of the PTH receptor in osteocytes, PTH fails to increase RANKL expression, and thereby osteoclastogenesis [45 • ]. In vitro assays using a co-culture system of osteocytes embedded in collagen gel and osteoclasts show that the osteocyte-derived RANKL reaches osteoclast precursors in its membrane-bound form through osteocyte dendritic processes [49]. …”

Peptide drugs accelerate BMP‐2‐induced calvarial bone regeneration and stimulate osteoblast differentiation through mTORC1 signaling

“…ST‐2 cells (a murine osteoblastic cell line [RIKEN, Ibaragi, Japan]), were maintained in α‐minimum essential medium (Sigma‐Aldrich) containing 10% fetal bovine serum (Moregate Biotech), penicillin (100 U/mL) and streptomycin (100 µg/mL) (Sigma‐Aldrich) 31, 32. After 12 hours of serum starvation, the cells were treated with vehicle or various concentrations of W9 or OP3‐4, as indicated, for 20 minutes.…”

“…After 12 hours of serum starvation, the cells were treated with vehicle or various concentrations of W9 or OP3‐4, as indicated, for 20 minutes. Total cell lysates were prepared and analyzed by Western blotting as described previously 31, 32. Briefly, ice‐cold PBS was used to stop the stimulation and the cells were immediately lysed lysis buffer (PBS, pH 7.4, 1.0% Triton X‐100) supplemented with protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) and phosphatase inhibitors (Sigma‐Aldrich).…”